Chemical ligation : tools for biomolecule synthesis and modification 9781119044093, 111904409X, 9781119044116, 1119044111, 9781119044130, 1119044138

Table of contents :
Content: Cover
Title Page
Copyright
Contents
List of Figures
List of Plates
List of Contributors
Preface
Chapter 1 Introduction to Chemical Ligation Reactions
1.1 Introduction
1.1.1 Chemical Synthesis of Proteins: From the Stepwise Synthesis to the Chemical Ligation Approach
1.1.2 Chemical Modification of Proteins: From Conventional Methods to Chemoselective Labeling by Chemical Ligation
1.2 Chemical Ligation Chemistries
1.3 Imine Ligations
1.3.1 Oxime Ligation
1.3.2 Hydrazone Ligation
1.3.3 Pictet-Spengler Ligation
1.3.4 Thiazolidine Ligation
1.4 Serine/Threonine Ligation (STL) 2.3.2 SEAon/off Concept and the Design of a One-Pot Three Peptide Segment Assembly Process2.3.3 SEA on/off Concept and the Solid-Phase Synthesis of Proteins in the N-to-C Direction
2.4 Chemical Synthesis of HGF/SF Subdomains for Deciphering the Functioning of HGF/SF-MET System
2.5 Conclusion
References
Chapter 3 Development of Serine/Threonine Ligation and Its Applications
3.1 Introduction
3.1.1 Protein Synthesis by SPPS
3.1.2 Native Chemical Ligation (and Extended Desulfurization)
3.1.3 KAHA Ligation
3.2 Serine/Threonine Ligation (STL)
3.2.1 SAL Ester Preparation 3.2.2 N-Terminal-Protecting Group for Successive C-to-N Ser/Thr Ligations3.2.3 Scope and Limitations
3.3 Application of STL in Protein Synthesis
3.3.1 Consecutive STL of Peptides/Proteins
3.3.2 STL-Mediated Peptide Cyclization
3.3.3 Thiol SAL Ester-Mediated Aminolysis in Peptide Cyclization
3.3.4 A Fluorogenic Probe for Recognizing 5-OH-Lys Inspired by STL
3.3.5 Expressed Protein Semisynthesis via Ser/Thr Ligation
3.4 Conclusion and Outlook
References
Chapter 4 Synthesis of Proteins by Native Chemical Ligation-Desulfurization Strategies
4.1 Introduction 4.2 Ligation-Desulfurization and Early Applications4.2.1 Metal‐Free Desulfurization
4.2.2 Ligation-Desulfurization toward the Synthesis of Proteins
4.3 Beyond Native Chemical Ligation at Cysteine - The Development of Thiolated Amino Acids and Their Application in Protein Synthesis
4.3.1 Phenylalanine
4.3.2 Valine
4.3.3 Lysine
4.3.4 Threonine
4.3.5 Leucine
4.3.6 Proline
4.3.7 Glutamine
4.3.8 Arginine
4.3.9 Aspartic Acid
4.3.10 Glutamic Acid
4.3.11 Tryptophan
4.3.12 GlcNAc-Asparagine
4.3.13 Asparagine
4.4 Ligation-Deselenization in the Chemical Synthesis of Proteins

Citation preview

Chemical Ligation

Chemical Ligation Tools for Biomolecule Synthesis and Modification

Edited by Luca D. D’Andrea and Alessandra Romanelli

This edition first published 2017 © 2017 John Wiley & Sons, Inc. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, except as permitted by law. Advice on how to obtain permission to reuse material from this title is available at http://www.wiley.com/go/permissions. The right of Luca D. D’Andrea and Alessandra Romanelli to be identified as the authors of this work has been asserted in accordance with law. Registered Offices John Wiley & Sons, Inc., 111 River Street, Hoboken, NJ 07030, USA Editorial Office 111 River Street, Hoboken, NJ 07030, USA For details of our global editorial offices, customer services, and more information about Wiley products visit us at www.wiley.com. Wiley also publishes its books in a variety of electronic formats and by print-on-demand. Some content that appears in standard print versions of this book may not be available in other formats. Limit of Liability/Disclaimer of Warranty The publisher and the authors make no representations or warranties with respect to the accuracy or completeness of the contents of this work and specifically disclaim all warranties; including without limitation any implied warranties of fitness for a particular purpose. This work is sold with the understanding that the publisher is not engaged in rendering professional services. The advice and strategies contained herein may not be suitable for every situation. In view of on-going research, equipment modifications, changes in governmental regulations, and the constant flow of information relating to the use of experimental reagents, equipment, and devices, the reader is urged to review and evaluate the information provided in the package insert or instructions for each chemical, piece of equipment, reagent, or device for, among other things, any changes in the instructions or indication of usage and for added warnings and precautions. The fact that an organization or website is referred to in this work as a citation and/or potential source of further information does not mean that the author or the publisher endorses the information the organization or website may provide or recommendations it may make. Further, readers should be aware that websites listed in this work may have changed or disappeared between when this works was written and when it is read. No warranty may be created or extended by any promotional statements for this work. Neither the publisher nor the author shall be liable for any damages arising here from. Library of Congress Cataloguing-in-Publication Data Names: D’Andrea, Luca D., editor. | Romanelli, Alessandra, editor. Title: Chemical ligation : tools for biomolecule synthesis and modification / edited by Luca D. D’Andrea, Alessandra Romanelli. Description: Hoboken, NJ : John Wiley & Sons, Inc., 2017. | Includes bibliographical references and index. Identifiers: LCCN 2016052028 (print) | LCCN 2016052635 (ebook) | ISBN 9781119044109 (cloth) | ISBN 9781119044093 (pdf ) | ISBN 9781119044130 (epub) Subjects: LCSH: Biosynthesis. | Peptides–Synthesis. | Proteins–Synthesis. | Bioorganic chemistry. | Chemistry, Organic. Classification: LCC QP551 .C51587 2017 (print) | LCC QP551 (ebook) | DDC 612/.015756–dc23 LC record available at https://lccn.loc.gov/2016052028 Cover image: © Catherine Peer / EyeEm/Gettyimages Cover design by Wiley Set in 10/12pt Warnock by SPi Global, Chennai, India

Printed in the United States of America 10 9 8 7 6 5 4 3 2 1

Contents List of Figures  xiii List of Plates  xxiii List of Contributors  xxix Preface   xxxiii 1

Introduction to Chemical Ligation Reactions  1 Lucia De Rosa, Alessandra Romanelli, and Luca Domenico D’Andrea

1.1 ­­Introduction  1 1.1.1 Chemical Synthesis of Proteins: From the Stepwise Synthesis to the Chemical Ligation Approach  2 1.1.2 Chemical Modification of Proteins: From Conventional Methods to Chemoselective Labeling by Chemical Ligation  5 1.2 ­Chemical Ligation Chemistries  6 1.3 ­­Imine Ligations  7 1.3.1 Oxime Ligation  7 1.3.2 Hydrazone Ligation  13 1.3.3 Pictet–Spengler Ligation  15 1.3.4 Thiazolidine Ligation  19 1.4 ­­Serine/Threonine Ligation (STL)  21 1.5 ­Thioether Ligation  24 1.6 ­­Thioester Ligation  25 1.6.1 Native Chemical Ligation (NCL)  26 1.6.2 Expressed Protein Ligation (EPL)  40 1.6.2.1 Protein trans‐Splicing (PTS)  43 1.6.3 Thioacid‐Mediated Ligation Strategies  44 1.7 ­ α‐Ketoacid‐Hydroxylamine (KAHA) Ligation  49 1.7.1 Acyltrifluoroborates and Hydroxylamines Ligation  51 1.8 ­Staudinger Ligation  52 1.9 ­Azide–Alkyne Cycloaddition  57 1.10 ­Diels–Alder Ligation  61 ­References  64

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Protein Chemical Synthesis by SEA Ligation  89 Oleg Melnyk, Claire Simonneau, and Jérôme Vicogne

2.1 ­Introduction  89 2.2 ­Essential Chemical Properties of SEA Group  93 2.3 ­Protein Total Synthesis Using SEA Chemistry – SEAon/off Concept  97 2.3.1 Synthesis of SEAoff Peptide Segments  97 2.3.2 SEAon/off Concept and the Design of a One-Pot Three Peptide Segment Assembly Process  99 2.3.3 SEAon/off Concept and the Solid-Phase Synthesis of Proteins in the N-to-C Direction  103 2.4 ­Chemical Synthesis of HGF/SF Subdomains for Deciphering the Functioning of HGF/SF-MET System  106 2.5 ­Conclusion  114 ­ References  114 3

Development of Serine/Threonine Ligation and Its Applications  125 Tianlu Li and Xuechen Li

3.1 ­Introduction  125 3.1.1 Protein Synthesis by SPPS  125 3.1.2 Native Chemical Ligation (and Extended Desulfurization)  125 3.1.3 KAHA Ligation  128 3.2 ­Serine/Threonine Ligation (STL)  130 3.2.1 SAL Ester Preparation  130 3.2.2 N‐Terminal‐Protecting Group for Successive C‐to‐N Ser/Thr Ligations  136 3.2.3 Scope and Limitations  137 3.2.3.1 Effect of Side‐Chain‐Unprotected Lys Residue  137 3.2.3.2 Effect of the C‐Terminal Amino Acid at Ligation Site  138 3.3 ­Application of STL in Protein Synthesis  140 3.3.1 Consecutive STL of Peptides/Proteins  140 3.3.1.1 Teriparatide (Forteo)  140 3.3.1.2 hGH‐RH 141 3.3.1.3 Human Erythrocyte Acylphosphatase (ACYP1)  142 3.3.1.4 MUC1 Glycopeptides  143 3.3.2 STL‐Mediated Peptide Cyclization  143 3.3.2.1 STL in Head‐to‐Tail Tetrapeptide Cyclization  143 3.3.2.2 STL in Head‐to‐Tail Cyclization of Peptides of Various Sizes  145 3.3.2.3 Total Synthesis of Daptomycin via Serine‐Ligation‐Mediated Peptide Cyclization  145 3.3.3 Thiol SAL Ester‐Mediated Aminolysis in Peptide Cyclization  147 3.3.4 A Fluorogenic Probe for Recognizing 5‐OH‐Lys Inspired by STL  149

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3.3.5 Expressed Protein Semisynthesis via Ser/Thr Ligation  151 3.4 ­Conclusion and Outlook  154 ­ References  154 4

Synthesis of Proteins by Native Chemical Ligation–Desulfurization Strategies  161 Bhavesh Premdjee and Richard J. Payne

4.1 ­Introduction  161 ­4.2 Ligation–Desulfurization and Early Applications  162 4.2.1 Metal‐Free Desulfurization  164 4.2.2 Ligation–Desulfurization toward the Synthesis of Proteins  166 4.3 ­Beyond Native Chemical Ligation at Cysteine – The Development of Thiolated Amino Acids and Their Application in Protein Synthesis  174 4.3.1 Phenylalanine  174 4.3.2 Valine  178 4.3.3 Lysine  179 4.3.4 Threonine  188 4.3.5 Leucine  188 4.3.6 Proline  193 4.3.7 Glutamine  195 4.3.8 Arginine  198 4.3.9 Aspartic Acid  198 4.3.10 Glutamic Acid  202 4.3.11 Tryptophan 206 4.3.12 GlcNAc‐Asparagine 206 4.3.13 Asparagine 206 4.4 ­Ligation–Deselenization in the Chemical Synthesis of Proteins  211 4.4.1 Selenol Amino Acids  214 4.5 ­Conclusions and Future Directions  216 ­References  218 5

Synthesis of Chemokines by Chemical Ligation  223 Nydia Panitz and Annette G. Beck‐Sickinger

5.1 ­Introduction – The Chemokine–Chemokine Receptor Multifunctional System  223 5.2 ­Synthesis of Chemokines by Native Chemical Ligation  224 5.3 ­Synthesis of Chemokines by Alternative Chemical Ligation  231 5.4 ­Semisynthesis of Chemokines by Expressed Protein Ligation  233 5.5 ­Prospects  241 ­References  243

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Chemical Synthesis of Glycoproteins by the Thioester Method  251 Hironobu Hojo

6.1­ Introduction  251 6.2­ Ligation Methods and Strategy of Glycoprotein Synthesis  252 6.3­ The Synthesis of the Extracellular Ig Domain of Emmprin  254 6.4 ­Synthesis of Basal Structure of MUC2  256 6.5­ N‐Alkylcysteine‐Assisted Thioesterification Method and Dendrimer Synthesis  257 6.6 ­Synthesis of TIM‐3  260 6.7­ Resynthesis of Emmprin Ig Domain  262 6.8­ Conclusion  264 ­References  264 7

Membrane Proteins: Chemical Synthesis and Ligation  269 Marc Dittman and Martin Engelhard

7.1 ­Introduction  269 7.2 ­Methods for the Synthesis and Purification of Membrane Proteins  270 7.2.1 Synthesis of Hydrophobic Peptides  270 7.2.2 Purification of Hydrophobic Peptides  272 7.3 ­Ligation and Refolding  273 7.3.1 Ligation Strategies  273 7.3.2 Refolding of Chemically Synthesized Hydrophobic Peptides and Membrane Proteins  275 7.4 ­Illustrative Examples  276 7.4.1 Diacylglycerol Kinase (DAGK) 276 7.4.2 Semisynthesis of the Sensory Rhodopsin/Transducer Complex  278 7.4.3 Semisynthesis of the Functional K+ Channel KcsA  279 ­ References  280 8

Chemoselective Modification of Proteins  285 Xi Chen, Stephanie Voss, and Yao-Wen Wu

8.1 ­Chemical Protein Synthesis  285 8.1.1 Native Chemical Ligation (NCL) and Expressed Protein Ligation (EPL)  285 8.1.2 Traceless Staudinger Ligation  286 8.2 ­Chemoselective and Bioorthogonal Reactions  287 8.2.1 Oxime/Hydrazone Ligation  287 8.2.2 Staudinger Ligations  294 8.2.3 Copper-Catalyzed Azide–Alkyne Cycloaddition (CuAAC)  294 8.2.4 Strain-Promoted Azide–Alkyne Cycloaddition (SPAAC)  297 8.2.5 Inverse Electron-Demand Diels–Alder Cycloaddition (DAINV)  300 8.2.6 Light-Induced Click Reactions  303

