Amino acids, peptides and proteins. Volume 42 978-1-78801-408-3, 1788014081, 978-1-78801-062-7, 978-1-78801-002-3

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Published on 29 November 2017 on http://pubs.rsc.org | doi:10.1039/9781788010627-FP001

Amino Acids, Peptides and Proteins Volume 42

Published on 29 November 2017 on http://pubs.rsc.org | doi:10.1039/9781788010627-FP001

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Published on 29 November 2017 on http://pubs.rsc.org | doi:10.1039/9781788010627-FP001

A Specialist Periodical Report

Amino Acids, Peptides and Proteins Volume 42

Editors Maxim Ryadnov, National Physical Laboratory, London, UK Ferenc Hudecz, Eo ¨ tvo ¨ s Lorand University, Budapest, Hungary Authors Kenichi Akaji, Kyoto Pharmaceutical University, Japan Zolta´n Ba´no ´ czi, Eo ¨ tvo ¨ s L. University, Hungary Annarita Falanga, University of Naples Federico II, Italy Stefania Galdiero, University of Naples Federico II, Italy Ferenc Hudecz, Eo ¨ tvo ¨ s L. University, Hungary Pirjo Laakkonen, University of Helsinki, Finland Vadim Le Joncour, University of Helsinki, Finland Marc-Philipp Pfeil, University of Oxford, UK, and Harvard Medical School, USA Ja´nos Szoloma´jer, University of Szeged, Hungary Anthony Watts, University of Oxford, UK Ma´rta Zara´ndi, University of Szeged, Hungary

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ISBN: 978-1-78801-002-3 PDF eISBN: 978-1-78801-062-7 EPUB ISBN: 978-1-78801-408-3 ISSN: 1361-5904 DOI: 10.1039/9781788010627 A catalogue record for this book is available from the British Library r The Royal Society of Chemistry 2018 All rights reserved Apart from fair dealing for the purposes of research for non-commercial purposes or for private study, criticism or review, as permitted under the Copyright, Designs and Patents Act 1988 and the Copyright and Related Rights Regulations 2003, this publication may not be reproduced, stored or transmitted, in any form or by any means, without the prior permission in writing of The Royal Society of Chemistry, or in the case of reproduction in accordance with the terms of licences issued by the Copyright Licensing Agency in the UK, or in accordance with the terms of the licences issued by the appropriate Reproduction Rights Organization outside the UK. Enquiries concerning reproduction outside the terms stated here should be sent to The Royal Society of Chemistry at the address printed on this page. Published by The Royal Society of Chemistry, Thomas Graham House, Science Park, Milton Road, Cambridge CB4 0WF, UK Registered Charity Number 207890 For further information see our web site at www.rsc.org Printed in the United Kingdom by CPI Group (UK) Ltd, Croydon, CR0 4YY, UK

Preface

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DOI: 10.1039/9781788010627-FP005

Since 1969 this series of specialist periodical reports has covered the area of protein and peptide science without favouring topical polularity. This approach has helped to keep abreast with achievements in seemingly less acknowledged areas that would have limited coverage in the mainstream scientific literature. As any other series of this type these reports are presented as research accounts that evolve owing to emerging research areas. This volume continues the tradition of bringing new and established science together. The book reviewes literature predominantly published over the last three years, with each chapter providing fundamental concepts and terminology. This 42nd volume opens with a detailed discussion of contemporary developments in the applied research of amino acids (Zarandi and Szolomajer). The review focuses on the chemistry, origin and physical properties of amino acids. It starts with naturally occuring derivatives, both L- and D-epimers, introducing fundamentals of structural diversity, which is logically followed by application highlights ranging from pharmaceuticals to food supplements. Mechanisms of biological action and biosynthetic routes towards particular derivatives are then described, bridging with chemical syntheses and low- and high-resolution analytical methods used for physico-chemical characterisation. The second chapter ramps up the discussion to the challenge of synthetic strategies for the bioconjugation of amino acids and peptides (Banoczi and Hudecz). Various bioconjugates are discussed as proven research tools and validated precursors for therapeutic agents. The chapter predominantly stresses the importance of synthetic and semi-synthetic approaches, explaining conjugation mechanisms and presenting linkers commonly used for ligation and labelling. Therapeutic agents are exemplified by conjugates with known biologically active peptide moieties including the cell-adhesion RGD motif and antimicrobial peptides. Selected areas of applications feature peptide conjugates as immunogens and cellpenetrating drug delivery systems. Mechanisms by which peptides interact with, cross or disrupt cellular membranes are given in the following chapter (Pfeil and Watts). This report tackles the challenge of elucidating inter- and intra-molecular interactions underpinning the biological function of antimicrobial peptides, synthetic and native. Stateof-the-art NMR and EPR methods and innovative approaches are detailed with regards to solving lipid-peptide ensembles in membrane bilayers. The chapter links the conserved physicochemical parameters that are characteristic of antimicrobial peptides with the physical basis of their biological action. Structure-activity relationships of naturally occuring antimicrobial peptides are discussed in the following chapter (Falanga and Galdiero), which relates to the preceding report with an attempt to find a generic rationale for developing such peptides as drugs. The chapter gives a contemporary overview of recent and on-going efforts to Amino Acids, Pept. Proteins, 2018, 42, v–vi | v

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commercialise antimicrobial peptides with specific references to drug developers active in the area. Pharmaceutical developments addressing the problem of infectious diseases are further discussed in the next chapter (Akaji), which reviews progress in the design of selective peptidebased ligands able to interact with 3C like proteases of coronaviruses. The chapter is written in a form of an R&D protocol that provides information as to the structure of the protein target, its function in viruses and mechanisms of its inhibition by a variety of peptidomimetics. Comparative efficacies of the peptidomimetics presented are tabulated into IC50 values obtained against coronoviruses causing Severe Acute and Middle East Respiratory Syndromes (SARS and MERS, respectively). The chapter is built around the traditional concept of ligand–protein targeting, which remains key for pharmaceutical industry. A complementary strategy is presented in the closing chapter of the volume (Le Joncour and Laakkonen), which drives the topic of peptide-based drugs to exploiting the intrinsic properties of the peptides to specifically target cancer cells. Screening combinatorial technologies, e.g. phage displays, are discussed as effective discovery platforms for novel and highly selective peptides targeting tumour cells. The chapter outlines pros and cons of ex vivo, in vivo and in vitro screening approaches, highlighting technical issues that are critical for further progress. The chapter then concentrates on identified peptides with known and unknown targets and the application of these peptides for both tumour imaging and therapy. The report concludes the volume with a neat analogy of a Swiss-Army Knife relating to the multi-purpose function of peptides. Maxim Ryadnov and Ferenc Hudecz

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CONTENTS

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Cover Front cover image courtesy of Michael Shaw and Angelo Bella. The image shows a colour-coded depth projection of self-assembled peptide fibres arranged into star-like structures.

Preface

v

Amino acids: chemistry, diversity and physical properties ´rta Zara ´ndi and Ja ´nos Szoloma ´jer Ma

1

1 Naturally occurring amino acids 2 Chemical synthesis and resolution of amino acids 3 Analytical methods Abbreviations References

1 12 51 66 67

Amino acid and peptide bioconjugates ´n Ba ´no´czi and Ferenc Hudecz Zolta 1 Introduction 2 Synthesis 3 Selected applications of peptide-bioconjugates Abbreviations Acknowledgements References

85

85 85 112 129 131 131

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Magnetic resonance studies of antimicrobial peptides in membranes Marc-Philipp Pfeil and Anthony Watts 1 Introduction: peptide–lipid interactions 2 Electron paramagnetic resonance (EPR) 3 Solid-state nuclear magnetic resonance (NMR) 4 Discussion Acknowledgements References

Emerging therapeutic agents on the basis of naturally occurring antimicrobial peptides

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146 152 166 179 181 181

190

A. Falanga and S. Galdiero 1 Introduction 2 AMPs origin and classification 3 Mechanism of action 4 SAR of AMPs 5 Design of novel AMPS with improved activity 6 Nano-Antimicrobials: delivery systems for AMPs 7 AMPs with other activities 8 AMPs in clinical applications 9 Conclusion References

190 193 195 199 203 208 211 212 218 219

Advances in the design of ligands interacting with 3CL protease of novel coronaviruses causing infectious respiratory syndrome

228

Kenichi Akaji 1 Introduction 2 Coronavirus 3CL protease 3 Inhibitors of 3CL protease 4 Conclusions References

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Targeting peptides, a Swiss-Army Knife against cancer Vadim Le Joncour and Pirjo Laakkonen 1 Introduction 2 The phage display technology: screening and characterisation of tumour targeting peptides 3 Targeting peptides for cancer imaging and therapy 4 Concluding remarks List of abbreviations References

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280 281 292 307 307 309

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A short guide to abbreviations and their use in peptide science Abbreviations, acronyms and symbolic representations are very much part of the language of peptide science – in conversational communication as much as in its literature. They are not only a convenience, either – they enable the necessary but distracting complexities of long chemical names and technical terms to be pushed into the background so the wood can be seen among the trees. Many of the abbreviations in use are so much in currency that they need no explanation. The main purpose of this editorial is to identify them and free authors from the hitherto tiresome requirement to define them in every paper. Those in the tables that follow – which will be updated from time to time – may in future be used in this Journal without explanation. All other abbreviations should be defined. Previously published usage should be followed unless it is manifestly clumsy or inappropriate. Where it is necessary to devise new abbreviations and symbols, the general principles behind established examples should be followed. Thus, new amino-acid symbols should be of form Abc, with due thought for possible ambiguities (Dap might be obvious for diaminoproprionic acid, for example, but what about diaminopimelic acid?). Where alternatives are indicated below, the first is preferred. Amino Acids Proteinogenic Amino Acids Ala Alanine Arg Arginine Asn Asparagine Asp Aspartic acid Asx Asn or Asp Cys Cysteine Gln Glutamine Glu Glutamic acid Glx Gln or Glu Gly Glycine His Histidine Ile Isoleucine Leu Leucine Lys Lysine Met Methionine Phe Phenylalanine Pro Proline Ser Serine Thr Threonine Trp Tryptophan

A R N D C Q E G H I L K M F P S T W

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Tyr Val

Tyrosine Valine

Y V

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Copyright & 1999 European Peptide Society and John Wiley & Sons, Ltd. Reproduced with permission from J. Peptide Sci., 1999, 5, 465–471.

Other Amino Acids Aad a-Aminoadipic acid bAad b-Aminoadipic acid Abu a-Aminobutyric acid Aib a-Aminoisobutyric acid; a-methylalanine bAla b-Alanine; 3-aminopropionic acid (avoid Bal) Asu a-Aminosuberic acid Aze Azetidine-2-carboxylic acid Cha b-cyclohexylalanine Cit Citrulline; 2-amino-5-ureidovaleric acid Dha Dehydroalanine (also DAla) Gla g-Carboxyglutamic acid Glp pyroglutamic acid; 5-oxoproline (also pGlu) Hph Homophenylalanine (Hse = homoserine, and so on). Caution is necessary over the use of the prefix homo in relation to a-amino-acid names and the symbols for homo-analogues. When the term first became current, it was applied to analogues in which a side-chain CH2 extension had been introduced. Thus homoserine has a side-chain CH2CH2OH, homoarginine CH2CH2CH2NHC(¼NH)NH2, and so on. In such cases, the convention is that a new three-letter symbol for the analogue is derived from the parent, by taking H for homo and combining it with the first two characters of the parental symbol – hence, Hse, Har and so on. Now, however, there is a considerable literature on b-amino acids which are analogues of a-amino acids in which a CH2 group has been inserted between the a-carbon and carboxyl group. These analogues have also been called homo-analogues, and there are instances for example not only of ‘homophenylalanine’, NH2CH(CH2CH2Ph)CO2H, abbreviated Hph, but also ‘homophenylalanine’, NH2CH(CH2Ph)CH2CO2H abbreviated Hph. Further, members of the analogue class with CH2 interpolated between the a-carbon and the carboxyl group of the parent a-amino acid structure have been called both ‘a-homo’and ‘b-homo’. Clearly great care is essential, and abbreviations for ‘homo’ analogues ought to be fully defined on every occasion. The term ‘b-homo’ seems preferable for backbone extension (emphasizing as it does that the residue has become a b-amino acid residue), with abbreviated symbolism as illustrated by bHph for NH2CH(CH2Ph)CH2CO2H. Hyl d-Hydroxylysine Hyp 4-Hydroxyproline aIle allo-Isoleucine; 2S, 3R in the L-series Lan Lanthionine; S-(2-amino-2-carboxyethyl)cysteine Amino Acids, Pept. Proteins, 2018, 42, x–xvii | xi

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MeAla

Nle Orn Phg Pip Sar Sta Thi Tic aThr Thz Xaa

N-Methylalanine (MeVal = N-methylvaline, and so on). This style should not be used for a-methyl residues, for which either a separate unique symbol (such as Aib for a-methylalanine) should be used, or the position of the methyl group should be made explicit as in aMeTyr for a-methyltyrosine. Norleucine; a-aminocaproic acid Ornithine; 2,5-diaminopentanoic acid Phenylglycine; 2-aminophenylacetic acid Pipecolic acid; piperidine-s-carboxylic acid Sarcosine; N-methylglycine Statine; (3S, 4S)-4-amino-3-hydroxy-6-methyl-heptanoic acid b-Thienylalanine 1,2,3,4-Tetrahydroisoquinoline-3-carboxylic acid allo-Threonine; 2S, 3S in the L-series Thiazolidine-4-carboxylic acid, thiaproline Unknown or unspecified (also Aaa)

The three-letter symbols should be used in accord with the IUPAC-IUB conventions, which have been published in many places (e.g. European J. Biochem. 1984; 138: 9–37), and which are (May 1999) also available with other relevant documents at: http://www.chem.qnw.ac.uk/iubmb/iubmb. html#03 It would be superfluous to attempt to repeat all the detail which can be found at the above address, and the ramifications are extensive, but a few remarks focussing on common misuses and confusions may assist. The three-letter symbol standing alone represents the unmodified intact amino acid, of the L-configuration unless otherwise stated (but the L-configuration may be indicated if desired for emphasis: e.g. L-Ala). The same three-letter symbol, however, also stands for the corresponding amino acid residue. The symbols can thus be used to represent peptides (e.g. AlaAla or Ala-Ala = alanylalanine). When nothing is shown attached to either side of the three-letter symbol it is meant to be understood that the amino group (always understood to be on the left) or carboxyl group is unmodified, but this can be emphasized, so AlaAla = H-AlaAla-OH. Note however that indicating free termini by presenting the terminal group in full is wrong; NH2AlaAlaCO2H implies a hydrazino group at one end and an a-keto acid derivative at the other. Representation of a free terminal carboxyl group by writing H on the right is also wrong because that implies a terminal aldehyde. Side chains are understood to be unsubstituted if nothing is shown, but a substituent can be indicated by use of brackets or attachment by a vertical bond up or down. Thus an O-methylserine residue could be shown as 1, 2, or 3.

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Note that the oxygen atom is not shown: it is contained in the threeletter symbol – showing it, as in Ser(OMe), would imply that a peroxy group was present. Bonds up or down should be used only for indicating side-chain substitution. Confusions may creep in if the three-letter symbols are used thoughtlessly in representations of cyclic peptides. Consider by way of example the hypothetical cyclopeptide threonylalanylalanylglutamic acid. It might be thought that this compound could be economically represented 4.

But this is wrong because the left hand vertical bond implies an ester link between the two side chains, and strictly speaking if the right hand vertical bond means anything it means that the two Ala a-carbons are linked by a CH2CH2 bridge. This objection could be circumvented by writing the structure as in 5.

But this is now ambiguous because the convention that the symbols are to be read as having the amino nitrogen to the left cannot be imposed on both lines. The direction of the peptide bond needs to be shown with an arrow pointing from CO to N, as in 6.

Actually the simplest representation is on one line, as in 7.

Substituents and Protecting Groups Ac Acetyl Acm Acetamidomethyl Adoc 1-Adamantyloxycarbonyl Alloc Allyloxycarbonyl Boc t-Butoxycarbonyl Bom p-Benzyloxymethyl Bpoc 2-(4-Biphenylyl)isopropoxycarbonyl Btm Benzylthiomethyl Bum p-t-Butoxymethyl Bui i-Butyl Bun n-Butyl But t-Butyl Bz Benzoyl Bzl Benzyl (also Bn); Bzl(OMe) = 4-methoxybenzyl and so on Cha Cyclohexylammonium salt Amino Acids, Pept. Proteins, 2018, 42, x–xvii | xiii

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Clt Dcha Dde Ddz Dnp Dpp Et Fmoc For Mbh Mbs Me Mob Mtr Nps OA11 OBt OcHx ONp OPcp OPfp OSu OTce OTcp Tmob Mtt Pac Ph Pht Scm Pmc Pri Prn Tfa Tos Troc Trt Xan Z

2-Chlorotrityl Dicyclohexylammonium salt 1-(4,4-Dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl 2-(3,5-Dimethoxyphenyl)-isopropoxycarbonyl 2,4-Dinitrophenyl Diphenylphosphinyl Ethyl 9-Fluorenylmethoxycarbonyl Formyl 4,4 0 -Dimethoxydiphenylmethyl, 4,4 0 -Dimethoxybenzhydryl 4-Methoxybenzenesulphonyl Methyl 4-Methoxybenzyl 2,3,6-Trimethyl,4-methoxybenzenesulphonyl 2-Nitrophenylsulphenyl Allyl ester 1-Benzotriazolyl ester Cyclohexyl ester 4-Nitrophenyl ester Pentachlorophenyl ester Pentafluorophenyl ester Succinimido ester 2,2,2-Trichloroethyl ester 2,4,5-Trichlorophenyl ester 2,4,5-Trimethoxybenzyl 4-Methyltrityl Phenacyl, PhCOCH2 (care! Pac also = PhCH2CO) Phenyl Phthaloyl Methoxycarbonylsulphenyl 2,2,5,7,8-Pentamethylchroman-6-sulphonyl i-Propyl n-Propyl Trifluoroacetyl 4-Toluenesulphonyl (also Ts) 2,2,2-Trichloroethoxycarbonyl Trityl, triphenylmethyl 9-Xanthydryl Benzyloxycarbonyl (also Cbz). Z(2C1) = 2-chlorobenzyloxycarbonyl and so on

Amino Acid Derivatives DKP Diketopiperazine NCA N-Carboxyanhydride PTH Phenylthiohydantoin UNCA Urethane N-carboxyanhydride Reagents and Solvents BOP 1-Benzotriazolyloxy-tris-dimethylamino-phosphonium hexafluorophosphate xiv | Amino Acids, Pept. Proteins, 2018, 42, x–xvii

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CDI DBU DCCI DCHU DCM DEAD DIPCI DIPEA DMA DMAP DMF DMS DMSO DPAA EEDQ HATU

Carbonyldiimidazole Diazabicyclo[5.4.0]-undec-7-ene Dicyclohexylcarbodiimide (also DCC) Dicyclohexylurea (also DCU) Dichloromethane Diethyl azodicarboxylate (DMAD = the dimethyl analogue) Diisopropylcarbodiimide (also DIC) Diisopropylethylamine (also DIEA) Dimethylacetamide 4-Dimethylaminopyridine Dimethylformamide Dimethylsulphide Dimethylsulphoxide Diphenylphosphoryl azide 2-Ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline This is the acronym for the ‘uronium’ coupling reagent derived from HOAt, which was originally thought to have the structure 8, the Hexafluorophosphate salt of the O-(7-Azabenzotriazol-lyl)-Tetramethyl Uronium cation.

In fact this reagent has the isomeric N-oxide structure 9 in the crystalline state, the unwieldy correct name of which does not conform logically with the acronym, but the acronym continues in use.

HMP HOAt HOBt HOCt NDMBA NMM PAM PEG

Similarly, the corresponding reagent derived from HOBt has the firmly attached label HBTU (the tetrafluoroborate salt is also used: TBTU), despite the fact that it is not actually a uronium salt. Hexamethylphosphoric triamide (also HMPA, HMPTA) 1-Hydroxy-7-azabenzotriazole 1-Hydroxybenzotriazole 1-Hydroxy-4-ethoxycarbonyl-1,2,3-triazole N,N 0 -Dimethylbarbituric acid N-Methylmorpholine Phenylacetamidomethyl resin Polyethylene glycol Amino Acids, Pept. Proteins, 2018, 42, x–xvii | xv

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PtBOP SDS TBAF TBTU TEA TFA TFE TFMSA THF WSCI Techniques CD COSY CZE ELISA ESI ESR FAB FT GLC hplc IR MALDI MS NMR nOe NOESY ORD PAGE RIA ROESY RP SPPS TLC TOCSY TOF UV

1-Benzotriazolyloxy-tris-pyrrolidinophosphonium hexafluorophosphate Sodium dodecyl sulphate Tetrabutylammonium fluoride See remarks under HATU above Triethylamine Trifluoroacetic acid Trifluoroethanol Trifluoromethanesulphonic acid Tetrahydrofuran Water soluble carbodiimide: 1-ethyl-3-(3 0 -dimethylaminopropyl)-carbodiimide hydrochloride (also EDC) Circular dichroism Correlated spectroscopy Capillary zone electrophoresis Enzyme-linked immunosorbent assay Electrospray ionization Electron spin resonance Fast atom bombardment Fourier transform Gas liquid chromatography High performance liquid chromatography Infra red Matrix-assisted laser desorption ionization Mass spectrometry Nuclear magnetic resonance Nuclear Overhauser effect Nuclear Overhauser enhanced spectroscopy Optical rotatory dispersion Polyacrylamide gel electrophoresis Radioimmunoassay Rotating frame nuclear Overhauser enhanced spectroscopy Reversed phase Solid phase peptide synthesis Thin layer chromatography Total correlation spectroscopy Time of flight Ultraviolet

Miscellaneous Ab Antibody ACE Angiotensin-converting enzyme ACTH Adrenocorticotropic hormone Ag Antigen AIDS Acquired immunodeficiency syndrome ANP Atrial natriuretic polypeptide ATP Adenosine triphosphate BK Bradykinin xvi | Amino Acids, Pept. Proteins, 2018, 42, x–xvii

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BSA CCK DNA FSH GH HIV LHRH MAP NPY OT PTH QSAR RNA TASP TRH VIP VP

Bovine serum albumin Cholecystokinin Deoxyribonucleic acid Follicle stimulating hormone Growth hormone Human immunodeficiency virus Luteinizing hormone releasing hormone Multiple antigen peptide Neuropeptide Y Oxytocin Parathyroid hormone Quantitative structure–activity relationship Ribonucleic acid Template-assembled synthetic protein Thyrotropin releasing hormone Vasoactive intestinal peptide Vasopressin J. H. Jones

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Amino acids: chemistry, diversity and physical propertiesy Ma´rta Zara´ndi* and Ja´nos Szoloma´jer Published on 29 November 2017 on http://pubs.rsc.org | doi:10.1039/9781788010627-00001

DOI: 10.1039/9781788010627-00001

The occurrence, chemistry, resolution, and analysis of amino acids published in the literature from 2013 finished with the year of 2016 are reviewed in this Chapter which is arranged in sections similar to previous Volumes in this Specialist Periodical report. Scientific Papers published during 2013–2016 have been sourced mainly from the Web of Science databases and Pubmed on the internet and from scanning a selection of major journals.

1

Naturally occurring amino acids

1.1 Occurrence of amino acids Several naturally occurring amino acid derivatives are more and more in the focus as sources for new agrochemicals. The 53 most important natural products of amino acids are presented in a review together with their natural sources, mode of action, and herbicidal, fungicidal, or insecticidal activities.3 It is known that Arg, the most basic amino acid, occurs less frequently than Lys in proteins. The few important proteins abundant in Arg have important roles in biological systems. A review collects the data of occurrence, functions, and the biological significance of these Arg-rich proteins.4 Leu is a potential signalling molecule to regulate cell growth and metabolism. Structure–activity relationships of Leu derivatives in HeLa S3 cells were investigated for cellular uptake and for the induction of phosphorylation. The results may provide a new insight into therapeutics targeting both L-amino acid transporters 1 and Leu sensor.5 Phenyl-Gly-type amino acids occur in a wide variety of natural peptides. The biosynthesis of 4-hydroxyphenyl-Gly, 3,5-dihydroxyphenyl-Gly, and phenyl-Gly was investigated. Structures and properties of phenyl-Gly containing natural products, the biosynthetic origin and incorporation of phenylglycines are discussed in a review.6 4-Methyl-Pro (4-mPro) is a rare nonproteinogenic amino acid produced by cyanobacteria. Eight biosynthetic gene clusters were found from available cyanobacteria genomes, showing that 4-mPro is a good marker to discover previously unknown nonribosomal peptides.7 Amino acids represent a fraction of organic matter in marine and freshwater ecosystems. The occurrence of D-amino acids is usually linked to the presence of bacteria. The distribution of L- and D-amino acids in ´m te´r 8, Hungary. Dept. of Medical Chemistry, University of Szeged, 6721 Szeged, Do E-mail: [email protected] y Papers published during 2013–2016 have been sourced mainly from the Web of Science databases1 and Pubmed2 on the internet and from scanning a selection of major journals. Amino Acids, Pept. Proteins, 2018, 42, 1–84 | 1  c

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the lacustrine environment of Terra Nova Bay, Antarctica was investigated.8 Microorganisms that utilize various D-amino acids were successfully isolated from deep-sea sediments. Some of the isolates exhibited high enantioselective degradation activities to various D-amino acids.9 An antialgal compound was isolated from the cultured broth of Streptomyces jiujiangensis JXJ 0074(T). Based on the data of different analytical methods, the active compound was identified as L-Val which showed antialgal activity. This is the first report showing that L-Val is active against cyanobacteria.10 Diverse D-amino acids have been found in mammalian tissues. The physiological functions of these D-amino acids are being gradually clarified. It has been demonstrated that D-Ser, D-Asp, D-Ala, and D-Cys play important roles in the nervous and endocrine systems. The investigations of metabolism and the physiological functions of D-amino acids provide new therapeutic and diagnostic strategies for diseases related to the nervous and endocrine systems.11 D-Ser is a key signalling molecule utilized by neurons and astroglia in the mammalian central nervous system. Alterations in the extracellular levels of D-Ser disrupt cell–cell signalling that leads to many chronic or acute neurological and psychiatric disorders, and are associated with addictive behaviour. Experimental data supports that astroglia and neurons use different pathways to regulate levels of extracellular D-Ser.12 A rare Gln derivative, hemerocallisamine I, was isolated from the flower buds of daylily. It was first reported that a Gln derivative with a pyrrole ring is found in natural plants.13 Increased reactive oxygen species are accompanied by 2-aminobutyric acid accumulation and compensatory maintenance of myocardial glutathione levels. It was demonstrated for the first time that 2-aminobutyric acid modulates glutathione homeostasis in the myocardium.14 b-Amino acids are components of complex natural products generating significant and unique biological functions. The de novo synthesis of b-amino acids and the mechanisms of b-amino acid incorporation into natural products are summarized.15 Mycobacterium tuberculosis (Mtb) is responsible for 9 million active tuberculosis cases annually, resulting in 1.5 million death cases per year worldwide. It is known that Mtb uses Arg as a nitrogen source in vitro, but the metabolic pathways have not been identified. It was found that, both nitrogen and carbons from Arg can be incorporated into the central metabolism of Mtb. The highly induced pathway for Arg utilization in Mtb differs from that of other bacteria including non-tuberculous mycobacteria.16

1.2 New amino acids and derivatives The increasing interest of modified peptides in chemical engineering of proteins and also as therapeutic agents has refreshed research toward the development of derivatives of new natural and non-natural amino acids. Oxidative stress plays an important role in the development of atrial fibrillation. Derivatives of Arg including asymmetric dimethyl-Arg (ADMA) are central to nitric oxide metabolism and nitrosative stress. 2 | Amino Acids, Pept. Proteins, 2018, 42, 1–84

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The circulating levels of ADMA, L-Arg, symmetric dimethyl-Arg (SDMA), and the ratio of L-Arg/ADMA to incidence of atrial fibrillation were investigated. The results revealed that circulating ADMA is not strongly associated with new-onset atrial fibrillation, and L-Arg and SDMA are also not predictive of long-term incidence of atrial fibrillation.17 A growing number of studies showed elevated concentrations of circulating ADMA and SDMA leading to mortality and cardiovascular diseases. A systematic review summarizes the evidence from studies of ADMA and SDMA with the risks of all-cause mortality and incident cardiovascular disease in meta-analyses. It was concluded that ADMA and SDMA are independent risk markers for all-cause mortality and cardiovascular disease across different populations and methodological approaches.18 In search for new drugs lowering arterial blood pressure, novel potential renin inhibitors based on human angiotensinogen, were designed and synthesized. All these inhibitors contain unnatural amino acids that are derivatives of N-alkylleucyl-b-hydroxy-g-amino acids. In vitro renin inhibitory activity of all obtained compounds was within the range 106–109 M.19 A series of different amino acid-bearing thieno[2,3-D]pyrimidine moiety and a tricyclic imidazothienopyrimidine of the Gly derivative were synthesized. All the obtained amino acid-derivatives were screened for their post-irradiation protective efficacy. Most of the newly synthesized derivatives showed significant protective effects against injuries induced by g-irradiation exposure, and they may be promising curative agents against g-irradiation induced oxidative stress and physiological disturbance in different organs.20 A novel series of biaryl-based phenylalanines with a carboxyl-benzene (or a carboxyl-thiophene ring) was designed, synthesized, and pharmacologically characterized in vitro. The structural modifications were focused on positions 5- and 4- of the Phe ring. Various combinations of small-sized groups, both polar and lipophilic, hydrogen bond donors and acceptors, were investigated. All the target amino acids were pharmacologically characterized by radioligand binding at native AMPA, kainate, and NMDA receptors, and the structure–activity relationships were also studied.21 Thiazolides are polypharmacologycal agents with at least three mechanisms of action against a broad spectrum of parasites, bacteria and viruses. New amino-acid ester thiazolide prodrugs were synthesized in order to improve their systemic absorption and tested.22 Oxazolidinone derivatives serve as important drugs among antibiotics, since they act on multidrug-resistant bacteria. A small library of compounds based on isoxazolidinone and dehydro-b-Pro was designed with the aim to obtain antibacterial agents and monoaminooxidase(MAO)-inhibitors.23 Substituted indolizidine and quinolizidine derivatives are readily assembled from cyclic amino acids (Pro or pipecolic acid) and g-nitroaldehydes by a simple decarboxylative annulation process.24 Derivatives of aminobenzoic acid were functionalized via a chemoselective carbene insertion manner without implementing protection and deprotection strategy under mild reaction conditions leading to carboxy and hydroxy functionalized a-amino esters (27 examples).25 The Amino Acids, Pept. Proteins, 2018, 42, 1–84 | 3

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neurotrophic effects of L-Glu and b-phenyl-Glu hydrochloride were compared. It was found that b-phenyl-Glu hydrochloride was more potent than L-Glu in neuroprotection and increasing survival rate.26 A new member of the dimethylallyl-Trp synthase superfamily catalyzes prenylations of both Tyr and Trp derivatives. The results enhance the relationship of Tyr O- and Trp C7-prenyltranferases and provide a new enzyme for production of prenylated derivatives.27 Arctigenin, a traditional medicine with many pharmacological activities, has been restricted due to its poor solubility in water. Five amino acid derivatives of arctigenin have been synthesized using Gly, Ala, Val, Leu, and Ile. The results showed that the amino acid derivatives have better solubility and exhibit higher anti-tumour activity than arctigenin.28 New amino acid derivatives with carbocycles of adamantine and quinaldic acid were developed; their in vitro antiviral activity against highly pathogenic influenza A virus (H5N1) was evaluated; and found that they suppressed viral replication.29 New N-(4-substituted phenyl)Gly derivatives have been synthesized. The intermediates (the chalcone and the thiosemicarbazone derivative) were derivatized and/or cyclized into different heterocyclic target derivatives and evaluated as potential anti-inflammatory agents.30 Novel rifamycins containing L-amino acid esters were produced, and their structure–activity relationships in solution were studied. The presence of the rifamycins’ structures influenced antibacterial properties.31 Schiff base compounds of cinnamaldehyde and amino acids have been synthesized and investigated for their antimicrobial activities. A total of 24 Schiff base compounds were synthesized using a simple approach with 3 cinnamaldehyde derivatives and 8 amino acids. Results from the structure–activity relationship suggest that both –p–Cl on benzene ring of cinnamaldehyde and the number of –COOK of amino acid salts significantly contributed to antimicrobial activity.32 A series of bile acid (cholic acid and deoxycholic acid) aryl/heteroaryl amides linked via a-amino acid were synthesized and tested against 3 human cancer cell-lines. Some of the conjugates showed promising results to be new anticancer agents with good in vitro results. They showed fairly good activity against the breast cancer cell line with respect to Cisplatin and comparable with respect to Doxorubicin and showed better activity against glioblastoma cancer cell line with respect to both Cisplatin and Doxorubicin drugs used as standards.33 A series of red-shifted azobenzene amino (Scheme 1) acids have been synthesized via a two-step procedure. Derivatives of Tyr were first oxidized to the corresponding quinonoidal spirolactones followed by the

Scheme 1 4 | Amino Acids, Pept. Proteins, 2018, 42, 1–84

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unsymmetrical azo formation with the substituted phenylhydrazines in the presence of the ceric ammonium nitrate catalyst.34 Kojic acid (5-hydroxy-2-hydroxymethyl-4H-pyran-4-one) is a natural product that is produced by many species of fungi. A simple and efficient method for the preparation of dialkylated kojic acid based a-amino acid derivatives was described. The novel derivatives might find application as tyrosinase inhibitors.35 Oleanolic acid or oleanic acid (3b-hydroxy-olean-12-en-28-oic acid) is a naturally occurring pentacyclic triterpenoid related to betulinic acid. It is widely distributed in food and plants where it exists as a free acid or as an aglycone of triterpenoid saponins and exhibits a wide range of pharmacological and biological activities. Four known and two new amino acid conjugates of oleanolic acid were prepared, and investigated for their cytotoxic effects. The results revealed that two derivatives showed significantly increased inhibition rates than the parent oleanolic acid.36 Novel derivatives of ligustrazine-oleanolic acid were designed, and synthesized by conjugating amino acids to the 3-hydroxy group of ligustrazine-oleanolic acid by ester bonds. Their cytotoxicity was evaluated on four cancer cell lines.37 Hepatic fibrosis is a naturally occurring wound-healing reaction, with an imbalance of extracellular matrix during tissue repair response, which can further deteriorate to hepatocellular carcinoma without timely treatment. It was found that Gly derivative of ligustrazine-oleanolic acid selectively inhibited the proliferation and induced apoptosis indicating that Gly derivative of ligustrazine-oleanolic acid might be a potential antifibrosis agent for the therapy of hepatic fibrosis.38

1.3 Miscellaneous Methylation of Lys is one of the important post-translational modifications of histones that produces N(e)-mono-, di-, or trimethyl Lys residues. Multiple, site-specific Lys methylations of histones are essential to define epigenetic statuses and control heterochromatin formation, DNA repair, and transcription regulation. A new method was developed for preparing histones bearing multiple N(e)-monomethyl Lys residues at specified positions that enables the installation of authentic N(e)monomethyl Lys at multiple positions within a protein for large-scale production.39 All 20 common natural proteinogenic and 4 other a-amino acidisosteric a-amino tetrazoles have been synthesized. Since the tetrazole group is bioisosteric to the carboxylic group, these derivatives are widely used in medicinal chemistry and drug design. The synthetic process involves the use of the Ugi tetrazole reaction followed by deprotection (Scheme 2).40 Amino acids with their variable side chains are ideal candidates for synthesizing biodegradable functional polyesters. The synthetic methods for poly-a-hydroxy esters derived from amino acids were reviewed.41 The efficient tRNA-mediated incorporation of the hydroxamate containing amino acid, N(e)-acetyl-N(e)-hydroxy-L-Lys into a transcription factor was reported. The tetrahydrofuranyl and tetrahydropyranyl O-protecting Amino Acids, Pept. Proteins, 2018, 42, 1–84 | 5

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Scheme 2

groups can be removed using mild acid conditions. These protecting groups can be used as valuable alternative for O-protection.42 Two new antifouling zwitterionic polymers, poly(lysine methacrylamide) and poly(ornithine methacrylamide) derived from natural amino acids Lys and Orn, respectively were developed and investigated. The super low fouling, biomimetic, and multifunctional properties of poly(lysine methacrylamide) and poly(ornithine methacrylamide) make them promising materials for a wide range of applications, such as implant coating, drug delivery and biosensing.43 Amino-acid-based chiral surfactants with polymerizable moieties are developed for the production of chiral nanoparticles. The synthesized particles are tested for their ability as nucleating agents in the enantioselective crystallization of amino acid conglomerate systems. The results demonstrate that only the chiral nanoparticles made of the polymerizable surfactant are able to act efficiently as nucleation agent in enantioselective crystallization.44 The preparation by a direct condensation method, characterization, and cytocompatibility of homo- and hetero-polyesters of a-hydroxy amino acid derivatives with or without lactic acid conjugation has been described. It was concluded that overall selective cytocompatibility and bioactivity might render a-hydroxy amino acid polymers useful as extracellular matrix-mimicking materials for tissue engineering.45 An asymmetric decarboxylative Csp(3)–Csp(2) cross-coupling has been achieved via a mild, operationally simple protocol with Ni-catalyst. Variety of naturally abundant a-amino acids and aryl halides are transformed into valuable chiral benzyl amines.46 A highly diastereoselective acid-catalyzed N,O-acetalization/intramolecular transcarbamoylation cascade of reactions between protected a-amino acid derivatives (Ser and Thr) and non-natural a-amino acid derivatives with tetramethoxyalkanes has been reported. The resulting oligocyclic N,O-acetals have been used as excellent chiral building blocks for asymmetric transformations. The complete diastereoselectivity achieved with natural amino acid precursors is completely lost with their non-natural analogues.47 Incorporation of a non-natural Arg analogue (guanidiniocarbonyl pyrrole) into a cyclic peptide is capable of completely altering the selfassembly properties of the peptide. In contrast to the peptide which does not self-assemble, guanidiniocarbonyl pyrrole-containing peptide forms cationic nanofibers of micrometer length that are capable of gene transfection.48 Several new functions of amino acids have been recently discovered that could result in new applications. E.g. oral stimulation by Glu triggers the cephalic phase response to prepare for food digestion. Branchedchain amino acids are the major components of muscles, and ingestion 6 | Amino Acids, Pept. Proteins, 2018, 42, 1–84

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of branched-chain amino acids has been found to be effective for decreasing muscle pain. Amino acids can be used in a novel clinical diagnostic method: the balance of amino acids in the blood could be an indicator of the risk of diseases such as cancer. The newly discovered functions of amino acids are discussed.49 It was observed that Gln levels in plasma were significantly lower just after cardiac surgery compared to pre-operative levels.50 The interactions of the side chains of Arg and Lys was investigated with each of the 19 non-Gly amino acids in proteins in the protein data bank. The guanidino group of Arg interacts with non-polar aromatic and aliphatic side chains above and below the guanidinium plane while hydrogen bonding with polar side chains is restricted to in-plane positions. In contrast, non-polar side chains interact with the aliphatic part of the lysine side chain. Molecular dynamics simulations underlie the preference for Arg as a mobile charge carrier in voltage-sensing domains.51 To understand the molecular mechanisms of effect of additives to stabilize proteins, molecular dynamics simulations of the surface residues lysozyme was performed in the presence of three commonly used additives: Arg, Lys, and guanidine that have different effects on stability of proteins and have different structures with some similarities. The investigations revealed that the internal dynamics, as well as the lifetimes of the hydrogen bonds within the protein changes depending on the additives.52 A three-step process involving Cu-catalyzed allylation of Ser-, Asp-, and Glu-derived organo-Zn reagents have been used for the synthesis of 7-oxo, 8-oxo, and 9-oxo amino acids.53 Amino acids derived steroidal and nonsteroidal architectures have also been developed. The benzofused, amino acid-derived steroidal and nonsteroidal molecules had promising biological activity in hormonal related disorders.54 Optically pure valinol was prepared via different o-transaminases from the corresponding prochiral hydroxy ketone. Reductive amination was performed in organic solvent using 2-propyl amine as amine donor, and Ala was applied in aqueous medium.55 Five simple rules of thumb are developed to summarize the adsorption properties of the proteinogenic a-amino acids as mediated by hydrogen bonding on silicon surface, and they are expected to provide a helpful guide to future studies of larger biomolecules and their potential applications.56 The adsorption of some amino acids by fullerene (C60) and fullerene nanowhiskers have been investigated. The aromatic group of fullerene did not interact with the hydrophobic side chains (alkyl chain, cyclic structures of Trp, Phe and Pro) of amino acids.57 The role of hydrophobicity of the side chain of amino acids in the formation of hydrogel was studied by using Fmoc-Nle and Fmoc-Met. The results indicate that Fmoc-Met forms reversible hydrogels in water, whereas Fmoc-Nle fails to display any gelation under similar conditions. The difference in the self-association behaviour of Fmoc-Met and FmocNle emphasise the importance of weak noncovalent interaction between side chains to stabilise supramolecular self-assembly of Fmoc-protected amino acids.58 Amino Acids, Pept. Proteins, 2018, 42, 1–84 | 7

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Unlike other Fmoc-functionalized amino acid gelators, FmocLys(Fmoc)-OH exhibits pH-controlled ambidextrous gelation. (hydrogelation at different pH values as well as organogelation). Spectroscopic analyses proved that the self-assembly of Fmoc-Lys(Fmoc)-OH was driven by aromatic p–p stacking and hydrogen bonding interactions in both hydrogels and organogels.59 Fmoc-Lys(Fmoc)-OH was demonstrated as gelators to gelate a variety of alcohols and aromatic solvents under the sonication conditions. The ultrasound-triggered organogel also exhibited thixotropic property. The gels with the fibrous 3D network structure were unravelled into sols. However, after standing, these sols returned to the gels showing a more ordered lamella-like packing structure.60 The nature of p–p interactions in the self- and coassembly of Fmoc-Phe-derived hydrogelators was investigated by systematically varying the electrondonating or electron-withdrawing nature of the side chain substituents and correlating these effects to the emergent assembly and gelation properties of the systems. The findings provide significant insight into the structure–function relationship for Fmoc-Phe-derived hydrogelators.61 A review summarizes the recent progress of self-organization of Fmoc-amino acids and Fmoc-modified di-, tri-, tetra- and pentapeptides.62 Gemini surfactants have been used for in vitro gene delivery. Amino acid-derived gemini surfactants combine the special aggregation properties with high biocompatibility and biodegradability. Novel Ser-derived gemini surfactants, differing in alkyl chain lengths and in the linker group bridging the spacer to the head groups (amine, amide and ester) were evaluated.63 The effects of the spacer chain length of amino acidbased gemini surfactants on the formation of wormlike micelles in aqueous solutions were investigated. The surfactants were synthesized by reacting dodecanoyl-Glu anhydride with diamine compounds. Formation of cation-rich molecular aggregates was not observed when the longest spacer analogue (8 methylene units) was used.64 A review reports the most important contributions in the structure, synthesis, physicochemical and biological properties (toxicity, antimicrobial activity, and biodegradation) of natural amino acid-based (Arg, Lys, Ser, Ala, Sar, Asp, and Cys) gemini surfactants and some potential applications. Amino acidbased gemini surfactants have some benefits compared with the classical gemini surfactants.65 The development of new antimicrobial agents is very important because of the rapid increase in the number of multiple drug resistant bacteria and fungi. Synthetic amino acid-based surfactants constitute a promising alternative to conventional antimicrobial compounds. In a review, the structural features that promote antimicrobial activity of amino acid-based surfactants and also the synthesis and basic physico-chemical properties are discussed. Cationic surfactants based on amino acids show excellent antimicrobial and antifungal properties.66 Anionic, cationic, and zwitterionic amphiphiles can be prepared as surfactants by using one of the 20 proteinogenic amino acids. A review gives examples of procedures and discusses important physicochemical properties and various applications of amino acid-based surfactants, as well as highlights concepts that are unique to amino acid-based 8 | Amino Acids, Pept. Proteins, 2018, 42, 1–84

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surfactants. E.g. Surfactants based on amino acids with two carboxyl groups are effective chelating agents; a surfactant based on Cys readily oxidizes into the corresponding cystine compound, which can be regarded as a gemini surfactant.67 Another review discusses surfactant-amino acids and surfactant–surfactant interactions in aqueous medium.68 Threonine aldolases catalyze the pyridoxal phosphate-dependent condensation between small amino acids (Gly) and aldehydes. A review briefly summarizes the reaction mechanism and lists all published synthetic reactions by threonine aldolases.69 A paper reviews the literature data on amino acid supplementation with Arg, Glu, and beta-hydroxy-beta-methylbutyric acid and wound healing in diabetic foot. A pilot study showed that administration with hydroxyproline (Hyp), a major component of collagen, improved the healing of diabetic foot wounds via increased collagen production.70 Determination of the 4-hydroxy-L-Pro concentration provides information for the diagnosis and prognosis of diseases caused by disorders of collagen metabolism. It was found that LC–MS method would be advantageous for measuring the hydroxyl-Pro concentration and for the diagnosis of diseases associated with abnormalities of collagen metabolism.71 4-Methylproline is a rare nonproteinogenic amino acid produced by cyanobacteria through the action of a zinc-dependent long-chain dehydrogenase. The use of biosynthetic genes of 4-methylproline helped to discover new bioactive compounds from cyanobacteria.7 Prodrugs in which the hydroxyl moiety is reversibly protected as a carbamate ester linked to the amino group of a natural amino acid (Ile or b-Ala) have been produced. Prodrugs having amino acids with hydrophobic side chains were readily absorbed after intragastric administration.72 Prodrugs of resveratrol in which the OH groups are engaged in an N-monosubstituted carbamate ester (–OC(O)NHR) linkage with a natural amino acid (Leu, Ile, Phe, Thr) to prevent conjugation and modulate the physicochemical properties of the molecule that was synthesized in high yield; characterized; and the stability and in vivo pharmacokinetic behaviour also was determined. It was concluded that prodrugs based on the N-monosubstituted carbamate ester bond have the appropriate stability profile for the systemic delivery of phenol compounds.73 The protonation constants, the protonation enthalpy changes, and the solubility of six natural a-amino acids (Gly, Ala, Val, Leu, Ser, and Phe) have been performed in NaCl and in (CH3)4NCl media with new potentiometric experiments.74 The protonation equilibrium concentrations of proton–ligand formation as a function of pH were investigated. Biologically active ligands like amino acids, peptides, DNA constituents, and amino acid esters in nonaqueous media have been investigated in a review.75 Complex formation of equilibrium for the binary complexes of Cu(II) with 1-aminocyclopropane carboxylic acid and 3,3-bis(1-methylimidazol-2-yl)propionic acid were studied. The ternary complexes are formed in a stepwise mechanism.76 Antibody-drug conjugates as potent antitumour drugs provide increased efficacy, specificity, and tolerability over chemotherapy for the Amino Acids, Pept. Proteins, 2018, 42, 1–84 | 9

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treatment of cancer. A novel method is described that enables sitespecific conjugation of toxins to antibodies using chemistry to produce homogeneous, potent, and highly stable conjugates. A cell-based mammalian expression system was used that is capable of site-specific integration of a non-natural amino acid containing an azide moiety. The azide-alkyne linkage has high stability.77 The hybrid molecules of antibody-drug conjugates consist of a tumour antigen-specific antibody coupled to a chemotherapeutic small molecule. A cell-free protein expression system was established for production of antibody drug conjugates through site-specific incorporation of para-azidomethyl-L-Phe. The resultant antibody-drug conjugates were highly potent in in vitro cell cytotoxicity assays.78 A novel amino acid racemase was discovered through exploration of natural variation in Arabidopsis thaliana. N-malonyl-D-allo-Ile and a novel amino acid racemase that is involved in its biosynthesis was identified. This finding provides the first functional characterization of a eukaryotic member of a large and widely conserved phenazine biosynthesis protein family, and a new D-amino acid racemase gene family is also identified.79 A simple theoretical model of amino acid similarity matrices, which allows splitting the matrix into two parts, was introduced. The new synthetic amino acid properties are derived from the pairwise similarities and used to reconstruct similarity matrices. The new properties derived from amino acid similarity matrices correlate highly with properties known to be important for molecular evolution such as hydrophobicity, size, shape, and charge of amino acids.80 A group of five amino acid based zwitterionic vinyl monomers, based on Ser, Lys, Orn, Glu, and Asp were developed for potential antifouling applications and grafted on gold chips by polymerization. Considering multiple applications (e.g. medical devices and drug delivery) of the antifouling materials, the cytotoxicity of monomers and polymer nanogels for all five materials at various concentrations were evaluated. Very low cytotoxicity was observed for all tested amino acid-based monomers and nanogels, which is comparable or even lower than the traditional and some newly developed antifouling materials.81 Genetically encoded electrophilic unnatural amino acid found to react with His and Lys. In addition to efficient cross-linking of proteins interand intramolecularly, the unnatural amino acid permits direct stapling of a protein a-helix and covalent binding of native membrane receptors.82 A review examines recent advances in substrate-controlled asymmetric reactions induced by the chirality of a-amino acid templates in natural product synthesis and related areas.83 Small molecules that target different components of the spliceosome represent valuable research tools to investigate this complicated macromolecule. N-palmitoyl-L-Leu was identified as a new splicing inhibitor that blocks a late stage of spliceosome assembly.84 Amino acid transporters are expressed in the body and form a series of channels to pump nutrients against concentration gradients into cells. Abnormal expression of amino acid transporters is often associated with 10 | Amino Acids, Pept. Proteins, 2018, 42, 1–84

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Scheme 3

cancer, addiction, and multiple mental diseases. A new class of amino acid mimics (boramino acids) (Scheme 3) are described that can serve as general imaging probes for amino acid transporters and show strong amino acid transporter specificity, can be labelled easily with 18Ffluorination, and should find wide application in the development of previously unavailable PET imaging probes for clinical diagnosis.85 The enzyme 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase is the first one which is responsible for the synthesis of aromatic amino acids in bacteria and plants, and a potential target for the development of antibiotics and herbicides. The enzyme from Mycobacterium tuberculosis displays an allosteric regulation with three interdependent allosteric binding sites and a ternary allosteric response to combinations of the aromatic amino acids L-Trp, L-Phe, and L-Tyr. The binding mode of D-amino acids was investigated using X-ray crystallography, site directed mutagenesis, and isothermal titration calorimetry, and the key differences in the binding mode were identified.86 The syntheses and photophysical characterization of three novel cyanotryptophans (6- and 7-cyano Trp, and N-methyl-7-cyano Trp) and their efficient incorporation into proteins as fluorescent probes are described. 6-Cyano Trp was also incorporated into two DNA binding proteins and an RNA recognition motif. The novel cyanotryptophans can be used for studying conformational changes of proteins and DNA–protein interactions.87 The controlled assembly of Trpzip b-hairpins was investigated. The results show how sensitive is peptide and protein aggregation to minor sequence variation and that it is possible to use a photolabile non-natural amino acid analogue of Lys to tune the rate of peptide aggregation and to control fibrillar structure.88 Different functionalized azabicyclo[X.Y.0]alkanone derivatives of amino acids have been developed via electrophilic transannular cyclizations of 8-, 9-, and 10-membered unsaturated macrocycles to form 5,5-, 6,5-, 7,5-, and 6,6-fused bicylic amino acids. Macrocycles were obtained by a sequence featuring peptide coupling of vinyl-, allyl-, homoallyl-, and homohomoallylglycine building blocks followed by ring-closing metathesis. The conformational preferences and the mechanism for the diastereoselective formation of specific azabicycloalkanone amino acids were also studied.89 Two pyridine-linked bis(b-cyclodextrin) copper(II) complexes have been reported that enantioselectively hydrolyse chiral esters of amino acids. It was demonstrated that the enantioselective hydrolysis was related to the cooperative roles of the intramolecular flanking chiral b-cyclodextrin cavities with the coordinated copper ion.90 Fluorescent sensors based on semiconductor quantum dots have been investigated for enantioselective molecular recognition. A versatile fluorescent sensor encapsulated with Amino Acids, Pept. Proteins, 2018, 42, 1–84 | 11

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cyclodextrin clicked silica via layer-by-layer modification was reported for chiral selection of amino acids.91 New stevia amino acid sweeteners, stevia Gly ethyl ester and stevia L-Ala methyl ester were synthesised and characterized. The novel sweeteners were stable in acidic, neutral, or basic aqueous solutions.92 Mycosporine-like amino acids (MAAs) are produced by organisms that live in environments with high volumes of sunlight, usually in marine environments. They are produced by a large variety of microorganisms including cyanobacteria, lichen, fungi, and marine micro- and macroalgae. They are water soluble, small intracellular secondary metabolites that absorb ultraviolet radiation, but their function is not limited to sun protection. MAAs and their derivatives are natural antioxidants, show immunomodulatory effects and anti-inflammation activities, promote wound healing, and inhibit collagenase.93,94 Over 30 MAAs have been resolved and all contain a central cyclohexenone or cyclohexenimine ring and a wide variety of substitutions. The biosynthesis of MAAs in the cyanobacterium Arthrospira sp. CU255 was investigated. The results indicate that the studied cyanobacterium may protect itself by synthesizing mycosporine-Gly, the UV-absorbing/screening compound as important defense mechanism.95 Identification of novel MAAs is important from a biotechnology perspective, and the identification of the genes responsible for biosynthesis of MAA is vital for future genetic engineering. A study confirmed first a biosynthetic gene cluster for MAA from Gram-positive bacteria. Structure elucidation revealed that the novel MAA is mycosporine-Gly-Ala, which substitutes L-Ala for the L-Ser of shinorine.96

2

Chemical synthesis and resolution of amino acids

2.1 Asymmetric and stereoselective synthesis Although a large number of methods are available for the synthesis of a-amino acids, the development of new, highly efficient, and enantioselective methods for the synthesis of many of the natural and unusual a-amino acids has been a long-standing goal of synthetic chemists. Much effort has focused on the enantioselective version of Strecker reaction resulting in the development of an assortment of effective metalbased and metal-free catalysts. Derivatives of a-amino acids have been synthesized via enantioselective addition of masked acyl cyanides to imines. This method works for the free amino acids rather than their N- or C-protected derivatives.97 Spontaneous formation of an enantioenriched a-amino nitrile which is a chiral precursor for Strecker amino acid synthesis was produced without addition of any chiral substances.98 Asymmetric synthesis of a wide variety of a-amino acids was achieved by alkylations of chiral or achiral Ni(II) complexes of Gly Schiff bases. Origin of diastereo-/enantioselectivity in the alkylations reactions, aspects of practicality, generality, and limitations of this method is critically discussed.99 A broad review discussed the data in the literature on 12 | Amino Acids, Pept. Proteins, 2018, 42, 1–84

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asymmetric synthesis of a-amino acids via Michael addition reactions involving Ni(II)-complexes and the practical aspects of the methodology.100 One of the most recent developments in asymmetric catalysis is to employ two or more catalysts under one-pot reaction conditions. A cooperative dual-catalytic protocol relying on the catalytic ability of dirhodium carbenoid (derived from rhodium(II) tetracarboxylate and a diazo compound) and a chiral spirophosphoric acid in an asymmetric N–H insertion reaction was investigated.101 The development of enantioselective C–H activation reactions by desymmetrization have been progressed in the past decade. Enantioselective C–H olefination of a-hydroxy and a-amino phenylacetic acids was achieved by kinetic resolution with the use of palladium(II) catalyst.102 N-Aryl glycine esters with terminal alkynes were synthesized via enantioselective crossdehydrogenative coupling by Cu catalyst.103 Asymmetric synthesis of N-Boc-(R)-silaproline was achieved via Rh-catalyzed intramolecular hydrosilylation of dehydroalanine and continuous flow N-alkylation with excellent yield and enantioselectivity.104 Due to its pharmaceutical importance, (1R,2S)-1-amino-2-vinylcyclopropane-carboxylic acid was prepared by advanced asymmetric procedure. The target amino acid was achieved via two-step SN2 and SN2 0 alkylation of novel axially chiral Ni(II) complex of Gly Schiff base with excellent yields and diastereoselectivity.105 A chiral amine-catalyzed highly stereoselective vinylogous allylic–allylic alkylation of Morita– Baylis–Hillman carbonates with olefin azlactone gives access to chiral multifunctional acyclic a-amino acid derivatives or protected cyclic quaternary a-amino acids.106 Despite significant advances in synthetic methodology, the efficient synthesis of enantiopure a-amino acids carrying complex side chains remains challenging. Palladium-catalyzed bidentate auxiliary-directed C–H functionalisation reactions for a-amino acid substrates have been investigated. A variety of a-amino acid precursors can undergo multiple modes of C(sp(3))–H functionalisation, including arylation, alkenylation, alkynylation, alkylation, alkoxylation, and intramolecular aminations at the b, g, and even d positions to form new a-amino acids.107 A highly efficient and enantioselective synthesis of g-lactams and g-amino acids by Rh-catalyzed asymmetric hydrogenation has been developed from cyano-substituted acrylate esters under mild conditions.108 Highly enantioselective synthesis of 5-substituted g-lactams was achieved by Ir-catalyzed sp(3) C–H alkylation of g-butyrolactam with alkenes. 5-substituted g-lactams were readily converted into chiral 4-substituted g-amino acids.109 Enantioselective aza-Diels–Alder reaction of oxodiazenes with a-chloroaldehydes by NHC catalysis leads to derivatives of a-amino acids with excellent yield and enantioselectivity.110 The difficult to access d(3)-amino acids can be produced by asymmetric cyclopropanation of conjugated cyanosulphones using a novel bifunctional organocatalyst.111 Phenylalanine ammonia lyases catalyze the synthesis of amino acids by 4-methylideneimidazole-5-one cofactor independent pathway.112 Amino Acids, Pept. Proteins, 2018, 42, 1–84 | 13

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Phenylalanine ammonia-lyase catalyzed the deamination of an acyclic amino acid. Enzyme mechanistic studies were achieved by a novel microreactor filled with magnetic nanoparticles.113 The simplest and minimal modification of a single amino acid or peptide bonds is represented by N-methylation. It is crucial to provide optically pure N-methyl-amino acids and N-methylated peptides structural and conformational studies. A review focuses on the results obtained in the field of chemical synthetic methodologies for the N-methylation of amino acids in the last decade.114 Another review specifically examines recent advances in substrate-controlled asymmetric reactions induced by the chirality of a-amino acid templates in natural product synthesis research and related areas.83 Studies on the diastereo- and enantioselective syntheses of anti-bhydroxy-a-amino acid esters using transition-metal-chiral-bisphosphine catalysts have been summarized. A variety of transition metals (Ru, Rh, Ir, and Ni) in combination with chiral bisphosphines, worked well as catalysts for the direct anti-selective asymmetric hydrogenation of a-amino-bketo ester hydrochlorides.115 1H-Imidazol-4(5H)-ones are introduced as novel nucleophilic a-amino acid equivalents in asymmetric synthesis. N,C(a),C(a)-trisubstituted a-amino acid derivatives were synthesized by using 1H-imidazol-4(5H)-ones.116 Esters of N-methyl-Pro and N-allyl-Pro were prepared and used for synthesis of chiral triazine based coupling reagents. The coupling reagent can be applied in the enantioselective incorporation of L- and D- amino acids directly from racemic substrate.117 Amino acids are produced at the multi-million-ton-scale with the economical fermentative production. Productivities of amino acid producing strains, e.g. Corynebacterium glutamicum and Escherichia coli are constantly improved by metabolic engineering. A review summarizes the new pathways for amino acid production as well as fermentative production of non-native compounds derived from amino acids or their metabolic precursors.118 The direct fermentation is an attractive method for production of L-Serine from sugars. However, low L-Ser production and superfluous by-product accumulation limit the industrial production on large scale. Several modifications demonstrated that combined metabolic and bioprocess engineering strategies could minimize byproduct accumulation and improve L-Ser productivity.119 The fermentative production of L-Thr and L-Ile with Corynebacterium glutamicum is usually accompanied by the by-production of L-Lys. An effective strategy was developed to reduce L-Lys by-production.120 A new system is used for continuous and direct production of poly-(gGlu) in a hybrid reactor system that integrated conventional fermentative production step with membrane-based downstream separation and purification. This integrated system provides compact, flexible, ecofriendly, and largely fouling-free ensuring steady and continuous production of poly-(g-Glu) directly from a renewable carbon source.121 A review focuses on the current trends and future perspectives of microbial poly(e-L-Lys) and contributes to the development of this novel homopoly(amino acid) and serve as a basis of studies on other biopolymers.122

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2.2 Natural a-amino acids and derivatives Many a-amino acids and their derivatives are found in addition to the 20 natural a-amino acids in various natural products. Constituting the structural basis of peptides and proteins, a-amino acids continue to be at the forefront of chemical synthesis research. The Strecker amino acid synthesis is an organic reaction used to convert an aldehyde or ketone and a primary amine or ammonia to an a-amino acid using a metal cyanide, acid catalyst, and water. The Strecker synthesis has long been considered for the synthesis of a-amino acids, but highly enantioenriched a-amino acids through this method remains a puzzle. Since nowadays enantiomeric purity is one of the major issues in a-amino acid synthesis, tremendous efforts have been put into the development of asymmetric versions of Strecker’s protocol. Near enantiopure L- and D- amino acids have been produced by asymmetrical Strecker reaction of an achiral imine at a single-crystal face.123 Replication of chiral a-amino acids has been achieved in combination with the asymmetric induction, amplification, and multiplication of their own chiral intermediates (L- and D-aminonitriles) in the solid-phase via Strecker reaction between three achiral components.124 A novel method for the synthesis of L- and D-amino a-amino acids by a Ni-catalyzed reductive cross-coupling of Ser/homo-Ser-derived iodides with aryl/acyl/alkyl halides is described. This method provides convenient access to varieties of enantiopure and functionalized amino acids.125 The reactivity of Pd-catalysts in the widely used C–H functionalisation strategies were investigated. It was demonstrated that the amino acid ligand plays crucial roles in the ligand-assisted Pd(II)-catalyzed C–H activation.126 Novel nonsymmetrically substituted N-protected b,b-diaryl-a-amino acids and esters have been produced through asymmetric hydrogenation of tetrasubstituted olefins by catalytic, asymmetric, and stereodivergent synthesis.127 Non-natural aliphatic a-amino acids were synthesized via asymmetric hydrogenation of b-alkyl (Z)-N-acetyldehydroamino esters employing rhodium-Duanphos complex catalyst with excellent yields and high enantioselectivities. An efficient approach for the synthesis of the intermediate of the orally administered anti-diabetic drugs Alogliptin and Linagliptin in the DPP-4 inhibitor class was also developed.128 Derivatives of a-amino acids have been prepared via enantioselective additions of masked acyl cyanide reagents to N-Boc-aldimines using modified cinchona alkaloid catalyst in excellent yields and high enantioselectivities.97 Three novel types of regio- and enantioselective multiple amino-functionalisations of terminal alkenes via cascade biocatalysis have been produced to get a-amino acids. The modular approach for engineering multi-step cascade biocatalysis is useful for developing other new types of one-pot biotransformations for chemical synthesis.129 N-alkyl aminomalonates undergo a fast and selective intramolecular C-N acyl rearrangement reaction in the presence of a strong base leading to N-protected glycinates in excellent yield. The reaction can be

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applied for the preparation of nonproteinogenic tertiary and quaternary N-alkyl a-amino acids in a very simple and reliable way.130 Highly functionalised azatricyclononanes were achieved by uncatalysed cycloaddition of substituted diaryldiazo compounds onto bicyclic unsaturated lactams derived from derivatives of pyroglutamic acid (Glp). The products provide rapid access to conformationally constrained amino acids and their analogues.131 The presence of NH function in ‘‘N–H’’ – Ni(II) complexes of Gly Schiff bases does not interfere with the homologation of the Gly residue. The practical application of these NH-type complexes were applied for the general synthesis of a-amino acids and in the asymmetric synthesis of various b-substituted Glp via Michael addition reactions with chiral Michael acceptors.132

2.3 a-Substituted analogues of amino acids Amino acids and their derivatives play a central role in the design of life. The relevance of the 20 proteinogenic L-amino acids as building blocks in peptides and proteins is self-evident, but the class of quaternary a-amino acids also called a,a-disubstituted amino acids has attracted the interest of organic chemists due to their biological importance. They play a crucial role and increasing interest in the development of unnatural peptides and peptidomimetics as therapeutics agents. Quaternary a-amino acids are present in a family of peptide antibiotics of fungal origin that could destabilize the membrane by formation of pores in the bilayer membrane. Quaternary a-amino acid residues such as a-aminoisobutyric acid and isovaline are present in peptaibol and peptaibiotics which constitute a family of peptide antibiotics of fungal origin. Peptaibiotic could destabilize the membrane by formation of pores in the bilayer membrane. Optically active a,a-disubstituted a-amino acids have generated increasing interest in biology, pharmacology, and in chemistry. Some of these compounds occur naturally or are structural components of natural products that have specific properties. The inclusion of a,a-disubstituted a-amino acids in a peptide may affect its secondary or tertiary structure by inducing a particular conformation. Biotechnological and biomedical chemical processes often use a,a-disubstituted a-amino acids. These compounds contain a quaternary stereo enter, which is challenging for stereoselective synthesis. The additional alkyl substituent at a-carbon could inhibit the free rotation of the peptide backbone leading to unique folding when incorporated into peptides. They can restrict the conformational freedom of the peptides and provide key information concerning the conformation responsible for biological recognition. Moreover, peptides containing a,a-disubstituted amino acid residues also tend to have an increased hydrophobicity, as well as an increased stability toward both chemical and enzymatic degradations. The first asymmetric synthesis of a-allyl-a-aryl a-amino acids is reported via a three-component coupling of a-iminoesters, Grignard 16 | Amino Acids, Pept. Proteins, 2018, 42, 1–84

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reagents, and cinnamyl acetate. The a-allyl group offers to generate further a-amino acid structures as exemplified by ring closing metathesis to generate a higher ring homologue of a-aryl-Pro.133 Asymmetric synthesis of a-allyl-a-aryl a-amino acids has also been achieved by tandem N-alkylation/p-allylation. Analogues of homo-Tyr and homo-Glu have been synthesized. This strategy can also be used to the asymmetric synthesis of acyclic and cyclic amino acid derivatives and higher ring homologues of enantioenriched a-substituted Pro.134 Allyl cyanate-toisocyanate rearrangement (Ichikawa rearrangement) can be the key step to achieve a,a-disubstituted a-amino acids.135 A Brønsted acid accelerated Pd-catalyzed direct asymmetric allylic alkylation of azlactones with simple allylic alcohols leads to quaternery allylic amino acid derivatives under mild reaction conditions in excellent yields and good enantioselectivities.136 Asymmetric allylic alkylation of azlactones with 4-arylvinyl-1,3-dioxolan-2-ones with Pd catalyst was developed leading to ‘‘branched’’ chiral a-amino acids with vicinal tertiary and quaternary stereocenters in high yields. Studies on mechanism revealed that the formation of a hydrogen bond between the Pd–allylic complex and azlactone isomer is responsible for the excellent regioselectivities.137 An electrophilic alkynylation of azlactones can lead to C(a)-tetrasubstituted a-amino acid derivatives in short reaction times (Scheme 4).138 An enantioselective synthesis of a,a-disubstituted a-amino acids have been developed via direct catalytic asymmetric addition of acetonitrile to a-iminoesters bearing an N-thiophosphinoyl group.139 (R) or (S)-a-benzyl-b-azido-Ala, a-benzyl-b-(1-pyrrolidinyl)-Ala, a-benzylb-(1-piperidinyl)-Ala, and a-benzyl-b-(4-morpholinyl)-Ala have been replaced by Phe in deltorphin I analogues. The potency, selectivity, affinity, and conformational behaviour were investigated.140 Asymmetric synthesis of a,a-disubstituted amino acids using regio- and stereocontrolled 1,3-dipolar cycloaddition reactions with vinyl ethers and a,a-dialkylketonitrones have

Scheme 4 Amino Acids, Pept. Proteins, 2018, 42, 1–84 | 17

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been reported. A series of a,a-disubstituted amino acid derivatives have been synthesized with a novel method using silver carbonate, diaryliodonium bromides as aryl sources, and azlactones.142 A review provides an overview on the synthesis and applications of a-quaternary a-ethynyl a-amino acids from 1977 to 2015.143 Alkylation of deprotonated a-aminonitriles derived by the Strecker reaction from (4S,5S)-5-amino2,2-dimethyl-4-phenyl-1,3-dioxane leads to a series of a-quaternary arylglycines in high optical purity (Scheme 5).144 Derivatives of a,a-disubstituted a-amino acid were produced by asymmetric Michael addition of azlactones to a,b-unsaturated trichloromethyl ketones with the use of commercially available quininederived thiourea catalyst in high yields and with excellent stereoselectivities.145 Pseudoephedrine-directed asymmetric a-arylation of a-amino acid derivatives leads to quaternary amino acids.146 Enantioenriched a,a-dialkyl substituted a-nitroacetates were produced by an enantioselective conjugate addition of a-alkyl substituted a-nitroacetates to phenyl vinyl selenone with a use of a novel Cinchona alkaloid catalyst. The Michael adducts were converted to various cyclic and acyclic quaternary a-amino acids.147 All-carbon quaternery stereogenic centres were achieved by asymmetric Michael reaction of nitroalkanes and b,b-disubstituted a,b-unsaturated aldehydes catalyzed by diphenylprolinol silyl ether. The reaction mechanism is also discussed.148 Asymmetric Michael addition of a-fluoro-a-nitro esters to nitroolefins leads to quaternary a-fluoro-a-substituted amino acids in excellent chemical yields and with high enantioselectivities.149 Quaternary a-nitroesters have been achieved by enantioselective Michael addition of tertiary a-nitroesters to b-unsubstituted vinyl ketones in the presence of an L-tertLeu-derived squaramide as organocatalyst. The products can be transformed to quaternary a-amino acids.150 Enantioselective C-2- and C-4-selective g-additions of oxazolones to 2,3butadienoates catalyzed by phosphine lead to 2-aryl-4-alkyloxazol-5-(4H)ones that provided rapid access to optically enriched a,a-disubstituted a-amino acid derivatives.151 A chiral phosphine can catalyze the stereoconvergent g-additions of racemic nucleophiles (racemic heterocycles) to racemic electrophiles. This method enables the synthesis of protected a,a-disubstituted a-amino acid derivatives in good yield, diastereoselectivity, and enantioselectivity.152 An enantioselective and diastereoselective asymmetric aldol reaction by memory of chirality starting from L-Ala provides a rapid access to enantiopure b-hydroxy quaternary a-amino acids in three steps.153

Scheme 5 18 | Amino Acids, Pept. Proteins, 2018, 42, 1–84

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A highly stereoselective multicomponent cascade reaction of ketones or unprotected ketohexoses and unprotected amino acids leads to quaternary stereogenic centre.154 The pyridoxal-5 0 -phosphate-dependent L-serine hydroxymethyltransferase from Streptococcus thermophilus was engineered to achieve the stereoselective synthesis of a broad structural variety of a,a-dialkyl-a-amino acids.155 Pyrrolidine-2-carboxylate esters substituted in the 3-, 4- or 5-positions were converted to their N 0 -aryl urea derivatives by intramolecular diastereoselective arylation. After several reaction steps, a range of enantiopure and enantioenriched quaternary a-aryl Pro derivatives have been achieved.156 1,4-Substituted triazole oligomers are made from derivatives of quaternary amino acids that present a conformational behaviour with similarities to that of natural peptides.157 A new and efficient method has been developed for the synthesis of racemic-protected a-ethynyl Phe starting from DL-2-benzyl-Ser in ten steps. The absolute configurations of the separated enantiomers were also determined.158

2.4 Aliphatic and cycloaliphatic a-amino acids Among the numerous general strategies that have commonly been used for the syntheses of a-amino acids, there are many that apply de novo synthesis focused on enantioselective bond construction around the a carbon and others that consider conversion of existing precursors of aamino acids carrying suitable functional groups on side chains. A strategy based on the selective functionalisation of side chain C–H bonds of various readily available precursors of a-amino acids may provide a straightforward and broadly applicable synthesis and transformation of a-amino acids. A systematic investigation of palladium-catalyzed bidentate auxiliary-directed C–H functionalisation reactions for a-amino acid substrates was carried out. The palladiumcatalyzed auxiliary-directed sp(3) C–H functionalisation reduces the synthetic difficulty for many a-amino acids leading to a wide range of b-monosubstituted a-amino acids that also can undergo further C–H functionalisation at the b-methylene position to generate various b-branched a-amino acids in a stereoselective way.107 Pd-catalyzed alkylation of methylene C(sp(3))–H bonds of aliphatic quinolyl carboxamides with a-haloacetate and methyl iodide have been applied in the stereoselective synthesis of various b-alkylated a-amino acids. This method also provides a convenient and powerful way to site-selectively incorporate isotopes into the carbon scaffolds of amino acids.159 Cyclopropane a-amino acid esters bearing quaternary carbon centres have been synthesized by cyclopropanation of 2-aminoacrylates with N-tosylhydrazones in high yields and diastereoselectivities.160 The literature data on asymmetric synthesis of a-amino acids via Michael addition reactions involving Ni(II)-complexes is reviewed. The discussion is divided into two groups dealing with applications of: (a) Ni(II)-complexes of Gly as C-nucleophiles and (b) Ni(II)-complexes of dehydro-Ala as Michael acceptors. The review focused on the practical Amino Acids, Pept. Proteins, 2018, 42, 1–84 | 19

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aspects of the methods. Asymmetric synthesis of a-allyl-a-aryl a-amino acid esters was developed. The syntheses of analogues of homo-Tyr and homo-Glu and derivatives of cyclic a-amino acids were completed.134 Synthesis of g-amino acids by Rh-catalyzed asymmetric hydrogenation of cyano-substituted acrylate esters has been developed by the use of Rh-(S,S)-f-spiroPhos complex with high yield and enantioselectivity.108