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8.2.7 1,2-Aminothiol Condensation  304 8.2.8 Transition-Metal-Catalyzed Couplings  305 8.2.9 Miscellaneous Protein-Labeling Reactions  306 8.3 ­Site-Selective Protein Modification Approaches  307 8.3.1 Site-Selective Modification of Native Proteins  307 8.3.1.1 Cysteine (Cys), dehydroalanine (Dha), and disulfides  307 8.3.1.2 N-Terminal Protein Labeling  309 8.3.1.3 Kinetically-Controlled Protein Labeling (KPL)  309 8.3.1.4 Affinity Labeling for Site-Specific Labeling of Native Proteins  310 8.3.2 Chemical Tags for Labeling Proteins in Live Cells  311 8.3.2.1 Self-Labeling Peptide Tags  312 8.3.2.2 Ligand-Binding Domains  317 8.3.2.3 Self-Labeling Enzymatic Domains  317 8.3.2.4 Enzymatic Modifications  318 8.3.2.5 Metal Chelation  318 8.3.3 Unnatural Amino Acid Mutagenesis  319 8.3.3.1 Residue-Specific UAA Mutagenesis Via SPI  319 8.3.3.2 Site-Specific UAA Incorporation  322 ­ References  322 9

Stable, Versatile Conjugation Chemistries for Modifying Aldehyde-Containing Biomolecules  339 Aaron E. Albers, Penelope M. Drake and David Rabuka

9.1 ­Introduction  339 9.2 ­Aldehyde as a Bioorthogonal Chemical Handle for Conjugation  339 9.3 ­Aldehyde Conjugation Chemistries  340 9.4 ­The Pictet–Spengler Ligation  341 9.5 ­The Hydrazinyl-Iso-Pictet–Spengler (HIPS) Ligation  341 9.6 ­The Trapped-Knoevenagel (thioPz) Ligation  343 9.7 ­Applications – Antibody–Drug Conjugates  346 9.8 ­Next-Generation HIPS Chemistry – AzaHIPS  348 9.9 ­Applications – Protein Engineering  349 9.10 ­Applications – Protein Labeling  349 9.11 ­Conclusions  351 ­ References  351 10

Thioamide Labeling of Proteins through a Combination of Semisynthetic Methods  355 Christopher R. Walters, John J. Ferrie, and E. James Petersson

10.1 ­Introduction  355 10.2 ­Thioamide Synthesis  356 10.3 ­Thioamide Incorporation into Peptides  357

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10.4 ­Synthesis of Full‐Sized Proteins Containing Thioamides  360 10.5 ­Applications  368 10.5.1 Structural Studies  368 10.5.2 Use as Photoswitches  371 10.5.3 Site‐Specific Circular Dichroism Labels  373 10.5.4 Fluorescence Quenching  374 10.5.5 Protein Folding in Model Systems  375 10.5.6 Monitoring Proteolysis  377 10.5.7 α‐Synuclein Misfolding Studies  379 10.6 ­Conclusions  381 ­Acknowledgments  381 ­References  382 11

Macrocyclic Organo-Peptide Hybrids by Intein-Mediated Ligation: Synthesis and Applications  391 John R. Frost and Rudi Fasan

11.1 ­Introduction  391 11.1.1 Naturally Occurring Macrocyclic Peptides  392 11.1.2 Natural Product Analogs via Reengineering of NRPS and PRPS Biosynthetic Pathways  395 11.2 ­Macrocyclic Organo-Peptide Hybrids as Natural-Product-Inspired Macrocycles  396 11.2.1 MOrPHs via CuAAC/Hydrazide-Mediated Ligation  398 11.2.2 Catalyst-Free MOrPH Synthesis via Oxime/AMA-Mediated Ligation  401 11.2.3 Structure–Reactivity Relationships in MOrPH Synthesis  401 11.2.4 Synthesis of MOrPH Libraries  404 11.2.5 Macrocyclization Mechanism  405 11.2.6 Bicyclic Organo-Peptide Hybrids  406 11.3 ­Application of MOrPHs for Targeting α-Helix-Mediated Protein– Protein Interactions  406 11.4 ­Conclusions  410 ­References  410 12

Protein Ligation by HINT Domains  421 Hideo Iwaï and A. Sesilja Aranko

12.1 ­Introduction  421 12.2 ­Protein Ligation by Protein Splicing  423 12.3 ­Naturally Occurring and Artificially Split Inteins for Protein Ligation  424 12.4 ­Conditional Protein Splicing  427 12.5 ­Inter- and Intramolecular Protein Splicing  429 12.6 ­Protein Ligation by Other HINT Domains  430

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12.7 ­Bottleneck of Protein Ligation by PTS  432 12.8 ­Comparison with Other Enzymatic Ligation Methods  432 12.9 ­Perspective of Protein Ligation by HINT Domains  437 12.10 ­Conclusions and Future Perspectives  438 ­Acknowledgment  438 ­References  438 13

Chemical Ligation for Molecular Imaging  447 Aurélien Godinat, Hacer Karatas, Ghyslain Budin, and Elena A. Dubikovskaya

13.1 ­Introduction  447 13.2 ­Chemical Ligation  448 13.2.1 Classical Chemical Ligation  448 13.2.2 Bioorthogonal Chemistry  450 13.2.2.1 Bioorthogonal Chemistry for Optical Imaging  454 13.2.2.2 Bioorthogonal Chemistry for Nuclear Imaging (PET, SPECT)  462 13.2.2.3 Bioorthogonal Chemistry for Magnetic Resonance Imaging (MRI)  469 13.3 ­Conclusion  470 ­References  473 14

Native Chemical Ligation in Structural Biology  485 Lucia De Rosa, Alessandra Romanelli, and Luca Domenico D’Andrea

14.1 ­Introduction  485 14.2 ­Protein (Semi)synthesis for Molecular Structure Determination  486 14.3 ­Protein (Semi)Synthesis for Understanding Protein Folding, Stability, and Interactions  494 14.4 ­Protein (Semi)Synthesis in Enzyme Chemistry  501 ­References  506 Index  517

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List of Figures Figure 1.1 Schematic representation of the solid‐phase peptide synthesis.  2 Figure 1.2 Chemical preparation of a polypeptide by convergent synthesis in solution phase.  4 Figure 1.3 The chemical ligation general concept. A single polypeptide chain is obtained by covalently joining two peptide segments through the reaction of two mutually reactive functional groups. The type of covalent bond generated at the junction site depends on the reactive groups employed for the ligation reaction.  4 Figure 1.4 Derivatization of the C‐terminus of a recombinant protein with an oxyamine functional group using intein chemistry.  12 Figure 1.5 The native chemical ligation reaction mechanism.  27 Figure 1.6 Amino acid analogs used in native chemical ligation–desulfurization synthetic strategies.  39 Figure 1.7 The mechanism of intein splicing.  40 Figure 1.8 Preparation of recombinant α‐thioester proteins (a) or N‐ terminal cysteinyl‐proteins (b) using engineered inteins.  42 Figure 1.9 Schematic mechanism of protein trans‐splicing. IntN and IntC are the halves, N‐ and C‐terminal, respectively, of a split intein.  44 Figure 2.1 Structure of the bis(2-sulfanylethyl)amido (SEA) group.  92 Figure 2.2 Protein synthesis by N-to-C solid-phase sequential ligation of unprotected peptide segments using the SEAoff group as a latent thioester surrogate. The peptide segment elongation cycle consists in activating the SEAoff group by a SEA–thiol exchange reaction and then performing an NCL reaction in the presence of MPAA (see also Scheme 2.11). (a) Synthesis of a model 135 amino acid polypeptide. A sample was treated with NaOH after each elongation cycle and analyzed by LC–MS to verify the completeness of the coupling step (right).

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(b) Characterization of the purified 135-amino-acid model polypeptide.  105 Figure 2.3 Design of the multivalent semisynthetic NB and K1Bstreptavidin scaffolds.  107 Figure 2.5 Binding of NB/S or K1B/S complexes to purified recombinant or endogenous MET receptor. (a) NB, K1B and MET-Fc binding assay: increasing concentrations of NB or K1B were mixed with extracellular MET domain fused with human IgG1-Fc (MET-Fc) and incubated with streptavidin AlphaScreen® donor beads and Protein A acceptor beads. Error bars correspond to standard error (±SD) of triplicates. (b) Endogenous MET capture. Streptavidin-coated beads loaded with NB or K1B were incubated with HeLa or CaPan1 total cell lysates. Input, flow-through, and elution fractions from NB or K1 loaded beads were analyzed by specific total MET Western blot.  111 Figure 2.6 HeLa cells were treated with increasing concentrations of mature HGF/SF, K1B/S, NK1, and K1B/Ab for 7 min. Activation levels of Akt (a) and ERK (b) were measured using HTRF technology and plotted as the 665/620 nm HTRF signal ratio. (c) Cell scattering assay. MDCK isolated cell islets were incubated for 18 h in culture media with HGF/SF (HGF), K1B, K1B/S, and NK1. Cells were then stained and observed under microscope (40×). (d) Angiogenesis. Mice were injected with a mixture of Matrigel and HGF/SF (HGF), VEGF, NK1, K1B/S, K1B, or S. Hemoglobin absorbance was measured and concentration was determined using a rate hemoglobin standard curve and plug weight.  112 Figure 5.1 Native chemical ligation reaction mechanism demonstrated for the synthesis of glycosylated XCL1. The first step is an intermolecular trans‐thioesterification, where the thioester is cleaved from the N‐terminal fragment. Next a spontaneous intramolecular S→N acyl transfer leads to the native peptide bond between both protein segments.  225 Figure 5.2 Generation of lipidated chemokines demonstrated for CXCL12. The first CXCL12 (1–33) fragment is synthesized by SPPS coupling the β‐carboxy group of an Asp‐Oallyl‐amino acid at position 33 on Rink amide resin. Next, the allyl protection group is removed and thioesterification of the segment is performed with ethyl‐2‐mercaptopropionate. The CXCL12 fragment is cleaved from the resin as amide leading to the native Asn amino acid at position 33. The fragment CXCL12 (34–68) that is lipidated is synthesized by SPPS with a

List of Figures

Dde protection at Lys68, the lipidation position. After Dde cleavage, Fmoc‐Glu is attached at that position via its γ‐ carboxy group. Fmoc is removed by piperidine, and the fatty acid is coupled at the amino function of the carboxyl group of Glu. Finally, the peptide is cleaved from the resin and NCL is performed.  228 Figure 5.3 The approach of N─C directed NCL demonstrated for the generation of [G49]‐CXCL14. For the N─C directed NCL, three fragments of CXCL14 have been synthesized, where the first and third segments are generated by Fmoc strategy. The second part is prepared by using the specific (N‐Fmoc‐ glycyl‐N‐sulfanylmethyl)aminobenzoic acid linker‐ incorporated resin. It is now possible to cleave the peptide as cysteinyl peptide thioacid by NaSH. The first NCL occurs between the first and second fragments using thiophenol as activator. Next, the thioacid at the C‐terminus is converted into a thioester by the application of Ellman’s reagent and KHCO3. The second NCL is performed by using this thioester and the Cys of the N‐terminus of the third part leading to the full length [G49]‐CXCL14.  229 Figure 5.4 Solid‐phase NCL demonstrated for the synthesis of the chemokine vCCL3 combined with a safety‐catch linker (SCAL). The SCAL is colored in gray and allows the synthesis of an amidated C‐terminus. Therefore, an agarose resin is coupled with a Cys, which can react with the thioester function of the SCAL linker. The three vCCL3 fragments have been prepared by Fmoc SPPS strategy, whereas the third fragment the SCAL is coupled to the resin, prior to the peptide synthesis. After chemical ligation of the SCAL with the modified agarose resin, C─N directed NCL reactions can be performed using the fragment vCCL3 (13–35) and next vCCL3 (1–12). At the end, the full‐length chemokine can be cleaved from the resin.  230 Figure 5.5 Mechanism of the tandem peptide ligation represented for the generation of the vCCL3 chemokine. The tandem peptide ligation is a combination of the formation of a pseudoproline and a subsequent NCL. For the generation of the pseudoproline bond, the N‐terminal amine of the second fragment reacts with the aldehyde function of the C‐terminal part of the first peptide segment. After this imine capture in acidic environment, the thiol interacts and a ring chain tautomerization occurs. The pseudoproline reaction is finished by an intramolecular O─N acyl transfer. The subsequent NCL is performed under mild conditions at pH ≥ 7.0. The typical reaction mechanism takes

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place leading to a CCL3 variant including a pseudoproline bond.  231 Figure 5.6 Reaction mechanism of the Ag+‐catalyzed (left) and non‐Ag+‐ catalyzed (right) chemical ligation illustrated for the generation of CCL27. CCL27 is synthesized into three fragments, where the second and the first segments contain C‐terminal linear thioesters. The sulfur interacts with the Ag+ and at the same time the hydroxyl function of the HOOBt with the carbonyl C‐ atom during the Ag+‐catalyzed reaction. The thioester is cleaved from the second fragment, and the amino function of the third fragment can react with the activated carbonyl C‐ atom leading to a native peptide bond. After Fmoc cleavage, the reaction is repeated with the first peptide fragment. In comparison to the Ag+‐catalyzed reaction, the Ag+‐free chemical ligation is performed with the new thioesterification at the C‐terminus using 4‐mercaptophenylacetic acid (MPAA). By the connection of MPAA to the C‐terminus, HOOBt can directly react with the carbonyl C‐atom using the same reaction mechanism that leads to the same product as for Ag+‐ catalyzed reaction. The difference to the silver‐catalyzed reaction is the necessity to additionally cleave the Acm protection groups from the side chains of Cys.  232 Figure 5.7 Expressed protein ligation mechanism demonstrated for the labeling of CXCL8. (a) One part of the protein is obtained by the use of the IMPACT™ system, where the target protein is linked to the intein‐CBD. The CBD can bind to the chitin column and subsequently purified. After an intramolecular N─S acyl transfer, the cleavage is performed by the addition of an excess of thiols such as MESNa. The second fragment [K69(CF)]‐CXCL8 (56–77) is synthesized by SPPS with an N‐ terminal Cys. In the final step, both fragments react with each other by NCL leading to a fluorescently labeled CXCL8 chemokine.  235 Figure 5.8 Mechanism of the controlled photoactivation of chemokines illustrated for CXCL12. The CXCL12 fragment M‐[A49]‐ CXCL12 (1–49) was expressed and purified by the IMPACT™ system, whereas the second fragment CXCL12 (50–68) is synthesized by SPPS and includes the exchange of position 56 or 58 by Nvoc‐serine and Nvoc‐homoserine, respectively. Coupling of amino acid 55 or 57 via the side chain leads to a depsipeptide bond. After NCL of both fragments and protein refolding, the Nvoc can be removed by UV irradiation. The free amino group attacks the carbonyl C‐atom and leads to a native

List of Figures

peptide bond, where the protein changes into its native active tertiary structure. Thus, a photoactivatable version of CXCL12 has been achieved.  239 Figure 6.1 Protein synthesis by the thioester method.  253 Figure 6.2 Synthetic route of glycoprotein by the thioester method.  253 Figure 6.3 Synthesis of the Ig domain of emmprin.  255 Figure 6.4 Synthesis of mucin model by the thioester method.  257 Figure 6.5 Synthesis of glycopeptide dendrimer.  259 Figure 6.6 Synthesis of TIM‐3 Ig domain by the one‐pot ligation.  261 Figure 6.7 Resynthesis of the Ig domain of emmprin by the one‐pot ligation method.  263 Figure 7.1 Zheng et al.’s two‐step conversion of peptides with removable Arg‐tagged backbone modification to the native peptide [32]. Source: Zheng et al. (2014) [32]. Reproduced with the permission of American Chemical Society.  275 Figure 8.2 UAAs used in residue-specific UAA mutagenesis.  320 Figure 8.3 UAAs used in site-specific UAA mutagenesis.  321 Figure 9.1 Conjugation of proteins modified with hydrazines or alkoxyamines.  340 Figure 9.2 Pharmacokinetic analysis of circulating CT-tagged α-HER2 thioPz-Glu-PEG2-Maytansine following a single 5 mg kg−1 i.v. bolus injection in mice. Analyte concentrations were determined by ELISA. Total antibody was captured with anti-human IgG and detected with anti-human Fc. Total ADC was captured with anti-human Fab and detected with a mouse anti-maytansine primary followed by an anti-mouse IgG1 secondary. Calculated ADC half-life: 7.6 days; error bars represent 1 SD; n = 3 mice/time point.  346 Figure 10.1 Synthesis of thioacylating monomers from commercially available Fmoc‐protected amino acids. (a) IBCF, NMM, THF, 0 °C, 15 min; N‐Boc‐OPD or 4‐nitro‐OPD; (b) Lawesson’s reagent or P4S10, CH2Cl2, or THF, 2–18 h; (c) R2═F, R3═H; phosgene or triphsogene, CH2Cl2; (d) R2═NO2, R3═H; NaNO2, 95:5 AcOH/H2O, 30 min; (e) R2═H, R3═Boc; 50:50 TFA/CH2Cl2, 0 °C, 2 h; then NaNO2, 95:5 AcOH/H2O, 30 min. IBCF = isobutyl chloroformate.  359 Figure 10.2 Synthesis of doubly labeled thioproteins. (a) Expressed protein fragment containing Cnf from Uaa mutagenesis and an N‐ terminal Cys. Ligation with a thiopeptide yields double‐labeled protein containing a free cysteine. (b) Expressed protein fragment containing Cnf as a Uaa and an N‐terminal Lys or Arg. Hcm is transferred to the N‐terminal residue via AaT. The resulting protein can undergo NCL with a thiopeptide and

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subsequent methylation to yield double‐labeled protein with a native methionine at the ligation site.  364 Figure 10.4 Photocontrol of RNase S hydrolysis of cytidine 2′,3′‐cyclic monophosphate. Binding of the thioamide containing RNase S peptide to the RNase S protein produces the active enzymatic complex. Irradiation of the thioamide results in trans to cis isomerization (represented by the lighter segment of the peptide) and halts catalytic hydrolysis. Thermal relaxation of the thioamide bond to the trans conformation revives activity.  372 Figure 10.5 Fluorogenic protease‐sensing peptides. (a) The LSLKAAμ peptide, where μ represents 7‐methoxycoumarin‐4‐ylalanine, is quenched by the proximal thioamide. Proteolytic cleavage at the Lys‐Ala bond liberates the coumarin‐containing peptide resulting in a turn‐on of fluorescence. (b) Dithioamide‐ containing peptides can be used to enhance the turn‐on of protease‐sensing peptides via increased fluorescence quenching in the uncleaved peptide.  378 Figure 10.6 FRET/PET quenching during α‐synuclein compaction. With increasing concentration of TMAO, α‐synuclein undergoes successive compation. During this process, changes in the distance between two regions of the protein have been monitor by FRET (a) where quenching of Cnf fluorescence by thioamide occurs in a distance‐dependent manner and PET (b) where van der Waals contact between fluorescein and thioamide is required for quenching.  380 Figure 11.1 Chemical structures of cyclopeptide natural products generate from nonribosomal (NRPS) and ribosomal (RiPPs) biosynthetic pathways and representative structures of macrocyclic organopeptide hybrids obtained according to the intein-based methodologies highlighted in the text.  393 Figure 11.2 General scheme for the modular synthesis of macrocyclic organo-peptide hybrids (MOrPHs) via a dual ligation reaction between a genetically encoded biosynthetic precursor (BP) and a chemically synthesized synthetic precursor (SP). UAA: unnatural amino acid.  397 Figure 11.3 Overview of the copper-dependent and the catalyst-free methods for the synthesis of macrocyclic organo-peptide hybrids (MOrPHs). (a) CuAAC/hydrazide-mediated ligation method. (b) Oxime/AMA-mediated ligation method. The different structures of the nonpeptidic components are illustrate in the boxes. CuAAC: copper-catalyzed alkyne/azide cycloaddition; AMA: 2-amino-mercaptomethyl-aryl.  399

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Figure 11.6 MOrPH-based inhibitors of p53:HDM2/X interaction. (a) Crystal structure of the complex between p53 transactivation domain and the p53-binding domain of HDM2. The triad of cofacial residues in p53 helix which insert into HDM2 binding cleft are labeled. The model of a representative MOrPH-based HDM2/X inhibitor is provided (i/i + 10 peptide cyclization with SP8). (b) Sequence and in vitro inhibitory activities for the linear p53-derived peptide and the MOrPHbased inhibitors. The structures of the linker SP6, SP8, and SP4 (negative control) are indicated.  408 Figure 12.1 Schematic presentation of the chemical mechanism of protein splicing and expressed protein ligation (EPL). (a) The four reaction steps involved in protein splicing by canonical inteins. Step 1, N─S(O) acyl shift: thiol or hydroxyl group of the first residue of the intein attacks the preceding peptide bond forming a (thio)ester bond; step 2, trans-(thio)esterification: thiol or hydroxyl group of the nucleophilic +1 residue replaces the (thio)ester bond formed by the first residue of the intein resulting in a “branched intermediate”; step 3, Asn cyclization: the intein is cleaved off from the exteins; step 4, S(O)─N acyl shift: the released branched (thio)ester intermediate undergoes spontaneous rearrangement to form a more energetically stable peptide bond between N- and C-exteins. (b) Chemical reaction steps of EPL. The precursor protein containing a protein of interest (POI) fused to the inactivated intein is immobilized on the column by an affinity tag for purification. Step 1, N─S acyl shift: the first step of EPL is N─S acyl shift induced by the partially inactivated intein; step 2, thiol modification and cleavage: thiol agent cleaves the thioester bond in the precursor to release POI with the C-terminal thioester; step 3, elution: cleaved POI with the C-terminal thioester is eluted from the column; step 4, NCL step: the released POI with the C-terminal thioester reacts with an N-terminal cysteinyl peptide to form a peptide bond by NCL. ExteinN and ExteinC stand for N- and C-exteins, respectively. Asterisks indicate the mutated intein.  422 Figure 12.2 Protein trans-splicing (PTS) and the split sites on the intein structure. (a) Schematic presentation of PTS. An intein is split into interacting halves, termed N- and C-inteins (IN and IC). (b) Cartoon presentation of the three-dimensional structure of a HINT domain (NpuDnaE intein, PDB ID: 2KEQ) showing the natural split site (“C35”) and two artificially engineered split sites (“C6” and “N11”). C35 site is located at the conserved

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HEN insertion site before 35 residues from the C-terminus. The C6 and N11 split inteins were created by shifting the split site toward either C- or N-termini to have 6-residue C-intein and 11-residue N-intein, respectively. The shorter fragments of the split inteins are shown in black. Black arrows indicate the split sites and the conserved HEN insertion site. N and C show the N- and C-termini, respectively.  424 Figure 12.3 Segmental isotope-labeling and protein semisynthesis by PTS. (a) Segmental isotopic labeling in vitro. The N- and C-terminal fragments of POI are fused to N- and C-inteins, respectively. One of the precursors is expressed in isotopic (15N)-labeled culture medium and the other precursor in unlabeled culture medium. The two precursors are purified separately and mixed together for PTS by refolding. (b) Protein semisynthesis. An N-terminal fragment of POI is fused with N-intein and recombinantly expressed. The C-terminal precursor containing C-intein and a chemical label is chemically prepared. The two precursor fragments are mixed to initiate PTS, thereby producing the semisynthetically ligated product. IN and IC stand for N- and C-inteins, respectively. POIN and POIC stand for the N- and C-terminal fragments, respectively, of POI.  426 Figure 12.4 Intra- and intermolecular applications with protein cis-splicing (PS) and trans-splicing (PTS). (a) Small-molecule induced CPS: Split intein halves with low affinity are fused with two interacting domains in the presence of a small molecule. Addition of a small molecule induces PTS upon association of the two precursors. (b) Light-induced CPS: light removes a protective group stalling PTS. (c) Three-fragment ligation. POI is split into three pieces (POIN, POIM, and POIC) and fused with two orthogonal split inteins (IN1/IC1 and IN2/IC2). (d) Inteinmediated protein alternative splicing (iPAS). Intermolecular protein splicing by protein 3D-domain swapping produces alternatively ligated products instead of cis-spliced products. (e) Cyclization and polymerization. Circularly permutated precursor bearing a split intein ligates the N- and C-termini of a protein by intramolecular protein splicing, thereby resulting in the backbone cyclization. The identical precursor protein could result in polymerization when intermolecular interactions dominate intramolecular interactions. IN and IC stand for N- and C-inteins, respectively.  428 Figure 12.5 HINT domains and protein splicing by BIL domain. (a) A superposition of the structures of DnaE intein from

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Synechocystis sp. PCC6803 (SspDnaBΔ275 intein, PDB ID: 1MI8) colored in light gray and BIL4 domain from Clostridium thermocellum (CthBIL4, PDB ID: 2LWY) colored in black. N- and C indicate the N- and C-termini. (b) The mechanism of protein splicing by BIL domains involves the following steps: step 1, N─S(O) acyl shift forming a (thio)ester bond; step 2, Asn cyclization cleaving off the C-flank from the BIL domain; step 3, aminolysis by the N-terminal amine group of the cleaved C-flank. FlankN and FlankC stand for the N- and C-terminal flanking sequences, respectively.  431 Figure 12.6 Junction sequence dependencies of different inteins. (a) Numbering of junction residues of an intein-containing precursor. The −1 and −2 positions are the first and second residues preceding the intein sequence. The +1 and +2 positions are the first and second residues following the intein sequence. The first residue of an intein is indicated as “1.” The +1 residue is the nucleophilic residue and either Cys or Ser or Thr in inteins. (b) Cis-splicing efficiencies of DnaE intein from Nostoc punctiforme (NpuDnaE intein) with 20 different amino-acid types at the N- and C-terminal junctions (the −1 and +2 positions). (c) Cis-splicing efficiencies of minimized DnaB intein from Nostoc punctiforme (NpuDnaBΔ290 intein) with 20 different amino-acid types. The left and right panels show the data for the −1 position and the +2 position, respectively. Amino-acid types are indicated by one-letter codes at the bottom. The standard deviations of the mean (n = 3) are shown as error bars.  433–434 Figure 13.1 (a) General principle of bioorthogonal reactions. X and Y moieties are orthogonal to the functional groups found in biological environment. (b) Chemical ligation reactions, reagents, and ligation products generated with the respective reaction.  449 Figure 13.2 (a) Staudinger reaction [12]. (b) Reaction mechanism for Staudinger ligation [13]. (c) Traceless Staudinger ligation [14–17].  451 Figure 13.5 (a) An example of SPAAC‐based synthesis of PET probes. 18 F‐DIBO (1) is conjugated to an azido‐modified targeting moiety such as geldanamycin (2), an inhibitor of Hsp90 protein, and TATE peptide (3), a somatostatin 2 agonist. Examples are taken from Bouvet et al. and Arumugam et al. [117,118]. (b, c) Antibody‐based in vivo pretargeting methodology using IED DA reaction for PET imaging. (b) IED DA reaction for conjugation of a TCO‐modified

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antibody (A33‐TCO) as targeting moiety and 64Cu–NOTA– Tetrazine construct (64Cu–Tz–Bn–NOTA) as tracer. (c) Schematic of the antibody‐based in vivo pretargeting methodology using IED DA reaction between A33‐TCO and 64 Cu–Tz–Bn–NOTA. Examples are taken from Zeglis et al. [119].  464 Figure 14.1 Segmental isotopic labeling. An isotopic-labeled domain and an unlabeled domain are separately prepared and then joined through a native chemical ligation reaction to form the native protein of interest.  489 Figure 14.3 Preparation of protein with an NMR-invisible solubility tag by protein trans-splicing. The protein of interest is fused to the solubility tag and a split intein (IntC) portion and expressed using a minimal medium with heavy isotopes (13C, 15N, etc.). The other split intein (IntN) portion is fused to the solubility tag and expressed in standard medium. Intein assembly and splicing exchange the solubility tag domains, appending an unlabeled tag domain to the labeled protein of interest.  493 Figure 14.4 Schematic procedure to prepare double-modified proteins combining EPL and chemoselective labeling in solution. Source: De Rosa, Lucia; Russomanno, Anna; Romanelli, Alessandra; D’Andrea, Luca D. 2013. “Semi-synthesis of labeled proteins for spectroscopic applications.” Molecules 18, no. 1: 440–465.  495 Figure 14.5 Schematic mechanism of intein splicing. Source: De Rosa, Lucia; Russomanno, Anna; Romanelli, Alessandra; D’Andrea, Luca D. 2013. “Semi-synthesis of labeled proteins for spectroscopic applications.” Molecules 18, no. 1: 440–465.  504