2.5 Amino acids with branched chain It is known for more than 40 years that trouble in metabolism of branched-chain amino acids leads to higher levels of Ile, Leu, and Val that is strongly associated with higher risk of type 2 diabetes, but it is not known whether this association is causal. Genome-wide association studies coupled with large-scale metabolomic measurements were used to investigate the etiologic relationship between metabolism of branchedchain amino acid and type 2 diabetes.161 A review on branched-chain amino acids in metabolic signalling and insulin resistance concluded that increased levels of branched-chain amino acids are more likely to be a marker of loss of insulin action and not causative.162 Plasma branchedchain amino acids are positively associated with incident diabetes and insulin resistance163 and underlying metabolic abnormalities.164 It was also shown that high consumption of branched-chain amino acids is associated with an increased risk of type 2 diabetes.165 Recently, a Mendelian randomisation study using genetic variants associated with circulating branched-chain amino acid levels and insulin resistance as instrumental variables suggests that higher levels of branched-chain amino acids do not have a causal effect on insulin resistance while increased insulin resistance drives higher circulating fasting branchedchain amino acid levels.166 Amino acids in plasma are valuable biomarkers to determine increased risk of mortality in patients with end-stage liver disease. Val concentrations and constellations composed of branched-chain and aromatic amino acids were strongly associated with prognosis.167 Elevated plasma levels of branched-chain amino acids also are associated with a greater than twofold increased risk of future pancreatic cancer diagnosis.168 Branched chain amino acids (Ile, Leu, Val) participate in protein synthesis in animals and humans and regulate many key signalling pathways that connect many diverse physiological and metabolic roles. Abnormally elevated levels of branched chain amino acids in the blood seem to be a good biomarker for the early detection of obesity, diabetes, and other metabolic diseases. A review provides some insights into these novel metabolic and physiological functions of branched chain amino acids.169 Conflicting data exist on the impact of dietary and circulating levels of branched chain amino acids on cardiometabolic health and it is unclear to what extent these relations are mediated by genetics. In a crosssectional study, female twins were examined. The results showed that higher intakes of branched chain amino acids were associated, independently of genetics, with lower insulin resistance, inflammation, blood 20 | Amino Acids, Pept. Proteins, 2018, 42, 1–84

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pressure, and adiposity-related metabolites. The associations between intakes of amino acids with known mechanistic links to cardiovascular health and direct measures of arterial stiffness, central blood pressure, and atherosclerosis were examined. It was found that higher intakes of Leu, Glu, and Tyr were most strongly associated with pulse wave velocity. The data provided evidence that intakes of several amino acids are associated with cardiovascular benefits beyond blood pressure reduction.171 A pharmacometabonomic study showed that branched-chain amino acids are predictors for individual differences of cisplatin nephrotoxicity in rats. This study could provide new insight into cisplatin nephrotoxicity and may help expedite personalized medicine of cisplatin or other antitumour drugs in future clinical studies.172 Production of syn-selective b-branched a-amino acids is based on the alkylation of Gly imine esters with secondary sulphonates. The optimized conditions enabled a straightforward preparation of a number of b-branched a-amino acids.173

2.6 Amino acids with halogen substitution Strategies to protect bioactive peptides from serum proteolytic degradation include incorporation of halogens into natural or nonnatural amino acids. Among the halogen-substituted amino acids, the most important ones have fluorine in the molecule. Fluorine became the ‘‘second-favourite heteroatom’’ after nitrogen in drug design due to the special properties of fluorine–containing compounds.174 It was empirically demonstrated that the presence of fluorine in compounds results in metabolic stability leading to improved bioactivity and bioavailability.175–177 Since compounds with fluorine mimic their parent compounds with hydrogen well, they fit into the same enzyme binding site.178 The highly electronegative fluorine atom forms a highly polarized bond of extraordinary strength with carbon.178 Combination of the unique physical and chemical properties of fluorine with amino acids represents a new approach for the design of biologically active peptides with improved pharmacological properties.179 Due to their unique electronic properties, fluorinated amino acids have huge effects on protein stability, protein–protein as well as ligand– receptor interactions, and the physical properties of protein- or peptide-based materials.176,178,179 In order to alter distinct properties of peptides and proteins (e.g. hydrophobicity, acidity/basicity, and conformation), it is an efficient strategy to incorporate amino acids with fluorinated side chains. Proteins and complexes with proteins can be stabilized by highly fluorinated amino acids via enhanced hydrophobicity, and provide novel methods for identification of specific molecular events in complex solutions.178,180 It was shown that specific incorporation of fluorinated amino acids into proteins can experimentally distinguish cation–p interactions between the p systems of aromatic residues and the positively charged portion of phospholipid head groups from membrane insertion of the aromatic side chains of amino acids in proteins which hydrophobically interact with Amino Acids, Pept. Proteins, 2018, 42, 1–84 | 21

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lipid tails. Fluorinated aromatic amino acids destabilize the cation–p interactions by altering electrostatics of the aromatic ring, whereas their increased hydrophobicity enhances membrane insertion.181 In the contrary, the majority of anion–p interacting residues are located in regions with helical secondary structure. The influence of anion–p interactions should not be neglected in supramolecular chemistry.182 Recently, it was found that incorporation of pentafluoro-Phe into human growth hormone releasing hormone analogues suppressed the growth of different human tumours in vitro and in vivo.183 Although 4-F-Thr is the only naturally occurring fluoro-amino acid to date,178 many synthetic methods are developed for the syntheses of mono- or poly-fluoro and other halogen amino acids and derivatives. As one of the possible fluorinated analogues of proteinogenic Ile, two diastereoisomers of 5,5,5-trifluoroisoleucine ((2S,3S)-5-F3Ile and (2R,3S)5-F3-allo-Ile) have been synthesized in enantiomerically pure form and the relationship of their side chain hydrophobicity and a-helix propensity were examined.184 g-(4S)-Trifluoromethyl Pro was synthesised by a modified literature protocol with improved yield and a multigram scale, and the conformational properties of the amide bond were characterised. Replacement of native Pro by g-trifluoromethyl Pro in the peptide antibiotic gramicidin S was shown to preserve the overall amphipathic peptide structure.185 The synthesis of (2S,4R)- and (2S,4S)-iodophenyl ethers of hydroxy-Pro has been described. These amino acid derivatives are capable of modification via rapid, specific Suzuki and Sonogashira reactions in water and provide new functionalisation for peptides with great control of conformation.186 Fluorinated Phe analogues (4-fluoro-, 2,4-difluoro-, or 4-trifluoromethylPhe) were incorporated into endomorphin-2-analogues. They showed strong antinociceptive effect indicating that they were able to cross the blood–brain barrier.187 Two all-cis, 2,3,5,6-tetrafluorocyclohexyl (S)phenylalanines having the 2,3,5,6-tetrafluorocyclohexyl group in meta and para positions at the aromatic ring of Phe have been synthesized as novel fluorinated a-amino acids for peptide synthesis.188 A stereoselective method has been developed for the synthesis of new b-fluorinated 2-aminocyclohexanecarboxylic acid derivatives with fluorine at position 4 of the ring.189 Substituted anti-b-fluorophenylalanines were produced from the corresponding enantiopure a-hydroxy-b-amino esters using a stereospecific XtalFluor-E promoted rearrangement procedure (Scheme 6) in good yield and high diastereoisomeric purity.190 Optically pure amino acids that have easily replaceable functional groups at the o-positions are highly important synthetic targets, as the

Scheme 6 22 | Amino Acids, Pept. Proteins, 2018, 42, 1–84

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Scheme 7

functional group at the o-position can be modified to the required group, which provide a series of non-natural amino acids. o,o-difluoroalkyl amino acid derivatives were synthesized by oxidative desulphurizationfluorination reactions of suitable arylthio-2-phthalimido butanoates and pentanoates. Mainly a,o-polyfluorinated amino acid derivatives were formed by additional sulphur-assisted a-fluorination.191 Fmoc-perfluorotert-butyl-Tyr was synthesized in two steps from commercially available Fmoc-4-NH2-Phe via diazotization followed by diazonium coupling reaction with perfluoro-tert-butanol.192 Fmoc-, Boc-, and free (2S,4R)- and (2S,4S)-perfluoro-tert-butyl 4hydroxy-Pro have been synthesized in 2-5 steps. The perfluoro-tert-butyl group was incorporated with perfluoro-tert-butanol in a Mitsunobu reaction. Peptides containing these amino acids were detected by (19)F NMR, suggesting their use in probes and medicinal chemistry.193 Trifluoromethyl-substituted cyclopropane a-amino acids could be obtained with a method that starts with cyclopropanation of trisubstituted olefinic azlactones with a stock solution of CF3CHN2 in CH3CN (Scheme 7). Azlacton rings having trifluoromethyl-substituted cyclopropanes are produced in good to high yields and diastereoselectivities.194 Fluorinated homoproline derivatives bearing three stereogenic centres are achieved by intramolecular aza-Michael reaction from 2-p-tolylbenzyl carbanions as a source of chiral benzyl nucleophiles. The selectivity of the cyclization process can easily be set by changing the reaction conditions.195 A radical based synthesis of a variety of protected enantiopure fluorinated derivatives of a-amino acids is described that lead to mercapto-a-amino acids (Scheme 8) for native chemical ligation.196 Pd(II)-catalyzed fluorination of inactivated methylene C(sp(3))–H bonds led to diastereoselective b-fluorinated a-amino acids and their derivatives.197 Highly site-selective and diastereoselective fluorination on inactivated sp(3) carbons were achieved by the use of palladium acetate as the catalyst leading to derivatives of b-fluorinated amino acids.198 It was shown for the first time that a quinoline-based ligand contributes to b-C(sp(3))–H fluorination. Nonnatural enantiopure fluorinated a-amino acids were obtained through sequential b-C(sp(3))–H arylation and subsequent stereoselective fluorination from L-Ala.199 Amino Acids, Pept. Proteins, 2018, 42, 1–84 | 23

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Scheme 8

Sometimes enzymes are applied as catalysts for the production of fluorinated amino acids or their derivatives. The production of nitro analogues of 4-F-DL-Trp and 5-F-L-Trp in large scale is based on the use of TxtE-based class I cytochrome P450 enzyme;200 this was the first biological nitration procedure. Arabidopsis thaliana (AtPAL2) proved to be a very good catalyst for the formation of (S)-3-F-Phe, (S)-4-F-Phe and (S)-2-Cl-Phe.201 These halogenated amino acids are valuable building blocks for the formation of various drug molecules. Many physiological and pathological processes (e.g. atherosclerosis and tumorigenesis) involve matrix metalloproteinases that belong to a group of zinkdependent endopeptidases located in the extracellular matrix. Fluorinated analogues of a-aminocarboxylic and a-amino hydroxamic acid-based matrix metalloproteinase inhibitors showed comparable or superior inhibiting potencies as compared to their non-fluorinated analogues. The corresponding a-aminocarboxylic acid derivatives are less potent than the a-amino hydroxamic acid analogues or were inactive.202 Multi-substituted b-lactams can be used as building blocks for the construction of b-amino acids. A review from Tarui summarizes the direct functionalisation of fluoro-b-lactams.203 The introduction of an electronegative fluorine atom to a b-lactam ring gave the corresponding fluoro-b-lactam that can be used for the preparation of electrophilic b-lactams, and these compounds can be converted to fluorinated b-amino acids.203 a-Fluorinated b-amino thioesters were obtained in high yields and stereoselectivities under mild reaction conditions by organocatalyzed addition reactions of a-fluorinated monothiomalonates to N-Cbzand N-Boc-protected imines. The addition products were used for coupling-reagent-free peptide synthesis.204 A diastereoselective addition of fluoroacetate or a-alkylated fluoroacetate to N-tert-butylsulfinyl imines results in a-fluoro-b-amino acids containing fluorinated quaternary stereogenic carbon centres (Scheme 9) with very good yields and high diastereoselectivities.205 A diastereoselective Mannich-type reaction of a-alkyl, a-aryl, and a-vinyl fluoroacetates with N-tert-butylsulfinyl imines provides a broad range of highly functionalized b-amino acids containing a-fluorinated quaternary stereogenic carbon centres. The stereochemical outcome of the present simple reaction is highly dependent on the steric and electronic properties of the fluorocarbon nucleophiles.206 Enantiomerically 24 | Amino Acids, Pept. Proteins, 2018, 42, 1–84

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Scheme 9

Scheme 10

pure (R)- or (S)-configured 3-amino-4,4,4-trifluorobutanoic acids were obtained by asymmetric Mannich reactions of malonic acid derivatives and (SS)-N-(tert-butanesulfinyl)-3,3,3-trifluoroacetaldimine with the use of inorganic or organic base as catalyst.207 Despite the growing demand for enantioenriched fluorine containing small molecules, a- and b-fluorinated carbonyl compounds have been neglected in direct enolization chemistry because of the competing and dominating defluorination pathway. Direct and highly stereoselective Mannich-type reactions of a- and b-fluorine-functionalized 7-azaindoline amides guarantee an efficient enolization, while suppressing undesired defluorination provide a series of fluorinated analogues of enantioenriched b-amino acids (Scheme 10).208 Highly fluorinated non-natural amino acids and derivatives were produced by using oxazolone enolate for a nucleophilic substitution of highly fluorinated (hetero)arenas.209 Fluorinated aminoanthranilamides, derivatives of non-native aromatic beta-amino acids have been developed by a highly regio-selective nucleophilic aromatic substitution of difluorinated nitrobenzoic acid. The findings opened unexplored routes to novel amino-acid structures.210 Novel fluorinated analogues of g-aminobutyric acid (b-polyfluoroalkylGABAs) have been synthesized with substituents b-CF3-b-OH, b-CF3; and b-CF2CF2H, these analogues are bioisosteres of Pregabalin (Lyricas). Biological investigations showed that fluorinated analogues of GABA are structural but not functional analogues of GABA.211 2.7 Hydroxy amino acids Chiral a-hydroxy amino acids and their derivatives play important role in preparation of pharmaceuticals and are involved in many biological processes. b-Hydroxy-a-amino acids are important chiral building blocks in chemical syntheses and serve as precursors to many important medicines. Amino Acids, Pept. Proteins, 2018, 42, 1–84 | 25

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In addition, they are also found in natural products and show antimicrobial or anti-cancer properties or inhibition of b-amyloid peptide release. Derivatives of b-hydroxy-a-amino acids have been produced by aldolization of pseudoephenamine glycinamide with lithium hexamethyldisilazide in the presence of LiCl followed by addition of an aldehyde or ketone. The stereoisomerically pure products can be transformed into b-hydroxy-a-amino acids by mild hydrolysis.212 The Cu-catalyzed asymmetric conjugate hydroboration reaction of b-substituted a-dehydroamino acid derivatives has been accomplished leading to enantioenriched synand anti-b-boronate-a-amino acid derivatives with excellent yields and enantioselectivities. The hydroboration products were converted into b-hydroxy-a-amino acid derivatives.213 b-hydroxy-a-amino acid derivatives have been produced by an enantioselective four-component reaction of a diazoketone, water, an aniline and ethyl glyoxylate in the presence of catalytic Rh2(OAc)4 and a chiral Brønsted acid in good yields and high diastereoselectivity and enantioselectivity.214 A review of Zhang Y. also summarizes other methods for the syntheses of b-hydroxy-a-amino acids.215 An asymmetric aldol reaction by memory of chirality is reported with a substrate control of stereoselectivity by aldehyde. Enantiopure b-hydroxy quaternary a-amino acids have been obtained by starting from L-Ala in three steps.153 A stereoselective synthesis of anti-b-hydroxy-a-amino acids is based on the palladium-catalyzed sequential C(sp(3))–H functionalisation by 8aminoquinoline, followed by a previously established monoarylation and/or alkylation of the b-methyl C(sp(3))–H of Ala derivative leading to various anti-b-hydroxy-a-amino acid derivatives. b-mercapto-a-amino acids that are very important for to chemical ligation can also be produced by this practical method.216 Natural L-a-amino acids having aliphatic, aromatic, or heteroaromatic moieties were transformed to the corresponding enantiopure (R)- or (S)-a- hydroxyacids by formal biocatalytic inversion or retention of absolute configuration. The one-pot transformation was carried out by a concurrent oxidation reduction cascade in aqueous media.217 N-hydroxy-Gly, N-hydroxy-Ser, L-homoserine, and a-hydroxy-Gly have been synthesized and tested for their water-holding capacities. Some predictive ‘rules’ for further design and refinement of chemical structures also have been developed.218 Threonine aldolases are very stereoselective for a-carbon and catalyze the pyridoxal phosphate-dependent condensation between amino acids and aldehydes. A review published the mechanism of reactions by threonine aldolases.69 Threonine aldolases also catalyse the unnatural aldol condensation with Gly to produce valuable b-hydroxy-a-amino acids. A review by Fesko summarizes the techniques and enzyme engineering and mutagenesis studies.219 Natural threonine aldolases were also used for the direct biochemical synthesis of tertiary a-amino acids. The novel L- and D-threonine aldolases catalyze the asymmetric synthesis of b-hydroxy a-methyl- and a-hydroxymethyl-a-amino acids with perfect enantioselectivity at a-carbon.220 A novel stereoselective synthesis of all four isomers of b- and g-hydroxy a-amino acids was achieved. The strategy is based on enzymatic kinetic 26 | Amino Acids, Pept. Proteins, 2018, 42, 1–84

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resolution and cyanate-to-isocyanate rearrangement as key steps. The proper choice of the starting hydroxyacid, the course of kinetic resolution, and the stereospecific sigmatropic rearrangement step resulted in stereo control with full chirality transfer.221 Phosphine ligand stabilized air-stable Cu(I) complexes have been successfully used to functionalize the aromatic aminobenzoic acids in a chemoselective manner without protection and deprotection strategy under mild reaction conditions. This chemoselective carbene insertion into –NH bond over –COOH and –OH bonds leads to a wide range of carboxy and hydroxy functionalized a-amino esters.25 Stereoselective synthesis of analogues of (2S,3R)-a-hydroxy-b-amino acid has been reported by Cu(I)-catalyzed reactions of (R)-glyceraldehyde acetonide and dibenzylamine with terminal alkynes leading to the corresponding (2S,3R)-a-amino alcohols with good-to-excellent diastereoselectivity. Subsequent chemical transformations provided easy access to alkynyl side-chain containing (2S,3R)-a-hydroxy-b-amino acids.222 Substituted a-hydroxy-b-amino amides have been synthesized by a carbamoyl anion-initiated cascade reaction with acylsilanes. A series of a-aryl-a-hydroxy-b-amino amides has been synthesized in high yields with excellent diastereoselectivities.223 A new method is developed for direct access to derivatives of isoserine from simple imines in a four-step, one-pot reaction. The strategy employs a hypervalent iodine(III)-catalyzed bromination/rearrangement/cyclization cascade reaction that leads to a broad range of structurally different lactams from cheap and easily available imides. This cascade reaction is furthermore extendable by an in situ ring-opening reaction, giving direct access to a-hydroxy b-amino acids from simple imines in a four-step, one-pot reaction (Scheme 11).224 Two isomeric forms of Hyp that is found in collagen and few other extracellular proteins (trans-4-hydroxy-L- and trans-3-hydroxy-L-Pro) play an important role in collagen synthesis and thermodynamic stability of the triple-helical conformation of collagen and associated tissues. Elevated level of Hyp is observed in several disorders, while its decreased level is a marker of poor wound-healing. A review summarizes the potential use of Hyp as a biochemical marker.225 An enzymatic method can be used to measure the content of T4LHyp (Hyp 2-epimerase) in the acid-hydrolysate of collagen or blood plasma.226 To date, there are few general methods that describe the O-arylation of L-Ser. This transformation has been achieved by nucleophilic aromatic substitution, but this protocol is limited by the use of strong bases (NaH and KHMDS) and the need for 1-fluoro-2-nitrobenzene substrates. An effective and practical protocol for O-arylation of b-hydroxy-a-amino acids (Thr and Ser) was achieved via Chan–Lam cross-coupling leading to novel

Scheme 11 Amino Acids, Pept. Proteins, 2018, 42, 1–84 | 27

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Scheme 12

unnatural derivatives of b-aryloxy-a-amino acids. This new Cu(II)catalyzed transformation involves mild conditions and is well-tolerated with a variety of protected (Boc-, Cbz-, Tr-, and Fmoc-) Ser and Thr derivatives and various potassium organotrifluoroborates and boronic acids.227 A synthetic approach towards (2S,3S)-3-hydroxyleucine that can be found in an increasing number of bioactive natural products was developed.228 A novel method showed that carbohydrates can be applied as starting materials to prepare amino acids. The synthesis of (2S,5R) and (2S,5S)-5-hydroxy-lysine was reported by using D-galactose as a chiral precursor with stereo retention.229 The mechanism of action of several hydroxy-Trp containing tritrpticin derivatives that has a strong microbial activity against Gram-positive and Gram-negative bacteria as well as fungi was studied. The addition of a hydroxyl group to the indole ring of Trp was able to modify the mechanism of action of the peptides. This study also shows that 5OH-Trp constitutes a new probe to modulate the antimicrobial activity and mechanism of action of other Trp-rich antimicrobial peptides.230 Gamma-hydroxy norvaline (Scheme 12) was produced in a one-pot organocatalytic Mannich reaction and an enzymatic ketone reduction by using 2-PrOH both as solvent and as reducing agent. Each of the four stereoisomers was achieved in high yield and excellent stereoselectivity.231 The b-turn inducer cyclic hydroxy-amino acids also are important building blocks for the development of new therapeutic drugs. During the last decades, the stereoselective synthesis of hydroxy-amino acids has been growing since some of their representatives show enzyme inhibitory activities. An enantioselective total synthesis of the amino acid (2S,4R,5R)-4,5-di-hydroxy-pipecolic acid starting from D-glucoheptono-1, 4-lactone is described that involved 12 steps with an overall yield of 19%. The structures of the compounds synthesized were elucidated on the basis of comprehensive spectroscopic (NMR and MS) and computational analysis.232 Four novel potential renin inhibitors have been synthesized. All these inhibitors contain unnatural moieties that are derivatives of N-methylleucyl-b-hydroxy-g-amino acids (4-[N-(N-methylleucyl)-amino]3-hydroxy-7-(3-nitroguanidino)-heptanoic acid, 4-[N-(N-methylleucyl)amino]-3-hydroxy-5-phenyl-pentanoic acid, or 4-[N-(N-methylleucyl)amino]-8-benzyloxycarbonylamino-3-hydroxyoctanoic acid).19 2.8 Unsaturated amino acids Unsaturated a-amino acids have turned out to be especially important building blocks for incorporation into proteins due to the diverse reactivities of the multiple bonds and their ability to introduce biologically active functionalities. These compounds are also used in peptide 28 | Amino Acids, Pept. Proteins, 2018, 42, 1–84

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Scheme 13

chemistry to confer b-turn secondary structure to induce new properties as enzyme inhibitors and peptidomimetics as therapeutic agents. A review of Boibessot T provides an overview of the literature from 1977 to 2015 concerning synthesis and applications of a-quaternary a-ethynyl a-amino acids (Scheme 13).143 Sterically constrained a-amino acids are important in drug industry. Asymmetric synthesis of (1R,2S)-1-amino-2-vinylcyclopropanecarboxylic acid was achieved via two-step SN2 alkylation of a novel chiral nucleophilic Gly equivalent with excellent yields and diastereoselectivity.105 Enantiopure a-amino phenylacetic acids (phenylglycines) are important structural motifs found in many pharmaceuticals and biologically active compounds, e.g. antibiotics. Phenylglycine (Phg) derivatives can also be used as catalysts and chiral building blocks in organic syntheses. Kinetic resolution of enantioselective C–H olefination of a-amino phenylacetic acid was developed by a palladium(II)-catalyzed enantioselective C–H activation and C–C bond formation.102 Stereoselective synthesis of unsaturated a-amino acids by asymmetric alkylation was summarized. An enantioselective approach induced by the Corey–Lygo catalyst under chiral phase transfer conditions or the hydroxypinanone chiral auxiliary, both implicating Schiff bases as substrate (Scheme 14). The use of a prochiral Schiff base gave higher enantiomeric excess and yield in the final desired amino acid.233 A transition metal-free, organocatalytic asymmetric synthesis for b-alkynyl-b-amino acids have been developed via a mild chiral Brønsted base-catalysed asymmetric Mannich-type reaction of in situ generated N-Boc C-alkynyl imines with a-substituted b-keto esters and less-acidic malonate (thio)esters as nucleophiles with high efficiency. The catalytic activation strategy may have broad use in catalysis and synthesis. This methodology could also be applied for the catalytic asymmetric synthesis of biologically important b-alkenyl-b-amino acids that are difficult to prepare by asymmetric catalysis and b-alkyl-b-amino acids.234 Palladium-catalyzed N-quinolyl carboxamide-directed olefination of the inactivated C(sp(3))–H bonds of phthaloyl Ala with vinyl iodides (Scheme 15) led to a wide range of b-vinyl a-amino acids.235 One-pot synthesis of highly functionalized a-vinylated g-oxo-b-amino esters from three-components is revealed. The cycloaddition of the 1-alkyne and sulphonyl azidetriazole with the use of Cu(I)-catalyst resulted in 1-sulfonyl-1,2,3-triazole. An a-imino Rh(II)-carbene generated from an open-chain a-imino diazo derivative of the triazole, reacts with g-hydroxy a,b-unsaturated esters to form allylic (Z)-amino vinyl ethers. The later one led, after rearrangement, to a-vinyl g-oxo-b-amino esters in high yields and diastereoselectivity.236 Amino Acids, Pept. Proteins, 2018, 42, 1–84 | 29

30 | Amino Acids, Pept. Proteins, 2018, 42, 1–84 Scheme 15

Scheme 14

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Photoredox a-vinylation of a-amino acids produced several natural products and a number of pharmacophores.237 Radical decarboxylative allylation of N-protected a-amino acids and esters has been accomplished via a combination of palladium and photoredox catalysis.238 Derivatives of all-carbon quaternary allylic amino acids have been produced by a Brønsted acid accelerated Pd-catalyzed asymmetric allylic alkylation of azlactones with simple allylic alcohols.136 Protected d,e-unsaturated a,b-diamino acids as templates have been used for the preparation of 12 new a,b-diamino acids.239 Derivatives of exomethylenic cyclohexane b-amino acid were selectively synthesized from unsaturated bicyclic b-lactams by transformation of the ring olefin bond through three different regio- and stereocontrolled hydroxylation techniques, followed by hydroxy group oxidation and oxo-methylene interconversion with a phosphorane.240 Dehydroalanines equipped with oxazolidin-2-one chiral auxiliaries have been prepared and applied for stereoselective synthesis of substituted tryptophans [(S)-2-methyltryptophan and (S)-5-fluoro-Trp].241 2.9 a-Amino acids with aromatic, heteroaromatic, and heterocyclic side-chains Non-covalent interactions involving aromatic amino acids are ubiquitous in nature and facilitate most of the chemical and biological processes. Aromatic amino acids play a diverse role in stabilization of a-helix and b-sheet of soluble and membrane proteins.242 The most often used strategy to increase potency, selectivity, and metabolic stability is constraining the conformation of flexible peptides. Not only constraining the backbone dihedral angles, but the correct orientation of the amino acid side chains is equally important. The applications of cyclized analogues of the aromatic amino acids (Phe, Tyr, Trp, and His) within peptide medicinal chemistry are showed with examples of enzyme inhibitors and ligands for G protein-coupled receptors.243 Significant advances in C–H bond functionalisations have been achieved with the discovery of new mechanisms in the past decade. Coordinating activation strategy using Nickel-catalysed radical oxidative cross-coupling between C(sp(3))–H bonds and (hetero)arylmethyl free radicals have been developed. Considering that the free radical acts as a real coupling partner, the reported transformation has further enriched the type of the oxidative cross-coupling reactions. This method can use many a-amino acids, (hetero)arylmethanes, arylmethylenes, and arylmethines leading to a large library of both a-tertiary and a-quaternary b-aromatic a-amino acids. In addition, this reaction can proceed well on a larger scale, and the coordinating group can be handled readily and removed easily.244 Intermolecular catalytic (Pd(II)) arylation of inactivated b-C(sp3)–H bonds in a-hydroxy aliphatic acid derivatives with aryl iodides was achieved. The feasibility of amino acid auxiliary as a directing group also was demonstrated.245 Direct palladium-catalyzed C(sp(3))–H arylation of amino acid derivatives with aryl iodides bearing different electronic Amino Acids, Pept. Proteins, 2018, 42, 1–84 | 31

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Scheme 16

properties resulted in the desired amino acid molecule by the cleavage of the tethered click-triazoles after the catalytic reaction. This provides a practical protocol for the production of both natural and synthetic amino acids.246 Complex b-aryl a-amino acids have been synthesized by palladium-catalyzed b-C(sp(3))–H arylation of phthaloyl Ala derivatives with aryl iodides. A variety of aryl iodides (e.g. bearing alkoxyl, carbonyl, nitro, and halogen groups in ortho position) can react with the 2-(2pyridyl) ethylamine-coupled phthaloyl Ala (Scheme 16). This method can be used for preparing complex b-aryl a-amino acids.247 A highly efficient monoarylation reaction by Pd-catalyzed b-methyl C(sp(3))–H of an Ala derivative with aryl iodides using an 8-aminoquinoline led to various b-aryl-a-amino acids with high efficiency and retention of chirality.248 The mechanism of amino acid ligand-assisted Pd(II)-catalyzed C–H activation and the important roles of the amino acid ligand and the CsF base was analyzed in a review.126 Fmoc-protected aryl/heteroaryl-substituted phenylalanines (Bip derivatives) using the nonaqueous palladium-catalyzed Suzuki–Miyaura cross-coupling reaction of Fmoc-protected bromo- or iodophenylalanines have been reported (Scheme 17). This method allows for the direct formation of a variety of unnatural biaryl-containing amino acids.249 Substituted D-phenylalanines have been synthesized starting from inexpensive cinnamic acids with a novel one-pot approach by coupling phenylalanine ammonia lyase amination with a chemoenzymatic deracemization (based on stereoselective oxidation and nonselective reduction). Multienzymatic cascade increased the yield and optical purity of the D-configured product.250 Novel nonsymmetrically substituted N-protected b,b-diaryl-a-amino acids and esters have been produced through the asymmetric hydrogenation of tetrasubstituted olefins. A variety of N-acetyl, N-methoxycarbonyl, and N-Boc b,b-diaryldehydroamino acids, containing a diverse and previously unreported series of heterocyclic and aryl substituted groups (24 examples) were obtained with high yields and excellent enantioselectivities.127 Eighteen substituted a-arylalanines were converted to their (S)-b-aryl and b-heteroaryl-b-amino acids in vivo that showed no toxicity to cells. The synthesized substituted b-aryl-b-amino acids can be used in redox and Stille-coupling reactions to make synthetic building blocks, or as bioisosteres in drug design.251 Aryloxyproline diastereomers can 32 | Amino Acids, Pept. Proteins, 2018, 42, 1–84

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Scheme 17

stereospecifically used in molecular design, medicinal chemistry, and catalysis. The synthesis of (2S,4R)- and (2S,4S)-iodophenyl ethers of hydroxy-Pro has been described that can be differentially applied in distinct structural contexts.186 It was reported that a pyridine-type ligand that overcomes the limitation of N-methoxyamide auxiliary leads to a significant improvement of b-arylation of a-amino acids. The arylation used in this method can be applied for syntheses of unnatural amino acids, bioactive molecules, and chiral bis(oxazoline) ligands.252 Conjugated unnatural a-amino acids bearing a 5-arylpyrazole sidechain has been prepared by Horner–Wadsworth–Emmons reaction of an aspartic acid derived b-keto phosphonate ester with aromatic aldehydes resulting in b-aryl a,b-unsaturated ketones. A further reaction with phenyl hydrazine followed by oxidation gave the regioselective synthesis of pyrazole derived-amino acids.253 A new method has been developed for the asymmetric synthesis of chiral heterocyclic amino acids via alkylation of the Ni(II) complex of Gly and alkyl halides (Scheme 18). The intermediate decomposes to form a series of chiral amino acids in high yields and with excellent diastereoselectivity.254 Enantiopure 1,2,3-triazolyl-b-amino acids were prepared from the corresponding alkynyl-b2-amino acids. The enantiopure products were obtained via diastereomeric salt formation and subsequent separation.255 The synthesis of novel unnatural N(a)-Fmoc pyrimidin-4-one amino acids is based on an aromatic nucleophilic substitution reaction between 4-[4-(benzyloxy)benzyloxy]-2-(benzylsulphonyl)pyrimidine and the nucleophilic side chain of several N(a)-Boc amino esters followed by a series of standard protecting group transformations.256 Indolesubstituted (S)-tryptophans have been produced from corresponding indoles with the use of (S)-methylbenzylamine and derivatives. The products utilize a chiral auxiliary-facilitated Strecker amino acid synthesis strategy. Eight optically pure (S)-Trp analogues were synthesized, which were subsequently used for the convergent synthesis of a potent antibacterial agent, argyrin A and its analogues.257 The amino acid His plays a significant role in the structure and function of proteins. Its functions include enzyme catalysis, metal binding activity, and involvement in cation–p, p–p, salt-bridge, and other types of noncovalent interactions. N–H  N hydrogen bonds involves imidazole nitrogen atom of His. Along with the predominant occurrence in loop segments, a new structural role for His in protein structures was proposed.258 Amino Acids, Pept. Proteins, 2018, 42, 1–84 | 33

Scheme 18

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Two tetrahydrofuran amino acid derivatives were synthesized to replace Tyr in neurotesin. The new compounds showed substantial neurotensin S2 receptor binding affinity and up to 1000-fold selectivity over neurotensin S1 receptor.259 All biological systems code strictly 20 canonical amino acids with few exceptions. Given the limited functionalities of 20 amino acids, biochemists have longed for an approached for the production of proteins with unique biochemical and biophysical handles. Pyrrolysine (Pyl), the 22nd proteinogenic amino acid, was restricted until recently to few organisms. Its translational use necessitates the presence of enzymes for synthesizing it from lysine. A review discusses the pyrrolysine (Pyl) incorporating system from its discovery to its applications. In about a decade after the Pyl incorporation mechanism was discovered, the system has been transferred to bacteria, yeast, and mammalian cells for the genetic incorporation of more than 100 non-canonical amino acids or a-hydroxy acids into proteins. With the magnificent tools developed, the field might diverge to focusing on a large variety of applications from basic study to biotechnological development.260 Functionalized Pyl analogues for site specific protein labelling and biochemical studies have been developed. Incorporating a wide array of noncanonical amino acids by the Pyl translational mechanism made possible the synthesis of recombinant proteins. The first use of this technology for the production of branched cyclic proteins have been reported.261 It was demonstrated how Pyl synthase can be used to oxidize various isopeptides to novel amino acids by combining chemical synthesis with enzyme kinetics and X-ray crystallography. A detailed description of the Pyl synthase reaction is suitable for the biosynthesis of pyrroline and tetrahydropyridine rings as constituents of Pyl analogues.262 The gasphase conformational potential energy surfaces of Pyl and related derivatives (neutral, deprotonated, and protonated) were extensively searched. The conformational electronic energies and thermochemical properties of proton affinity/dissociation energy and gas-phase acidity/ basicity were also determined.263 2.10 N-Substituted amino acids The development of an efficient synthesis of N-hydroxy-a-amino acids and derivatives represents a challenging goal in organic synthesis since these compounds are key intermediates in metabolic pathways and can be found in human and animal tumours. In peptoids the side chain is connected to the nitrogen of the peptide backbone, instead of the a-carbon as in peptides. Like D-peptides and b peptides, peptoids are completely resistant to proteolysis, and are therefore advantageous for therapeutic applications where proteolysis is a major issue. N-alkylated N-arylsulfonylglycines were prepared. The procedures gave access to peptoid monomers bearing a wide variety of functional groups. The synthesized N-substituted N-arylsulfonylglycines were used as monomers in solid-phase synthesis to receive peptoid oligomers and peptide–peptoid hybrids.264 Accelerated submonomer Amino Acids, Pept. Proteins, 2018, 42, 1–84 | 35

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solid-phase synthesis of peptoids was achieved by incorporating multiple substituted N-aryl Gly monomers. This method enables the rapid room temperature synthesis of a wide variety of N-aryl Gly-rich peptoid oligomers in good yields.265 Catalytic enentioselective synthesis of N,C(a),C(a)-trisubstituted derivatives of a-amino acids was achieved by using 1H-imidazol-4(5H)ones as novel nucleophilic a-amino acid equivalents. These compounds provide direct access to N-substituted (alkyl, allyl, aryl) a-amino acid derivatives.116 3-[(2-Hydroxyphenyl)amino]butanoic and 3-[(2-hydroxy-5methyl(chloro)phenyl)amino]butanoic acids were converted to a series of derivatives containing 2-hydroxyphenyl, hydrazide, pyrrole, and chloroquinoxaline moieties. The new compounds were tested for their antimicrobial activities.266 N-Substituted acyclic b-amino acids were synthesized and investigated for their effect as GABA uptake inhibitors.267 N-aralkylpyroglutamic acids of substituted bispidine were prepared and evaluated for their ability to inhibit collagen induced platelet aggregation.268 A series of dithiocarbamate ligands derived from N-substituted amino acids reacted with different diorganotin dichlorides to give 18 diorganotin complexes. The variations in the molecular conformation, shape, and cavity size of the macrocycles depending on the aliphatic spacer was investigated.269

2.11 Amino acids containing sulphur or selenium The interactions of the side chains of the highly polarizable sulphur containing amino acids Met and Cys have received little attention, in contrast to aromatic–aromatic, aromatic–aliphatic, or/and aliphatic– aliphatic interactions. It was found that sulphur containing amino acids form stronger interactions than aromatic or aliphatic amino acids. Thus, these amino acids may provide additional driving forces for maintaining the structure of membrane proteins and may provide functional specificity.270 The side chain of Met exhibits different degrees of susceptibility to oxidation depending on solvent accessibility. Different sets of oxidation-sensitive and oxidation-resistant methionines contained in human proteins are investigated. The examination of the sequence surrounding of the non-oxidized Met revealed a preference for neighbouring Tyr and Trp residues, but not for Phe. The results showed that the S-aromatic motif, which decreases the reactivity of the involved sulphur towards oxidants is important.271 Pd(II)-Met methyl ester complex was synthesized and characterized by physicochemical measurements, and interaction with biorelevant ligands was investigated.272 Amino acids containing sulphur are important in Maillard reaction. The reaction conditions for the blue Maillard reaction products were only studied by few research groups. A study investigates the characteristic colour formation and antioxidant activities in four different Maillard reaction model systems and the optimum reaction conditions for the blue colour formation were investigated in a xylose-Gly model system.273 Glucosamine and Cys were also studied in aqueous model systems to 36 | Amino Acids, Pept. Proteins, 2018, 42, 1–84

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investigate the effects of reaction temperature, initial pH, reaction time, and concentration ratio of glucosamine and Cys. The optimum condition for colour change was also measured.274 Plasma homocysteine (Hcy) is an important risk factor for various diseases. A novel redox-sensitive fluorescent probe was developed for the selective detection of Hcy.275 The [4-thio]-S-ribosylhomocysteine analogues were synthesized by coupling of 4-thioribose with a thiolate generated from the protected Hcy.276 S-ribosylhomocysteinase enzyme (EC 4.4.1.21) catalyses the cleavage of the thioether linkage of S-ribosyl-Hcy producing Hcy and 4,5-dihydroxy-2,3-pentanedione which is the precursor of the signalling molecules known as autoinducer 2 responsible for the bacterial cell to cell communication. Isomeric analogues of S-ribosyl-Hcy with Hcy unit attached to C2 of ribose ring via C2-sulfur bond have been synthesized.277 Proteins are often attacked by hydroxyl radicals, that are the most reactive oxygen species, and this oxidation causes diseases. Several oxidized products have been experimentally characterized, but the reaction pathways remain unclear. Sulphur-containing amino acids would be oxidized more easily than OH-containing amino acids. This was proven by the stability of the sulphur radical intermediates.278 Multiple inputs control the synthesis of sulphur-containing amino acids Met and Cys in Saccharomyces cerevisiae.279 A novel chemoselective and stereochemically defined synthetic method to phosphorylate the side-chain of Cys is reported. This work provides a novel synthetic strategy to incorporate native phosphorylated Cys (pCys) residues into unprotected peptides. The results show that the presented chemical and analytical tools are highly valuable in accessing endogenous pCys peptides and thereby open the possibility to identify new Cys phosphorylation sites from biological samples.280 Met adenosyltransferase catalyses the metabolism of Met in the liver by converting it to S-adenosylmethionine. The importance of homeostasis in the metabolism of sulphur-containing amino acids with a particular focus on the transsulphuration pathway which could be a promising therapeutic target in liver injury is reviewed.281 The antioxidative activities of free L-amino acids against Fenton system-mediated hydroxyl radical (HO( )) production in aqueous solution was compared, and the relation between amino acids and a set of physicochemical properties was examined. Sulphur-containing free L-amino acids generated different secondary reactive products, which were discriminated by electron paramagnetic resonance spin-trapping spectroscopy. The findings are important for the understanding of oxidation processes in natural and waste waters.282 Sulphur-containing amino acids have dual role in the mechanochemical synthesis of IV–VI semiconductor nanocrystals from lead acetate and L-cystine.283 Enantiopure fluorinated analogues of Met and Cys (L-trifluoro-Met and L-S-(trifluoromethyl)-Cys) have been synthesized by using a cheap and user-friendly radical trifluoromethylation approach. The protected amino acid derivatives were used in peptide syntheses both liquid- and solidphase.284 Cinchona alkaloids functionalized with a hydrogen bond Amino Acids, Pept. Proteins, 2018, 42, 1–84 | 37

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donating group at the C6 0 position enantioselectively catalyse sulfaMichael additions to a,b-unsaturated N-acylated oxazolidin-2-ones and related a,b-unsaturated a-amino acid derivatives. The products were subsequently converted in high yields to enantiopure b-functionalized cysteines suitable for native chemical ligation.285 A dithiol amino acid (Dtaa) that can form two disulphide bridges at a single amino acid site have been developed. Application of Dtaas to a serine protease inhibitor and a nicotinic acetylcholine receptor inhibitor, that contain disulphide constraints, enhanced their inhibitory activities.286 Selenium initially was described as a toxin and was shown to be essential for health and development. By the mid-1990s selenium emerged as one of the most promising cancer chemopreventive agents, but subsequent human clinical trials yielded contradictory results. Selenocysteine (Sec) is a naturally occurring proteogenic amino acid that is encoded in the genomic sequence of relatively abundant proteins in many of the model species commonly used for biomedical research.287 Sec is incorporated into proteins by unique synthetic mechanisms. Regulation of Sec incorporation into the selenium transport protein was investigated. It was revealed that Sec incorporation requires both cisand trans-acting factors, which are known to be sufficient for Sec incorporation in vitro. It was suggested a role for yet unidentified mammalian-specific processes or factors.288 It was also shown that the subcellular location of most human selenoproteins containing Sec has impact on their function.289 Mammalian selenoproteins contain single Sec residues, with the exception of selenoprotein P that has 7–15 Sec residues depending on species. The regulation of Sec content of human selenoprotein P was investigated.290 Chiral a-selenoamino acid derivatives were prepared from N-acetoxyphthalimide derivatives of Asp and Glu as visible light photoredox chiral sources and diorganyl diselenides as radical acceptors. This simple method with mild reaction conditions and high efficiency provides an important strategy for the synthesis of chiral molecules.291 2,2 0 -dithiobis5-nitropyridine dissolved in trifluoroacetic acid (with or without thioanisole) was used previously for the removal of 4-methoxybenzyl and acetamidomethyl protecting groups from Cys and selenocysteine (Sec). Disadvantage of this method is that excess thiol must be used to drive the reaction to completion and then removed before using the Cyscontaining or Sec-containing peptide in further applications. Advancement of this method shows that ascorbate at pH 4.5 and 25 1C reduces the selenosulphide to selenol. This method might find a number of other applications.292 Malfunctions of selenoproteins can lead to various human disorders including cancer.293 The naturally available Sec exhibits novel anticancer activities against human cancer cell lines. The roles of Sec in cancer, health, and development was summarized.294 Investigations clearly demonstrate that selenoproteins that contain Sec can act as oncosuppressors, but can also favour the formation of malignant tumours.295 In another study, the understanding on the molecular mechanisms of Sec in human glioma treatment was revealed.296 Castration-resistant 38 | Amino Acids, Pept. Proteins, 2018, 42, 1–84

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progression of prostate cancer after androgen deprivation therapy remains a critical challenge in the clinical management of prostate cancer. A study showed the preclinical efficacy of methyl-Sec in delaying castration-resistant progression of prostate cancer.297 An oxidized Sec (Se–S bond) which has electrophilic character reacts with a nucleophilic arylboronic acid to provide the arylated Sec. The arylated derivatives are more stable under oxidative conditions than the corresponding alkylated Sec. This reaction is unique to Sec, and a wide range of boronic acids can react with different biorelevant functional groups.298 The synthesis of Sec derivatives which bear TFA-labile sidechain protecting groups was reported from a bis Fmoc-protected Sec precursor. The new compounds (Fmoc-Sec(Xan)-OH and Fmoc-Sec(Trt)-OH) are useful and practical alternatives to the traditional Fmoc-Sec-OH derivatives currently available to the peptide chemist.299 Sec is synthesized from a serine precursor in a series of reactions that require Sec tRNA. It was shown that allosteric regulation might play an important role in regulation of Sec and selenoprotein synthesis.300 Twenty one derivatives of Cys and Sec are synthesized from easily prepared protected dichalcogenide precursors in high yield. The crude form of these derivatives were precipitated from petroleum ether in sufficient purity for direct use for peptide synthesis.301 Site-specific dual antibody conjugation via Cys and Sec was described for the production of homogeneous antibody-drug conjugates with improved therapeutic effects.302 The important biological redox mediators for two-electron transfers (thioredoxin reductases) contain either 2 cysteines or a Cys and a Sec at the active site. In case of an accidental one-electron transfer to a S–S or a S–Se bond during catalysis, a thiyl or a selanyl radical would be formed, respectively. The thiyl radical can abstract a hydrogen from the protein backbone, which subsequently leads to the inactivation of the protein. But a selanyl radical will not abstract a hydrogen. Therefore, formation of Sec radicals in the active site will less likely result in the destruction of a protein.303 A variation of native chemical ligation involves the reaction of peptides bearing an N-terminal Sec residue with peptide thioesters, which proceeds through the same mechanism as the parent reaction. It was discovered that Sec within peptides can be chemoselectively deselenized without the concomitant desulfurization of Cys residues. A review summarizes the use of Sec in ligation chemistry and investigations of chemoselective ligation-deselenization chemistry at other selenol-derived amino acids.304 The use of native chemical ligation at selenocysteine residues with peptide thioesters and a one-pot oxidative deselenization chemistry that uses native chemical ligation at Sec residues with peptide thioesters and additive-free selenocystine ligation is described. This simple and rapid method leads to native peptides with Ser in place of Sec at the ligation junction.305 Trifluoroselenomethionine (TFSeM), a new unnatural amino acid, was synthesized in seven steps from N-(tert-butoxycarbonyl)-l-aspartic acid tert-butyl ester. TFSeM shows enhanced methioninase-induced Amino Acids, Pept. Proteins, 2018, 42, 1–84 | 39

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cytotoxicity against HCT-116 cells derived from human colon cancer. It was shown that transformation of TFSeM into seleno-Met is enzymatically catalysed by E. coli extracts, but TFSeM is not a substrate of E. coli Met adenosyltransferase.306

2.12 Phosphorus-containing amino acids Phosphorylation of Ser, Thr, and Tyr is a well-known posttranslational modification of proteins, but phosphorylation of other amino acid side chains is underappreciated and minimally characterized by comparison. In case of the O-phosphorylated amino acids, synthetic constructs were critical to assessing their stability and developing tools for their study. A review summarizes the synthetic chemical approaches of labile amino acid phosphorylation.307 In recent years, substantial progress has been made in the field of asymmetric phosphine catalysis; many new reactions have been discovered; and numerous enantioselective processes have been reported. New families of powerful amino acid-derived bifunctional phosphines were developed for new modes of phosphine activation, unknown reactions, and more enantioselective transformation.308 Important a-amino phosphonic acids and its derivatives have been synthesized by using copper-catalysed electrophilic a-amination of phosphonates and phosphine oxides with O-acyl hydroxylamines. This amination provides the first example of C–N bond formation which directly introduces acyclic and cyclic amines to the a-position of phosphonates in one step.309 Diphenyl (a-aminoalkyl)phosphonates act as inhibitors against serine proteases by forming a covalent bond with the hydroxy group of Ser in the active centre. The stereochemical effect of the diphenyl phosphonate moiety on the selective chemical modification was evaluated. The results demonstrate that the peptidyl derivatives bearing an optically active diphenyl phosphonate moiety can be used as affinity labelling probes in protein bioconjugation.310 A simple, efficient, and versatile organocatalytic asymmetric 1,2-addition reactions of a-isothiocyanato phosphonate were developed. Through these processes, derivatives of b-hydroxy or b-amino substituted a-amino phosphonic acid and a,b-diamino phosphonic acid derivatives were produced. This new method provides a novel route for the enantioselective functionalization of a-phosphonic acid derivatives.311 A series of phosphonodipeptides containing C-terminal a-aminoalkylphosphonic acids has been synthesized from 2-(N-benzyloxycarbonylamino)alkanamides, aldehydes, and phosphorus trichloride via Mannich-type reaction and subsequent sequential hydrolysis in good yields. The reaction mechanism was proposed and verified.312 Activated carbon-based amino phosphonic acid chelating resin was produced, and its adsorption properties for Ce(III) removal was investigated. The chelating resin based on activated carbon adsorbent was prepared from activated carbon followed by oxidation, silane coupling, ammoniation and phosphorylation, and characterized.313 Fifty phosphorus-containing amino acids (phosphonic and phosphinic acids) were screened for inhibition of human endoplasmic reticulum aminopeptidases. The SAR studies 40 | Amino Acids, Pept. Proteins, 2018, 42, 1–84

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revealed several potent compounds, particularly among the phosphinic dipeptide analogues, that were strong inhibitors.314 Highly efficient and chemoselective phosphorylation of amino acid derivatives was achieved with the use of phosphoryl chlorides catalysed by 2-aryl-4-(dimethylamino)pyridine-N-oxides.315 The strength of hydrogen-bond in phosphorylated and sulphated amino acids were investigated. The findings showed that for phospho-Tyr (pTyr), bidentate interactions with Arg are particularly dominant, as has been previously demonstrated for pSer. The interactions of sulfo-Tyr with Arg are significantly weaker, even as compared to the same interactions made by Glu.316 4-Phosphothiophen-2-yl Ala, a novel five-membered ring analogue of pTyr was developed. The new analogue showed high selectivity for pTyr and no cross-reactivity with other phosphorylated amino acids.317 4-Phosphopyrazol-2-yl Ala, a non-hydrolysable analogue of phosphoHis, was also synthesized.318 The interactions between O-phosphorylated and standard amino acid side-chain models in water were investigated by theoretical studies.319 Recently, peptides and proteins containing N-phosphorylated amino acids such as phospho-Arg (pArg), phospho-His (pHis), and phospho-Lys (pLys) have gained interest because of their different chemical properties and stability profiles. A direct synthetic approach to incorporate pLys residues in a site-specific manner into peptides in solution was reported by taking advantage of the chemoselectivity of the Staudinger-phosphite reaction.320 Aminoacyl adenylates constitute essential intermediates of protein biosynthesis. Their polymerization in aqueous solution is a potential route to abiotic peptides despite a highly efficient CO2-promoted pathway of hydrolysis. The efficiency and relevance of this frequently overlooked pathway from model amino acid phosphate mixed anhydrides including aminoacyl adenylates were reported. The evolutionary importance of the intramolecular pathways of hydrolysis of phosphate ester mixed anhydrides with amino acids and peptides was also discussed.321

2.13 Labelled amino acids Radiolabelled peptides with high specificity and affinity towards receptors that are overexpressed by tumour cells are used in nuclear medicine for diagnosis and therapy of cancer. Since Met is prone to oxidation during radiolabelling procedures and the formation of oxidative side products can affect the purity of the final radiopharmaceutical and affinity towards the corresponding receptor, it is important to substitute Met with oxidation resistant amino acid analogues. Replacement of Met with Nle preserves the length of the side chain of amino acid that is important for hydrophobic interactions, but not its hydrogen-bonding properties. Methoxinine (Mox), a non-canonical amino acid resembles more closely the electronic properties of Met than Nle and represents a suitable, oxidation-stable amino acid substitute of Met in radiolabelled peptide conjugates.322 A mild and selective photocatalytic synthesis of oncological positron emission tomography (PET) imaging agents was developed by Amino Acids, Pept. Proteins, 2018, 42, 1–84 | 41

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18

F-fluorination of inactivated C–H bonds in branched aliphatic amino acids. The biodistribution and uptake of three 18F-labelled Leu analogues in several cancer cell lines is reported.323 For tumour imaging with PET, 18 F-labelled Trp derivatives were synthesized by electrophilic 18 F-fluorination or by introducing a [18F]fluoroalkyl group in 3-steps.324 Two simultaneous click reactions in one-pot with a simple solid-phase extraction purification method were developed for the synthesis of new [18F]fluorinated 1,2,3-triazolyl amino acid derivatives. The final product was completely pure in terms of radiochemical (495%) and chemical purity with high radiochemical yields.325 A novel synthetic method was developed to achieve 6-18F-fluoro-3,4-dihydroxy-L-Phe (18F-DOPA). The method involved the nucleophilic substitution of a diaryliodonium salt precursor with non-carrier-added 18F-fluoride.326 New developments in radiochemistry are summarized in a review. The new methods provide solutions to long standing problems involved in the synthesis of the important but elusive radiotracer [18F]6-fluoro-3,4-dihydroxy-L-Phe ([18F]F-DOPA). Advances in nucleophilic synthesis have been achieved by optimising multistep strategies and using both hypervalent iodine chemistry and transition metal-mediated fluorinations.327 A novel 18Flabelled a,a-disubstituted amino acid-based tracer has been synthesized for brain tumour imaging with a long alkyl side chain to increase brain availability via L-amino acid transport system. Biodistribution and in vitro uptake assays showed that both (R)- and (S) 2-amino-5-[18F]fluoro-2methylpentanoic acid have good tumour imaging properties with the (S)-enantiomer providing higher tumour uptake and tumour to brain ratios.328 Four structurally related non-natural 18F-labelled amino acids in (R)- and (S)-configuration have been prepared and evaluated for their potential in brain and systemic tumour imaging with PET. All four tracers showed moderate to high levels of uptake by the cancer cell lines tested. The accumulation of these tracers was higher in tumour than most normal tissues.329 New fluoroethoxy Trp analogues were synthesized and evaluated in vivo. Radiosynthesis was accomplished by no-carrier-added nucleophilic 18F-fluorination following either an indirect or a direct approach using a protected mesyl precursor.330 Two 18F-labelled analogues of 5-hydroxy-L-[b-11C]Trp have been synthesized as tracers for tumour imaging.331 Amino acid transport is an attractive target for imaging in oncology. Despite a high demand of cancer cells for cationic amino acids, their potential as PET probes remains unexplored. The synthesis and preliminary biological evaluation of a new cationic tumour imaging agent O-2((2-[18F]fluoroethyl)methylamino)ethyl-Tyr were reported.332 It was previously reported that high tumour uptake is shown by 11 C-labelled 2-amino-2-methyl-butanoic acid (Iva). 11C-Iva is a promising PET probe for non-invasive tumour imaging.333 D-isomers of some radiolabelled amino acids are potential PET tracers for tumour imaging, e.g. the D-isomer of S-11C-methyl-D-Cys that was synthesized and evaluated.334 The radiolabelled serotonin precursor [11C]5-hydroxy-Trp showed significant accumulation in the pancreas of healthy volunteers by dynamic PET. In the contrary, a substantial and highly significant reduction in the pancreatic uptake was seen in type 1 diabetic patients. 42 | Amino Acids, Pept. Proteins, 2018, 42, 1–84

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Retention of [ C]5-hydroxy-Trp seems to be a useful non-invasive surrogate marker for the human endocrine pancreas.335 The 11C-methylation of Schiff-base-activated a-amino acid derivatives has been optimized for the radiosynthesis of various a-11C-methyl amino acids.336 A convenient alternative to Fluorine-18 and Carbon-11 can be Nitrogen13. A one-pot, enzymatic and non-carrier-added synthesis of the 13Nlabelled amino acids (L-[13N]Ala, [13N]Gly, and L-[13N]Ser) was achieved by using L-Ala dehydrogenase from Bacillus subtilis. After optimization of the experimental conditions, the radiochemical yields were sufficient.337 The 13C-enrichment measurements were used as a direct calibration to calculate the original 13C/12C ratios of individual amino acids. The number of non-analyte added carbon atoms and assess the nonstoichiometrical recovery due to their incomplete oxidation was determined for the main proteinogenic amino acids.338 L-[4-13C]Gln was prepared by Wittig reaction. This method resulted in fewer steps and higher yield than previously reported methods.339 Leu, Val, and Ile were exclusively 13C labelled on methyl groups. This isotopic labelling strategy represents an easily obtainable unambiguous long-range distance restraints in protein solid-state NMR studies.340 Aromatic amino acids such as Phe, Trp, 3 0 ,4 0 -dihydroxy-L-Phe (L-DOPA), and their derivatives play an essential role in human metabolic processes. Incorrect or slow biotransformation of these compounds leads to some metabolic dysfunctions and neurodegenerative diseases. The mechanisms of biotransformation of the above amino acids was investigated by using kinetic and solvent isotope effect methods.341 Halogen derivatives of L-Trp (4 0 -F-, 7 0 -F-, 5 0 -Cl- and 7 0 -Br-L-Trp) were specifically labelled with deuterium in a-position of the side chain. The labelled compounds were obtained by enzymatic coupling of the corresponding halogenated derivatives of indole with S-methyl-L-Cys in H2O, catalysed by enzyme tryptophanase. 100% deuterium labelling was observed in the a-position.342 Halogenated derivatives of L-Trp, L-Tyr, and L-Phe were labelled with tritium and doubly with deuterium and tritium. Enzymatic methods were used for the synthesis with tritiated water and heavy water (D2O).343 Four new 68Ga-labelled 1,4,7,10-cyclododeca-1,4,7,10-tetraacetic acid/1,4,7triazacyclononane-1,4,7-triacetic acid derived glycine/hippurate conjugates have been produced for PET renography. The 68Ga labelling was achieved by reacting an excess of the non-metallated conjugate with 68Ga14C.344 Amino acids labelled with 76Br are attractive positron emitters with relatively long half-life, and they could potentially be used as tumour imaging tracers. Two 76Br-labelled L-Phe derivatives were produced and investigated. The findings suggest that 2-76Br-bromo-a-methyl-L-Phe could constitute a potential new PET tracer for tumour imaging.345 The novel compound, (S)-amino-2-methyl-4-76Br]bromo-3-(E)-butenoic acid was synthesized and characterized. The above compound is a promising PET tracer for brain tumour imaging and lead compound for a mixed transport substrate.346 Fluorescence spectroscopy has become a powerful tool for probing a range of complex biological processes including enzyme mechanisms Amino Acids, Pept. Proteins, 2018, 42, 1–84 | 43

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and protein–protein interactions. A review describes advances in design, properties, and applications of fluorescent amino acids.347 Another review highlights the recent synthetic methods developed for the incorporation of highly conjugated chromophores (e.g. coumarin, flavone, and polyaromatic derived chromophores) into the side-chain of a-amino acids and the application of these compounds as probes for imaging in medicine and biology.348 A fluorescent L-amino acid bearing the 4 0 methoxy-3-hydroxyflavone fluorophore that shows dual emission has been synthesized. The fluorescent amino acid undergoes excited state intramolecular proton transfer for site-specific probing and imaging of peptide interactions.349 Small fluorescence probes are useful for tracking changes in the interior space of proteins. Fluorescence donors of six unnatural amino acids (structurally related to Trp) are described and show how they can be efficiently incorporated into a protein as fluorescence probes.350 The design and synthesis of microenvironment sensitive fluorescent triazolyl unnatural amino acids were reported that are decorated with donor and/ or acceptor aromatic chromophores via click chemistry. The synthesized fluorescent amino acids show interesting solvatochromic characteristic and/or intramolecular charge transfer feature. One of the amino acid (triazolyl-perylene amino acid) has been exploited for studying interaction with BSA and found that it is able to sense BSA with an enhancement of fluorescence intensity.351 New demands for imaging technologies and in vivo diagnostics are increasing. There is a desire for new reporter molecules that can provide strong signals, are non-toxic, and can be tailored to diagnose or monitor the progression of several diseases. Aequorin is a non-toxic photoprotein that can be used as a sensitive marker for bioluminescence in vivo imaging. The sensitivity of aequorin is due to the fact that bioluminescence is a rare phenomenon in nature and, therefore, it does not suffer from autofluorescence, which contributes to background emission.352

2.14 Resolution of amino acids (Enantioseparation) The first purely chemical method has been reported for the dynamic kinetic resolution of unprotected racemic a-amino acids. This method can challenge the economic efficiency of the enzymatic reactions.353 Chemical dynamic thermodynamic resolution and S/R interconversion of unprotected tailor-made a-amino acids was developed through intermediate formation of the corresponding Ni(II)-chelated Schiff bases. This advanced method works well in convenient conditions and allows the large-scale preparation of target a-amino acids in enantiomerically pure form.354 Highly efficient chiral resolution of D,L-Arg was carried out by cocrystal formation followed by recrystallization under preferential enrichment conditions.355 By utilizing the preferential enrichment technique, an improved enantiomeric resolution of DL-leucine (Leu) was achieved using a 1 : 1 cocrystal of D,L-Leu and oxalic acid.356 Aqueous two-phase systems based on tropine type chiral ionic liquids and inorganic salt solution were designed and prepared for 44 | Amino Acids, Pept. Proteins, 2018, 42, 1–84

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the enantiomeric separation of racemic Phe. The results indicate that D-enantiomer of Phe interacts more strongly with chiral ionic liquids and Cu21.357 A new, recyclable solid–liquid resolution system was developed based on tropin ionic liquids for the enantiomeric resolution of several racemic amino acids (Phe, Trp, Tyr, and phenyl Gly). The system provides high resolution without organic solvent and recycle of all chemical materials.358 Tropine-type chiral ionic liquid with proline anion was immobilized on silica gel as adsorbent for separation by chemical modification method for the first time. The static experiment showed that adsorption rate of two enantiomers of the racemic amino acids was different.359 An aqueous two-phase system was designed and prepared for the enantiomeric separation of racemic amino acids. The system used task-specific hydrophilic ionic liquid and inorganic salt solution. The mechanism of separation was also studied.360 A novel solid-liquid twophase system was developed for the chiral separation of racemic Phe with new dication imidazolium-based chiral ionic liquids in a solid-liquid twophase system where copper ions represented the solid phase. The results indicated that L-enantiomer of Phe interacts more strongly with chiral ionic liquids and Cu21.361 Diasteromerically pure g-oxyfunctionalized a-amino acids were produced by a multi-enzymatic cascade reaction. Racemic N-acetylmethionine was quantitatively converted into L-methionine-(S)-sulfoxide by the cascade reaction under optimized conditions.362 Research on application of amino acids and amino acid amides as chiral auxiliaries in cyanuric chloride based chiral derivatizing agents for enantioseparation was discussed in a review. The reagents gain advantage as chiral derivatizing agents (in terms of mild derivatization conditions for synthesis), stability of derivative, and high resolution over several other reagents.363 A new and efficient method was developed for the synthesis of racemic protected a-ethynyl Phe from D,L-2-benzyl Ser, in ten steps. Resolution was performed by HPLC using a chiral stationary phase.158 Structurally simple, recyclable, and inexpensive chiral tridentate ligands were used for non-enzymatic dynamic kinetic resolution of unprotected racemic tailor-made a-amino acids.364 Enzymatic dynamic kinetic resolution of different racemic N-formyl- and N-carbamoyl-amino acids using a dynamic kinetic resolution approach was achieved. The enzymes (L–N-carbamoylase and N-succinyl-amino acid racemase) were immobilized on two different solid supports.365 Since racemases allow racemization in one reaction step, using enzymatic catalysis for racemization can be very beneficial. The natural roles of racemases and their occurrence, the applications, and the biochemistry and engineering of this promising class of biocatalysts was summarized.366 Pyridine-linked bis(b-cyclodextrin) copper(II) complexes were reported that enantioselectively hydrolyse chiral amino acid esters.90 Monoterpene nitroso chlorides with a-amino acid derivatives form terpene-amino acid hybrids. Reaction with an excess of racemic DL-amino acids and their derivatives induces partial resolution of the amino acid components and formation of the diastereomeric mixtures of the terpene-amino acids hybrids.367 Amino Acids, Pept. Proteins, 2018, 42, 1–84 | 45

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Resolution of N,C-unprotected b-amino acids was accomplished through enantioselective formation and disassembly of nickel(II) complexes under convenient conditions in good yields and excellent enantioselectivity. The method can be used for resolution of b-aryl, b-heteroaryl, and b-alkyl-derived b-amino acids.368 Peptide-catalysed conversion of racemic oxazol-5(4H)-ones into enantiomerically enriched a-amino acid derivatives was reported.369 Racemic amino acids can be enantiomerically resolved by materials composed from polysaccharide containing cellulose and chitosan.370

2.15 Sugar amino acids Sugar amino acids represent an important class of building blocks for the generation of peptide scaffolds and constrained peptidomimetics, owing to the presence of a relatively rigid furanoid or pyranoid ring decorated with space-oriented substituents. Glycosylation is one type of posttranslational modification of proteins. Understanding on the molecular level of the structural features of glycoproteins that are recognized by various enzymes and receptors would be valuable in developing inhibitorbased strategies to control carbohydrate-mediated cellular processes. This fundamental understanding leads to new therapeutic strategies for conditions that are characterized by abnormal glycosylation. Amino acids and sugars are involved in complex biological processes such as catalysis and highly selective molecular recognition. Four ranunculins, novel hybrids of amino acids and sugars from Ranunculus ternatus Thunb, a plant used in traditional Chinese Medicine was reported. These four sugar amino acids possess a g-aminobutyric acid (GABA) moiety and monosaccharide or disaccharide moieties and have potential tail to tail ether-connected (6,6-ether-connected) bonds. This represents an unprecedented structural phenomenon for the connections of sugars with the participation of g-amino acids.371 Branched-chain and aromatic amino acids are transformed into higher alcohols with yeast Saccharomyces cerevisiae in the Ehrlich pathway. Five specific metabolites of glycated amino acids were synthesized and characterized. It was shown for the first time that S. cerevisiae can use glycated amino acids as a nitrogen source and transform them into new metabolites, provided that the substances can be transported across the cell membrane.372 Cyclic functionalised sugar amino acids and their 3- to 6-membered nitrogen heterocyclic and carbocyclic analogues are used in the synthesis of peptidomimetics and glycomimetics. These derivatives of sugar amino acids provide access to hydrophilic and hydrophobic peptide isosteres.373 A new synthetic route, useful derivatives, and coupling strategies were published for obtaining C-3 epimers of sugar amino acids as foldameric building blocks.374 A systematic conformational search was carried out for monomers and homohexamers of furanoid b-amino acids. The results show that hexamers of cis-furanoid b-amino acids show great variability, while hexamers of hydrophobic aminocyclopentane carboxylic acid and hydrophilic xylose sugar amino acid foldamers favour two different 46 | Amino Acids, Pept. Proteins, 2018, 42, 1–84

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Scheme 19

zigzagged conformations, the backbone fold turns into a helix in case of hexamer of b-aminotetrahydrofurancarboxylic acid.375 The synthesis, purification, and characterization of a new lactosylderivative, i.e. a lactosyl thiophenyl-substituted triazolyl-thione L-Ala was described. This amino acid-sugar conjugate was prepared by solution synthesis analogue to the natural fructosyl-amino acids, tested oncologically, and showed significantly higher anticancer effect.376 C-2 Deoxy glycosides and amino acid conjugates were synthesized by mediation of hypervalent iodine. E.g. 2-iodo serinyl glycosides was produced by this simple, efficient, and practical method (Scheme 19).377 S-Glycosylated b(2,2)-amino acids were achieved with a sulphurcontaining nucleophile by using 1-thio-b-D-glucopyranose derivatives. The glycosylation occurred with inversion of configuration at the quaternary centre.378 2.16 b-Amino acids and derivatives Enantiopure b-amino acids represent interesting scaffolds for peptidomimetics, foldamers, and bioactive compounds. Because of their high biological potential, cyclic b-amino acids are of importance in medicinal chemistry. These compounds are both elements of bioactive products and building blocks in peptide research. Since b-amino acids are components of complex natural products; produce structural diversity in natural products; and provide characteristic architectures beyond those of a-L-amino acids, they have significant and unique biological functions in nature. The known bioactive b-amino acid-containing natural products (nonribosomal peptides, macrolactam polyketides, and nucleoside-bamino acid hybrids), the biosynthetic enzymes that form b-amino acids from a-amino acids, the de novo synthesis of b-amino acids, and the mechanisms of b-amino acid incorporation into natural products are summarized in a review.15 A mild organocatalyzed Mannich reaction provides direct and highly stereoselective access to acyclic b(2)- and b(2,3,3)-amino thioesters with adjacent tertiary and quaternary stereocenters. Mechanistic studies provided insight into the high stereochemical differentiation between the two ester moieties and showed that the stereochemistry can be controlled by the choice of the substrate. The b-amino thioesters can be used in coupling-reagent-free peptide synthesis.379 Highly stereoselective Mannich-type addition reactions of a-fluorinated monothiomalonates with N-Cbz- and N-Boc-protected imines under mild organocatalytic conditions resulted in a-fluorinated b-amino thioesters. Optimization of Amino Acids, Pept. Proteins, 2018, 42, 1–84 | 47

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the stereoelectronic properties of the thioester moiety allowed to tune the reactivity of the a-fluoro-b-amino thioesters and enabled their couplingreagent-free incorporation into peptides.204 Enantioselective Mannich reactions of aldehydes with in situ generated N-carbamoyl imines followed by a Horner–Wadsworth–Emmons reaction lead to chiral vinylogous b-amino acids that are useful building blocks for the construction of combinatorial libraries of peptidomimetic compounds.380 A broad range of highly functionalized b-amino acids containing afluorinated quaternary stereogenic carbon centres have been synthesized via a diastereoselective Mannich-type reaction of a-alkyl, a-aryl, and a-vinyl fluoroacetates with N-tert-butylsulfinyl imines. The stereochemical outcome of the reaction is highly dependent on the steric and electronic properties of the fluorocarbon nucleophiles.206 A diastereoselective addition reaction of fluoroacetate and a-alkylated fluoroacetate to N-tert-butylsulfinyl imines provides a-fluoro-b-amino acids.205 b- and b(3)-amino acid analogues have been prepared by a method using copper(I)-catalyst. The substituted dihydropyrimidin-4-ones from propargyl amides led to the intermediate ketenimine under mild reaction conditions. The obtained substituted dihydropyrimidin-4-ones were elegantly transformed into the corresponding b- and b(3)-amino acid analogues.381 The challenging intermolecular amination of inactivated C(sp(3))–H bonds has been achieved via Pd(II) – catalysed intermolecular amination leading to a variety of unnatural b(2)-amino acid analogues. The reaction works without using of phosphine ligand or external oxidant.382 Rhodium-catalysed enantioselective hydrogenation of tetrasubstitued aacetoxy b-enamido esters leads to chiral derivatives of a-hydroxyl-b-amino acid in excellent enantioselectivities.383 An organocatalytic asymmetric aminomethylation of a,b-unsaturated aldehydes by N-heterocyclic carbene and Brønsted acid cooperative catalysis resulted in b-amino esters in good yields and high enantioselectivities. This redox-neutral strategy is conducted under mild reaction conditions and is suitable for the scalable production of enantiomerically enriched b-amino acids bearing various substituents.384 Asymmetric Friedel–Crafts alkylation of a-substituted b-nitroacrylates led to b(2,2)amino acids bearing indolic all-carbon quaternary stereocenters.385 Novel stereoisomerically pure b 0 -hydroxy-b-amino acid derivatives have been produced by an easy, mild, and efficient method using dibutylboron triflate-mediated aldol reaction of suitably protected b-amino acids bearing chiral oxazolidinone. Both a,b-syn and a,b-anti isomers are accessible through the choice of the oxazolidinone chirality.386 It was reported a new method for the synthesis of conformationally restricted b-amino acids involving a Vilsmeier–Haack reaction with nonaromatic carbon nucleophiles. The tertiary and all-carbon quaternary centres was successfully used to generate several b(2,2,3)-amino esters (e.g. derivatives of homoproline, homoalanine, and homopipecolinic esters).387 A new type of arylboronic acid equipped with chiral aminothiourea was used for the first intermolecular asymmetric Michael addition of 48 | Amino Acids, Pept. Proteins, 2018, 42, 1–84

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nitrogen-nucleophiles to a,b-unsaturated carboxylic acids. BnONH2 as a nucleophile gives a range of enantioenriched b-(benzyloxy)amino acid derivatives in good yields and with high enantioselectivity.388 Stereoselective synthesis of enantioenriched a-(silyloxy)-b-amino amides was developed by a silyllithium-initiated coupling of a-ketoamides with tertbutanesulfinylimines. The use of a-ketoamides is critical for achieving high yields and diastereoselectivities in the resulting a-hydroxy-b-amino acid derivatives.389 Four structures of oxoindolyl a-hydroxy-b-amino acid derivatives have been synthesized. The diastereoselectivity of the chemical reaction involving a-diazoesters and isatin imines in the presence of benzyl alcohol is confirmed through the relative configuration of the two stereogenic centres.390 The selective preparation of 1,4-disubstituted 1,2,3triazoles attached to b-amino acids has been summarized from the corresponding alkynyl-b2-amino acids according to Huisgen’s coppercatalysed 1,3-dipolar cycloaddition under mild conditions and with very high efficiency.255 Spontaneous rearrangement of 4-carboxy-2-oxoazepane a,a-amino acids resulted in 2’-oxopiperidine-containing b(2,3,3)-amino acids, upon basic or acid hydrolysis of the 2-oxoazepane a,a-amino acid ester. The reordering process involved the spontaneous breakdown of an amide bond, which typically requires strong conditions, and the formation of a new bond leading to the six-membered heterocycle.391 N-substituted-b-amino acid derivatives containing 2-hydroxyphenyl, benzo[b]phenoxazine and quinoxaline moieties have been synthesized from 3-[(2-hydroxyphenyl)amino]butanoic and 3-[(2-hydroxy-5-methyl(chloro)phenyl)amino]butanoic acids. The novel compounds were tested for their antimicrobial and antifungal activities.266 Cyclic b-amino acids are of importance in medicinal chemistry since they are elements of bioactive products and building blocks in peptide research. Catalytic cycloaddition of enecarbamates with electrophilic metalloenolcarbenes yields chiral cyclopentyl b-amino esters in excellent enantio- and diastereocontrol.392 Protected Orn-derived (3S,4S)-b-lactam was used as intermediate in the preparation of conformationally constrained (3S,4S)-2-oxoazepane a,a- and (2S,3S)-2-oxopiperidine-b(2,3,3)amino acid derivatives. In the synthesis of these heterocyclic amino acids, the incorporation of a p-methoxyphenyl moiety is crucial for the excellent stereochemical purity.393 Diastereoselective synthesis of bheteroaryl syn-a-methyl-b-amino acid derivatives (esters and amides) have been achieved via an addition reaction to five- and six-membered heterocyclic tert-butyl sulfinimines.394 The non-proteinogenic b2,2-amino acid was produced via an enantioselective Rh(I)-catalyzed conjugate addition reaction of a-substituted b-nitroacrylates with various arylboronic acids by using chiral Rh(I) diene catalysts under mild conditions in a range of common organic solvents.395

2.17 Non-natural amino acids The research in the field of design and synthesis of unnatural amino acids is growing fast because of the increasing demand of proteins of Amino Acids, Pept. Proteins, 2018, 42, 1–84 | 49

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potential therapeutics. Molecules of non-proteinogenic amino acids (e.g. b-N-methylamino-L-Ala) participate in various physiological processes and can produce adverse ecological effects. It is known that accumulation of b-N-methylamino-L-Ala via the food chain can lead to development of neurodegenerative diseases in humans. Natural sources of b-N-methylamino-L-Ala, methods for its detection, and possible mechanisms of toxicity in different living organisms are discussed in a review.396 Although the asymmetric synthesis provides some efficient protocols, it is attractive to make unnatural chiral a-amino acids from available natural a-amino acids through keeping of the existing chiral a-carbon. 83 unnatural chiral a-amino acids were prepared at room temperature under visible-light assistance from derivatives of L-Asp and L-Glu.397 Efficient asymmetric synthesis of unnatural alkenyl amino acids has been achieved using alkylation of a fluorine-modified Ni(II) Schiff base complex as the key step.398 New methods for the synthesis of unusual amino acids using copper(I)-catalyzed click reactions was summarized. This method is efficient for the synthesis of peptides and amino acids conjugated with carbohydrates, thymidine, and ferrocene.399 An improved second-generation synthesis of the unnatural amino acids AHMOD and AMD, components of the anticancer peptaibol culicinin D has been developed. A novel Wittig reagent for one-carbon homologation of aldehydes is also reported.400 The effect of replacing Trp with the non-natural analogue b-(1-azulenyl)-L-Ala was investigated in the well-known beevenom peptide melittin. The results showed that b-(1-azulenyl)-L-Ala can serve as a solvent insensitive alternative to Trp that does not have significant impacts on structure or function of membrane interacting peptides.401 The non-proteinogenic amino acids phenyl-Gly and hydroxyphenyl-Gly are crucial components of certain peptidic natural products and are important for the preparation of various medicines. The conformational behaviour of these two non-proteinogenic amino acids was investigated that might help in designing of bioactive peptides and peptide based drugs.402 Effects (on structure and function) of incorporation of non-natural amino acids into core region of enzyme was investigated.403 An ironcatalyzed diastereoselective synthesis of unnatural chiral (S)-a-amino acids with g-quaternary carbon centres has been developed. The method shows some advantages: simple and wide substrates, mild conditions, high diastereoselectivity, and easy workup procedures.404 The synthesis of non-natural amino acid 2-amino-3,3,4-trimethyl-pentanoic acid has been developed via Cu(I) chloride Michael addition, followed by a Curtius rearrangement.405 Non-natural aryl Gly amino acids are achieved in three steps from aromatic aldehydes with N-(trimethylsilyl)imines as enantioselective catalyst.406 Ligand-controlled C(sp(3))–H bond arylation and olefination was developed for the synthesis of unnatural chiral a-amino acids with transition metal catalysts.407 Novel hydrophilic transcyclooctenenylated noncanonical amino acids have been designed, synthesized, tested, and incorporated into proteins.408 Silicon-containing unnatural amino acids are becoming an interesting new class of building blocks. A review summarizes the different methods 50 | Amino Acids, Pept. Proteins, 2018, 42, 1–84

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used to prepare silicon-containing amino acids and their implications on conformational structures and biological applications.409 The dengue virus and West Nile Virus proteases are attractive targets for the development of dual-acting therapeutics against these viral pathogens. The synthesis and biological evaluation of inhibitors that contain benzyl ethers of 4-hydroxyphenyl-Gly as non-natural peptidic building blocks was published.410

3

Analytical methods

3.1 Gas–Liquid chromatography Nitrogen isotopic composition of amino acids has been widely applied to biochemical, ecological, archaeological, and biogeochemical studies to trace nitrogen source and transformation processes. For accurate isotope analysis of individual amino acids, a preparative method was validated involving the isolation of underivatized amino acids by ion-pair chromatographic separation and confirmed the consistency of nitrogen isotope composition. Ion-pair reversed-phase liquid chromatography coupled with electrospray ionization mass spectrometry (LC–ESI/MS) and gas chromatography/combustion coupled with isotope ratio mass spectrometry (GC/C/IRMS) were conducted for the purpose of separation of underivatized amino acids and nitrogen isotopic analysis, respectively.411 An extra-facile chiral liquid chromatography–time of flight mass spectrometry (LC–TOF/MS) analytical method of amino acid enantiomers has been developed without a derivatization process. The enantioseparation of eighteen proteinogenic amino acids (except for Pro) was simultaneously performed using a combination of a chiral column (CROWNPAK CR-I(þ)) and TOF/MS. An isocratic condition of a simple mobile phase comprising acetonitrile/water/trifluoroacetic acid gave baseline separation of all underivatized amino acid enantiomers on the chiral column.412 Amino acids are an important and highly dynamic fraction of organic N in soils and their determination in soil without derivatization is challenging due to the difficulties in separation and detection of trace amounts of these polar analytes. An analytical method to quantify 20 free amino acids in aqueous soil extracts without derivatization was developed.413 The quantitation of free amino acids from physiologic samples is essential for diagnosing and monitoring patients with inherited metabolic disorders. Current methods are hindered by long preparative and/or analysis times, expensive reagents, and often suboptimal performance characteristics. An improved method for amino acid analysis using liquid chromatography–tandem mass spectrometry (LC–MS/MS) has been developed and validated. Chromatographic separation of amino acids occurred using two columns. Eluted compounds were detected by selective reaction monitoring (SRM). Results showed excellent correlation with the Biochrom 30 amino acid analyser. The results show that the method is extremely sensitive, specific, reproducible and represents an improvement over other currently available technologies.414 A procedure Amino Acids, Pept. Proteins, 2018, 42, 1–84 | 51

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was developed for the direct determination of dissolved free amino acids (DFAAs) in freshwater samples employing ion-pairing liquid chromatography and mass spectrometry. The approach allowed accurate quantification of subnanomolar concentrations of DFAAs without derivatization or sample clean-up steps. Ala, Ser, Glu, Arg, and Gly constituted 65% of the total pool, while Met and Trp occurred at sub-nM concentrations only. The composition of DFAAs significantly differed at all examined spatial scales, and this could be mainly attributed to Ala, Asp, and Gly. The method equals or outperforms existing ones in terms of sensitivity and reproducibility, and it is superior for the high-throughput analysis of freshwater samples.415 A novel triazine-type chiral derivatization reagent, i.e., (S)-2,5-dioxopyrrolidin-1-yl-1-(4,6-dimethoxy-1,3,5-triazin-2-yl) pyrrolidine-2-carboxylate (DMT-(S)-Pro-OSu), was developed for the highly sensitive and selective detection of chiral amines and amino acids by ultra high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) analysis. The enantiomers of amino acids were easily labelled with the reagents at room temperature. The diastereomers derived from proteolytic amino acids, except Ser, were well separated under isocratic elution conditions by reversed-phase chromatography. A highly sensitive detection was obtained from the SRM chromatogram. The chiral amines (e.g., adrenaline and noradrenaline) labelled with DMT-(S)-Pro-OSu were also well separated. Furthermore, D-Ala and D-Pro were also detected in relatively high concentrations. Consequently, DMT-(S)-ProOSu seems to be a useful chiral derivatization reagent for the determination of amines and amino acids in biological samples.416 An improved analytical procedure was developed for the resolution and quantification of amino acid enantiomers by multidimensional GC. The procedure contains a derivatization step, by which amino acids were transformed into N(O,S)-ethoxycarbonylheptafluorobutyl esters. This highly sensitive method was tested on a sample of the Murchison meteorite, for which obtained chromatograms show excellent peak resolution, minimal co-elution and peak overlap. The comprehensive two dimensional chromatography, in combination with the optimized derivatization method is a highly suitable technique for the analysis of samples with very limited quantities.417 Stereoisomers (enantiomers and diastereoisomers) of synthetic, non-protein amino acids comprising a-, b-, and g-amino acids, including a,a-dialkyl amino acids, were converted into the respective N-trifluoroacetyl-O-methyl esters, analysed and resolved by GC on a commercial fused silica capillary column coated with the chiral stationary phase octakis(3-O-butyryl-2,6-di-O-pentyl)-gcyclodextrin. The chromatographic method presented is highly suitable for the elucidation of the stereochemistry of non-proteinogenic amino acids.418 Rapid, easy, and reliable quantification of amino acids is crucial in research on plant amino acid metabolism and nutritional improvement of crops via enrichment of essential amino acids. A recently reported analytical method, based on solid phase extraction (SPE), derivatization with methyl chloroformate and gas chromatography–mass spectrometry 52 | Amino Acids, Pept. Proteins, 2018, 42, 1–84

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(GC-MS) was optimized and tested on three-week-old Arabidopsis thaliana leaf tissues. Of the 16 selected amino acids, 14 were quantified successfully.419 Recently, the demand for D-amino acid profiling has been drastically increasing. The present methodologies for D-amino acid profiling are still unsatisfactory. A novel method for D-amino acid profiling was developed by using a combination of a chiral column and TOF/MS. Based on the literature this method has the best performance for D-amino acid analysis that also includes the shortest analytical time, the highest enantioseparability without derivatization, and the largest coverage for analytical targets.420 Most chiral amino acid separation techniques require complicated derivatization procedures to achieve the desirable chromatographic behaviour and detectability. A highly sensitive analytical method for the enantioseparation of chiral amino acids without any derivatization process using LC–MS/MS was developed. The results demonstrated the applicability and feasibility of the LC–MS/MS method as a novel, effective tool for D-amino acid measurement in various biological samples.421

3.2 Ion-exchange chromatography Ion-exchange chromatography (IEX) is a historical technique widely used for the detailed characterization of therapeutic proteins and can be considered as a reference and powerful method for the qualitative and quantitative evaluation of charge heterogeneity. Several analytical techniques have been used in amino acid geochronology to measure the relative abundances of D- and L-enantiomers. During the past two decades, reverse-phase (RP) liquid chromatography has become most common, whereas ion-exchange (IE) liquid chromatography was widely used prior to the mid-1990s. A new method was proposed based on intralab paired analyses of RP and IE liquid chromatography to mathematically convert A/I (allo-isoleucine: isoleucine) values determined with IE to corresponding D/L values for comparison with new RP results. Regression equations are provided to convert A/I to an equivalent D/L value for five amino acids, thereby enabling the large literature base of AAR results from IE chromatography to be compared and integrated with AAR results from RP chromatography.422 The natural Cinchona alkaloid quinidine as chiral selector in chiral ligand-exchange chromatography was systematically studied. Chromatographic conditions for enantioseparation of twenty a-amino acids were first time studied by changing mobile phase parameters such as pH, concentration of organic solvent, type of salt, ligand to metal ratio and column temperature. Maximum retention and enantioselectivity factors were observed at the region close to pH ¼ 8, since the tertiary amine (the quinuclidinic nitrogen) of the quinidine is protonated only in a small degree, and therefore is available for the chelate formation.423 Specific food proteins, peptides, and amino acids released by enzymatic hydrolysis have demonstrated several biological activities, therefore they represent an interesting perspective in agri-food, biopharmaceutical, Amino Acids, Pept. Proteins, 2018, 42, 1–84 | 53

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and nutraceutical industries. However, these biomolecules are present at significantly lower concentration in a complex mixture of different sizes, conformations, and net charges. The conventional pressure-driven membrane processes are widely used as a first choice for the separation and purification of proteins, peptides, and amino acids. Innovative electro membrane processes such as EMF and EDFM are recently gaining much attentions and are regarded as favourable alternatives to the conventional methods. These methods could be potentially used for the efficient separation of charged compounds like proteins, peptides, and amino acids.424 A conventional nonchiral column was used for the enantioseparation of several racemic a-amino acids (native and derivatized) using Cinchona alkaloids as chiral selectors along with Cu(II) ions in chiral ligandexchange chromatography. The mobile phase composition modulated retention and enantioselectivity. Good enantioseparation of many amino acids was obtained using equimolar amounts of Cu(II) and either cinchonidine, quinine, or quinidine as chiral selectors in the mobile phase. Good correlations were obtained between experimental enantioselectivity factors and calculated energetic differences.425 The enantiomeric purity of N-a-Fmoc-protected amino acids is crucial from the viewpoint of peptide synthesis; therefore, a sensitive HPLC protocol was developed for the identification and quantification of enantiomeric impurities of commercially available N-a-Fmoc-protected amino acids on Cinchona alkaloid quinine-and quinidine-based weak anion exchangertype chiral stationary phases. During the evaluation of the chiral chromatographic method, the effect of the mobile phase composition, nature, and concentration of different additives were optimized. The method permits detection of less than 0.01% enantiomeric impurity in the presence of the major enantiomer.426 Stereoselective HPLC and subcritical fluid chromatographic separations of 19 N-Fmoc proteinogenic amino acid enantiomers were carried out by using Quinidine-based zwitterionic and anion-exchanger-type chiral stationary phases Chiralpak ZWIX(-) and QD-AX. The effect of column temperature on the enantioseparation was investigated and thermodynamic parameters were calculated. The thermodynamic parameters revealed that the enantioseparations were enthalpy-driven. The elution sequence was determined in all cases and with the exception of Fmoc-Cys(Trt)-OH, it was identical on both chiral stationary phases whereby the L-enantiomers eluted before the D-enantiomers.427

3.3 Thin-layer chromatography (TLC) and high performance thin layer chromatography (HPTLC) Thin-layer chromatography (TLC) is a simple and inexpensive technique permitting several samples to be handled simultaneously, thus yielding a higher precision than sequential analysis. The inert character of the thinlayer material makes it ideally suitable for use with strong corrosive reagents. Certain groups of interest can be chemically bonded to the reactive groups of support material, for example silanization for 54 | Amino Acids, Pept. Proteins, 2018, 42, 1–84

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reversed-phase studies. Impregnation of the adsorbent with a variety of reagents adds an additional feature for influencing the adsorption characteristics without covalently affecting the inert character of the adsorbent. TLC is also successful in providing direct resolution of enantiomers of a variety of compounds by the proper manipulation of the support material.428 High performance thin layer chromatography (HPTLC) is becoming increasingly popular amongst analysts for its simplicity of operation and capability of running several samples simultaneously. The introduction of newer spectroscopic detection methods and the combination of HPTLC with mass spectrometry (HPTLC-MS) has made this method useful for analysts. That is why HPTLC finds numerous applications in the pharmaceutical field. All analyses reported with HPLC are now being performed by HPTLC with a saving of time and cost.429 Impregnation of a stationary phase by organic and inorganic agents in HPTLC may result in higher separation selectivity and resolution. The influence of polymer structure in the stationary phase and a method of modification of the stationary phase on the efficiency of vitamin and amino acid determination and on the enantioselectivity factors of beta-blockers separation were investigated. It was established that such polymers can be used as modifying agents of HPTLC systems for on-line preconcentration of vitamins (B2) and amino acids (Lys, Trp and Glu). These polymers can also be recommended as chiral selectors for the effective TLC separation of beta-blockers.430 In recent years, protein chemistry tends toward the analysis of more complex proteins, proteoforms, and posttranslational protein modifications. Although MS developed quite fast, sample preparation and separation of these analytes is still a major issue. For many years, electrophoresis seemed to be the method of choice. Similarly, twodimensional separation can also be performed with TLC. As the revival of TLC developed enormously in the last decade, it seems to be also an alternative to use HPTLC for the separation of proteins. An HPTLC system was established, that allows a separation of protein mixtures over a broad polarity range, or if necessary allowing to modify the conditions with only few steps to improve the separation for a specific scope. Several layers and solvent systems have been evaluated to reach a fully utilized and optimized separation system.431

3.4 High-performance liquid chromatography (HPLC) A new analytical method for the analysis of 18 amino acids in natural waters using SPE followed by LC–MS/MS operated in multiple reaction monitoring mode was developed. Two different preconcentration methods, SPE, and concentration under reduced pressure were tested. SPE was a suitable extraction method for real samples due to the lower matrix effects. The SPE method still incorporates a broad sample cleanup and decreased endogenous matrix effects by reducing interferences originating from real water samples. The method limits of quantification (MLQ) for the SPE LC–MS/MS method in ultrapure water ranged from Amino Acids, Pept. Proteins, 2018, 42, 1–84 | 55

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0.1 to 100 mg L as N for the different amino acids. The SPE LC–MS/MS method was successfully applied to the analysis of amino acids in 3 different drinking water source.432 CHIRALPAK ZWIX(þ) and ZWIX() are cinchona alkaloid-derived zwitterionic chiral stationary phases (CSPs) containing a chiral sulfonic acid motif which serves as negatively charged interaction site. An extensive experimental work aimed at developing schemes for an efficient generic screening and proposing straightforward approaches for method optimization on these ZWIX columns. Various chromatographic parameters were investigated using a large series of diverse amino acids and analogues. The involvement of acetonitrile (ACN) or tetrahydrofuran (THF) can help for adjusting retention time and selectivity. The presence of water in a low percentage is beneficial for peak shape, resolution, analysis speed, sample solubility, and MS detection performance.433 Unusual amino acids play fundamental roles in many scientific fields. Since the single enantiomeric form can cause different and often serious response of organisms, chiral separations of unusual amino acids are irreplaceable tools. Two types of chiral stationary phases, two teicoplaninbased and four polysaccharide-based columns, were used. Separation conditions of RP mode, polar organic mode, and hydrophilic interaction chromatography were evaluated and compared. Teicoplanin-based chiral stationary phases, especially Chirobiotic T column, were able to separate almost all enantiomers tested, with the exception of Z-D,L-4-F-Phe ethyl ester.434 The successful enantioseparation of axially chiral amino acid derivatives containing a cyclohexylidene moiety on an analytical and semipreparative scale was achieved by HPLC using polysaccharide-based chiral stationary phases. Racemic methyl N-benzoylamino esters, easily obtained by methanolysis of the corresponding 5(4H)-oxazolones, were subjected to chiral HPLC resolution using chiral stationary phases based on immobilized 3,5-dimethylphenylcarbamate derivatives of amylase or cellulose. The behaviour of both selectors under different elution conditions was evaluated and compared.435 Trp and its eight derivatives are biologically important compounds. Since their enantiomers can exhibit different behaviour, efficient enantioselective separation methods are needed for both analytical and preparative purposes. In capillary electrophoresis (CE), cyclodextrins and their derivatives were proved to be suitable chiral selectors. Two pH values of background electrolytes were tested to affect ionization of the analytes and consequently their enantioseparation. Despite of the CE method was suitable for the enantioseparation, different separation systems/conditions were required. In HPLC, various separation modes and columns were used. The best results of enantioseparation of Trp and its amphoteric derivatives were achieved with teicoplanin based chiral stationary phases and methanol as a mobile phase.436 The growing scientific attention in the biological function of D-amino acids leads to an increasing analytical interest for enantiomeric amino acid separation, which is challenging due to the lack of sufficiently sensitive, high-throughput analytical methods. A reversed phase ultra high performance liquid chromatography coupled with quadrupole–quadrupole 56 | Amino Acids, Pept. Proteins, 2018, 42, 1–84

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time of flight mass spectrometry (RP-UHPLC-QqToF/MS) method using pre-column derivatization for very precise discrimination of amino acids enantiomers was developed. The method shows a superb sensitivity with limits of detection in the range of several pmol/l.437 The intrinsic D-amino acid profile of mouse macrophages was analysed using HPLC. Six D-amino acids (D-Ser, D-Asp, D-Leu, D-Ala, D-Lys, and D-Gln) were detected in cell lysates of mouse macrophages. The D-amino acid composition of RAW 264.7 cells, which is a model macrophage cell line, was like that of the mouse macrophage. The results suggest that macrophages and RAW 264.7 cells with macrophage-like functions have a similar D-amino acid profile.438 A fully automated two-dimensional HPLC (2D-HPLC) was established by using silica-based monolithic ODS column as the first-dimension column with acetonitrile-trifluoro acetic acid–water as the mobile phase, micro Chiralpak QD-1-AX column as the enantiomer separation column with 10 mM citric acid in methanol-acetonitrile as the mobile phase for the second-dimension separation, and 4-fluoro-7-nitro-2,1,3- benzoxadiazole as the fluorometrical derivative reagent. The method has a higher separation efficiency and a higher detection sensitivity than that of existing methods in the determination of acidic amino acid enantiomers.439 A highly sensitive analytical method for the enantioseparation of chiral amino acids without any derivatization process using LC-MS/MS was developed. By optimizing MS/MS parameters, a quantification method that allowed the simultaneous analysis of 18 D-amino acids with high sensitivity and reproducibility was established.421 A chromatographic analytical method for the direct determination of amino acids by hydrophilic interaction liquid chromatography (HILIC) was developed. A dual gradient simultaneously varying the pH 3.2 ammonium formate buffer concentration and level of acetonitrile in the mobile phase was employed. Analyte chromatographic parameters such as the sensitivity of retention to the water fraction in the mobile phase values HILIC were determined as part of method development. A degradation product of Gln (5-pyrrolidone-2-carboxylic acid) was observed and resolved chromatographically with no method modifications. The separation was used to quantitate amino acid content in acid hydrolysates of various protein samples.440 Rapid and simple quantitative analysis of intracellular metabolites is a critical tool for monitoring the alteration of biologically significant metabolites. An UHPLC method was established, equipped with HILIC column coupled to MS/MS. 19 amino acids and 2 related derivatives in human cell lines were simultaneously determined. Chromatographic separation was achieved within 20 min using a BEH amide column, with ammonium acetate and ammonium hydroxide as aqueous mobile phase, and acetonitrile as the organic mobile phase. Amino acids were analysed in positive ion multiple reaction monitoring (MRM) mode without the need of derivatization. The method was successfully applied to simultaneously detect the 21 compounds in a human colon cancer cell line DLD1.441 The analysis of human plasma free amino acids is important for diagnosing the health of individuals, because their concentrations vary Amino Acids, Pept. Proteins, 2018, 42, 1–84 | 57

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with various diseases. An amino acid analytical method was developed based on HPLC–ESI/MS. The method was also validated for 21 major types of free amino acids in human plasma samples. The results of the specificity, linearity, accuracy, repeatability, intermediate precision, reproducibility, and limits of detections were sufficient for the measurement of amino acids in human plasma samples and should be suitable for use in clinical fields.442 As a common derivatization reagent, phenyl isothiocyanate (PITC) is widely used in the field of amino acid analysis. However, researchers have long faced the problem of coupling efficient separation with simultaneous, sensitive MS detection. A novel HPLC–ESI/MS method based on PITC derivatization was established for detecting amino acids. Complete separation of 15 amino acid derivatives was achieved. Therefore, the proposed method is a simple alternative to current methods of detecting amino acids.443 A reference measurement procedure for amino acid quantification in blood samples was established based on deproteinization with 5-sulfosalicylic acid (SSA) and an isotope dilution-ultra performance LC–MS method. Five model amino acids (Val, Ile, Leu, Tyr, and Phe) in the SSAtreated samples showed ionization enhancement as well as stable background signals without significant ion suppression effects. Five analytes were clearly separated within 3 min using gradient elution and ion-pair chromatography. The method was applied to various blood samples including serum, whole blood, and plasma.444 Ion-exchange HPLC generally fails as a method to determine low levels of free amino acids in body fluids. A modified RP-HPLC protocol for the determination of amino acids in body fluids and its application in mood disorder patients were developed. A previous research protocol was improved. The combination of the modifications, together with fluorescence detection (FLD), allows sensitive and practical determination of free amino acid levels in body fluids of depressive patients.445 A sensitive RP liquid chromatography method for the determination and investigation of amino acids in wolfberry fruit (Lycium barbarum) after solid-phase extraction-derivatization was established. The investigation illustrated that each tested wolfberry fruit contained at least 16 amino acids and the main amino acids were Glu, Asp, Pro, Ala, Ser, Gly, Lys, and Tyr.446 An RP-HPLC method was developed for determining free amino acids in burley tobacco. The test was done with OPA/3mercaptopropionic acid as the pre-column derivatizing reagent. The results of the determination show that seventeen kinds of free amino acids in burley leaves were produced.447 An analytical method for the simultaneous determination of free amino acids and biogenic amino acids (BA) in Cannonau and Vermentino wines was developed by using selective derivatization with dansyl chloride followed by HPLC with fluorescence detection. Thirty-two compounds were identified in the wines analysed. High levels of AA were found, with Pro being the most abundant. His was never detected in any Vermentino wines. gAminobutyric acid, 4-hydroxy-Pro, Gly, Leu, Ile, and putrescine proved to be useful for differentiating Cannonau wines from Vermentino wines.448 58 | Amino Acids, Pept. Proteins, 2018, 42, 1–84

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A simple analytical method was proposed and validated for determining Trp in some cereal and legume samples. In the method, alkaline hydrolysis of proteins was used due to the destruction of Trp structure during acid hydrolysis. Separation and detection of Trp were performed on a RP column with fluorescence detection by using a mobile phase of acetonitrile and acetate buffer of pH 6.3.449

3.5 Mass spectrometry (MS) The analysis of amino acids has become a central task in many aspects. While amino acid analysis has traditionally mainly been carried out using either GC in combination with flame ionization detection or LC with either post-column derivatization using ninhydrin or pre-column derivatization using o-phthalaldehyde, many of today’s analysis platforms are based on chromatography in combination with MS. While derivatization is mandatory for the GC-based analysis of amino acids, several LC platforms have emerged, e.g. HILIC coupled to MS, allowing the analysis of underivatized amino acids. Among the numerous analytical methods available for amino acid analysis today, three prominent approaches, GC–MS, LC–MS, and HILIC–MS were compared.450 A novel method was developed for the direct detection of amino acids in biological fluids by extractive electrospray ionization (EESI) tandem MS using minimal sample pretreatment. The EESI/MS conditions were optimized using representative amino acid standards. The methanol– water solvent was electrosprayed at positive ion detection mode. The temperature of the heated capillary was optimized. Collision induced dissociation (CID) experiments were performed by applying 17%–25% of the collision energy. The limit of detection (LOD) for the amino acids was in the range of 0.14-26.2 mg L1. The average time for a single amino acid analysis was less than 0.5 min. The results showed that EESI/MS is a powerful tool for the rapid, sensitive, and quantitative detection of amino acids in complex biological samples.451 A LC–MS analytical procedure has been developed for the detection and quantitative determination of underivatized amino acids at low concentrations in a standard reference material-urban dust. An accelerated solvent extraction followed by a solid phase extraction was applied prior to LC–MS/MS. Fourteen amino acids were separated by high resolution LC, detected and quantified by MRM on a triple quadrupole. This methodology avoids derivatization and allows the amino acid quantification in a complex matrix, and represent a good method suitable to analyse this class of compounds in atmospheric aerosol.452 A simple capillary electrophoresis (CE)-MS/MS method was developed for the analysis of free amino acids in commercial royal jelly (RJ) products. All 16 amino acids were determined without derivatization. The CE separation was achieved in an uncoated fused-silica capillary using a 1 M formic acid solution as the electrolyte, followed by MS/MS detection.453 A preliminary investigation was undertaken to assess the performance of a new chromatography column technology in applications involving Amino Acids, Pept. Proteins, 2018, 42, 1–84 | 59

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LC coupled to MS. The new column design allows mobile phase and solute to be extracted from the radial central region of the column, which reduces the solvent load to the mass spectrometer and improves separation efficiency.454 A novel method based on the strategy of N-phosphorylation labelling was developed for quantification of twenty natural amino acids in human serum by RP-LC/ESI/MS/MS. Twenty N-phosphoryl amino acids were separated on an RP-C18 column within 20 min by isocratic elution. At the same time, MRM/MS enabled quantitation of twenty natural amino acids in human serum. All twenty amino acids were successfully detected in human serum samples.455 Nitric oxide (NO) is a regulatory molecule involved in many biological processes. NO is produced by NO synthase by conversion of L-Arg to L-citrulline. Methylated derivatives of L-Arg, ADMA and SDMA, regulate L-Arg availability and the activity of NO synthase. A new multistep analytical methodology based on LC combined with MS was established for the accurate identification of the above compounds that were measured as stable 2,3,4,5,6-pentafluorobenzoyl chloride derivatives, which allows for simultaneous analysis of all compounds through chromatographic separation of ADMA and SDMA using a reverse phase column. This robust and fast LC-ESI/MS method may be a useful tool in quantitative analysis of L-Arg, ADMA, SDMA, and L-citrulline.456 Trp is an essential amino-acid and the precursor of many biologically active substances such as kynurenine (KYN) and serotonin (5HT). Its metabolism is involved in different physiopathological cases, such as cardiovascular diseases, cancer, immunomodulation, or depression. A simple method was developed for quantification of Trp and 8 of its metabolites, involved in both KYN and 5HT pathways, using LC coupled to tandem MS. The method was also validated in human plasma samples. The method enables the detailed analysis of these metabolic pathways, which are thought to be involved in a number of pathological conditions.457 Phthalic acid was introduced as a mobile phase additive to quantify free amino acids by HILIC coupled to ESI/MS/MS. The addition of phthalic acid significantly increased the signal intensity of protonated amino acid ions. Meanwhile, the shapes of chromatographic peaks of amino acids were optimized. This simple method was validated and successfully applied to the analysis of twenty-four free amino acids in human thyroid carcinoma and para-carcinoma tissues.458 A new analytical method for the analysis of 18 amino acids in natural waters using SPE followed by LC–MS/MS was performed. The SPE LC–MS/ MS method was successfully applied to the analysis of amino acids in 3 different drinking water source waters. Among the 18 amino acids analysed, the most abundant amino acids were found to be Tyr, Leu and Ile.432 An improved method for free amino acid analysis using LC–MS/MS has been developed and validated. Chromatographic separation of amino acids occurred using two columns, and the eluted compounds were detected by SRM, and quantitated by relating peak areas of amino acids to externally run standards.414 60 | Amino Acids, Pept. Proteins, 2018, 42, 1–84

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Regular and accurate monitoring the levels of Phe and Tyr in blood is prerequisite for a successful management of patients with hyperphenylalaninemia (HPA). The MS/MS and the amino acid analyser (AAA) as methods to measure blood Phe and Tyr levels and Phe/Tyr ratio was compared. Venous blood samples were collected for the AAA analysis. Capillary blood was spotted directly on filter paper for the MS/MS analysis. 207 pairs of measurements were performed. The Phe and Tyr levels obtained by the MS/MS were on average 26.1% and 15.5% lower, respectively compared to those obtained by the AAA. The Phe/Tyr ratio by the MS/MS was on average 10.6% lower. Due to the considerable interassay variability, a single method is preferable for long-term follow-up of patients.459 An RP-UHPLC-QqToF/MS method using pre-column o-phthalaldehyde/ isobutyryl-L-cysteine (OPA/IBLC) derivatization for very precise discrimination of amino acid enantiomers was developed. The method shows a superb sensitivity with limits of detection in the range of several pmol/l.437 A procedure for the direct determination of DFAAs in freshwater samples was developed employing ion-pairing LC–MS. The method approach accurate quantification of subnanomolar concentrations of DFAAs without prior concentration, derivatization or sample clean-up steps. DFAAs were separated on a C-18 resin using tridecafluoroheptanoic acid as an ion-pairing agent.415 The sensitivity of coupled enantioselective capillary electrophoresis mass spectrometry (CE–MS) of amino acids is often blocked by the chiral selectors in the background electrolyte (BGE). A new CE–MS method is presented in which the use of a chiral selector is circumvented by involving (þ)-1-(9-fluorenyl)ethyl chloroformate as chiral amino acid derivatizing agent and ammonium perfluorooctanoate (APFO) as a volatile pseudostationary phase for separation of the formed diastereomers.460 An automated precolumn derivatization amino acid analytical method was developed based on HPLC–ESI/MS. This method enabled the separation of at least 38 types of physiological amino acids within 8 min. The method was also validated for 21 major types of free amino acids in human plasma samples.442 A novel method for D-amino acid profiling using a combination of a chiral column and TOF/MS was developed. Based on the literature this method has the best performance for D-amino acid analysis.420 During the ESI process, ions move through a heated capillary aperture to be detected on arrival at a mass analyser. However, the ESI process an ion cloud with an area larger than that of the heated capillary aperture, significantly contributing to an ion loss of 50% due to coulombic repulsion. To improve ion transmission, a novel method was proposed using a home-made golf ball positioned between the ion source and the inlet of the mass analyser to hydrodynamically focus the ions passing through the golf ball. The ion plume produced by the ESI that passes through the golf ball will reduce the size of the ion cloud then be focused and most of them flowed into the mass analyser. 20 trace amino acids in complex samples, including tea, urine and serum were Amino Acids, Pept. Proteins, 2018, 42, 1–84 | 61

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determined. The results showed that the analytical performance of the determination of the 20 amino acids in samples using the home-made golf ball-assisted ESI source is better than that of a commercial ESI source.461 Accurate assessment of mass isotopomer distributions (MIDs) of intracellular metabolites, such as free amino acids, is crucial for quantifying in vivo fluxes. A high-throughput LC–MS/MS method allowing the quantification of the levels and labelling of free amino acids was developed and validated. Sensitivity in the order of the femtomol was achieved using MRM mode. The method was applied to the determination of the 13C-labelling abundance in free amino acids extracted from maize embryos cultured with 13C-Gln or 13C-glucose. Although Cys was below the limit of detection in these biological samples, the MIDs of 18 free amino acids were successfully determined. This novel method will enable the assessment of more complete and accurate labelling information of intracellular amino acids.462 Quantitative structure-retention relationships (QSRR) were constructed based on data obtained by a LC–ESI/QTOF/MS/MS method for the determination of amino acid analogues, following their derivatization via chloroformate esters. Chromatographic separation is based on gradient elution using methanol/water mixtures. The group of examined molecules was diverse, including mainly a-amino acids, but also b- and gamino acids and their analogues, decarboxylated and phosphorylated analogues, and dipeptides. Through stratified random sampling procedures, 57 compounds were split to a training set and a test set. Based on models, simplified equivalent was then created using a multi-linear regression (MLR) method. The suggested models are considered useful for the estimation of retention times of amino acid analogues for a series of applications.463 An analytical method to quantify 20 free amino acids in aqueous soil extracts without derivatization was developed. The method employed HILIC–MS/MS technique combined with a cation exchange SPE. Good separation of 20 underivatized amino acids was achieved within 12 min. The method with high throughout and high analyte specificity shows great promise for consistent analysis of free amino acids.413 An UHPLC– HILIC–MS/MS method has been developed and validated for the quantification of 21 amino acids (20 proteinogenic amino acids and Cys) in their free form (FAA) and as protein constituents (total amino acids, TAA) in a rich protein food matrix such as lyophilized mussels samples. FAAs were analysed after suspending the samples in the presence of trichloroacetic acid, while TAAs were determined after acid hydrolysis with 6 M HCl. In hydrolysed samples 17 amino acids could be determined since Trp, Cys, cystine, and Asn were degraded during acid hydrolysis. The method proved to be a fast and reliable tool for acquiring information on free and total amino acids profile in high protein content foodstuffs such as mussels.464 Cisplatin (cis-[PtCl2(NH3)2]) is an important platinum-containing anticancer drug for treatment of malignancies. Probing the interactions between cisplatin and free amino acids are beneficial for investigation of 62 | Amino Acids, Pept. Proteins, 2018, 42, 1–84

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its pharmacology and side effects. The interactions of twenty amino acids with cisplatin in water, acidic, neutral and basic solutions were studied using ESI/MS. Adducts of cisplatin and amino acids were recognized. Multiple tandem mass spectrometry was employed to probe the affinities of nucleophilic functional groups, such as side chains, a-NH2 groups and a-COOH groups on amino acids for cisplatin in water. The results indicated that the chelated complexes formed were stable in aqueous solution.465 Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that affects both lower and upper motor neurons, leading to muscle atrophy, paralysis, and death caused by respiratory failure or infectious complications. Altered levels of hCy, Cys, Met, and Glu have been observed in plasma of ALS patients. A method for determination of these potential biomarkers in plasma by CE–MS/MS was established. The validated method was applied to the analysis of plasma samples from a group of healthy individuals and patients with ALS, showing the potential of Glu and homocysteine metabolites as biomarkers for ALS.466

3.6 Capillary electrophoresis (CE) Capillary array electrophoresis (CAE) is a promising technique for multiple enantiomeric separations. Carboxytetramethylrhodamine succinimidyl ester (TAMRA SE), a rhodamine-core fluorescent probe, has rarely been applied as an original precolumn derivatization reagent for chiral amino acid analysis so far. High-throughput enantiomeric separations of 12 TAMRA SE-AAs by a home-made 532 nm CAE-LIF scanner are presented. The effect of cyclodextrins and a variety of organic modifiers was quickly investigated. Multiple determination of the enantiomeric excess in non-racemic mixtures of Ala is successfully presented.467 A new CE–MS method is developed in which the use of a chiral selector is circumvented by involving (þ)-1-(9-fluorenyl)ethyl chloroformate as chiral amino acid derivatizing agent and ammonium perfluorooctanoate as a volatile pseudostationary phase for separation of the formed diastereomers. Selective detection and quantification of 14 chiral proteinogenic amino acids were achieved with chiral resolution. Asp and Glu were detected, but not enantioseparated.460 New kinds of amino acid ionic liquids (AAILs) with pyridinium as cations and l-Lys as anion have been developed as the available chiral ligands coordinated with Zn(II) in chiral ligand-exchange CE (CLE-CE). Four kinds of AAILs, including [1-ethylpyridinium][l-lysine], 1-butylpyridinium][l-lysine], [1-hexylpyridinium][l-lysine] and 1-[octylpyridinium][llysine], were successfully synthesized and characterized by nuclear magnetic resonance (NMR) and MS. Compared with other AAILs, the best chiral separation of L-amino acids could be achieved when [1-ethylpyridinium][l-lysine] was chosen as the chiral ligand.468 A CLE-CE method using Zn(II) as the central ion and l-4-hydroxy-Pro as the chiral ligand coordinating with g-cyclodextrin was developed for the enantioseparation of amino acids and dipeptides. After optimization, it has been found that eight pairs of labelled amino acids and six pairs of labelled dipeptides Amino Acids, Pept. Proteins, 2018, 42, 1–84 | 63

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could be baseline-separated. Furthermore, the proposed method was applied in determining the enantiomeric purity of amino acids and dipeptides.469 CE with ultraviolet detection was applied to determine underivatized amino acids in beer, based on the coordination interaction of Cu ions and amino acids. Using the United Nations Food Agriculture Organization/World Health Organization model of essential amino acid pattern and flavour of amino acids, the quality and taste in three kinds of beer were evaluated. It was found that the content of Phe, Pro, Ser and Ile was relatively large in all three kinds of beers with a great influence on beer flavour. This method was shown to be applicable to the separation of amino acids in beer and to perform quantitative analysis directly without derivatization.470 A novel CE–MS/MS was established for the enantioseparation of enantiomers without derivatization for clinical purposes. Vancomycin chloride was used as an efficient chiral selector for the discrimination of the enantiomers by capillary electrophoresis employed with complete capillary filling method. Hyphenation of CE with MS/MS allows a reliable identification of separated enantiomers as well as their quantification.471 Simple and inexpensive CE with UV-detection method (CE-UV) was optimized and validated for determination of six amino acids (Ala, Asn, Gln, Pro, Ser, and Val) for Sudanese food. Amino acids in the samples were derivatized with 4-chloro-7-nitro-2,1,3-benzoxadiazole (NBD-Cl) prior to CE-UV analysis. The validated method was successfully applied for the determination of amino acids for Sudanese food samples.472 A method for determination of hCy, Cys, Met, and Glu as potential biomarkers of ALS in plasma by CE–MS/MS was established. All amino acids were separated within 25 min.466

3.7 Nuclear magnetic resonance (NMR) A method using 1H NMR spectroscopy has been developed to quantify simultaneously thirteen analytes in honeys without previous separation or pre-concentration steps. The method has been successfully applied to determine carboxylic acids, amino acids (Ala, Phe, Pro and Tyr), carbohydrates, ethanol, and hydroxymethylfurfural in eucalyptus, heather, lavender, orange blossom, thyme, and rosemary honeys. Quantification was performed by using the area of the signal of each analyte in the honey spectra, together with external standards. Good precision, with relative standard deviations over the range of 0.78–5.21% is obtained.473 The amino acid composition of Nephila clavipes dragline silk fiber was determined by conducting 1H NMR spectroscopy experiments on acidhydrolyzed material. This silk was found to consist of Gly, Ala, Glx, Leu, Tyr, Ser, Pro, Arg, Asx, Val, Thr, Phe, and Ile. Compared with standard chromatography-based AAA, the chemical resolution of NMR allows for an amino acid solution to be characterized without separation. In general, this 1H NMR AAA technique is applicable to a large range of proteins and peptides for precise composition characterization, especially when the 64 | Amino Acids, Pept. Proteins, 2018, 42, 1–84

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precise content of a minor component is critical and relatively large amounts of sample are available (microgram to milligram quantities).474 Amino acids, fully or partially neutralized with amines, are a new class of CO2 absorbents that have the possibility to combine the ionic character of amino acids with the better CO2 absorbing properties of the amines to form a more energy efficient solvent with improved environmental footprint. Equimolar mixtures of the amino acids, Gly, L-Ala, L-Pro, taurine, and L-Ser, with alkanolamines, monoethanolamine (MEA) and 2-amino-2-methyl-1-propanol (AMP), were quantitatively studied by 13C NMR at 25 1C. It is observed that in amino acid-MEA blended systems, carbamate formation from both MEA and the amino acids occur. The experimental data show that not only the pKa value of the amino acid is important but that also steric effects play a role in determining the preferred carbamate forming position. In the amino acid-AMP systems almost all carbamate is formed on the amino acid amine group due to the steric hindrance in AMP. Generally, addition of amine to an aqueous amino acid solution increases the amino acid solubility. NMR spectroscopy analysis showed that the precipitate consists mainly of amino acid and bicarbonate/carbonate with very small amounts of amine.475 Amino acid ionic liquids (AAILs) have attracted significant attention in the recent literature owing to their ubiquitous applications in diversifying areas of modern chemistry, materials science, and biosciences. Noncovalent interactions accompanying Phe, Trp, and Tyr AAILs composed of 1-methyl-3-butyl-imidazole and its methyl-substituted derivative as cations have been analysed employing the dispersion corrected density functional theory. It has been shown that cation–anion binding in these bioionic ILs is primarily facilitated through hydrogen bonding in addition to lp—p and CH—p interactions those arising from aromatic moieties which can be probed through (1)H and (13)C NMR spectra calculated from the gauge independent atomic orbital method. It has been demonstrated that indirect spin–spin coupling constants across the hydrogen bonds correlate linearly with hydrogen bond distances. Besides the direction of frequency shifts of characteristic C¼O and NH stretching vibrations in the calculated vibrational spectra has been rationalized.476 Binding interactions between twisted cucurbit[14]uril (tQ[14]) and twenty standard amino acids have been investigated by NMR spectroscopy and isothermal titration calorimetry (ITC) in aqueous HCl solutions and in DMSO. The results showed that tQ[14] displays weaker binding affinity for amino acids with hydrophobic or polar side chains and clear binding affinity for amino acids with a positively charged side chain or containing an aromatic ring, with the binding mode depending on the type of side chain present in the amino acids.477 (2S,4R)- and (2S,4S)-perfluoro-tert-butyl 4-hydroxy-Pro were synthesized (as Fmoc-, Boc-, and free amino acids) in 2-5 steps. The key step of each synthesis was the incorporation of perfluoro-tert-butyl group, with nine chemically equivalent fluorines. Both amino acids were incorporated in model a-helical and poly-Pro helix peptides. Each amino acid exhibited distinct conformational preferences, with (2S,4R)-perfluoro-tert-butyl Amino Acids, Pept. Proteins, 2018, 42, 1–84 | 65

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4-hydroxyproline promoting poly-Pro helix. Peptides containing these amino acids were sensitively detected by (19)F NMR, suggesting their use in probes and medicinal chemistry.193 The genetically encoded amino acid Sec and its dimeric form, selenocystine, are both utilized by nature. They are found in active sites of selenoproteins, enzymes that facilitate a diverse range of reactions, including the detoxification of reactive oxygen species and regulation of redox pathways. Due to Sec and selenocystine’s specialized biological roles, it is of interest to examine their (77)Se NMR properties and how those can in turn be employed to study biological systems. A solid-state (77)Se NMR measurements was established of the L-selenocystine chemical shift tensor, which provides the first experimental chemical shift tensor information on Sec-containing systems. Quantum chemical calculations of L-Sec models were performed to help understand various structural effects on (77)Se L-selenocystine’s chemical shift tensor. These results suggest that the dihedral information may be deduced for a protein with appropriate structural models. These first-time experimental and theoretical results will facilitate future NMR studies of seleniumcontaining compounds and proteins.478

Abbreviations AAA ADMA ALS AMPA CE CLE DFAA Dtaa EESI GABA GC HILIC HPTLC IEX LC–ESI/MS LC–MS/MS LC–TOF/MS MAA MAO Mox 4-mPro MRM Mtb NMDA

Amino acid analyser Asymmetric dimethyl-Arg Amyotrophic lateral sclerosis 2-Amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid Capillary electrophoresis Chiral ligand-exchange Dissolved free amino acid Dithiol amino acid Extractive electrospray ionization g-Aminobutyric acid Gas chromatography Hydrophilic interaction liquid chromatography High performance thin layer chromatography Ion-exchange chromatography Liquid chromatography–electrospray ionization mass spectrometry Liquid chromatography-tandem mass spectrometry Liquid chromatography–time of flight mass spectrometry Mycosporin-like amino acid Monoaminooxidase Methoxinine 4-Methylproline Multiple reaction monitoring Mycobacterium tuberculosis N-Methyl D-aspartic acid

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pArg PET pHis pLys pTyr RP-UHPLCQqToF/MS SAR SDMA Sec SRM SPE TFSeM TLC-MS UPLC-MS/MS

Phosphoarginine Positron emission tomography Phosphohistidine Phospholysine Phosphotyrosine Reversed phase ultra-high performance liquid chromatography–quadrupole–quadrupole time of flight mass spectrometry Structure–activity relationship Symmetric dimethylarginine Selenocysteine Selected reaction monitoring solid phase extraction Trifluoroselenomethionine Thin layer chromatography–mass spectrometry Ultra-high performance liquid chromatography– tandem mass spectrometry.

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W. S. Qi, Q. Guan, T. Q. Sun, Y. J. Cao, L. Zhang and Y. L. Guo, Anal. Chim. Acta, 2015, 870, 75–82. U. Groselj, S. Murko, M. Z. Tansek, J. Kovac, A. T. Bakija, B. R. Lampret and T. Battelino, Clin. Biochem., 2015, 48, 14–18. A. Prior, R. C. Moldovan, J. Crommen, A. C. Servais, M. Fillet, G. J. de Jong and G. W. Somsen, Anal. Chim. Acta, 2016, 940, 150–158. Y. H. Li, C. Y. Chen, C. H. Kuo and M. R. Lee, Anal. Chim. Acta, 2016, 938, 98–105. J. C. Cocuron, E. Tsogtbaatar and A. P. Alonso, J. Chromatogr. A, 2017, 1490, 148–155. N. Kritikos, A. Tsantili-Kakoulidou, Y. L. Loukas and Y. Dotsikas, J. Chromatogr. A, 2015, 1403, 70–80. E. D. Tsochatzis, O. Begou, H. G. Gika, P. Karayannakidis and S. Kalogiannis, J. Chromatogr. B: Anal. Technol. Biomed. Life Sci., 2017, 1047, 197–206. H. Liu, N. B. Zhang, M. Cui, Z. Q. Liu and S. Y. Liu, Int. J. Mass Spectrom., 2016, 409, 59–66. Z. Cieslarova, F. S. Lopes, C. L. do Lago, M. C. Franca and A. V. C. Simionato, Talanta, 2017, 170, 63–68. K. Y. Liu and L. Wang, J. Chromatogr. A, 2013, 1295, 142–146. B. B. Sun, X. Y. Mu and L. Qi, Anal. Chim. Acta, 2014, 821, 97–102. X. Y. Mu, L. Qi, J. Qiao, X. Z. Yang and H. M. Ma, Anal. Chim. Acta, 2014, 846, 68–74. T. Luo, J. Ke, Y. Xie and Y. Dong, J. Food Drug Anal., 2017, 25, 789–797. M. Svidrnoch, A. Pribylka, V. Bekarek, J. Sevcik, V. Smolka and V. Maier, J. Chromatogr. A, 2016, 1467, 383–390. M. M. A. Omar, A. A. Elbashir and O. J. Schmitz, Food Chem., 2017, 214, 300–307. G. del Campo, J. Zuriarrain, A. Zuriarrain and I. Berregi, Food Chem., 2016, 196, 1031–1039. X. Y. Shi, G. P. Holland and J. L. Yarger, Anal. Biochem., 2013, 440, 150–157. A. F. Ciftja, A. Hartono and H. F. Svendsen, Int. J. Greenhouse Gas Control, 2014, 27, 169–177. S. S. Rao and S. P. Gejji, J. Phys. Chem. A, 2016, 120, 5665–5684. J. Zhang, Y. Y. Xi, Q. Li, Q. Tang, R. B. Wang, Y. Huang, Z. Tao, S. F. Xue, L. F. Lindoy and G. Wei, Chem. – Asian J., 2016, 11, 2250–2254. J. Struppe, Y. Zhang and S. Rozovsky, J. Phys. Chem. B, 2015, 119, 3643–3650.

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Amino acid and peptide bioconjugates Zolta´n Ba´no ´ czi and Ferenc Hudecz*

Published on 29 November 2017 on http://pubs.rsc.org | doi:10.1039/9781788010627-00085

DOI: 10.1039/9781788010627-00085

The increasing number of papers in which bioconjugates of oligopeptides and/or amino acids are present indicates the importance and potential of these constructs. In this chapter there is an attempt to provide an overview concerning the preparation, functional characterization and application of conjugates described in the literature since 2012. The first part covers the synthetic aspects, like chemical reactions used, while later dozens of conjugates as selected examples of wide range applications are presented.

1

Introduction

The complexity of the challenges in front of researchers is increasing, the compounds are used to study processes within cell, in vivo should meet more and more requirements. The publication activity on bioconjugates shows that conjugation of biologically relevant compounds may be proper response for these challenges. If these partners are covalently attached to each other and preserve their activity, the product is called ‘‘bioconjugate’’. In the development of efficient bioconjugate means the answer of two main questions. First, what is the good position(s) of the partners for coupling? This problem is often very complicated because of the high number of possible sites and orientations. Secondly, what kind of covalent linkage is the best choice? Unfortunately, there are no universal solutions, but the increasing experience may help. In the literature thousand and thousand examples can be found for very divergent bioconjugates and conjugation methods. Amino acids and peptides could have a lot of different biological functions and therefore these compounds are often conjugated with other molecules for basic studies as well as to improve the efficacy. As there are well-established synthetic routes and well-characterised modifications, peptides can easily be transformed into the desired form for conjugation. In this chapter we made an effort to provide a comprehensive overview of the chemistry and application of only amino acid and/or peptide based bioconjugates. Considering the huge number of publications and the diversity of the field, we focused on new as well as the more widely used methods for synthesis and indication of trends in applications.

2

Synthesis

The wide diversity of side chain of amino acids provides the advantages of fine tuning the biological properties of bioconjugates and the high number of possible conjugation strategies. But, on the other side, it may be a limitation factor in the synthesis of well-characterised conjugates. ¨tvo ¨s L. University and Research Group of Peptide Department of Organic Chemistry, Eo ¨tvo ¨s L. University, Pa ´zma ´ny Pe´ter Chemistry, Hungarian Academy of Sciences, Eo ´ny 1/A, Budapest, Hungary. E-mail: [email protected]; [email protected] Se´ta Amino Acids, Pept. Proteins, 2018, 42, 85–145 | 85  c

The Royal Society of Chemistry 2018

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The presence of groups with similar reactivity or more same functionalities makes necessary the use of protection strategy or use very selective reactions. When peptides as reaction partners are used there is a need to have mild reaction conditions and ambient temperature. The most reactive groups are the amino, carboxyl and thiol functionality in peptides and proteins. Therefore these are often used in conjugation reactions. As an alternative, these functions can be modified with at least bifunctional reagent, one for modification the functional group in the sequence, the other for the conjugation reaction. Beside this, a dozen of unnatural amino acids are known with specific functionality in the side chain, which makes possible very selective conjugation reaction. These derivatives can be incorporated into the peptide by Solid-Phase Peptide Synthesis (SPPS) or into proteins by genetic modification. These modifications widen the range of the available chemical reactions as conjugation strategies. 2.1 Amide bond forming reactions The amide bond as the natural connection in proteins and peptides is one of the very often formed covalent linkage in amino acid and peptide bioconjugates. This bond offers advantages for both the bioconjugates – for example stability, biosimilarity – and for the conjugation chemistry. There is at least one carboxyl or amino group in all amino acid or peptide which can be used for the conjugation and thus only the partner should be modified. From the first successful years of peptide synthesis the chemistry of amide bond formation have been precisely developed. These standard reactions are fast, efficient and in some circumstances racemisation free. 2.1.1 Coupling reagents. The most preferred way to form an amide bond is the use of coupling reagents. These reagents activate the carboxyl group mainly via the formation of in situ active esters and repress the racemisation during the coupling. Since the beginning several very active reagents are developed, but new compounds (Fig. 1) or methods turn up in the literature from time to time. For example in solution, bis(4,6-dimethoxy-1,3,5-triazin-2-yl) ether could be used to couple carboxylic acids which are lipophilic and

Fig. 1 Structure of bis(4,6-dimethoxy-1,3,5-triazin-2-yl) ether (a), ethyl 2-cyano-2(2-nitrobenzenesulfonyloxyimino)acetate (b), 9-silafluorenyl dichloride (c), bis(2-oxo3-oxazolidinyl)phosphorodiamidic chloride (d), 1,1-dichlorocycloheptatriene (e). 86 | Amino Acids, Pept. Proteins, 2018, 42, 85–145

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1

sterically hindered. The ethyl 2-cyano-2-(2-nitrobenzenesulfonyloxyimino)acetate (o-NosylOXY) is the first coupling reagent which can be recovered by synthesis from the byproducts.2 Recently developed reagent is the 9-silafluorenyl dichloride.3 Coupling with this compound does not require pre-activation neither the carboxyl nor the amino group. It brings close the reaction partners like as a chemical ligation reagent. The 1,1-dichlorocycloheptatriene (chlorotropylium chloride) transforms the corresponding carboxylic acid to acid chloride and allows one-pot coupling reaction with alcohols or amines.4 Another stand-alone coupling reagents were formed from the bis(2-oxo-3-oxazolidinyl)phosphorodiamidic chloride.5 This compound was reacted with N-hydroxides; HOBt, HOAt and ethyl 2-cyano-2-(hydroxyimino)acetate (Oxyma) (wellknown additives in coupling reactions). The reagents above are used mainly in peptide synthesis in solution or solid phase. When the amide bond between the partners cannot be formed during the SPPS on resin, a solution phase conjugation is needed. This is mainly performed in water or water containing solution, because of the good water solubility of peptides. The main problem during these syntheses is the racemisation. Some additives, like HOBt and HOAt are used to decrease the level of racemisation, but this is limited by organic solvent solubility and the explosive nature. The Oxyma as a coupling additive was developed with a lower risk of explosion (Fig. 2a).6 The reagent showed the same effectiveness in coupling reactions using DIC as HOBt or HOAt in DMF. In order to expand its use in water, several water soluble derivatives were tested.7 Among these compounds, the (2,2-dimethyl-1,3dioxolan-4-yl)methyl 2-cyano-2-(hydroxyimino)acetate (Glycero-Oxyma) (Fig. 2b) was the most appropriate. The presence of this reagent resulted in racemisation free coupling of Z-, Fmoc-, Boc- or Ac-protected amino acid with partner amino acids or oligopeptides in water in the presence of water soluble carbodiimide and NaHCO3 as base. It was used in a SAR study to conjugate pleuromutilin derivative – as antibacterial drug – with amino acids (Orn or Gly).8 It was also the additive in the synthesis of Muraymycin D1, an aminoribosyl-uridyl peptide with antimycobacterial activity.9 The Glycero-Oxyma was further modified to increase the yield of coupling of N-acetyl or N-formyl a-amino acids or to solve the racemisation free coupling of peptide segments.10 The diethylphosphorylglyceroacetonide-oxyma (Fig. 2c) was used successfully in coupling of segments in DMF.

Fig. 2 Structure of ethyl 2-cyano-2-(hydroxyimino)acetate (a), (2,2-dimethyl-1,3dioxolan-4-yl)methyl 2-cyano-2-(hydroxyimino)acetate (b) and diethylphosphorylglyceroacetonide-oxyma (c). Amino Acids, Pept. Proteins, 2018, 42, 85–145 | 87

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2.1.2 Ligation methods. These methods provide selective reactions between the introduced functionalities resulting in amide bond, very often ‘‘native’’ peptide bond formation. The main application of this strategy is the chemical synthesis of proteins, but are also used in synthesis of amino acid or peptide bioconjugates. The basic techniques are developed since decades, but are often fine-tuned to expand the scope of their use. 2.1.2.1 Native chemical ligation (NCL). This was the first technique, which allowed the formation of ‘‘native’’ amide bond between two unprotected peptide segments.11 NCL is used mainly for the synthesis of proteins, but there are several elegant examples of the use in bioconjugation. A dual labelled ubiquitin was synthesized using NCL to detect the deubiquitinase’s activity.12 Fluorescent molecule was attached to the Cys-Lys dipeptide and this construct was ligated to the C-terminal of expressed protein. In the other study, a unique method was described for reconstruction of adenosine A2A receptor in synthetic liposomes.13 The reconstruction occurred during the formation of lipid by NCL from dodecyl maltose thioester and cysteine-functionalised oleoyl lysophosphatidylcholine. The NCL has been used very successfully in the synthesis of large-size peptides, proteins; but there is some restriction in its use. One is the synthesis of peptide-thioester or its activation for the fast reaction. In this method the reaction of a peptide thioester with an N-terminal Cys-peptide is occurred in the presence of an excess of thiol additives (for example 4-mercaptophenylacetic acid (MPAA)).14 If there are more Cys residues in the peptide fragments (‘‘cysteine rich peptides’’), the ligation proceeds without any thiol-additives,15 combination of this behaviour with alanine replacement with cysteine and desulfurization16 after the ligation results in the desire peptide/protein.17 Unfortunately, the thioester linkage is unstable under alkaline condition. Therefore the Fmoc-chemistry is not suitable for peptide thioester synthesis. This limitation can be avoided with synthesis of peptidederivatives, which are transformable into thioester (see more in reviews18,19). One type of the potential derivatives is the N-sulfanylethylanilide peptides (SEAlide peptide). These compounds can be transformed into thioester, after the peptide synthesis or on resin under acidic condition20 or in the presence of phosphate salt.21 The latter allows its use in sequential NCL. In a very interesting study SEAlide unit was used for affinity labelling of proteins (Fig. 3a).22 The fundamental concept can be summarized as follows: the SEAlide unit transformed into the reactive thioester derivative after the binding, which is directed by the built in ligand (Fig. 3b). Without protein binding no activation could occur. This method was used with success to the preparation of labelled human carbonic anhydrase and cyclooxygenase 1 enzymes and this unit was also applied as traceable linker for enrichment and selective labelling of target proteins.23 The other solution for thioester synthesis is the use of peptidehydrazide as thioester surrogates.24 It can be easily transformed into 88 | Amino Acids, Pept. Proteins, 2018, 42, 85–145

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Fig. 3 Structure of affinity label with SEAlide unit (a) and its rearrangement (b) after binding to target protein resulting in covalent labelling.22

peptide-thioester using NaNO2 and thiol additive (e.g. MPAA). This twostep reaction can be carried out in one-pot. First, NaNO2 as oxidant presumably transforms the hydrazide into azide, which then forms thioester. Using this derivative the C-terminal of ubiquitin was labelled with 7-amino-4-methylcoumarin.25 Several techniques are developed for NCL without Cys residue. In case of the ‘‘Auxiliary Mediated Ligation’’, a group is introduced at the N-terminus of peptide instead of Cys, which can be cleaved after the ligation. In the synthesis of azabenzene26 or hemithioindigo27 – photochromic compounds – containing peptide bioconjugates, the 4,5,6trimethoxy-2-mercaptobenzyl as auxiliary was utilized in NCL (Scheme 1). This auxiliary compound was also involved in the synthesis of N-glycopeptide.28 The sugar moiety was modified with the auxiliary and was ligated with peptides containing asparagine thioester at the N-terminus. Expressed proteins were also conjugated with ubiquitin peptides using this method.29 The conjugation was carried out at the e-amino group of target Lys residue in the protein sequence. The Lys residue was genetically replaced by azidonorleucine and was reduced when the other amino group of lysines in the sequence were protected with Boc. Then the free e-amino group was acylated by auxiliary containing Gly and this construct was ligated with ubiquitin peptide. Application of selenocysteine for the NCL results in a selenopeptide/ protein, which can be transformed into native sequence by selective deselenization.30 It is important to note that the Cys residues are stable under this condition. The selenocysteine can be transformed not only into Ala, but Ser residue too. By this method large size glycosylated MUC polypeptides were produced from glycosylated peptide fragments.31 2.1.2.2 Chemoselective ligation. The a-ketoacid–hydroxylamine amide ligation (KAHA ligation) was developed into a robust methods to synthesize proteins.32,33 The reaction between a-ketoacid and hydroxylamine group prefers aprotic polar solvents such as DMF, DMA, or Amino Acids, Pept. Proteins, 2018, 42, 85–145 | 89

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Scheme 1 Schematic illustration of ‘‘Auxiliary Mediated Ligation’’ using 4,5,6-trimethoxy2-mercaptobenzyl group.

Fig. 4 Structure of 5-oxaproline (a) and oxazetidine (b).

DMSO. The presence of water decreased significantly the reaction rate. For the reaction in water, the benzyl-O-hydroxilamine derivatives are preferred. But these are not compatible with a-peptides. As a good solution for the problem 5-oxaproline residue was proposed as ring closed hydroxylamine derivative.34 Unfortunately, this ligation results in homoserine at the conjugation site. To avoid this, oxazetidinecontaining peptide is described (Fig. 4).35 Tri-functionalized MMP2 FRET probes were built up by KAHA ligation.36 In this study, a new N-hydroxylamine derivative of Lys was applied for the reaction with methoxycoumarin acetic acid derivative containing trifluoroacetyl oxazolone moiety as a ketoacid precursor. The Staudinger and the mainly traceless Staudinger ligation are very useful method in protein synthesis.37 The traceless Staudinger ligation was successful in the synthesis of a-N-ribosyl-asparagine and a-N-ribosylglutamine;38 and also of glycosylphosphatidylinositol glycan-peptide conjugates.39 This ligation was also used to label G protein–coupled receptors (GPCRs) on living cells.40 The receptor protein was genetically modified by an unnatural amino acid, p-azido-L-phenylalanine (azF), 90 | Amino Acids, Pept. Proteins, 2018, 42, 85–145

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substitution. A FLAG octapeptide epitope (H-Asp-Tyr-Lys-Asp-Asp-AspAsp-Lys-OH) conjugated with a triarylphosphine was selectively ligated to the azF in GPCR and the presence of the epitope was verified by ELISA. A Ser/Thr ligation was developed by Li et al. to prepare protein using peptide O-salicylaldehyde ester and peptide with Ser/Thr at its N-terminus41 as reviewed in details.42 In the recent years by this method several peptide conjugates were produced. Peptides with serine at N-terminus and peptidomimetic O-salicylaldehyde esters were chemoselectively conjugated43 and peptoid-protein hybrids were reported.44 Teixobactin, a recently discovered new antibiotics, effective against multidrug resistant bacteria and has a core ring with an exocyclic 7-mer peptide tail. In a systematic study to identify efficient analogues, the peptide tail was conjugated to the core ring using Ser/Thr ligation.45 2.1.2.3 Enzymatic ligation. Enzymes can catalyse very selective and efficient reactions between partner molecules. These properties make these proteins ideal to assist conjugation reactions mainly in the synthesis of site specific coupling to a protein. Microbial transglutaminase enzymes (MTG) available in pure form have easy use for bioconjugation forming isopeptide bond as reviewed.46 The enzyme catalyses the amide bond formation between the g-carboxamide group of Gln and an amino group (e.g. e-amino group of Lys in proteins or peptides). Unfortunately, it has broad substrate tolerance, thus experiments are the best way to identify the reactive glutamine or lysine side chains in a protein. For example the possible Lys conjugation sites in Interferon Alpha-2b were identified by using Z-Gln-Gly dipeptide or its polyethylene glycol derivative; while the Gln conjugation sites were found by dansyl-cadaverine or amino-polyethylene glycol as amine source.47 The Z-Gln-Gly dipeptide is an excellent substrate of MTG enzyme, thus it was involved to attach PEG to cytochrome c,48 and for multiple protein conjugations to a DNA aptamer.49 The sortase A (SrtA) enzyme from Staphylococcus aureus is a transpeptidase, which catalyses the attachment of the target protein to the bacterial cell wall.50 It recognises the LPXTG amino acid sequence, cleaves the Thr-Gly bond and forms thioester with the carboxyl group of Thr residue to be conjugated with the amino group of oligo(Gly) branch in the peptidoglycan. This reaction is suitable for labelling of proteins.51 T-cell epitope peptides were also ligated to a monoclonal antibody against the C-type lectin DEC205. The LPETG peptide was introduced to the C-terminus of the antibody and the epitope peptides carried three glycines at their N-terminus.52 In another convincing study, the membrane of red blood cells was modified with the disease associated autoantigens (12-20 residue peptides) using sortase A ligation.53 Dasgupta et al. demonstrated that this enzyme is able to form isopeptide bond too.54 In this reaction the first step is the same, but in the second step, the e-amino group of Lys is acting as the nucleophile. The utility of this method in conjugation was proved by Bellucci et al.55 Due to the reversible nature of the sortase A guided ligation, one of the reaction partners should be in excess. To avoid this drawback Row et al. developed a novel strategy with Amino Acids, Pept. Proteins, 2018, 42, 85–145 | 91

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Scheme 2 Butilase 1 enzyme mediated ligation.

a modified peptide sequence (LPETGGHG) for sortase A recognition.56 Thus the enzymatic cleavage released the GGHG tetrapeptide, which could bind Ni21 ion very tightly. After complexation the nuclophile nature of the peptide was lost and the sortase A mediated ligation became irreversible. Butilase, an asparagine/aspartate (Asx) peptide ligase, isolated from Clitoria ternatea57 is involved in the macrocylization of cyclotides. Beside this activity, as a ligase this enzyme could conjugate a peptide with C-terminal Asn/Asp-His-Val motif with the N-terminus of another peptide (except terminal Pro) resulting in a peptide bond between Asx and the N-terminal amino acid.58 The authors proved that this ligation could be performed irreversible and with high yield using thiodepsipeptide (Scheme 2). Peptide dendrimer conjugates were prepared by this enzyme catalysis with thiodepsipeptides as substrate.59 In a recent study the bacterial cell wall was decorated with reporter molecules (e.g. fluorescein and/or biotin), a tumour-associated monoglycosylated peptide (MUC 1 glycopeptide) and mCherry protein using butilase 1 enzyme.60 The NHV (AsnHis-Val) butilase recognition motif was incorporated at the C-terminus of an anchoring protein OmpA, which thus presented it on the cell surface of E. coli. The cargos were built in a short peptide sequence containing Gly-Ile using the e-amino group of Lys residue or the N-terminus of glycopeptide was extended with this dipeptide. 2.2 Reactions yielding a non-amide bond 2.2.1 Hydrazone ligation. Hydrazino group can condensate with an aldehyde/ketone group resulting in hydrazone bond. This covalent linkage between the two components of the conjugate is pH sensitive and at decreased pH it opens. The pH dependent stability makes it suitable for targeting/delivering of drugs coupled to vehicles via hydrazone bond and released in environment with lower pH. Quader et al. used three-component conjugate against glioblastoma multiform.61 PEG-b-poly(Asp) copolymer was modified on its terminal aldehyde group by cyclic RGD (cyclo(Arg-Gly-Asp-Phe-Lys)) via thiazolidine ring formation of the SH group of cysteine – attached to the e-amino group of Lys using 5-aminopentanoic acid linker. The benzyl ester groups of poly(Asp) part was reacted with hydrazine and then the formed hydrazide groups were ligated with the keto group of epirubicin, one of the most potent antiglioblastoma anthracycline, to form acid-sensitive hydrazone bond. In another study 5-aminolevulinic acid and an MMP2 92 | Amino Acids, Pept. Proteins, 2018, 42, 85–145

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enzyme activable cell-penetrating peptide (CPP) were bound to the surface of gold nanoparticles via hydrazone linkage.62 A dual-targeting hybrid peptide containing an extracellular signalregulated kinases peptide inhibitor, a thiol spacer and transferrin receptor binding peptide was conjugated with doxorubicin (DOX) via hydrazone bond.63 DOX was transformed into hydrazine derivative with a bifunctional linker, (6-maleimidocaproyl) hydrazide, and was conjugated with the peptide bearing Cys via thioether bond. Bifunctional ligand was also synthesized via hydrazone linkage containing a typical opioid peptide and a fragment with neurokinin-1 receptor antagonist property.64 The tetrapeptide hydrazide was reacted with the antagonist (3,5-bis(trifluoromethyl)benzaldehyde) in ethanol to form hydrazone bond between the reaction partners. The acid sensitivity of hydrazone bond has the advantage to control the behaviour of biologically active compounds involved. Koren et al. described multicomponent liposomes with CPP, monoclonal antibody (mAb), PEG and drug load.65 Long chain PEG modified cancer-specific mAb, PEG2k and Tat as CPP with short linker (PEG1k) were attached to lipids and formed liposomes. The PEG2k-aldehyde was first reacted with 4-(4-N-maleimidophenyl) butyric acid hydrazide as a bifunctional linker. Then the hydrazone derivative of PEG2k was conjugated to the 1,2dipalmitoyl-sn-glycero-3-phosphothioethanolamine.66 Another approach is to conjugate a shielding molecule with biologically active compound via hydrazone bond as a pH sensitive switch. Xiang et al. prepared liposomes conjugated with activable cell-penetrating peptide.67 A positively charged CPP was conjugated with negatively charged peptide via hydrazone bond. For the synthesis a bifunctional linker was prepared with two maleimide moieties bridging by hydrazone bond (Fig. 5). The two peptides were attached via their Cys residues followed by coupling this construct with PEGylated lipids. Han et al. used activable peptide HAIYPRH (T7) as a transferrin receptor ligand to induce cellular-uptake of plasmid DNA packed in dendrigraft of poly[L-Lys].68 The T7 peptide hydrazide was coupled to the dendrimers via bifunctional PEG. Diethylenetriaminepentaacetic acid (DTPA) was reacted with 1-aminoacetone and this derivative was conjugated to the peptide via hydrazone bond. Kelly et al. described the synthesis of PEG based conjugates containing one or two types of prodrugs simultaneously.69 In these conjugates an antimicrobial peptide (AMP, an a-helical amphipathic peptide, a hybrid sequence of cecropin A(1–8) and magainin 2(1–12)) and DOX were

Fig. 5 Structure of bifunctional linker with hydrazone bond. Amino Acids, Pept. Proteins, 2018, 42, 85–145 | 93

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attached to the termini of bi-functional linear PEG. To prepare prodrug the AMP was elongated its N-terminal with a cathepsin B tetrapeptide substrate. This peptide was amidated by an amino-PEG-acid on the resin. Then the amino-group of PEG was transformed into succinic hydrazide, cleaved from the resin and reacted with DOX. Polysaccharides like pullulan may be promising drug carriers due to beneficial properties (e.g. water solubility, biocompatibility) and availability of multiple sites for drug and/or targeting moiety conjugation. Balasso et al. modified pullulan by oxidation to form aldehyde functions,70 and used two ligation strategies to attach bioactive compounds. The preS121–47 peptide, targeting SERPINB3 receptor on hepatocellular carcinoma cells, and cysteamine were attached to the oxidised pullulan by reductive amination via a PEG spacer. The DOX was modified by e-maleimidocaproic acid hydrazide forming hydrazone bond before the conjugation with cysteamine containing pullanan. Liposomes for drug delivery often hampered by the entrapment in the endosome. To avoid this Oude Blenke et al. compared different ligation methods to fine-tuning the lytic activity of melittin towards endosome membrane.71 The melittin was attached to the liposomes via PEG-lipids with its N-terminus using various conjugation techniques, like maleimidethiol, click and aldehyde-hydrazide chemistry. The hydrazone linkage was the best as it was stable at physiological pH, but cleaved at lower pH. 2.2.2 Oxime ligaton. An O-alkyl hydroxylamine can be reacted very selectively and efficiently with aldehydes or ketones resulting in oxime bond which is stable in physiological conditions. This reaction is frequently used for the synthesis of bioconjugates as reviewed.72,73 The hydroxyamino functionality can be introduced into synthetic peptides on solid support using aminooxyacetic acid.74 In case of expressed protein this group can be incorporated by genetically using unnatural amino acid, aminooxy-L-Lys75 or using NCL to build into C-terminal.76 In these examples the aminooxy protected peptide/protein was ubiquitinated using aldehyde functionalised ubiquitine. The oxime ligation often performed in water using buffer at acidic pH. In a recently published study Chelushkin et al. showed that ketones, reacting much slower, can be ligated in short time, almost with quantitative conversion in acetic acid.77 Model peptides containing aminooxyacetyl (Aoa) group were reacted with polyvinylpyrrolidone-based polymers, RGDpeptide (Arg-Gly-Asp-Phe-OH) both carried pyruvoyl groups and with steroid too. The Aoa group is often introduced by coupling with N-Boc-2(aminooxy)acetic acid or its diprotected form, N,N-(Boc)2-Aoa-OH. The latter helps to avoid the over-acylation of Aoa during the coupling reaction.78 Shao et al. described the isopropylidene protected Aoa as an alternative for synthesis of peptide derivatives.79 This compound can be prepared easily, stable and removable in mild conditions. Enyedi et al. also used isopropylidene protected Aoa for the synthesis of dual targeting peptide-drug conjugates.80 The daunomycin and targeting NGR cyclopeptide conjugates were prepared via oxime bond. After the SPPS and cyclisation the isopropylidene group was cleaved by methoxyamine. 94 | Amino Acids, Pept. Proteins, 2018, 42, 85–145

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The 1,3-dichloroacetone is often used in macrocyclisation of linear peptides with two Cys residues and also labelling the macrocyclic peptide derivative.81 It should be noted that parallel with macrocyclisation a ketone group is incorporated suitable for oxime-ligation. To prove the applicability of the approach, fluorescent molecules, biotin and peptides were conjugated to study this reaction after macrocylisation.81 This method was adopted for the macrocyclisation and conjugation of phage displayed peptides with biotin or various carbohydrates.82 The 1,3-dichloroacetone and aminooxy moiety were reacted and the cyclisation and conjugation could occur in a single step. In another study, di(bromomethyl)-benzyl derivatives were used for cyclisation of linear peptides.83 These derivatives contained protected aldehyde or hydroxylamine groups (Fig. 6). Then the cyclised peptides could be conjugated via oxime ligation, after the deprotection. Since oxime ligation is very specific it is often combined with other functionality to perform sequential ligation. Scaffolds are useful to manipulate the spatial arrangement of often more than two conjugated partners. A cyclo(Lys-Lys) peptide was modified with diverse ‘clickable’ functionalities like hydroxyamine, azide, alkyne and aldehyde. An Abbinding motif, the KLVFFA peptide and the curcumine were ligated to this scaffold.84 In the other research G-quadruplex conformations were stabilized using cyclopeptide scaffold (Fig. 7) and specific sequential ligations; oxime, thioether formation and copper-catalyzed azide-alkyne cycloaddition.85

Fig. 6 Structure of di(bromomethyl)-benzyl reagent with protected aldehyde (a) or protected hydroxyl-amine group (b) for peptide cyclisation and labelling.