List of Plates Figure 2.4 Chemical synthesis of NB and K1B proteins using the one-pot three peptide segment assembly process.  109 Figure 2.7 (a) FVB mice were injected intravenously with anti-Fas monoclonal antibody (AbFAS) mixed with K1B, K1B/S, NK1, or HGF/SF or PBS. A second injection without anti-Fas was performed 90 min later. Livers were extracted and fixed in formalin after three additional hours. (b) FVB mice were injected with an increased concentration of K1B/S complex, K1B, or NK1. After 10 min, livers were extracted, snap-frozen, and crushed. Cell lysates were analyzed by specific total MET, Akt and ERK or phospho-MET, phospho-Akt, and phosphoERK Western blot.  113 Figure 7.2 Retrosynthesis scheme for the synthesis of DAGK. Figure was kindly provided by C. F.W. Becker. Source: Lahiri et al. (2011) [16]. Reproduced with the permission of John Wiley & Sons.  277 Figure 7.3 (a) Ribbon diagram of the crystal structure of the transmembrane part of the NpSRII/NpHtrII complex [2]. (b) Scheme of the synthesis of the NpHtrII1‐114 domain containing 114 amino acids. Source: Dittmann et al. (2014) [14]. Reproduced with the permission of John Wiley & Sons.  278 Figure 7.4 The selectivity filter of K+ channels. (a) Ribbon representation of two opposite subunits of the KcsA channel. Green spheres illustrate the potassium ions in their binding sites. (b) Close‐up view of the selectivity filter. In a functional channel, sites 1 and 3 are in equilibrium with sites 2 and 4. Source: Valiyaveetil et al. (2006) [45]. Reproduced with the permission of American Chemical Society.  279 Figure 8.1 Reagents and products of 1,2-aminothiol condensations. Source: Conley et al. (2012) [131]. Reproduced with the permission of John Wiley & Sons.  305

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Figure 10.3 Future directions for incorporation of thioamides into full‐ length proteins. (a) Three‐part ligation. The intein generated protein–thioester 1 undergoes ligation with 2 to generate thioprotein fragment 3 after oxidation and treatment with a thiol. The C‐terminal fragment is expressed with an N‐terminal Lys, and Hcm is transferred by AaT to yield 5. Ligation proceeds between 3 and 5, followed by methylation of the two Hcs residues with CH3I to yield a full‐length thioprotein with Met residues at each ligation site. (b) Expressed protein containing a side‐chain thioamide and N‐terminal Cys is ligated with a backbone thiopeptide to result in a side chain– backbone dithioamide protein. (c) A peptide fragment containing an N‐terminal cysteine surrogate undergoes NCL with a thiopeptide. Subsequent desulfurization yields a thiopeptide with a non‐Cys amino acid at the ligation site.  366 Figure 11.4 Representative structure-reactivity data for oxime/AMAmediated ligation method. (a) Percentage of MOrPH product upon reaction of different oxyamino/amino-thiol synthetic precursors (SP4–SP7) with biosynthetic precursors containing peptide target sequences from 4 to 15-amino acid residues long (12 hour time point). (b) Reactivity of the 20 biosynthetic precursor I-1 variants. Left: Plot of the amount of full-length protein after purification from Escherichia coli versus yield of SP4-induced macrocyclization (5 h). Right: Overall yield of MOrPH product as given by the (% of full-length protein) × (% of SP-induced macrocyclization) yield from full-length precursor protein. (c) Amount of SP4-induced protein splicing for the proline-scan and glycine-scan biosynthetic precursor libraries and the reference X5T library. The target sequence, in which X corresponds to a fully randomized position (NNK codon), is specified.  403 Figure 11.5 Bicyclic MOrPHs. (a) Strategy for synthesis of bicyclic organopeptide hybrids via post-cyclization oxidation of MOrPHs derived from cysteine-containing target sequences. The oxyamine/amino-thiol synthetic precursor, the unnatural amino acid (p-acetyl-Phe), and the reactive cysteine residue within the target peptide sequence are highlighted in blue, green, and red, respectively. (b) Amino acid sequences of the precursor polypeptides utilized for synthesis of bicyclic MOrPHs. (c) Representative MALDI-MS spectra illustrating the clean formation of the macrocyclic (“m”) and bicyclic product (“b”) before and after the oxidation step.  407

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Figure 13.3 (a) Metabolic introduction of chemical reporters on biomolecules in cell. Cells are incubated with unnatural metabolic precursors bearing the chemical reported (e.g., azide) and are incorporated in biomolecules using the metabolic machinery of the cells. The reporter is selectively reacted with an exogenous probe using a compatible bioorthogonal reaction. (b) Fluorescence microscopy of CHO cells labeled with Cy5.5–phosphine conjugate. Cells, incubated with Ac4ManNAz (100 μM) for 3 days, were treated with Cy5.5–phosphine conjugate (200 μM) for 2 h at 37 °C. The cells were then fixed and permeabilized with MeOH and stained with DAPI before imaging. Red: Cy5.5 channel. Blue: DAPI channel. Scale bar: 20 µm. Source: Chang et al. (2007) [41]. Reproduced with the permission of American Chemical Society.  453 Figure 13.4 (a) Principle of caged‐luciferin approach. d‐Luciferin can be modified with caging groups on the phenol, amino, or carboxylic acid group prohibiting the interaction with Fluc until caging moiety is removed. (b) Use of a phosphine– luciferin conjugate (caged luciferin) for bioluminescence imaging of cell‐surface azido‐sugars. The phosphine–luciferin conjugate releases d‐luciferin upon Staudinger ligation with azides. Then, free d‐luciferin diffuses into cells, and luciferase‐ catalyzed conversion of d‐luciferin to oxyluciferin yields a photon of light, which is detected using CCD camera. (c) Imaging of cell‐surface azido‐sugars with phosphine– luciferin conjugate. LNCaP‐luc cells, a prostate cancer cell line stably transfected with luciferase, were incubated for 2 days at various concentrations of the following azido‐modified sugars: Ac4ManNAz, Ac4GalNAz, Ac4GlcNAz, or media. The cells were washed thrice with 200 μL of PBS and then treated with the phosphine–luciferin probe (100 μM) for 120 min. Error bars represent the standard deviation of the mean for three replicate experiments. Source: (b, c) Cohen et al. (2010) [26]. Reproduced with the permission of American Chemical Society. (d) The split luciferin reaction. Condensation reaction between 6‐amino or 6‐hydroxy‐2‐cyanobenzothiazole and d‐ cysteine forming d‐aminoluciferin or d‐luciferin, respectively, as product. (e) Caged d‐cysteine for caspase‐3/7 imaging using the split luciferin reaction. (f ) Imaging caspase‐3/7 in FVB‐ Luc+ mice using the DEVD‐(d‐Cys) peptide and H2N‐CBT. Graph represents the total luminescence over 1 h from transgenic reporter mice treated with either PBS

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(control group) or combination of LPS (100 µg kg−1 in 50 μL of PBS) and d‐GalN (267 mg kg−1 in 50 μL of PBS). Six hours posttreatment, the animals received IP injections of a combination of DEVD‐(d‐Cys) peptide (22.6 mg kg−1 in 100 μL of PBS) and H2N‐CBT (6.8 mg kg−1 in 20 μL of DMSO). Statistical analyses were performed with a two‐tailed Student’s t‐test. **P  CO-SAlk Peptide1 — CO-SAr

Cys-Peptide2 — CO-SAlk

NCL Peptide1 -Cys-Peptide2 —CO-SAlk NCL

Scheme 2.3  The kinetically controlled ligation concept relies on the use of fast- and slow-reacting peptide thioesters.

can be reacted subsequently with another Cys peptide. A substantial limitation of the KCL approach resides in the possibility for the slow-reacting thioester functionality to react during the first ligation step [66]. The KCL method developed by Kent’s group relies on the differential reactivity of arylthioester (fast-reacting) and alkylthioester (slow-reacting) functional groups in the absence of arylthiol catalyst [67, 68]. These recent years have shown the raise in power of N,S-acyl shift systems [60, 69, 70] as thioester surrogates for protein total synthesis. Some of these systems have demonstrated the capacity to substantially simplify the synthesis of peptide thioesters but also the assembly of the peptide segments giving access to the target proteins. Indeed, several KCL methods were developed by using thioester surrogates based on N,S-acyl shift systems [71–74]. The bis(2sulfanylethyl)amido (SEA) group belongs to this family and was introduced in 2010 by Ollivier et  al. (Figure 2.1) [27]. Importantly, its 1,7-dithiol structure confers to the SEA group several particular chemical properties in comparison with the other N,S-acyl shift systems, all of which feature only one thiol group in their structure. As demonstrated by several examples, SEA peptides (sometimes, very large ones >50 AA) can be produced from easy-to-make solid supports. Some of O N Peptide1

SH SH

SEA peptide

Figure 2.1  Structure of the bis(2-sulfanylethyl)amido (SEA) group.

2.2 ­Essential Chemical Properties of SEA Grou

these are now commercially available and can be applied on conventional automated Fmoc-SPPS synthesizers [23, 24, 27]. Besides this convenient adaptation, the powerfulness of the SEA group for protein total synthesis resides in three major chemical properties, which will be discussed in the first section of this book chapter. The first essential aspect of SEA group is its capacity to act as a thioester surrogate in water at neutral pH [27]. The SEA ligation process, which is the reaction of an SEA peptide with a Cys peptide, results in the chemoselective formation of a native peptide bond to cysteine. The second important property is the possibility to convert SEA peptides into peptide thioesters using mild aqueous experimental conditions, thereby allowing SEA and NCL chemistries to synergize [51, 52]. Last but not least, the SEA group is a dithiol, which can be inactivated by intramolecular disulfide bond formation and kept in this latent form in the presence of the cysteine thiols or of the excess of aryl thiol used for catalyzing the NCL reaction [23, 24, 75–79]. The second part of this chapter details how the combination of these chemical properties enabled the conception of one-pot methods allowing the assembly of protein domains or large cyclic peptides. We also discuss the potential of SEA solid-phase protein synthesis, that is, SEA SPPS, for accessing large peptides. The final part of this chapter presents the application of SEA chemistry to the synthesis of functional protein domains derived from the hepatocyte growth factor/scatter factor (HGF/SF). We explain how the manipulation of these synthetic domains allowed us to clarify essential aspects of the HGF/ SF-MET interaction and to propose a novel strategy for designing strong MET agonists, which have a great potential in regenerative medicine.

2.2 ­Essential Chemical Properties of SEA Group During the last decade, several N,S-acyl shift systems based on the N-(2sulfanylethyl) amide structure have been designed as thioester surrogates [70]. The primary objective was to exploit the propensity of these systems to undergo an acyl migration reaction from nitrogen to sulfur in strong acidic media for accessing peptide thioesters [39, 70, 73, 80–83]. Indeed, the fact that N-(2-­ sulfanylethyl) amides rearrange spontaneously into thioesters in strongly acidic aqueous solutions has been known for a long time. For example, the capacity of N-acetyl-β-mercaptoethylamine to rearrange into S-acetyl-β-mercaptoethylamine was noticed by Martin et al. in the late 1950s [84, 85] and studied later on by Barnett and coworkers [86, 87]. Recently, it has been observed that some N-(2-sulfanylethyl) amides could also behave as thioester surrogates in water at neutral pH. The first report going in this direction introduced the SEA N,S-acyl shift system and the SEA native peptide ligation reaction (Scheme 2.4) [27]. Fundamentally, SEA ligation is a transamidation process whereby the bis(2-sulfanylethyl)amino group is

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2  Protein Chemical Synthesis by SEA Ligation TCEP, DTT pH 5.5–7

O

SH SH

N Peptide1

O

MPAA (excess)

S S

N Peptide1

I2, diamide, DMSO SEAon peptide

SEAoff peptide MPAA (excess) TCEP pH 5.5–7

N,S-acyl shift O O Peptide1

O

H

O

OH pH 2 37–65°C

S N

Peptide1

O N

S

HS SEA transient thioester

CO2H S Thiazolidine thioester peptides

MPA (excess) TCEP, pH 4 37°C O S

Peptide1

NH SH

O

NH2

Peptide2

SEA ligation

Peptide1

MPA thioester peptides HO2C O OH MPA

SH

O

CO2H

Peptide1

HS

SH SH

P

Peptide2

N H

O CO2H

CO2H

HS MPAA

CO2H TCEP

Scheme 2.4  Main features of SEA chemistry, which are exploited for peptide thioester and protein synthesis.

replaced by the N-terminal amino group of the Cys peptide segment [69]. The reaction proceeds efficiently in water at neutral pH in the presence of 4-­mercaptophenylacetic acid (MPAA [67]) and is accelerated at mildly acidic pH (pH 5.5) [51, 78]. SEA ligation used as such [88, 89] or in combination with the NCL reaction enabled the total synthesis of several functional proteins [23, 24, 72, 75, 77, 90]. Intramolecular SEA ligation is also a useful reaction for accessing tail-to-side chain [78] or head-to-tail cyclic peptides [63]. Later on, other studies showed that the capacity of SEA peptides to be transamidated by N-terminal Cys peptides in water at a pH close to neutrality is shared by other N,S-acyl shift systems [74, 91, 92]. However, one unique feature of the SEA group is the presence of two thiol groups in a 1,7 relationship. This property allows for reversibly blocking the N,S-acyl shift process by intra­ molecular disulfide bond formation. The resulting N-acyl perhydro-1,2,5-­ dithiazepine [79] moiety is called SEAoff for the sake of simplicity, while the

2.2 ­Essential Chemical Properties of SEA Grou

N,S-acyl shift-active SEA dithiol form is called SEAon (Scheme 2.4). SEAon and SEAoff groups can be rapidly and quantitatively interconverted by oxidation/ reduction using a variety of popular oxidants (iodine, DMSO) or reducing agents (dithiothreitol, DTT; tris(2-carboxyethyl)phosphine, TCEP). Importantly, acyclic disulfides such as cystine bonds or Cys(StBu) residue can be selectively reduced by MPAA in the presence of SEAoff group due to the large difference in the reducing potential between 1,7-dithiols and simple alkylthiols [93]. These redox properties are central to the solution [23, 24, 60, 76, 77] or solid-phase [76] assembly methods which are discussed in the next section. Finally, the SEA group was also shown to be a useful precursor for peptide thioester synthesis (Scheme 2.4), thereby allowing for combining the powerfulness of SEA and thioester peptide chemistries for protein total synthesis [23, 24, 39, 51, 52]. One simple method for accessing peptide thioesters is to exchange the bis(2-sulfanylethyl)amino group by an exogenous alkylthiol such as MPA [39]. This reaction proceeds efficiently under mildly acidic conditions (pH 4, Scheme 2.4) and can give access to large peptide thioester segments (~50 AA) [23, 24]. The experimental conditions for the exchange reaction are described in detail in recent protocol articles [24]. Another method for preparing peptide thioesters starting from SEA peptides is to trap the transient SEA thioester form produced by N,S-acyl shift with glyoxylic acid (Scheme 2.4) [52]. Indeed, the transient SEA thioester features a β-aminothiol moiety, which can react under acidic conditions with glyoxylic acid to produce a stable thiazolidine (for a recent review on the chemistry of glyoxylic acid, see Ref. [94]). The method can be applied to the synthesis of peptidyl prolyl thioesters, although in this case, thiazolidine formation requires higher temperatures (65 °C) compared to other C-terminal amino acid residues such as valine or tyrosine [52]. The resulting thiazolidine thioesters (TT) can be purified by HPLC as usual. TT peptides were shown to react significantly faster than peptide thioesters derived from MPA. These peptide derivatives are, therefore, of interest for forming difficult junctions. For example, the halftime (t½, first-order kinetic law) for the NCL reaction with a model thiazolidine thioester peptide featuring a C-terminal valine residue was only 10 h, while the t½ for the MPA thioester analog was 44 h (MPAA 10 mM, 37 °C, pH 7.4) [52]. Recently, the exceptional reactivity of TT peptides was illustrated in a study examining the formation of Pro-Cys junctions; such formation is known to be difficult [51]. This study revealed that the NCL reaction with peptidyl prolyl thioesters is accompanied by the formation of a two amino acid deletion side product in variable amounts depending on the type of thioester used (MPA, TT, SEA), on the nature of the C-terminal amino acid, and on the pH of the reaction (Scheme 2.5). The formation of this side product was confirmed independently by Nakamura and coworkers [95]. This side reaction proceeds probably through the formation of a transient peptidyl diketopiperazine intermediate (Scheme 2.5), which reacts further with MPAA to yield a C-terminal MPAA

95

96

2  Protein Chemical Synthesis by SEA Ligation Pro Xaa

O

O R Peptide1

LG

N NH

O Peptidyl prolyl thioester or thioester surrogate MPAA TCEP

MPAA TCEP

Cys-Peptide2

O R

Peptide1-Xaa-Pro-Cys-Peptide2 Peptide1 ArS-

LG SEA

TT

MPA

O

S S N

O

R CO2H

S

O

Peptidyl diketopiperazine intermediate

N S

N N

S CO2H

SAr

Peptide1

N

HN O

O Cys-Peptide2 Peptide1-Cys-Peptide2

Two amino acids deletion side product

Scheme 2.5  The NCL reaction with peptidyl prolyl thioesters is accompanied by the formation of a two amino acid deletion side product. The amount of side product formed is significantly lower with SEA or TT peptides than with MPA thioester peptides.

thioester lacking two amino acid residues at the C-terminus. Finally, the MPAA thioester is expected to react with the Cys peptide segment to produce the two-amino-acid deletion side product. This side reaction is potentially a serious problem for those who intend to produce proteins featuring a Pro-Cys peptide bond, since the two-amino-acid deletion side product is expected to be difficult to separate from the target polypeptide. Interestingly, TT peptides proved superior to MPA thioester analogs by minimizing substantially the extent of side product formation, while the rate of the ligation was significantly higher. SEA peptides also proved useful in reducing the amount of side

2.3 ­Protein Total Synthesis Using SEA Chemistry – SEAon/off Concept

product formation, albeit in this case, the ligation had to be performed at 65 °C and pH 5.5 to obtain synthetically useful kinetic rates.

2.3 ­Protein Total Synthesis Using SEA Chemistry – SEAon/off Concept 2.3.1  Synthesis of SEAoff Peptide Segments

In their original report, Ollivier and coworkers described the preparation of a bis(2-sulfanylethyl)amino polystyrene solid support, that is, SEA PS, for the preparation of SEA peptides using conventional Fmoc-SPPS (Scheme 2.6). In this work, the trifluoroacetate ammonium salt derived from bis(2-sulfanylethyl)amine was loaded onto chlorotrityl PS resin in DMF by an SN1 nucleophilic substitution mechanism. The use of a 10-fold molar excess of chlorotrityl Cl +H N 2

Cl– Cl 1. Trt-SH, DBU 2. TFA, TIS SH

+H

2N

CF3CO2SH

Cl—Trt

HO—Trt

1.

1. Cl—Trt Polystyrene (1.4 mmolg–1)

HO—Trt ChemMatrix (0.45 mmolg–1) CHCl3, TFA

DMF 2. 2,6-Lutidine HN

S S

Trt

Trt Polystyrene (0.15 mmolg–1)

HN

2. 2,6-Lutidine Trt S S

Trt

ChemMatrix (0.18–0.20 mmolg–1)

Scheme 2.6  Preparation of SEA polystyrene (cross-linked with 1% of divinylbenzene) or SEA ChemMatrix® resins.

97

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2  Protein Chemical Synthesis by SEA Ligation

PS resin relative to the bis(2-sulfanylethyl)amine allowed the efficient protection of both thiol groups. This procedure yields a highly stable solid support, which is ready to be used for the automated Fmoc-SPPS. A detailed protocol for its preparation is available [96]. Later on, an alternative solid support based on ChemMatrix® resin [97] has been prepared with the aim to produce large SEA peptides. In this case, the trifluoroacetate ammonium salt derived from bis(2-sulfanylethyl)amine was loaded onto hydroxytrityl ChemMatrix® resin under acidic conditions (10% TFA in chloroform). The protection of both thiol groups was found to be quantitative as judged by the negative Ellman assay, although the molar excess of hydroxytrityl groups relative to the bis(2-sulfanylethyl)amine was only ~2. A detailed protocol describing the preparation of SEA ChemMatrix® resin has been published recently [23, 24]. This solid support enables the preparation of ~50 AA SEA peptides and thus is potentially useful for accessing challenging protein targets. The Fmoc-SPPS of SEAoff peptides is detailed in Scheme 2.7. The coupling  of the first amino acid requires a strong activating reagent such as N-[(1H-1,2,3-triazolo[4,5-b]pyridin-1-yloxy)(dimethylamino)methylene]N-methylmethanaminium hexafluorophosphate (HATU) in the presence of

S

HN

S 1. Fmoc-SPPS 2. TFA, scavengers

O

O S

Peptide HS

SH

N

NH2+

SH

Peptide

SEAon amide

SEAon thioester

I2, AcOH, or air oxidation, pH 8.5 or TMAD, pH 7 or DMSO pH 6 O N Peptide

S S SEAoff

Scheme 2.7  Fmoc-SPPS of SEAoff peptides.

2.3 ­Protein Total Synthesis Using SEA Chemistry – SEAon/off Concept

N,N-diisopropylethylamine (DIEA) due to the reduced nucleophilicity of the supported secondary amine. Usually, the first residue is coupled manually to allow the determination of the loading by quantification of the dibenzofulvene–piperidine adduct formed upon treatment with piperidine in DMF. Standard automated Fmoc-SPPS and deprotection in TFA with the appropriate scavengers yield the target SEAon peptide, which must be purified by HPLC. The HPLC purification of SEAon peptides is feasible. However, the SEAon amide form can equilibrate with the SEAon transient thioester form during the HPLC purification step, a phenomenon that can result in significant mass losses (see Scheme 2.4) [27]. A simple solution to this problem is to convert the SEAon peptide into the SEAoff derivative prior to the HPLC purification step using a thiol-oxidizing reagent such as iodine in aqueous acetic acid [27], molecular oxygen under slightly basic conditions, N,N,N’,N’-tetramethyl-azodicarboxamide (TMAD) [63, 76] at neutral pH, or DMSO at pH 6 [63]. An important advantage in isolating SEAoff peptide segments is their excellent stability upon storage as a lyophilized powder or in solution, thanks to their tertiary amide structure. 2.3.2 SEAon/off Concept and the Design of a One-Pot Three Peptide Segment Assembly Process

As already mentioned, one unique feature of the SEA group is the presence of two thiol groups in a 1,7 relationship that allows for reversibly blocking the N,S-acyl shift process by intramolecular disulfide bond formation. The unique redox properties of SEAon/SEAoff chemical system are central to the peptide assembly methods which are discussed in this section. The thiol–disulfide interchange reaction has been extensively studied in the past (Scheme 2.8) [93]. Similar to DTT, some 1,7-dithiols are strongly reducing although the value of the thiol–disulfide exchange can vary significantly with their structure and, in particular, the nature of the moiety in position β relative to the thiol groups. The thiol–disulfide interchange equilibrium constant for the SEAon dithiol was not determined. We can predict that Keq determination can be complicated by the spontaneous N,S-acyl shift reaction of SEAon system as well as the thiol–thioester exchange reaction of the transient SEAon thioester by mercaptoethanol (MESH). Note that MESH is not the best choice for such a study, as in this case, the reaction will inevitably be complicated by the formation of the corresponding O-ester, which is formed by S,O-acyl shift of the intermediate S-(2-hydroxyethyl)thioester [98–100]. Since thiophenol is significantly less reducing compared to MESH (for thiophenol Keq = 0.31, see Ref. [93]), we hypothesized that reduction of SEAoff group by an excess of MPAA at pH 7 would be an unfavorable process. This proved to be the case for a large variety of SEAoff peptide segments, even those featuring a C-terminal glycine residue, which is one of the fastest reacting amino acids in the SEA ligation

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2  Protein Chemical Synthesis by SEA Ligation

Keq

Dithiol DTT S

X

+

S

SH

2 HO

O O

Dithioloff

SH

MESH

SH

+

SH Dithiolon

63 M

S SH

14 M

Keq

X

180 M SH

SH SH HO

S

S

OH

6.7 M

S SH SH

MES–S

6.1 M

O Keq

=

[dithioloff][MESH]2 [dithiolon][MES-S]

SH SH 3.6 M SH

Scheme 2.8  Thiol–disulfide equilibrium constants for some 1,7-dithiols (data taken from Ref. [93]).

reaction. In particular, SEAoff peptides featuring an N-terminal cysteine residue do not cyclize in the presence of MPAA at neutral or mildly acidic pH, although intramolecular SEA ligation in the presence of TCEP is a highly efficient process [63, 78]. In contrast, MPAA used in excess at neutral pH reduces aliphatic disulfides to a significant extent. For example, MPAA removes the tert-butylsulfenyl group from Cys(StBu) residues and cleaves at least partially cystine bonds. Thus, the use of an excess of MPAA at neutral pH allows the NCL reaction to proceed efficiently, provided the ligation is performed in an inert atmosphere. If not, MPAA and Cys thiols are rapidly oxidized into disulfides, which are unable to participate in the NCL reaction. The capacity of SEAoff group to act as a latent thioester surrogate under these mild reducing conditions enabled the design of the N-to-C one-pot three peptide segments assembly process depicted in Scheme 2.9. It is based on a sequential NCL/SEA native peptide ligation process. Note that the presence of TCEP or DTT during the first ligation step cannot be tolerated, since these strong reducing agents would activate the SEAoff group into SEAon. It is important to mention that the first NCL reaction is not kinetically controlled [65]. The internal peptide segment 2, that is, Cys-peptide2SEAoff, is unable to cyclize or oligomerize even for long reaction times. Similarly,

2.3 ­Protein Total Synthesis Using SEA Chemistry – SEAon/off Concept N-to-C assembly O

StBu Peptide1—CO-SR

+

Cys-Peptide2

N

S SEAoff S

Ligation 1

NCL (MPAA, pH 7) O N

Peptide1-Cys-Peptide2 Activation

S S

TCEP O N

Peptide1-Cys-peptide2 SEAon Ligation 2

SH SH Cys–peptide3 SEA ligation (pH 5.5–7)

peptide1 -Cys-peptide2-Cys-peptide3

Scheme 2.9  N-to-C one-pot three peptide segments assembly process.

the intermediate ligation product, that is, peptide1-Cys-peptide2-SEAoff, is unable to react with the internal peptide segment 2 during the course of the reaction or to hydrolyze. Very often, mixed disulfides between Cys thiols and MPAA are observed at this stage due to the very mild reducing potential of MPAA. Formation of mixed disulfides is reversible and does not impair the NCL reaction to go to completion. The formation of mixed disulfides can potentially complicate the monitoring of the NCL reaction, but a simple solution to this problem is to treat the aliquot with TCEP and extract the MPAA with diethylether before running the HPLC analysis. A dedicated procedure is available in a recent article describing the total synthesis of a biotinylated analog of the HGF/SF N domain [75]. Once the first ligation step is finished, the addition of TCEP and of the third peptide segment triggers the SEA native peptide ligation step and yields the full-length polypeptide. The SEA ligation step can be performed in the pH range of 5.5–7.5, but lowering the pH down to 5.5 can be advantageous since the ligation proceeds faster at this pH. The pH of the second SEA ligation step can also be varied to take into account some solubility problems. Note that MPAA whose pKa is 6.6 tends to precipitate below pH 5.5.

101

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2  Protein Chemical Synthesis by SEA Ligation

The one-pot three peptide segment assembly process described in Scheme 2.9 enabled the successful synthesis of several functional proteins including the wild-type [77] or biotinylated K1 HGF/SF domain [90], the biotinylated N HGF/ SF domain [75], or, very recently, a SUMO-1 protein peptide conjugate [23, 24]. Fundamentally, the same concepts are behind the one-pot process depicted in Scheme 2.10, which has been designed for accessing large cyclic peptides  [63]. The peptide thioester segment features an N-terminal protected cysteine residue, which is deprotected in-between the NCL and cyclative SEA ligation steps. The N-acetoacetyl [61, 62] (AcA) protecting group was found to be particularly useful in this case due to its compatibility with the SEA– thiol exchange reaction allowing the preparation of peptide thioesters (see Scheme 2.4). Moreover, the AcA is removed in a few minutes with only 2 equiv of hydroxylamine at acidic pH in the presence of TCEP, which acts as a O AcA-Cys-peptide1—CO-SR

N

H-Cys(StBu)–peptide2

S S

Ligation 1

NCL (MPAA) O N

AcA-Cys–peptide1-Cys-peptide2

S S

H2NOH O N

Cys–peptide1-Cys-peptide2

S

AcA O O

S Activation SEAoff SEAon

TCEP O

Cys–Peptide1-Cys-peptide2

Ligation 2

N

SH SH

Cyclative SEA ligation

peptide1-Cys-peptide2 HN O

NH O SH

Scheme 2.10  Synthesis of cyclic peptides by a one-pot two peptide segments ligation/ cyclization process (AcA: acetoacetyl).