Fig. 7 The structure of cyclopeptide with three different functions for ligation. Amino Acids, Pept. Proteins, 2018, 42, 85–145 | 95

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Scheme 3 The cycle of oxim ligation and cargo release.

91

Azide functional group is not compatible with solid-phase oligonucleotide synthesis. Therefore it should be introduced after the nucleotide synthesis for example by using oxime ligation.86 Then the azide group can participate in other ligation e.g. strain-promoted [2 þ 3] cycloaddition of peptide-oligonucleotide conjugate. Labelling peptides with radioactive group needs fast, efficient and stable ligation mode. Oxime bond formation fulfils these requirements. In general, an aldehyde moiety could be created on the radioactive component and it is reacted with hydroxyamino group containing peptide. [18F]Fluorobenzalydehyde,87,88 2-[18F]-fluoro-3-pyridinecarboxaldehyde89 and 2-[18F]fluoro-2-deoxyglucose90 was successfully conjugated to targeting peptides. Park et al. described a very dynamic system to ligate and to release a partner.91 Hydroquinone oxidised to benzoquinone, which can react with hydroxyamino group in oxime ligation (Scheme 3). After a new reduction step, amino-phenol is formed and the conjugated molecule is released from the construct. 2.2.3 Cycloadditions. During the last decades several cycloaddition reactions were introduced into the synthesis of bioconjugates, but the most frequently used one is the cycloaddition reaction between an azide and an alkyne. This reaction is very specific, fast and does not need protection. It can occur at ambient temperature even in water solution. The huge number of studies in which this ‘‘click’’ reaction was used demonstrates its good applicability in bioconjugation. Two variants can be distinguished: Cu(I)-catalyzed (CuAAC) and strain promoted (SPAAC) cycloadditions. 2.2.3.1 Cu(I)-catalyzed azide-alkyne cycloaddition. Since Sharpless92 and Meldal93 independently described that Cu(I) catalysis dramatically 96 | Amino Acids, Pept. Proteins, 2018, 42, 85–145

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increase the rate of [3 þ 2] Huisgen cycloaddition and markedly improved the regioselectivity; a dozen of bioconjugates were synthesized. Li and Zhang reviewed recently the developments in the field.94 Peptide-oligonucleotide conjugates (POCs) could have improved biological functions. CuAAC is a convenient method to prepare POCs. In the first examples the peptide was attached to the terminus of oligonucleotide (ON).95,96 The effect of position of functionalities was studied; both partner bearing the alkyne or azide group, but the conjugation was successful with 3 0 -azido-modified oligonucleotide and an alkyne-modified Tat peptide.95 In another case the oligonucleotide was extended with alkyne functionality at its 5 0 terminus using p-(N-propynoylamino)toluic acid (PATA) ‘‘an activated triple bond linker’’.96 This modified oligonucleotide was reacted with azide functionalised peptide (PLG; GAAKRVKLD (c-myc) and PKKKRKVG (SV40)) on the solid support. The PATA linker was used for biotinylation of peptides (C-myc) and ONs too.97 The biotin was modified with this linker and this derivative was conjugated with azido-peptide and/or nucleotide. If the peptide is planned to conjugate internally new strategy is required. Enkephalin peptides (YGGFM (Met-enkephalin); YGGFL (Leuenkephalin) with azide group on the N-terminus were coupled to a locked DNA oligonucleotides containing one or two internal built in 2 0 -alkyne-2 0 amino-locked nucleic acid monomer(s) (Fig. 8a).98,99 Alternatively, the alkynyl moiety was introduced onto the phosphate group of nucleotides and 5 0 -O-dimethoxytrityl-2 0 -deoxyribonucleoside 3 0 -O-ethynylphosphinoamidite synthons were used for the oligonucleotide synthesis (Fig. 8b).100 The peptide component (N3-Gly-Arg-NH2; Ac-Lys(N3)-Lys-LysArg-Gly-NH2; Ac-Lys(N3)-Arg-Arg-Arg-Gly-NH2; Ac-Lys(N3)-Lys-Lys-His-HisHis-NH2) for enhancing cellular-uptake was attached by CuAAc. Alkynylated antisense oligonucleotides were conjugated with azide modified Tat peptide by a very similar approach for increased cellular-uptake recently.101 When this alkynylated nucleotides are built in only the labelling with same compounds is possible. Carell et al. described an efficient method to introduce up to three different ligands into the ON chain.102 The authors used unprotected and protected alkyne moieties for the synthesis. The click reaction was carried out on the solid phase or in solution. There are some limitations of this method. Therefore Honcharenko et al. demonstrated another route.103 A linker was used for multiple

Fig. 8 Structure of synthons used in the synthesis of POCs. 2 0 -alkyne-2 0 -amino-locked nucleic acid monomer (a), 5 0 -O-Dimethoxytrityl-2 0 -deoxyribonucleoside 3 0 -O-ethynylphosphinoamidite (b) and 2-cyanoethylphosphoramidite (c) derivatives. Amino Acids, Pept. Proteins, 2018, 42, 85–145 | 97

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Fig. 9 Structure of linker for sequential labelling of the oligonucleotide chain.

functionalization H-phosphonate which can be introduced into the 5 0 -end of ON on solid support (Fig. 9). Then it was ligated with the azido partner and a new one was built in and so on. Peptide, carbohydrate and fluorophore were attached in parallel to the ON chain. The high concentration of Cu(I) and its toxicity are often prohibit in vitro/in vivo application of click CuAAC. In a recently published paper a protein mediated click reaction was described.104 ONs containing 2 0 -alkynylated nucleotide (Fig. 8c) were conjugated with dye, peptide and carbohydrate azide derivatives in ultra-mild conditions. Human Cu(I)binding chaperon Cox17 was used to promote the CuAAC reaction. In these reactions the chemically synthesized oligonucleotides were modified for the ligation. The two-step phosphoramidation is a versatile method for post-synthetic modification of ONs with phosphate at the 5 0 termini.105,106 This reaction was used by Su et al. to prepare amino group containing ONs using diamine spacers.107 Then the amino-group was coupled with alkyne or azide derivatives and was ligated with fluorophore or Tat peptide using CuAAc or SPAAC. The azide-alkyne click reaction is often used in glycochemistry as reviewed recently.108,109 The S-propargylated Cys containing peptides are suitable for conjugation. Vala et al. demonstrated that these derivatives can react with azido-carbohydrate in azide-alkyne reaction.110,111 Peptides modified with propargylglycine can also be used in click ligation. Garcı´a et al. synthesized diazido cyclodextrin derivatives and reacted these compounds in CuAAC with two C-terminally progargylglycine modified peptides; the dimerization scaffold and the basic region of GCN4 yeast transcriptional activator.112 An A,D-substituted diazido cyclodextrin was included in a similar study to demonstrate the suitability of CuAAc reaction in cyclodextrin modification.113 Kitagishi et al. ligated mono azido cyclodextrin with octaarginine.114 This conjugate could effectively deliver physical bound anionic porphyrin. Maschauer et al. applied the same strategy in the synthesis of radiolabelled octreotide peptide to image somatostatin (SST) receptors in vivo.115 The 5-hexynoic acid modified peptides were ligated with 6-deoxy-6fluoroglucosyl azide by CuAAC. Not only peptides, but also carbohydrates can carry the propargyl group. These modified carbohydrates, for examples were conjugated with a cyclopeptide as a dendritic core.116 In additionally using oxime-ligation this strategy was improved to prepare dendrimers. Calix[4]arenes and calix[6]arenes are very interesting scaffolds to be modified via the phenolic hydroxyl groups. CuAAC as a ‘‘second level’’ 98 | Amino Acids, Pept. Proteins, 2018, 42, 85–145

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reaction is proved to be very useful for introduction functionalities into calixarens. Since the first successful modification117 more than hundred papers have been published. In a recent paper O-propargylated calixarens are altered with peptides to increase cation complexation ability.118 The steroids also have well-defined structure and functional group for conjugation thus they may be utilized as scaffolds. Garcia et al. used deoxycholic acid derivatives and GCN4 binding region peptides (DPAALKRARNTEAARRSRARKLQ) in the synthesis of DNA binding conjugates.119 For this azide or alkyne modified deoxycholic acid derivatives and peptides were reacted. For CuAAC conjugation in living cell the decreased concentration of Cu catalyst is needed. Kuang et al. found that azide partner, which can chelate copper reacts faster in CuAAC reactions.120 Based on this observation Uttamapinant et al. proved that these copper-chelating azides can applied for fast and living cell-compatible CuAAC.121 The authors labelled cell-surface or intracellular protein and intracellular RNAs with picolyl azide derivatives by chelation-assisted CuAAC. To improve this conjugation new azides including modified picolyl derivative (Fig. 10a) were examined.122 Jiang et al. were able to detected newly synthesized glycoprotein on the surface of or in mammalian cells and early zebrafish embryogenesis.123 Diverse oligosaccharides were attached to MUC1 peptide or to DNA by this way.124 The reducing oligosaccharides were modified a picolyl derivative (Fig. 10b) for conjugation. Nishihara et al. reported the click chemistry based synthesis of macrocylic helix-turn-helix peptid labelled by fluorescein to generate targeting peptides.125 One peptide contained propargylglycine at its C-terminus and the other was azidoacetylated at the N-terminus. After that, the two peptides were ligated and the formed oligomer was cyclised by thioether formation with 1,3-dibromomethyl-5-propargyloxybenzene. The alkyne functionality of linker was used for labelling by click reaction. Bonache et al. also attached two peptides to obtain large molecules with increase biological activity.126 Two calmodulin binding peptides were elongated with an azide or alkyne modified oligoethylene glycol followed by ligation. Conjugation of disulfide bridge containing peptide, for examples cy¨nay and Klok published a method clopeptide, is a challenging task. Gu suitable for the conjugation of such cyclic peptide to poly(N-(2-hydroxypropyl)methacrylamide) copolymer.127 The poly(pentafluorophenyl methacrylate) precursor was reacted with propargylamine and this modified copolymer was coupled with azido peptide partner by CuAAC.

Fig. 10 Structure of 4-((6-(azidomethyl)pyridin-3-yl)amino)-4-oxobutanoic acid (a) and thiopicolyl azide (b). Amino Acids, Pept. Proteins, 2018, 42, 85–145 | 99

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Microwave assisted CuAAC was used in the synthesis of AMP – scaffold conjugates with128 or without metallocene (ferrocene or ruthenocen) (Fig. 11).129 Ferreira et al. conjugated ubiquicidin – an AMP – with 3-azido-7diethylaminocoumarin by CuAAC.130 AMP was acetylated at its N-terminus by 4-pentynoic acid on resin. The CuAAC was carried out with ´rio et al. attached the 3-azidoresin bound peptide. In the other study Hila 7-diethylaminocoumarin by CuAAC to a tetrapeptide which may increase the internalisation of coumarin derivative.131 Barbosa et al. covalently attached LLLFLLKKRKKRKY (Dhvar-5), a well-known AMP to chitosan via CuAAC.132 The propargyl-Gly modified Dhvar-5 was ligated to the azido-chitosan. The alkyne chain was introduced into the N-terminus of the peptide via 6-aminohexanoic acid (Ahx) spacer. Amanitin, a natural bicydic octapeptide was modified with N-propargyl-Asn for CuAAC.133 This derivative was then ligated with azido-rhodamine and azido-RGD peptide derivative (cyclo[Lys(N3)-ArgGly-Asp-D-Phe]). Pilkington-Miksa et al. also used RGD derivatives (Fig. 12) for targeting delivery of paclitaxel (PTX) as antitumour agent.134

Fig. 11 Structure of mono-, di- and trivalent scaffold (a) and a conjugate with trivalent scaffold (b).

Fig. 12 Structure of cyclic RGD derivatives with amino or azido group. 100 | Amino Acids, Pept. Proteins, 2018, 42, 85–145

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Several PTX-conjugates with different spacer and connection mode between the peptide and drug were prepared. In a most active conjugate the RGD derivative was connected to PTX via triazole ring and ethylene glycol spacer. Conjugates with similar structure, but containing multiple RGD moieties (Fig. 12) were also reported.135 Kim et al. connected a fibronectin extra domain B (EDB)-specific aptide (high-affinity peptide) with docetaxel.136 The aptide was alkynylated and the drug contained azide group for the CuAAC reaction. Graphene oxide was modified by azide and protected alkyne functionalities and then was reacted in sequential CuAAC reactions.137 First, propargyl-modified angiopep-2 peptide (TFFYGGSRGKRNNFKTEEYG) was reacted with azide group and then the alkyne group was deprotected and ligated with N3–PEG–N3. 2.2.3.2 Strain-promoted azide/alkyne cycloaddition. Cu(I) catalyst in the CuAAC could be a drawback in some circumstances. Since this catalyst should be present in high concentration and this often results in toxicity towards living organisms. One excellent solution for copper free click reaction is the strain-promoted azide/alkyne cycloaddition (SPAAC).138 Although there is no need of Cu(I), this approach has its own drawbacks, which could hamper its widespread application. The reaction rate is low in comparison with other click reactions as outlined by Dommerholt et al.139 An option to accelerate the reaction rate is to modify the cycloalkyne, but Dommerholt et al. followed other strategy.140 The reactivity of different aromatic azides with an aliphatic cyclooctyne; bicyclo[6.1.0]non-4-yne (BCN) (Fig. 13a) and a dibenzoannulated cyclooctyne; dibenzo-aza-cyclooctyne (DIBAC) (Fig. 13b) was compared. It was found that aromatic azides and aliphatic cyclooctynes react via an inverse electron-demand mechanism, and BCN can react with electrondeficient azide 40 times faster than DIBAC. The difference between the reactivity of aliphatic and dibenzoannulated cyclooctyne was utilized in one-pot, three-component protein labelling inserting a bivalent spacer with either aliphatic and aromatic azide. The reaction rate of SPAAC could be increased by micellar catalysis as well.141 The reaction rate of reaction between DIBAC and a hydrophilic or hydrophobic azide in the presence or absence of anionic, cationic, and non-ionic surfactants was also studied. The anionic and cationic surfactants were the most efficient catalyst. Tian et al. obtained very similar results during the labelling a G

Fig. 13 Structure of bicyclo[6.1.0]non-4-yne (a) and dibenzo-aza-cyclooctyne (b). Amino Acids, Pept. Proteins, 2018, 42, 85–145 | 101

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protein-coupled receptor, genetically modified by p-azido-phenylalanine with dibenzocyclooctyne-modified fluorescence dye.142 It was found that incorporation of azF close to the membrane region of the receptor protein resulted in enhanced reaction rate in comparison with azF in water-exposed position. This could be due to the hydrophobicity of the modified fluorophore present at higher concentration next to the membrane. For labelling more hydrophilic, water-exposed azide BCN seemed to be a better choice. Unfortunately it can react with the thiol group of cysteine too producing a high level unspecific labelling.143 However, this undesirable side reaction can be significantly reduced by addition of a small amount of b-mercaptoethanol to the reaction mixture and thus the genetically-encoded azido group in rhodopsin was labelled selectively.142 As the copper ion can be bound to the chelator, the Cu free ligation methods, like SPAAC are the best choice for the synthesis of conjugates with chelator as demonstrated by Schultz et al.144 Monoflourosubstituted cyclooctyne attached to DOTA was ligated with a neuropeptide Y analogue containing an internal azide-modified Lys residue. In a next study the synthesis of ‘‘clickable’’ DOTA derivative was improved.145 For labelling SST analogues with 64Cu isotope, a phosphonate-containing cross-bridged chelator was conjugated via SPAAC.146 This method was also used to prepare 18F labelled peptides for PET imaging. Bombesin, binding to gastrin-releasing peptide receptor, was acetylated at Lys3 residue by DIBAC derivative.147 This construct was reacted with three different 18F-containing azides. In another study, DIBAC was attached to a structure containing one or two cyclic RGD derivatives (c[RGDyK] or H-E(c[RGDyK])2) and was modified with 18F labelled aliphatic azide.148 Naganathan et al. reported on scanning the accessibility of amino acids on the surface of living cells.149 First, human CC chemokine receptor 5 was genetically modified by azF at different positions. The expressed receptor protein containing cells were treated with DIBAC labelled FLAG epitope peptide and based on immunosorbent assay the amount of conjugated epitope peptide was determined. Stefan et al. used the SPAAC to prepare a toxin prodrug.150 The truncated exotoxin A of Pseudomonas was modified with two azidohomoalanine residues at distinct positions and was reacted with DIBAC labelled enzyme-cleavable peptide conjugated with PEG. The PEGylation decreased dramatically the toxicity of toxin, but after the enzymatic cleavage of the PEG chains the toxicity recovered. Pam3CysSK4 is a Toll-like receptor agonist, which was conjugated with a peptide comprising a T-cell epitope. This conjugate was fluorescently labelled by SPAAC.151 An azido-Lys was incorporated into the C-terminus of the peptide capable to react with BCN modified fluorescence dye. Peptide-PEG-lipid conjugates could increase the stability and cellularuptake of cationic liposome-nucleic acid complexes. Ewert et al. prepared the conjugate on the resin by SPPS or in solution by maleimide-thiol or SPAAC ligation.152 POCs were prepared also by SPAAC chemistry as presented recently.153 Either the 3 0 or 5 0 end phosphate of ONs were conjugated with a tag containing BCN and these ONs were ligated with peptides (e.g. 102 | Amino Acids, Pept. Proteins, 2018, 42, 85–145

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YEVEALEKKVAALESKVQALEKKVEALEHG-NH2) elongated with 6-azido hexanoyl group at its N-terminus. 2.2.3.3 Other cycloaddition reactions Strain-promoted oxidation-controlled cycloaddition. The above described click ligations are very promising methods for the synthesis of peptide bioconjugates with all advantages for conjugation in biological compatible environment. Ayyadurai et al. developed a simple conjugation technique to prepare protein-polysaccharide conjugates in which genetically incorporated unnatural amino acid and mild periodate oxidation are combined.154 Tyr was replaced by 3,4-dihydroxy-L-phenylalanine (L-DOPA) in proteins and in the presence of periodate the side chain could react with the amino-function of amino polysaccharide, e.g. chitosan. During the periodate oxidation ortho-quinone is generated capable to accept the attack of the amino groups of chitosan. Borrmann et al. reported a new temporal controlled ligation method ‘‘Strain-Promoted Oxidation-Controlled Cyclooctyne–1,2-Quinone Cycloaddition (SPOCQ)’’.155 BCN derivative of biotin could react with a model peptide (LYKAG) containing a catechol moiety at the N-terminus only after oxidation. The ortho-quinone containing peptide could react selectively in the presence of a model peptide with azide at the N-terminus (Scheme 4). Only the product of the reaction between BCN and ortho-quinone was observed. Without oxidation the SPAAC reaction happened between the BCN and azide group. Based on these results, the authors prepared a bifunctional linker (Fig. 14) carrying an azide as well as a catechol moiety for fluorescence labelling of green fluorescence protein (GFP) and its dimerization. In connection with this reaction mushroom tyrosinase oxidised Tyr residue into intermediate 1,2-quinone which then reacted with BCN.156 This transformation can be executed with proteins (e.g. antibodies) with N-terminal Tyr. Tetrazine ligation with trans-cyclooctene. This very fast, highly selective and biocompatible reaction described first by Blackman et al.157 is suitable for the synthesis of bioconjugates in different media. The reaction

Scheme 4 Reaction of oxidised catechol moiety with BCN.

Fig. 14 Structure of bifunctional linker with azido and a catechol groups. Amino Acids, Pept. Proteins, 2018, 42, 85–145 | 103

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happens between a cyclooctene and a tetrazine in an inverse-demand Diels-Alder reaction followed by a retro-Diels-Alder reaction. An attractive method was reported by Rao et al. to synthesize ternary conjugates.158 Simple modification of tetrazine structure with two chlorine atoms leads to two new conjugation sites. The chlorine atoms of 3,6-dichloro-1,2,4,5-tetrazine can be replaced in SNAr reaction with different nucleophiles. Then the tetrazine moiety can be reacted in the tetrazine ligation. After the description of tetrazine ligation Fox et al. proved its usability in radiolabelling,159 by reaction between 3,6-diaryl-s-tetrazines and 18 F-labeled trans-cyclooctene. By this ligation exendin-4 a GLP-1 receptor ligand,160 an RGD peptide (c(RGDyC)) as well as VEGF protein161 were 18F-labelled for in vivo imaging. In both cases the peptide/protein contained a Cys residue modified with a bifunctional tetrazine linker (Fig. 15a) and then ligated with the 18F trans-cyclooctene (Fig. 15b). Zeglis et al. described another synthetic rout.162 The tetrazine was introduced into the peptide as a Lys derivative during the solid-phase peptide synthesis. To illustrate this technology in the synthesis of compounds for PET imaging a model pentapeptide was ligated with transcyclooctene-bearing derivative of the 89Zr41 chelator desferrioxamine. There is also an example for the synthesis of 125I-labelled conjugate using tetrazine ligation.163 The cyclic RGD peptide (c[RGDyK]) modified with trans-cyclooctene was conjugated with tetrazine derivatives substituted by 125 I isotope. An Escherichia coli enzyme, lipoic acid ligase (LplA) used in PRIME164 was mutated to incorporate trans-cyclooctene into a specific position of the peptide.165 The 13-amino acid peptide LAP was genetically inserted into protein and modified by LplA reaction. Such proteins on the cell surface or in the cytosol of living cells can be labelled by tetrazine modified fluorophore. The tetrazine ligation is orthogonal to CuAAC and these reactions can be performed to attach two different moieties to a peptide or protein as reported by Pagel et al.166 The aim was to cover titanium surface with cell adhesion promoting conjugate to increase its biocompatibility. Since the

Fig. 15 Structure of bifunctional linker with tetrazin and maleimido groups (a) and the 18F labelled ligation partner (b). 104 | Amino Acids, Pept. Proteins, 2018, 42, 85–145

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mussel-derived peptide containing DOPA can strongly bind to the surface of titanium, it was conjugated with cyclic RGD (c[RGDfK]) by tetrazin ligation and with heparin binding peptide by CuAAC. In another study titanium binding peptide was ligated with cyclic RGD (c[RGDfK]) on resin via tetrazine reaction.167 Ameta et al. studied the effect of dienophile on the reaction condition in the synthesis of peptide-RNA conjugate.168 The dienophiles were coupled to the phosphate group of GMP/ AMP and these modified nucleotides were enzymatically incorporated to the 5 0 -end of RNA. The tetrazine derivative was attached to the N-terminal of the peptide. The best dionephile was the bicyclononyne, although the synthesis of trans-cyclooctene containing derivative was not successful. Recently several studies demonstrated that tetrazine could be replaced by 1,2,4-triazins and efficient reaction takes place with BCN169 or transcyclooctene,170 but no reaction was observed with norbornene. Siegl et al. reported a systematic study on the reactivity of triazine derivatives in ligation reaction with trans-cyclooctene.171 It was found that the positively charged pyridinium triazine (Fig. 16) was reactive, stable, had a good solubility and leaded to fluorescent product after the reaction with trans-cyclooctenes. Several bifunctional linkers were also prepared with modified alkyl chain at the N-atom (Fig. 16). In pre-targeted radioimmunotherapy the antibody should capture the radioactive compound in a very fast and selective reaction. In vivo, the trans-cyclooctene may be isomerized to the cis isomer, which has low reactivity due to its interaction with Cu-containing proteins.172 The application of tetrazin ligation for labelling target molecules on the surface of and in the cytosol of living cell was reviewed by Devaraj and Weissleder.173 Detailed review is also available on inverse electron demand Diels–Alder reactions.174 Strain-promoted alkyne–nitrone cycloaddition. A novel bioorthogonal reaction, alkyne-nitrone cycloadition was described by Ning et al.175 in 2010 and the advancement focusing on kinetic studies and the structurereactivity relationship was recently reviewed by MacKenzie et al.176 The nitrone can be incorporated into the N-terminal of peptides or proteins with the modification of Ser residue. First, the N-terminal Ser is oxidized by sodium periodate and the excess of the reagent is quenched by benzenethiol. Then, without work-up an N-alkylated hydroxylamine is added, often in the presence of p-anisidine, and the nitrone is generated in situ. This procedure could provide a good chance for dual labelling of peptides or proteins as reported by Temming et al.177 The authors introduced two entities, chloroquine and a stearyl group, into a CPP; or

Fig. 16 Structures of positively charged pyridinium triazine derivatives with different functional group. Amino Acids, Pept. Proteins, 2018, 42, 85–145 | 105

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Fig. 17 Structure of nitrone-tag modified D-Ala derivatives.

biotin and a terminal alkyne for further CuAAC. The N-alkanyl group in the hydroxyl-amine was replaced by the stearyl or propargyl moieties. The chloroquine or biotin were built into in the BCN. MacKenzie et al. reported on the labelling living bacteria using alkyne– nitrone cycloaddition.178 D-Lys and D-ALA derivatives bearing endocyclic nitrones were applied for metabolic incorporation of this functionality into cell-wall peptidoglycans. The nitrones moieties were reacted with fluorescently modified dibenzocyclooctyne. This concept was further developed by Sherratt et al.179 Two different derivatives of D-Ala (D-AlaDMImo and D-Ala-CMPO) were used for simultaneous labelling two different bacteria. The derivatives were distinct in their nitrone tags with different reactivity (Fig. 17). Dibenzocyclooctyne can react with both derivatives; D-Ala-DMImo and D-Ala-CMPO while BCN can react only with D-Ala-DMImO. Miscellaneous reactions. This year Finbloom et al. published an interesting study on the catalytic effect of cucurbit[6]uril on azide-alkyne cycloaddition.180 This compound could accelerate the reaction of an azidoethylamine and propargylamine derivatives via complex-formation. Brun et al. reported a comprehensive study on the synthesis of polyamide conjugates via Diels–Alder cycloaddition on resin.181 The conjugation of peptide and peptide-nucleic acid (PNA) was studied. The reaction partners were modified with (E)-4,6-heptadienoic acid or 3-maleimidopropanoic acid, then they were conjugated on the resin. The partner on resin either peptide or PNA carried the (E)-4,6-heptadienoic acid. 2.2.4 Conjugation via Cysteine Disulfide bond formation. Oxidation of the thiol group results in a disulfide bond. This bond is stable, but under reductive environment of cytosol it opens up. Unfortunately, the formation of homodimer is favourable during the oxidation reaction. Therefore to achieve a heterodimer one of the thiol groups should be activated. This activation can be performed after the synthesis or a protecting group (e.g. during the SPPS) can act as activating group. A smart peptide conjugate of DOX with disulfide bond was synthesized to overcome multi-drug resistance.182 The active thiol derivative of DOX was prepared by acylation with 2-pyridyl-2-carboxyethyl disulfide (Fig. 18) and the DOX derivative was conjugated with Cys containing pHLIP, a 36-amino acid peptide derived from the C-helix of bacteriorhodopsin. DOX was also coupled with a CPP, octaarginine.183 Two copies of DOX were conjugated to a spacer carrying 2-pyridyl disulfide goups as activated disulphide via oxim-ligation. This dimer was reacted with octaarginine containing N-acetyl Cys at its N-terminal. In another study the 106 | Amino Acids, Pept. Proteins, 2018, 42, 85–145

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Fig. 18 Structure of 2-pyridyl-2-carboxyethyl disulfide.

PTX, inhibiting the dissociation of microtubules during cell division, was conjugated to a tumour targeting peptide, degarelix.184 The Trtprotecting mercaptopropionic acid was introduced into the amino groups at different positions of the peptide. A hydroxyl group of PTX was acylated 2-pyridyl disulfide p-nitrophenyl carbonate and this activated thiol reacted with the free thiol group of the peptide. The 2-pyridyl-2-carboxyethyl disulfide (Fig. 18) was also used to modify kidney targeting peptide.185 Muguruma et al. described a new way of antibody targeted delivery of plinabulin, a microtubule depolymerization agent.186 The thiol containing plinabulin was synthesized as inactive sulfide by CuAAC and transformed into active disulfide by 3-nitro-2-pyridinesulfenyl chloride containing resin. This resin attached activated disulfide was reacted with a 33-mer peptide (Z33) derived from Protein A, which has a strong and selective binding to the Fc region of human IgG1. A four-component conjugate was prepared to form redox-responsive micelles for intracellular delivery of DOX.187 Stearyl amine was conjugated with chitosan using 2-carboxyethyl disulfide linker. While targeting peptide was PEGylated at its C-terminus. This PEGylated peptide was also attached to the amino group of chitosan containing stearyl amine. This four-component conjugates could form micelles and packed DOX. A small antigenic peptide (SIINFEKL) derived from ovalbumin was coupled with biotin via disulfide-bridge and this conjugate was physically associated with streptavidine presented on the surface of a novel microparticle (MIS416) with vaccine adjuvant-cargo co-delivery properties.188 With proper stability, the disulfide bound cargo can be delivered not only into the cytosol, but into mitochondria too. Lei and Kelley studied different linker to optimize conjugates with mitochondrial release.189 The mitochondria-penetrating peptide was conjugated with Luminespib, an HSP90 inhibitor under development as anticancer agent by Novartis via disulfide linkers. The linker was attached to the N-terminal of the peptide and a FRET pair was involved to analyse the efficacy of mitochondrial delivery. Luminespib was conjugated to the peptide via optimised monosubstituted disulfide linker (Fig. 19a). The presence and hydrolysis of an ester bond in the conjugate lead to traceless release of the drug (Fig. 19b). Covalent immobilization of Dhvar5 with defined orientation resulted in higher antiadherence effect as compared with physical adsorption.190 Chitosan was modified first with N-acetyl-Cys to obtain thiol derivative and was reacted with peptide Dhvar5 with N- or C-terminally added Cys with different incorporated linkers. Disulfide bond between chitosan and the peptide was formed in oxidative conditions (20% DMSO). A promising combination of a peptide and antibiotic drug was also reported by Amino Acids, Pept. Proteins, 2018, 42, 85–145 | 107

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Fig. 19 Structure of optimised linker for mitochondrial delivery (a) and release of the ester bond cargo after reduction of disulfide bond (b).

Brezden et al.191 A CPP with intrinsic antimicrobial activity was conjugated with aminoglycoside antibiotic kanamycin via disulfide linkage. The two components were conjugated using 4,4 0 -dithiobutyric acid as linker. Lee and Lim described an in solution fragment coupling method to prepare peptide-DNA conjugates.192 A peptide chimera (RGD dimer ((ArgGly-Asp)2) and p53 peptide segment) was conjugated with a 5 0 -end thiol modified oligonucleotide. The ON was preactivated with 2,2 0 -dipyridyl disulfide and was reacted with the Cys-peptide chimera. Dirin et al. reported a similar solution phase conjugation, – but with 2 0 -end thiol derivative of ON (2 0 -thioethyl arm).193 The pre-activation of one thiol group was avoided by using S-sulfonate protecting Cys residue during the peptide synthesis. This group is stable in Fmoc/tBu strategy and also during the final peptide cleavage step. It can undergo thiolysis forming disulphide bond containing conjugates with appropriate partner. As the solution phase conjugation strategy has some limitations (e.g. the cumbersome purification, limited applicability due to solubility problems), Dirin et al. further developed the conjugation process into a versatile method to synthesize oligonucleotide conjugate on solid phase.194 The same building blocks for ON synthesis was utilized, but with other protecting group (-StBu instead of Trt group). This protection can be selectively removed even on the glass surface and the free thiol could react with the peptide containing S-sulfonate protected Cys. The disulfide linkage was used also to produce protein-peptide conjugates for protein delivery.195 The nlsEGFP (the enhanced GFP modified by nuclear localisation signal sequence) was linked to a three-arm cationic oligomeric carrier via disulfide bridge linkage. The protein was activated using the succinimidyl derivative of 2-pyridyl-2-carboxyethyl disulfide and conjugated with the oligomeric carrier built up form artificial amino acid (succinyl-tetraethylene pentamine) and Cys. A peptide inhibiting transthyretin aggregate-induced cytotoxicity was conjugated to PEG via disulfide and amide bond.196 The PEG carried 108 | Amino Acids, Pept. Proteins, 2018, 42, 85–145

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Fig. 20 Structure of cleavable linker for the synthesis of disulfide containing peptide conjugate.

thiol groups at both ends, which were activated with 2-piridylsulphenyl group. Patil et al. developed a linker to mimic the formation of disulfide bond in insulin and used for the synthesis of heterodimer peptides with disulfide bridge (Fig. 20).197 This linker is stable during the SPPS with Fmoc/tBu strategy, the work-up procedure and also disulfide formation, but can be cleaved with hydrazine treatment. 2,2 0 -dipyridyl disulfide as thiol activating reagent was applied. Thioether bond formation. The thiol group can be alkylated/arylated to form stable covalent connection between the partners. The presence of Cys in the peptide/proteins is often the source of thiol group(s). Most frequently the other partner contains either the maleimido or haloacetyl (mainly chloroacetyl) group. The Michael-type addition reaction between the maleimido and thiol group is fast, selective and reliable therefore often used in bioconjugation. The influence of conditions (solvent, initiator and thiol) on this reaction was recently summarized.198 B-cell epitope peptides were conjugated with T-cell epitope containing dendrimer peptide via maleimido-thiol reaction.199 A Cys was attached to the C-terminal of B-cell epitope peptide and conjugated to the Lys core via maleimido-propionyl group. Proteins can be ligated with peptide using this method too. Cas9 protein, extended with a Cys residue at its C-terminal was successfully ligated with a CPP containing maleimido group at the N-terminal, while the expression of fused construct did not produced enough amount of conjugate.200 POCs were also prepared using this strategy. For example fibrin/filaggrin citrullinated peptides were synthesized by Fmoc/tBu strategy with maleimido group at the N-terminal and ONs with hexyl thiol group at the 5 0 -end.201 The two partners were coupled in solution, with moderate yield. Gold nanoparticles (AuNPs) were functionalised by peptides originated from hepatitis B virus-PreS1 protein for targeting hepatocellular carcinoma cells.202 The surface of the AuNPs was partially covered with a thiol containing amino group for maleimide derivatisation with 3-(maleimido)propionic acid N-hydroxysuccinimide ester. The Cys residue Amino Acids, Pept. Proteins, 2018, 42, 85–145 | 109

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was added to the C-terminal of peptide. The surface of silica NPs was modified with an AMP (Cecropin P1) containing Cys at the C-terminal.203 For the ligation the surface of the silica NPs was covalently modified by a maleimido-PEG linker. Polymer based NPs (poly(lactic-co-glycolic acid) (PLGA)) was also conjugated with peptide via thioether linkage.204 Maleimido-PEG-PLGA was incorporated into the NPs and they were reacted with fluorescein isothiocyanate (FITC)-(Asp)7-Cys peptide. Liposomes were conjugated to the targeting peptide, cyclic RGDyC (c[RGDyC]) sequence by thioether ligation.205 The liposomes contained a few percent maleimido group modified lipids and the cyclic targeting sequence was attached to these lipids via thiol group of the added Cys. Pt complexes were conjugated with EGFR targeting peptide.206 The peptide was extended at the N-terminal with Cys attached mini PEG spacer, while the cisplatin and oxaliplatin componants were modified with the isocyanate derivative of maleimido-propinoic acid. This method is also suitable to prepare imaging probes with radioisotopes. For example Cys modified chelator, NOTA was attached to the N-terminal amino group of peptide YHWYGYTPQNVI, specifically recognizing EGFR) via Ahx spacer.207 The c(RGDyK) peptide was acylated with maleimidopropyl group at the e-amino function of Lys. Fluorophores were attached to heterodimer peptide conjugate also via this reaction.208 Apoptosis inducing peptide was attached to CPP and this conjugate, containing Cys at its C-terminal was ligated with maleimido derivative of Alexa fluorescence dye. Li et al. applied thioether bond formation in the functionalisation of biodegradable polyester by peptide conjugation.209 Polyester was first modified by maleimido groups and was reacted with peptide with added C-terminal Cys (EPLQLKMC) recognizing specifically bone marrowderived mesenchymal stem cells. Similarly, a Cys was added to the C-terminal of exendin-4, a GLP-1 receptor agonist and conjugated with maleimido containing linear or trimeric PEG.210 Haloacetyl group can be incorporated into a peptide by acylation of amino group(s). The simplest way is to create an N-terminal amino group, but the side chain of Lys can also be acylated during the SPPS. Although the reactivity is higher in case of bromine or iodine, due to the lack of stability the use of chloroacetyl group is wide spread. It reacts selectively with thiol group at slightly alkaline pH. The alkaline condition is the main drawback of this technique, because the dimerization side reaction of thiol containing component is unavoidable. The reaction may be dependent on the position of the Cys residue. As solution one can dissolve the chloroacetylated partner in the alkaline buffer and add the thiol partner in small portion to the reaction mixture or use reductive conditions during the conjugation. Multimeric peptide antigens were prepared by the reaction of thiolated multiple antigenic peptide and chloroacetylated epitope peptides.211 The reaction was carried out in 0.02 M NaHCO3, pH 7.5 and to reduce the disulfide bond formation tris(2-carboxyethyl)phosphine was added to the reaction mixture. 110 | Amino Acids, Pept. Proteins, 2018, 42, 85–145

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The surface of chitosan micro or nanoparticles was chloroacetylated by chloroacetic anhydride followed by the addition of epitope-LHRH peptide chimera containing N-terminal Cys.212 CPPs with N-terminal Cys were ligated to chloroacetylated core peptide (linear or dendrimer)213 in 50% aqueous DMF using an excess of N,N-diisopropylamine (DIEA) and KI to generate iodoacetyl groups in situ. Dinuclear thiolato-bridged arene Ru complexes were conjugated with peptides via thioether bond formation.214 The thiol containing Ru complexes were reacted with N-terminal choloracetylated peptides in water– acetonitril ¼ 1 : 1 (v/v) also in the presence of DIEA and KI. Cys containing glycopeptide was also conjugated with chloroacetylated dendritic peptide in DMF-water in the presence of DIEA and KI.215 Fiore et al. synthesized carbohydrate-peptide conjugates using thioether ligation.216 The cyclodecapeptide with six Lys residues as a scaffold and two different thiolated carbohydrates were conjugated. Four lysine were modified with Alloc protecting group, while two were chloroacetylated (Fig. 21) or vice versa, which resulted in the same modification on one side of cyclopeptide. The thiolated carbohydrates were ligated in chloroacetyl-thiol or thiol-ene reaction. The two-step conjugation could be performed in one-pot reaction with reasonable yield, if the chloroacetyl group reacts first then the alkene. Wang et al. reported a method for specific and covalent protein labelling via thioether bond formation.217 Two coiled-coil peptides were engineered; one of them contained a free Cys, while the side chain of the other was chloroacetylated in the proper position. When peptide 1 attached to the protein and peptide 2 with labelling moiety bind to it thioether linkage could form spontaneously. Zhang et al. described a new strategy for site selective labelling of proteins or peptides.218 The authors observed that the Xaa-Cys-Pro-Xaa motif (where Xaa is an aromatic amino acid residue) increases the reactivity of thiol group of the Cys residue towards perfluoroaryl probe (Scheme 5). This probe may comprise very divergent compounds.

Fig. 21 Multivalent cyclopeptide scaffold with two different kinds of ligation site. Amino Acids, Pept. Proteins, 2018, 42, 85–145 | 111

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Scheme 5 Reaction of thiol containing peptide with perfuloroaryl compounds.

Calce and De Luca reviewed the methodologies of S-alkylation of Cys.219 Tedaldi et al. described bromomaleimides as a new set of reagents for thioether formation and thus modification of Cys.220 The reaction is very selective towards Cys and the product can be opened or transformed into dehydroalanine. Its applicability in bioconjugation was also demonstrated221 and reviewed recently.222 It was also noticed that dibromomaleimide can react with disulfide bridge and gives two thioether bonds. Stefanucci et al. reported the synthesis of fluorophore labelled opioid peptides.223 The dibromomaleimide derivative of fluorescein was reacted with cyclic peptide containing disulfide bond or linear peptide with two thiol groups. These reactions resulted in fluorescently labelled cyclic peptides. In the synthesis of oxytocin-polymer conjugates the polymer was also modified with dithiophenol maleimide groups.224

3

Selected applications of peptide-bioconjugates

Oligopeptide based bioconjugates are widely applied in various branches of biomedicinal/biomaterial research. Three main area could be identified: (a) drug/reporter molecule conjugates to improve efficacy/selectivity for potential therapeutic/imaging use by targeting (Chapter 3.1), (b) conjugates can be utilized as delivery systems to alter solubility, biodistribution, to increase circulation half-life and/or bioavailability, degradation of the peptide attached bioactive molecule, which might result in decreased toxic side effects, pyrogenicity and/or modified immunogenicity (Chapter 3.2), (c) bioconjugates with peptides can function as synthetic antigens/immunogens to understand better immunrecognition and to develop efficient synthetic vaccines and/or antigens for sensitive immunodiagnosis (Chapter 3.3). A separate group of compounds has been developing very fast recent years in which oligopeptides are covalently attached to nanostructures (e.g. liposomes, nanoparticles) or nanostructures made of peptide bioconjugates for the above applications (Chapter 3.4). The common feature of these constructs is the carefully design to combine the beneficial characteristics of the peptide partner as well as of the coupled moiety. Covalent linkage between the partners could be 112 | Amino Acids, Pept. Proteins, 2018, 42, 85–145

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direct or a ‘‘spacer’’ unit is inserted for structural (e.g. steric accessibility) and/or functional purpose (e.g. facilitate enzymatic release, binding). Here we are providing some examples from recent years only belonging one of the above described categories. This short and necessarily very sketchy outline could be indicative of the importance of bioconjugates both in basic biomolecular science and also in the more applied drug/ imaging/vaccine research and documents its originality and international recognition. 3.1 Targeting by peptide bioconjugates Peptide bioconjugates have realised and also potential use in ‘‘active targeting’’ of various, but mainly anticancer/antimicrobial drugs, reporter molecules (e.g. radionuclides, optical probes, biotin), enzymes, proteins, oligonucleotides and of genes. Peptide bioconjugates can be classified as molecules with (chapter 3.1.1) or without (known) (chapter 3.1.2) recognition unit, which enables specific binding to cell–surface receptor (e.g. hormone, homing peptide) and/or intracellular targets (e.g. mitochondria, DNA) and/or efficient cellular uptake (e.g. CPPs). The properties of the peptide component will largely influence the fate of the conjugate, and the conjugate will differ from those of free compounds administered by the same route. For example regulatory peptides that exert action through their specific high-affinity binding receptors seem to be particularly good candidates for targeting. Many of these receptors in certain pathological conditions like cancer, inflammation, vascular and infectious diseases were shown to be massively overexpressed and thus target in diagnosis and therapy. 3.1.1 Targeting based on specific recognition. The most frequently applied targeting structure include peptides related to SST, integrin, gonadotropin-releasing hormone (GnRH), bombesin, neurotensin, vasoactive intestinal peptide, neurotensin (NT), neuropeptide Y (NPY), a-melanocyte-stimulating hormone (a-MSH) and tumour-homing peptides identified by phage display or combinatorial approaches. The importance of the linking structure between the partners, in both drug and reporter conjugates, if any, is well-documented by a recent study.225 Concerning the cargo entity of bioconjugates predominantly molecules with antitumour or with signalling/reporting properties are present in the literature. Some papers are also available with intracellular targeting in microbial diseases. Drug targeting (chemotherapy). In cancer research, for tumour chemo/ radiotherapy – among others – SST, integrin, gastrin-releasing peptide (GRP), GnRH, a-MSH, and GLP-1 receptors, just to mention a few, have been successfully applied. Recently, several conjugates were published with oligopeptides identified by phage display approach.226–229 Results of oligopeptide (e.g. LHRH, ICAM-1) conjugates with DOX, methotrexate (MTX), or camptothecin (CPT) in connection with anticancer research were reviewed by Majumdar and Siahaan recently230 and lately, a wider coverage of drug-targeting with special focus on linker Amino Acids, Pept. Proteins, 2018, 42, 85–145 | 113

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inserted between the drug (e.g. also PTX, and metal based complexes) and the peptide moiety was published.231,232 DOX coupled with tumour-targeting SST analogue, octreotide via a cleavable disulfide-intercalating cross-linking reagent successfully interacted with the relevant cell surface receptors and, the conjugate suppressed the ACTH secretion in AtT-20 pituitary tumour cells. The authors concluded that the conjugate, unlike the free octreotide, can be applied as potent and selective antitumour agent.233 For receptor-mediated tumour-targeting Wang et al. have conjugated PTX with a GnRH antagonist, degarelix [Ac-D-Nal-D-Cpa-D-Pal-SerAph(L-Hor)-D-Aph(Cbm)-Leu-ILys-Pro-D-Ala-NH2]. In the five compounds 2-pyridyl disulfide PTX carbonate via disulfide bond was attached to positions 1 or/and 6 of degarelix with/without SH functionalized Arg-Gln spacer. All PTX–degarelix conjugates had elongated half-life in human serum and two variants exhibited an antagonism efficacy similar to that of free degarelix. The in vitro cytostatic effect of conjugates was markedly higher on MCF-7 human breast and HT-29 human colon cancer cells, than on 3T3 mouse embryonic fibroblast cells.184 Karampelas et al. shown that the agonist GnRH analogue peptide (Glp-HWSYlysLRPG) with covalently attached gemcitabine at the Lys e-NH2 group modified by succinic anhydride is efficiently recognize GnRH receptor overexpressed in prostate cancer, and the conjugate (GSG) as a prodrug of gemcitabine also prevents its rapid inactivation by cytidine deaminase.234 GSG preserved the peptide partner related agonist properties in vivo and in vitro and conjugate can release gemcitabine intracellularly.235 Several conjugates were also reported on the antitumour activities of novel conjugates with GnRH-III hormone analogue monomer or dimer peptide with daunomycin.236–238 By the use of N-terminal FITC-labelling and SPR, Soudy et al. have demonstrated that a 12-mer peptide VPWMEPAYQRFL (p160) and also its enzymatically stable 10-mer analogue (WxEAAYQrFL) specifically recognize keratin 1 as the target receptor highly expressed on MCF-7 breast cancer cells and taken up via endocytosis. A conjugate of the 10-mer peptide with a pro-apoptotic antimicrobial peptide, microcin J25 (GGAGHVPEYFVGIGTPISFY) at the N-terminal exhibited selective cytotoxicity toward breast cancer cells and inhibited the growth of MDAMB-435 MDR multidrug-resistant cells with an IC50 value comparable to that of non-resistant cells.229 Concerning antimicrobial research, targeting of intracellular bacterial pathogens (e.g. M. tuberculosis, Salmonella and Brucella) with cleavable conjugate could overcome the inability of antibiotics to traverse eukaryotic membranes. Accordingly, Brezden et al. prepared peptide conjugate of the aminoglycoside antibiotic kanamycin with a de novo proline rich, non-membrane lytic, broad-spectrum AMP (P14LRR) forming a cationic amphiphilic polyproline helix with efficient mammalian cell penetration. Two partners were linked by disulphide bond that breaks down in the reducing environment within cells. Indeed, the authors reported that the P14KanS conjugate effectively cleared various intracellular pathogenic bacteria within J774A.1 macrophage cells more potently than that of a 114 | Amino Acids, Pept. Proteins, 2018, 42, 85–145

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191,239

conjugate lacking the disulfide moiety. In response to the emergence of antibiotic resistance and the ability of resistant pathogens to form biofilms Mohamed et al. have reported the above kanamycin conjugate possessed rapid bactericidal activity against stationary phases of both Gram-positive and Gram-negative pathogens and was superior in disrupting adherent bacterial biofilms and in killing intracellular pathogens as compared to conventional antibiotics in vitro and in vivo.240 New pyridopyrimidine derivatives with in vitro antimycobacterial activity were conjugated with a novel heterotrimer oligotuftsin derivative (TKPR[TKPKG]2) as targeting moiety at Lys8 and Lys13 positions.241 The membrane activities of the conjugates on mono- and bilayer lipid models were studied in the light of their in vitro antimicrobial, cytotoxic and cellular uptake activities on MonMac6 cell line. Correlation was established between membrane affinity properties and in vitro biological characteristics. The free compounds showed little membrane affinity to lipid mono and bilayer models, but their conjugation dramatically enhanced modelmembrane interaction as well as cellular uptake rate in vitro.242 Rodriguez et al. synthesized a cationic AMP (CAMP)-conjugate with small antibiotics, fluoroquinolone (levofloxacin, LVFX) at all Na – and Ne – amino groups of the peptide RGRRSSRRKK-NH2 (Pep-4) derived from the C-terminal portion of human beta defensin-3. The conjugate with three copies of LVFX significantly enhanced the effectiveness against the model Gram-negative bacterium E. coli and Gram-positive bacterium B. cereus under physiologically relevant salt concentrations as compared with that of free Pep-4. These findings suggest that conjugation of CAMPs to auxiliary compounds, such as small molecule antibiotics to generate hybrid compounds could be a promising strategy for increasing peptide therapeutic potential.243 One of the most promising antimicrobial oligopeptides derived from ubiquicidin (ribosomal protein S30) murine microbicidal protein present in the cytosolic fraction of macrophages244 was conjugated with coumarin derivative.130 The conjugate of the peptide amide (RAKRRMQYNH2) exhibited antimicrobial activity against C. gattii and C. neoformans, including a fluconazole-resistant strain of C. gattii.130 It is interesting to note that a non-cleavable conjugate of MTX and a short cell penetrating peptide was demonstrated to target intracellular Listeria monocytogenes.245 Like antimicrobial photodynamic therapy,246 the related sonodynamic therapy (SDT) has received considerable attention since the low-intensity ultrasound is able to penetrate human tissue and could be utilized to treat deep-seated infections. Johnson et al. reported on the synthesis of the AMP (KLAKLAK)2 conjugate in which the covalently attached hydrophilic eosin Y, as sensitizer inactivated Gram positive and Gram negative bacteria after photodynamic therapy with negligible photodamage observed to host cells.247,248 Inspired by these papers Costley et al. conjugated a C(KLAKLAK)2 peptide with N-terminal Rose Bengal sensitizer and used in antimicrobial SDT. After treatment with the conjugate and subsequently explosion of the samples to ultrasound, marked reduction was observed in numbers of microbes (Staphylococcus aureus, Pseudomonas aeruginosa). The conjugate also displayed improved uptake by Amino Acids, Pept. Proteins, 2018, 42, 85–145 | 115

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bacterial cells as compared with a mammalian cell line (Pr0.01). The results of in vivo preliminary experiments with wounded mice were also promising.249 Reporter molecule targeting (imaging). Molecular imaging by peptide bioconjugates provides the non-invasive visualization and also quantification of biological events at cellular level within living systems. Recently, a significant progress could be observed in the application: besides imaging tumour related structures, events antimicrobial peptide conjugates with fluorophore have been highly attractive for imaging bacterial infections in animals as reviewed.250,251 By interaction between physiological target and ligand labelled structures on the cell surface and also within the cell can also be identified. Thus bioconjugates with fluorophores or radionuclides represent a highly useful class of tools in early diagnosis, treatment and in therapy monitoring as well as in fundamental research of various diseases. In order to avoid loss in functional properties (e.g. binding, signalling efficacy), appropriate spacer unit is frequently incorporated between the two partners in this group of bioconjugates. Probably the two most frequently applied classes of reporter molecules for molecular visualization are the radionuclides for nuclear and fluorescent compounds for optical imaging. The combination of these modalities led to the development of peptide heterodimer bioconjugates as dual-targeting molecular probes as well as compounds with both imaging entities. Nuclear imaging is the premier clinical method. The most suitable techniques are the radionuclide-based PET and SPECT. Several radionuclides, including 99mTc, 111In, 64Cu, 68Ga and 18F for diagnosis or 90Y and 177Lu for therapeutic use, have been employed for radiolabelling. Hosseinimehr et al. reviewed results concerning liver uptake and excretion of most studied radiolabelled peptides (e.g. bombesin, monomeric/ dimeric cyclic RGD peptide [c(RGDfK), c(RGDfK)2] a-MSH, octreotide), which are important considerations for the design and further development of peptide conjugate based imaging agents.252 Several additional excellent reviews are available on the peptide receptor radionuclide therapy (PRRT) using SST receptor antagonists/agonists peptideconjugates with radionuclides as effective treatment modality for advanced neuroendocrine tumours (NETs).253,254 Grob et al. proposed to replace the oxidation sensitive Met by methoxinine (Mox) an oxidation-stable amino acid substitute in peptide conjugates with radiolabel. The authors showed that 177Lu-DOTA-labelled derivatives of the tumour targeting peptide bombesin (BB, Pyr-Gln-Arg-Leu-Gly-AsnGln-Trp-Ala-Val-Gly-His-Leu-Met-NH2), namely 177Lu-DOTA-PEG4-[Mox14] BB(6-14) and the corresponding BB(7-14) analogue, exhibited superior in vitro properties and in vivo tumour-targeting performances in comparison to the [Met14]- and even [Nle14]- analogues.255 Similar observations were reported on radiolabelled minigastrin conjugates (177LuDOTA[X15]MG11 (where MG11 ¼ H-D-Glu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2 and X ¼ Met, Nle or Mox) regarding cell internalization/externalization properties, receptor affinity (IC50), blood plasma stability).256 116 | Amino Acids, Pept. Proteins, 2018, 42, 85–145

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Neurotensin, a 13 amino acid peptide, is the native ligand for NTR1 receptor overexpressed in different cancers. A novel set conjugate with DOTA, as chelator at the N-terminus of a ligand analogue and different lengths of spacers was developed. Jia et al. found that the presence of DOTA has negatively impact the binding affinity of the NTR1 ligand. However, the optimal binding was restored by incorporation of a b-Ala linker. The best conjugate, DOTA-b-Ala-Lys-Pro-(N-Me)Arg-Arg-Pro-DmtTle-Leu-OH, reported with 177Lu demonstrated substantial tumour accumulation.257 Peptide GGNKRTR derived from the a tumour-homing c(CGNKRTRGC) peptide could specifically bind to NRP-1 overexpressed in various cancer cells. Tyr was added at the N-terminus and the truncated linear peptide and radiolabelled with 131I. The conjugate was localized in NRP-1 positive cancer cells in vitro and in tumour xenografts in vivo, indicating its potential to detect NRP-1 positive tumours.258 For imaging of non-small-cell lung cancer overexpressing of EGF receptor, a conjugate with hydrazinonicotinamide (HYNIC) and targeting hexapeptide was prepared [HYNIC-(Ser)3-(Leu-Ala-Arg-Leu-Leu-Thr)] and labelled with 99mTc. The ligand of EGF receptor (Leu-Ala-Arg-Leu-LeuThr) was identified from a virtual peptide library of putative EGF-R binding peptides by screening against the EGF-R X-ray crystal structure in silico and in vitro.259 For metastatic melanoma imaging new, 99mTc-labeled lactam bridgecyclized a-MSH hexapeptides [c(DHfRWK)-CONH2] using four different bifunctional chelating agents. In between a Gly-Gly-Nle tripeptide spacer was inserted. Receptor binding affinities to melanocortin-1 (MC1) and biodistribution studies in B16/F1 melanoma-bearing C57 mice showed that melanoma lesions were clearly visualized by SPECT/CT and the best compound was the conjugate with HYNIC chelator.260,261 Conjugate with further optimized structure was reported in which a Gly-extended cyclic MSH heptapeptide [c(DHfRWGK)-CONH2] and gamma aminobutyric acid-Nle dipeptide spacer was applied.262 In recent years, bifunctional chelators have found multiple applications in the field of medical imaging and therapy. To minimize the decomplexation in vivo, a novel macrocyclic bifunctional chelator incorporating propylene cross-bridge was invented recently by Pandya et al. and conjugated with cyclic RGD motif containing peptide [c(RGDyK)] by using isothiocyanate linkage. The conjugate formed strong, stable complexes with radiometal 64Cu (II) ions allowing both PET imaging and radiotherapy. Biodistribution, in vivo stability, and animalPET imaging studies confirmed the excellent tumour targeting efficacy of the c(RGDyK) conjugate.263 Oxboel et al. compared of two new three-party conjugates for PET imaging of tumour-angiogenesis in vivo using human xenograft tumours in mice. In these constructs dimers [c(RGDyK)2] were bridged with Glu residue having a chelator moiety at the end of its side chain and labelled with 64Cu or 68Ga (64Cu-NODAGA-E[c(RGDyK)]2 and 68Ga-NODAGAE[c(RGDyK)]2 respectively). Based on studies with human xenograft tumours, the authors proposed the use of 68Ga derivative for imaging.264 Amino Acids, Pept. Proteins, 2018, 42, 85–145 | 117

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Recently, in clinical settings preliminary PET imaging of integrin avb3 expression in breast cancer patients was demonstrated with the proposed conjugate.265 In order to identify a suitable chelating agent for 68Ga labeling, Kumar et al. compared the labelling kinetics, stability, binding properties and biodistribution of peptide conjugates composed of a 27-mer pituitary adenylate cyclase activating peptide [PACAP] analogue with high affinity for VPAC1 receptors expressed human breast cancer cells (T47D) and C-terminal chelating moiety (DOTA or NODAGA).266 In vitro and in vivo studies with NODAGA-peptide conjugate showed more convenient radiolabelling features and was a suitable radiotracer to PET/CT image in mice bearing BT474 breast cancer.267 Optical imaging methods provide advantages over nuclear imaging modalities including high sensitivity, molecular level monitoring, the use of nonradioactive agent, experimental flexibility, wide range of compounds with different spectroscopic properties. A great number of novel fluorescent probes are available and the development of click chemistry is providing a great deal of simplicity in labelling. However, it should be considered that fluorophores in general are sensitive to the environment (e.g. solvent, pH, ionic strength) and the selection typically differs for in vitro and in vivo applications. To label endogenous synaptic protein PSD-95 (postsynaptic density protein 95), peptide RVRLQTSV, identified by a phage display approach, with high affinity for PDZ domain 3 of PSD-95 was first elongated by RGGG tetrapeptide spacer (RGGGRVRLQTSV), and then conjugated at the N-terminal with squaraine rotaxanes (SeTau-647) with attractive fluorescence properties (e.g. high quantum yields in water, high extinction coefficients, high photostability, low sensitivity for oxygen species) ideal for in vivo two-photon imaging. The authors have demonstrated that the non-toxic, stable conjugate allowed long-term neuronal imaging for in vivo two-photon microscopy.227 To evaluate the feasibility of ApoPep-1 (Apoptosis-targeting peptide1,CQRPPR) as an imaging tool for apoptosis in AD it was conjugated with fluorescein at the N-terminus of peptide using amino caproic acid as linker. Lee et al. have shown that ApoPep-1 peptide bound to primary cultured apoptotic cells under conditions simulating AD, as well as to apoptotic brain cells from AD mice. Thus the conjugate could detect apoptotic cell death in brain tissue and could be an effective tool for optical imaging of apoptosis both in vivo and ex vivo.268 Dual (bifunctional/bimodal) targeting. Peptide-based radiopharmaceuticals can be tailored to specifically target biomarkers expressed predominantly on tumour cells for both diagnostic and therapeutic purposes. The emerging concept of simultaneous, multi-receptor targeting with a single radiocompound using rationally designed heterodimeric peptide conjugates has shown improved molecular imaging properties when compared to monomeric peptide constructs. Combination of two different functions (diagnosis and therapy) within a single bioconjugate resulted in two different ‘‘cargo’’ (e.g. a reporter 118 | Amino Acids, Pept. Proteins, 2018, 42, 85–145

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molecule and drug) are attached to a targeting oligopeptide. These compounds, frequently defined as ‘‘theranostics,’’ offer promising tools capable of multiple functions in vivo, including targeting, image contrast and drug delivery. Jackson et al. prepared peptide-radiopharmaceuticals for dual targeting with utility for PET imaging of prostate tumour. In this construct, aVb3 integrin and a gastrin releasing peptide receptor (GRPr) targeting peptide, c(Arg-Gly-Asp-D-Tyr-Lys) and Gln-Trp-Ala-Val-Gly-His-Leu-MetNH2, an agonist of shortened (7–14) bombesin, were connected by using Glu-6-Ahx spacer, conjugated with NOTA at the side chain of Glu and radiolabelled with 64Cu.269 Later, Durkan et al. published a similar construct in which the bombesin agonist was replace by an antagonist peptide (D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2) recognizing GRPr.270 Recently, a further modified conjugate was reported in which NOTA was replaced by DOTA, labelled with 111In, 177Lu or 90Y and tested not only for imaging, but also for radiotherapy in murine U87-MG/PC-3 xenograft model.271 Dual-receptor molecular imaging probe with avb3 integrin/EGFR-targeting was published in which c(RGDyK) and YHWYGYTPQNVI, as EGF receptor ligand, identified by the phage display approach, were joined by 6-Ahx-Cys dipeptide spacer at the side chain of Cys, conjugated with NOTA at the N-terminal and radiolabelled with 64Cu.228 Dual receptor targeting could mean also to utilize a membrane (e.g. SST receptor) and an intracellular structure (e.g. nucleic acid). Based on the hypothesis that simultaneous down-regulation of a tumour survival gene and delivery of internally emitted cytotoxic radiation will be more effective than either treatment modality alone, a three party conjugate comprising octreotide, a SST analogue, an anti-bcl-2-PNA (peptide nucleic acid) and DOTA chelator with 177Lu label was constructed for targeted combined antisense radiotherapy.272 The labeled DOTAanti-bcl-2-PNA-Tyr3-octreotate antisense conjugate effectively inhibited tumour progression in a mouse model with human non-Hodgkin’s lymphoma tumour xenografts.273 Conjugates with modified PNA sequence (Gly at T14 was replaced by Ser/Asp) and labelled with 64Cu exhibited significantly reduced live uptake (Ser) and increased tumour-toblood ratio.274 Similarly, for intracellular nuclei targeting a fluorescent bispecific peptide conjugate of Her2-binding peptide ligand (NH2-GSGKCCYSL), NLP peptide (CGYGPKKKRKVGG) and of Cy5.5 fluorophor at the N-terminal was tested for nuclear localization in breast cancer cells. The peptide components were linked by a hexa(ethylene glycol) spacer. The fluorescent/confocal microscopic imaging demonstrated the presence of the signal in the cytoplasm of Her2/neu positive breast cancer cells. However, no fluorescent signals were detected within the nuclei suggesting poor nuclear localization of the conjugates within the cytoplasm after cellular uptake.275 The dual targeting strategy for Auger endoradiotherapy using first receptor-mediated internalization by SST receptors, followed by binding of labelled DNA ligand-peptide conjugate has been demonstrated. The Amino Acids, Pept. Proteins, 2018, 42, 85–145 | 119

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authors prepared an octreotide conjugate with a DNA targeting, minor groove binding bis-benzimidazole, attached to the N-terminal amino group of the SST analogue by amide bond and labelled with an Auger electron emitting radionuclide isotope 125I. The limited extent of intracellular cleavage of the conjugate suggested the need of a less stable linkage between the DNA ligand and the peptide.276 For lysosome targeting, Au nanoparticles (AuNPs) with or without FITC label were conjugated with CPP having N-terminal Cys (penetratin, CRQILIWFQNRRMKWKK or Tat, CYGRKKRRQRRR) and with lysosomal sorting peptide (YQRLC or CNPGY). The authors observed efficiently and selectively delivered of nanoconjugtates into the lysosomes with minimal cytotoxic effects.277 Hyaluronic acid (HA) solutions could effectively lubricate the ocular surface and are in use for the treatment of dry eye related symptoms. In order to prevent the rapid clearance of HA due to limited adhesion, Lee et al. developed a novel approach by using a bifunctional peptide-polymer bioconjugate. In these constructs ocular surface tissues protein binding peptide [(GGSPYGRC, SABpep) or type I collagen binding peptide from decorin (SYIRIADTNIT, ColBpep)] was positioned at the N-terminal of the poly(ethylene glycol) (PEG, Mw 1000 Da), while the peptide with HA binding capacity (HABpep, STMMSRSHKTRSHHVGC) was present at the C-terminal of the conjugate. The delivery of HA to the ocular surface by sialic acid binding peptide as an anchor resulted in significantly prolonged retention time ex vivo and in vivo models.278 Viehweger et al. constructed a three-party bioconjugate for bimodal (PET/fluorescence) imaging by targeting the EGF receptor. This compound is consist of a hexapeptide (LARLLT) recognizing the receptor, a sulfonated near-infrared fluorescent cyanine dye (‘‘sulfo-Cy5’’) attached to the N-terminus via a b-homopropargylglycine-Cys-b-Ala-b-Ala spacer for study the EGF receptor-binding ability of the probe by laser-fluorescence spectroscopy and a 64Cu(II)-chelating moiety introduced at the side chain of the spacer unit for use in vivo PET. The conjugate proved to behave as non-competitive antagonist and preserved properties related to the other two functions and seems to be suitable for future development of more complex nanoparticle-based multimodal imaging agents.279 [18F]-organotrifluoroborate was coupled with fluorescent bis-(c(RGDyK) peptide using appropriate spacer enabled for dual-mode fluorescence/ PET imaging of neuroendocrine tumours. The conjugate for PET imaging was prepared as single-step labelling and reported in details reviewing of the design principles and preclinical applications.280–282 Similar peptide conjugates were described with SST analogue, octreotide,283 bombesin derivative284 and kallidin.285 A novel three-component conjugate was invented by Welling et al. for specific identification of bacterial infections. This construct was composed of a hybrid label of DTPA of chelating the radioisotope 111 In and Cy5 fluorescent dye with an AMP derived from ubiquicidin (TGRAKRRMQYNRR, UBI29–41) at its N-terminal. Preclinical in vitro and in vivo evaluation emphasized its ability to visualize individual bacteria by microscopy in excised tissues, as well as bacterial infections.286 120 | Amino Acids, Pept. Proteins, 2018, 42, 85–145

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A photosensitizer protophophyrin IX was conjugated with one or two copies of AMP, YVLWKRKRKFCFI-NH2 recognizing lipopolysaccharide for the effective fluorescent imaging and photodynamic inactivation of Gram-negative strains. The disubstituted conjugate exhibited potent activity against drug-resistant bacterial pathogens and thus served not only as a promising fluorescent probe, but also as potent photosensitizer for photodynamic inactivation of drug resistant Gram-negative bacterial strains.287 A novel nucleoside-peptide conjugate composed of systemin, an 18-mer tomato plant signalling peptide (AVQSKPPSKRDPPKMQTD) naturally produced in response to wounding or pathogen attack and with N-terminal 3-azido-2,3-dideoxythymidine (AZT) proved to be an effective transporter of low-molecular mass molecules (cargo) in a tomato plant to the study of mechanisms underlying plant peptide-dependent signalling pathways.288 3.1.2 Targeting by cell penetrating peptides (CPP). Bioconjugates with CPP enable efficient cellular uptake of covalently or even noncovalently attached cargo (e.g. nucleotide, nucleic acid, organometallic compounds.232,289–292 Advances in the field were summarized in recent reviews with outlook on the factors influencing the cellular uptake mechanism293–295 and discuss the limitations of CPP conjugates (e.g. low cellular/tissue selectivity). However, to enhance selectivity novel constructs were introduced by incorporating of intracellular/nuclear localization sequences for cell organelles (e.g. mitochondria). Novel ‘‘hybrid’’ CPP conjugates could transport of drugs through blood–brain barrier, through the conjunctiva of eyes and skin.296 A comparative study with two novel DOX-conjugates with cleavable linkage containing peptide differing in net charge and secondary structure (octaarginine, [Ac-CR8] vs proline rich amphipathic peptide [AcCGGWVELPPPVELPPPVELPPP-NH2]) showed that each peptide transport DOX into the cytosol of MCF-7 and HT-29 cancer cell lines.297,298 Pemetrexed (Pem), a novel antimetabolite anticancer drug inhibiting enzymes involved in the folate pathway, for which the presence of its free carboxylic groups is necessary, was conjugated with a targeting peptide Ile-Glu-Leu-Leu-Gln-Ala-Arg-NH2 identified by phage peptide library or with cell-penetrating octaarginine by thioether bond. The conjugates exhibited cytostatic effect in vitro on non-small cell lung carcinoma as well as on human leukemia cell lines. Furthermore, data show that the cytostatic effect of the free Pem was essentially maintained after conjugation.226 MTX and its pentaglutamylated MTX derivatives with CPP (penetratin or octaarginine) with/without GFLG spacer were prepared. MTX-Glu5penetratin(desMet) was effective on resistant MDA-MB-231 tumour cells suggesting that the translocation of polyglutamylated MTX may be a new way to treat resistant tumours.299 Zhang et al. reported on the effect of the sequential position of DOX on cellular uptake and cytotoxicity against both drug-sensitive and multidrug resistant tumour cells. The presence of DOX with an enzyme Amino Acids, Pept. Proteins, 2018, 42, 85–145 | 121

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cleavable GFLG tetrapeptide unit at the C-terminal of the Tat peptide (GRKKRRQRRRPPQ) resulted in markedly higher penetration and cytotoxicity by multidrug resistant cervical cancer cell line, as compared to the conjugate with N-terminal DOX-GFLG.300 It has been demonstrated that conjugation of a triplex-forming PNA to the C-terminal of penetratin (RQIKIWFQNRRMKWKK) elongated by a Lys trimer unit can modify a target chromosomal gene in the hematopoietic progenitor cells of mice following systemic i.p. administration. This indicates the potential of PNA-peptide conjugates for in vivo gene targeting in multiple tissue compartments.301 A rapid parallel strategy was developed for the synthesis of PNA-peptide conjugates recently. The PNA cargo containing a C-terminal Cys and two flanking Lys residues (Lys-CCTCTTACCTCAGTTACA-Lys-Cys) was coupled with alkyne functionalized CPPs (16 analogues of Pip5h, [RXRRXRILFQY-RXRRXR]) with terminal alkyne and successfully screened in a splicing redirection assay in HeLa p Luc705 cells.302 Arouri et al. reported the in vitro effect of a conjugate with platinum binding chelator MBL-III-7 attached to the N-terminal of the D isomer of L-maurocalcine, a 33-mer peptide isolated from the venom of a Tunisian chactid scorpion, Scorpio maurus palmatus with pronounced cell penetrating properties. The conjugate, especially at low doses, induced intracellular oxidative stress in human glioma U87 cells and it is considered as candidate for a preclinical assessment of Pt-based therapy for treatment of glioblastoma multiforme, a highly malignant and aggressive primary brain tumour.303 Deferasirox (DFX), a clinically recently approved and orally active iron chelator used extensively for the treatment of iron overload was coupled with peptide YGRKKRRQRRR [Tat(47–57)] at its N-terminal. Goswami et al. reported improved water solubility and the conjugate was able to remove iron ions from overloaded sera as well as from cytosol. Both free and the iron bound DFX-conjugate translocated through RBE4 cell line, an in vitro model of the blood–brain barrier.304 A novel mitochondrial localizing Ru(II)-peptide conjugate capable of sensing dynamic changes in local O2 concentrations within living cells was described by Martin et al. The luminescent dinuclear Ru(II) polypyridyl complex bridged across a mitochondrial penetrating peptide, FrFKFrFK-CONH2 or Arg8 (as control CPP) N-acylated with Ahx permitted semiquantitative determination of oxygen concentration at the mitochondria and also elevated reactive oxygen species. This indicates the potential of targeted binuclear complex in cell biology where understanding the metabolic status of the mitochondria is required. It is interesting to note that the construct with Arg8 was effective in transporting Ru(II) complexes into cytoplasmic regions without organelle specificity.305 CPP conjugates for molecular imaging were also reported during the last years. Splith et al. have developed three-party CPP conjugate (Gly-Leu-Arg-Lys-Arg-Leu-Arg-Lys-Phe-Arg-Asn-Lys-Ile-Lys-Glu-Lys, sC18)306 with a hypoxic radiosensitizer (2-(2-nitroimidazol-1-yl)acetic acid, NIA), and a radioactive label. Under in vivo conditions accumulation of the 122 | Amino Acids, Pept. Proteins, 2018, 42, 85–145