2.3 ­Protein Total Synthesis Using SEA Chemistry – SEAon/off Concept

powerful catalyst for its removal. The method was illustrated with the synthesis of a 45 amino acid cyclic peptide and might potentially give access to small cyclic proteins composed of ~100 amino acids, considering the actual limits of the Fmoc-SPPS of SEA peptides [23, 24]. Note that AcA protecting group is a useful alternative to thiazolidine protection for N-terminal cysteine residue. 2.3.3  SEA on/off Concept and the Solid-Phase Synthesis of Proteins in the N-to-C Direction

Today, several one-pot three peptide segments sequential assembly methods are available [24, 59, 60, 64, 65, 77]. These methods save one intermediate isolation step and simplify the access to proteins composed of up to ~150 amino acids. The synthesis of large proteins (>200 amino acids) often requires the assembly of more than three peptide segments. The design of one-pot approaches for assembling four peptide segments (or more) is highly challenging as the need to perform all the chemical steps in a single reactor restricts considerably the diversity of protecting group strategies and of ligation chemistries which can be combined. Also, such one-pot methods are less desirable due to the inevitable accumulation of unreacted peptide segments or deleted polypeptides, which complicate the final purification step. A potential solution to this problem is to perform the ligations on a solid support. In this case, reagents and unreacted peptide segments can be easily removed with simple washing steps. In addition, an excess of the reagents or the peptide segments can be used to force a particular reaction. Moreover, the immobilization of the growing peptide chain on a water-compatible solid support allows circumventing the poor aqueous solubility of some intermediates. Finally, a substantial advantage of the solid-phase approach is the possibility of automating the elongation process (for a recent review on the solid-phase synthesis of proteins, see Ref. [101]). Two chemical properties of the SEAoff group are central to the peptide segment elongation cycle depicted in Scheme 2.11. The capacity of SEAoff peptides to be easily converted into peptide thioesters is used in the activation step of the elongation cycle. The latent peptide thioester properties of the SEAoff group are exploited in the subsequent coupling step, which is identical to the first chemical step of the one-pot three peptide segments assembly process discussed in the previous section. Note that in solution, the SEA–thiol exchange reaction cannot be integrated into the one-pot process since it requires the presence of TCEP, which is not compatible with the first step of the one-pot method. In contrast, the removal of TCEP in the solid-phase approach corresponds to a simple washing step, which is performed before running the coupling reaction with the next SEAoff peptide. With this elongation cycle, the peptide chain is elongated in the N-to-C direction. This method is thus complementary to the N-to-C solid-phase strategy designed by Canne and coworkers, which relies on the latent thioester properties of the thiocarboxylate group [102].

103

104

2  Protein Chemical Synthesis by SEA Ligation SEA SPPS Elongation cycle O

O N

Linker–Peptidei

Linker-Peptidei-Cys-Peptidei+1

S S

N

Step 4

S S

Wash

Step 1

SEA-thiol exchange

MPA, TCEP pH 4

MPAA, pH7

Coupling

Activation

O

StBu

Step 2 Wash

O

Linker–Peptidei

O

Cys–peptidei+1

Step 3 NCL

N

S S

OH

S

Scheme 2.11  SEA SPPS elongation cycle.

The implementation of the SEA SPPS method requires that the first SEAoff peptide segment is attached to the solid phase through its N-terminus. This attachment was achieved by using the copper-catalyzed or the strain-promoted alkyne azide cycloaddition reaction, that is, CuAAC [103, 104] and SPAAC [105], respectively (Scheme 2.12). Therefore, the first SEAoff peptide segment was modified on the N-terminus with an azido-functionalized ethylsulfonyl-2-ethyloxycarbonyl (Esoc) handle using the method developed by Aucagne et al. [106], while the solid support was modified by a terminal alkyne or a cyclooctyne

N3

O

S S

O segment1 N S O O O O off N3–Esoc-segment1-SEA

Alkyne PEGA resin , CuAAC or O Cyclooctyne PEGA resin, SPAAC

Triazole

O

S O O

segment1

O O

N

S S

O off

PEGA-Esoc-segment1-SEA –1 (>95%, loading 70–100 µmolg )

Scheme 2.12  Chemoselective immobilization of the first SEAoff peptide segment on PEGA resin using CuAAC or SPAAC.

2.3 ­Protein Total Synthesis Using SEA Chemistry – SEAon/off Concep

Figure 2.2  Protein synthesis by N-to-C solid-phase sequential ligation of unprotected peptide segments using the SEAoff group as a latent thioester surrogate. The peptide segment elongation cycle consists in activating the SEAoff group by a SEA–thiol exchange reaction and then performing an NCL reaction in the presence of MPAA (see also Scheme 2.11). (a) Synthesis of a model 135 amino acid polypeptide. A sample was treated with NaOH after each elongation cycle and analyzed by LC–MS to verify the completeness of the coupling step (right). (b) Characterization of the purified 135-amino-acid model polypeptide.

105

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2  Protein Chemical Synthesis by SEA Ligation

derivative. The Esoc linker, which is reminiscent of the Msc group [107], is stable under the neutral or mildly acidic conditions used for the elongation cycle but is rapidly cleaved with aqueous base (pH ~ 11). The latter property was used for monitoring the elongation process or for performing the final cleavage step. The method was illustrated with the synthesis of a 135 amino acid polypeptide by ligating sequentially five peptide segments as shown in Figure 2.2a. A sample of the peptidyl resin was treated with NaOH after each elongation cycle and analyzed by LC-MS to verify the completeness of the coupling step (Figure 2.2a, right). Note that, in contrast to peptide thioesters, SEAoff peptides are stable under the basic conditions used for the cleavage step. The overall isolated yield for the target 135-amino-acid polypeptide was 6.5%, including the HPLC purification step (Figure 2.2b). Nine chemical steps were performed in the solid phase, meaning an average yield of 74% per step. Interestingly, the high stability of the SEAoff group under the basic conditions used for separating the peptide from the solid support permitted the isolation of a large SEAoff peptide (60 amino acids, 22% overall yield). The SEAoff peptide was produced by ligating the peptide segments as shown in Figure 2.2a and constitutes, when attached to the solid support, an intermediate in the synthesis of the 135 amino acid polypeptide discussed earlier.

2.4 ­Chemical Synthesis of HGF/SF Subdomains for Deciphering the Functioning of HGF/SF-MET System To illustrate the usefulness of SEA chemistry for accessing complex protein targets and for understanding essential biological mechanisms, we discuss a recent work that has been undertaken to decipher the individual role of the N and K1 domains from HGF/SF in MET receptor binding and activation [108]. HGF/SF is a secreted ~90 kDa protein with an unusually large size and complex structure compared to other growth factors. HGF/SF is synthesized as an inactive monomeric precursor that is subsequently converted to an α/β active disulfide-linked heterodimer by proteolytic cleavage (Figure 2.3) [109,110]. The α chain of HGF/SF consists of an N-terminal domain (N) and four kringle domains (K1, K2, K3, and K4). In the mature HGF/SF molecule, the β chain is linked to the K4 domain through a disulfide bond and corresponds to a catalytically inactive serine proteinase homology domain (SPH). HGF/SF is a bivalent ligand that contains a high-affinity binding site for MET in the α chain and a secondary low-affinity binding site in the SPH domain. Before this study, the high-affinity HGF/SF binding site for MET was thought to require both N and K1 domains, with critical residues located in the K1 portion [111]. Importantly, NK1 protein, which includes the N and K1 domains, is a natural HGF/SF

2.4 ­Chemical Synthesis of HGF/SF Subdomains for Deciphering the Functioning of HGF/SF-MET Syste

Figure 2.3  Design of the multivalent semisynthetic NB and K1B-streptavidin scaffolds.

variant generated by a premature transcription arrest. Independent studies suggest that the NK1 binding site on MET can be located on the SEMA domain [112] or within immunoglobulin-like domains 3 and 4 [113]. The SPH domain binds MET with a well-defined interface onto the SEMA domain [114]. The crystal structure of NK1 protein shows a “head-to-tail” noncovalent  homodimer, which is believed to be responsible for MET dimerization (Figure 2.3) [115]. NK1 protein possesses a partial agonist activity but requires the presence of heparan sulfate (HS) to promote an efficient activation of MET

107

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2  Protein Chemical Synthesis by SEA Ligation

receptor. In contrast, the isolated K1 domain has a weak micromolar MET agonist activity, whereas the isolated N domain has no activity [110]. K1 is a weak MET binder (Kd ~ μM), while opposing results were reported for the capacity of N domain to bind MET [110, 116]. Thus, the controversy regarding the location of NK1 binding site on MET together with the role of individual of N and K1 domains makes difficult the building of a consensus model for the HGF/SF-MET interaction. The work described hereinafter has been stimulated by the conviction that monomeric N and K1 domains are not optimal for interrogating the functioning of the NK1-MET system, which, by nature, is a complex multivalent system. Protein chemical synthesis allowed the production of site-specifically biotinylated N and K1 (NB and K1B) domains with the aim of preparing semisynthetic and multivalent scaffolds using streptavidin (S) technology (Figure 2.3). Indeed, analysis of the relative positions of N and K1 domains in the crystal structure of NK1 homodimer reveals that the C-termini of the two N and K1 domains are separated by 1.3–2 nm, a distance that is close to that between the individual biotin binding sites in the streptavidin homotetramer (~2.0–3.5 nm). Therefore, streptavidin was envisioned as a good template for presenting pairs of N or K1 domains as observed in the NK1 crystal structure but independently of each other. Such a design is extremely hard to propose by classical recombinant approaches. In addition, chemical synthesis ensured the production of proteins with an atom-by-atom control of the semisynthetic scaffold structure and devoid of any biological contaminants, such as HS or heparin, which could generate heterogeneity and artifacts in the in vitro and/or in vivo assays. The linear NB [75] and K1B [77, 90] polypeptides were produced by total chemical synthesis using the one-pot three peptide segments assembly strategy (Figure 2.4a). The folded N domain of HGF/SF is composed of 97 amino acid residues (HGF/SF 31–127) and is stabilized by two disulfide bonds (Figure 2.4b). The folded K1 domain is composed of 85 amino acid residues (125–209) and is stabilized by three disulfide bonds (Figure 2.4c). In NB and K1B, the primary structure was extended at the C-terminus with a lysine residue modified on its side chain with a biotin group. Both linear polypeptides were folded successfully using the glutathione–glutathione disulfide redox system and their tertiary structure and disulfide bond pattern validated by proteomic analysis and circular dichroism (CD) spectroscopy. The functionality of individual NB and K1B proteins was verified using various biochemical and in  vitro cellular assays. In particular, NB showed no MET agonist activity despite its ability to bind HS or heparin, a property that is critically dependent on the tertiary structure, while K1B behaved as a weak micromolar MET agonist. In other words, at this stage, the synthetic NB and K1B proteins behaved as reported by others using recombinant proteins. Next, MET binding properties of NB/S and K1B/S scaffolds were evaluated using AlphaScreen® technology and pull-down experiments (Figure 2.5). NB or K1B was loaded on streptavidin-coated donor beads and incubated with

2.4 ­Chemical Synthesis of HGF/SF Subdomains for Deciphering the Functioning of HGF/SF-MET Syste

Figure 2.4  Chemical synthesis of NB and K1B proteins using the one-pot three peptide segment assembly process. (See color plate section for the color representation of this figure.)

109

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recombinant MET-Fc chimera (human MET ectodomain fused with hIgG1 Fc fragment) loaded on Protein A coated acceptor beads. If K1B/S or NB/S donor beads interact with MET-Fc/Protein A acceptor beads, a chemical energy transfer is possible between the beads, leading to fluorescence emission upon laser excitation. In saturation experiments, NB showed background signal intensities, whereas K1B induced strong signal intensities (Figure 2.5a). Competition assays confirmed an apparent Kd around 15 nM for the K1B/S-MET interaction, a value that is 100-fold lower than the Kd reported for K1-MET interaction [110]. This study was complemented by examining the binding of NB/S and K1B/S complexes to endogenous MET from a whole cell lysate (Figure 2.5b). Streptavidin-coated agarose beads were incubated with NB or K1B to form immobilized complexes. The whole lysate from HeLa or CaPan1 cells, that is, containing solubilized endogenous MET receptor, was applied on the beads. Western blot analysis of the eluted material showed that only K1B/S complexes were able to capture endogenous MET from cell lysates, whereas NB/S failed. Altogether, these binding experiments demonstrate that N domain does not bind endogenous or recombinant MET receptor to the contrary of K1B/S complex, which binds MET at low nanomolar concentration. Importantly, the high affinity of K1B/S for MET shows the critical role played by multivalency in the K1-MET interaction system. Next, the biological activity of NB/S or K1B/S complexes was examined using various in vitro cellular phenotypic assays. NB/S proved to be inactive, whereas K1B/S showed a strong MET agonistic activity on the human HeLa and canine Madin Darby canine kidney (MDCK) cell lines. The activity on the MET downstream signaling cascade (extracellular signal regulated kinase, ERK and oncogene of the AKT8 (AKR mouse thymoma-8) virus, Akt pathways) has been measured using HTRF™ technology (Figure 2.6a and b). Impressively, K1B/S complex was able to trigger significant ERK and Akt phosphorylation levels down to a low nM range and thus displayed an agonist activity similar to recombinant NK1 protein. The fact that activation of MET by monomeric K1B was detected only for micromolar concentrations, as reported in the literature, highlights again the critical role played by multivalency for achieving strong receptor activation. The ability of K1B/S to induce cell scattering was evaluated in MDCK cells, the gold standard cell line for this phenotypic assay (Figure 2.6c). In the presence of HGF/SF or NK1 for 18 h, MDCK cells acquired a mesenchymal-like phenotype and scattered as expected. Impressively, K1B/S complex induced a similar phenotype, whereas scattering induced by K1B was weak. To extend this observation in a more physiological assay, K1B/S was injected subcutaneously with Matrigel® plugs into immunodeficient SCID mice in order to induce angiogenesis. Indeed, HGF/SF is a potent angiogenic factor that stimulates endothelial cell proliferation and migration. The plugs were extracted after 11 days to determine the quantity of hemoglobin infiltrated into the plug as a measure of induced angiogenesis (Figure 2.6d). As expected, VEGF or

2.4 ­Chemical Synthesis of HGF/SF Subdomains for Deciphering the Functioning of HGF/SF-MET Syste

(a)

Figure 2.5  Binding of NB/S or K1B/S complexes to purified recombinant or endogenous MET receptor. (a) NB, K1B and MET-Fc binding assay: increasing concentrations of NB or K1B were mixed with extracellular MET domain fused with human IgG1-Fc (MET-Fc) and incubated with streptavidin AlphaScreen® donor beads and Protein A acceptor beads. Error bars correspond to standard error (±SD) of triplicates. (b) Endogenous MET capture. Streptavidin-coated beads loaded with NB or K1B were incubated with HeLa or CaPan1 total cell lysates. Input, flow-through, and elution fractions from NB or K1 loaded beads were analyzed by specific total MET Western blot.