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conjugates was observed in hypoxic tumour regions. In order to prevent fast enzymatic degradation and clearance, to improve tumour accumulation and to optimize labelling procedure and yield a novel group of conjugates were synthesized with all-D version of the sC18 peptide, with more than one NIA moiety and DOTA was replaced by NODAGA as bifunctional chelator. The constructs, exhibiting high stability in vitro as well as in vivo, were radiolabelled by positron emitting radionuclides 64 CuII or 68GaIII. Bergman et al. observed marked differences: the use of 64 CuII with longer half-life and the low positron energy permitted longer PET experiments and resulted in images with higher resolution than compounds with 68GaIII radionuclide.307 To improve the poor aqueous solubility and cell permeability of Ir(III) complexes, Dolan et al. conjugated, for first time, the novel Ir(III) luminophore [cyclometalated iridium(III) polypyridyl complex; iridium(III) bis(2-(2,4-difluorophenyl)pyridinato-N, C2 0 )-2(4-carboxylphenyl)imidazo [4,5-f][1,10]phenanthroline] to octaarginine at the N-terminal. The conjugated complex exhibited rapid cellular uptake by SP2 and CHO cells and evidence from imaging suggests its penetration to the nucleus whereas the free compound does not.308 A three-party conjugate was prepared first by coupling a Cy7-like fluorescent dye, 3-mercapto-propioniccyclohexenyl-Cy7-bis-norbornenylbromide to CPP (CRQIKIWFQNRRMKWKK) followed by the attachment of two copies of 4-methyl-5-oxo-2,3,4,6,8-pentazabicyclo[4.3.0]nona-2,7,9triene-9-carboxamide (temozolomide, TMZ). This prototype theranostic conjugate acted as both a contrast agent (Cy7) and as a therapeutic molecule (TMZ), enabled the monitoring of cellular phenotype change and cancer cell killing effects by local enrichment of a cytotoxic agent.309

3.2 Delivery systems A number of potent therapeutic agents cannot enter into clinical evaluation, due to poor delivery, low bioavailability. Peptide bioconjugates can either be considered as soluble drug derivatives for ‘‘passive targeting’’ or modifiers of the bioactive component resulting in altered pharmacokinetics, decreased non-specific toxicity and even diminished immunogenicity etc. Bioconjugation opened an alternative route of delivery. The GLP-1 receptor agonist analogue [GLP-1 (7–37)] with A8G and K34R substitutions and with chloroacetylated linker of 2, 4, or 6 ethylene-glycol units at 26Lys was conjugated with a 5 kDa albumin-binding domain (ABD) derived from streptococcal protein G to extend the in vivo half-life. The conjugate preserved glucose-dependent insulin release of the free peptide in mouse islets.310 Clardy-James et al. reported on vitamin B12 conjugate with a GLP-1 (7–36) amide analogue, namely K34R-GLP-1. The replacement of Lys (K) with Arg (R) at position 34 has no effect on GLP-1 activity and allows selective conjugation at the K26 residue. In vitro data demonstrated that B12 attachment has little negative effect on the insulinotropic nature of GLP-1.311 B12 was also attached to peptide YY (3–36), known as pancreatic peptide, involved in appetite regulation. The Amino Acids, Pept. Proteins, 2018, 42, 85–145 | 123

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conjugate has considerable therapeutic potential through improved pharmacokinetic/ pharmacodynamic properties. It has similar activity to unconjugated peptide YY(3–36) in vitro, but improved function upon subcutaneous administration in vivo in rat model was reported.312,313 For the generation of long-acting peptide therapeutics, stabilization mechanisms were studied by Angelini et al. using the in vitro evolved bicyclic peptide inhibitor of the urokinase-type plasminogenactivator (uPA). The authors found when the bicyclic peptide was linked to an albumin-binding peptide, the bicyclic peptide conjugate not only circulated 50-fold longer (t1/2 ¼ 24 h), but also became entirely resistant to proteolysis when tethered to the long-lived serum protein.314 The small bicyclic peptides conjugated with albumin-binding domain reached high nanomolar concentrations in solid tumours, thus bicyclic peptides offer a new promising format for targeting.315 [D-Ala2, Leu5, Cys6]-enkephalin was attached to thiolated carboxymethyl dextran-cysteine (CMD-Cys) via disulfide bond and acted as prodrug by improving half-life and decreasing plasma clearance in rats.316 Also for altered pharmacokinetics and tumour accumulation in vivo, a hybrid peptide EGFR2R-lytic-Cys [(YRWYGYTPQNVIGGGKLLLKLLKKLLKLLKKK)Cys] was conjugated with CMD-Lys using disulfide bonds.317 To improve the poor pharmacokinetic properties of neuromedin (FRVDEEFQSPFASQSRGYFLFRPRN, NMU), an endogenous neuropeptide considered as a lead for the treatment of obesity/diabetes, it was conjugated with HSA at position Cys.34 Comparison of two conjugates prepared by maleimidobutyryl-(Ttds)2-NMU (where Ttds ¼ 1-amino-4,7,10-trioxa13-tridecanamine succinic acid) or by haloacetyl derivative, iodoacetyl(Ttds)2-NMU suggested that the haloacetyl–thiol chemistry lead to a stable thioether bond between the partners and yields a metabolically stable compound, with a long-lasting, robust body-weight lowering effect and improves glucose tolerance.318 Peptide inhibitors are derived frequently from the sequence of proteins with repeating units of seven amino acids (heptad repeat, HR) specialized in viral fusion. Antiviral potency could be increased by cholesterol conjugation to peptide inhibitors to augment affinity for the cholesterol-rich lipid rafts of the membranes, as proposed by Ingallinella et al.319 The presence of cholesterol dramatically increase the antiviral potency of HRderived inhibitors as demonstrated by Pessi et al. by using 34–36-mer peptides from HIV or HPIV proteins with C-terminal Cys residue and maleimide-functionalized cholesterol.320 Further examples (e.g. with mono- or dimer peptide from measles virus fusion protein with GSGSG and tetra-ethylene glycol spacers321 and cholesterol at the N-terminus of the fusion-inhibitory peptide of Newcastle disease virus or infectious bronchitis virus322 demonstrated increased antiviral potency, and simultaneously improved pharmacokinetics as reviewed by Pessi.323

3.3 Peptide-bioconjugates as synthetic antigens/immunogens In order to produce synthetic antigens/immunogens with optimal immunrecognition properties the structure of epitope peptides/haptens is 124 | Amino Acids, Pept. Proteins, 2018, 42, 85–145

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frequently manipulated by conjugation to oligo/polypeptide or with macromolecules.324 Considering prophylactic immunisation against polycyclic aromatic hydrocarbon benzo[a]pyrene (B[a]P), Schellenberger et al. investigated the immune response of 18 peptide conjugates composed of promiscuous T cell epitope of tetanus toxin and N-terminal B[a]P, in mice. Depending on the T cell epitope peptide, the conjugates induced very different levels of hapten-specific antibodies, thus a vaccination against B[a]P using pre-selected promiscuous T cell epitope of tetanus toxin as carriers is feasible.325 To study the carrier–adjuvant potency of Salmonella flagellin, the recombinant protein (rFliC) was coupled with B-cell epitope peptide from Plasmodium falciparum exported protein-1. The peptide had an N-terminal Cys residue (CDNNLVSGP) for the thioether linkage with the protein, modified with maleimide groups. The conjugate induced robust epitope peptide-specific antibody responses in mice and the immune sera recognized the native protein of the malaria parasite. The data clearly demonstrated that rFliC is a potent carrier with built-in adjuvant properties for antigenic peptide attached indicating its potential in conjugate vaccine research.326,327 N-terminus of glycoprotein B (AD-2) of human cytomegalovirus contains neutralizing linear epitopes. Peptides with 68NETIYNTTLKY78 epitope core and various flanking sequences at the C- and/or N-terminal were conjugated with a recombinant protein, a nontoxic mutant of the diphtheria toxin carrier, via Ahx at the C-terminus, and glutamic acids (E or EEE). Finnefrock et al. demonstrated that such a conjugate could produce strong binding titers specific to AD-2 in mice/rats, but was unable to generate significant virus neutralization.328 NGR-mTNF is a recombinant protein derived from the fusion of CNGRCG tumour homing peptide with the N-terminal of mTNF tumour necrosis factor. The CNGRCG peptide interacts with the CD13 (aminopeptidase N) receptor expressed by angiogenic vessels. NGR-hTNF is currently being tested in phase II and III clinical studies involving different types of tumours as immunmonotherapy and in combination with chemotherapeutic agents.329 Risini et al. reported on two the Ce3 domain derived human IgE peptide epitope-conjugates. The sequence of peptide P as well as of peptide Y (ADSNPRGVSAYLSRPSPGGC and QCRVTHPHLPRALMRS, respectively) were extended by Cys and conjugated with virus-like particle (VLP) derived from Qb bacteriophage by using the bifunctional linker (succinimidyl-6-[b-maleimidopropionamido] hexanoate). During preclinical evaluation in non-human primates, the induction of anti-peptide antibodies recognizing the intact human IgE and substantial lowering in circulating IgE levels were observed. A decision was made to advance this anti-IgE vaccine into human clinical testing.330 Liu et al. developed non-toxic, self-assembling polyacrylate tbutyl esterbased (PtBA) amphiphilic linear polymer or dendrimeric constructs with epitope peptides to induce T cell mediated immunity against E7 oncoprotein human papilloma virus related cancer. Four conjugates with Amino Acids, Pept. Proteins, 2018, 42, 85–145 | 125

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epitope peptide QAEPDRAHYNIVTF in different topography (linear, branched and dendritic) reduced tumour growth and eradicated E7positive TC-1 tumours in 40% of tested animals.331 Lately, the same group reported on dendrimeric conjugates with two different peptide epitopes, 744QAEPDRAHYNIVTF757of E7 and 643QLLRREVYDFAFRDL657 of E6 HPV oncoproteins.332 To develop transdermal immunotherapy of Duchenne’s muscular dystrophy, a conjugate of antigenic peptide (50VFLQKYPHTHLVHQA64) of myostatin was prepared with low molecular mass (17 kDa) hyaluronate derivative carrying aldehyde groups involving both a- and e-amino groups of the peptide. In this proof of concept study, Kong et al. found that the transdermal immunization of mdx mice, in the absence of adjuvant, significantly increased myostatin specific antibody responses and statistically marked improvement in the biochemical/pathological status of skeletal musculature/functional behaviours was observed.333 Biotin conjugate of antigenic peptides are in use for the analysis of epitope binding properties. Among those studies, Maas reported on the engineered, selective T lymphocyte-associated antigen 4 (CTLA-4, CD152) binders based on Cys-knot peptide, a trypsin inhibitor from Momordica cochinchinensis. The best variant was conjugated with biotin and assembled by folding. Its binding properties (specificity, affinity) to human (CTLA-4) was used for the analysis/screening of oligomeric variants.334 Babos et al. studied the effect of epitope orientation on autoantibody binding properties using new sets of rheumatoid arthritis (RA) specific biotin-peptide conjugates derived from filaggrin. The biotin was attached to the Cit/Arg containing epitope core (311TXGRS315) or epitope region (306SHQESTXGXSXGRSGRSGS324) peptide (where X ¼ Cit) at the N- or C-terminal of the peptides. The authors found that autoantibodies from RA patients bound specifically both filaggrin epitope conjugates, but the position of biotin in the peptide core sequence profoundly determined antibody recognition.335 3.4 Bioconjugates with nanoparticles Nanostructures in relation with peptide bioconjugates were reported in an increased number of papers recent years. These constructs could be classified as two groups. In group 1, pre-formed nanostructures are modified by covalent coupling of peptides of interest, while in group 2 nanostructures are made of peptide bioconjugates. These nanoconstructs could be utilized for biomedicinal applications (e.g. diagnosis, therapy) using the peptide attached components like, drugs, reporter molecules etc. Besides their unique physical properties, nanoparticles also afford a large surface area allowing the presentation of different types of functional groups and could serve as platform for combining multiple functions, like targeting ligands, imaging probes, and/or therapeutic drugs for simultaneous diagnosis, treatment, and monitoring of therapeutic responses by sensing different cellular signals. 3.4.1 Nanostructures modified by bioconjugation. Recently, Foubert et al. reviewed the most frequently used conjugation strategies for the 126 | Amino Acids, Pept. Proteins, 2018, 42, 85–145

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covalent modification of the functionalized (mainly carboxyl, amino and thiol) surface of quantum dots (QDs) with amino acids (e.g. Lys, Cys) and/or oligopeptides even proteins. Such QD based peptide conjugates have a wide range of applications as labels for various immunoassay detection methodologies, in vitro and in vivo cellular labelling, biosensing considering their high photostability and adjustable photoluminescence properties.336 Rationally designed peptide/protein nanospheres with covalently attached peptides/drugs have the potential to serve as biodegradable biomaterials for therapeutic applications. Hashizume et al. demonstrated that recombinant protein nanoparticles modified by genetic engineering with vascular endothelial cell binding peptide can efficiently target cancer cells.337 PLGA nanoparticples were prepared with a hexamer mitochondria penetrating peptide (Cha-Arg-Cha-Arg-Cha-Arg-NH2) where Cha ¼ cyclohexylalanine. Since the access to mitochondria can be difficult the potential use of this non-toxic nanoconjugate the pharmacological application for cancer and oxidative pathologies was discussed.338 Pozsgay et al. showed, for the first time, that bifunctional PLGA nanoparticle conjugate containing multiple copies of both targeting B-cell epitope peptide (from b chain of fibrin) and the complement activating effector peptide (from HIV1 gp120 protein) on the surface, significantly reduce b60–74Cit peptide specific ex vivo autoantibody level specific for citrullinated proteins production by inducing complement dependent lysis of specific B cells of seropositive RA patients.339 The green luminescent FITC or the near infrared fluorescent dye (MPA) was attached to AuNPs through matriptase enzyme peptide substrate-bridge equipped with C-terminal Cys (GRQSRAGC-amide) for in vitro and in vivo detection of the enzyme expression on tumour cells. The peptide substrate served as the linker between the dye(donor) and AuNP (acceptor). In this configuration, the dye fluorescence was quenched by AuNP under physiological conditions and recovered after selective cleavage of the GRQSRAGC by matriptase.340 Another gold nanosensor was fabricated by using AuNP-peptide conjugate for detecting luteinizing hormone (LH) of sheep. The peptide with Nterminal Cys (CDHPPLPDILFL) was attached in uniform orientation and detected by competitive binding of AuNP-LH conjugate and free LH toward anti-LH polyclonal antibody.341 A novel concept of folate receptor targeting by liposomes, a conjugate of folic acid with oligopeptide (QAAWFSQY) representing a hydrophobic fragment of pulmonary surfactant protein D was designed containing a tetrapeptide spacer (DRDD). The insertion of the peptide conjugate (folic acid – DRDDQAAWFSQY) deeply into the lipid bilayer without affecting liposomal integrity resulted in high stability and specific recognition. This delivery system was more efficient in Caco-2 cells than liposomes having folate integrated by non-peptide, PEG containing moiety.342 3.4.2 Nanostructures based on peptide bioconjugates. Peptide conjugates has been utilized for establishment of nanostructures (e.g. filaments, capsules, nanotubes, nanofibrils, organogels) to be used as Amino Acids, Pept. Proteins, 2018, 42, 85–145 | 127

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theranostics. Self-assembled of pro-drugs with oligopeptide-drug conjugate forming nanofibers have received considerable attentions in recent years.343 Various self-delivery systems of various therapeutic agents were reported: PTX conjugate with glutathione344 or with Tau peptide, AcCGVQIVYKK;345 camptothecin conjugate with various oligopeptides346 including Tau peptide347 or peptide FFYGE-ss-EEE.348 Similar constructs are described using oligopeptide substrate 2-Nap-FFKY(PO3H2) of an alkaline phosphatase enzyme conjugate with cisplatin,349 with gemcitabine conjugated to the N-terminal 4-formylbenzoic acid moiety of peptide GFFYGRGD350 or benzoic acid nitrogen mustard derivative carrying a short amphipathic peptide (AAAK).351 Nakamura et al. have constructed nanostructure by taking an advantage of the self-assembling potential of a synthetic 24-mer b-annulus peptide (INHVGGTGGAIMAPVAVTRQLVQS) of tomato bushy stunt virus capsid protein. Virus-like nanocapsules (artificial viral capsids) of approximately 45–160 nm in size were formed with homopolymeric 20-mer conjugate of oligodeoxyadenosine or oligothymidine with C-terminally attached b-annulus peptide.352 A special group of conjugates have been developed based on the potential of aromatic Phe-Phe dipeptide and its conjugates to form nanostructures as reviewed.353 Recently, Castillo et al. reported on a folic acid conjugate of Phe-Phe dipeptide nanotubes used as coat on the surface of graphene electrode to detect of folate receptor on human cervical cancer cells.354 Due to biostability, D-peptides are emerging as an important set of compounds for biomedical applications.355 A recent study demonstrated that covalently attached taurine via an enzymatic cleavable linker could promote the cellular uptake of D-Phe-D-Phe dipeptides labelled with N-terminal 4-nitro-2,1,3-benzoxadiazole (NBD) fluorophore. The conjugate enters cell, the intracellular esterase removes taurine from the ester, thus the resulted D-peptide conjugate self-assembles.356 This observation could make possible to trigger intracellular self-assembly in mammalian cells providing a new strategy to achieving intracellular molecular selfassembly. Diaferia et al. described a set of conjugates capable to aggregate for diagnostic agents in MRI. These compounds contain three components: di- or tetrapeptide of Phe for promoting the self-assembly, a linear DTPA or branched DOTA bifunctional chelating agent for complexing Gd ion and an inserted linker of di- or hexaethylene glycol units.357 The replacement of Phe with the non-coded amino acid, 2-naphthylalanine (2Nal) resulted in conjugate, whose Gd complex (Gd-DOTA-L6-(2Nal)2) – in contrast to the Phe2 analogue – were able to self-assemble in long fibrillary nanostructures spontaneously in water solution.358 Lock et al. developed an MRI-based theranostic filament hydrogel system that directly uses conjugate of Pem with tetrapeptide FFEE at the N-terminal as both building units and label-free MRI contrast agents. The conjugate can spontaneously form supramolecular filament hydrogels under physiological conditions for injectable delivery into brain tumour sites, serving as both a therapeutic and diagnostic agent.359 128 | Amino Acids, Pept. Proteins, 2018, 42, 85–145

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Nanoparticles, containing chitosane-GRKKRRQRRPPQC (Tat peptide) conjugate with amide linkage and also chitosane-thioglycolic acid conjugate showed synergistic effect in transfection of pDNA gene expressing GFP into the HEK293 cells. The authors concluded that the combination of a CPP with thiomers with different degree of thiolation could provide a promising tool for enhanced non-viral transfection efficiency as well as desired controlled release properties for clinical applications in gene therapy.360 3.5 Future perspectives of bioconjugates Bioconjugate research is a dynamic trans-disciplinary field of science with fast development. During the last years the main focus has been the chemical synthesis and functional characterization of two- or three, even multi-component systems in which the partners including molecules as well as nanostructures are attached by covalent bond and preserve relevant functional properties (e.g. biological response profile, ‘‘reporter properties’’) after conjugation. The main field of application is still in biomedical sciences covering biomaterial research. Bioconjugates are important molecular tools for the analysis of interaction based phenomena including cell membrane and intracellular structure (e.g. mitochondria) binding. Also these constructs are markedly present in the prophylaxis, early diagnosis, treatment and in therapy-monitoring of various diseases mostly cancer, infectious disease, but more recently metabolic diseases. The novel challenge is the design, preparation and reliable structural and functional characterization of conjugates combining components suitable for both diagnostic and therapeutic. Future studies, markedly in the light of personalized medication, will provide realistic analysis regarding this concept for simultaneous delivering therapy and monitoring the pharmaceutical effect. There is hope that the reader, by consulting this chapter, perhaps together with the previous one from volume 37 in 2012,361 will have a chance to sense the trends, successes and failures in the field, which requires analytical as well as synthetic thinking and could also provide enjoyment and generate enthusiasm.

Abbreviations 2Nal AD AD-2 Ahx AMP Aoa Aph(Cbm) Aph(Hor) ApoPep-1 azF

2-naphthylalanine Alzheimer’s disease N-terminus of glycoprotein B 6-aminohexanoic acid antimicrobial peptide aminooxyacetic acid 4-(aminocarbonyl)amino-phenylalanine 4-(4S)-hexahydro-2,6-dioxo-4-pyrimidinylcarbonylamino-L-phenylalanine Apoptosis-targetingpeptide-1 p-azido-L-phenylalanine Amino Acids, Pept. Proteins, 2018, 42, 85–145 | 129

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AZT B[a]P BB BCN CAMP CMD Cpa CPP CPT CT CuAAC DFX DIBAC Dmt DOPA DOTA DOX DTPA EGF FITC FRET GFP GLP-1 GnRH GPCR GRP GRPr HA Her2 HR HYNIC ILys KAHA ligation LH LplA LVFX mAb MMP-2 Mox MPAA MRI a-MSH MTG MTX Nap NCL NIA

3-azido-2,3-dideoxythymidine benzo[a]pyrene bombesin bicyclo[6.1.0]non-4yne cationic antimicrobial peptide carboxymethyl dextran 4-chlorophenylalanine cell-penetrating peptide camptothecin computed tomography Cu(I)-catalyzed azide-alkyne cycloaddition deferasirox dibenzo-aza-cyclooctyne 2 0 ,6 0 -dimethyl-L-tyrosine 3,4-dihydroxy-L-phenylalanine 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid doxorubicin diethylenetriaminepentaacetic acid epidermal growth factor fluorescein isothiocyanate ¨ster resonance energy transfer Fro green fluorescent protein glucagon-like peptide-1 gonadotropin-releasing hormone G protein-coupled receptor gastrin-releasing peptide gastrin releasing peptide receptor hyaluronic acid human epidermal growth factor receptor 2 heptad repeat hydrazinonicotinamide N6-isopropyl-L-Lys a-ketoacid–hydroxylamine amide ligation luteinizing hormone lipoic acid ligase levofloxacin monoclonal antibody matrix metalloproteinase-2 methoxinine 4-mercaptophenylacetic acid Magnetic Resonance Imaging a-melanocyte-stimulating hormone microbial transglutaminases methotrexate 2-naphthaleneacetic acid native chemical ligation 2-(2-nitroimidazol-1-yl)acetic acid

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NMU NODAGA NOTA NPs NRP-1 NT ON Oxima Pal PATA Pem PET PLGA PNA POCs PSD-95 PTX QDs rFliC SAR SEAlide peptide SPAAC SPECT SPR SrtA SST TBP Tle TMZ TNF Ttds

neuromedin peptide 1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid 1,4,7-triazacyclononane-N,N 0 ,N00 -triacetic acid nanoparticles neurophilin 1 neurotensin oligonucleotide ethyl 2-cyano-2-(hydroxyimino)acetate 3-(3-pyridyl)-alanine p-(N-propynoylamino)toluic acid pemetrexed positron emission tomography poly(lactide-co-glycolide) peptide nucleic acid peptide–oligonucleotide conjugates postsynaptic density protein 95 paclitaxel quantum dots Salmonella flagellin recombinant protein structure-activity relationship N-sulfanylethylanilide peptide strain promoted azide-alkyne cycloaddition single photon emission computed tomography surface plasmon resonance sortase A enzyme from Staphylococcus aureus somatostatin titanium binding peptide L-tert-leucine, 2-amino-3,3-dimethylbutyric acid temozolomide tumor necrosis factor 1-amino-4,7,10-trioxa-13-tridecanamine succinic acid

Acknowledgements Supported by grant from Hungarian Research Fund (OTKA K 104928). Special thanks are due to Dr N. Mihala for helping to prepare the illustrations.

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Magnetic resonance studies of antimicrobial peptides in membranes Marc-Philipp Pfeila,b and Anthony Watts*a Published on 29 November 2017 on http://pubs.rsc.org | doi:10.1039/9781788010627-00146

DOI: 10.1039/9781788010627-00146

The development of microbial resistance to common small molecule antibiotics is continuing to be a thread to modern medicine and drives the search for novel antibiotics for the clinic. Antimicrobial peptides (AMPs), part of the innate immune system, are versatile and show multifaceted antimicrobial activity that has attracted recent focus as only limited cases of resistance are reported to date. AMPs primarily develop their potency at the pathogenic membrane, where they adapt different modes of action that compromise membrane integrity and result in cell death. While the primary peptide structure and overall biophysical characteristics are important for AMP activity, a precise structure–function relationship is still unresolved. The destructive membrane-activity and large conformational space exploited by AMPs is a challenge for biophysical characterisation and typically a battery of different techniques is required to study precise AMP activity. Magnetic resonance methods are outlined and reviewed that span the relevant time scales of the highly dynamic peptide–membrane interaction in a native-like environment. An overview of electron paramagnetic resonance (EPR) and uniaxially oriented nuclear magnetic resonance (NMR) methodologies is presented, which in combination resolve the peptide–lipid interaction in detail; understanding of the bilateral relationship of lipids and peptides is essential for the precise characterisation of native and synthetic AMP sequences.

1

Introduction: peptide–lipid interactions

Peptides fulfil a range of different purposes in biology: they are involved in cellular signaling, form part of large assemblies on membranes, can act as bilayer-disrupting agents or pass through a membrane. In the search for novel antibiotics, a particular interest has been focused on antimicrobial peptides (AMPs), also known as ‘‘host defence peptides’’, that form a class of membrane-lytic peptides constituting part of the innate immune system.1–4 Most AMPs are thought to act by compromising membrane integrity through distinct mechanisms that eventually cause cell lysis. The first contact of AMPs with a biological membrane is most often mediated by electrostatic interactions. Most evident is the charge-driven interaction between the anionic lipid headgroups and the cationic residues of the peptide. AMPs displace the divalent cations found in the LPS layer of gramnegative bacteria, destabilising the outer membrane.5 Similarly, grampositive bacteria that lack an outer membrane contain additional negatively charged teichoic and teichuronic acid adding to AMP interactions.6 The large electrochemical gradient across a bacterial membrane further drives the AMP–pathogen interaction. Early studies showed an a

Department of Biochemistry, University of Oxford, Oxford OX1 3QU, UK. E-mail: [email protected] b Department of Micro- and Immunobiology, Harvard Medical School, Boston, MA 02115, USA 146 | Amino Acids, Pept. Proteins, 2018, 42, 146–189  c

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increased membrane activity correlating to a larger electrochemical gradient.7 Initial membrane contact and the resulting interfacial electrostatic peptide interaction, triggers folding of the peptide, which is unstructured in solution.8 This action is thought to be followed by one of three distinct mechanisms that disrupt membrane integrity (Fig. 1): by forming a barrelstave pore (A), a toroidal pore (B) or a less organised carpet mechanism (C). Peptide–lipid interactions are important in order to understand AMPs mechanistically and establish molecular structure–function relationships. However, peptide–lipid interactions observed on pathogens are complex. Hence, the molecular mechanisms of AMP action is often described using simplified models. Peptide–lipid interactions represent a bilateral relationship where lipids and peptides impact each other, each of equal importance to the holistic mechanism. Lipids can cause changes of

Fig. 1 Schematic illustration of the most common modes of AMP action. AMPs tend to be unstructured in solution and adopt secondary structural elements upon membrane interaction, most commonly folding into their characteristic amphipathic arrangement (special separation of hydrophobic and hydrophilic phases are illustrated by different shades). The membrane selectivity of AMPs is mainly dependent on an interplay between the overall charge and hydrophobicity. Commonly AMPs are thought to compromise membrane integrity by one of three ways: (A) barrel-stave pore, (B) toroidal pore or (C) a less organised carpet mechanisms. All depicted mechanisms induce membrane lesion. Amino Acids, Pept. Proteins, 2018, 42, 146–189 | 147

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protein backbone topology, oligomerisation, rotational and tilt angles of transmembrane domains as well as changes of side chain orientations of domains or individual segments (Fig. 2).9 Lipid-interaction can promote selective 2D distribution of peptides facilitating different quaternary

Fig. 2 Possible peptide and lipid adaptations to both positive and negative hydrophobic mismatch. (A) A hydrophobic peptide, represented by a cylinder, is embedded into a schematic bilayer with positive hydrophobic mismatch. Part I: Peptide adaptations to bilayers with positive hydrophobic mismatch: (B) Peptide tilting to accommodate the entire hydrophobic mismatch to the hydrophobic membrane core; (C) Peptide oligomerisation to minimise the overall exposed hydrophobic region; (D) Changes in peptide backbone configuration to adapt to the bilayer thickness; (E) Changes in selective side chain orientations and conformations to adapt to the hydrophobic mismatch. Part II: Lipid adaptations to hydrophobic mismatch: (F) Lateral phase separation of a bilayer of mixed lipids due peptide interactions with lipids of best hydrophobic thickness match; (G) Lipids can stretch to cause a local bilayer thickening to accommodate the peptide; (H) In a negative hydrophobic mismatch scenario lipid acyl chains can become disordered, and in extreme cases forming non-lamellar lipid phases e.g. cubic phase.9 148 | Amino Acids, Pept. Proteins, 2018, 42, 146–189

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10

peptide structures (e.g. PG-1 ). On the other hand peptides can impact bilayer thickness and lateral lipid organisation or facilitate lipid movement across leaflets.9,11,12 Complex proteins or oligomerised peptides with multiple transmembrane domains, can show multifaceted lipid interactions and thus are highly complex and still poorly understood. Despite some alternative targets, most AMPs possess a certain degree of membrane activity and primarily act by causing membrane lesion. The structural properties of AMPs relate to their mode of action and mode of membrane insertion, yet a precise structure–function relationship for AMPs is mostly unresolved. Here the importance of magnetic resonance studies in understanding peptide-bilayer systems in the liquid crystalline phase is discussed illustrating examples that advanced the understanding of the underlying biophysical principles of AMPs. It has become increasingly evident that the action of AMPs depends on the structural elements of inherent charge, peptide conformation, polar angle, relative hydrophobicity and amphipathicity (summarised in Table 1). It is the interplay between these factors, which are dictated by the precise amino acid sequence, that determines the fate of AMP function.

1.1 Magnetic resonance studies of AMPs The complex nature of peptide–lipid interactions is accessible to a range of biophysical techniques. Magnetic resonance studies have emerged as a suitable tool to investigate peptide–lipid systems as they span the relevant timescales associated with dynamic peptides associated with a membrane and thus report on molecular structure and order (Fig. 3). For example, the impact of hydrophobic mismatch (Fig. 2), an imperfect alignment of the membrane hydrophobic core and the hydrophobic transmembrane domain, on both lipid and protein organisation has been extensively studied using the model peptide WALP (a simple peptide a repeated alanine and leucine cluster;12). The alanine rich transmembrane cluster is ideal for the characterisation by deuterium (2H) NMR (see geometric analysis of multiple labelled alanines (GALA);13,14 Section 3.2), resolving conformational peptide features such as tilt and rotational pitch.15,16 2H NMR studies of acyl chain deuterated phospholipids showed a slight but consistent acyl chain ordering upon WALP interaction suggesting local lipid stretching. Nevertheless a net overall thickening of the membrane could not be confirmed.12,17 Gramicidin, an AMP from bacillus brevis, has been reported to increase or decrease bilayer thickness in cases of positive and negative hydrophobic mismatch respectively.18 In this case the precise peptide–lipid interaction depends on the nature of the membrane of interest. In addition to hydrophobic mismatch, electrostatic interactions can lead to lipid segregation and cause global reorganisation. Cardiotoxin II, a cationic peptide, was previously shown to segregate anionic from zwitterionic lipids. Changes in the quadrupolar splitting of headgroup deuterated POPC (2H NMR) indicated altered electrostatics at the interface.19 Similar changes of obscured electrostatics were observed in the Amino Acids, Pept. Proteins, 2018, 42, 146–189 | 149

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Table 1 Summary of key biophysical properties considered for antimicrobial activity of AMPs. The precise mode of action of an individual AMP depends on the interplay of all or a subset of these features and tends to be complex.134 Biophysical characteristics of AMPs 1. Conformation

Despite the vast sequence variety of AMPs most peptides display an alpha helical or beta sheet secondary structure. Mixed alpha helical and beta sheet secondary structure (defensin) or non-structured (indolicidins) AMPs have also been reported. Alpha helical peptides most commonly exist in unstructured conformations in solution and adopt their secondary structure upon membrane insertion.

2. Charge

Most AMPs possess an intrinsic net positive charge ranging from þ2 – þ9. The charged residues often form defined patches and are thought to be critical for an interaction with the anionic membrane of most microorganisms. The membrane electrostatics differ between mammalian and pathogenic bacterial membranes and electrostatic selectivity is proposed (Fig. 1).

3. Amphipathicity

An alpha helix is normally based on 3.6 residues per turn and thus forms an optimal cluster for residue segregation in periodicity of 3–4. Beta sheet amphipathicity also exists with alternating strands showing different net characteristics. Overall amphipathicity is typically measured by the hydrophobic moment, which is important for interaction with zwitterionic membranes and thus hemolysis.

4. Hydrophobicity

Most AMPs have a relative hydrophobicity of ca. 50%. Hydrophobicity is important for membrane activity yet it can result in a high degree of cytotoxicity and an interplay with positive charged residues is important to achieve selectivity, which depends on the exact composition of the targeted pathogenic membrane.

5. Polar angle

The polar angle is a relative measurement of polar and non-polar segregation around an alpha helix. If one half of the cylinder-like helical surface is composed of polar residues and the other half of non-polar residue then the polar angle is 1801. It is thought that a smaller angle corresponds to greater membrane permeabilising abilities resulting in readily formed pore structures. However, the stability of such pores is also suggested to be low compared to pores formed by peptides with a larger polar angle. For example the polar angle of PGLa is 1001 and has a permeabilising potential then magainin 2 with a polar angle of 1801.

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Fig. 3 Timescale of different peptide/protein and lipid motions commonly observed membrane assemblies. Note that the timescales are an indication, actual time scales depend on the molecular characteristics of an individual membrane. The distinct dynamical profile of each individual process requires matched experimental timescales for structural investigation. The EPR experiments described here are especially suited to processes ranging from 108–109 s of lipid mobility within a model bilayer;135 whereas NMR experiments tend to be on a slower timescale of ca. 106 s; together allowing experimental accessibility for investigation of various dynamical membrane-associated processes.

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membrane interaction of melittin, a peptide found in bee venom, and showed altered macroscopic lipid phases.20 Headgroups of phosphatidylcholine and phosphatidylserine adapt their conformation upon melittin interactions most likely due to changes in the overall surface charge upon cationic peptide binding as illustrated by 2H and 31P NMR.21 Destructive membrane active peptides sample a large conformational space and thus a battery of biophysical methods is often required to describe the complete nature of precise peptide mechanistic.22,23 Nevertheless, the focus here is on the structural and dynamic peptide– lipid interactions as elucidated by magnetic resonance, which are complimentary to results obtained by other (often static) methods like atomic force or confocal microscopy: (i) Electron paramagnetic resonance (EPR) has emerged as a powerful technique to study precise lipid dynamics at different depths of the hydrophobic core, exploiting nitroxide radicals selectively placed at segments of the acyl chain.24 Further to this EPR has been used to investigate lipid flip–flop, peptide tilt, peptide structure and peptide oligomeriastion including stoichiometric orientation.25,26 The high sensitivity of EPR methods and small sample sizes required is an advantage. (ii) Oriented nuclear magnetic resonance (NMR) studies are highly complimentary to EPR investigations probing the peptide system of interest on a slower timescale. Besides important information on dynamics, oriented NMR methods exploit the orientational selectivity of NMR interactions and directly report on peptide orientation without the need for non-native spin-labels.

2

Electron paramagnetic resonance (EPR)

EPR, also known as electron spin resonance (ESR), is a technique that exploits the high sensitivity of unpaired electron spins. However, being a probe method, unlike for nuclear spins, site-directed spin-labels need to be incorporated at specific sites in the system of interest, mostly in the form of nitroxide radicals (an exception are non-silent metals, such as, for example in some haem groups). EPR studies on membranes use nitroxide spin-labels incorporated to the lipid acyl chain or headgroup region as paramagnetically active probes. Continuous wave EPR (cwEPR), where a continuous wave microwave radiation is applied under a varying magnetic field, allows for the detection of dynamics via anisotropy averaging of the electron–nitrogen hyperfine interaction (BGHz), on the timescale of ns at the site of the spin label. Pulsed EPR techniques apply short (ns) pulses that can, for instance, resolve dipolar couplings between two individual electron spins. Thus, pulsed doubleelectron–electron-resonance (DEER) resolves the distance dependence of dipolar couplings of up to 6 nm for membrane proteins, and hence is a powerful technique to measure precise distances between localised spins.27 152 | Amino Acids, Pept. Proteins, 2018, 42, 146–189

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2.1 Continuous-wave EPR on spin-labelled lipids Membranes are macromolecular assemblies of lipids, integral and peripheral proteins (often glycosylated), as well as peptides. The dynamic range of molecular and macromolecular motions in a membrane spans a wide range of timescales, from fast motions of specific acyl chain segments on the timescale of ps-ns, to more global reorganization events on slower (ms-s) timescales. The cwEPR timescale (ns-ms) is well matched to the rotational and vibrational motions of lipids contained in a membrane, as well as side chain and backbone motions of proteins and peptides. Thus, alterations of lipid dynamics upon peptide interaction, as well as lateral dynamic segregation of distinct membrane environments become evident in the EPR spectrum. The EPR spectrum of a typical nitroxide radical reflects three energy transitions at distinct magnetic field strengths (and irradiation by a constant microwave frequency) due to the hyperfine interaction of the free electron. The hyperfine interaction arises due to a partial localisation of the unpaired electron in the pz orbital of the 14N nucleus with spin I ¼ 1, which results in three hyperfine energy splittings of the þ1/2 and 1/2 Zeeman energy splittings respectively (spin states m ¼ 1, 0 and 1; Fig. 4). Under a constant application of microwave frequency and a magnetic field sweep, each transition becomes possible at the corresponding magnetic field strength. Due to the quantum mechanical selection rules this results in three possible energy transitions, represented by the low-, central- and high field lines (Fig. 4B). Depending on the application, nitroxide spin labels are attached at various segments along the acyl-chain of different phospholipids or stearic acid in order to probe order profiles at selected membrane crosssections (Fig. 5). For fully transmembrane peptides and proteins a spin

Fig. 4 Basic underlying EPR principles of a nitroxide spin. (A) The characteristic energy landscape of a nitroxide spin resulting from the Zeeman splitting of the I ¼ 1/2 of an electron and the hyperfine interaction with the local 14N with I ¼ 1. (B) A typical cwEPR spectrum of 1st derivative of the absorption spectrum is shown, illustrating the three energy transitions during a magnetic field sweep at a constant microwave frequency, corresponding to the low- , central- , and high-field lines. Amino Acids, Pept. Proteins, 2018, 42, 146–189 | 153

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154 | Amino Acids, Pept. Proteins, 2018, 42, 146–189 Fig. 5 Common spin-labelled lipids used for EPR studies. The nitroxide radical is selectively placed at segment five (C-n) for representation purposes. It can however be chemically placed at any segment of the acyl chain. 5-SASL: n-(4,4-dimethyloxazolidinyl-N-oxyl)stearic acid; 5-PCSL: 1-acyl-2-[n-(4,4-dimethyloxazolidinyl-N-oxyl)stearoyl]-sn-glycero-3-phosphocholine; 5-PESL: 1-acyl-2-[n-(4,4-dimethyloxazolidinyl-N-oxyl)stearoyl]-sn-glycero-3-phosphoethanolamine; 5-PGSL: 1-acyl-2-[n-(4,4-dimethyloxazolidinyl-N-oxyl)stearoyl]-sn-glycero-3-phosphoglycerol; 5-PSSL: 1-acyl-2-[n-(4,4-dimethyloxazolidinyl-N-oxyl)stearoyl]phosphoserine; 5-PISL: 1-acyl-2-[n-(4,4-dimethyloxazolidinyl-N-oxyl)stearoyl]-phospho-sn-1-myo-inisotol; 5-CLSL:1-(3-sn-phosphatidyl)-3-[1-acyl-2—[n-(4,4dimethyloxazolidinyl-N-oxyl)stearoyl]-sn-glycero-3-phospho]-sn-glycerol; 5-SMSL: N-[n-(4,4-dimethyloxazolidinyl-N-oxyl)stearoyl]-spingosine-1-phosphocholine.

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label in the lower part of the chain (segment 14–16 seems optimal) allows for the detection of two-component spectra representing peptide associated and bulk lipids respectively.28 Restricted lipid motion due to peptide interactions may give rise to a second broadened spectral component within the composite EPR spectrum,12,29 if the peptide complex is large enough (ca.4four monomers) and is diffusing sufficiently slow compared with lipid diffusion on the regime of ns. A second broad spectral component can also be an indication for domain or raft formation on the EPR timescale.30 Such a composite spectrum can be deconvoluted by spectral subtraction using a recorded library of spin labelled lipids at various temperatures or alternatively using spectral simulation. The relative contributing proportions are quantified according to the integral of the absorption spectrum. Using this methodology, the associated number of lipids with a peptide or protein can be estimated, which under certain assumptions can be related to the overall circumference of the aggregate and thus an ensemble oligomeric state can be approximated.31,32 To implement spectral subtraction recorded single-component spectra of a library of the same spin-labelled lipid in the absence of any peptide, which represent the motional profile of the bulk lipids is used. The EPR single-component spectra are recorded at a temperature range with a narrow step size to closely match the motion profile of the bulk lipids in the presence of peptide, which can differ due altered biophysical membrane characteristics like additional lateral pressure. The complementary slow mobility component of the peptide interacting lipid can be obtained from small unilamellar vesicles (SUVs) of a lipid, for example dimyristoylphosphocholine (DMPC) recorded in the gel phase below the phase transition. The single component spectra are recorded at a temperature range in order to find a good match of the motional profile of peptide associated lipids. For some samples it can be advantageous to sonicate the lipid dispersion prior to measurement, which results in an increased linewidth and depending on the sample of interest might match the lineshape more closely for both the immobile and mobile components respectively. Spectra are normalized to the integral of the absorption spectrum prior deconvolution. Following spectral subtraction the resulting difference component is compared to the complementary single component spectrum for a good match. Subtraction is an iterative process in order to chose the most closely matching single-component spectra and relative normalized proportions.28 The respective spectral proportions ( f ) are obtained by comparison of the integral of the absorption spectrum of the respective single component deconvoluted spectra to the composite spectrum. Alternatively the composite spectrum can be simulated using computational approaches (e.g. EasySpin33), where typically each individual component is represented by a nitroxide spin described by a set of parameters. Different iterative fitting algorithms are implemented in order to closely match the line shape of the experimental spectrum. Similar to the library approach, the relative proportions ( f ) are reported Amino Acids, Pept. Proteins, 2018, 42, 146–189 | 155

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according to the relative proportion of the integral of the absorption spectrum (Fig. 6). Relative spin-label correlation times at the local site can be approximated. Determining the fraction of restricted lipids ( f ) by spectral deconvolution allows for quantification of lipid kinetics and annular lipid binding sites (eqn (1); Fig. 6B). The final protein to lipid ratio (Nt) in the measured sample can be experimentally determined and the motionally restricted proportion ( f ) can then be related to an absolute number of lipid association sites at the transmembrane interface.28,34,35   1f 1 Nt ¼ 1 ; (1) f K r Nb where Kr relates to the association constant of the spin-labelled lipid within the sample when compared to the non-labelled lipids. Kr ¼ 1 if the relative association of nitroxide labelled lipids and non-labelled lipids is identical. Previous studies show no difference (i.e. Kr ¼ 1) in samples containing the same background lipids as the spin-labelled lipid species (e.g. DMPC and nitroxide-DMPC), and thus the presence of a spin-label does not seem to alter the membrane thermodynamics within the sample.28

Fig. 6 Spectral deconvolution by simulation using EasySpin. (A) The experimental twocomponent spectrum illustrating the slow and the fast mobility components (left); the experimental spectrum is overlaid with the individual deconvoluted slow and fast mobility components (middle); the experimental and simulated composite spectra are overlaid (right). (B) Schematic illustration of the annular lipid shell corresponding to the slow mobility EPR component and the bulk lipid corresponding to the fast mobility EPR component in a hypothetical peptide containing membrane (all shown as a top view onto a membrane). 156 | Amino Acids, Pept. Proteins, 2018, 42, 146–189

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Similarly, lipid selectivity for peptide–lipid interactions localized to the annular lipid shell of a peptide ((P); Fig. 6B) can be measured based on the exchange equilibrium of selectively labelled lipids (L*) with nonlabelled lipids (L) at the annular lipid shell with a set number of binding sites (Nb).36 The equilibrium association is described by: P  LNb þ L*

Kr

! P  LNb1 L* þ L

Where the association constant Kr is described by: Kr ¼

½L*P ½L ½LP ½L*

Kr in turn can be related to the free energy of association of a specific lipid type (DGass(L*)): DDGass ¼ DGass(L*)  DGass(L) ¼ kBln(Kr) where T and kb describe the absolute temperature and the Boltzmann constant respectively. Free energies of association are commonly reported as a relative measure compared to PC-lipids (an example of differential lipid associations and associate free energies of associations is given for the potassium channel KcsA, Table 237) and in light of cationic AMP function can shine light on preferred lipid interactions that can lead to lateral lipid phase separation based on preferred charge interactions, which can drive AMP–lipid interactions and membrane destabilisation. It is noteworthy that bacterial membranes tend to be enriched in anionic lipids like PG or teichoic acid fostering preferred cationic peptide interactions.6 Such EPR studies have been used extensively to characterize the AMPs interacting with model membranes and give valuable information on preferred lipid interactions and the overall scope of the annular lipid shell. In the case for gramicidin it was found that this corresponds to 3.6 (0.3) lipids per peptide.31 Using different spin labelled lipids and analysis of the relative proportion of immobilized lipids selective lipid binding preferences as well as relative association constants can be characterized.35 The 27-amino acid IsK-channel associated peptide K27 was found to associate with 2.2 lipids per peptide in its beta sheet conformation. The study further identified a deletion mutation that strongly alters the specificity for probed negatively charged lipids found in the Table 2 Relative lipid affinities to KcsA described by the association constant Kb and a free energy of association.37 Lipid

Kb(mole fraction)

DGassNA/kbT

CL PA PS PG

7.3  1.2 4.6  2.1 7.1  2.7 3.0  0.7

 2.0  0.2  1.5  0.5  2.0  0.4  1.1  0.2

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annular peptide shell which might have implementations for antimicrobial activity.32 A more detailed review on the interpretation of lipid stoichiometry and lipid kinetics is given elsewhere.28 2.2 Amplitude of segmental acyl chain motion The structural effects on lipid dynamics of peptide–membrane interactions are investigated using selectively spin-labelled lipids at different acyl chain positions, which act as specific probes for lipid perturbations at different regions of the bilayer. Lipid acyl chains undergo high frequency segmental side-to-side motions; and lipids as a whole undergo rotational diffusion around its long axis. Both types of motions are fast (to109 s). For dynamics in this time regime two anisotropic interactions can be directly measured from the EPR spectrum: A8 and A> (Fig. 7). These parameters effectively describe the angular motions of the spin label at the local site and it follows that an order parameter (Seff) can be defined in the most basic description as A8  A>. It should be noted that for EPR studies the precise lipid

Fig. 7 The EPR lineshapes of spin labelled DPPC at different acyl chain segments. The nitroxide anisotropy becomes averaged at the more dynamic lower acyl chain segments leading to narrow lines (12 – DPPC). The hyperfine splittings, A8 and A>, used to describe the segmental amplitude of motion are illustrated (Figure taken from ref. 136). 158 | Amino Acids, Pept. Proteins, 2018, 42, 146–189

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morphology is not important as vesicles of small and large sizes tumble slow on the EPR timescale (B108 s). The hyperfine splittings, A8 and A> of the nitroxide spin-labelled stearic acid or phospholipid relate to the anisotropic averaging of the spin label motion at its segmental position of the acyl chain, and thus the order parameter (Seff) can be related to S ¼ 12 ð3 cos2 y  1Þ, where y is the amplitude of chain motion at the label position, assuming a fast motional rate in fluid bilayers (tc1 4B108 s).38 Seff is a measure of the experimentally determined spectral anisotropy and can be calculated according to:24 Seff ¼

Ak  A? a0  0; 1 Azz  2 ðAxx þ Ayy Þ a0

(2)

with line positions A8 and A> shown in Fig. 7; a00 and a0, the polarity parameters are given by: a00 ¼

1  ðAzz þ Ayy þ Axx Þ 3

(3)

1 ðAk þ 2  A? Þ 3

(4)

a0 ¼

The principal hyperfine splittings Azz, Ayy and Axx for the spin label were previously characterised in oriented crystals, with principle hyperfine couplings found to be 5.9, 5.4 and 32.9 gauss.39 The polarity normalization term of a00 = a0 is introduced in the calculations to compensate for the different polarities at the specific membrane environments of the spin label.40 The amplitude of the segmental acyl chain motion has a dynamical gradient along the acyl chain of a given lipid species, with segments located close to the head group being more rigid compared to segments buried in the hydrophobic core i.e. the amplitude of motion at the hydrophobic core is larger compared to regions close to the interface region. This leads to averaged EPR spectra for nitroxide spin labels located at the hydrophobic core and slow mobility spectra for nitroxides close to the interface and headgroup region (Fig. 7). The molecular order changes due to AMP activity at selected acyl chain segments can be closely monitored using EPR order profiles. Further to this it becomes possible to investigate localized effects through the crosssection, along the bilayer normal, of a membrane and thus give information on the preferred location of antimicrobial activity (e.g. the interface region). Expected local order parameter changes for different schematic peptide insertion modes are illustrated in Fig. 8. It should however be noted that similar to the depicted full transmembrane peptide configurations, aggregation states within individual leaflets can also be monitored and as recently demonstrated can be sufficient to destabilize a pathogenic membrane.22 Amino Acids, Pept. Proteins, 2018, 42, 146–189 | 159

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Fig. 8 Geometric schematic of peptide–membrane interactions and the expected changes in local lipid order. Increased order is shown by straight acyl chains and decreased order by highly curved acyl chains. Note that net order of systems (A) and (B) remains unchanged; nonetheless composite EPR spectra for system B might be observed. (C), (D) and (E) represent systems with a net change of the system order. Note that these are individual scenarios and it is highly likely that different modes coexist in biological scenarios making unambiguous analysis of order profiles difficult.36 Note that equally to the here depicted transmembrane motives single leaflet motives can also exist, which indeed were recently shown to exist and cause antimicrobial activity by disruption of a single leaflet.22

Changes in lipid dynamics have been observed for various peptides once interacting with a lipid bilayer. Magainin led to increased lipid order upon membrane interaction.41 Similarly the peptide lipo-cyclic-gAApeptide induced reduced spin label motion, which was observed most prominently in the lipid headgroup region reported by 5-SASL indicated by a broadened EPR lineshape upon peptide interaction.42 Lateral lipid ordering was then confirmed by the high frequency EPR line shape (W band).42 The reduced dynamics are hypothesised to be due to an increased lateral pressure upon peptide binding. Reduced lipid dynamics have also been reported for other AMPs like cecropins, CM15 and Gramacidin.43–45 Peptide insertion most probably leads to increased lateral pressure accompanied with membrane thinning and reduced segmental acyl chain motions as part of the antimicrobial process. Information on lipid order profiles on the nanosecond timescale, together with information on global lipid phase organization is highly complementary to NMR derived lipid order profiles on the slower microsecond timescale and thus can aid the holistic understanding of dynamic lipid behaviour. It should be noted that the general bilayer fingerprint of the dynamic behaviour differs slightly between crosssectional order profiles derived by EPR and NMR, these differences mainly arise in the ‘‘plateau region’’ (segments closely located to the membrane interface region) and may be attributed to differences in the experimental timescales. cwEPR studies are typically performed in fully hydrated vesicles systems whereas NMR order profiles are most commonly derived from oriented samples supported by glass slides at near complete hydration. It should be noted that NMR order profiles can also be derived from fully hydrated vesicle systems using post experimental 160 | Amino Acids, Pept. Proteins, 2018, 42, 146–189

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processing by dePaking (an example of dePaking by a weighted Fourier Transformation can be found here46). 2.3 Spin label correlation times In addition to the angular motions reported by A8 and A>, the EPR spectrum also provides information on absolute correlation times of the spin label as reported by the central linewidth (H0). The central linewidth is a peak-to-peak measure of the central energy transition reported in the first derivative of the absorption spectrum. Unlike the hyperfine splitting of the spectrum, the central linewidth reports on the frequency of the segmental acyl chain motion and thus reports on relative correlation times rather then overall amplitude of segmental motions.47 A spectral broadening that indicates restricted spin label motion typically results in an increased central linewidth. The nitroxide spin-label rotational correlation time can be related to line widths and intensities: sffiffiffiffiffiffiffiffiffiffiffiffiffi ! hð0Þ 10 1 ; (5) tr ¼ 6:5  10 DH0  hð1Þ where H0 is the peak-to-peak width of the central line and h(0) and h(1) are the heights of the lines at the central and high field transition respectively.47 2.4 EPR on spin-labelled peptides In most cases peptides do not contain paramagnetic centres and thus these have to be chemically introduced to the system of interest. For peptide systems paramagnetic spin labels are commonly introduced by chemical synthesis or by site directed spin labelling (SDSL). The development of 2,2,6,6-tetramethyl-piperdine-1-oxyl-4-amino-4carboxylic acid (TOAC, Fig. 9), which can be incorporated into peptide sequences using both solution or solid-phase synthesis, has been instrumental to the study of peptides by EPR. The direct spiro linkage of the nitroxide to the Ca results in small rotamer populations and thus comparably narrow lines when investigated by EPR.48,49 The small rotamer population is also beneficial for investigations of dynamical aspects of peptides by cwEPR as only limited additional dynamics are introduced by otherwise extended side chains (e.g. as for site directed spin labelling). The common alternative is site directed mutagenesis and spin labelling using primary amine reactive (1-oxyl-2,2,5,5-tetramethylpyrroline-3carboxylate N-hydroxysuccinimide ester) or thiol reactive nitroxide spin labels (S-(1-oxyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-yl)methyl methanesulfonothioate (MTSL, Fig. 9)), both containing a linker between the nitroxide and Ca. Site directed spin labelling has been recently reviewed elsewhere.50 2.4.1 Continuous wave EPR of paramagentically labelled peptides. TOAC spin-labelled peptides are useful to investigate peptide aggregation states both in the fluid and gel phase. Aggregates of spin-labelled Amino Acids, Pept. Proteins, 2018, 42, 146–189 | 161

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Fig. 9 Commonly used nitroxide spin-labels and peptide orientations in a membrane. (A) The geometric orientations of a TOAC labelled peptide that are used for orientational analysis of cwEPR spectra in aligned membrane systems. (B) TOAC. (C) MTSL.

peptides are expected to give rise to spin–spin interaction that are reported in the EPR spectrum. Spin–spin interactions are observed as an underlying component that leads to line broadening and a distorted baseline in the EPR spectrum. Heisenberg exchange is the phenomenon of spin exchange of close unpaired electrons via partial orbital overlap. The upper distance limitation for Heisenberg exchange is 1.5 nm in most solids, however this can be larger for highly delocalised spin systems. In liquid samples, Heisenberg exchange is strongly dependent on paramagnetic centre collisions that cause strong orbital overlap for a limited amount of time. Heisenberg coupling is a combination of isotropic and anisotropic contributions, that are described by the exchange coupling tensor (J):51 ˆ ¼ S 1  J  S 2, H

(6)

where S1 and S2 are the individual spins. Note that additionally (to Heisenberg exchange) dipolar broadening due to spin interactions can add to the observed spectral broadening. Alamethicin is a 19 amino acid long peptide that forms conduction channels in a bilayer with various conducting states being reported, which are most likely correlated to the number of peptides forming part of the aggregation state. Indeed spin–spin interactions were observed for TOAC-labelled alamethicin in membranes below the phase transition temperature suggesting a close interaction; the spectral broadening was however lost once the membrane went through the phase transition, indicating a more dispersed peptide distribution in a fluid bilayer.52 Similar observations were made for alamethicin containing a more flexible spin label at either the N- or C- terminus.53,54 162 | Amino Acids, Pept. Proteins, 2018, 42, 146–189

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The a0 isotropic hyperfine coupling (described by eqn (4)) is independent of local motions and reports on the local polarity of the spin label environment.55,56 The polarity gives valuable insight into the location of the spin label when interacting with a membrane, and if multiple labels along the helical axis sites are available, can be used in order to investigate potential transmembrane vs. interfacial peptide locations, which in turn allows to differentiate between distinct antimicrobial modes of actions (Fig. 1). Indeed, TOAC labels at residues 16 and 19 of alamethicin show low values for a0 and thus are located in a more hydrophobic environment (i.e. buried deeper into the hydrophobic core).57 It should be noted that TOAC located at the interface region will still result in a lower a0 value when compared to TOAC in water and thus a membrane association can still be reported for such a mode of membrane activity, which for example was reported for the surface localized GV II peptide.58 Analogous to the order parameters for spin labelled lipids (Seff) an order parameter can be obtained for TOAC spin-labelled peptides: Szz ¼

Axx þ Ayy þ Azz Azz  A?  1 Ak  2 ðAxx  Ayy Þ 3a0

(7)

Axx þ Ayy þ Azz is a polarity correction. In the case of a constant 3a0 value for a0 across the probed temperature range, motional narrowing theory can be applied allowing for the estimation of angular order parameters. For axially symmetric spectra the reported order parameters are an estimation for the time averaged angular peptide motions. The order parameters obtained for TOAC labels in alamethicin correlated to the repective a0 parameter; i.e. for labelled sites that were exposed to a higher hydrophobic environment a higher order profile was observed. Similarly the torsional backbone librations of membrane associated alamethicin were investigated using time-resolved echo detected EPR of TOAC spin-labels at three sites along the peptide sequence.59 The results show torsional librations in the sub-ns to ns timescale, which might be of importance for ion conductance of alamethicin channels. The reported experiments were performed in a DMPC bilayer at low temperature and it should be taken into account that peptide dynamics, that often correlate with the degree of hydrophobic mismatch, can vary across different lipid environments and temperatures.36 Note that time resolved EPR methods have also been employed for analysis of librational motions of acyl chain labelled lipids in a bilayer environment60,61 (echo detected EPR experiments are recorded at low temperature with the lipid environment being present in the gel phase). More generally, alameticin was found to facilitate the coexistence of multiple conductance states in artificial membranes when imaged using droplet interface bilayer systems.62 This suggests that alamethicin might exist as multiple substates and alamethicin poration might not be accurately described by a single distinct model, which in turn seems to depend strongly on experimental conditions. Indeed it has been where,

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suggested that the different conductance states of alamethicin are occurring due to molecular rearrangements within the bilayer environment compared to changes in the net association of peptide; by EPR investigation it was suggested that the lateral surface pressure and associated lipid curvature might be of importance for the relative peptide aggregates and associated conductance states.36 2.4.2 Macroscopic aligned samples for cwEPR. The large anisotropy of nitroxide spin labels can also be exploited to obtain angular constraints relative to the membrane normal for oriented lipid systems on quartz substrates. Oriented systems can be used to relate peptide orientations to the laboratory and the relative membrane frame. Under uniaxial motional averaging, as can be assumed for alphahelical peptides, the order parameter of TOAC can be described by the following equation: Szz ¼ P2h(cosg)i  P2(cosyz),

(8)

where g describes the angle between the peptide long axis and the membrane normal, or in other words the angle of the principle axis of motional averaging (parameters are defined in Fig. 9). The assumption that the spin label motion is in the fast motional scheme and thus temperature independent allows for a relation of the order parameter to the overall peptide tilt relative to the motional references axis (B0), and hence this is in large similar to the common orientation selective NMR interactions used to obtain angular peptide restraints. This method was successfully used to describe tilt angles at various positions along the long axis of alamethicin, which ranged from 201 to 301 relative to the bilayer normal, broadly in agreement with a transmembrane configuration in liquid crystalline membranes of DMPC.52 2.5 Pulsed EPR (double electron–electron resonance (DEER)) Pulsed EPR is analogous to NMR, where under a constant magnetic field, nitroxide spins are excited by high power microwave pulses at a given frequency. DEER, also known as pulsed electron–electron double resonance (PELDOR), is based around a simple Hahn echo pulse sequence (Fig. 10) at a constant magnetic field, where the spin population is pulsed at two individual frequencies in order to separate electron dipolar couplings reported by transverse relaxation, which is represented in the time domain of the DEER experiment. A Hahn echo experiment creates a spin echo by two consecutive pulses: a p/2 or 901 pulse followed by a p or 1801 pulse. In the rotary frame representation with an equilibrium setting of the bulk magnetisation (B1 being aligned with the z-axis), a p/2 pulse flips the magnetisation into the xy-plane. This is followed by dephasing of the individual spins. A p-pulse then flips all individual spins by 1801 to rephase the spins, which then relax back to their initial alignment along z and generate the measured spin echo. An additional p pulse is applied at a frequency offset, selectively exciting a separate sub-population of spins in the same sample, the pump frequency (vpump). The frequency offset 164 | Amino Acids, Pept. Proteins, 2018, 42, 146–189

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Fig. 10 The pulse sequence of a 3p DEER (A) and a 4p DEER experiment (B) are illustrated. The top pulse train is applied at the vobs and the bottom pulse sequence is applied at vpump respectively.

between vobs and vpump needs to be large enough to avoid overlap of the two excitation windows. If the two separately pulsed spin populations are coupled, the pulse applied at vpump will affect the local field of the observer spins. By varying t, the offset time at which the pump pulse is applied, the dipolar coupling of the two stimulated spin populations can be resolved. The basic experiment described above is known as a 3-pulse (3p) DEER experiment.63 In a 4p DEER experiment an additional p is applied at vobs following the last p of the Hahn echo sequence (Fig. 1.7B). This creates a transient echo as part of the pulse train. The pump pulse applied at vpump can now be applied at t ¼ 0, at the refocused echo, without pulse overlap with any pulses applied at vobs. This allows for accurate data collection at short t-times at the cost of a slightly reduced signal amplitude. A combination of a 3p and 4p experiment has been proposed, where by post experimental processing the deadtime free 4p and the longer higher signal to noise 3p trace are combined by least square fitting yielding an accurate high sensitivity DEER time domain.64 DEER has benefitted from the development of site directed spin labelling of peptides and proteins that naturally don’t possess a paramagnetic centre and has been successfully used to resolve dipolar interaction of up to 6 nm of membrane associated protein systems.27 ¨rster resonance energy transfer (FRET), however This is comparable to Fo the use of a single comparably small labels allows for precise distance Amino Acids, Pept. Proteins, 2018, 42, 146–189 | 165

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measurements reporting a distance distribution yielding information on subpopulations and conformers, which are less accessible to FRET.65 Pulsed EPR has been used to study peptide conformations of biradically labelled synthetic peptides and shows the potential of yielding conformational information of peptides at high resolution.66 Further to this, single labelled peptides allow for investigation of intermolecular interactions and aggregation states of peptides in well defined quaternary assemblies.67–71 Recently it was also demonstrated that orientaional restraints can be obtained for spin labelled membrane active peptides when analyzed on mechanically uniaxially aligned bilayers. Both the inter-spin label distance and the relative orientation of both spin labels have been analyzed using a rigorous simulation based approach.72,73 The advantage of this methodology is that from a single sample both distance constraints and relative orientation to the bilayer normal can be obtained, which is essential for analysis of distinct mechanistic insertion motifs of antimicrobial peptides. Similar distance-dependent continuous wave approaches are limited to observed spin–spin interactions with a limit of dipolar broadening of o2 nm. However, cwEPR and DEER can also be used in combination to investigate peptide oligomers. TOAC-labelled alamathecin in solution (chloroform:toluene (1 : 1)), showed spin label correlation times in the regime of ca. 1 ns and ca. 6 ns for monomeric and aggregated alamethicin respectively. The cwEPR correlation times together with dipole–dipole interactions observed by DEER resulted in the characterization of the aggregation number to be no9 peptides in these solubilizing conditions. Using MD approaches a dimer of two antiparallel tetramers is proposed.74 Alternatives to the commonly used nitroxide paramagnetic probe were recently demonstrated to be suitable for pulsed EPR, for example the dipolar interactions between nitroxides and Cu21 or two Cu21 paramagnetic centres is possible.75 Additionally, gadolinium has recently been demonstrated as a paramagnetic centre for EPR studies giving an increased sensitivity due to the large zero field splitting and accessibility to large magnetic fields. Gadolinium spin interactions do not result in multi-spin artifacts, beneficial for the analysis of large oligomeric complexes. This has recently been exploited to characterise the oligomeric state proteorhodopsin as hexameric complexes.76 Nevertheless, similar oligomeric investigations have yet to be applied to antimicrobial peptide systems. The theory and implications of gadolinium based EPR have been recently reviewed elsewhere;77,78 it is however noteworthy to point out its applicability and high potential to oligomeric assemblies79 as frequently hypothesised for AMPs.

3

Solid-state nuclear magnetic resonance (NMR)

Besides the common magic angle spinning (MAS), solid-state NMR experiments of uniaxially aligned samples have become a complementary NMR technique to resolve anisotropic and thus orientationally selective interactions. Angular constraints can be elucidated and related to the 166 | Amino Acids, Pept. Proteins, 2018, 42, 146–189

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external magnetic field. Most commonly these methodologies resolve the quadrupolar interaction of spins I41/2 or the orientation-selective chemical shift anisotropy (CSA) together with dipolar coupling, which allow for orientational and structural approximations to be derived. This approach requires peptides to be reconstituted into uniaxially oriented bilayers achieved by controlled rehydration of lipid mixtures on microscope cover slides or spontaneous alignment of magnetically aligned bicelles; allowing for structural characterisation in a hydrated liquidcrystalline membrane environment at elevated temperatures, resembling a native-like environment. Uniaxially aligned NMR approaches have been developed around antimicrobial peptides from the beginning of the methodology onwards. Gramacidin A and magainins were first characterised using oriented NMR.15,80,81 More recently, the methodology has also been proven useful for larger integral membrane proteins and membrane spanning sequences such as the transmembrane domain of VpU or bacteriorhodopsin.82,83 For larger integral membrane proteins the two dimensional polarisation inversion spin exchange at the magic angle (PISEMA) is commonly used.84 For smaller AMP systems a combination of multiple one dimensional experiments can equally be applied.13,15,85,86 For peptide orientation these most commonly include the orientation selective chemical shift anisotropy (CSA) of selectively 15N labelled amide bonds or the quadrupolar splittings of strategically placed deuterium (2H). A combination of both constraints has also been used for detailed structural insight of peptide tilt and rotational pitch. Further to this, 2H and phosphorous (31P) NMR provide information on lipid perturbation upon peptide interaction; here the hydrocarbon segments of a single acyl chain are perdeuterated and report on orientational lipid order and peptide-induced disorder, whereas the orientation selective CSA of headgroup 31P provides more global information on lipid phase and orientation, which in turn can be related to specific peptide–lipid interaction motifs. In order to obtain a holistic picture of peptide–membrane interaction a combination of all three nuclei (15N, 2H and 31P) and corresponding NMR experiments is useful in order to report on rotational pitch and tilt relative to the external magnetic field (and bilayer normal) as well as membrane and lipid perturbations, complimentary to EPR investigations spanning various timescales. It is noteworthy to point out that different NMR active nuclei possess distinct timescales and thus can provide varied information on different phenomena. 3.1 31P NMR of lipid–peptide systems The large 31P abundance of close to 100% in biological samples and especially in phospholipids makes it an important nucleus for biological membrane sample characterisation, which renders isotopical labelling unnecessary. The CSA dependance of 31P (Fig. 11) is largely exploited to investigate membrane structure, distinct macromolecular lipid phases, which give rise to specific 31P line shapes. Additionally the 31P line shape yields information on local lipid perturbations in bilayers upon Amino Acids, Pept. Proteins, 2018, 42, 146–189 | 167

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Fig. 11 (A) Illustration of the origin of a 31P powder pattern as observed for lipid vesicles. (B) Simulated static 31P NMR spectra: for an anisotropic powder, a uniform alignment at 901 and at 01. A chemical shift anisotropy of 44 ppm was assumed roughly corresponding to DOPC, lines were broadened by 250 Hz and an external magnetic field of 400 MHz was used.137 The powder component was rescaled by a factor of four for plotting. Simulations were performed using SIMPSON.138,139

protein/peptide interaction.87 In the past 31P NMR has been extensively used to characterise peptide–lipid interactions; for example the phase dependent lipid binding of melittin to phospholipid bilayers has been elucidated by 31P NMR.88,89 31P is also important to evaluate lipid alignment for macroscopically aligned samples as quality control for sample preparation. A high degree of alignment is required for a welldefined axis framework to which orientational peptide constraints can then be related. The characteristic orientation-selective powder pattern exploited by most 31P solid-state NMR applications (Fig. 11) is best explained by assuming a sphere ‘‘A’’ with a vertical z  axis passing through the centre of mass and a radius r ¼ 1. The sphere ‘‘A’’ is covered in lipid headgroups, each represented by small spheres ‘‘B’’ themselves, and a vector ~ y connecting the centres of mass of ‘‘A’’ and ‘‘B’’. If the centre of mass of ‘‘A’’ is centreed to the xy  plane, then the radius of the circle describing the sphere relative to the xy  plane is largest at z ¼ 0, in other words at a 901 orientation of the vector ~ y relative to the z  axis. This radius approaches 0 at increasing |z|; ~ y and z only align at z ¼ 1 and the angle between ~ y and z is 01, in an ideal scenario this is only the case for a single sphere ‘‘B’’. Hence, the observed powder pattern is a function of the radius of the sphere ‘‘A’’ in terms of the xy  plane, when moving it along the z  axis, and thus the 901 orientation is populated the most (see Fig. 4). Note that this is only true for a large molecular assembly tumbling slowly on the NMR timescale; for fast tumbling samples, a single isotropic resonance at 0 ppm is observed. Both larger vesicles and membranes in the hexagonal phase will give rise to a powder shape as illustrated in Fig. 11B. Membrane regions of high curvature or the cubic phase result in central sharp resonances.87 Uniformly oriented systems of single lipid species results in a single resonance; upon the addition of multiple lipid species the chemical shift 168 | Amino Acids, Pept. Proteins, 2018, 42, 146–189

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differences are resolved. It should be noted that chemical shift differences are more pronounced in the 901 compared to the 01 sample orientation for macroscopically aligned samples. Upon peptide interaction membrane modulations are induced that can range from thinning, local dents and interdigitation to complete disintegration and formation of micellar structures. Peptide-induced membrane deformations cause changes in the relative lipid head group orientation and these changes are reflected the in the 31P line shape of macroscopically aligned samples. Peptide-induced lipid affects are illustrated for cases of toroidal and barrel-stave pores (Fig. 12B); the lipid headgroup orientations, represented by small arrows, differ in the two described poration mechanisms, and since the 31P chemical shift is orientation dependent this can be detected in uniformly aligned membrane systems. The larger orientational headgroup spread in cases of toroidal pores leads to a raised plateau between the lines representing the 01 and 901 orientations. In such a scenario, the exact 31P line shape depends also on the lateral lipid diffusion rates, under fast diffusion a single averaged line at a CSA offset is observed.90 In a slow diffusion environment the different probability populations (that are in slow

Fig. 12 Schematic comparison of lipid arrangements and resulting 31P NMR spectra in initial peptide association (A) and pore formation (B). The vectoral arrangement of the lipid headgroup is illustrated using an arrow. Curved lipid regions are emphasized using a dashed line. Peptides are depicted as simplified cylinders. All membranes in this illustration are orthogonally aligned to the external magnetic field with the membrane normal and B0 parallel.91 Amino Acids, Pept. Proteins, 2018, 42, 146–189 | 169

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exchange) are resolved. The expected line shape for a barrel-stave pore is similar to what is expected for a ‘‘local dent’’ and differs in the plateau region compared to a spectrum of a toroidal pore (Fig. 12B). The exact line shape depends on the dimensions of the curved section of the pore and lipid diffusion rates; it is noteworthy that under certain conditions powder-like line shapes are expected for samples corresponding to toroidal pores. The line shape sensitivity, for example was previously illustrated by the observed line shape differences of toroidal pores formed by magainin-2 and aurein-3.3 respectively. These differences might be attributed to variations in elliptic pore dimensions possibly due to differences in the peptide insertion depth/orientaion despite toroidal characteristics for both peptides.90 These differences can be analysed by rigorous simulation approaches. The chemical shift of the 31 P lipid headgroup can be combined with chemical shift analysis of 15N labelled peptide to yield holistic scenario of the peptide–lipid orientational interaction motif. 31 P NMR of lipid headgroups together with single constraints of 15N labelled peptide and a simulation-based analysis successfully described the insertion motif of alamethicin and novicidin.91 Distinct 31P line shapes are reported distinct pores of alamethicin and novicidin at high peptide to lipid ratios. The information on lipid orientation (31P NMR) was combined with 15N constraints of selectively labelled peptide that directly report on the peptide orientation and describe a barrel-stave and a toroidal pore for alamathecin and novicidin respectively.91 It is noteworthy that recently it was also demonstrated that a full transmembrane peptide orientation is not needed for transmembrane poration. Novicidin showed a 15N chemical shift indicative of a planar peptide orientation despite forming toroidal pores.91 Similarly a in planar-oriented melittin peptide was recently suggested to stabilize a perturbed toroidal pore by binding to the pore rim and thus remaining in a parallel orientation to the bilayer plane;92 illustrating the diverse nature of peptide–lipid interaction and the importance to jointly understand both lipid and peptide behavior. 3.2 2H NMR on selectively labelled peptides Quadrupolar nuclei possess a non-spherical charge distribution and thus can be affected by electric field gradients. Nuclei with I 4 1/2 possess an inherent unbalanced charge distribution, which results in asymmetric electric fields intrinsic to the nucleus. This is known as a quadrupole moment and is in addition to the common dipole moment. A quadrupole moment interacts with local electric field gradients and perturbs the individual spin energy levels accordingly, which becomes detectable in a quadrupolar echo experiment, consisting of two consecutive 901 pulses. For nuclei with I ¼  1, two single quantum energy transitions need to be considered and the quadrupolar splitting can be measured as the absolute separation between the two resonance frequencies. The same (3 cos2 Y  1) relationship as for dipolar coupling and CSA applies 170 | Amino Acids, Pept. Proteins, 2018, 42, 146–189

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Fig. 13 Quadrupolar splittings of a single nucleus defined by Euler angles [0.0, tilt, 0.0]. The FID of the individual traces are plotted and overlaid (above). The individual quadrupolar splittings for each configuration are shown illustrating the angular dependence (below). Note the central peak at an initial helix tilt of 54.71 is the magic angle at which the quadrupolar splitting collapses due to the averaging of the anisotropic dependence of (3 cos2y  1) to 0.

(Fig. 13). The quadrupolar moment depends on the relative orientation to B0, which can be exploited in uniformly aligned samples to obtain orientational constraints of selectively deuterated peptides relative to the bilayer normal (aligned parallel or antiparallel to B0). The quadrupolar splitting is dependent on both the relative orientation and the dynamics of the nucleus on the NMR timescale (in the range of ms). Distinct quadrupole splittings, resolved for selectively labelled alanine positions (note that a single quadrupolar splitting is observed per –CD3 group due to fast rotational averaging) within an alpha helix give a bond vector with relative orientation of the Ca  Cb side-chain vector to the bilayer normal that is aligned with B0. Note that Ca forms part of the peptide backbone and thus the quadrupolar splitting is directly related to the longitude peptide orientation (Fig. 14). The sensitivity profile of the quadrupolar splitting relative to tilt and peptide rotation is shown in Fig. 12C. Accordingly, multiple quadrupolar splittings can be fitted to an Amino Acids, Pept. Proteins, 2018, 42, 146–189 | 171

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overall tilt and rotational peptide angle using the basic geometric relationship (GALA):13,14

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DvQ ¼

1 DvQðtheo:Þ ½3 cos2 bðcost  sint cosd tanbÞ2  1 2

(9)

where DvQ(theo.), is the theoretical maximum splitting and is defined as: DvQ(theo.) ¼ (e2qQ/h)S

(10)

where (e2qQ/h) is the quadrupolar coupling constant and S refers to a structural order parameter that resembles intrinsic motion that can cause averaging of the quadrupolar interaction.85 d and t correspond to the helical rotation angle and overall tilt angle respectively. b is the angle between the side-chain orientation defined by Ca  Cb and the helix axis (for graphical illustration see Fig. 14A and B). For a carbon deuterium bond the quadrupolar splitting constant is 167 kHz,93 which in methyl groups (as contained in the alanine side chain) was initially thought to be averaged by a factor of three due to fast rotation around the C–2H bond.81 It was later suggested that for peptides 84 kHz is a more accurate description of the quadrupolar coupling constant of an alanine side chain in a static environment, yet in hydrated system the quadrupolar coupling constant tends to be o80 kHz due to motional averaging.93,94 A maximum quadrupolar splitting of 74 kHz was observed for dry peptide powder (PGLa) and for [C-2D3] – labelled Plexiglas, showing some intrinsic peptide motions leading to partial averaging in a powder form.13 Additional motions can cause further reduction of the quadrupolar splitting constant and are described by ‘‘S’’. It should be noted that different models exist that attempt to describe the local peptide dynamics including a variable model for peptide dynamics that resulted in a good RMSD fits for WALP23 and PGLa in various membrane insertion states.13 The experimentally measured quadrupolar splitting is reported as an absolute value since the sign cannot be determined from a simple deuterium spectrum. The individual components expected in a 2H spectrum of a peptide containing a selectively deuterated alanine side-chain are (I) a small powder line shape corresponding to unaligned or aggregated peptide (B38 kHz), (II) the splitting of partially aligned water of naturally abundant deuterium at the membrane interface (B1.5–2 kHz) and (III) the splitting representing the relative orientation of the rotationally averaged –CD3 side chain contained in the peptide sequence (Fig. 15). Examples of four labelled sites along the peptide sequence of tilamin are shown in Fig. 18B, each giving rise to a distinct quadrupolar splitting due to different rotational pitches of the labelled residues. Detection of deuterated alanine side chains has successfully been used to describe peptide insertion modes of various peptides ranging from fully inserted to tilted oblique angles including WALP, MSI-103, hF19W, PGLa and tilamin peptides in native-like lipid compositions that aided the holistic description of antimicrobial peptide activity.13,15,22,86,95 172 | Amino Acids, Pept. Proteins, 2018, 42, 146–189

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Fig. 14 The relative angles that relate the helix orientation to the external magnetic field (B0). (A) A side view of an alpha helix illustrated as a cylinder. The overall side chain tilt is shown by y, the rotational pitch by d, the side chain tilt by b and the helical tilt by t.85 Reproduced from ref. 22 with permission from The Royal Society of Chemistry. (B) A top view of a model alpha helix is shown. The relative rotational pitch angle is described relative to a defined reference axis. r is the overall helical pitch angle where a describes the additional pitch due to the intrinsic side chain pitch, which needs consideration.85 Reproduced from ref. 22 with permission from The Royal Society of Chemistry. (C) The theoretical quadrupolar splitting is plotted as a function of rotational and helical tilt angles. The quadrupolar splitting (DvQ) is plotted on the z-axis, tilt and rotational angles are plotted as x- and y- values respectively. The graphical surface representation was generated using Matlab.

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Fig. 15 A deconvoluted 2H spectrum of an individually labelled peptide (Ala-d3). The individual spectral components that are expected for a deuterated peptide in a uniformly aligned sample with a parallel bilayer normal to B0 are illustrated. The experimental spectrum was analysed by fitting Lorentzians to the individual components in order to define respective line widths. The experimental spectrum, the de-convoluted spectrum and the residuals are shown. Spectrum is recorded at 20 1C and a deuterium frequency of 122.78 MHz. Reproduced from ref. 22 with permission from The Royal Society of Chemistry.

3.3 2H NMR on perdeuterated lipid acyl chains The deuterium spectrum of mechanically oriented samples of lipids with perdeuteraded acyl chains is a superposition of individual Pake doublets, each corresponding to a deuterated segment of the acyl chain (Fig. 16B).96,97 The axis of motional symmetry of lipids in a bilayer is perpendicular to the bilayer plane. The C–D bond reorients itself rapidly at a near perpendicular orientation relative to B0 and thus results in relatively well resolved peaks. The quadrupolar splitting (DvQ) of the carbon deuterium bond is described by:97 DvQ ¼

3 e2 qQ  SCD 4 h

(11)

where e2qQ is the quadrupolar coupling constant and segmental acyl chain order parameter is given by SCD: SCD ¼

1  ð3 cos2 yCD  1Þ 2

(12)

In the NMR spectrum an average of eqn (12) is observed, where YCD is the relative orientation between the carbon–deuterium bond vector and the external magnetic field. The Pake doublets corresponding to the more dynamic acyl segments, located close to the bilayer centre, are well resolved as narrow doublets of 174 | Amino Acids, Pept. Proteins, 2018, 42, 146–189

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small absolute quadrupolar splittings in the centre of the recorded spectrum. The less dynamic segments located close to the interface region are less well resolved and often show significant overlap. Smoothened order parameters assume a monochromatic decay of dynamics in this region as information on individual acyl positions is lost, nevertheless the dynamics of the acyl chain as a whole can be characterised.97,98 Generally a larger membrane disordering is observed for peptides associated to the membrane in an in-planar orientation. A transmembrane peptide spans the bilayer more evenly and does not allow for the introduction of ‘‘space’’. Peptides that preferentially sit at the interface or the glycerol backbone region allow for additional space in the acyl chain region. In a graphical illustration peptides interacting with the interface region are often portrayed as ‘‘spacers’’ between the individual head groups leading to motional freedom of the acyl chains and disorganisation (Fig. 8E). These effects are often partially compensated by head group tilting, acyl chain interdigitation and trans–gauche isomerisations usually seen in disordered bilayers in an attempt to fill ‘‘voids’’ leading to membrane thinning.87 The volume and hydrophobic moment of the distinct peptide affect the extent of these deformations.99,100 Membrane thinning as a consequence to membrane disordering has been reported in literature and different relationships have been established between the structural order parameter and absolute membrane thickness. Most of these are based on a set of assumptions, that are discussed elsewhere.96,101,102 The net change of the bilayer thickness is most reliably described by:99 Dd ¼ 2L0Dh|S|i,

(13)

where L0 is the the length of the all-trans acyl chain and h|S|i is the average change in the order parameter across the bilayer. Note that this can only be seen as an approximation of the mean change in bilayer thickness and localised effects might be plausible. The difference in experimental timescales (NMR order parameters on ms vs. EPR order parameters on ns) when compared to the segmental order parameters calculated from cwEPR spectra, can lead to seemingly conflicting results where on the ns time scale lipid ordering is observed and experiments on the slower microsecond timescale might indicate disordering, this was recently observed for the synthetic peptide, tilamin, that orients in a near in-planar orientation upon membrane interaction (Fig. 16A).22 Quadrupolar splittings also reflect a possible reorientation of the lipids due to membrane thinning or selective lipid curvature, which are only observed indirectly by cwEPR. A reorientation or an overall disordering on the ms time scale (observed by 2H NMR (Fig. 16B) can indeed lead to a more dense acyl chain environment due to clustering and reduced amplitude of motion on the ns time scale as probed here by cwEPR using 5-PCSL (Fig. 16A and D). Further to the overall lipid perturbations it is evident that the disordering effect as observed by 2H-NMR (Fig. 16C) is more pronounced in the hydrocarbon acyl chain of POPG rather then POPC (that also shows some disordering), which suggests a preferred interaction between the Amino Acids, Pept. Proteins, 2018, 42, 146–189 | 175

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Fig. 16 (A) Proposed mechanism for antimicrobial monolayer poration as observed for tilamin (tilamin is simplified as cylinders). The regions showing increased order together with associated membrane thinning (on the ns timescale of EPR) upon tilamin interaction are highlighted . (B) 2H spectra of chain-deuteratated POPC and POPG probes. Note that the last deuterated segment (in the acyl chain) is a –CD3 methyl group. The 2H spectrum of POPC : POPG (3 : 1, molar ratio) in the absence of tilamin is shown (bottom); tilamin interaction is probed at two molar ratios and less distinct deuterium resonance are observed upon peptide addition at different lipid to peptide ratios (P/L), indicating bilayer perturbation (reproduced from ref. 22 with permission from The Royal Society of Chemistry). (C) The calculated segmental order parameters as probed by chain-deuterated POPC and POPG. The order parameters are smoothened for the last six positions closest to the glycerol backbone, where a monochromatic decay has been assumed (reproduced from ref. 22 with permission from The Royal Society of Chemistry). (D) Segmental order parameters Seff calculated from cwEPR spectra are plotted as a function of temperature. The plotted Seff value is calculated from hyperfine splittings of 5-PCSL upon tilamin interaction (reproduced from ref. 22 with permission from The Royal Society of Chemistry).

cationic peptide interaction and the negatively charged lipid headgroups. Such a peptide interaction could lead to lateral segregation of the individual lipid species in the model bilayer. Similar preferential chargedriven lipid interactions have also been observed for other cationic peptide systems, an example being pleurocidin, which also showed preferential interaction with the anionic PG component in mixed lipid systems.103 176 | Amino Acids, Pept. Proteins, 2018, 42, 146–189

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15

3.4 N NMR of selectively labelled peptides Similarly to the quadrupolar interaction of 2H the orientational sensitivity of 15N chemical shift (CSA) can be exploited to obtain angular peptide constraints. In a peptide that contains a single 15N nucleus, the inter nucleus (15N) distances and spin interactions are large enough to be ignored and an uncoupled spin can be assumed. In addition, the surrounding protons are decoupled by high field irradiation at the 1H frequency. If such a spin system is placed into a large magnetic field under static and unoriented conditions, a typical powder pattern of ca. 160 ppm CSA is observed;104 the CSA relates to the static main tensor elements that are described by the discontinuities within the 15N powder lineshape. The CSA interaction of 15N can be described by a principal axis system, where the orthogonal elements of the second rank tensor persist and accurately describe the chemical shift interaction. s11, s22 and s33 describe the static main tensor elements (Fig. 17). The tensor elements are molecule- and nucleus- specific and relate the internal molecular arrangements to the external magnetic field. The main tensor elements vary between amino acids and also depend on secondary structural elements. The best characterised tensor elements are for glycine and alanine residues and are found to be 55, 80 and 225 (each 5 ppm).105–107 However, these can vary slightly depending on the side chain nature; for example the main tensor elements for 15N-Tyr are: s11 ¼ 52.1 ppm, s22 ¼ 77.1 ppm and s33 ¼ 209.3 ppm.108 The tensor elements can be related to the coordinate frame, in macroscopically aligned sample the bilayer normal is typically parallel or orthogonal to B0, related to the external magnetic field via the Euler angles (F, Y, C):104 szz ¼ s11 sin2Y cos2F þ s22 sin2Y sin2F þ s33 cos2Y,

(14)

where szz is the measured chemical shift in an uniformly oriented system (Fig. 17B). When considering the molecular frame of an alpha helix it becomes evident that the CSA tensors of the 15N–1H bond vector are oriented only

Fig. 17 (A) Graphical representation of the 15N amide of CSA of residue 11 in a straight alpha helix at a relative tilt of 301 to B0. The representation was created using SIMMOL.105 (B) A schematic of the 15N CSA illustrating the main tensor elements s11, s22 and s33 and the experimentally observed orientation selective szz. Amino Acids, Pept. Proteins, 2018, 42, 146–189 | 177

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178 | Amino Acids, Pept. Proteins, 2018, 42, 146–189 Fig. 18 Orientation analysis of single angular constraints of tilamin by solid-state NMR. (A) A 15N spectrum of selectively backbone labelled (15N) tilamin at position 15 (Tyr). (B) Quadrupolar splittings of four selectively side-chain deuterated alanines at positions 5, 9, 16 and 19 along the peptide sequence. (C) Combined analysis of the single 15N constraint and an underlying RMSD plot representing the four individually recorded quadrupolar splittings (contour lines for 7 kHz, 6 kH, 5 kHz and 4 kHz). The overlapping conformational solutions are numbers. Reproduced from ref. 22 with permission from The Royal Society of Chemistry.

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within a few degrees relative to the helix axis and thus able to report on peptide orientation (Fig. 17A). The CSA of 15N is highly complimentary to orientational information obtained from quadrupolar splitting of motionally averaged CD3 groups and help to eliminate and narrow possible solutions in the conformational space. However, it should be noted that the quadrupolar interaction is more sensitive to sample heterogeneity and mosaic spread.86 Further to this, the 15N nucleus can be incorporated into a variety of amino acids and reports directly on the backbone conformation thus obeying the necessity of alanines in the peptide sequence. A complimentary analysis of individual 15N constraints and 2H quadrupolar splittings is illustrated in Fig. 18 on the example of tilamin.22 It should be noted that similar approaches combining 15N and 2H analysis have been used for a hydrophobic model peptide hF19W,86 the heterodimeric peptide distinctin,109 and the huntingtin membrane anchor (1–17).110 In the illustrated example four quadrupolar constraints are fitted to a single peptide orientation assuming a straight alpha helix (based on circular dichroism, data not shown) by RMSD analysis (Fig. 18C), which is combined with a single angular backbone constraint of 15N (Fig. 18A and C) to yield three combined orientation solutions. The inclusion of more quadrupolar splittings at different peptide locations can further narrow the possible solutions, yet in the presented example this would have resulted in necessary mutations which in turn can alter peptide–lipid interactions and functions. However, it should be noted that using multiple deuterated alanine residues, single orientational solutions could be obtained for some peptide systems e.g. WALP, PGLa and MSI-103.15,111

4 Discussion Antimicrobial peptides exploit a large conformational space.112 They are predominantly unstructured in solution and adopt regular secondary structures upon membrane interaction. The membrane interaction itself can be highly versatile and multifaceted. The here described magnetic resonance approaches have been extensively used to characterize this interaction and refine the conformational space to gain information on plausible mechanisms of antimicrobial activity. Further to this the different experimental timescales allow for selective probing of distinct peptide and lipid dynamics that greatly aid the description of the dynamic membrane activity of AMPs; including information on peptide topology and orientation, heterogeneity in their corresponding membrane-interaction as well as associated dynamics/order profiles of both peptide and lipids. However, NMR unlike EPR, suffers of the inherently low sensitivity of NMR active nuclei and in case of membrane active-peptides the nuclei of interest are diluted in lipid to 1–4 mol%. The recent development of dynamic nuclear polarisation (DNP) allows for magnetization transfer from polarized electrons contained in bi-radicals to NMR-active nuclei leading to a larger overall polarization and signal Amino Acids, Pept. Proteins, 2018, 42, 146–189 | 179

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enhancements. Recently it was demonstrated, using a custom built probe, that such methodology is also accessible to oriented solid-state NMR and antimicrobial peptides.113–115 In case of single labeled PGLa in DMPC/DMPG (3 : 1, molar ratio) and an estimated signal enhancement of 17 – fold was achieved, a considerable improvement on the otherwise long measurement times (up to multiple days).115 Development of DNP methodologies makes uniaxially oriented NMR of orientational constraint more accessible including 2D experiments like PISEMA, yet DNP requires frozen samples efficient polarization transfer.116 Besides technological advances, it is also becoming increasingly evident that antimicrobial peptide action might be more versatile then previously thought including alternative motifs (e.g. selective monolayer motifs22) or variations of the established motifs including intermediates. For example it was recently demonstrated that in the case of novicidin an in-planer peptide configuration can result in the formation of toroidal pores.91 A distorted toroidal pore was observed in the case of melittin and a magainin analog where lipids are curved, yet these models show a more stochastic peptide distribution.117–119 Further complexity is added by varying lifetime of toroidal pores.120 AMPs can also act in synergy, which was successfully shown for PGLa and megainin 2. Individually these peptides are either near-parallel to the bilayer plane or slightly tilted, however at equimolar ratio PGLa changes to a fully inserted transmembrane peptide.121,122 This synergistic behaviour translates into increased antimicrobial activity and resulted in excess of 20-fold increased carboxyfluoresein leakage from vesicles treated by a combination of magainin-2 and PGLa.123 A recent theoretical approximation demonstrated that the relative high partition constants and MIC values in the mM range can indeed translate to local membrane concentrations that are about 10 000 times higher than in solution, yielding scenarios close to complete membrane coverage (e.g. Melittin: a MIC of 9–18 mM and a partitioning constant of 6104 results in an estimated lipid to peptide ratio 2.5 : 1 at the membrane124–126). Such high membrane coverage can result in fluctuations in peptide structure and behaviour as well as localised modulations and transient non-cooperative peptide-peptide interactions that potentially can result in spontaneous pore formation. These phenomena are in the absence of well characterised peptide–peptide interactions and thus challenging to biophysically characterize. Such seemingly chaotic local peptide behaviours might be an advantage and might be the underlying principle to the often multistep mutation processes needed to develop resistance.127 At the same time it is these characteristics that challenge biophysical characterisation. The outline methodologies give valuable ensemble information on orientation and dynamics yet lack resolution on the single molecule level; it is therefore that for holistic AMP characterisation requires a spectrum of different techniques at the interface of biophysics that investigate both lipid and peptide behaviours. The combinational approach of distinct EPR and NMR techniques can yield discrete insight crucial for the understanding of AMPs, yet there are also some limitations associated with magnetic resonance methodology. 180 | Amino Acids, Pept. Proteins, 2018, 42, 146–189

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For example, despite of using multiple FIDs (up to thousands) for a single NMR spectrum, the spectra often remain broad leading to an experimental error when measuring CSA or quadrupolar interactions that cannot be ignored; spectral simulation might be beneficial in difficult cases. Further to this, the chemical shift tensor used for analysis of 15N CSA are often approximations and variations of 5–7 ppm can exist depending on the local environment.128 Most NMR interactions are dependent on dynamics that are often approximated for analysis and thus leading to deviations in the presented data; e.g. peptide fluctuation of a Gaussian distribution of 121 have been suggested for WALP in DMPC,129 and AMPs are expected to possess similar fluctuation dynamics. For uniaxially oriented NMR sample orientation on glass slides is essential as well as orientation of the stacked sample within the magnetic field both prone to error resulting in overall offsets of the measured parameters. Further to this, sample preparation is limited to model membrane systems that resemble overall biophysical characteristics of bacterial membranes yet are limited to the phospholipid component excluding asymmetries including LPS and peptidoglycan. However, relative progress has been made in the isolation and orientation of bacterial extracts of E. coli and M. luteus for oriented solid-state NMR,130,131 which is a step closer to the native bacterial cell envelope, yet the intrinsic membrane asymmetry is lost during sample preparation. Asymmetric membranes resembling the gram-negative outer membrane and include an LPS layer were recently synthetically assembled on a supported bilayer for neutron reflectrometry, a much needed step for investigation of peptide– membrane interactions in a native-like environment.132 Besides the current difficulties due to experimental limitations, antimicrobial peptides, show great promise for future development into antibiotics for the clinic and magnetic resonance will be an important contribution to this. The broad activity against bacterial membrane integrity as a whole makes the development of resistance difficult and is only reported in rare cases, which is in contrast to conventional antibiotics that target specific pathways and proteins.133 A precise understanding of sequence identity could advance the field towards modular peptide design principles allowing for a large arsenal of potent peptide antibiotics, possibly leading to peptide-based therapeutics for personalised medicine.

Acknowledgements The support from BBSRC studentship (BB/J012815/1) in collaboration with the National Physical Laboratory (NPL, Teddington, UK), supervision by Peter J. Judge (University of Oxford, UK) and Maxim G. Ryadnov (NPL, Teddington) are acknowledged.

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Emerging therapeutic agents on the basis of naturally occurring antimicrobial peptides Published on 29 November 2017 on http://pubs.rsc.org | doi:10.1039/9781788010627-00190

A. Falanga and S. Galdiero* DOI: 10.1039/9781788010627-00190

Antimicrobial peptides (AMPs) are found virtually in every living organism providing an immediate defence against pathogen invasion and constitute a challenging opportunity to be developed as therapeutical molecules. Many efforts are actually devoted to bring AMPs into clinical use especially because of the rapidly worldwide resistance development to conventional antibiotics. With no attempt to present a comprehensive overview of all AMPs being assessed as potential pharmacological agents, in this chapter we provide an overview of the biological role, classification, and mode of action of AMPs; besides antimicrobial activity, we also briefly describe the modulation of the immune response, the enhancement of angiogenesis and wound healing, and the prevention of post-surgical adhesions. We tackle the challenges of developing AMPs for clinical applications, and present an overview of innovative formulation/delivery strategies. In particular, we illustrate the relationships between their biophysical features and activities. The current status and future directions in the development of AMP therapeutics with efforts spanning a multitude of disciplines from chemistry, physics, biology to materials and contributing to the development of highly active AMPs are also discussed.

1

Introduction

The spread of bacteria resistant toward conventional antibiotics has become a major global health threat, causing considerable mortality and morbidity worldwide and, consequently, substantial increases in healthcare costs; raising concerns about antibiotic resistance as a seriously threat for the medical advancements made possible by antibiotics.1,2 Nowadays a huge number of people acquires methicillinresistant Staphylococcus aureus (MRSA) infections worldwide and more people die annually from MRSA infections than from HIV/AIDS, emphysema and Parkinson’s disease. The World Health Organization (WHO) has classified multidrug-resistant (MDR) bacteria as a top priority threat to human health, and incentives have been proposed for antibacterial drug development;3 moreover, resistant strains are regularly found for almost every antimicrobial used in clinics. Several factors are responsible of antibiotics resistance; such as the indiscriminate use worldwide, the massive employment for industrial purposes including food production as well as animal farming industry and pet animals, the presence of pathogenic bacteria in health care facilities, the lack of knowledge of basic hygienic procedures, the absence of antimicrobial drug prescription guidelines, the unavailability of a fast and effective diagnostic analysis for identifying the pathogenic isolates and choose the appropriate antibiotic. Thus, antibiotic resistance is a priority disease Department of Pharmacy, University of Naples Federico II, Via Mezzocannone 16, Napoli 80134, Italy. E-mail: [email protected] 190 | Amino Acids, Pept. Proteins, 2018, 42, 190–227  c

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and it is highly important to avoid unnecessary and improper consumption of antibiotics in human and animal health care, to enforce regulations and health-care procedures. The need of effective antibiotic treatment concerns mainly the ageing population and the immunocompromised patients;4 although there is an increasing fear for the rapid development of drug-resistant strains outside hospitals.5 Once bacteria have become resistant to antibiotics, treatments need to be switched to more costly second-line antibiotics such as vancomycin;6 however, already in 1996 a first vancomycin resistant Staphylococcus aureus strain was identified in Japan.6,7 Any introduced therapy relying only upon a bacteriostatic/bactericidal mechanism is expected to develop resistance; in fact, clinically significant resistance may appear sooner or later following introduction of a new antibiotic into the clinics. As a matter of fact, no later than one year from the introduction of daptomycin into the clinic in 2003, the emergence of resistance in patients with Enterococcus faecium and MRSA infections was reported. Although the development of new antibiotics may still represent a possible strategy against MDR bacterial infections, only two new classes of antibiotics have been introduced into the market in the last twenty years, which nonetheless are not significantly active against Gram-negative bacteria and will be as well as other antibiotics highly susceptible to bacterial resistance mechanisms and thus have only a limited life. A challenging health issue is thus to develop novel antimicrobial molecules for the treatment of infections. As a result of addressing this urgency, the main priority is the search for novel antimicrobial compounds with different mechanisms of action that may prevent the return to a pre-antibiotic era. Antimicrobial peptides (AMPs) in nature play a key protective role against a broad spectrum of bacterial and fungal pathogens establishing a defence system that may react in a fast and efficient manner.8 Interestingly, AMPs constitute a challenging class of novel drug candidates, which may also be able to overcome pathogen resistance and thus represent excellent candidates for clinical exploitation.9,10 AMPs are or are designed from natural molecules; in fact, they have been found in almost all organisms of the animal and plant kingdoms and display remarkable structural and functional diversity.11,12 In higher organisms, they are key components of the innate immunity, protecting the host against infections;1,13 nonetheless also bacteria produce AMPs to kill other bacteria competing for the same function/ place in the ecosystem. Constitutively expressed AMPs are stored as inactive precursors and are released at the site of infection and inflammation; others are expressed in response to pathogen-associated molecular patterns (PAMPs). AMPs exhibit broad-spectrum activity both in vitro and in vivo against various microorganisms, including Grampositive and Gram-negative bacteria, fungi, viruses and some are active against multi-drug resistant (MDR) bacteria and hold low susceptibility to develop resistance. Moreover, they can be used alone or in combination with other antibiotics.12 Several AMPs, apart from antimicrobial activity, have also the ability to modulate the innate immune responses Amino Acids, Pept. Proteins, 2018, 42, 190–227 | 191

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of the host and to indirectly promote pathogen clearance. There are some promising examples of AMPs in clinical trials or already introduced into the market. Several processes are responsible of antibiotic resistance such as inhibition of the interaction between the drug and the target, modification of the drug-binding site, discharge of the drug from target cells, alteration of microorganism genetic patterns as a result of environmental changes using sensor-transducer response systems.14 Thanks to their distinguished mode of action, which involves the direct interaction with the membrane of the pathogen, AMPs are less likely to induce bacterial resistance; and in fact, membrane mutation would likely be costly for the pathogen.13 The gathering of AMPs on the membrane surface is due to the selective interaction of their positive charges with the negatively charged surfaces of bacterial membranes. Following the initial electrostatic interaction, their hydrophobic domains are responsible for the interaction with the hydrophobic parts of the membrane. This set of complicated electrostatic and hydrophobic interactions is responsible of major rearrangements in the membrane structure which eventually leads to formation of peptide-lipid specific interactions, to peptide translocation across the membrane and interaction with intracellular targets or to membrane lysis (which is the most common process among AMPs). This mechanism is clearly different from that of standard antibiotics and given that the primary target of AMPs is the bacterial membrane, microbes should face a great challenge in order to preserve the cell membrane functional and structural integrity while avoiding the membrane-disrupting activity of AMPs. However, considerable concern has recently emerged about possible bacterial resistance which may be developed under selection pressure in vitro, deserving further investigations of the potential risks.15,16 Recently, several resistance mechanisms have been described which may render difficult their use and effectiveness as therapeutic agents.15,16 Notwithstanding this, AMPs represent an encouraging alternative to conventional antibiotics to fight the current drug resistance crisis. Their clinical and commercial development still holds some drawbacks, such as toxicity, susceptibility to proteases, and cost of production; thus, extensive efforts are devoted to overcoming those impediments. Native AMPs provide templates for the design of medically relevant anti-infective agents, which are easier to produce and/or more potent analogues and may represent an emerging strategy against numerous pathogens. Unusual amino acids or peptidomimetics are developed to avoid proteolytic degradation, while the obtainment of shorter peptides retaining activities represents a solution for the cost issue. With no intention to provide a comprehensive overview of all AMPs, this chapter focuses on their mechanism of action with an emphasis on AMPs being evaluated as potential therapeutic agents. Moreover, several design procedures will be highlighted such as combination with established antibiotics and attachments of nanosystems of various nature. 192 | Amino Acids, Pept. Proteins, 2018, 42, 190–227

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2

AMPs origin and classification

Several databases exist for natural AMPs, with more than 2500 peptides.17 AMPs are commonly arranged in different classes according to their structure: a-helical and b-sheet peptides, or peptides with extended/ random-coil structure.18–20 (Fig. 1) The a-helical AMPs, including magainin,21 cecropin,22 pexiganan,23 temporins24 and melittin25,26 constitute the most well-known class of AMPs. Although being unstructured in aqueous solution, these peptides are able to adopt an amphipathic a-helical structure when in contact with biological membranes or membrane-mimicking environments. a-helical AMPs are essentially cationic and amphipathic and have preferential activity against Gram-positive bacteria and fungi. The activity of most a-helical AMPs is attributed to the disruption of bacterial membranes, and several mechanisms of action have been proposed as will be described in the next paragraph. The b-sheet AMPs, such as a-/b-defensins,27 and protegrin,28 are stabilized by disulfide bridges, and form relatively rigid amphipathic structures. Most of the b-sheet AMPs exert their antimicrobial activities by disrupting bacterial membranes. The rigid structure of b-sheet peptides renders them more ordered in aqueous solution and differently from helical peptides, they do not undergo drastic conformational

Fig. 1 Structure of an example of AMP for each class. Panel A: NMR-structure of magainin (2LSA); Panel B: X Ray structure of human beta defensin 2; Panel C: NMR-structure of indolicidin (1QXQ). Amino Acids, Pept. Proteins, 2018, 42, 190–227 | 193

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changes upon membrane interaction. These peptides are perpendicularly inserted or tilted into the lipid bilayer, and their hydrophilic regions are able to interact with the head groups of the membranes. The third class comprises extended peptides which are often rich in specific amino acid residues such as proline, tryptophan, arginine, and histidine and lack secondary structure. Among peptides belonging to this group are (i) human salivary histatin, rich in His residues and with significant activity against several bacteria or fungi;29 (ii) the proline-rich peptides, originally isolated from insects;30 (iii) indolicidin and tritrpticin, which are small cationic peptides rich in Trp and Arg with a broad spectrum activity.31,32 They usually fold into amphipathic structures when in contact with a membrane. While some extended peptides (such as indolicidin) are membrane active and their activity is correlated to membrane leakage, others are not active against the membranes of pathogens, but they can penetrate across the membranes, interact with bacterial proteins inside and in this way achieve their antimicrobial activities. Beside cationic AMPs, also some anionic peptides have been isolated which are usually found in surfactant extracts, bronchoalveolar lavage, and airway epithelium cells; anionic AMPs require zinc as a cofactor for their antimicrobial activity. Examples of anionic AMPs are maximin H5 from amphibians,33 small anionic peptides rich in glutamic and aspartic acid isolated from ovine, bovine, and human tissues,34 and human dermicidin.35 These peptides usually exploit a lipid anchor to promote binding and cationic divalent ions to reduce any repulsive force and lead to an overall enhancement of the binding. Even though AMPs differ in primary sequence and secondary structure, they share some common physico-chemical features which are major drivers of membrane interactions, such as the overall positive charge, the significant fraction of hydrophobic amino acids, the amphipathic character and structural conformational flexibility.36,37 Most AMPs are rather short, they are made of 10–50 aminoacids, display an overall positive charge ranging from þ2 to þ11, essentially because of the presence of a high number of lysines and/or arginines and contain a substantial proportion (typically between 30–50%) of hydrophobic residues playing a vital role in the attainment of the typical amphiphilic structure38 while the negatively charged amino acids (Glu and Asp) are less represented. At physiological conditions, Arg is more represented than Lys, probably because it can form more stable bidentate hydrogen bonds with phosphate groups of the membrane. Moreover, hydrophobic amino acids are usually more abundant than polar residues and there is a high content of aromatic amino acids. The role of hydrophobic residues is to enhance penetration and disorganization of the lipid tail region of the bilayer. Notwithstanding the similarities in amino acid content and the differences in secondary structure and length, AMPs share a common three-dimensional amphiphilic scaffold which is characterized by the presence of defined hydrophilic and hydrophobic fragments/domains.39 These structural properties allow them to interact with the membranes of 194 | Amino Acids, Pept. Proteins, 2018, 42, 190–227

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bacteria, which contain high quantities of negatively charged phospholipids and non-cholesterol. One common property of most AMPs is their cationic nature which determines their higher targeting to negatively charged lipids present on the outer leaflet of bacterial membranes compared to zwitterionic mammalian membranes.40 AMPs can also bind to lipopolysaccharide (LPS) which is another negatively charged essential constituent of the outer membrane of Gram-negative bacteria41 and they may determine perturbation in the LPS layer. For instance, polymyxin B firstly establishes an electrostatic interaction with LPS, and after internalization, it binds to the bacterial membrane leaflet rich in phosphatidylglycerol, inducing the leakage of cytoplasmic content.42

3

Mechanism of action

Membrane interaction is a key requirement for achieving direct and rapid antimicrobial activity when the membrane is targeted or when an intracellular target must be reached through translocation across the membrane into the cytoplasm. The combined cationic and hydrophobic natures of AMPs represent critical determinants for the interaction and perturbation of the microbial cytoplasmic membranes, with the presence of polar phospholipid head groups on the membrane surface and the charge distribution of the peptide being key factors.43 To understand their mechanism of interaction with the target membrane, it is pivotal to familiarize with the structure and physical properties of the bacterial membranes which allows the classification in Gram-positive and Gram-negative bacteria (Fig. 2). While the inner membranes of both types of bacteria are analogous, Gram-positive bacteria present a cytoplasmic membrane surrounded by a thick peptidoglycan layer with negatively charged teichoic and lipoteichoic acid;

Fig. 2 Structural representation of membrane of Gram-negative (left) and Gram-positive (right) bacteria. Amino Acids, Pept. Proteins, 2018, 42, 190–227 | 195

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whereas Gram-negative bacteria possess a cytoplasmic membrane surrounded by a thin and less cross-linked peptidoglycan layer as well as an additional outer membrane rich in LPS. LPS molecules, being highly negatively charged thanks to the high number of phosphate groups, provide the primary barrier to most hydrophobic antibiotics. The inner membranes of Gram-positive and Gram-negative bacteria are rich in phosphatidylglycerol (POPG), cardiolipin (CL), and phosphatidylethanolamine (POPE), with negatively charged head groups; additional negative charge to the bacterial surface is provided by teichoic acids present in the cell wall of Gram-positive bacteria and LPS in the outer membrane of Gram-negative bacteria. The differences between Gram-positive and Gram-negative bacteria indicate beyond any doubt that the details of how AMPs penetrate into different bacteria must vary; in particular, there is a more direct interaction for Gram-positive bacteria, because AMPs have only to permeabilize/diffuse through one membrane, while two layers have to be permeabilized in the case of Gram-negative bacteria, resulting in a two-step process. As a matter of fact, most AMPs display specific activities and the composition of the membrane drives the specificity of AMPs; as an example, daptomycin is active against Gram-positive bacteria but not against Gram-negative bacteria, because it is unable to permeabilize their outer membrane.44 On the contrary, mammalian membranes are characterized by a neutral net charge because they are rich in zwitterionic phospholipids such as POPE, phosphatidylcholine (POPC), and sphingomyelin (SM). A typical feature of mammalian membranes is the asymmetric distribution of phospholipids with zwitterionic phospholipids present in the outer leaflet, and negatively charged phospholipids eventually present in the inner leaflet. Thus, AMPs, being cationic, recognize the anionic lipids exposed on the outer surface of the bacterial membrane; on the contrary, interactions with mammalian membranes take place mainly through hydrophobic interactions, which are somewhat weaker compared to the electrostatic interactions. Furthermore, mammalian membranes, containing a high content of cholesterol, determine a reduced activity of AMPs through the stabilization of the phospholipid bilayer. AMPs interaction with the bilayer can induce lipid rearrangements. In fact, highly cationic peptides once adsorbed on the bacterial membrane are also able to recruit other anionic lipids in the outer leaflet of the membrane modifying the fluidity and the structure of the membrane.45–47 ‘‘Flip-flop of anionic lipids from the inner to the outer leaflet produce an asymmetric distribution of charged lipids further reducing the membrane potential and inducing its destabilization’’.48 Although the detailed mode of interaction of AMPs with bacteria is poorly understood; in order to accomplish their function, the first step is to accumulate on the membrane surface up to a critical concentration which is regulated by the favourable interactions between the negatively charged surface of bacteria and the positive charges of AMPs. In addition, the complex structure of the LPS molecules renders the interaction with Gram-negative bacteria even more complex. Thus, the interaction with the bacterial membranes is regulated by several features of AMPs which 196 | Amino Acids, Pept. Proteins, 2018, 42, 190–227

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include: length of the sequence, number of positive charges, density of positive charges, secondary structure, hydrophobicity; with electrostatic and hydrophobic interactions being the two main driving forces that direct an AMP toward and into the bacterial membrane. The initial interaction between peptides and bacterial surfaces involves the binding step to the target cell by a purely non-specific interaction with charged bacterial membranes which is responsible of their broad spectrum activity over time. Most AMPs are not already structured is acqueous solution, but once in contact with the cytoplasmic membrane, they adopt an amphipathic secondary structure which is key for their biophysical activity. The charged domains are responsible for the interaction with the phospholipid hydrophilic head groups, while the peptide hydrophobic domains interact with the hydrophobic core of the lipid bilayer, thereby guiding the AMP deeper into the membrane. Literature reports on the many different methods and instruments used to unravel AMPs mechanism of action and several models have been proposed in order to describe how antibacterial peptides are inserted into bacterial membranes and produce bacterial membrane permeabilization.49 Among these the most represented are: the barrel-stave model,50 the toroidal pore model21 and the carpet model.51 Each of them depends on the nature of the AMP involved and differs mainly in the peptide attachment mode to bacterial cell exterior and its insertion into the cell membrane (Fig. 3).

Fig. 3 Principal mechanisms of interaction between AMP and lipid bilayer. A: barrel-stave model; B: toroidal pore model; C:detergent-like model. Amino Acids, Pept. Proteins, 2018, 42, 190–227 | 197

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Many AMPs form membrane pores and according to the geometry of the pores as well as the interactions of AMPs with the lipids, we can describe the barrel-stave and the toroidal models. The ‘‘barrel-stave model’’ involves the perpendicular insertion of the peptides into the bilayer with subsequent oligomerization resulting in the establishment of a peptide-lined transmembrane pore or channel. a-helical amphipathic peptides are able to form barrel-like transmembrane bundles, which form amphipathic pores (barrel-stave model). In this pore, the bundles of amphipathic helices are aligned with the hydrophobic face oriented towards the membrane hydrophobic core and the hydrophilic face toward the interior of the pore. The addition of monomeric molecules determines the increase of the pore size, the leakage of cell contents and hence cell death. The activity of alamethicin, dermcidin, pardaxin is attributed to this model.52 In the ‘‘toroidal pore model’’ AMPs generally assume a helical conformation and insert into the membrane. The lipids are tilted from the normal lamellar structure having the two leaflets of the membrane connected and forming a continuous bent tract reminiscent of a toroidal hole. Hydrophilic regions of the peptides and lipid head groups interact together forming a pore structure, which is generally larger than the barrel stave type pore.21 Magainins, protegrins and melittins use this mechanism.53 The principal difference between the two models is the morphology of the pore. In the toroidal-pore model, the pore is lined by both peptides and phospholipid headgroups, because peptide insertion forces the phospholipid to bend constantly from one leaflet to the other. This model is characterized by significant lipid disorder and membrane curvature change which are accompanied by superior membrane hydration; also electrostatic forces govern this mechanism because peptides hold less hydrophobic contacts with the tails.54 In the barrelstave model, the hydration of the membrane remains almost unchanged, the membrane does not present significant curvature and both electrostatic and hydrophobic interactions are key. In both models, the morphology of the pores depends on the pore diameter, the lipid composition and the lipid conformation around the pore, the life time of the pore (transient versus permanent), the peptide/lipid ratio and the number of peptide molecules necessary to stabilize the pore. Lipid composition is a key factor; as a matter of fact, most AMPs are able to induce formation of pores also in mammalian cells but at a much higher concentration because mammalian membrane are significantly more rigid and less prone to form pores while bacterial membranes rich in POPE with a small head group present a high tendency to produce a negative curvature. The third model is typical of AMPs that do not induce pore formation, and their interaction can be described with the ‘‘carpet mechanism’’. In this model, peptides bind parallel to the surface without inserting into the hydrophobic core and very strong ionic interactions with anionic groups from the outer leaflet of the bacterial cell membrane take place. At high concentrations, peptides adsorbs onto the bilayer surface and orient parallel to the membrane in a ‘‘carpet-like’’ fashion, while the inner leaflet is free of peptide binding, creating a large disparity across the 198 | Amino Acids, Pept. Proteins, 2018, 42, 190–227

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membrane of both charge and surface tension. When this accumulation of peptide molecules on the lipid membrane reaches a critical value, AMPs produce a degeneration of the bacterial membrane by disrupting the integrity of the molecular structure of the membrane and causing tension in the bilayer that leads to disruption of the membrane and formation of micelles.50,55 This model was first used to describe the action of dermaseptins.56 In the three models, membrane permeabilization first leads to leakage of ions and metabolites, depolarization of the transmembrane potential and subsequently to rupture and lysis of microbial cells. Besides leading to membrane lysis, studies indicate that some peptides present other modes of action. In fact, membrane permeabilization is also responsible for translocation of some AMPs into the cytoplasm without the formation of stable pores; this translocation without extensive permeabilization is not surprising considering the physical similarity between AMPs and some cell penetrating peptides.57–61 In the cytoplasm, AMPs may target key cellular processes that are not mediated by membrane permeabilization62 such as nucleic acid63 and protein synthesis,64,65 protein folding, enzymatic activity, and/or cell wall synthesis.38,66 Some authors also report that AMPs can interfere with the pathogen’s metabolism; they can trigger the synthesis of virulence factors, for instance, hyaluronic acid and capsular polysaccharide;67 or they can interfere in vitro or in vivo with cytokines of bacterial cells. It is likely that bacterial death is achieved through multiple and complementary processes, referred to as multi-hit mechanisms which all participate in enhancing their activity and in evading resistance development. Moreover, although the mode of action of each individual AMP may depend on several key parameters like peptide concentration, target bacterial species, tissue localization and growth phase of the bacteria, the antibacterial activity is strongly relying on the mechanism of interaction with the microbial membranes; notwithstanding the multitude of models proposed, the mechanism of action still remains unknown and it has been demonstrated that some peptides can suppress microbial growth according to an ensemble of different mechanisms resulting in cell death.62

4 SAR of AMPs Structure activity relationship analysis (SAR) plays a major role in the interpretation of the biological activity of any biomolecule. In particular, SAR studies have produced key information relating the structural features to the activity of AMPs showing that these are governed by charge and hydrophobicity, and have also facilitated the design of novel molecules with improved activities. Most studies on AMPs are performed on model membrane systems such as lipid vesicles in order to determine their mechanism of action and to design peptides with improved activity. The main structural features of AMPs are: (i) a positive net charge; (ii) conformational flexibility; (iii) hydrophobicity; (iv) amphipathicity; (v) the Amino Acids, Pept. Proteins, 2018, 42, 190–227 | 199

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presence of a hinge which allows the peptide to flip into the bacterial bilayer. The key to design novel antimicrobial molecules is to understand how AMPs function and how particular structural features contribute to a given mode of action. The aim of most works therefore is to precisely define the effects of structure on their biophysical properties and to relate these to the efficacy and mechanism of action of the peptide. Initially their antimicrobial activity was mainly attributed to their helical structures,68 but it is now widely accepted that both helical/beta structures as well as conformational flexibility are critical features which determine their potency and selectivity. The intrinsic structural flexibility plays a key role in AMPs activity. This feature seems also to be in common with other membrane interacting sequences such as viral fusion peptides;49,69–75 in fact, fusion seems to involve conformationally disordered peptides with pronounced structural plasticity. It is now widely accepted that AMPs interact with the surface of the membrane and this process is coupled with the induction of a secondary structure (helical or beta). Magainins, pleurocidin, PGLa and melittin form highly ordered helices in membrane bilayers, while adopting an extended conformation when in water solution.76–78 Temporins are a family of short amphipathic a-helical AMPs (10–14 residues), containing a large proportion of non-polar residues and only a few cationic amino acids in their structure and are mainly active on Gram-positive bacteria.24 They are characterized by random conformations in water solution and amphipathic a-helical structures in lipid.24 Indolicidin is a cationic antimicrobial peptide isolated from the bovine neutrophils79 which is unstructured in aqueous solution, and folds mainly in b-turn conformations when interacting with bacterial membranes80 or in boat-like structures when binding to zwitterionic micelles.81 Most of the biophysical studies performed on a-helical peptides were done on cecropins (cecropins A and B), which were isolated from the Hyalophora cecropia lysozyme in 1980.82 It was found that in mature cecropins, the polar and hydrophobic side chains at the N terminus were interspaced in a regular pattern;82 this amino acid distribution is likely to stabilize two fully ordered amphipathic a-helices interrupted by a hinge region containing a Gly-Pro sequence and the same structural motif was later observed also in many other AMPs (Fig. 4). Incorporation of the

Fig. 4 Crystal structure of the trimeric anti-microbial peptide channel dermycidin (2YMK). 200 | Amino Acids, Pept. Proteins, 2018, 42, 190–227

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a-helix breaking residue proline plays a critical role on the conformational flexibility of the peptide. Proline residues determine a hinged region that is crucial for antibacterial potency and selectivity;83 probably this is due to the ability of proline-containing peptides to resist selfassociation and interact selectively with the anionic lipids of the bacterial membrane. Moreover, in buforin II, the proline residue has been proposed to drive the translocation necessary for the inhibition of intracellular functions.84 Proline, not only causes a localized disruption of a-helix conformation, but it is believed to have a number of additional effects on cationic a-helical antimicrobial peptide activity: to prevent peptide selfassociation,85 to lead to improved access through bacterial lipopolysaccharides to the bacterial inner membrane,86 to perturb the charged segment in the helical wheel, a key determinant of activity and selectivity,87 to translocate across the inner membrane, opening up intracellular modes of bactericidal action. The several mechanisms leading to cell death (which were described in the previous paragraph) may be discriminated according to the secondary structure of the peptide. A peptide assuming a continuous helix provides less flexibility and may induce a reduced penetration into the lipid bilayer, leading to the disruption of the membrane structure by interaction with the lipid headgroups parallel to the membrane surface, the consequences being a leaky membrane and cell death. On the contrary, helix-bend-helix structure, derived from a central position of the proline residue, favor pore formation. Particularly interesting is distinctin,88,89 which is a peptide derived from the frog Phyllomedusa distincta and consists of two linear peptides linked by a disulfide bridge. Unlike other AMPs, both chains are amphipathic a-helices in aqueous solution and form a non-covalent parallel four helical bundle. Pore formation has been hypothesized and attributed to the tilted orientation of the shorter chain inside the bilayer.88,89 Interestingly, some AMPs produced by bacteria themselves and referred to as bacteriocins are composed of rare amino acids. These AMPs produced by the host bacteria act selectively against a broad spectrum of bacteria without harming the producer. Among them, nisin is produced by Lactococcus lactis, and is able to inhibit growth of a wide range of Gram-positive bacteria; it contains several posttranslationally modified amino acid residues: (a,b)-di-dehydroalanine, (a,b)-8-didehydrobutyrine, m-lanthionine and (2S,3S,6R)-3-methyllanthionine. The presence of the residues which form thioether bridges produces a series of cyclic molecules.90,91 Nisin is characterized by conformational flexibility with no experimental evidence of preferred overall conformation in aqueous solution whereas, in lipophilic solutions it adopts a more rigid, helical structure. Nisin is also widely used in food preservation. As already said another key structural principle is amphiphilicity, which is the ability to adopt a structure characterized by segregation of hydrophobic and hydrophilic (mainly cationic) amino acids spatially organized in discrete domains. For instance, cecropin22 and magainin92 assume an amphipathic a-helical secondary structure;93 bactenecin94 Amino Acids, Pept. Proteins, 2018, 42, 190–227 | 201

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and defensins assume a somewhat rigid anti-parallel b-sheet constrained by disulphide bonds with separated domains of cationic and hydrophobic residues. In particular, defensins are a class of b-sheet AMPs with positive charges and hydrophobic amino acid residues which are held in a three dimensional amphipathic structure by three internal disulphide bonds. Defensins (classified in a, b and y) are able to disrupt the structure of the microorganism bilayer, moreover they can inhibit the synthesis of the DNA, affect the metabolism turnover and possess an ample antimicrobial activity versus several microorganisms, like Grampositive and Gram-negative bacteria, fungi, and viruses.96 Considering the structure of the membrane, peptide amphiphilicity is expected to have a high impact on the membrane affinity and may be involved in determining the kind of mechanism used to lyse the bacterial membrane; amphipathicity drives the partitioning of AMPs into microbial membranes through the hydrophobic surfaces and stabilization of these interactions through electrostatic interactions with head group domains of these membranes. As reported in the previous paragraph, a-helical amphiphilic peptides locate at the membrane surface with their cationic face interacting with polar head groups while the hydrophobic face penetrates into the lipid tail domain causing perturbation at the membrane water interface; this mechanism is usually described by the carpet model. Peptides which are amphiphilic because of the presence of a cationic region followed by a hydrophobic region, usually adopt a transmembrane conformation driven by hydrophobic interactions with lipid tails and they usually oligomerize forming pores into the membrane (barrel stave or toroidal models) (Fig. 2). There are some AMPs which are not folded into regular a-helix or b-sheet structures and are rich of Arg/Lys, Trp, Pro or Cys residues. Interestingly, some of these AMPs are not active on bacterial membranes and they do not display a defined peptide structure and do not possess an amphipathic character, and their antibacterial activity has been attributed to intracellular interactions involving DNA binding.97 It is also possible that a single peptide can interact with a membrane surface in different ways enabling more than one mode of attacking the bacterial cell and this may be correlated also to the lack of membrane selectivity of some AMPs. The presence of cationic residues is also key for the activity and in fact, most AMPs are cationic. However, the overall positive charge is not the only determinant for activity, but the position of the charged residues in the sequence is also fundamental. Moreover, the number of cationic residues also presents a limit beyond which an increased charge does not correspond to increased activity. The basic amino acids Lys and Arg are involved in the interaction with the negatively charged surface of the bacteria. The guanidinium side chain of the Arg allows the formation of multiple interactions compared to single charged side group of the Lys. Moreover, poly-Arg peptides are able to cross membranes more efficiently than poly-Lys suggesting that the guanidinium group is a critical component also for determining the penetration mode. Although many studies have supported the view that there is a preference for Arg 202 | Amino Acids, Pept. Proteins, 2018, 42, 190–227

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residues over Lys in terms of enhancement of antimicrobial activity, still some peptides prefer Lys, suggesting that there is not a general rule.98 The Trp residue has a strong membrane disruption potential thanks to its ability to locate at the interface of the membrane bilayer with the aqueous solution and to anchor the peptide to the surface of the bilayer, which is correlated to its unique side chain containing a flat and rigid indole ring.99 Trp is also widely found in other membrane interacting sequences such as cell penetrating peptides and viral fusion peptides.49,57,59,69 Cys plays a key role in AMPs being involved in the formation of disulfide bridges and plays a role in the achievement of the overall structural fold of the peptide and in its proteolytic stability. Loss of antibacterial activity is observed upon removal of Cys residues; while reduced and oxidized form may present the same activity as in the case of b-defensin100 indicating that the peptide will oxidize and assume the correct folding upon interaction with the membrane bilayer. Pro also plays a key role, as already described, for the formation of a kink in the helix which is fundamental for activity. Pro is a residue which reduces the structural flexibility of the polypeptide chain because of its ring structure and is generally considered a helix breaker. Their high antimicrobial activity has been attributed to their ability to translocate across bacterial membranes followed by intracellular targeting. Apidaecin, drosocin, and Bac7, Bac5 are representatives of Pro-rich AMPs.101 Interestingly, there are several His rich AMPs.102 His-rich peptides have been proved to have enhanced activity (both membranolytic and antimicrobial) at low pH which seem to involve these residues. In fact, His is uncharged at physiological pH while it is fully positively charged at low pH, enhancing the interaction with anionic bacterial membranes in acid conditions. One example of this is constituted by human salivary histatins, which display significant activity against parasites103 or fungi such as Candida albicans and Cryptococcus neoformans.104 Besides these structural features already described, there are AMPs that are naturally found in cyclic conformations (either through disulfide bonds or backbone cyclization). Cyclic AMPs have been proved to be strongly active against different pathogenic bacteria however with poor selectivity. SAR studies have been performed to understand their structural determinants with the intention to improve their therapeutic profiles.105 In conclusion, the activity of AMPs depends on the appropriate balance of hydrophobic versus charged residues, the amphipathicity and the conformational flexibility of the sequence.

5

Design of novel AMPS with improved activity

Understanding structure activity relationships of native AMPs provides templates and essential information for the rational design of easier to Amino Acids, Pept. Proteins, 2018, 42, 190–227 | 203

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produce and/or more potent analogues, representing an emerging strategy against a wide array of pathogenic microorganisms.43 In particular, three dimensional structures of AMPs contribute to a better comprehension of the mechanism of action and can provide multiple information for optimization of metabolic stability, bioavailability and for safety and immunogenicity as well as reduction of production costs, fundamental for pharmaceutical applications. In fact, although AMPs have a great potential as drug candidates, because of their peptide nature they suffer some limitations: they are susceptible to inactivation by environmental conditions (pH, presence of salts and divalent cations, etc.) and action of proteases or other plasma components.106,107 Moreover, they are potentially cytotoxic to human cells, producing in some cases allergies and present high manufacturing costs at large scale.11,108,109 As a matter of fact only a few AMPs reach clinical trials and usually for topical applications mainly because they are toxic when delivered systemically. The wide recent literature devoted to the design of novel AMPs powerful against microbes but non-toxic to mammalian cells demonstrates that this represents a challenging goal of many research groups.11,109 First attempts to develop novel AMPs were mainly performed by trial and error modifications (such as sequence sorting and alanine scanning) of existing natural molecules due to the poor knowledge of their mechanism of interaction with the membranes. Alanine scanning has proved extremely useful for identifying critical residues in drug design.74,110 Subsequently, several bioinformatics tools have been developed with the diffusion of AMP databases (such as AMPer: http://marray.cmdr.ubc.ca/ cgi-bin/amp.pl), which allow to retrieve key biophysical features from the training data set which are then used to predict the antibacterial activity of unknown sequences. More recently with the growing literature describing the mechanism of action of AMPs, rational design has been applied to guide in the design of novel molecules. Rational design strategies include shortening of the native sequence,111 chemical modification of terminal ends of peptides,112 development of analogues containing unnatural amino acids,113,114 modifications of their amphipathic balance.115 Many novel strategies have also been introduced recently, such as polymeric nano-encapsulation,116 synthesis of dendrimeric peptides,117 which have helped in the improvement of peptide stability and reduction of cytotoxicity. Combinatorial chemistry and high-throughput screening are also effective tools which may enable the identification of families of peptides, with broad-spectrum antimicrobial activity. Several papers report on the modulation of the physicochemical parameters such as hydrophobicity, length, helicity and net charge,118,119 showing that peptides with similar physical features may have outstanding different antibacterial activities, determined by their different modes of action. In particular, the design of AMPs with higher potency, necessitates modulation/optimization of length, backbone, side chain, charge, hydrophobicity and amphipathicity. The analogues possess enhanced bioavailability and metabolic stability and retain activity and selectivity profiles of native AMPs.120 204 | Amino Acids, Pept. Proteins, 2018, 42, 190–227

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Modification of secondary structures is critical and the change of the a-helical content in the structure of peptides by covalent modifications has been extensively exploited. The preformation of a-helical structures in solution was proved to be unfavorable in some cases;121 in fact, the formation of the a-helix before engaging membrane interactions may not improve activity; on the contrary, the antimicrobial activity may be attributed to the inducibility of the a-helical conformation in a lipid bilayer rather than to the intrinsic helical stability.118,119,122,123 The preferential binding of cationic AMPs to the negatively charged membrane of bacteria over mammalian cells,124,125 is likely to induce conformational changes and often helix formation,118,119,122,126,127 which might also assist penetration. It is their amphipathic conformation not their secondary structure responsible for the insertion of the hydrophobic face into the bilayer.100,119,122,123 The combination of electrostatic and hydrophobic interactions allows AMPs to be not only highly water soluble but also able to interact with the lipid tails in the bilayer.118,119,122,128 Being the charge fundamental for activity, the substitution of Lys on the polar face with Ala reduces activity. Sometimes, there are some negatively charged residues on the polar face of several AMPs, but this feature is still not understood.118,119,122 The positive charge is the result of a high percentage of Arg and Lys, with Arg rich peptides thought to have stronger interactions with membranes. In fact, as already described, the side chain of Arg residues enhances the action of antimicrobial peptides more than the side chain of Lys, even though this is not a general rule. Accordingly, several Arg-rich AMPs have been designed such as (RW)n.129 The antibacterial activity of dermaseptin S4 against Escherichia coli was enhanced by incorporation of Lys and acylation;130 however, peptide selectivity and antibacterial activity were reduced by the increase of hydrophobicity and of the number of ionic residues.131 Antibacterial and hemolytic activities clearly depend on the type/ percentage of cationic and hydrophobic residues, which is further supporting the view that the appropriate weighing of hydrophobicity, amphipathicity and positive charge is fundamental for augmentation of the therapeutic potential. Hydrophobicity is a feature that strongly affects the interaction with membranes of different compositions and also determines the degree of peptide partitioning into the lipid bilayer. A decrease of hydrophobicity is usually related to a reduction of mammalian cell interactions while positive charges are related to the targeting of bacterial cell membranes. In conclusion, increased hydrophobicity is correlated with loss of antibacterial activity and enhanced toxicity towards mammalian cells and this parameter has to be well tuned when designing novel antimicrobial peptides. For instance, the effect of hydrophobicity was analysed through a set of peptides (KW)n, where the addition of repeats resulted in increase of hydrophobicity and antibacterial activity but with (KW)5 the increase also determined a decrease of antibacterial activity due to greater hydrophobicity and self-aggregation.132 A key role is played by peptide concentration on the membrane surface, thus several approaches have been exploited to boost surface Amino Acids, Pept. Proteins, 2018, 42, 190–227 | 205

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concentration though peptide self-aggregation which leads to more effective membrane disruption compared to monomers as demonstrated also for other membranotropic peptides such as viral peptides.133 The peptides have to self-aggregate in both the pore forming mechanisms and the carpet mechanism, but being highly cationic, the selfaggregation is prevented by electrostatic repulsion. In such cases, covalent linking (cross linking) can be exploited to generate covalent aggregates.43 The conjugation with fatty acids was also exploited to analyse the effect of self-assembling.134,135 In particular, results clearly demonstrated that increased length of the fatty acid tails, enhanced the interactions between the peptide and the membrane and thus increased antibacterial activity; however, longer fatty acid tails induced selfassembling of the peptides into nanofibers which produced reduced antibacterial activities. AMPs could be modified to achieve self-assembling properties which are essentially mediated by intermolecular interactions such as hydrogen bonding, electrostatic force, hydrophobic interaction and p–p stacking interaction. The obtained systems could present physicochemical properties different from their monomers; in particular, AMPs could display unique features such as sustained release and improved stability. Both charges and secondary structures can be modified when self-assembling occurs and could influence the interaction with cell membranes.136 Which is the functional three-dimensional structures of AMPs, is a key information for the rational design of synthetic peptides. Often AMPs are rather large and expensive to synthetize; minimalistic de novo approaches to design model amphipathic helical peptides are important. In fact, most AMPs are expensive to synthetize and there have been several successful examples of peptide engineering to reduce the peptide size with enhanced activity. The eleven-residue fragment of gaegurin 5 (GGN5N11) is inactive but it recovers its antimicrobial activity by a single Trp substitution at the hydrophobic-hydrophilic interface of the amphipathic helix.137 Moreover, ultrashort peptides with only 3–4 amino acids have been designed. Their modes of interaction still involves permeation of the membrane and involves cationic amino acids; most of them are also conjugated to fatty acid tails to provide additional hydrophobicity for antibacterial activity.138 LK peptides are 14- or 15 amino acid sequences composed of only Leu and Lys residues which were shown to possess antimicrobial activity. The introduction of a single Trp residue, stabilizes the helical conformation and enhances membrane interactions; 9 to 11 amino acid LKW peptides exhibited enhanced antimicrobial activity. The specific position of the incorporation was determined from the helical wheel projection, by aids of structural information. Defensins are stabilized mainly by the presence of the three disulfide bonds and their mechanism of action certainly involves contact with and permeabilization of the bilayer. Production costs of human b-defensin are quite high, furthermore human b-defensin 3 (hBD3) is known to be 206 | Amino Acids, Pept. Proteins, 2018, 42, 190–227

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active also at high concentration of sodium chloride which represents an extremely important issue for many pathologies such as cystic fibrosis. Analogues of hBD1 and hBD3 allowed to identify key domains involved in the antibacterial, antiviral and chemotactic activities. The charged C-terminal domain (RRKK) of hBD3 and the internal domain of hBD1 (PIFTKIQGT) were proved to be essential while deletion of the six N-terminal residues of hBD3 did not significantly affect activity.139 These SAR studies allowed the de novo design of a smaller cyclic peptide named AMC which contained both the active domains of hBD1 and hBD3 and showed similar antibacterial, antiviral and chemotactic activities as the native molecules but significantly reduced production costs and greater enzymatic stability.140 In fact, cyclic peptides determine the amphipathicity of the molecule and present greater enzymatic stability and cyclization has been widely considered in the design of novel AMPs.141 For instance, the activity against Gram-negative bacteria of indolicidin was improved and its protease susceptibility decreased through synthesis of a cyclic analog, named CP-11.142 Another widely used strategy is the incorporation of unnatural amino acids which provide enhanced proteolytic stability. A very common strategy is the change from L to D amino acids. D amino acids are very rare in nature and their inclusion is sufficient to change the side chain and backbone properties of the peptide sequence. Chemical modification enables tuning of the proper balance between hydrophobicity and hydrophilicity. It has been reported that modification of Phe with an additional benzene ring significantly enhances the activity of the peptide FRFR-NH2 while maintaining its low toxicity for mammalian cells.143 The most promising modifications include: a-peptoids, b-peptoids, b-peptides, apeptide/b-peptide hybrids, lipo-AA-peptides, and peptide/ peptoid hybrids.120,144–150 The use of b-amino acids involves a change in the backbone of the native structure without changing the side chain chemistry.120 N-substituted amino acids are characterized by the relocation of the side chain from the a-carbon to the nitrogen; decreasing net hydrophobicity and altering surface topology; the modification enhances proteolytic stability and membrane permeability.151 N-alkyl amino acids (e.g. N-methylation) are useful also to scan for positions of functional interest; in fact, their location regulates the degree of protection towards proteolytic agents, and affects the conformational freedom of the peptide backbone. Addition of lipid moieties is another strategy to improve peptide interaction with the bacterial membrane and their overall antimicrobial properties.152 A key role is played by the length of the fatty acid; in fact, too long acyl chains increased aggregation and self-assembly and reduced activity. The ideal fatty acid length is C14–C18 which may also promote formation of secondary structure when in contact with the membrane.135 Modification of the peptide MP196 (RWRWRW) with lipids of different length either at the N or C terminus showed that the C8 chain produced the most effective antimicrobial activity and that the N-terminal lipidation produced not only antibacterial activity but also hemolytic effects.153 Amino Acids, Pept. Proteins, 2018, 42, 190–227 | 207

View Online Table 1 Selection of peptide modifications for rational drug design with possible effect. Arg/Lys substitutions

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D-enantiomers Unnatural amino acids Lipidation

Cyclization

Specificity in membrane targeting (bacterial vs. mammalian) Increased solubility Increased metabolic stability Increased metabolic stability Increased metabolic stability Increased membrane association Control of the peptide orientation inside the membrane Increased metabolic stability Blocking the peptide in the active conformation

The marine environment is rich in potential new molecules with antibacterial activity. Myxinidin is active against a broad range of bacteria and yeast pathogens.154 The alanine scanning analysis155,156 showed that optimized bactericidal potency could be obtained by the addition of a tryptophan residue at the N-terminus and by the simultaneous substitutions of some residues with Arg.155–157 Pexiganan (also known as MSI-78), an analogue of magainin 2, is one of the most widely studied molecules for drug development. The solution structure of pexiganan showed that the peptide forms a dimeric antiparallel a-helical structure in a membrane mimetic environment with the side chains of three phenylalanine residues playing a key role for the selfdimerization and with its orientation in the membrane also being critical to understand the exact mode of membrane interaction. Interestingly, one strategy to modulate activity has been to design AMPs conjugated to antibiotics to induce a synergistic effect and thus enhance antibacterial activity, reduce administration doses and thus lower the risk of adverse side effects. One recent example is the coupling of the cationic peptides UBI (with antibacterial activity against E. coli, S. aureus, P. aeruginosa) and the antibiotic chloramphenicol. The obtained compound showed enhanced activity against E. coli and S. aureus and reduced toxicity against human cells.158 A list of possible modifications are reported in Table 1.