HGF/SF showed potent angiogenic properties compared to control plugs. Remarkably, K1B/S induced the formation of vessels with a hemoglobin content comparable to that of VEGF and significantly higher than those induced by NK1 or K1B. Thus, while NK1 and K1B/S displayed similar potencies in in vitro cell assays, their angiogenic properties were significantly different in vivo.

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Figure 2.6  HeLa cells were treated with increasing concentrations of mature HGF/SF, K1B/S, NK1, and K1B/Ab for 7 min. Activation levels of Akt (a) and ERK (b) were measured using HTRF technology and plotted as the 665/620 nm HTRF signal ratio. (c) Cell scattering assay. MDCK isolated cell islets were incubated for 18 h in culture media with HGF/SF (HGF), K1B, K1B/S, and NK1. Cells were then stained and observed under microscope (40×). (d) Angiogenesis. Mice were injected with a mixture of Matrigel and HGF/SF (HGF), VEGF, NK1, K1B/S, K1B, or S. Hemoglobin absorbance was measured and concentration was determined using a rate hemoglobin standard curve and plug weight.

Finally, the in vivo activity of K1B/S complex was evaluated in an acute and integrated mouse model. It was particularly interesting to check if K1B/S complex could promote hepatocyte survival when an apoptotic stress was induced in the liver. Indeed, injection of an anti-Fas antibody (anti-CD95) in mice quickly induces a massive hepatocellular apoptosis leading to fulminant hepatitis and death of the animals. Previous studies showed that HGF/SF was able to abrogate Fas-induced fulminant hepatitis but required prohibitive amounts to show significant effects (usually 1 nmol, i.e., 100 µg per mouse). In our assay, anti-Fas antibody was coinjected with 25 pmol of K1B, K1B/S, or NK1 or 2.5 pmol of mature HGF/SF per g of body weight. After 90 min, a second

2.4 ­Chemical Synthesis of HGF/SF Subdomains for Deciphering the Functioning of HGF/SF-MET Syste

Figure 2.7  (a) FVB mice were injected intravenously with anti-Fas monoclonal antibody (AbFAS) mixed with K1B, K1B/S, NK1, or HGF/SF or PBS. A second injection without anti-Fas was performed 90 min later. Livers were extracted and fixed in formalin after three additional hours. (b) FVB mice were injected with an increased concentration of K1B/S complex, K1B, or NK1. After 10 min, livers were extracted, snap-frozen, and crushed. Cell lysates were analyzed by specific total MET, Akt and ERK or phospho-MET, phospho-Akt, and phospho-ERK Western blot. (See color plate section for the color representation of this figure.)

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injection of each agonist protein was performed to sustain signaling. Livers were extracted after three additional hours for histological and molecular analysis. Macroscopically, mice treated with anti-Fas antibody and K1B, NK1, or mature HGF/SF presented an altered liver, retaining a deep brown color even after PBS perfusion and elimination of vascular blood content (Figure 2.7a). Remarkably, mice treated with K1B/S maintained a clear liver, almost structurally intact. Histological analysis demonstrated that the dark color ­displayed by altered livers was mostly induced by a vascular congestion attributable to a massive hepatocyte loss and subsequent blood infiltration. Further analysis confirmed that these disorganized regions corresponded to large clusters of apoptotic hepatocytes. Molecular analysis of signaling in hepatocyte confirms that resistance to Fasinduced apoptosis is due to MET activation. Indeed, K1B/S is able to promote strong MET signaling for at least 30 min at doses as low as 2.5 pmol per gram of body weight, that is, 0.5 µg of K1B per gram of body weight (Figure 2.7b). This study using synthetic proteins and semisynthetic scaffolds demonstrated that N domain does not bind MET receptor, in contrast to some previous reports, and is not necessary for engineering strong MET agonists. Previous studies with recombinant N domain might be perturbed by the presence of contaminants such as HS or heparin molecules for which N domain and MET have a strong affinity. We showed for the first time that the K1 domain is sufficient by itself for designing strong MET agonists, provided it is presented multivalently.

2.5 ­Conclusion The work on N and K1 HGF/SF domains is another illustration of the value of protein chemical synthesis in biological research. Given the growing importance of protein total synthesis in biology but also in other fields of research, it is important to facilitate the access to small functional proteins by developing novel chemoselective amide-bond-forming reactions and by designing efficient peptide segment assembly strategies. The SEA chemical toolbox relies on Fmoc-SPPS-compatible methods and contains a set of robust chemical methods, which can be useful to the peptide or protein chemists. Importantly, SEA chemistry can be easily combined with the NCL reaction for accessing challenging protein targets or unusual peptide-based scaffolds.

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chemical synthesis of calstabin 2 protein. In Peptides 2012 Proceedings of the Thirty-second European Peptide Symposium; Kokotos, G., KonstantinouKokotou, V., Matsoukas, J., Eds.; University of Athens: Athens, 2012; pp 642–643. Hou, W.; Zhang, X.; Li, F.; Liu, C. F.: Peptidyl N,N-bis(2-mercaptoethyl)amides as thioester precursors for native chemical ligation. Org Lett 2011, 13, 386–389. Ancot, F.; Leroy, C.; Muharram, G.; Lefebvre, J.; Vicogne, J.; Lemiere, A.; Kherrouche, Z.; Foveau, B.; Pourtier, A.; Melnyk, O.; Giordano, S.; Chotteau-Lelievre, A.; Tulasne, D.: Shedding-generated Met receptor fragments can be routed to either the proteasomal or the lysosomal degradation pathway. Traffic 2012, 13, 1261–1272. Ruff, Y.; Garavini, V.; Giuseppone, N.: Reversible native chemical ligation: a facile access to dynamic covalent peptides. J Am Chem Soc 2014, 136, 6333–6339. Burlina, F.; Papageorgiou, G.; Morris, C.; White, P. D.; Offer, J.: In situ thioester formation for protein ligation using α-methylcysteine. Chem Sci 2014, 5, 766–770. Lees, W. J.; Whitesides, G. M.: Equilibrium constants for thiol-disulfide interchange reactions: a coherent, corrected set. J Org Chem 1993, 58, 642–647. El-Mahdi, O.; Melnyk, O.: Alpha-oxo aldehyde or glyoxylyl group chemistry in peptide bioconjugation. Bioconjugate Chem 2013, 24, 735–765. Nakamura, T.; Shigenaga, A.; Sato, K.; Tsuda, Y.; Sakamoto, K.; Otaka, A.: Examination of native chemical ligation using peptidyl prolyl thioesters. Chem Commun (Camb) 2014, 50, 58–60. Ollivier, N.; Raibaut, L.; Blanpain, A.; Desmet, R.; Dheur, J.; Mhidia, R.; Boll, E.; Drobecq, H.; Pira, S. L.; Melnyk, O.: Tidbits for the synthesis of bis(2-sulfanylethyl)amido (SEA) polystyrene resin, SEA peptides and peptide thioesters. J Pept Sci 2014, 20, 92–97. Garcia-Martin, F.; Quintanar-Audelo, M.; Garcia-Ramos, Y.; Cruz, L. J.; Gravel, C.; Furic, R.; Cote, S.; Tulla-Puche, J.; Albericio, F.: ChemMatrix, a poly(ethylene glycol)-based support for the solid-phase synthesis of complex peptides. J Comb Chem 2006, 8, 213–220. Botti, P.; Villain, M.; Manganiello, S.; Gaertner, H.: Native chemical ligation through in situ O to S acyl shift. Org Lett 2004, 6, 4861–4864. Tofteng, A. P.; Jensen, K. J.; Hoeg-Jensen, T.: Peptide dithiodiethanol esters for in situ generation of thioesters for use in native ligation. Tetrahedron Lett 2007, 48, 2105–2107. Zheng, J. S.; Cui, H. K.; Fang, G. M.; Xi, W. X.; Liu, L.: Chemical protein synthesis by kinetically controlled ligation of peptide O-esters. ChemBioChem 2010, 11, 511–515.

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101 Raibaut, L.; El Mahdi, O.; Melnyk, O.: Solid phase protein chemical synthesis.

Topics Curr Chem 2015, DOI 10.1007/128-2014-609.

102 Canne, L. E.; Botti, P.; Simon, R. J.; Chen, Y.; Dennis, E. A.; Kent, S. B. H.:

103

104

105

106

107

108

109 110

111

112

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Chemical protein synthesis by solid phase ligation of unprotected peptide segments. J Am Chem Soc 1999, 121, 8720–8727. Tornoe, C. W.; Christensen, C.; Meldal, M.: Peptidotriazoles on solid phase: [1,2,3]-triazoles by regiospecific copper(I)-catalyzed 1,3-dipolar cycloadditions of terminal alkynes to azides. J Org Chem 2002, 67, 3057–3064. Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B.: A stepwise Huisgen cycloaddition process: copper(I)-catalyzed regioselective “ligation” of azides and terminal alkynes. Angew Chem Int Ed Engl 2002, 41, 2596–2599. Agard, N. J.; Prescher, J. A.; Bertozzi, C. R.: A strain-promoted [3 + 2] azide-alkyne cycloaddition for covalent modification of biomolecules in living systems. J Am Chem Soc 2004, 126, 15046–15047. Aucagne, V.; Valverde, I. E.; Marceau, P.; Galibert, M.; Dendane, N.; Delmas, A. F.: Towards the simplification of protein synthesis: iterative solid-supported ligations with concomitant purifications. Angew Chem Int Ed Engl 2012, 51, 11320–11324. Tesser, G. I.; Balvert-Geers, I. C.: The methylsulfonylethyloxycarbonyl group, a new and versatile amino protective function. Int J Pept Protein Res 1975, 7, 295–305. Simonneau, C.; Berenice, L.; Mougel, A.; Adriaenssens, E.; Paquet, C.; Raibaut, L.; Ollivier, N.; Drobecq, H.; Marcoux, J.; Cianferani, S.; Tulasne, D.; de Jonge, H.; Melnyk, O.; Vicogne, J.: Semi-synthesis of a HGF/SF kringle one (K1) domain scaffold generates a potent in vivo MET receptor agonist. Chem Sci 2015, DOI: 10.1039/c4sc03856h. Nakamura, T.: Structure and function of hepatocyte growth factor. Prog Growth Factor Res 1991, 3, 67–85. Holmes, O.; Pillozzi, S.; Deakin, J. A.; Carafoli, F.; Kemp, L.; Butler, P. J.; Lyon, M.; Gherardi, E.: Insights into the structure/function of hepatocyte growth factor/scatter factor from studies with individual domains. J Mol Biol 2007, 367, 395–408. Lokker, N. A.; Presta, L. G.; Godowski, P. J.: Mutational analysis and molecular modeling of the N-terminal kringle-containing domain of hepatocyte growth factor identifies amino acid side chains important for interaction with the c-Met receptor. Protein Eng 1994, 7, 895–903. Kong-Beltran, M.; Stamos, J.; Wickramasinghe, D.: The Sema domain of Met is necessary for receptor dimerization and activation. Cancer Cell 2004, 6, 75–84. Basilico, C.; Arnesano, A.; Galluzzo, M.; Comoglio, P. M.; Michieli, P.: A high affinity hepatocyte growth factor-binding site in the immunoglobulin-like region of Met. J Biol Chem 2008, 283, 21267–21277.

 ­Reference

114 Stamos, J.; Lazarus, R. A.; Yao, X.; Kirchhofer, D.; Wiesmann, C.: Crystal

structure of the HGF beta-chain in complex with the Sema domain of the Met receptor. Embo J 2004, 23, 2325–2335. 115 Chirgadze, D. Y.; Hepple, J. P.; Zhou, H.; Byrd, R. A.; Blundell, T. L.; Gherardi, E.: Crystal structure of the NK1 fragment of HGF/SF suggests a novel mode for growth factor dimerization and receptor binding. Nat Struct Biol 1999, 6, 72–79. 116 Merkulova-Rainon, T.; England, P.; Ding, S.; Demerens, C.; Tobelem, G.: The N-terminal domain of hepatocyte growth factor inhibits the angiogenic behavior of endothelial cells independently from binding to the c-met receptor. J Biol Chem 2003, 278, 37400–37408.

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3 Development of Serine/Threonine Ligation and Its Applications Tianlu Li and Xuechen Li Department of Chemistry, The University of Hong Kong, Hong Kong SAR, PR China

3.1 ­Introduction 3.1.1  Protein Synthesis by SPPS

The introduction of solid‐phase peptide synthesis (SPPS) has revolutionized synthetic peptide chemistry [1, 2]. Compared to the classical organic synthesis in solution, SPPS tremendously expedites the reaction process, improves reaction yield, and liberates manpower from repetitive work and laborious purification. However, as the peptide chain grows, the problems such as decreased solubility, poor conversion, and resultant accumulation of truncated products arise, which are hardly separable from the target molecule; thus, peptides of ~50 amino acid length represent the practical limit of SPPS [3] that could be reliably produced in modest yields as a homogeneous molecular species of defined covalent structure. Not to mention, each activation step during SPPS subjects the incoming amino acid to the potential of epimerization, given that an assortment of coupling reagents has been developed generation after generation. Novel protein chemistry approach is in urgent need. 3.1.2  Native Chemical Ligation (and Extended Desulfurization)

To circumvent the drawback of the stepwise assembly of linear peptide, a convergent synthetic strategy is needed. The convergent peptide coupling reaction requires high demand on the selectivity and reactivity. Firstly, the reaction has to be highly chemoselective, in the presence of versatile side‐chain functionalities within peptides. Secondly, the coupling reaction has to be regioselective, only reacting at the termini. Thirdly, the reaction should be highly if not absolutely stereoselective, preserving the stereo integrity of the reacting C‐terminal residue. Finally, the reaction needs to be very reactive, working with low Chemical Ligation: Tools for Biomolecule Synthesis and Modification, First Edition. Edited by Luca D. D’Andrea and Alessandra Romanelli. © 2017 John Wiley & Sons, Inc. Published 2017 by John Wiley & Sons, Inc.