6

Nano-Antimicrobials: delivery systems for AMPs

Recent research is highly devoted to the enhancement of stability, safety, and activity of AMPs via the design of innovative drug delivery systems;152 in fact, these problems may be addressed through the choice of the proper drug delivery system. Rapid peptide degradation and loss of activity is very common in infected tissues which are characterized by: high proteolytic activity mediated by both bacterial and human defence cell proteases; binding to serum proteins following to parenteral administration, with consequent clearance from blood stream circulation; difficulty in reaching bacteria that are localized intracellularly; impossibility to control AMP release at the infection site. In this context, nanotechnology offers the opportunity to target the delivery of AMPs to a specific site with controlled release overtime, thus minimizing sideeffects and increasing activity. Nanocarriers present several unique 208 | Amino Acids, Pept. Proteins, 2018, 42, 190–227

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advantages; firstly they can be produced from a large variety of materials which allows the obtainment of different shapes, sizes and surfaces; secondly they present a large surface area for adsorption/encapsulation and may prevent self-aggregation of the peptides; thirdly, attachment of AMPs to nanoparticles may avoid any dissemination into the environment. In recent years, various nanosystems have been optimized to improve antimicrobial activity and delivery of therapeutic molecules. Lipids, liposomes, polymers, micelles, nanoparticles (NPs), nanocapsules and other colloidal drug delivery systems of dimensions up to a few hundred nanometers can be loaded with AMPs and exploited to deliver them to infected sites. The key role of the nanosystem is to reduce chemical or biological degradation of AMPs, thus increasing the concentration at the site of the infection for an improved activity at lower doses, and a reduction of the side effects, controlling the release, allowing the biofilm penetration. Interestingly, most nanocarriers may present antimicrobial activity on their own, thereby enhancing the effect of the AMPs. A key feature of nanomaterials is the multivalency; and in fact, several examples of AMP dendrimers (branched polymers with peptides attached centrally to a core matrix) endowed with antimicrobial activity have been reported.104,128–130,159 Multivalency of dendrimers is critical for the design of novel AMPs because they amplify both cationic charges and hydrophobic clusters as the numbers of branches increases, mimicking the mechanism used by AMPs.117 SB041 is a tetra-branched antimicrobial peptide in which four identical peptides were coupled to a lysine core and was proved to be effective against Gram-negative bacteria.160 Preferred nanocarriers are biocompatible and biodegradable materials such as cellulose, chitosan, hyaluronic acid, polylactic-co-glycolic acid (PLGA), and polylactic acid (PLA). For example, hyaluronic acid nanogels were effectively used to encapsulate an analogue of LL-37 (LLKKK18) and showed enhanced activity against mycobacteria compared to the peptide alone both in vitro and in vivo and ability to stabilize the peptide against proteolytic degradation and reduce toxicity. Chitosan coated and colestin loaded PLGA nanoparticles were used for pulmonary delivery for the treatment of cystic fibrosis, where transport through the mucus layer and eradication of Pseudomonas aeruginosa biofilm is extremely difficult.161 Inorganic nanomaterials have attracted significant attention with numerous nanomaterials displaying antimicrobial activities also on their own which may provide additive or synergistic effects. Mesoporous silica nanoparticles (MSN) allowed fine-tuning of the release pattern of AMPs and in fact surface properties and porosity were shown to influence the distribution of LL-37, which had a significant impact on membrane adsorption, antimicrobial activity, toxicity and stability against proteolytic degradation. LL-37 and the small antimicrobial compound chlorhexidine were also incorporated into mesoporous silica monoliths.162 They both were characterized by a slow drug release (approximatively 200 h) and a high antibacterial activity Amino Acids, Pept. Proteins, 2018, 42, 190–227 | 209

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against S. aureus and E. coli but the nanosystem containing LL-37 showed a significant lower toxicity against human cells. Paramagnetic silica nanoparticles functionalized with magainin-I have been used to obtain antibacterial and magnetic properties and may find an application in the disinfection of aqueous solutions or in vivo for localized antibacterial action exploiting the magnetic field.163 Also metal nanoparticles (e.g. Ag, Au and Cu nanoparticles) offer interesting opportunities of development. Unlike traditional antibiotics, many antimicrobial nanomaterials based on metal nanoparticles, have shown significant toxicity towards a broad range of microorganisms with low toxicity to humans; moreover, many traditional disinfectants are toxic to humans because of their non-specific action against the specific microorganism and thus they may be harmful for the environment (plants and animals).164–168 The antimicrobial properties of some metals and metal compounds have been exploited since ancient times when Ag-based compounds were used for water treatment and wound healing. Their distinctive antimicrobial activity may be attributed to different mechanisms such as a large contact area with bacteria that leads to direct membrane lysis and damage of respiration functions, binding to sulfhydryl groups of metabolic enzymes, binding to microbial DNA, release of toxic metal ions, generation of reactive oxygen species.58,168,169 Today the most widely commercially used antimicrobial nanomaterials are Ag nanoparticles which are effective against a broad range of bacteria. Decoration of the nanoparticle surface with the peptide may not only produce increased activity but also determine a reduction of the eventual toxicity of silver thanks to the protecting covering effect of the AMPs. As an example, silver nanoclusters conjugated with daptomycin improved bacterial killing.170 Ag nanoparticles functionalized with polymixin B displayed higher antimicrobial activity against Vibrio fluvialis and P. aeruginosa.171 Antimicrobial gold nanodots were prepared immobilizing surfactin (SFT) on Au nanodots.172 The synergistic effect of peptides and gold nanodots enhanced antibacterial activity by disruption of the membrane in vitro; the nanosystem was also tested in vivo and showed activity against MDR S. aureus infections; moreover, it promoted collagen production in wounded skin of rats infected with MDR S. aureus. Au-indolicidin nanoparticles were used against S. cerevisiae and showed to have potential clinical applications because of their lower toxicity to mammalian cells.173 Quantum Dots (QDs) coated with polymyxin B displayed higher antimicrobial activity against E. coli compared to the peptide alone.174 The investigation of the antimicrobial activity and toxicity of QDs in presence and absence of indolicidin coating demonstrated improved antibacterial activity against S. aureus, P. aeruginosa, E. coli and K. pneumoniae and reduced toxicity against Daphnia magna;175 although the results of the study suggest that effects of chronic exposure cannot be ignored.176 Results of this study show the importance of testing multigenerational impacts of nanomaterials and of acquiring toxicity information on 210 | Amino Acids, Pept. Proteins, 2018, 42, 190–227

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nanomaterials impact on early developmental stages of an organism and future generations.176 The production of self-disinfecting surfaces, such as children toys, medical devices or hospital surfaces has become a priority. One main area of research is devoted to contaminated surfaces such as medical devices (prostheses, catheters etc) which constitute a significant source of possible infections for humans. Nosocomial infections are of particular concern, mainly because most hospital-acquired bacterial infections are due to multi-resistant bacteria, and spreading of antibiotics on all available surfaces in the hospital environment, would only increase the emergence and diffusion of multi-resistant microorganisms. The use of AMPs, in such cases, may be the optimal solution to prevent the attachment of microorganisms to surfaces. In fact, the broad-spectrum antimicrobial activity displayed by many AMPs and their activity even at low concentrations make AMPs excellent candidates for the coating of medical devices and implants.177 Ocular infections related to the use of contact lens are extremely frequent and of difficult therapeutic resolution. Contact lenses containing the peptide melimine on their surface have been developed.178 Melimine contains the active regions of protamine (from salmon sperm) and melittin (from bee venom). Melimine-coated contact lens produced a significant reduction of microbial activity against fungi Acanthamoeba and multidrug resistant S. aureus and P. aeruginosa with no effect on the lenses parameters (such as diameter, central thickness or curvature). Two short RKW-rich AMPs (namely, RK1 and RK2) were used for the surface coating of silicone to be used to prevent catheter-associated infections.179 The immobilized peptides showed good antibiofilm activity, and a considerable activity against E. coli, S. aureus and Candida albicans. An interesting attempt to incorporate additives containing AMPs that may be compatible with water-dispersed acrylate coatings (latex paint), showed a good antibacterial activity and the possibility of a broad spectrum of applications.180 An important challenge in orthopedic surgery and dental care is the prevention of bacterial colonization and biofilm formation; a stable immobilization of GL13K to titanium dental implants was developed against Porphyromonas gingivalis.181 GL13K is a peptide derived from the parotid secretory protein with bactericidal activity in vitro and anti-endotoxin activity in vivo. Titanium coated with GL13K showed bactericidal activity and significant cytocompatibility with osteoblasts and human gingival fibroblasts. A multilayered coating with HHC-36 on titanium showed a controlled release of AMP for the prevention of implant-associated infections in orthopedic surgery against both Gram-positive and Gram-negative bacteria.182

7

AMPs with other activities

Besides their antimicrobial activity, some AMPs also have other key roles in immune regulation, inflammation, anticancer, sepsis and wound healing.183 Amino Acids, Pept. Proteins, 2018, 42, 190–227 | 211

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Their activities include stimulation of chemotaxis, modulation of immune cell differentiation, initiation of adaptive immunity, contribution to bacterial clearance. LL-37 and bovine lactoferricin inhibit the LPS(TLR4)-induced secretion of TNF-a and IL-6 in THP-1 cells and LL-37 suppresses the LTA (TLR2)- and LPS(TLR4)-induced production of TNF-a, IL-1b, IL-6, and IL-8 in primary monocytes.1 Cancer cells are characterized by a higher content of anionic lipids (phosphatidylserine), thus opening a new opportunity for the application of cationic AMPs which may be exploited as novel drugs with cancer-selective activity which may also avoid resistance mechanisms.152,184,185 It is possible to improve the therapeutic index of current chemotherapeutic agents, combining AMPs with a chemotherapeutic agent which may help in reducing the dosage and thus result in lower toxicity. An example of this application is provided by the antimicrobial peptide KLA ([KLAKLAK]2) that was shown to have also anticancer activity, destabilizing the mitochondrial membrane potential and triggering apoptotic cell death programs.186 Hybrid cecropinmelittin peptides were used in combination with the chlorambucil. The coupling increased cytotoxicity against several cancer cell lines between six and nine fold.187 LL37 is in Phase 2 clinical trial for melanoma (lesions at least 10 mm and not completely resectable) by intratumoral injections in patients with no known immune deficiency; LTX-315 is in phase 1 trial for treatment of multiple types of transdermally accessible tumors.184

8 AMPs in clinical applications AMPs are promising candidates for therapeutic applications. AMPs perform many actions, which may represent an advantage in therapeutic cases such as complicated skin and soft tissue infections, where both Gram-positive and Gram-negative organisms are involved (for an updated database of AMPs see DRAMP: http://dramp.cpu-bioinfor.org/). Nevertheless, only a few are approved for clinical use. In spite of the large number of patents or related scientific articles, there is still a significant divergence between the list of AMPs and the real outcomes of clinical trials. The rational design of AMPs usually starts with in vitro screening using standard MIC or MMC assays, which is often followed by in vivo assays. However, the activity is highly sensitive to environmental conditions, which results in poor correlation and also controversial data between in vitro vs. in vivo activity and makes any prediction very difficult. Peptides may be inactive or modestly active in vitro while showing a relevant activity when assayed in vivo in experimental animal models; in in vitro assays, the mammalian ionic environment is not replicated and the bacterial susceptibility to AMPs may be significantly reduced. On the other hand, AMPs with high in vitro activities may lack activity in vivo because of their rapid proteolytic degradation and/or protein binding in the body. The low metabolic stability may also reduce their clinical applicability; peptides are characterized by low oral bioavailability because of pre-systemic enzymatic degradation 212 | Amino Acids, Pept. Proteins, 2018, 42, 190–227

View Online Table 2 AMPs in the market. Antimicrobial peptide/commercial name

Published on 29 November 2017 on http://pubs.rsc.org | doi:10.1039/9781788010627-00190

Daptomycin/Cubicin

Colistin/Coly-Mycin M Parenteral and Colomycin injection Gramicidin S

Nisin

Indication

Company

Intravenous administration Cubist MDR Gram-positive infections Pharmaceuticals/ Novartis Intravenous administration Parkdale MDR Gram-positive and Pharmaceuticals Gram-negative infections and Xellia Topical use Gram positive and Gramnegative infections Food preservative DuPont Gram positive bacteria

and poor penetration of the intestinal mucosa, which makes oral administration usually impossible. Furthermore, systemic administration through intravenous injection is limited by their rapid degradation by proteolytic enzymes in blood plasma and rapid removal from the circulation by the liver (hepatic clearance) and kidneys (renal clearance).1,188 Thus, local application (creams, emollients, nasal sprays etc.) constitutes the preferable administration route.1 However, tissue proteolytic enzymes can degrade peptides even upon local delivery. Unfortunately, only a few antimicrobial peptides are approved and commercialized so far, despite the excellent results obtained in in vitro analysis. Many AMPs with therapeutic potential based on magainin, protegrin, indolicidin and histatins have entered clinical trials, but only a few have attained the approval from the Food and Drug Administration (FDA).12 In particular, only four AMPs have been approved and are actually on the market (Table 2). These are daptomycin, colistin, gramicidin S and nisin. Daptomicin is a lipopeptide for systemic and life-threatening Gram-positive bacterial infections that was first approved for clinical use in 2004 in the US. It is marketed under the trade name Cubicin by Cubist Pharmaceuticals in US and by Novartis in EU.189 Colistin is also a lipopeptide belonging to the polymyxin group with activity essentially against Gram-negative organisms.190 Two forms are commercially available: colistin sulfate produced by Parkdale Pharmaceuticals (Coly-Mycin M Parenteral) and colistimethate sodium manufactured by Xellia (Colomycin injection). Gramicidin S is a cationic anti-parallel b-sheet cyclic decapeptide. It is applied in the treatment of infected surface wounds and nasal ocular and throat infections.191 It is hemolytic at even low concentrations, thus it is only used as topical applications. Gramicidin S has been used as a spermicide and therapeutic for genital ulcers produced by sexually transmitted disease. The last compound on the market is nisin.90 Nisin is a polycyclic antibacterial peptide which is active against several Gram-positive bacteria. It has been approved as an additive for food use in the USA in the late 1960s and is owned by DuPont. Amino Acids, Pept. Proteins, 2018, 42, 190–227 | 213

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Here we describe a selection of AMPs under clinical development for treatments against various bacterial pathogens (Table 3). One of the first AMPs to be developed for therapeutic purposes was an analogue of the Xenopus laevis peptide magainin, named Pexiganan (MSI-78) (Dipexium Pharma/MacroChem/Genaera).192–194 Pexiganan is a 22-amino-acid peptide in phase III clinical trials as a topical cream for treatment of diabetic foot ulcers. Although additional clinical trials are currently ongoing, after completion of phase III it has not yet received the FDA approval. Another AMP currently in phase III is Omiganam (Micrologix Biotech). Omiganam is an a-helical peptide (MBI-226) derived from indolicidin which was isolated from bovine neutrophils;195 it has proved to be effective as a broad antibacterial in vitro, and has been assessed as a topical cream for catheter infections (phase III) and rosacea (phase III) (Mallinckrodt/Cutanea Life Sciences, Inc.). Phase III clinical trials failed to significantly reduce venous catheter-related bloodstream infections, although a good reduction of colonization of the catheter itself was obtained; therefore it may represent a valid option for direct sterilization of medical devices. Another application in clinical trial is as topical cream for the treatment of usual type vulvar intraepithelial neoplasia/ moderate to severe inflammatory acne vulgaris/mild to moderate atopic dermatitis (Cutanea Life Sciences, Inc.).195,196 Another indolicidin analog is MBI 594AN, which was designed for the topical treatment of acne. Preclinical studies showed a high activity against Propionibacterium acnes, both sensitive and antibiotic-resistant strains in vitro. Topical application of the cream containing 2.5% of MBI 594AN twice a day is in phase II clinical study and seems to be useful to decrease symptomatology.197 SGX942 (also referred to as SGX-94) contains the 5-amino acid peptide dusquetide which is an analogue of indolicidin having higher solubility and stability (Inimex and subsequently Soligenix). It presents broadspectrum activity against Gram-negative and Gram-positive, intracellular or extracellular bacteria and presents also synergic activity with standard antibiotics.198 It received Fast Track designation from the FDA for the treatment of oral mucositis as a result of radiation and/or chemotherapy treatment in head and neck cancer patients.199 LTX-109 (Lytix Biopharma) is a synthetic peptide, used for local application in uncomplicated Gram-positive skin infections, impetigo, and in nasal eradication of staphylococcus. It is effective against a broad range of bacteria including E. coli and S. aureus200 and also against a panel of drug-resistant Gram-positive bacteria and is in phase II clinical trials.201 hLF-11 (AM Pharma) is a cationic fragment comprising the first eleven N-terminal amino acids of human lactoferricin, used for intravenous treatment of life-threatening bacterial and fungal infections in immunocompromised stem cell transplant recipients. PXL01 (Pergamum AB) is another peptide derived from human lactoferricin; the hyaluronic acid-based gel formulation has been evaluated in phase II clinical trials for prevention of post-surgical adhesion formation in hand surgery.1,202 214 | Amino Acids, Pept. Proteins, 2018, 42, 190–227

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Table 3 Selection of AMPs in clinical trials or development.

Amino Acids, Pept. Proteins, 2018, 42, 190–227 | 215

Antimicrobial peptide

Indication

Phase

Company

Pexiganan (MSI-78) a derivative of magainin

Topical cream for diabetic foot ulcers

III

Omiganam (MBI-226) an analogue of indolicidin

Topical cream for the treatment of skin antisepsis, prevention of catheter infections/Rosacea

III

Topical cream for the treatment of usual type vulvar intraepithelial neoplasia/moderate to severe inflammatory acne vulgaris/ mild to moderate atopic dermatitis Topical treatment of acne Treatment of oral mucosis due to radiation and/or chemotherapy in head and neck cancer Topical antibiotic for the treatment of nasal carriers of MRSA/ Topical cream for the treatment of infections due to Gram-positive bacteria Intravenous administration for the treatment of neutropenic stem cell transplantation patients. Prevention of bacteraemia and fungal infections Prevention of post-surgical adhesion formation in hand surgery Treatment of meningococcal septicemia Brush on treatment for fungal infections of the toenail Treatment of chronich leg ulcers

III

Dipexium Pharma/ MacroChem/ Genaera Mallinckrodt/ Cutanea Life Sciences, Inc. Cutanea Life Sciences, Inc.

MBI 594AN an analogue of indolicidin SGX942 (SGX-94) contains dusquetide an analogue of indolicidin LTX-109 synthetic AMP

hLF-11 a derivative of lactoferrin

PXL01 a derivative of lactoferrin Neuprex Novexatin synthetic AMP designed on defensins LL-37

II Fast Track

Inimex /Soligenix

II

Lytix Biopharma

I/II

AM Pharma

II

Pergamum AB Xoma ltd NovaBiotics Pergamum AB

I/II II

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Table 3 (Continued) Antimicrobial peptide

Indication

Phase

Company

Iseganan (IB-367), a derivative of protegrin 1

Mouthwash for the treatment of chemotherapy induced oral mucositis

III

Aerosol for the treatment of infections of cystic fibrosis patients

III

POL7080 a cyclic analogue of protegrin PAC-113 (P-113)

Treatment of non-cystic fibrosis broncjiectasis Oral gel for treatment of candidosis of HIV infected patients

II I/ II

PL-5 synthetic peptide

Skin infections

DPK-060 Brilacidin, (PMX-30063), a defensin mimetic

Topical application for atopic dermatitis Intravenous administration for the treatment of acute bacterial skin and skin structure Infection caused by Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA) Oral rinse for the treatment of ulcerative mucositis associated with chemo/radiation therapy of cancer

II III

Ardea Biosciences/ National Cancer Institute IntraBiotics Pharmaceuticals Polyphor/Roche Periodontics/Pacgen Biopharmaceuticals ProLight Pharmaceuticals Pergamum AB Cellceutix

II

Cellceutix

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It shows inhibitory activity against most important targets of adhesion formation by inhibiting secretion of proinflammatory cytokines while promoting fibrinolysis. PXL01 regulates the production of the mucinous glycoprotein lubricin in surgery, providing a mechanism to enhance its adhesion-preventive properties.202,203 Neuprex, (Xoma ltd) is derived from the cleavage of the bactericidal/ permeability increasing protein (BPI); it was used in a study on children affected with severe meningococcal septicemia and results were very promising.204 Novexatin (NovaBiotics), is a synthetic cyclic and highly positively charged peptide based on human a- and b-defensins, targeting stubborn toenail fungal infections. Cathelicidins are widely exploited because they are able to reduce the effects of septic shock in mouse models of staphylococcal sepsis.205 Human cathelicidin is synthesized as an inactive precursor, which is made of a highly conserved N-terminal signal sequence, a conserved cathelin domain, and a small antimicrobial C terminal domain. The small antimicrobial C-terminal domain is known as LL-37 (Pergamum AB) and is effective in mouse urogenital tract infections due to E. coli.206 LL-37 is undergoing phase II for treatment of chronic leg ulcers. The clinical phase results show that LL-37 has a significantly improved healing rate compared to placebo. The mechanism of wound healing promoted by LL-37 is not fully understood, and probably involves several wound repair processes such as re-epithelialization, angiogenesis, and inflammation. Re-epithelialization is stimulated by the chemoattractant effects of LL-37 on epithelial cells while vascularization is thought to be regulated by LL-37 stimulating endothelial tube formation and production of angiogenic factors.207 Iseganan (Ardea Biosciences) is a synthetic analogue of protegrin I (IBL-367)208 which is in phase III clinical trials for patients in radiation therapy and affected by oral mucositis as a mouth wash. A further phase III clinical study involved cystic fibrosis patients suffering from lung infections which were treated with aerosolized Iseganan. Both studies did not prove a significant efficacy. A cyclic analogue of protegrin I named POL7080 (Polyphor and Roche) is active against Gram-negative bacteria and its mechanism of action implicates the inhibition of a homolog of the b-barrel protein LptD which is involved in the assembly of LPS in the outer leaflet of the outer membrane. POL7080 is highly active on many clinical isolates including the multi-drug resistant Pseudomonas. It showed remarkable in vivo efficacy in septicemia, lung and thigh infection models. In 2015, POL7080 has competed a phase II trial in 20 patients with non-cystic fibrosis bronchiectasis.199 Roche discontinued its involvement and thus its development has been terminated during phase II. The peptide PAC-113 (P113) (Periodontics/PACgen Biopharmaceuticals), a derivative of histatin 3 and histatin 5, has been used in HIV infected patients having oral candidosis as a component of a mouthwash in the phase I/II of clinical trials. It was shown to decrease the symptoms of gingivitis and gum bleeding without any side effects. Actually, there Amino Acids, Pept. Proteins, 2018, 42, 190–227 | 217

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are significant data supporting its use for the treatment of lung infections caused by P. aeruginosa.209 PL-5 (ProteLight Pharmaceuticals) is an a-helical peptide which obtained approval from the China Food and Drug Administration (CFDA) to enter clinical trials for skin infection. It presents low toxicity and highly potent activity against a broad spectrum of drug-resistant bacteria, and also shows synergic activity with conventional antibiotics and improves antibacterial activity in vitro and in vivo against Gram-positive and Gram-negative bacteria.210 DPK-060 (Pergamum AB) is a cationic peptide with broad spectrum activity against both Gram-positive and Gram-negative bacteria; it has recently completed a Phase II studies for topical application in atopic dermatitis; moreover, Phase II clinical trials have been performed for outer ear infections and showed a significant improvement in a 10-day cure rate compared to placebo and also showed safety and tolerability; however, no recent reports of DPK-060 development have been forthcoming.211 Brilacidin (PMX-30063) (Polymedix Inc. and subsequently Cellceutix corp.) is an arylamide analogue of defensins with high antimicrobial activity against a broad range of drug-susceptible and multidrug-resistant Gram-negative and Gram-positive bacteria.212 Brilacidin has completed phase III for the treatment of acute S. aureus skin infections; it shows minimal irritation and is equally efficacious as vancomycin;213 and phase II trials for ulcerative mucositis associated with chemo/radiation therapy of cancer.

9

Conclusion

As a result of the continuous increase of bacterial pathogens resistant to many antibiotics, search for drugs that use alternative mechanisms of action has become an urgent imperative. In recent years, AMPs have stepped forward as an important alternative to conventional antibiotics and technological development enabled a multidisciplinary approach that provided a valuable insight for the discovery of new AMPs for therapeutic use. Since their discovery, no other class of compounds has shown the same versatility as multifunctional compounds against polymicrobial infections (bacterial and viral) and cancer. A selection of peptides in the drug pipeline and currently in clinical use have been described, and this represents a good promise for the near future. In general, AMPs show different folding and a high dynamic flexibility that allows them to structure in the presence of lipids, modifying their mechanism of action. The biological properties are determined by their noticeable amphipatic behavior and structure-activity relationship studies have shown that it is possible to chemically modify natural sequences to obtain AMPs with improved antibacterial activity and lower cellular toxicity. The multifunctionality determined by a typical AMP structure suggests that no single property is completely optimized in natural AMPs and rational peptide engineering is essential to AMP development for 218 | Amino Acids, Pept. Proteins, 2018, 42, 190–227

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clinical applications. A key point is to dissect the structural determinants of each property to uncouple each function for optimization of specific applications; moreover, these studies need to be conducted in a systematic way to allow significant exploitation of the clinical potential of AMPs as a novel class of therapeutic agents. Notwithstanding the recent advances, the number of AMPs in clinical use is still low; in fact, translation into effective antibiotics requires the overcoming of important issues such as the high cost of production and the marked sensitivity to proteases that generally makes peptide drugs poorly stable, toxicity and serum binding. These problems can, however, be overcome by a rational design increasingly focused on defining formulations and delivery systems that can be effective in vivo and minimize toxicity. This will enable a rapid development of AMPs to eventually represent the alternative to conventional antibiotics we all are eagerly awaiting. Unfortunately, microbial resistance is also not a negligible factor. As a matter of fact, bacterial resistance development toward AMPs is also starting to be observed; it was believed that bacterial resistance was more difficult to develop because of the AMPs’ mechanism of action involving attacking multiple rather than one defined, high-affinity target, typical of conventional antibiotics. In fact, being the bacterial membrane the primary target of AMPs, this renders difficult for microbes to preserve the membrane functionally and structurally and at the same time avoid AMPs activity; however, recently, it has been reported that bacteria may develop resistance toward AMPs; further studies concern the potential that AMP therapy induces cross-resistance toward AMPs that are effectors of our innate immune system, and thus compromising our natural defence against pathogens.16

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Advances in the design of ligands interacting with 3CL protease of novel coronaviruses causing infectious respiratory syndrome Kenichi Akaji DOI: 10.1039/9781788010627-00228

Two newly isolated coronaviruses (CoVs) cause the severe pneumonia-like respiratory illnesses, Severe Acute Respiratory Syndrome (SARS) and Middle East Respiratory Syndrome (MERS). Neither therapeutic agents nor vaccines have been developed thus far, and even future pandemics of related infectious diseases are expected through zoonotic virus infections. Since the 3C like (3CL) protease of SARS/MERS CoV, which has structural similarities with the 3C protease of rhinovirus causing common cold in humans, is essential to the proliferation of SARS/MERS CoV, inhibition of the 3CL protease (3CLpro) is thought to be an ideal target for the development of therapeutic agents against SARS and MERS. This article describes the recent achievements in the development of inhibitors of the SARS/MERS 3CLpro mainly based on two different approaches: one by combining a peptide-like structure with a reactive functional group, a so-called ‘‘warhead,’’ and a second one by combining virtual screening and high-throughput screening of a real compound library. A recent approach based on the structure-based rational design of a novel inhibitor scaffold for 3CLpro is also included.

1

Introduction

Coronaviruses (CoVs) are enveloped, positive-strand RNA viruses that infect various vertebrates including bats, poultry, and humans. The name ‘‘coronavirus’’ is derived from the crown-like spikes on their surface. There are four main sub-groups of coronaviruses based on phylogenetic analysis of the genome, known as alpha, beta, gamma, and delta. Infectious bronchitis virus (IBV) is the first coronavirus discovered and was isolated from chicken embryos in 1937.1 In the 1960s, two human coronaviruses, human alpha coronavirus 229E (HCoV-229E) and human beta coronavirus OC43 (HCoV-OC43), were discovered.2,3 These human coronaviruses usually cause disorders such as common cold and/or respiratory illnesses of mild to moderate severity. In 2003, a new human beta coronavirus (Severe Acute Respiratory Syndrome or SARS CoV) was identified.4–6 Recently, three additional human coronaviruses were identified: alpha coronavirus NL637–9 and the beta coronavirus HKU110,11 and MERS-CoV (also known as HCoV-EMC, the coronavirus that causes Middle East Respiratory Syndrome, MERS)12,13 (Fig. 1). Among these human CoVs, SARS- and MERS-CoV, in contrast to HCoV-229E and HCoV-OC43, cause a life-threatening atypical pneumonia, termed severe acute respiratory syndrome. The origin of both SARS- and MERS-CoV are strongly suspected to be zoonotic viruses that infect bats or camels. Department of Medicinal Chemistry, Kyoto Pharmaceutical University, Japan. E-mail: [email protected] 228 | Amino Acids, Pept. Proteins, 2018, 42, 228–279  c

The Royal Society of Chemistry 2018

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Fig. 1 Structure of MERS-CoV: S, spike protein; M, membrane protein; E, envelope protein; N, nucleocapsid protein.

In 2003, SARS spread worldwide from its likely origin in southern China in a short period of time, showing that the causative CoV, SARS-CoV, is a great threat to human health. The SARS epidemic affected about 8500 patients with more than 800 fatalities, which boosted coronavirus research in all directions. Nevertheless, in 2012, a new respiratory illness similar to SARS was identified in Europe and the Middle East. This respiratory syndrome called Middle East Respiratory Syndrome, MERS, had affected more than 1800 patients with a fatality rate of 36%. Even after these pandemics, no effective therapy exists for infections with these coronaviruses which may cause re-emergence of SARS/MERS or other related severe diseases. SARS- and MERS-CoVs recognize a specific receptor on the host cell membrane using the spike (S) protein of the virus. Angiotensinconverting enzyme 214 (ACE2) is a functional receptor for the SARS CoV, and dipeptidyl peptidase 415 (DPP4, also known as CD26) is a functional receptor for the MERS CoV. Interaction of the S1 domain of the viral S protein with the host cell receptors is followed by membrane fusion of the virus and host cell to transport the virus RNA into the host cell. Thus, anti-ACE2 or anti-DPP4 antibodies block viral infections of the host cells, strongly suggesting that agents such as a corresponding soluble receptor (ACE2 or DPP4) or its antibody could be promising inhibitors of the viruscell interactions.14,15 The viral genome is translated and processed to virus-derived structural proteins such as spike (S), envelope (E), membrane (M), and nucleocapsid (N) proteins. A set of nonstructural proteins including virus-derived proteases are also processed by the viral protease. Thus, inhibitors of the processing reaction required to yield proteins necessary for viral replications are other promising agents to suppress the proliferations of the infectious viruses. In this review article, recent efforts to develop therapeutic agents for SARS/MERS and related respiratory syndromes are discussed focusing on a coronavirus-specific protease, 3CL (chymotrypsin like) protease. The contents cover the following headings: 1. Introduction (this section) 2. Coronavirus 3CL protease 2.1. 3CL protease of SARS-CoV and MERS-CoV Amino Acids, Pept. Proteins, 2018, 42, 228–279 | 229

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2.2. Crystal structure of 3CL protease 2.3. Maturation of the 3CL protease and enzymatic activity 3. Inhibitors of 3CL protease 3.1. Peptide-mimetic inhibitors 3.2. Small-molecule inhibitors 4. Conclusion In a recent overview regarding SARS-CoV 3CL protease inhibitors,16 a cumulative source of SARS 3CL protease inhibitors is described in detail. Thus, in this article, the author will focus on several typical inhibitors based on the inhibitory mechanism as well as the structural interactions with the 3CL protease. First, the protein chemistry of the corona virus 3CL protease is described as the base of structure analyses of proteaseinhibitor interactions.

2

Coronavirus 3CL protease

2.1 3CL protease of SARS-CoV and MERS-CoV The 29.7 kb positive-strand RNA genome of SARS-CoV is complexed with the basic nucleocapsid (N) protein to form a helical capsid (Fig. 1). The virus membrane is complexed with three viral proteins: a glycoprotein called spike (S) protein, a membrane-spanning protein called membrane (M) protein, and a hydrophobic envelope (E) protein that covers the entire structure of the CoV. The SARS-CoV genome contains two open reading frames (ORFs 1a and 1b; Fig. 2) encoding two large replicative polyproteins, pp1a (486 kDa) and pp1ab (790 kDa).17,18 Expression of the ORF1b-encoded region of pp1ab is involved in the ORF produced by ribosomal frameshifting into the 1 frame just upstream of the ORF1a translation termination codon. The pp1a and pp1ab polyproteins are processed by a viral protease to yield the functional components. SARS-CoV encodes two proteases for this processing: papain like cysteine protease (PLpro) and 3C-like cysteine protease (3CLpro, also called main protease, Mpro). The name of 3C-like comes from picornavirus 3C proteases encoded on the non-capsid region 3C since the substrate specificity of both proteases is similar.19 PLpro cleaves at three sites in the N-terminal region of pp1a/pp1ab, and 3CLpro

Fig. 2 SARS-CoV genome organization and expression. 230 | Amino Acids, Pept. Proteins, 2018, 42, 228–279

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20

cleaves at eleven sites in the C-terminal region (Fig. 3). The SARS-CoV 3CLpro cleaves the precursor protein at three times more sites than PLpro. In addition, 3CLpro is indispensable for viral replication but not found in the host cell.21,22 These features of the SARS 3CLpro make it as an ideal target for antiviral agents. SARS 3CLpro consists of 306 amino acid residues and is a cysteine protease containing the catalytic dyad defined by His41 and Cys145 (Fig. 4). The N-terminal part (1-184, domains I and II) is composed of a two-b-barrel fold similar to that of a chymotrypsin-type protease. The C-terminal part (201–303, domain III) containing five a-helices takes on a globular fold (details of the three-dimensional structures are discussed in the following section). Enzyme activity-concentration relationship studies support the proposal obtained by previous studies on other CoVs that the 33 kDa monomer of the SARS 3CLpro shows remarkably weak enzymatic activity, whereas the dimer of the 3CLpro is the active form.

Fig. 3 Cleavage sites of PLpro and 3CLpro in the two SARS-CoV precursor proteins pp1a and pp1ab.

Fig. 4

Amino acid sequence of SARS 3CLpro. Amino Acids, Pept. Proteins, 2018, 42, 228–279 | 231

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Most CoV derived 3CL proteases have a conserved (Leu/Ile)-Gln-k-(Ser, Ala, or Gly) core sequence (canonical sequence, the cleavage site is indicated by -k-). In addition to the canonical sequences, SARS 3CLpro recognizes three noncanonical cleavage sites with Phe, Val, or Met in the P2 position and one non-canonical site with Asn in the P1 0 -position.23 Comparison of cleavage efficiencies of synthetic substrates containing the eleven cleavage sites of SARS 3CLpro confirmed that the most suitable substrate is the P1/P2 site, the N-terminal site of the SARS 3CLpro itself. The second one derives from the P2/P3 site, the C-terminal site of the SARS 3CLpro, suggesting that SARS 3CLpro cleaves itself most efficiently. The maturation process of the protease will be discussed later. It was also confirmed that Phe in the P2 position can be a substrate similar to the canonical substrate with Leu in the P2 position, indicating that the corresponding S2 pocket of the SARS 3CLpro is rather large and hydrophobic. SARS 3CLpro uses its thiol group as a nucleophile for proteolysis (Fig. 5). The initial step is deprotonation of Cys145-thiol by an imidazole group of His41. The resulting nucleophilic sulfur atom attacks the substrate carbonyl carbon. Thus, SARS 3CLpro shows the highest enzymatic activity at around pH 7 and the activity decrease to 10% at pH 5 because of the protonation of His41. Then, the N-terminal substrate fragment is released from the enzyme, while the imidazole group of His41 is restored to the deprotonated form. Next, the resulting thioester consisting of the

Fig. 5 Hydrolysis of the substrate at the active site of SARS 3CLpro. 232 | Amino Acids, Pept. Proteins, 2018, 42, 228–279

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enzyme and the C-terminus of the substrate is hydrolyzed by nucleophilic attack of a deprotonated water molecule. The following release of a carboxylic acid regenerates the free enzyme. Thus, compounds containing a warhead interacting with the thiol group of Cys145 could be a promising agent against SARS. MERS 3CLpro is closely related to SARS 3CLpro. It is a cysteine protease consisting of 306 amino acid residues, and like SARS 3CLpro, it has three domains. The catalytic dyad Cys148-His41 and the substrate binding site are located in the cleft between domains I and II in the active dimer form. Substrate specificities are also similar to other b-CoVs. The enzymatic activity however is low and it is 5-fold less efficient compared to SARS 3CLpro. Indeed, MERS 3CLpro is the least efficient protease among the bCoVs (MERS, HKU5, and HKU4).

2.2 Crystal structure of 3CL protease In 2003, the X-ray structure of the SARS 3CLpro complexed with a peptide CMK (chloromethylketone, 1; Fig. 6) inhibitor was reported.24 In the same year, the crystal structure of the free 3CLpro of HCoV-229E and porcine transmissible gastroenteritis (corona)virus (TGEV) 3CLpro complexed with 1 were elucidated.21 In addition, compound 2 (AG7088, Fig. 6), known as a human rhinovirus 3Cpro inhibitor, is supposed to interact similarly with CoV-3CLpro as with 3Cpro as suggested by superimposition of several crystal structures of 3CLpro/3Cpro-inhibitor complexes. Because the substrate specificity of picornavirus 3Cpro is similar to that of CoV-3CLpro, compound 1 and 2 had been used as a starting point for the design of peptide-based 3CLpro inhibitors. Following these initial studies25,26 on the crystal structure of 3CLpro, more than hundred structures of SARS 3CLpro with or without inhibitors are registered in the PDB at present. The SARS 3CLpro monomer is comprised of three domains (Fig. 7a). Domains I and II are six-stranded antiparallel b-barrels forming the chymotrypsin-like architecture as in picornavirus 3Cpro. The substratebinding site is located in a cleft between these two domains. A long loop connects domain II to domain III, a globular cluster of five helices. The amino acid sequence of SARS 3CLpro displays 40 and 44% sequence identity to 3CLpro of HCoV 229E and TGEV, respectively (Fig. 7b). Especially, the sequences in domains I and II, the catalytic domains, show a higher degree of sequence conservation in coronavirus 3CL proteases than does domain III. The MERS 3CLpro monomer also shows high structural and sequence similarities to the SARS 3CLpro. SARS 3CLpro forms a tight dimer in the crystal structure (Fig. 8), whereas the protease is expected to exist as a mixture of monomers and dimers in solution and their ratio is dependent on the protease concentration. Several kinetic and biophysical studies have demonstrated that SARS 3CLpro is only active in vitro as a tightly associated dimer. Domain III plays an essential role in dimer formation, since domain III alone is able to form a tight dimer.27 In the mature dimer form, the N-terminal amino acid residues (N-finger) are squeezed in between Amino Acids, Pept. Proteins, 2018, 42, 228–279 | 233

Fig. 6 Structures of inhibitors 1 and 2.

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Fig. 7 Structure of coronavirus 3CLpro. (a) Monomer of SARS 3CLpro. a-helices are labeled A to F according to their occurrence from the N-terminus, with an additional one-turn A 0 a-helix in the N-terminal segment. b–strands are labeled a to f in domain I and II. Thick bars above the sequences indicate a-helices, and horizontal arrows indicate b-strands (labeled a to f, followed by the domain to which they belong). The N- and C-termini are labeled N and C, respectively. Side chain structures of the catalytic dyad, Cys145 and His41, are indicated using a ball and stick model. (b) Structure-based sequence alignment of HCoV-229E, TGEV (porcine transmissible gastroenteritis virus), BatCoV (bat coronavirus), and MERS-CoV 3CL proteases. The auto-cleavage sites of the proteases are marked by vertical arrows above the sequences. Four residues each of the viral polyprotein N-terminal P1/P2 and C-terminal P2/P3 auto-cleavage sites are also shown. Residue numbers for SARS 3CLpro are given below the sequence. Catalytic-site residues Cys145 and His41 are shaded.

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Fig. 8 Dimer of SARS 3CLpro. N- and C-termini of the monomer are labeled as spheres and the letters N and C, respectively.

domains II and III of the partner monomer to hold the specific dimer interactions. Thus, the exact placement of the N-terminus has a structural role to hold the mature 3CLpro, because deletion of residues 1 to 5 led to a decrease in activity. Addition of amino acids via a His-tag at either the N- or C-terminus also drastically reduces the enzymatic activity due to the decreased ability to maintain the dimer structure.28 MERS 3CLpro also shows a similar dimer form in the crystal structure. The protein-protein interactions to stabilize the dimer, however, are rather weak compared to those of SARS 3CLpro, which causes the lower enzymatic ability of MERS 3CLpro compared to SARS 3CLpro. Differences in the maturation process, an auto-processing of the precursor protein, are thought to largely contribute to this discrepancy as discussed in the following section. 2.3 Maturation of the 3CL protease and enzymatic activities Results of analytical gel-filtration and analytical ultracentrifugation (AUC) analyses indicated that a large fusion protein, which has additional protein sequences at the N- and C-terminus of SARS 3CLpro, exists as a monomer in solution. The monomer polyprotein, however, can still retain its enzymatic activity to some extent in vitro,29 although the mature SARS 3CLpro functions in the tight dimer form instead of the monomer. To address this dilemma, formation of a substrate-induced immature ‘‘intermediate’’ dimer is expected to be a key mechanism, which also presents a possible mode of SARS 3CLpro auto-release from the precursor polyprotein in vivo. The proposed auto-release mode of SARS 3CLpro involves four steps (Fig. 9).30,31 At first, along with the polyprotein synthesis following the viral replication, two ‘‘immature’’ 3CLpro precursor monomers approach one another, and their domain III forms an ‘‘intermediate’’ dimer 236 | Amino Acids, Pept. Proteins, 2018, 42, 228–279

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Fig. 9 A proposed mode of SARS-CoV 3CLpro auto-release from the precursor polyproteins; domains I, II, and III of 3CLpro are shown as boxes and cylinders, respectively.

structure, which triggers the substrate-induced dimerization and insertion of their uncleaved N-termini into the substrate-binding pockets of the opposite monomer (Step 1). Next, with a substrate induced-fit mechanism, the active site at the interface of domains I and II is set to catalyze the cleavage of the N-terminal precursor sequences. This results in the N-terminal fingers slipping away from the active sites to their final positions (Fig. 8) to yield a dimer with ‘‘uncleaved’’ C-termini (Step 2). The product of this cleavage is a dimer which has the mature N-terminal sequence and increased catalytic ability compared to the ‘‘intermediate’’ dimer. Then, the ‘‘uncleaved’’ C-terminus of the resulting dimer can insert into an active site of another dimer to proceed trans-cleavage (Step 3). Once the C-terminus is processed, the final mature dimer with authentic N- and C-termini is formed, which is observed in the crystal structure of highly active wild-type SARS 3CLpro (Step 4). Evaluation of the monomer-dimer dissociation constant, Kd, for MERS 3CLpro revealed that the capacity of MERS 3CLpro to dimerize is 130-fold weaker than SARS 3CLpro.32 Analytical ultracentrifugation sedimentation velocity (AUC-SV) studies support the weak association of the MERS 3CLpro dimer, which strongly suggests that the enzyme exists mainly as a less active monomer in solution. The AUC-SV studies also revealed that the addition of low concentrations of ligand to the MERS 3CLpro remarkably increases the ratio of the dimer as well as the catalytic Amino Acids, Pept. Proteins, 2018, 42, 228–279 | 237

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Fig. 10 A proposed kinetic model for activation of MERS 3CLpro via ligand-induced dimerization.

ability. Based on these data, the activation mechanism of MERS 3CLpro can be explained by the kinetic model shown in Fig. 10. The mature dimer of MERS 3CLpro, produced from two immature MERS 3CLpro monomers according to the route shown in Fig. 9, dissociates into inactive monomers in the absence of any ligand (substrate, inhibitor, or 3CLpro cleavage sites in the precursor polyprotein). Binding of ligand to the monomer promotes the monomer to dimer switch. The substrate binds to the resulting dimer at the second active site and is cleaved catalytically. A potent inhibitor can competitively bind to the active site, or directly compete with the ligand for binding to free dimer active sites.

3

Inhibitors of 3CL protease

Inhibition of the catalytic activity of coronavirus 3CLpro is thought to be the most promising mechanism to prevent virus proliferation, since the 3CLpro is essential for the virus’s viability as described above. Thus, after the SARS pandemic in 2003, numerous studies focused on the development of potent inhibitors for SARS 3CLpro. These inhibitors are structurally classified into two types: peptide-mimetic inhibitors and non-peptide small-molecule inhibitors. Considering the mode of interaction, both types of inhibitors can be classified into two types, inhibitors forming a covalent bond with 3CLpro (irreversible inhibitor) and noncovalent type inhibitors (reversible inhibitor). Typical inhibitors of the first generation reported after the first outbreak of SARS are summarized in Fig. 11.33–36 These peptide-mimetic and non-peptide inhibitors provided valuable insights into further modifications based on structurebased design. In the following sections, several typical inhibitory mechanisms as well as the structural modifications of peptide-mimetic and non-peptide inhibitors are described. 238 | Amino Acids, Pept. Proteins, 2018, 42, 228–279

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Fig. 11 Representative first generation SARS 3CLpro inhibitors; (a) peptide inhibitors, (b) non-peptide inhibitors.

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3.1 Peptide-mimetic inhibitors Generally, peptide-mimetic inhibitors of proteases can be designed combining a substrate-like sequence with a functional group (a so-called ‘‘warhead’’) targeting the catalytic center of the target enzyme. The substrate-like sequence of peptide-mimetic inhibitors for the SARS 3CLpro is designed to optimize the specific interactions at the S1 0 site and the S1 to S4 non-prime sites of the 3CLpro. Since the SARS 3CLpro is a cysteine protease, functional groups that can interact with the thiol group are selected as the chemical ‘‘warhead’’. These warhead groups include a Michael acceptor, aldehyde, halomethyl ketone, epoxy, and others. 3.1.1 Peptides with a Michael acceptor. The general strategy for the design of a peptide-mimetic inhibitor containing a Michael acceptor involves the replacement of a substrate’s scissile amide bond with an appropriate Michael acceptor. At the active center of the SARS 3CLpro, the nucleophilicity of the thiol group of the catalytic center Cys145 is increased by a proton-withdrawing effect caused by His41 at the catalytic dyad, which promotes a typical 1,4-addition to the a,b-unsaturated structure of the Michael acceptor (Fig. 12). The resulting protonated His41 gives the proton to an unstable intermediate anion to form a 3CLpro covalently bound to the inhibitor. Thus, the Michael acceptor type compound acts as a suicide substrate to abolish the catalytic activity of the enzyme. Starting from a prototype compound 2 (Fig. 6) targeting rhinovirus 3CLpro, optimizations of side-chain structures at the P1 0 to P4 sites were conducted to develop SARS 3CLpro specific inhibitors (Table 1).37–39

Fig. 12 Inhibition of cysteine proteases by a Michael acceptor type compound. 240 | Amino Acids, Pept. Proteins, 2018, 42, 228–279

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Table 1 Inhibitory activities of Michael type inhibitors against 3CL

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Compounds

. Ki(mM) for SARS 3CLpro

420

13.2

9.1

0.88

0.038

0.099

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pro

Interactions of the SARS 3CL with some of these inhibitors were evaluated based on X-ray crystal structure analyses of the 3CLpro complexed with the inhibitor (for example, PDB codes 2ZU4 and 2ZU5). These structure analyses confirmed that Cys145 attacks the a-carbon of the Michael acceptor (Fig. 12) at the P1 0 site to form a covalent C-S bond of 1.99 Å. Glutamine and a five-membered lactam ring were favored as P1 site substitutes with the five-membered lactam ring being much better. For the P2 site, a cyclohexyl or isopropyl substitute was preferred over a phenyl substitute, which suggests that rigid and planar properties were not favorable for binding in the large hydrophobic S2 pocket of SARS 3CLpro. The P3 group is directed toward the bulk solvent and was predicted to have no specificity for binding. A lipophilic tert-butyl group at this site, however, increased the inhibitory activity, probably due to shifting the substituent toward the P4 site, which induced hydrophobic interactions with the 3CLpro at the phenyl ring of N-terminal benzoyl group. In another study,40 the effect of methylene insertion between the reactive a,b-unsaturated structure and the P1 position was investigated (Table 2). The Michael type analogs elongated toward the prime site showed no inhibitory activities. These results suggest that the recognition of the prime-site structure is strict and no linker structure inserted between the scissile site and neighboring prime site would be tolerated. By comparing the inhibitory activities of both type inhibitors containing the same peptide sequence, it was also suggested that an aldehyde group would be more effective as a warhead than the a,b-unsaturated structure. 3.1.2 Peptide aldehydes. An aldehyde group is another effective functional group involved in a nucleophilic addition reaction to yield an alcohol (Fig. 13). Thus, combined with a substrate peptide sequence, an aldehyde group instead of a Michael acceptor can be used as an effective warhead of SARS 3CLpro inhibitors. Combining the structural analyses (PDB code 3SN8) of the SARS 3CLpro complexed with prototype compound 16 (Fig. 14) and structure-activity relationship (SAR) studies of Michael-type inhibitors (Table 1), a potent peptide aldehyde inhibitor 17 (Ki ¼ 53 nM) was developed.41,42 Analyses of the crystal structure of SARS 3CLpro complexed with 17 (PDB code 2GX4) revealed that the thiol group of Cys145 attacks the carbonyl carbon of the aldehyde of 17 to form a covalent C-S bond (1.24 Å). The resulting active site consisting of a Cys145-His45 dyad at the S1 0 pocket, an oxyanion hole formed by the aldehyde oxygen and N-H of Cys145 and Gly143 at the S1 pocket, as well as a large hydrophobic cavity at the S2 to S4 pockets were clearly observed. In addition, at the P1 and P4 sites, hydrogen bonds served to hold the interactions between the 3CLpro and inhibitor 17, whereas the P2 site cyclohexyl group formed extensive hydrophobic contacts at the S2 pocket. During our studies on the SARS 3CLpro and its inhibitors, we found that the mature SRAS 3CLpro is susceptible to auto-degradation at the Arg188/Gln189 site, which causes a loss of activity.43 Mutation of Arg to Ile remarkably increased the stability while keeping the almost same three-dimensional structure (PDB code 3AW1) as native SARS 3CLpro. 242 | Amino Acids, Pept. Proteins, 2018, 42, 228–279

View Online Table 2 Effects of methylene linkers connecting the P1 position and active center.

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Compounds

IC50 (mM) for SARS 3CLpro

37

330

No inhibition

No inhibition

Probably due to this stability, the mutant protease (R188I SARS 3CLpro) showed much higher activity than the mature protease. Use of this highly active mutant protease made it possible to quantitatively evaluate peptide-aldehyde inhibitors using a conventional HPLC system combined with a non-modified substrate peptide instead of peptide derivatives with fluorescent substituents. Initial SAR studies of a substrate-based peptide aldehyde inhibitor revealed that a P1 site imidazole substituent increases the inhibitory activity more than 6-times compared to a simple substrate-based inhibitor, thus yielding inhibitor 20 (IC50 ¼ 5.7 mM; Table 3) from inhibitor 12 (IC50 ¼ 37 mM). Structural analyses of the SARS 3CLpro complexed with inhibitor 20 (PDB code 3AW0) demonstrated that the large hydrophobic S2 pocket is not fully occupied and the side Amino Acids, Pept. Proteins, 2018, 42, 228–279 | 243

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Fig. 13 Nucleophilic addition reaction to aldehyde group.

Fig. 14 Structures of peptide aldehydes 16 and 17.

chain functional group at the P5 site is not involved in the interactions. Structure-based optimizations provided the potent tetra-peptide aldehyde inhibitor 22 (IC50 ¼ 98 nM).40 X-ray crystal structure analysis of the SARS 3CLpro complexed with 22 clearly showed the tight hydrophobic interactions of the cyclohexyl group at the P2 site as well as an additional hydrogen-bond interaction at the b-hydroxyl group of the P4 site Thr. In these X-ray structural analyses of inhibitors complexed with the mutant protease, the carbonyl carbon of the aldehyde was detected at a distance of 2.30 Å in 20 and 2.48 Å in 22 from the thiol of Cys145 at the catalytic center (Fig. 15). The electron density of the aldehyde group could be fitted to an expected sp2 carbon. In addition, no significant difference was detected between the IC50 value obtained after preincubation of the inhibitor with the protease prior to the addition of the substrate and that obtained by simultaneous mixing of the inhibitor, 244 | Amino Acids, Pept. Proteins, 2018, 42, 228–279

View Online Table 3 Optimization of a substrate-based peptide aldehyde inhibitor.

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Compounds

IC50(mM) for SARS 3CLpro

37

62

48

5.7

0.065

0.098

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246 | Amino Acids, Pept. Proteins, 2018, 42, 228–279 Fig. 15 X-ray structure of inhibitor 22 bound to the R188I SARS 3CLpro.

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protease, and substrate. These results strongly suggested that no stable covalent bonds are formed with the protease. Kinetic data for 22 obtained from Lineweaver–Burk plots also suggested that inhibitor 22 containing an aldehyde functions as a competitive inhibitor without forming a covalent bond. Based on an initial study on peptide-mimetic inhibitors of 3Cpro from enterovirus 71 (EV71), three peptide-aldehyde inhibitors were identified as MERS 3CLpro inhibitors.44 Compounds 23, 24, and 25 (Table 4) showed IC50 values of 2.4, 4.7, and 1.7 mM against MERS 3CLpro, respectively. These compounds also inhibited SARS 3CLpro at lower IC50 values. In silico molecular docking of 25 against MERS 3CLpro suggested that the g-sulfur of Cys148 forms a covalent bond with the aldehyde carbon of 25 and the resulting oxyanion is stabilized by His41. In a cytopathic inhibition assay using MERS CoV infected Huh-7 cells, peptide aldehyde inhibitors 23, 24, and 25 suppressed viral replication with EC50 values of 1.4, 1.2, and 0.6 mM, respectively. The lower EC50 than IC50 value was considered to be due to the high concentration of MERS 3CLpro used for in vitro enzymatic assay because of the weak dimerization ability of MERS Table 4 Inhibition of MERS 3CLpro by peptide-aldehyde inhibitors. IC50 (mM) Compounds

MERS 3CLpro

SARS 3CLpro

2.4

0.7

4.7

0.5

1.7

0.2

Amino Acids, Pept. Proteins, 2018, 42, 228–279 | 247

View Online pro

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3CL . These peptide-aldehyde inhibitors were also cytotoxic for other human CoV, 229E and OC43, although they were not as potent as for inhibiting SARS- and MERS-CoV. 3.1.3 Peptides with halomethyl ketone or an electrophilic substituent. Halomethyl ketone is another warhead which can form a covalent bond by an apparent alkylation reaction. The halomethyl group makes the adjacent ketone group more susceptible to a nucleophilic attack, and the initial nucleophilic attack of a thiolate of Cys145 of 3CLpro toward the carbonyl group of the inhibitor (I) leads to reversible formation of a thiohemiketal (E–I*) holding the tetrahedral conformation that resembles the enzyme-substrate intermediate in the catalytic cleavage (Fig. 16). Intramolecular rearrangement leads to the alkylation product (E–I) as the final product. Experimental data on the kinetics indicate that the above mechanism is more conceivable than a direct mechanism in which the thiolate ion attacks the halomethyl carbon adjacent to the warhead carbonyl leading to the alkylated product. Initial studies of N,N-dimethyl glutaminyl inhibitors with a fluoromethyl ketone group showed that a halogenated methyl group functions as an effective and promising warhead (Table 5).45 These compounds were designed based on their caspase inhibitory activities. Antiviral activity assessed by cytopathic effect (CPE) inhibition in SARS-CoV infected Vero cell cultures revealed that compound 26 can protect the cells against SARS infection with an EC50 value of 2.5 mM and showed

Fig. 16 A possible mechanism for the inactivation by a halomethyl ketone inhibitor. 248 | Amino Acids, Pept. Proteins, 2018, 42, 228–279

View Online Table 5 Structures of compounds 26–29 containing a fuluoromethyl ketone warhead. EC50(mM)

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Compounds

Vero

CaCo2

2.5

2.4

5.3

8.8

6.6

13

4100

4100

low toxicity in mice. P2-Leu of 26 can be replaced by Ile or Val to yield additional compounds 27 and 28 with slightly lower EC50 values. These active compounds were inactive against rhinovirus type-2 in a cell-based assay, which suggests that these compounds are specific against SARS-CoV. Analyses of the catalytic mechanism based on kinetic evaluation revealed an effect of the P1 site substituent as well as a dependence on a halogen atom in the warhead for the inhibitory mechanism shown in Fig. 16. Kinetic inhibition data of compounds 30–33 containing different P1 site substituents and halogen atoms of the warhead for the SARS 3CLpro are summarized in Table 6.46 Hydrophobic substituents such as Amino Acids, Pept. Proteins, 2018, 42, 228–279 | 249

View Online Table 6 Structures and kinetic inhibition data for compounds 30–33. For SARS 3CLpro

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Compounds

Ki (mM)

k3 (102 s1)

0.31

1.5

0.37

2.8

0.38

1.8

0.40

{0.005

aromatic groups (phenyl, naphthyl, or parafluorophenyl) as well as an aliphatic bulky group at P1 site are tolerated, although Gln is traditionally present at this position. This tolerance is consistent with previous results reporting modifications at the P1 site with a lactam ring, keto-glutamine analogs, and an a,b-unsaturated ester at P1 site. Altering the halogen atom of the warhead had a substantial effect on the kinetic property of the inhibitors. The rate of the irreversible inactivation step (k3 in Fig. 16) is related to the ability of the warhead to accept a nucleophilic attack by the active center thiol leading to the eventual alkylation. The value of k3 of compound 31 (2.8102 s1) was almost twice that of 30 (1.5102 s1) and 32 (1.8102 s1), which suggests that the larger substituent at the P1 site may orient the warhead in a favorable conformation for the interaction with the thiol group of Cys145. In contrast, the k3 value of compound 33 was too small to be measured 250 | Amino Acids, Pept. Proteins, 2018, 42, 228–279

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accurately, indicating that the irreversible step is very slow and that compound 33 behaves as a reversible inhibitor for several hours. Irreversible inhibition of the enzyme activity was only noticed after a 12 h incubation with compound 33 at a high concentration. The structure of the wild-type SARS 3CLpro complexed with compound 33 was determined using X-ray crystallography (PDB code 3D62). Although the electron density for only a portion of compound 33 was observed, a thioether bond (1.7 Å) between the carbon that was originally bound to bromine of 33 and the sulfur atom of Cys145 was clearly detected. This structural analysis is consistent with a time-dependent bimodal mode of inhibition for this compound: initial formation of a reversible complex (E–I*) followed by rearrangement to an irreversible complex (E–I) after at least 6 h incubation. The phthalhydrazide group is another functional group which makes the adjacent ketone carbonyl carbon more susceptible to a nucleophilic attack. Extending the initial studies of a new class of inhibitors against hepatitis A virus (HAV) 3Cpro, several phthalhydrazide type inhibitors for SARS 3CLpro were developed (Fig. 17).47 The phthalyhydrazide warhead functions well against the SARS 3CLpro, especially in combination with a g-lactam substitute at the P1 site (Fig. 17; 34–37 vs. 38–41). In the inhibitory reaction by compound 38, no kinetic evidence of covalent inhibition was observed when 10 mM 3CLpro was pre-incubated with 100 mM of 38 for 15 to 60 min. Electron spray ionization-mass spectrometry (ESI-MS) analysis of a SARS 3CLpro crystal complexed with compound 38, however, revealed that the product is a covalent adduct between the 3CLpro and compound 38 without the phthalhydrazide moiety. Detailed analyses of the crystal structures of the SARS 3CLproinhibitor complex obtained via the co-crystallization method (PDB code 2Z3E) and via the soaking method (2Z3C) suggested the intermediacy of the episulfide linkage in solution (Fig. 18).48 In these crystal structures, the bond length between the sulfur atom of the Cys145 thiol and the carbon atom of the P1 carbonyl was 1.81 Å in the thioacyl-like structure and 1.83 Å in episulfide structure. The structures obtained from the electron densities also indicated that transformation of the SARS 3CLprobound episulfide cation to the thiomethyl ketone structure requires a conformational change which is more attainable in aqueous solution than in crystals. 3.1.4 Peptides with trifluoromethyl ketone or other electrophilic substituents. A trifluoromethyl group is another warhead making a neighboring carbonyl group susceptible to a nucleophilic attack. The substrate-based trifluoromethylketone inhibitor 46 inhibited the SARS 3CLpro (IC50 ¼ 10 mM) more effectively than a series of low molecule inhibitors (Table 7).49 A Lineweaver-Burk plot showed that the inhibition with 46 was competitive in the initial 4 h reaction. Prolonged incubation of the 3CLpro with 46, however, exhibited a timedependent decrease in the enzymatic activity as a function of the inhibitor concentration. This time-dependent tightening of inhibition was assumed to be caused by the slow formation of a covalent adduct Amino Acids, Pept. Proteins, 2018, 42, 228–279 | 251

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252 | Amino Acids, Pept. Proteins, 2018, 42, 228–279 Fig. 17 Inhibitory activities of phthalhydrazide containing compounds.

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Fig. 18 Proposed intermediacy of the episulfide species in the inhibitory reaction by 38.

through the nucleophilic attack of the thiol of Cys145 on the carbonyl carbon (Fig. 19). Although the X-ray crystal analyses were not successful on this compound, computational molecular modeling strongly suggested a covalent bond formation in the final stage of the inhibition. A substrate-based trifluoromethyl ketone inhibitor containing Glu or Gln at the P1 site (47, Fig. 20) showed only moderate inhibitory activity due to the formation of a cyclic structure which is expected to hardly interact with the active site of SARS 3CLpro.50 In order to block the cyclization, the side chain was modified to increase the bulkiness, yielding the more potent inhibitor 47. Replacement of the trifluoromethylketone group with an electrophilic thiazolyl ketone 49 further increased the inhibitory activity ten times. Following structure optimization at the P4 site combined with a benzothiazole warhead yielded inhibitors 50 and 51, both having low-nano-molar IC50 values.51,52 3.1.5 Aza-epoxide and aziridine peptides. The natural product E-64 (Fig. 21) from Aspergillus japonicus has been used as a reference inhibitor for many clan CA cysteine proteases including papain, cathepsins, and calpains. The epoxysuccinate structure is a key functional group for the inhibitory activity, but E-64 is not effective in the inhibition of clan CD protease. During the investigations of inhibitors effective against clan CD proteases, aza-peptide epoxides were designed by replacing the nitrogen atom of the scissile amide bond with a reactive epoxy group to receive a nucleophilic attack by an active center Cys.53 Additional conversion of the a-carbon of an amino acid at the P1 site into a nitrogen results in an aza-epoxy peptide (Fig. 21). This replacement induces a trigonal planar geometry to the a-atom of the P1 residue and reduces the electrophilicity of the carbonyl carbon of the P1 Amino Acids, Pept. Proteins, 2018, 42, 228–279 | 253

View Online Table 7 Inhibition of substrate-based trifluoromethyl ketone compounds against SARS 3CLpro.

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Compounds

IC50(mM) for SARS 3CLpro

15

20

40

40

10

Fig. 19 Proposed mechanism of inhibition of SARS 3CLpro by compound 46. 254 | Amino Acids, Pept. Proteins, 2018, 42, 228–279

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Fig. 20 Inhibition with peptides with trifluoromethyl ketone or thiazolyl ketone substituents.

Fig. 21 Structures of E-64, aza-peptide epoxide, and aziridinyl peptides.

residue, which makes the carbonyl group resistant to the nucleophilic attack. Another screening study of peptide derivatives containing electrophilic building blocks indicated that the trans-configured Amino Acids, Pept. Proteins, 2018, 42, 228–279 | 255

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aziridine-glycylglycylate 52 has moderate inhibitory activity against SARS 3CLpro.54 Following studies focusing on inhibitors of the SARS 3CLpro, an azaepoxy peptide possessing an aza-glutamine at the P1 residue 53 was found that inhibited the 3CLpro with a Ki value of 18 mM. The S, S diastereomer of the epoxide strongly inhibited the cleavage of a substrate peptide, while the R, R diastereomer did not detectably inhibit the 3CLpro. Evaluation of the crystal structure of mature SARS 3CLpro complexed with 53 confirmed the formation of a covalent bond with a distance of 2.01 Å between the sulfur atom of Cys145 and the epoxide C3 atom of 53 (Fig. 22).55,56 Although the length of a normal C-S bond is about 1.8 Å, the difference between the refined (2.01 Å) and expected (B1.8 Å) distance was not considered significant since the estimated overall coordinate error for the structure analysis was 0.12. Further analyses of the crystal structures revealed an induced-fit binding of an aza-epoxy peptide to the SARS 3CLpro. In the unbound form, the active site and the S1 pocket of the 3CLpro is in a collapsed conformation, whereas they are in an open conformation in the inhibitor-bound form. 3.1.6 Nitrile-based peptidemimetic inhibitor. A nitrile group, a wellknown warhead group of DPP4 inhibitor vildagliptin used as an antidiabetes agent, was incorporated in the substrate sequence of SARS 3CLpro to develop nitrile-based inhibitors (Fig. 23). Inhibitory activities of four nitrile-based inhibitors with different protecting groups, 5-methylisoxazole-3-carboxyl (Mic), tert-butyloxycarbonyl (Boc), and carboxybenzyl (Cbz) were evaluated.57 The Cbz-tetrapeptide inhibitor Cbz-AVLQ-CN 56 was ten-times more potent (IC50 ¼ 4.6 mM) than the other inhibitors including the Cbz-hexapeptide inhibitor Cbz-TSAVLQCN 57, which suggests that the Cbz group at the P4 position contributed to the suitable interactions at the S4 pocket of SARS 3CLpro. The crystal structures of the SARS 3CLpro in complex with the nitrilebased inhibitor (PDB codes 3VB7, 3VB4, 3VB5, 3VB6) demonstrated that the inhibitor was covalently bonded to the thiol group of Cys145 via the carbon atom of the nitrile warhead (Fig. 24). In addition, the tetrapeptide inhibitor Cbz-AVLQ-CN 56 can inhibit 3CLpro from human coronavirus strains 229E (IC50 ¼ 2.3 mM), NL63 (IC50 ¼ 2.8 mM), OC43 (IC50 ¼ 1.6 mM), and HKU1 (IC50 ¼ 1.3 mM), while the same inhibitor had no observable inhibitory effect on caspases. These results suggest that the nitrile-based inhibitor is specific to 3CLpro from coronaviruses and exhibits broadspectrum inhibition against the 3CLpro. 3.2 Small-molecule inhibitors In general, a low molecular weight inhibitor for infectious viruses and bacteria is considered a promising agent against infectious diseases. Usually, small-molecule inhibitors have various chemical structures even when the same protein/protease is targeted. Thus, many of these small-molecule inhibitors were discovered from natural products and/or related derivatives or by high-throughput screening of synthetic compound libraries. In this section, several examples of small-molecule 256 | Amino Acids, Pept. Proteins, 2018, 42, 228–279

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Fig. 22 Formation of a covalent bond by an aza-epoxy peptide.

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Fig. 23 Structures and IC50 values of the nitrile-based inhibitors against SARS 3CLpro.

Fig. 24 Interactions of the nitrile-based inhibitor with SARS 3CLpro.

inhibitors of SARS/MERS 3CLpro found by these procedures are included. 3.2.1 Natural products and related derivatives. Isatin (2, 3-dioxindole) is an indole derivative found in many plants, such as Isatis tinctoria and Calanthe discolor. It is known that certain isatin compounds are potent inhibitors of rhinovirus 3Cpro. Because the active site architecture of the 3Cpro is similar to that of SARS 3CLpro, isatin derivatives were expected to be good candidates for SARS 3CLpro inhibitors. Using 258 | Amino Acids, Pept. Proteins, 2018, 42, 228–279

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initial studies on isatin derivatives which have N-1 and C-5 substituents, potent inhibitors for SARS 3CLpro 58 (IC50 ¼ 0.37 mM) and 59 (IC50 ¼ 0.95 mM) were developed (Fig. 25).58,59 Computer modeling analyses suggested that the isatin scaffold is docked in the S1 site, and the N-1 substituent is located in the S2 site of the 3CLpro. The sulfur atom of Cys145 in the active center of the 3CLpro is estimated to be located in hydrogen-bond distance from the isatin oxygen at C-3. Related high-throughput screening revealed that 5-bromoisatin was a potent inhibitor of the 3CLpro and could be soaked into a crystal of the 3CLpro. Based on these results, a replacement of the carboxamide group using a series of sulfonamide groups was achieved, and isatin 5-sulfonylamide derivatives 60 (IC50 ¼ 1.18 mM) and 61 (IC50 ¼ 1.04 mM) were identified as promising inhibitors for SARS 3CLpro (Fig. 26).60 Docking studies, however, suggested a mode of docking different from that expected for the above derivatives 58 and 59. The 2,3-dioxindole scaffold of the isatin 5-sulfonylamide derivatives is docked at the S1 0 site of SARS 3CLpro instead of the S1 site for the above derivatives. The sulfonamide substituent and N-1 substituent were located at the S2 and S1 sites, respectively. These docking model could support the differences of inhibitory activities in a series of isatin 5-sulfonamide derivatives. During the search for positional anti-SARS CoV agents from medicinal plants and foodstuffs, a series of chalcones isolated from an ethanol extract of Angelica keiskei were found to be moderate inhibitors of SARS 3CLpro. Among the 13 isolated and structure-determined chalcones,

Fig. 25 Structures of isatin derivatives 58 and 59 showing inhibitory activities against SARS 3CLpro.

Fig. 26 Structures of isatin 5-sulfonylamide derivatives 60 and 61 showing inhibitory activities for SARS 3CLpro. Amino Acids, Pept. Proteins, 2018, 42, 228–279 | 259

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Fig. 27 Structures of chalcone derivatives 62 and 63 showing inhibitory activities against SARS 3CLpro.

compounds 62 and 63 (Fig. 27)61 showed relatively high inhibitory potencies for the SARS 3CLpro (IC50 ¼ 27 mM for 62 and 11 mM for 63). Comparing the inhibitory activities of both compounds, the perhydroxyl group was expected to be more effective in the interactions with the hydrogen bonding site of the 3CLpro. Lineweaver-Burk and Dixon plots suggest that both chalcones 62 and 63 are competitive inhibitors with Ki values of 35 mM and 16 mM, respectively. In an in silico docking simulation, both chalcones fitted nicely into the substrate-binding pocket of the 3CLpro. The perhydroxyl group of compound 63 formed strong hydrogen-bonds with Cys145 (3.45 and 2.72 Å) as one of the key motifs of this inhibitor. In a separate study on natural products isolated from Broussonetia papyrifera, a few polyphenols were found to be moderate inhibitors for SARS and MERS 3CLpro with IC50 values of 28B65 mM (Fig. 28).62 3.2.2 Ester and ketone analogs. A series of active heterocyclic ester analogs was identified as a novel class of mechanism-based irreversible inhibitors with activities in the nanomolar range.63 The possible irreversible acylation of Cys145 by the typical ester derivatives 67 and 68 was verified by ESI-MS (Fig. 29).64,65 The acylation by the ester ligand was also confirmed by an X-ray crystal structure of the SARS 3CLpro complexed with the benzotriazole ester.66 Further studies on 3-chloropyridyl ester-based inhibitors revealed that the position of the carboxylic acid ester is critical for potent inhibitory activity (Fig. 30).67 Carboxylate substitution on indole rings at the 4-position gave the most potent inhibitor 69 with an IC50 of 30 nM. This inhibitor also showed a SARS CoV antiviral activity with an EC50 value of 6.9mM. Covalent modification by this inhibitor was confirmed by MatrixAssisted Laser Desorption Ionization (MALDI) time of flight (TOF) MS analysis of the 3CLpro obtained after incubation with the inhibitor. Although the above mentioned active-ester analogs act as potent covalent inhibitors of the 3CLpro, they are supposed to be susceptible to hydrolysis catalyzed by various enzymes such as esterases, lipases, and other enzymes in mammalian cells. These ester analogs initially bind competitively to the active site and are then hydrolyzed by the 3CLpro as a suicide substrate. Thus, the corresponding ketone derivative might form a hemithioacetal with the thiol of Cys145 to act as a reversible inhibitor. Based on this idea, a series of methylene ketones and fluoro-methylene 260 | Amino Acids, Pept. Proteins, 2018, 42, 228–279

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Fig. 28

Structures of polyphenol derivatives showing inhibitory activities toward SARS and MERS 3CLpro.

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262 | Amino Acids, Pept. Proteins, 2018, 42, 228–279 Fig. 29 Covalent bond formation by an active-ester type inhibitor.

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Fig. 30 Active 5-chloropyridine ester analogs.

ketones were synthesized and those inhibitory activities were evaluated (Fig. 31).68 The methylene ketone 73 and its fluorinated methylene ketone analogs 74 and 75 were good inhibitors of the 3CLpro with IC50 values of 13–57 mM. ESI-MS analysis of the reaction mixture strongly suggested that these compounds utilize a non-covalent reversible mechanism of inhibition. Docking model studies also suggest that these compounds interact with the 3CLpro in a S4–S1 binding mode using the three-aromatic ring structure to block the entry of substrates into the active site. 3.2.3 Metal-conjugated inhibitors. Metal-conjugated compounds have been used as pharmaceutical excipients and antimicrobial preservatives in parenteral and topical pharmaceutical formulations. From the screening of a compound library consisting of 960 commercially available drugs and biologically active substances, some metal ions (Cu21, Hg1, Zn21) and their metal-conjugated compounds [phenylmercuric acetate (PMA), toluene-3,4-dithiolato zinc (TDT), and N-ethyl-Nphenyldithiocarbamic acid zinc (EPDTC)] were identified to show inhibitory activity against SARS 3CLpro with Ki values of around micromolar and sub-micromolar levels (Fig. 32).69 Crystal structure analyses of these compounds as well as two additional zinc-based inhibitors complexed with SARS 3CLpro revealed the binding mode with the 3CLpro (PDB codes 2Z9J, 2Z9K, 2Z9L, 2Z9G, and 2Z94).39,70 Hg1-PMA was coordinated with Cys44, Met49, and Tyr54 with a square planar geometry at the S3 pocket, whereas each Zn21 of the four zinc-inhibitors was tetrahedrally coordinated with the His41–Cys145 catalytic dyad. Although the electron density of the bulky substituent of the metal ion was not Amino Acids, Pept. Proteins, 2018, 42, 228–279 | 263

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264 | Amino Acids, Pept. Proteins, 2018, 42, 228–279 Fig. 31 Halomethyl pyridyl ketone analogs and their inhibitory activities.

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Fig. 32 Structures and inhibitory parameters of metal conjugated inhibitors.

detected, computer modeling study showed the entire EPDTC could be accommodated in the active site pocket of the 3CLpro. 3.2.4 Compounds found by high throughput screening. Screening of a compound library gives an opportunity to identify a novel scaffold or unexpected activity of old compounds, while experimental screening of real compounds generally provides clear-cut data on the potential activity and experimental base for rational structure optimization. A few examples of the SARS 3CLpro inhibitors found by virtual or highthroughput screening are listed in Fig. 33 and the screening processes are summarized in the following sections. Cinanserin (Fig. 11), a well-characterized serotonin antagonist, was identified as an inhibitor of the SARS 3CLpro by virtual screening of a database.35 The substrate-binding pocket formed by residues within a radius of 6 Å around the catalytic dyad (His41 and Cys145) was used as the target site for virtual screening using a docking program. The comprehensive Medicinal Chemistry Database of Molecular Design Limited (MDL-CMC) containing the structure information of more than 8000 compounds was searched, and finally cinanserin was selected as a potential candidate compound. After the selection, it was confirmed that cinanserin can indeed inhibit the activity of SARS 3CLpro with an IC50 value of 5 mM. The binding affinity (KD ¼ 49.4 mM) of cinanserin was Amino Acids, Pept. Proteins, 2018, 42, 228–279 | 265

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Fig. 33 Several compounds found by virtual or high-throughput screening.

determined by kinetic analyses using surface plasmon resonance (SPR). The antiviral activity of cinanserin was further evaluated in tissue culture assays. The level of virus RNA and infectious particles was reduced by up to 4 log units, with IC50 values ranging from 19 to 34 mM. In a study on the screening of small molecular inhibitors for SARS 3CLpro conducted after the above work, it was reported that cinanserin showed no inhibition of the SARS 3CLpro.71 This discrepancy was assumed to stem from the protease preparation used in each experiment: the N-terminal extended 3CLpro was used in the above initial screening work, whereas mature 3CLpro without the incorporation of additional sequences at the N-terminus was used in the latter small-molecule inhibitor screening experiment. Thus, cinanserin may be effective at inhibiting a non-dimeric form of the SARS 3CLpro. In another study, structure-based virtual screening of 308 307 chemical compounds was performed using the computation tool Autodock 3.0.5 on a WISDOM (Wide In Silico Docking On Malaria) production environment.72 From the top 1,468 ranked compounds selected through the hydrogen bond interaction at the active site of the 3CLpro, 53 compounds were tested in an in vitro assay. Two potent 3CLpro inhibitors 81 and 82 (Fig. 33) were finally identified as competitive inhibitors of 3CLpro with Ki values of 9.11 and 9.93 mM, respectively. Another validated docking protocol based on the Gold Docking Program was used for a virtual screening of 120 000 compounds to select 108 candidates to be tested in vitro.73 Two compounds, 83 and 84 (Fig. 33), were finally selected as promising inhibitors for SARS 3CLpro with IC50 values of 18 and 17 mM, respectively. Using the docking simulation carried out in the screening procedure, these two inhibitors were expected to show occupancy of the S1 0 , S1, and S2 pockets of the SARS 3CLpro, but not the S4 pocket. 266 | Amino Acids, Pept. Proteins, 2018, 42, 228–279

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A combination of virtual screening (VS) and high-throughput screening (HTS) techniques identified a novel, non-peptide small molecule inhibitor 85 (Fig. 33) of SARS 3CLpro.74 A structure-based VS approach integrating docking and pharmacophore based methods was used to screen 621 000 compounds from the ZINC library, a free database of commercially-available compounds for virtual screening. The screening protocol was validated using known 3CLpro inhibitors and was further optimized. Subsequently, a fluorescence-based enzymatic HTS assay was used to screen approximately 41 000 compounds chosen based on the VS results. After eliminating the false positives from the initial HTS hits by a secondary orthogonal analysis using SPR, the final candidate compound 85 was identified as a reversible small molecule inhibitor exhibiting mixed-type inhibition with an IC50 value of 13.9 mM and a Ki value of 11.1 mM. In a study of Jacobs and co-workers71 on a search of noncovalent smallmolecule inhibitors of the SARS 3CLpro, an initial high-throughput screening of the NIH molecular libraries sample collection (B293 000 compounds) at the Scripps Research Institute Molecular Screening Center (SRIMSC) produced 406 hits. The second screen of these initial hits produced 136 active compounds, and the following dose-response testing yielded 44 active compounds with IC50 values below 10 mM. Among the several scaffold clusters obtained by structural clustering analysis of these active compounds, dipeptide compound 86 was selected for further structure-activity relationship studies (Fig. 34). Initial structure optimization, however, only produced one compound 87 with slightly weaker inhibitory activity than 86. X-ray crystal structure of the SARS 3CLpro complexed with compound 87, however, revealed that the binding orientation of 87 is overall similar to a known covalent peptidemimetic inhibitor. Inhibitor 87 preferentially occupied the S3-S1 0 pockets of the 3CLpro as the R enantiomer (Fig. 34). The catalytic Cys145 was positioned beneath both the amide carbonyl carbon and the furan oxygen at a distance of 3.5 Å. Based on these structural analyses, a second chemical library focusing on P1 0 and P1 replacement was constructed and the inhibitory activities were evaluated. Following enantiomer separation of the selected active compound finally provided 87-R as a potent SARS 3CLpro inhibitor with an IC50 value of 1.5 mM (Fig. 34). The mechanism of inhibition was determined to be a competitive mode with a Ki value of 1.6 mM. Compound 87-R can effectively inhibit SARS CoV replication in cell culture with an EC50 value of 12.9 mM. Further screening of the above NIH molecular library gave the related diamide compound 88 having an IC50 value of 6.2 mM (Fig. 35).75 X-ray crystal structure analysis of the mature SARS 3CLpro complexed with 88 revealed a unique induced-fit reorganization of the S2–S4 binding pockets. This induced fit accommodated the syn N-methyl pyrrole and anilido acetamide moieties of the inhibitor within the pockets that can be characterized as S2–S4 and S2–S1 0 pockets, respectively. Additional SAR studies afforded compound 89 with a P3 truncated structure as a minimum pharmacophore as a noncovalent nanomolar inhibitor. Amino Acids, Pept. Proteins, 2018, 42, 228–279 | 267

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268 | Amino Acids, Pept. Proteins, 2018, 42, 228–279 Fig. 34 Structure optimization of 86 found by high-throughput screening.

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Fig. 35

Structures of diamide type inhibitor 88 and the P3 truncated inhibitor 89.

Fig. 36 Structure of a parent peptide aldehyde inhibitor 22 and concept for a serine derivative.

3.2.5 Rational design based on structure analyses. Few examples of structure-based rational design of inhibitors for SARS 3CLpro have been reported, and the inhibitory activities are still moderate. Nevertheless, the rational approach would largely contribute to designing a novel scaffold of a non-peptide low molecular inhibitor of the SARS/MERS 3CLpro which has a higher potential as a therapeutic agent than peptide-based inhibitors. In this section, two approaches for the design of non-peptide inhibitors starting from a potent peptide aldehyde inhibitor are included. For the structure-based rational design of a SARS 3CLpro inhibitor, Konno et al.76 selected a serine-based scaffold as a suitable candidate for small-molecule inhibitors. A highly potent peptide-based inhibitor was selected as a starting derivative for the design. Side chain structures at the P1, P2, and P4 sites of tetrapeptide inhibitor 22 were used for the design of the serine derivative, since serine, a commercially available proteinogenic amino acid, has three variant reaction sites: an alcohol, an amino, and a carboxylic acid moiety, which can be orthogonally connected to various functional groups (Fig. 36). Various molecular mechanics calculations with SPARTAN from Wavefunction and docking simulations of protein interactions by GOLD were carried out to determine whether a series of serine Amino Acids, Pept. Proteins, 2018, 42, 228–279 | 269

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derivatives could adopt an energetically favorable conformation mimicking parent inhibitor 22. The initial trial however gave a result contrary to expectations: the cyclohexyl group of the serine derivative occupied the S1 pocket instead of the expected S2 pocket of the 3CLpro. In contrast, a cinnamoyl derivative reported by Bai et al. was located deep inside of the S1 0 , S1, and S2 pockets with appropriate cinnamoyl functionalities, which was identified via simulation using Autodock 3.0. Combining the contrary results of these simulations, a hybrid scaffold with Bai’s derivative and the serine derivative was designed for the following SAR studies (Fig. 37). Detailed SAR studies at the P1 0 and P4 positions provided two optimized compounds, 94 and 95, as novel scaffolds of small molecular inhibitors for the SARS 3CLpro with IC50 values of 30 and 65 mM, respectively. Docking simulations of these compounds confirmed the expected interactions at the S1 0 , S1, and S4 pockets (Fig. 38). In a separate study,77 another approach starting from the same peptide inhibitor 22 was examined. Previous analyses of the crystal structure of the SARS 3CLpro complexed with inhibitor 22 (PDB code 3ATW) revealed that a cyclohexyl substituent at the P2 site was well packed in the corresponding S2 pocket of the 3CLpro, an interaction critical for making 22 a potent competitive inhibitor. Detailed analyses of this hydrophobic interaction at the S2 pockets revealed that the P2 site cyclohexyl structure was situated rather close to the peptide backbone in the active site cleft. The distance of the a-amide nitrogen of the P2 cyclohexylalanine (Cha) to the position 2 carbon (C2) of the cyclohexyl ring of Cha was estimated to be 3.48 Å in the crystal structure. Thus, connecting the C2 carbon of the cyclohexyl ring to an a-nitrogen of the P2 site Cha via a methylene linker is considered to yield a novel fused ring structure acting as a core hydrophobic substituent at the P2 position (Fig. 39). The resulting decahydroisoquinoline scaffold is expected to maintain the hydrophobic interactions at the cyclohexyl ring of 22 in the S2 pocket. It is also assumed that this fused ring scaffold will be able to arrange the P1 site imidazole and active site functional aldehyde at each required position. The acyl substituent on the nitrogen in the decahydroisoquinoline scaffold may add an extra position for additional interactions with the SARS 3CLpro. Compounds containing different enantiomers of the fused ring structure were separately prepared to examine the effect of configurations at the fused ring scaffold. For these preparations, a combination of the enantiomer resolution by salt formation and Pd-catalyzed stereoselective cyclization reaction was used. As summarized in Table 8, the synthesized decahydroisoquinoline derivatives all showed moderate but clear inhibitory activities toward SARS 3CLpro, which strongly suggests that the decahydroisoquinoline can function as a hydrophobic core structure at the P2 site. Clear differences of the inhibitory activities due to the difference of stereostructure at the fused ring moiety were also observed. In addition, a limited range of differences of the inhibitory activity caused by the substituent on the nitrogen atom of the decahydroisoquinoline scaffold were also observed. 270 | Amino Acids, Pept. Proteins, 2018, 42, 228–279

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Fig. 37 Design of the serine derivative as an inhibitor for SARS 3CLpro based on virtual screening.

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Fig. 38 Optimized inhibitors 94 and 95.

Fig. 39 Design of a decahydroisoquinoline scaffold.

These differences of the inhibitory activities were rationalized by X-ray crystal structure analyses of the SARS 3CLpro complexed with decahydroisoquinoline derivatives, compounds 96, 101, and 97 (PDB code 4TWY, 4TWW, and 4WY3). The decahydroisoquinoline inhibitor 101 was at the active site cleft of the 3CLpro as observed in the parent peptide aldehyde inhibitor 22. The carbonyl carbon of the aldehyde group of 101 was detected at a distance of 2.31 Å from the thiol of Cys145, suggesting that the decahydroisoquinoline inhibitor would function as a competitive inhibitor to the parent peptide aldehyde inhibitor 22 (Fig. 40). It was also clearly confirmed that the decahydroisoquinoline scaffold of 101 took a trans-fused configuration and was inserted into the large S2 pocket of the 3CLpro. Most of the S2 pocket was occupied by the fused-ring structure. The nitrogen atom of the P1 site imidazole of 101 formed hydrogen 272 | Amino Acids, Pept. Proteins, 2018, 42, 228–279

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Table 8 Inhibitory activities of the decahydroisoquinoline derivatives.

IC50 R

(3S, 4aR, 8aS)

(3R, 4aS, 8aR)

96 108 mM

97 240 mM

98 135 mM

99 135 mM

100 68 mM

101 63 mM

102 175 mM

103 57 mM

bonds with nearly the same mode as the parent inhibitor 22, resulting in close fitting at the S1 pocket. Thus, the hydrophobic interaction of the decahydroisoquinoline scaffold in the S2 pocket functions to hold the P1 site imidazole and terminal aldehyde group inside the active site cleft. Crystal structures of the 3CLpro complexed with 96 and 97 were compared to evaluate the effects of the decahydroisoquinoline scaffold configuration (Fig. 41). Compared to the interaction of the active compound 96, the decahydroisoquinoline scaffold having less active configuration (4aS, 8aR) in 97 was located in the S2 pocket in a more twisted position. Amino Acids, Pept. Proteins, 2018, 42, 228–279 | 273

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Fig. 40 Interactions of inhibitor 101 at the active center and P1 site (a), and at P2 site (b) of SARS 3CLpro.

Fig. 41 X-ray crystal structure analyses of the 3CLpro complexed with 96 (PDB code 4TWY) and 97 (4WY3).

This conformational change of the fused ring in the S2 pocket was transferred in the direction of the N-substituent of the fused ring scaffold, and the N-substituent of the less active compound 97 was directed toward the outside of the surface of the 3CLpro. In contrast, the substituent of the active compound 96 was located on the surface of the 3CLpro, where an additional interaction with the protease might be possible. These conformational differences at the N-substituent derived from the configurational change at the decahydroisoquinoline scaffold would explain the discrepancy in the inhibitory activity between 96 and 97.

4 Conclusions Two procedures have been used in the development of potent inhibitors of SARS 3CLpro: (i) a combination of substrate-based peptide structures and effective warheads and (ii) a combination of in silico virtual screening and high-throughput screening of actual compound libraries. The data obtained through these approaches summarized in this article are the basis for the development of therapeutic agents for the infectious SARS coronavirus. Inhibitory potencies against the proliferation of the CoV in cells as well as the inhibitory effects towards the 3CLpro were 274 | Amino Acids, Pept. Proteins, 2018, 42, 228–279

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confirmed for several candidate compounds including peptide-based inhibitors and small-molecule inhibitors. The low toxicity for cells was also examined for a few compounds; however, further in vivo studies should be conducted. These inhibitors are potential starting points for the design of inhibitors as they have high inhibitory potency against the CoV as well as good physicochemical and pharmacodynamics properties necessary for in vivo use. Recent studies on the rational design of a novel scaffold starting from a peptide-based inhibitor revealed a possibility to yield inhibitors based on another category. Compounds with good viral inhibitory activity can also be discovered by screening a library composed of approved drugs or therapeutics still in clinical development. MERS 3CLpro has proteochemical properties different from SARS 3CLpro, especially regarding the stability of the dimer structure. This might interfere with the exact evaluation and design of specific inhibitors for MERS 3CLpro. Instead, broad-spectrum inhibitors might be promising since the sequence similarity of the 3CLpro of SARS and MERS is estimated to be more than 60%. Development of novel anti-SARS/MERS CoV inhibitors with drug-like properties can be attained based on the achievements summarized in this article.

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Targeting peptides, a Swiss-Army Knife against cancer Vadim Le Joncour and Pirjo Laakkonen* Published on 29 November 2017 on http://pubs.rsc.org | doi:10.1039/9781788010627-00280

DOI: 10.1039/9781788010627-00280

Characterised by both great selectivity and flexibility, peptides are also considered safe and usually quite well tolerated by patients. During the past two decades, development of anti-cancer peptide therapeutics has formed sustained activity in the research and development (R&D) departments of pharmaceutical industry. Cancer is the emerging leading cause of death worldwide often without a satisfactory therapeutic option available. To overcome the often-acquired chemotherapeutic resistance and side effects, novel drugs that are specifically attacking cancer cells and less toxic to normal cells are required. Thus, tumour targeting peptides constitute an appealing tool for cancer detection and treatment. In this chapter, we will summarise the identification of novel cancer targeting peptides and also address some of the most promising candidates currently under preclinical investigation.

1

Introduction

Natural peptides account for up to 7000 different entities with central roles in mammalian physiology. They act as neurotransmitters, hormones, growth factors, and channel protein ligands as well as antimicrobial agents. Classically, they bind to their specific receptors, e.g. a G-protein coupled receptor (GPCR) or an ion channel, and trigger intracellular signalling cascades responsible for a wide range of cell activities such as proliferation, migration or programmed death. The binding of a peptide to its receptor target is known to be highly selective and efficacious. In the context of drug development, peptides have displayed great tolerability, excellent targeting, and safety in patients. The so-called targeting peptides are thus constituting a weapon of choice for the specific targeting of imaging and therapeutic agents for complex diseases, such as tumours. Cancer related deaths are estimated to reach 11.5 million in 2030.1 For many patients, the burden of the disease is aggravated by the absence of efficient treatments, systemic toxicity caused by the chemotherapy and/or development of resistance. Indeed, many of the available non-targeted drugs cause unwanted adverse effects. For instance, cytotoxic chemotherapies take advantage of the high division rate of cancer cells to damage the exposed DNA or block the cell proliferation. Unfortunately, normal dividing cells are also drastically affected by such drugs, causing notable side-effects to the patients. Recently, targeted therapies have been proposed to offer one of the possible solutions to this challenge.2 They mostly consist of small molecule inhibitors and antibodies specific to essential molecular mechanisms of the tumour growth. For instance, University of Helsinki, Research Programs Unit, Translational Cancer Biology, Haartmaninkatu 8, 00014, Helsinki, Finland. E-mail: [email protected]; [email protected] 280 | Amino Acids, Pept. Proteins, 2018, 42, 280–319  c

The Royal Society of Chemistry 2018

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several tyrosine kinase inhibitors have been identified or developed with the aim of blocking the activity of the key growth factor receptors responsible for tumour cell proliferation and metastasis.3 The U.S. Food and Drug Administration approved the use of a tyrosine kinase inhibitor called erlotinib (Tarceva) that targets the epidermal growth factor receptor (EGFR) overexpressed in pancreatic or lung cancers and has shown to benefit the patients.4,5 Moreover, several chimeric antibodies have been developed such as bevacizumab. This therapeutic antibody binds specifically to the vascular endothelial growth factor (VEGF) and prevents its binding to the receptor and induction of the angiogenic process. In the absence of VEGF signalling, tumour mass collapsing is observed in patients due to an inadequate nurturing of tumour via the blood flow.6 However, the high complexity of production associated with high cost remain the major limitations of the protein/antibody-based biopharmaceuticals. Therefore, peptides appear at the interface of small molecules and proteins in regard of the production complexity and costs. Thus, they have been intensively investigated in pre-clinical studies for the development of new imaging moieties and drug conjugates to visualize and destroy tumour cells.7 We discuss in this chapter some aspects of the R&D set up to screen and characterise tumour targeting peptides.

2 The phage display technology: screening and characterisation of tumour targeting peptides 2.1 Viral tools A targeting peptide can be discovered by using different approaches. These include the development of derivatives inspired by the natural protein sequences,8 identification of short binding sequences to molecular targets overexpressed by neoplastic cells,9 isolation of natural peptides with targeting properties10 or the one-bead one-compound (OBOC) peptide libraries.11 One breakthrough in the identification of new targeting peptides came from the idea of screening peptide libraries composed of billions of short random amino acid sequences displayed on viral particles.12,13 The first demonstration of such viral engineering was published in 1985.14 In this article, the author has generated genetically engineered DNA-containing filamentous bacterial viruses called bacteriophages (phage). The used virions were modified to express extra amino acid sequences on their protein coat. A phage-displayed library is actually a heterogeneous melting-pot of phage clones each containing an individual foreign DNA sequence and therefore displaying an individual peptide on its surface.15 The peptide library consists of random synthetic peptides that are prepared according to the NNK rule where N can be any nucleotide (A, T, C or G) and K can be either T or G. The exclusion of A or C in the third position decreases the possibility of stop codons in the peptide sequence from 3 to 1. In case of the filamentous phage the peptide is inserted in the N-terminus of the pIII/pVIII capsid protein. More recently, inverted Amino Acids, Pept. Proteins, 2018, 42, 280–319 | 281

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pVIII proteins have been used to display peptides with free C-terminus.16 Another technique called pVIII landscape allows the display of peptides on all the pVIII coat proteins.17 The filamentous viruses used for phage display include the M13 phage characterised by high replication rate and capacity to allow foreign DNA limited to short peptide sequences (7 to 12mer) within its DNA. M13 is a slowly growing, long (900 nm) rod-shaped phage that infects Escherichia coli (E. coli) with pilus. The fd filamentous phage, sharing structural and genomic similarities with the M13, is also used for the pIII and pVIII displays.13 More recently, the lytic T7 phage was engineered and used for phage displayed peptide library construction. T7 is a small (about 65 nm in diameter) icosahedral phage, can accommodate longer peptides, i.e. 6–20mer, infects E. coli without pilus, and grows faster than the non-lytic M13 phage.

2.2 Production of phage displayed peptide libraries A brief description of the T7 engineering is shown in the Fig. 1A. Each oligonucleotide encoding one random peptide sequence is inserted in an T7 phage vector creating a fusion between the gene encoding the capsid protein and peptide. Thus, the first cDNA library is constituted of billions of plasmids encoding the phage capsid proteins and differing only on the random peptide sequence. The final phage library is then obtained after an in vitro packaging, i.e. transduction of E. coli cells with the cDNA allowing the phage particle assembly in the host bacteria, its release by bacterial lysis followed by infection of more bacterial cells and amplification of phage. The phage displayed libraries are currently widely used for combinational selections. Within a library each phage clone displays one unique peptide in multiple copies and the diversity of peptide libraries can reach up to 109 permutations. As illustrated on Fig. 1B, the screening process per se is performed by introduction of the phage library to the target molecules (i) in vitro as isolated single proteins/receptors on microtiter wells or beads (Table 1), (ii) expressed by cultured cells (Table 2), (iii) ex vivo present in cell or tissue extracts/cells, and (iv) in vivo in whole animals (Table 2). Solely the phage with binding specificity towards the target will be selected and enriched in a process that is analogous to natural selection.

2.3 In vivo phage display for tumour-specific peptide identification The interaction between the peptide and target allows the discrimination and washing off the unspecific/unbound phage. Specificallybound phage are rescued and used for production of more phage by new bacterial infections to obtain a phage mixture (pool) enriched with the different binding phage. Repeated cycle of these steps is referred to as ‘‘biopanning’’. When sufficient enrichment has 282 | Amino Acids, Pept. Proteins, 2018, 42, 280–319

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Fig. 1 Summary of the phage displayed library production and subsequent screening. A, left panel, the displayed peptide is cloned in the C-terminus of 10B gene encoding the capsid protein in vector that contains the T7 cDNA. When transduced in E. coli, the produced protein is a chimera of the coat protein and the targeting peptide, ultimately displayed outside of the phage (middle panel) where it is accessible to the environment. The final phage library contains up to 109 peptide sequences (right panel). B, Once the library has been prepared, phage can be incubated on isolated proteins on microtiter wells or bound to streptavidin beads (left panel), cultured cells or isolated tissues as well as extracts from both (middle panel) or directly injected into tumour-bearing animals (right panel, see more details on Fig. 2). Unbound phage particles are washed and the bound ones are rescued, amplified and re-introduced on biological samples during multiple cycles of selection. This biopanning procedure enriches the phage population to retain only the bound one(s).

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occurred, individual phage are isolated and the part of the cDNA encoding the peptide is sequenced to identify the binding peptides. When considering finding new cancer-associated molecular targets, the combined ex vivo/in vivo screening is recommended,18,19 since it highly increases the chances of isolating specific tumour homing peptides. Indeed, the tumour microenvironment, which is absent in the cell culture, orchestrates the expression of cancer specific molecular markers expressed by tumour cells and the associated vasculature responsible for the tumour growth, metastatic escape20 or immunosuppressive status.21 Moreover, major remodelling of the microenvironment22 that may participate in the development of treatment resistance and tumour relapse is observed after cycles of radiotherapy. Therefore, the xenograft models consisting of grafted patient derived cells or fragments in immunocompromised animals offer recapitulation of the microenvironment containing stromal cells as well as tumour induced angiogenesis and lymphangiogenesis. However, it is noteworthy that these models lack some of the immune cells present in human tumours. As summarised in the Fig. 2, the in vivo screening of polypeptides displayed on phage requires injection of the libraries intravenously, intraperitoneally or intracardially in the living animals followed by circulation of the phage for up to 2 h to ensure the proper distribution and binding to the molecular targets. Then the tissue of interest is harvested and the bound phage rescued from the dispersed tissue. The recovered and amplified phage pool is then re-injected for additional cycles of selection before the analysis of the binding motifs. Phage clones displaying peptides interacting with non-target tissues, i.e., non-tumour organs or ubiquitous cell surface receptors and plasma proteins, are progressively depleted from the collected phage population. It is also important to note that only peptides that exhibit both resistance to the degradation in the blood and proper accessibility to their targets can be selected and propagated. During the in vivo screening, positive and negative selections occurs simultaneously leading to removal of phage particles that bind to non-tumour organs. However, non-specific uptake of particles by the reticuloendothelial system removes phage from the blood stream and some of the possible binders may be lost. Therefore, the in vivo biopanning is recommended to be preceded by the ex vivo round(s) (Fig. 2)23 where the initial phage library is incubated with a tumour-derived cell suspension prepared from the resected xenografts. If peptides binding to a specific cell population within the tumour is required, magnetic beads coated with antibodies directed against molecular markers of desired cells are used to pull-down these cells. This method can also be used to remove the unwanted cells from the population (e.g. host endothelial cells found in the tumour vasculature, infiltrates of immune cells or fibroblasts) and consequently, deplete the nontumour-cell specific phage from the pool. The beads are added to the cell suspension after the phage binding and wash-off the unspecific phage. 284 | Amino Acids, Pept. Proteins, 2018, 42, 280–319

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Table 1 Sequences and molecular targets of peptides isolated from libraries screened on purified receptors.a,b Application ref.

Amino Acids, Pept. Proteins, 2018, 42, 280–319 | 285

Sequence

Acronym

Target protein

Library

Imaging

Therapy

Year

DHLASLWWGTEL NYSKPTDRQYHF AC*AQKLDGC*SYISWSC*G AC*SGWWPKC*QGYIPGC*G AC¤APGVYRC¤NQNFIWC¤G IPLPPPSRPFFK LMNPNNHPRTPR CHHNLTHAC CLHHYHGSC CHHALTHAC SPRPRHTLRLSL TMGFTAPRFPHY RMWPSSTVNLSAGRR NGYEIEWYSWVTHGMY FRSFESCLAKSH YHWYGYTPQNVI QHYNIVNTQSRV QRHKPRE HSQAAVP AGNWTPI PLLQATL LSLITRL CRGDCL GACRGDCLGA CRRETAWAC GACRRETAWACGA VSWFSRHRYSPFAVS CDCRGDCFC

TJ12P1 APP1 BC1 BC2 BC3

GPC3 PD-L1

Ph.D.-12

27 28

28

2016 2015 2016

b-Catenin

ACX6CX6CG

PDGFRb PKCd

Ph.D.-12

PTPRJ

Ph.D.-C7C

PKC-bp PTPRJ-pep19 PTPRJ-pep24 PTPRJ-pep19.4 B18 P1 SP5.2

TfR 1 Tie 2 CD-21 VEGFR1 (Flt-1) IL-10Ra

SP5.2

Ph.D.-12 15mer 16mer Ph.D.-12

EGFR P12 AP8 P7 S7

FGF8b aFGF bFGF IL-6Ra a5b1

P3 RGD-4C

a6b1 avb3/avb5

Ph.D.-7

29 30 31 32

2013 2016 2012

33 34

53

2013 2013 2009 2006 2003 2013 2005–2013 2014 2015 2013 2014 2008 2005 1993

54

1994

35 36 37 38 39, 40 47 48 49 50 51

39, 41–46

52 6mer sp CX7C sp 15mer CX9

55 56–58

57–61

1996 1998–2011

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286 | Amino Acids, Pept. Proteins, 2018, 42, 280–319

Table 1 (Continued) Application ref. Sequence RTDLDSLRTYTL CTTHWGFTLC APSPMIW, LQNAPRS SWTLYTPSGQSK SWELYYPLRANL WQPDTAHHWATL CSDSWHYWC WHWLPNLRHYAS WHTEILKSYPHE, LPAFFVTNQTQD YNTNHVPLSPKY YSAYPDSVPMMS TNYLFSPNGPIA CLSYYPSYC CVGVLPSQDAIGIC CGPLPVDWYWC CEWKFDPGLGQARC CDYMTDGRAASKIC KCCYSL MARSGL, MARAKE, MSRTMS WTGWCLNPEESTWGFCTGSF MCGVCLSAQRWT, SGLWWLGVDILG NPGTCKDKWIECLLNG DPRHCQKRVLPCPAWL, FRERCDKHPQKCTKFL GGVSCMQTSPVCENNL ANTPCGPYTHDCPVKR, PQNSKIPGPTFLDPH

Acronym CTT

P1 peptide III CAIX-P1 YSA TNYL L-26-19 L-26-24 N-12-1 N-12-2 p6.1

Target protein

Library

avb6 MMP-9 CD133 N-cadherin E-cadherin PSMA VEGFR-3

Ph.D.-12 CX5-8C Ph.D.-7

EGFRvIII/EGFR CAIX EphA2 EphB4 PS

Ph.D.-12 Ph.D.-C7C

Ph.D.-12

CX7C

Imaging 63–66 71 72 73 74 75 76 77 78 79 83 85 86

Therapy

Year

62 63, 66–70

2007 1999–2011 2008 2008 2009 2006 2007 2015 2010 2010 2009–2013 2011, 2012 2008 2011

80–82 84

Ph.D.-12 HER2 6mer

EC-1

20mer Ph.D.-12

A3-10

TAG-72

A2-6 G3-C12 G3-A9

Galectin-3

16mer

87–89 90 91 92 93, 94 95 96 97, 98

2007–2010 2001 2004 2010 2007–2011 2011 2008 2008, 2009

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IVWHRWYAWSPASRI HGRFILPWWYAFSPS CGLIIQKNEC CNAGESSKNC AESGDDYCVLVFTDSAWTKICDWSHFRN MQLPLAT CRALLRGAPFHLAEC IELLQAR TLTYTWS

P30-1 P-30 CTL1 CTL2 C19

CVAYCIEHHCWTC AC*ERYQGC*FSVGGYC*G THENWPA WHPWSYLWTQQA VLWLKNR CTVRTSADC

C-4 NRR17 CV-1 RP-1 FP16 ZD2

AAAPLAQPHMWA SHSLLSS ALWPPNLHAWVP

sAPRIL-BP1

a

T antigen

15mer

Fibrin-fibronectin complexes

CX8C

FGFR E-selectin MMP2-processed collagen IV PSA Notch1 NRR CD44 FGF3 Extradomain-B fibronectin APRIL p16 pre-miR-21

26mer Ph.D.-7 15mer Ph.D.-7

99 100 101 102 103 104

2011 1997 2006 102

106

1999 2002 1999 2000 2009

CX3CX4CX2C ACX6CX6CG Ph.D.-7 Ph.D.-12 Ph.D.-7 Ph.D.-C7C

107 108 109 110 111 112

2000 2015 2016 2015 2015 2015

Ph.D.-12 Ph.D.-7 Ph.D.-12

114 115

105

113

2015 2015 2015

Table 1 note: disulfide bonds between two cysteine residues are highlighted in bold in the peptide sequence; *bicyclic peptide obtained by 1,3,5tris(bromomethyl)benzene; ¤bicyclic peptide obtained by 1,3,5-triacrylolyl-1,3,5-triaziane. Centered references in italics describe the peptide discovery without imaging or therapeutic application. b GPC3, glypican 3; PD-L1, programmed death-ligand 1; PDGFRb, platelet-derived growth factor receptor b; PKCd, protein kinase C d; PTPRJ, receptor-type tyrosineprotein phosphatase J; TfR1, transferrin receptor 1; Tie2, angiopoietin receptor 2; CD-21, complement receptor type 2; VEGFR1/3, vascular endothelial growth factor receptor type 1/3; IL-10Ra, interleukin-10 receptor a; EGFR/vIII, epidermal growth factor receptor/variant III; FGF8b, fibroblast growth factor 8b; a/bFGF, acidic/basic fibroblast growth factor; IL-6Ra, interleukin-6 receptor a; MMP-9, matrix metallopeptidase-9; CD133, prominin-1; PSMA, prostate-specific membrane antigen; CAIX, carbonic anhydrase IX; Eph A2/B4, ephrin type-A/B receptor 2/4; PS, phosphatidyl serine; HER2, human epidermal growth factor receptor-2; TAG-72, tumour-associated glycoprotein 72; FGFR, fibroblast growth factor receptor; PSA, prostate-specific antigen; Notch1NRR, negative regulatory region in Notch1; CD44, lymphocyte homing receptor; FGF3, fibroblast growth factor 3; APRIL, a proliferation-inducing ligand.

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Table 2 Sequences, molecular targets and cancer type of homing peptides isolated from libraries screened on cells and/or in vivo.a,b Peptide Sequence LTVSPWY SSMDIVLRAPLM QQLPSSSTSTYP FPMFNHWEQWPP SYPIPDT HTSDQTN CLFMRLAWC

Methodology Acronym

CSSRTMHHC CPIEDRPMC RGDLATLRQLAQEDGVVG-VR SPRGDLAVLGHK SPRGDLAVLGHKY CQQSNRGDRKRC CMGNKCRSAKRP CGEMGWVRC

Library

Setup

Biological sample

In In In In In

SKBR3 HCT116 SiHa U-87MG A431

In vitro

pHCT74 CSP-3

HER2

P1 P2

EGFR

Ph.D.-7 Ph.D.-12 Ph.D.-12 Ph.D.-12 Ph.D.-7

MUC18

CX7C

DMPGTVLP DWRGDSMDS VPTDTDYS VEEGGYIAA VTWTPQAWFQWV AQYLNPS

Targeted receptor

Nucleolin

VTW

RPMrel H2009.1 HBP HBP-1 RMS-I RMS-II

GP130 Nestin Cadherins a5b1

vitro vitro vitro vitro vitro

Landscape phage display In vitro libraries f8/8 & f8/9 Ph.D.-12 In vitro Ph.D.-7 In vitro

B16 cells þ B-1 lymphocytes

Cancer type

Imaging

Breast Colorectal Cervical Glioblastoma Epidermoid carcinoma Melanoma

116, 117 118, 119 120 121 122 123

Therapy Year 2007, 2012 2015 2017 2012 2012

124

2008 2010

MCF-7

Breast

125, 126

U-87MG Glioma stem cells B16-F10-Nex2 HT29 H2009 HNO223

Glioblastoma Glioblastoma

127, 128 129

2013, 2015 2011

130 132 134–139

2010 2003, 2004 2004–2013 2009

avb6

CX7C CX7C Ph.D.-20 Ph.D.-12

In In In In

avb3

CX7–10C

In vitro

RD

IL-13Ra2

Ph.D.-C7C

In vitro

G26-H2 & SnB19-pcDNA

vitro vitro vitro vitro

Application ref.

Melanoma Colon NSCLC Head & neck Rhabdomyosarcoma Glioblastoma

131 133 140 141

2009

142

2012

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GFRFGALHEYNS

VP2

CTLPHLKMC ASGALSPSRLDT SWDIAWPPLKVP

OSP-1

CTVALPGGYVRVC ETAPLSTMLSPY GIRLRG

Pep42 GMBP1

VPAC1

Ph.D.-12

In vitro

IGHC HSPG Adenoviral receptor

CX7C Ph.D.-12 Ph.D.-12

In vitro In vitro In vitro

CX3-12C Ph.D.-12 Ph.D.-7

In vitro In vitro In vivo

GRP78

Amino Acids, Pept. Proteins, 2018, 42, 280–319 | 289

CPGPEGAGC

APP

CX7C

In vivo

CGRRAGGSC

IL-11Ra

CX7C

In vivo

CRGRRST

RGR

CD40

CX7C

In vivo

CNGRCVSGCAGRC

NGR

APN

CX9

CGNKRTRGC

LyP-1

p32/gC1qR

CX7C

TIP-1 a2bb3

Ph.D.-7 Ph.D.-7

Ex vivo, in vivo Ex vivo, in vivo In vivo In vivo

a3b1 NRP-1

Ph.D.-7 Ph.D.-7

In vivo In vitro

HVGGSSV RGDGSSV

SWKLPPS GGKRPAR

P4

VPAC1transfected CHO-K1 Raji 143B A172 Me6652/4 SGC7901 Irradiated GL261 Patient mammary tissue Patient prostate

Angiogenic islets in RIP1Tag2 mice MDA-MB435, U-87MG MDA-MB435, U-87MG Irradiated and/ or SU11248treated LLC & GL261 AZ-P7a PC-1

Colorectal

143

2013

Lymphoma Osteosarcoma Glioblastoma

144 145 146

2008 2010 2014

Melanoma Gastric Murine glioblastoma Breast

¨m Waldenstro macroglobulinemia Pancreatic islet

Breast & Glioblastoma Breast & Glioblastoma Lewis Lung Carcinoma (LLC) & murine glioma Gastric Prostate

150

147, 148 149 150

151

152

2006, 2008 2013 2010 2002

153

2004, 2007

154

2008

155–162

2000–2011

164–168

2004–2011

169–172 169, 171 173 173

2008, 2011 2003

163

174 175

2006 2011

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Table 2 (Continued) Peptide

Methodology

Sequence

Acronym

RIGRPLR CGFYWLRSC RPARPAR

P7

TLTYTWS

Targeted receptor

Library

Setup

CX7C Ph.D.-7

In vitro Ex vivo In vivo In vivo In vitro

MMP2processed collagen IV

Ph.D.-7

SSQPFWS

VAV3

Ph.D.-7

In vivo

YRCTLNSPFFWEDMTHEC-HA KTLLPTP PTP

CRKL Plectin-1

20mer Ph.D.-7

In vivo Ex vivo

CGLSGLGVA

MDGI

CX7C

Ex vivo, In vivo

CooP

Application ref.

Biological sample

Cancer type

Imaging

Molt-4 PC-1

Lymphoma Prostate

175

LLC MMP2processed collagen IV Patient glioblastoma DU145 PDAC cells from the Kras/p53 mouse model

LLC

HIFko murine astrocytes

Glioblastoma

Therapy Year 176

2011 2011

106

2009

177

Prostate Pancreatic ductal 179, adeno180 carcinoma (PDAC) Murine glioma 181 181

2014 178

181–183

2009 2008, 2010

2014 2013–2015

a Table 2 note: disulfide bonds between two cysteine residues are highlighted in bold in the peptide sequence. Centered references in italics describe the peptide discovery without imaging or therapeutic application. Cancer cells or tissue used for the screening are human unless otherwise specified. b HER2, human epidermal growth factor receptor-2; EGFR, epidermal growth factor receptor; MUC18, melanoma cell adhesion molecule; GP130, glycoprotein 130; IL-13Ra2, Interleukin 13 receptor a2; VPAC1, vasoactive intestinal polypeptide receptor 1; IGHC, immunoglobulin heavy locus C; HSPG, heparin sulfate proteoglycan; GRP78, glucose-regulated protein78; APP, amyloid precursor protein; IL-11Ra, interleukin-11 receptor a; PDGFRb, platelet-derived growth factor receptor b; APP, aminopeptidase P; TIP-1, tax interacting protein-1; NRP-1, neuropilin-1; MMP-2, metallopeptidase-2; VAV3, guanine nucleotide exchange factor; CRKL, Crk-like protein; MDGI, mammary-derived growth inhibitor; LLC, Lewis lung carcinoma; NSCLC, non-small cell lung cancer; PDAC, pancreatic ductal adenocarcinoma; HIFko, hypoxiainducible factor 1 knockout cells.

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Fig. 2 Methodology of the in vivo phage display screening. The most commonly used tumour-bearing rodent models allow the engraftment of human neoplastic tissue that is essential for the biopanning. Libraries can be applied as such or via library enrichment reached by a preliminary step of ex vivo round(s) (left).

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2.4 Remarks on the methodology As discussed shortly above, phage displayed libraries have several advantages: 1. they can display combinational peptide libraries with billions of peptide sequences; 2. the length of the displayed peptide is flexible; 3. they can accommodate a known protein fold as in the case of conotoxin, immunoglobulins, disulphide bonds or zinc-fingers. This can be achieved by grafting random oligopeptides on the tertiary sites; 4. the methodology is efficient, inexpensive and covers a wide range of peptides, i.e. short or long sequences, linear or simple cyclic peptides; 5. at least the M13 libraries (Ph.D.-7, Ph.D.-12 and a cyclic Ph.D.-C7C library) are commercially available; 6. the adaptability of the library allows the use of in vivo models for the screening; 7. the identification of novel ligands does not require any knowledge of the identity of the targets expressed by tumours; 8. fine chemical manipulation of the phage library by using thiolreactive compounds or cross-linkers allows the display of peptides with limited cyclization and derivatization.24,25 However, despite the list of advantages, the biological libraries are suffering from some limitations: 1. restriction to 20 eukaryote L-amino acids that can be inserted in the phage; 2. the increased risk of proteolysis in the absence of N- and C-terminal protection; 3. the unwanted enrichment of target-unrelated peptides with no real affinity for the target;26 4. the principle of the phage displayed library is only applicable to binding and some functional assays like protease substrate determination or affinity maturation; 5. increased complexity of the peptide structure, i.e. bicyclic, branched structures, compacted scaffolding and peptides requiring fine chemistry of cyclisation, might not be achievable in a phage library; 6. optimisation of the sequence in order to preserve the peptide from proteolysis often compromises the binding activity and the specificity to the targeted tumour specific receptor. 7. The inherent stickiness of the phage particle brings background to the system.

3

Targeting peptides for cancer imaging and therapy

3.1 Diversity of the tumour targeting peptides The biopanning of phage displayed peptide libraries has uncovered numerous tumour targeting peptides binding/homing to tumour cell surface proteins as well as tumour associated blood and lymphatic 292 | Amino Acids, Pept. Proteins, 2018, 42, 280–319

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vasculature. These identified peptides target tumours from multiple origins: lungs, skin, breast, thyroid, liver, head and neck, prostate, bladder, colorectal and gastric cancers, osteosarcoma, glioblastoma, pancreatic ductal adenocarcinoma and squamous cell carcinoma. Table 1 displays a list of targeting peptides characterised using the in vitro biopanning on purified receptors and Table 2 summarises peptides identified using cells, tissues or animals. Although the tables recapitulate the major discoveries of the past twenty years, in the text we are focusing on the more recent or more widely used cancer-specific sequences. Homing peptides are classified according to their tumour targets, i.e. the blood and lymphatic molecular markers, the microenvironment/immune cell infiltrates and finally the cancer cell specific receptors.

3.2 Peptides targeting the blood and lymphatic vessel specific receptors 3.2.1 Peptides binding to the neuropilin-1. During development neuropilin-1 (NRP-1) plays a central role in axon guidance and angiogenesis.184 In addition, NRP-1 is also implicated in the primary immune response.185 In the case of vascular development, NRP-1 binds the VEGF-A165 isotype and acts as a co-receptor for its receptor VEGFR-2, whose activation serves as a sensor for the endothelial cell guidance during expansion of the blood vessel network and migration of cells towards a molecular gradient.186 NRP-1 expression is upregulated during the angiogenic response induced by wound healing or hypoxia.187 When taking cancer into account, NRP-1 is highly expressed in a variety of neoplasms including breast, lung, pancreatic, prostate, ovarian, and gastrointestinal carcinomas.188 Acute lymphoblastic and acute myelogenous leukaemia also express high levels of NRP-1.176 Globally, increased expression of NRP-1 correlates with metastatic progression in patients and tumour growth and angiogenesis in animal models. Thus, the hypothesis of using NRP-1 targeting peptides in a receptormediated drug delivery system has been raised up in several studies. One of the first NRP-1 targeting peptide, RPARPAR, identified in the Ruoslahti lab via an ex vivo screening of T7 phage libraries189 has been subsequently joined by the GGKRPAR and RIGRPLR sequences. Both are exhibiting a higher binding affinity and specificity compared to RPARPAR when they were tested in a microfluidic phage selection channel system (MIPS).175 This MIPS system consist of cells expressing the receptor of interest cultured at a minimal density in a microchamber equipped with channels. Such channels can be perfused by a peristaltic pump with medium containing the library, creating a more stringent selection condition than classical 2D cell cultures. The affinity and specificity is then determined after washing and lysis of the cells. Usage of targeting sequences such as CLKADKAKC (CK3), containing a cryptic C-end rule (CendR) motif described by Teesalu et al.189 that is essential for the binding to NRP-1, has been recently assessed as a potential tool for in vivo imaging. Peptide conjugates CK3-HYNIC and CK3-Cy5 have Amino Acids, Pept. Proteins, 2018, 42, 280–319 | 293

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been used for micro single-photon emission computed tomography (microSPECT) and near-infrared fluorescence (NIRF) imaging, respectively, of human breast cancer xenografts in mice, respectively.190 Both conjugates showed specific accumulation within the neoplastic mass associated with excellent tumour contouring with both SPECT and NIRF imaging modalities. Interestingly, the heptapeptide ATWLPPR191 exhibiting targeting properties towards NRP-1 has been used by Wu et al. in a heterodimeric probe linked to an RGD peptide. The [18F]-labelled heterodimer showed higher tumour uptake compared to single peptide sequences in a subcutaneous model of human glioblastoma grafted in rodents.192 In the search of novel targeting peptides with therapeutic applications, the screening of a CX7C phage displayed library on human T-cell lymphomas revealed a new motif F(F/Y)xLRS (x ¼ random amino acid) that targets NRP-1.176 Karjalainen et al. then developed a cyclic peptide CGFYWLRSC (C residues forming disulfide bonds in bold) linked to a pro-apoptotic (KLAKLAK)2 peptide prepared using D-amino acids.193 This engineered peptide diminished lymphoma cell viability whereas a mixture of both unlinked peptides was not cytotoxic at the same micro-molar range concentration. More recently, the group of Kim tested thirty prescreened tumour homing motifs reported in the Tumor Homing Peptide database (TumorHoPe194) to find pre-identified sequences that would possess cytotoxic activity on cancer cells. This led to discovery of a sequence that bound to NRP-1 called TU17.195 This sequence linked to the mitochondrial targeting domain (MTD) KLLNLISKLF, generated a novel TU17:MTD therapy that specifically targets, penetrates and kills more efficiently human colorectal cancer in vitro and in vivo than the TU17:(KLAKLAK)2 conjugate. Glioblastoma, the most common subtype of glial-derived brain neoplasms is characterised by intense tumour angiogenesis. Therefore, therapeutic functionalised nanoparticles were decorated with the previously mentioned ATWLPPR191 peptide to use NRP-1 as an entryway, in addition to the heparin sulphate binding peptide CGKRK196 targeting both the endothelium and the neoplastic cells.197 When loaded with the cytotoxic chemotherapy Paclitaxel, the authors reported a greatly increased accumulation and penetration of the nanocarriers within the tumour. Moreover, homing of these nanoparticles correlated with a significantly prolonged survival of treated mice intracranially implanted with human glioma cells compared to the plain drug Paclitaxel. 3.2.2 Homing peptides binding to the vascular endothelial growth factor receptor-3. Vascular endothelial growth factor receptor-3 (VEGFR-3) is a type III tyrosine kinase receptor widely expressed in the embryonic endothelium and whose expression gradually decreases during the development in the blood vessels but remains high in the lymphatic vasculature.198 VEGFR-3 appears to be upregulated in many human cancers and is essential for the angiogenic sprouting of tumour blood and lymphatic vessels.199,200 One of the first homing peptides with affinity towards VEGFR-3 was identified using the phage display library on the isolated receptor.75 The sequence CSDSWHYWC (peptide P1) specifically binds to 294 | Amino Acids, Pept. Proteins, 2018, 42, 280–319

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VEGFR-3 expressed by various carcinoma cells. In addition to P1, Shi et al. also isolated the WHWLPNLRHYAS or peptide III from a phage displayed library screened on the recombinant VEGFR-3 and showed that it co-localises with endothelial cells lining lymphatics and it could be a useful tool for the microSPECT imaging as demonstrated using xenografted ovarian tumours.76 3.2.3 LyP-1 homing peptide. The peptide sequence CGNKRTRGC known as LyP-1 has an interesting profile. Discovered by Laakkonen et al., LyP-1 is capable of homing to tumour lymphatics, tumour cells, and tumour-associated macrophages that express its receptor p3223,201 in primary and metastatic breast cancer xenografts165 as illustrated in the Fig. 3. Its targeting activity is interestingly accompanied by cell penetrating properties and cytotoxic effect on human breast cancer cells.165 LyP-1 was very recently used as an imaging moiety for human pancreatic cancer xenografts in immunocompromised rodents.202 In this study, mesoporous silicate nano-spheres (SiO2) decorated with LyP-1 specifically bound to the mitochondrial p32 and accumulated in neoplastic pancreatic tissue but not in the normal pancreas. This LyP-1 nanoparticle model has been further engineered by addition of ferrous oxide (Fe3O4) for the T2-weighted magnetic resonance (T2-MR) imaging in animals. As a proof-of-concept, the Fe3O4-SiO2-LyP-1 particles showed a long lasting (up to 24 h) accumulation in the pancreatic tumour tissue. A similar rationale has been adopted to develop a LyP-1/ micelle/drug-conjugate. Such engineered particle allows combination of (i) photothermal (PTT), (ii) photodynamic (PDT), and (iii) chemotherapy

Fig. 3 Targeting the tumour lymphatics using the CGNKRTRGC (LyP-1) peptide. Nude mice xenografted with MDA-MB-435 cells (human breast cancer) were intravenously administered with a fluorescein-labelled LyP-1 to determine the intra-tumoural distribution of LyP-1 using histological analyses. Labelling with antibodies directed against MECA-32 (left panel), a blood vessel marker, or LYVE-1 (right panel), a lymphatic vessel marker, was then performed. Arrows indicate the homing of LyP-1 to breast cancer cells. The dashed arrow in left panel points the absence of blood vessel targeting while the arrowheads in the right panel identify the co-localisation of LyP-1 with the tumour lymphatics. Laakkonen et al. unpublished images. Amino Acids, Pept. Proteins, 2018, 42, 280–319 | 295

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203

modalities. Shortly, PTT and PDT consist of laser-excitable probes such as IR820 that can locally increase the temperature (up to 45 degrees of Celsius) and/or release singlet oxygen when laser-irradiated at 808 nm and kill the surrounding cells. The authors demonstrated that LyP-1 conjugates allow the homing of the targeted therapy within human breast tumours engrafted in murine mammary fat pads. Irradiation of the neoplastic mass permits a local and efficient PTT that slowed down tumour growth and prolonged survival of tumour-bearing mice. Eventually, LyP-1 conjugated to a conventional cytotoxic chemotherapy, doxorubicin (dox), has been assessed as a potential targeted therapy for human breast cancer.204 For instance, when incubated on cultured human breast cancer cells, LyP-1-dox showed significantly higher cytotoxicity than the free dox. In addition, LyP-1 conjugated liposomes loaded with dox reduced number of tumour-associated lymphatics and decreased lymph node metastases.205 More recently, Roth et al. demonstrated that a truncated sequence (from CGNKRTRGC to CGNKRTR) of the LyP-1 (tLyP-1) that still contains the CendR sequence motif (ref. 189 for review) allowing peptide binding to NRP-1 and internalisation.206 The tLyP-1 surprisingly exhibited better potency than the parent peptide LyP-1 and enhanced the extravasation of co-injected nanoparticles in human breast cancer xenografts. Moreover, Dong et al. have recently tested a [131I]-labelled tLyP-1 for targeted imaging of human non-small cell lung cancer (NSCLC) xenografts in nude mice.207 The detection of probe showed an intense accumulation of the radiolabelled peptide within the neoplastic mass six hours postinjection, suggesting a possibility of translating this targeting agent for clinical imaging. Similar observation has been reported on subcutaneously implanted human gliomas in immunocompromised mice.208 The development of a [18F]-tLyP-1 imaging probe for the microSPECT shows intense uptake of the radioactivity by the tumour mass that can be specifically washed away by the intravenously injected free cold ligand. Drug conjugates using the tLyP-1 have been developed during the past three years, mostly using nanoparticles. For instance, in a setup of a dual targeting of tLyP-1 and lactoferrin (Lf), a globular glycoprotein from the transferrin family that is particularly expressed by endothelial cells in the blood-brain-barrier and acts as transcytosis enhancer for the nanocarriers. The tLyf-1 peptide was used as a glioma-targeting peptide was co-administered with Lf-nanoparticles containing the chemotherapy Paclitaxel. Experiments on cells showed excellent penetration of the nano-therapies in cultured gliospheres and studies with mice bearing murine gliomas demonstrated higher accumulation and deeper penetration of the nanoparticles in the brain tumours compared to the particles without co-administration with tLyP-1 or Lf.209 3.2.4 Other peptides homing to tumour vessels. Among the major discoveries in the homing peptide domain, the tripeptide RGD targeting the integrin avb3 identified in the late 80’s210 is still used as a targeting moiety for imaging211 or drug delivery.212 The avb3 is highly expressed by the angiogenic blood vessels213 and at the surface of 296 | Amino Acids, Pept. Proteins, 2018, 42, 280–319

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cancer cells (the reader can refer to 3.4.1). One interesting use of the RGD was published by Peiris et al.214 The detection of micro-metastases derived from peripheral cancers, including breast cancer, can be particularly challenging due to the reduced size of the neoplastic mass, sometimes composed of only hundreds of cells. The authors developed gold-nanoparticles (Au-NP) displaying RGD peptides to target the metastatic vascular niche at an early stage of the disease. They successfully visualised hotspots of micro-metastases by using a [99Tc]-Au-NP-RGD particles and the microSPECT imaging. Sites of metastasis within the organs were confirmed by detection of the GFP expressing fluorescent tumour cells. Disseminated GFP-expressing breast cancer cells of the orthotopically xenografted immunocompromised mice were detected in the endothelium of lungs, kidneys, intestine, and liver at sites that overexpressed avb3 allowing the detection by the probe.214 Novel binding sequences specific to the tumour endothelium are discovered each year, often orphan by their molecular targets. For instance, ASSHN was discovered via in vitro biopanning on human endothelial cells using the M13 library displaying random pentapeptides.215 The authors developed dox containing liposomes (Lip) conjugated to ASSHN to demonstrate the targeting and therapeutic potential of the peptide. Mice bearing human colon cancer and intravenously injected twice with the ASSHN-Lip-dox showed statistically significant diminution of the tumour size. This observation is quite promising, considering that most of the drug conjugates require heavier dosing up to daily injections.

3.3 Peptides targeting tumour microenvironment 3.3.1 Peptides homing to the immunosuppressive environment within tumours. The implication of the immunosuppressive microenvironment of tumours and the development of novel therapies to counteract it, is a relatively new topic in cancer biology.216 Thus, targeting peptides isolated from phage libraries are relatively sparse. Nevertheless, some elegant approaches using synthetic peptides for imaging purposes have demonstrated encouraging results. For instance, a preclinical study conducted by Larimer et al.217 brilliantly demonstrated that a serine-protease granzyme B, that plays a major role in cancer cell death and is released by T-cells, can be used as a biomarker for early response prediction of tumour immunotherapy. They developed a synthetic granzyme B peptide (GZP) conjugated to [68Ga]-DOTA. By using microSPECT imaging on immunocompromised mice bearing human melanoma xenografts, they were able to predict, which animals treated with several immune checkpoint blockers would respond to the therapy, based on the binding of the GZP-peptide to the released granzyme B. GZP is designed to irreversibly bind granzyme B and the subsequent imaging allows the discrimination of treatment responsive tumours from the resistant ones based on the granzyme B levels visualised by the peptide (Fig. 4). Amino Acids, Pept. Proteins, 2018, 42, 280–319 | 297

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298 | Amino Acids, Pept. Proteins, 2018, 42, 280–319 Fig. 4 Usage of a granzyme-B targeting peptide to follow-up the melanoma response to treatment. A, Melanoma-bearing immunocompromised mice are injected with a cocktail of immune checkpoint blockers. If responsive to treatment, T-cells release massive amounts of granzyme B that kills melanoma cells. The GZP peptide labelled with [68Ga]-DOTA allows PET/CT imaging of the granzyme B levels and thus correlates with the treatment response. B, Accordingly, monotherapy or non-successful combination therapy generates low T-cells response, less granzyme B secretion and thus lower radiolabelling of the tumour mass. C, Absence of treatment allows melanoma progression and induces total absence of intra-tumour radioactivity detected in PET.

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Other studies have attempted direct targeting of the immune cell population that participate in tumour progression. For instance, M2 macrophages or tumour-associated macrophages (TAMs) are known to facilitate the progression of various diseases including cancer unlike the tumour suppressor M1 macrophages.218 It was recently demonstrated that targeted inactivation of these tumour-infiltrating macrophages using small molecule inhibitors significantly diminished the number of tumour-initiating cells, number of metastases, and abolished drug resistance.219 Researchers successfully isolated a peptide sequence, YEQDPWGVKWWY (M2pep) via the biopanning of a phage library on macrophages. They had prior the screening polarised murine bonemarrow-derived macrophages, using stimulation either with interferon-g and lipopolysaccharide (to obtain the M1 phenotype) or interleukin-4 (to induce the M2 phenotype). The generated M2 macrophages were then used for the in vitro biopanning. Furthermore, mice bearing fibroblastic carcinomas (CT-26) were injected with the fluorescence-labelled Alexa660-M2pep to confirm the intra-tumour homing and specificity to the M2 macrophages. Translational therapeutic application was assessed by linking a cytotoxic (KLAKLAK)2 sequence to the M2pep and administration of the fusion peptide allowed longer survival time of the treated animals compared to controls. Reduction of the TAM number within the tumour confirmed the targeting efficacy of M2pep.220 Further development of M2pep to increase the valency such as divalent M2pep linked to the divalent (KLAKLAK)2 potentiated the depletion of tumour-associated M2 macrophages.221 Most of the constructs showed increased selectivity and toxicity towards M2 but not M1 macrophages in the CT-26 mouse model. Interestingly, modifications of the peptide structure by cyclisation chemistry (ref. 222 for reference) also increased the peptide stability in the serum and binding to M2 macrophages. Eventually, as the authors state, the receptor of M2pep associated with M2 TAMs is yet to be identified to fully dissect the binding mechanism. Identification of the receptor will also facilitate development of further means to inactivate, repolarize and/or deplete the tumour promoting macrophages. 3.3.2 Peptides homing to the extracellular matrix proteins. Cancer cells obtain their invasion/metastatic potential not solely from genetic mutations but they are also capable of modifying their biophysical and biomechanical features and adapting to the surrounding microenvironment. The extracellular matrix (ECM) is a central component of the tumour. It provides a mechanical support for the tissue, controls the cell-microenvironment interactions and plays an important role in tumour cell invasiveness and metastasis. Tumour cell invasion is a multistep process including the cell transition from epithelial morphology to a mesenchymal one, allowed by the high plasticity of the cells and the close interaction of specialized membrane receptors with the extracellular environment.223 It also requires a complete re-orchestration of the ECM, coordinated by numerous matrix metalloproteinases (MMPs).224 Those endopeptidases pave the way, i.e. degrade the collagens, fibronectin and other ECM protein networks, allowing the tumour cells to Amino Acids, Pept. Proteins, 2018, 42, 280–319 | 299

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225

infiltrate the surrounding tissue. In solid metastatic cancer, the two often overexpressed MMPs are the type 2 and 9.226 It is also noteworthy to mention that MMPs can be cell membrane-bound or secreted and they are produced in an inactive pro-form that is activated by a propeptide cleavage.227 This suggests that (i) the membrane-bound proforms of MMP-2 and -9 can be used to monitor/image invading cells and (ii) the activated forms of MMPs, responsible for the proteolytic modifications of the ECM within and around the neoplastic mass, can be imaged to follow-up the tumorigenic modifications of the microenvironment. CTTHWGFTLC (CTT-1), identified from a phage display library screen on activated MMP-9-coated microtiter wells, is one of the first peptides targeting the MMP-9. Interestingly, CTT-1 displays not only high affinity towards MMP-9 but binds also to MMP-2. This peptide inhibits migration of normal endothelial and tumour cells (in vitro) and tumour progression in animal models.63 The group of Kairemo produced a modified version of the CTT peptide, called CTT-2, by adding a peptide linker (a tyrosine moiety) for iodination purposes and a short spacer sequence GRENYHG, enhancing the freedom of the peptide when conjugated to liposomes. The GRENYHGCTTHWGFTLC-micelles can be radio-iodinated with [125I] for nuclear imaging, histological distribution and/or quantification, and the liposomal part can be loaded with cytotoxic drugs such as dox. In a preclinical model of ovarian carcinoma, the authors demonstrated higher accumulation of CTT-2-micelles into the neoplastic mass compared to normal organs. Notably, the therapeutically engineered liposomes containing dox were also capable of prolonging the survival of the xenografted animals of more than forty days compared to the plain dox.61 Crisp et al. recently proposed another model allowing the dual targeting of MMP-2 and the integrin avb3 for the optical imaging of various tumours including glioma, ovarian carcinoma, and breast cancer orthotopically implanted in immunocompromised mice.228 The authors propose that an optimisation of the substrate-enzyme interaction inspired by the natural inclusion of an exosite-binding domain adjacent to a protease cleavage site in the substrate, improves its affinity and specificity for the enzyme. They used this strategy to improve the targeting properties of an activatable anionic cell penetrating peptide (ACPP) via addition of a specific MMP-2 recognition sequence that has to be cleaved to allow the internalisation of the CPP. The addition of a cyclic-RGD to the ACPP fragment was inspired by the reported binding of the integrin avb3 to the hemopexin domain of MMP-2 and the involvement of avb3 in the activation of the MMP-2.229 Thus, the cyclic-RGD was added as a co-targeting moiety by attaching it to the inhibitory portion of the ACPP. This moiety is subsequently attacked by the proteases to allow the CPP separation from the cyclic-RGD-polyglutamate fragment of the conjugate (Fig. 5). The authors observed improved cellular uptake compared to unmodified ACPP. Fluorescence imaging of the probe within the tumour mass in animals very interestingly showed not only excellent tumour specificity but the probe allowed detection of pulmonary metastatic lesions of mammary adenocarcinoma as small as 0.5 mm. 300 | Amino Acids, Pept. Proteins, 2018, 42, 280–319

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Fig. 5 Rationale of the dual targeting to MMP-2 and the integrin avb3, using a cyclic-RGD-PLGC(Me)AG-ACPP.

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Eventually, the pre-clinical therapeutic application by using the cytotoxic monoethylauristatin E (MMAE) conjugated to the ACPP-RGD showed encouraging results. The authors reported a volume diminution of the breast cancer, orthotopically implanted in the mammary fat pads of nude mice as well as greatly enhanced survival compared to the free-drug injected animals (300 days vs. 20 days). However, prudence is required with the usage of ACPP in the detection of tumours exhibiting intense angiogenesis. Indeed, it has been shown that ACPP can be activated by the vasculature even before reaching the tumour. In addition, false positive results were obtained in highly angiogenic tumours expressing low levels of MMP-2/9 by the tumour cells.230 The tumour stroma and the ECM established after a wound share a predominant fibrotic composition. Moreover, clotting of plasma proteins is known to leak in the extravascular space of tumours and other nonneoplastic lesions. Pilch et al. identified, by using phage display, two peptide sequences CGLIIQKNEC (CLT1) and CNAGESSKNC (CLT2) that specifically accumulate into the fibrillar meshwork of the tumour stroma, injured arteries, the skin and the sceletal muscle.101 The characterised target appears to be the ECM protein fibronectin. In human prostate and bladder cancer cells, CLT1 forms complexes with fibronectin and is internalised. CLT1 can also bind fibrinogen but that prevents its internalisation.230 Interestingly, in bladder cancer cells, CLT1 induced cell death due to the accumulation of cytotoxic CLT1-fibronectin coaggregates as previously observed in the growing blood vessels.231 In cancer cells the co-aggregate can activate the integrin a5b1 causing its internalisation and triggering an autophagic cell death via the endoplasmic reticulum stress in cooperation with an intracellular chloride channel protein called CLIC3. Tumour cells express high amounts of both a5b1 and CLIC3 making the CLT1 induced cell death an attractive candidate for anti-cancer peptide therapy.

3.4 Peptides targeting cancer cells 3.4.1 Peptides homing to tumour specific membrane proteins. Tumour cells are known to overexpress a large collection of GPCRs, tyrosine kinase receptors, cell adhesion molecules and ion channels. Among all these markers, some cannonballs are found to be overexpressed in a variety of human cancer cells, such as the epidermal growth factor receptor (ErbB1 or EGFR). One of the peptides binding specifically and efficiently to the EGFR is YHWYGYTPQNVI or GE11. Its dissociation constant is around 22 nM and it shows mitogenic activity at significantly lower concentration than the natural ligand EGF.41 GE11 accumulates into the EGFR overexpressing human hepatoma xenografts when administered intravenously. Furthermore, GE11 is internalised by tumour cells. This characteristic can be used for the gene delivery as was demonstrated with the apoptosis-inducing hTRAIL gene in a preclinical study of laryngeal cancer, a cancer type known to express high amounts of EGFR.232 A very exciting approach of exosome engineering by Ohno et al. demonstrated the possibility to produce 302 | Amino Acids, Pept. Proteins, 2018, 42, 280–319

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extracellular vesicles in human embryonic kidney cells (HEK) that display external GE11 residues as cancer targeting agent.233 GE11 decoration was achieved by its fusion with the platelet-derived growth factor receptor (PDGFR) abundantly found in the exosome membrane (for review see ref. 234). They brilliantly demonstrated the targeting by using fluorescence imaging of the exosomes internalized by the tumour cells. Delivery of the let-7a microRNA (miRNA) into the human breast cancer xenografts, overexpressing EGFR, served as an additional proof for the internalization. As let-7a is known to alter the cell cycle and thus act as a tumour suppressor, xenografts that incorporated the miRNA displayed significant reduction in the tumour volume compared to the animals injected with the plain, non-engineered exosomes naturally produced by the HEK cells. Other EGFR-binding peptides have been isolated on epidermoid carcinoma cells from the M13-based Ph.D.-7 phage displayed peptide library, such as SYPIPDT (P1) and HTSDQTN (P2).123 The two peptides inhibited the EGF-induced phosphorylation of EGFR in a dose-dependent fashion. Binding studies also demonstrated that phage displaying either P1 or P2 competed with EGF binding on EGFR-expressing human epidermoid carcinoma cells. However, more studies with other cell lines and tumours are necessary to validate the targeting potential of P1 and P2. The platelet-derived growth factor receptor b (PDGFRb) is a transmembrane glycoprotein from the tyrosine kinase receptor superfamily. PDGFRb is a central receptor in the coordination of cell proliferation, differentiation, tissue growth, and development. In cancer, PDGFRb participates in tumour growth through its pro-angiogenic and profibrotic functions. This receptor is often found upregulated in solid neoplasms and its activation plays a role in the regulation of interstitial fluid pressure in tumours.235 Therefore, it constitutes a potentially valuable target for imaging and treatment in oncology or management of fibrotic diseases. The peptide IPLPPPSRPFFK was identified by biopanning of a Ph.D.-12 phage displayed peptide library on the recombinant extracellular domain of PDGFRb.30 A synthetic version, IPLPPPSRPFFKYNH2 or PDGFR-P1 wears an additional tyrosine residue at the C-terminal end, allowing its radiolabelling by iodination for imaging purposes. In vitro validation was performed on high vs. low PDGFRb expressing cell lines and binding of [I125]-PDGFR-P1 was inhibited up to 90% by the cold PDGFR-P1. Biodistribution in human pancreatic adenocarcinomabearing animals showed higher accumulation in the tumour compared to control normal organs. Integrins are another class of highly represented membrane receptors in cancer.236 The integrin avb3 binds to ECM proteins exhibiting a RGD sequence237 and plays key roles in angiogenesis and the metastatic spread of human cancers. Therefore, multiple peptide sequences containing the RGD motif have been identified within phage displaying peptides libraries. For instance, RMS-1 (CQQSNRGDRKRC) discovered by biopanning on human rhabdomyosarcoma cells naturally expressing avb3 is capable of selectively bind to tumour cells but not the equivalent non-neoplastic tissue i.e. skeletal muscle cells and fibroblasts.141 Further Amino Acids, Pept. Proteins, 2018, 42, 280–319 | 303

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optimisation of the RGD cyclopeptides can be achieved by modifying the ring size, the amino acid chirality or constrained sequence blocks introduced by N-methylated amino acids.238 Such modifications allowed the discovery of Cilengitide (EMD121974), a head-to-tail cyclic peptide with remarkable anti-tumour activity demonstrated in intracranially implanted human gliomas in rodents.239 Cilengitide has been included in several clinical trials with patients suffering from high grade gliomas. A first trial using the peptide monotherapy showed encouraging results,240 unfortunately not confirmed by another phase III study. In the latter, Cilengitide was administered in combination with the reference chemotherapy for glioblastoma (Temozolomide) but did not show any beneficial effect on the cancer management or improvement of the patient survival.241 Cancer metastasis is a real therapeutic challenge and for some tumours, its molecular mechanism is not fully deciphered. Nevertheless, new molecular targets are discovered every year such as the mammaryderived growth inhibitor (MDGI). Originally isolated in the late 80s from the bovine mammary gland as a fatty acid binding protein,242 MDGI is suggested to be involved in the mammary gland differentiation.243 However, either its deletion or overexpression affect the mammary gland development.244 Involvement of MDGI in tumour suppression in breast cancer was deducted from the gene location. Indeed, the 1p32-p35 chromosome region containing the MDGI gene appears to be very frequently deleted in sporadic breast neoplasms.245 However, its cancer-suppressive role is questioned by several studies, indicating for instance that MDGI is involved in the metabolic switch to lipogenesis when hypoxia occurs in breast cancer and glioblastoma cells.246 Moreover, in non-small cell lung carcinomas a strong correlation between metastasis and diminished survival of the patients was observed when tumours overexpressed MDGI.247 Using a CX7C phage displayed peptide library and a two-steps biopanning (two ¨nen et al. identified a sequence ex vivo and several in vivo rounds), Hyvo CGLSGLGVA capable of homing to invasive glioma cells in vivo.181 One of the selected tumour cell model consisted transformed murine astrocytes, which lacked the hypoxia-inducible factor 1 gene (HIFko) and thus incapable of triggering angiogenesis.248 In order to have access to nutrients and oxygen, HIFko cells implanted in murine brains have to coopt the pre-existing brain capillaries. HIFko cells also use the vasculature to invade distant brain areas. This phenomenon is often observed in patients suffering from high grade gliomas and it may be responsible for the tumour relapse after surgery. Therefore, the CGLSGLGVA sequence was baptised CooP in reference to its targeting selectivity to co-optive cancer satellites (Fig. 6A). CooP have been used in several preclinical studies as an imaging and/or drug delivery moiety. Radiolabelled CooP allowed the microSPECT imaging of intracranially grafted human gliomas in nude mice181 (Fig. 6B). In addition, intravenously administered CooP-based delivery of a cytotoxic drug, Chlorambucil, prolonged the survival of intracranial tumour bearing animals compared to the free drug.181 Moreover, functionalised porous silicon nanovectors coated with 304 | Amino Acids, Pept. Proteins, 2018, 42, 280–319

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Fig. 6 Illustration of the MDGI expression in patient-derived glioblastoma xenografts (GBM PDX) and usage of CooP as a targeting peptide for intracranial tumours. A, micro-photography of a patient-derived glioblastoma implanted in nude mice brain. Middle panel focuses on the presence of invasive single cells (arrows) and left panels on the co-option of blood vessels (arrowheads). MDGI is preferentially overexpressed by tumour blood vessels, invasive tumour cells escaping from the frontline of the main tumour mass and by tumour cells co-opting the blood vessels. B, microSPECT imaging of nude mice bearing orthotopically implanted human glioblastoma cells. Control peptide (left panel) is devoid of homing capability to the tumours whereas CooP (right panel, arrow) accumulates in tumours, allowing the contouring of the tumour mass by radio-imaging. A Le Joncour et al., and B Hyvo ¨ nen et al., unpublished data.

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the CooP peptide were capable of homing to the MDGI-expressing human breast cancer cells xenografted in animals.182 Furthermore, Feng et al. demonstrated that the paclitaxel-loaded nanoparticles displaying external CooP penetrated deeply into the brain and targeted human glioma cells in animals following the intravenous administration. Beneficial effects on the animal survival compared to the plain drug was also reported.183 3.4.2 Peptides homing to intracellular targets. This part of the chapter describes some attempts to take advantage of the cell penetrating peptides (CPP) as delivery vehicles. The CPPs are short, often non-toxic peptides with cationic and/or amphipathic properties allowing them to cross the cellular membranes.249 They are used for the delivery of a wide variety of compounds like proteins, oligonucleotides, and therapeutic molecules into cells. However, as such they lack any targeting properties and are able to enter any cell they come into contact with. In order to perform an intracellular biopanning using a phage displayed library, the viral particles need to access the cytosolic compartment. A novel phage displayed peptide library based on the M13 filamentous phage called iPhage was very recently developed. The penetratin (pen) sequence RQIKIWFQNRRMKWKK was ligated with the pVIII capsid protein of the phage to allow the internalisation of the library. Complete protocol for the constitution of a iPhage-based library can be found in the publications from Rangel et al.250 and Dobroff et al.251 Interestingly, it appears that the two tryptophan residues of the pen sequence are critical for the membrane translocation, as their replacement by alanine amino acids induced a total loss-of-function.252 In the same article, the authors inserted either a mitochondrial localisation signal of cytochrome c oxidase or a peptide sequence YKWYYRGAA in the iPhage to allow targeting to mitochondria or endoplasmic reticulum, respectively. Binding of the viral particles to their respective target organelles induced ultrastructural alterations such as cytoplasmic vacuoles, swollen mitochondria, and chromatin condensation, signs associated with cell death. An interesting method of selecting targeting peptides for the intracellular compartment has been described by Umlauf et al.253 Biopanning of the phage displayed peptide library was performed on cultured human NSCLC cells treated or not with chlorpromazine to block the clathrinmediated endocytosis. The authors isolated two peptide sequences, ATEPRKQYATPRVFWTDAPG (15.1), previously identified also on a different human NSCLC cell line, and LQWRRDDNVHNFGVWARYRL or H1299.3. The 15.1 peptide is capable of entering the cells independent of clathrin and therefore enters both the treated and non-treated cells. The H1299.3 was shown to be endocytosed preferentially via clathrindependent manner in cancer cells. These two peptides clearly targeted different organelles inside the NSCLC cells: 15.1 accumulated in the perinuclear space, while H1299.3 appeared to be selectively targeting and accumulating within the lysosomes. 306 | Amino Acids, Pept. Proteins, 2018, 42, 280–319

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Such demonstration provides a proof-of-concept that cancer targets are no more restricted to the plasma membrane or the extracellular domain. It is now possible to trigger a tumour-cell specific death by targeting essential organelles, constituting a promising cannonball that ignores the tumour heterogeneity or the patient-specific cancer mutations.

4 Concluding remarks Phage display is a very powerful selection tool to identify novel tumour homing peptides. The Swiss-Army Knife does not have the reputation of being the deadliest weapon, but its multi-purpose usage has saved many lives worldwide. Similarly, peptide sequences can be used in different ways to increase the specificity of delivery of imaging probes and therapeutic drugs to their targets in order to increase the efficacy and decrease side effects. Tumour homing peptides bind to targets on various cell types within the tumour tissue. In addition to tumour cells peptides recognise tumour-associated macrophages, blood and lymphatic endothelial cells, and extracellular matrix proteins. Tumour homing peptides can also be used to identify novel tumour specific targets that can be used for further development of innovated targeted therapeutics. Advantages of peptides include the small size, relatively easy and inexpensive production, flexibility, low immunogenicity, and they rarely show species specificity. Peptides can also be conjugated to myriad of different molecules, therapeutics, proteins and/or nanocarriers allowing the development of novel therapeutic options, which in future will hopefully benefit patients suffering from cancer.

List of abbreviations a/bFGF ACPP APP APP APRIL CAIX CD133 CD-21 CD44 CendR CLIC3 CPP CRKL DAPI DNA Dox ECM EGF EGFR/ErbB1 EGFRvIII

acidic/basic fibroblast growth factor activatable anionic cell penetrating peptide amyloid precursor protein aminopeptidase P a proliferation-inducing ligand carbonic anhydrase IX prominin-1 complement receptor type 2 lymphocyte homing receptor cryptic C-end rule motif chloride intracellular channel protein 3 cell penetrating peptide Crk-like protein 4 0 ,6-diamidino-2-phenylindole deoxy ribonucleic acid Doxorubicin extracellular matrix epidermal growth factor epidermal growth factor receptor epidermal growth factor receptor variant III Amino Acids, Pept. Proteins, 2018, 42, 280–319 | 307

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Eph A2/B4 E. coli FGF3 FGF8b FGFR GBM GFP GP130 GPC3 GPCR GRP78 GZP HEK HER2 HIFko HSPG hTRAIL IGHC IL-10Ra IL-11Ra IL-13Ra2 IL-6Ra Lf LLC LYVE-1 MDGI MECA-32 microSPECT MIPS miRNA MMAE MMP-2/9 MTD MUC18 NIRF Notch1NRR NP NRP-1 NSCLC p32 PDAC PDGFRb PD-L1 PDT PDX Pen PET/CT

ephrin type-A/B receptor 2/4 Escherichia coli fibroblast growth factor 3 fibroblast growth factor 8b fibroblast growth factor receptor glioblastoma multiforme green fluorescent protein glycoprotein 130 glypican 3 g-protein coupled receptor glucose-regulated protein78 granzyme B peptide human embryonic kidney human epidermal growth factor receptor-2 hypoxia-inducible factor 1 knockout cells heparin sulfate proteoglycan human tumour necrosis factor-related apoptosisinducing ligand immunoglobulin heavy locus C interleukin-10 receptor a interleukin-11 receptor a Interleukin 13 receptor a2 interleukin-6 receptor a lactoferrin Lewis lung carcinoma lymphatic vessel endothelial hyaluronan receptor-1 mammary-derived growth inhibitor aka PLVAP, plasmalemma vesicle associated protein single-photon emission computed tomography for small animals microfluidic phage selection channel system micro ribonucleic acid monoethylauristatin E matrix metallopeptidase-2/9 mitochondrial targeting domain melanoma cell adhesion molecule near infrared fluorescence negative regulatory region in Notch1 nanoparticle neuropilin-1 non-small cell lung cancer general protein kinase C-binding protein pancreatic ductal adenocarcinoma platelet-derived growth factor receptor b programmed death-ligand photodynamic patient-derived xenograft penetratin positron emission tomography/computed tomography

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PKCd PS PSA PSMA PTPRJ PTT R&D T2-MR TAG-72 TAM TfR1 Tie2 TIP-1 TumorHoPE VAV3 VEGF VEGF-A165 VEGFR1/3 VPAC1

protein kinase C d phosphatidyl serine prostate-specific antigen prostate-specific membrane antigen receptor-type tyrosine-protein phosphatase J photothermal research and development T2-weighted magnetic resonance tumour-associated glycoprotein 72 tumour-associated macrophages transferrin receptor 1 angiopoietin receptor 2 tax interacting protein-1 Tumour Homing Peptide database (http://crdd.osdd. net/raghava/tumorhope/) guanine nucleotide exchange factor vascular endothelial growth factor vascular endothelial growth factor-A165 isotype vascular endothelial growth factor receptor type 1/3 vasoactive intestinal polypeptide receptor 1

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