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concentration of the substrate (e.g., mM). Traditionally, peptide condensation has been used to construct long peptides, using side‐chain‐protected peptide fragments. Protected peptides often have poor solubility in any solvent, resulting in handling difficulties. In addition, activation of the C‐terminal residue with coupling reagents is needed for peptide condensation, which readily leads to epimerization at the nonglycine or proline C‐terminus [4]. Gratifyingly, the development of native chemical ligation (NCL) (Scheme 3.1) has significantly expanded the scope of synthetic protein chemistry [5]. The reaction involves a thiol(ate)–thioester exchange between a C‐terminal thioester and an N‐terminal Cys residue, which spontaneously undergoes intramolecular rearrangement to give a native amide bond at the ligation site. The reaction is performed with side‐chain‐unprotected peptide segment and does not involve in situ activation of C‐terminal amino acid. While the former step is freely reversible, the latter is definitely irreversible due to the favorable geometric arrangement of the α‐NH2 moiety with respect to the thioester formed in the initial chemoselective ligation reaction. The regioselectivity is also demonstrated, in that only the Cys at N‐terminus bears the 1,2‐thiolamine functionality. Taking advantage of the solubilizing agents such as urea and guanidine hydrochloride, the reaction takes place under mild, aqueous condition at room temperature. In summary, the method is theoretically plausible and practically useful, as has been demonstrated by numerous successes over the past two decades since its introduction. It is fair to say that the NCL technique brings about the new era of prosperity to the peptide chemistry community, where synthetic chemistry almost lost the battle to recombinant DNA technique due to insuperable shortcomings of SPPS in handling large peptides/proteins. While NCL relies on the N‐terminal Cys residue within the peptide sequence, the low abundance of Cys limits its wide application; furthermore, sometimes, Cys does not situate at a proper site for convergent synthesis. Introduction of thiol auxiliary, in this case, provides more choices of ligation sites and expands the scope of NCL. The most straightforward case is the mutation of Ala into Cys for NCL, which is then converted back postligation. To name a few, ligation at amino acid residues such as Ala, Phe, Leu, and Val is currently accessible, all of which involve the reduction of thiol via metal‐based or metal‐free desulfurization (Scheme 3.2). In 2001, Yan and Dawson first combined NCL and desulfurization for converting Cys‐containing peptide into its Ala‐containing counterpart [6], in the synthesis of cyclic antibiotic microcin J25, streptococcal protein G B1 domain (PGB1), and a variant of the 110‐amino‐acid ribonuclease, barnase. The method received wide attention, and later on, in 2007, Kent’s group found that these metal‐based desulfurization protocols could effectively accommodate acetamidomethyl (Acm) functionality [8], as is demonstrated by the effective  synthesis of a 37‐residue peptide hormone, [A24P,S27P,S28P]amylin. Despite these successes, drawbacks such as requirement of a large excess of

O Unprotected peptide-1

SR

+

+H N 3

Transthiolesterification

O Unprotected peptide-2

O Unprotected peptide-1

–S

S H2N

Spontaneous rearrangement (S-to-N acyl shift) HS O Unprotected peptide-1

N H

Unprotected peptide-2

O

Scheme 3.1  The principle of native chemical ligation.

Unprotected peptide-2

O

128

3  Development of Serine/Threonine Ligation and Its Applications Kent and coworkers [6] O Unprotected peptide-1

O + SR2 + H3N –

S

HS O

NCL Unprotected peptide-2

N H

Unprotected peptide-1

R1

R1 Unprotected peptide-2

O Metal-based Yan and Dawson [5] Raney Nickel or Pd/Al2O3

R1 = amino acid functionalities Desulfurization

Metal-free Wan and Danishefsky [7] TECP, VA-044, tBuSH

R1

O Unprotected peptide-1

N H

Unprotected peptide-2

O

Scheme 3.2  The evolution of the chemical ligation–desulfurization [5–7].

nickel and poor accommodation with Trp, thiols, thioethers, thioesters, and thiazolidine (Thz) limited the generality of this approach. To expand the scope further, Wan and Danishefsky established the radical‐ based, metal‐free desulfurization protocol [7], based on an unexpected desulfurization observed when trimethylphosphine or tris(2‐carboxyethyl) phosphine (TCEP) was used to reduce disulfide bonds in aqueous phase [9]. With TECP as phosphine source and VA‐044 as radical initiator, the desulfurization was carried out in water in the presence of tBuSH, which acts as a hydride donor to accelerate the reaction [10]. The reaction was fast and clean, under mild condition, and accommodated various functional groups including Met, Cys(Acm), Thz, and biotin. NCL desulfurization method boosts the synthetic protein world to an unprecedented level. One of the most fantastic examples is the total synthesis of homogeneous erythropoietin (EPO) by Danishefsky’s group [11, 12]. Many researchers dedicated to such a chemically difficult yet theoretically demanding task, and they eventually made it with quadruple cysteine NCLs from four glyco‐containing peptide fragments, one global metal‐free desulfurization, and a final folding step. The synthetic EPO was confirmed in vitro activity. The accomplishment of such a complex glycoprotein with sophisticated chemistry and multiple rounds of NCL bodes well for the applicability of this powerful method. 3.1.3  KAHA Ligation

Alternatively, the α‐ketoacid hydroxylamine (KAHA) ligation (Scheme 3.3) launched by Bode et al. offers another opportunity for the chemical synthesis of peptides. The ligation employs C‐terminal peptide α‐ketoacid and N‐terminal peptide hydroxylamines or derivatives [13], rendering innocuous homoserine depsipeptide at the ligation site. The ester is then, if necessary, treated with basic aqueous solution to give the acyl‐transferred product, which adopts the

R4 R1 H 2N

N H

Unprotected peptide

S CN O

Oxidation of cyanosulfur ylides HO R1 H2N

Protected peptide

N H O

O

H N

O

Unprotected peptide

COOH

Free hydroxylamines O

R3

MeO Protected α-keto acids

O

H O N R2

R1 H 2N

N H

R2

Unprotected peptide N H Keto Acid

+

O

O

H N

OH

N H

Unprotected peptide

O-substituted hydroxylamines

O N H

Unprotected peptide

Cyclic alkoxyamines

O Hydroxyl Amine

KAHA ligation

R1 H2N

Unprotected peptide

N H

O

H N O

R2

N H

COOH

Unprotected peptide

Scheme 3.3  Overview of different functional groups used in KAHA ligation.

COOH

COOH

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3  Development of Serine/Threonine Ligation and Its Applications

native amide bond [14]. To date, KAHA ligation has established its success with a number of proteins such as prokaryotic ubiquitin‐like protein (Pup) [15], portable cold‐shock protein (CspA) [15], ubiquitin‐fold modifier 1 (UFM1) [16], interferon‐induced transmembrane protein 3 (IFITM3), SUMO2, and SUMO3 [17]. Furthermore, it has been demonstrated that when the ring size of the N‐terminal hydroxylamine is decreased, that is, oxazetidine instead of oxaproline, the reactivity is largely improved, and the limitation of KAHA ligation was reduced. In this optimized way, the ligation operates at lower concentration and milder temperature and yields product within a shorter reaction period. The utility of this strategy is demonstrated by the synthesis of S100A4 (metastasis), a 12 kDa calcium‐binding protein [18].

3.2 ­Serine/Threonine Ligation (STL) In an analogous yet totally different manner, Li’s group has devised an alternative toward the convergent synthesis of peptides/proteins, termed Ser/Thr ligation (Scheme 3.4). According to the principle of this ligation strategy, the bifunctional nucleophilic 1,2‐hydroxylamino group on the N‐terminal Ser/Thr could serve to initiate a chemoselective capture step, which brings two side‐ chain‐unprotected peptide segments in proximity, thus making it possible for a following intramolecular acyl transfer leading to a native amide bond at the ligation junction. Based on the seminal imine‐formation‐initiated ligation by Kemp [19] and Tam [20, 21], the peptide containing a salicylaldehyde (SAL) ester at the C‐terminus was shown to chemoselectively react with N‐terminal Ser or Thr residue of another peptide segment to give rise to a ligated product, and the resultant N,O‐benzylidene moiety formed at the ligation site is found to be very acid‐labile to reveal a native peptidic peptide linkage. This reaction is highly chemoselective, tolerating the side‐chain functionalities present in the peptide sequence, regioselective, and stereoselective, maintaining the chiral integrity of the reacting C‐terminal amino acid [22, 23]. The N,O‐benzylidene intermediate proves to be stable (to NMR analysis as well as HPLC purification and lyophilization); on the other hand, the acidolytic cleavage of this auxiliary is efficient. Even dilute as 50% TFA can rapidly remove benzylidene acetal within 5 min; treatment with 5–10% TFA completes the cleavage within 4 h. Under 1% TFA condition, the reaction is relatively slow [24]. 3.2.1  SAL Ester Preparation

Similar to the peptide thioester in NCL, the peptide SAL ester is a requisite segment in serine/threonine ligation. So far, several strategies have been developed for the generation of peptide SAL esters via SPPS. Since the peptide phenolic ester is labile to piperidine treatment during Fmoc‐SPPS, the peptide

O +

O

O

H2N

CHO

HO

O

R

HN

R

O

R = H, Ser R = Me, Thr 5-endo-trig O O

N

1,5 O-to-N acyl transfer

R OH

O H+

O N H HO

R

N O

R

OH N, O-benzylidene acetal intermediate

Scheme 3.4  Principle of Ser/Thr ligation.

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3  Development of Serine/Threonine Ligation and Its Applications

SAL ester can be synthesized either through Boc‐SPPS or post Fmoc‐SPPS modification. Firstly, the peptide SAL ester can be synthesized via direct coupling of SAL dimethyl acetal with the protected peptide acid obtained from the Fmoc‐SPPS on a 2‐chlorotrityl resin (Scheme 3.5). The coupling is mediated by either DCC/DMAP or PyBOP/DIEA. Upon TFA‐mediated global deprotection, the unprotected peptide SAL ester is thus obtained. However, this strategy works only when the C‐terminal amino acid is glycine or proline, owing to the potential epimerization issue of other amino acids at the C‐terminus under the coupling condition. Secondly, in seeking for a Boc‐SPPS method of preparing the peptide SAL ester, Li’s group [25] and Liu’s group [26] independently worked it out in the Boc mode, making good use of the key intermediate (E)‐3‐(2‐hydroxyphenyl) acrylic acid (i.e., trans‐2‐hydroxycinnamic acid). The C═C herein serves as a precursor since most protected forms of the aldehyde are acid‐labile and thus not compatible with the strong acidic conditions employed in Boc SPPS. Liu’s group immobilized the free acid on the amine‐functionalized 4‐methylbenzhydrylamine (MBHA) resin (Scheme 3.6). The peptide was elongated via the phenol group and cleaved from the resin via trifluorometha nesulfonic acid (TFMSA)/TFA or HF. It was then subjected to ozonolysis in CH3CN/H2O (5:1) as the best solvent. In Li’s design, the acetyl‐protected SAL undergoes the Wittig reaction followed by TFA liberation to give this intermediate, which is loaded to aminomethyl resin. After removal of acetyl protection, the phenol group is ready for peptide chain elongation under Boc‐SPPS condition. Upon completion, TMSOTf/TFA/thioanisole cocktail removes the side‐chain protection; finally, ozonolysis in CH2Cl2/TFA (95:5) mixture released the resin‐bound peptide with an SAL ester at the C‐terminus. This method proved to be efficient and convenient in the application to the synthesis of cyclic peptide, including tetrapeptide and the ones with various sizes (from 5 to 9 residues). It should be noted that ozone is a powerful oxidant that hardly tolerates Cys, Met, and Trp residues. Finally, the preparation of SAL ester via Fmoc‐SPPS has also been devised (Scheme 3.7), based on the N‐acylbenzimidazolone (Nbz) linker developed by Dawson’s group [27]. The resin is derivatized with 3,4‐diaminobenzoic acid (Dbz), to which the peptide is elongated. To prevent the potential acylation of the unprotected o‐aminoanilide, especially in Gly‐rich sequences in the presence of excess base [28], it can be reversibly protected with Alloc group [29]. In an analogous manner to Dawson’s approach to prepare peptide thioester with the Nbz linker, the phenolysis of the peptide–Nbz with salicylaldehyde would generate the peptide SAL ester in solution or on resin. In contrast to the sluggish phenolysis in solution, on‐resin phenolysis was shown to yield the desired product. After a close investigation in terms of the

PG

Fmoc SPPS

Cl

O

2-Chlorotrityl chloride resin

N

FmocHN

O

FmocHN O

PG

O

HGEGTψ(Phe,Thr)

O

PG

O O

CH2Cl2/TFE/AcOH

PG FmocHN

PG

HGEGTψ(Phe,Thr) PG

O OH

O

O OH

TFA/H2O/PhOH DCC/DMAP

Scheme 3.5  Direct coupling of SAL ester at Ψ(Phe, Thr) site.

FmocHN

HGEGTFT

O CHO

trans-2-Hydroxycinnamic acid OH

O OH

H2N

OH

PG

O

Boc-SPPS N H

PyBOP, DIEA

H2N

O

An...A2A1

O

N H

PG

MBHA resin

O

O Cleavage HF

H2N

An...A2A1

O

O3

O NH2

H2N

An...A2A1

1–2 min

Scheme 3.6  SAL ester generation developed by Liu’s group.

O

O

H O

3.2 ­Serine/Threonine Ligation (STL OH

Ac2O Pyridine

CHO

OAc CHO

Ph3P

OtBu

O

OAc

O

OtBu

THF 1. TFA/H2O (95:5) Aminomethyl O resin

O

PG

O

An...A2A1

2. PyBOP, DIEA DMAP, DMF

N H

PG

OAc

1) 20% piperidine/DMF

O N H

2) Boc-SPPS

1) TMSOTf/TFA/thioanisole, 0°C 2) O3, –78°C; then Me2S PG

O An...A2A1

O

1. SAL dimethyl acetal, Na2CO3, THF/DCM

O H

2. TFA/H2O(95:5)

An...A2A1 PG

O O

N

O

N H

N H Peptide SAL ester

1) p-Nitrophenyl chloroformate, CH2Cl2 2) DIEA, DMF

Fmoc A1 HN H2N

O O

Rink amide resin N H

PG Fmoc SPPS

O

An...A2A1 PG

HN H 2N

O N H

Scheme 3.7  Generation of SAL ester via (upper) Boc‐ or (lower) Fmoc pathway.

type of base, solvent, aldehyde alternatives, it is clear that SAL masked as dimethyl acetal displays increased nucleophilicity and decreased side reaction, compared to SAL; displacement takes place in THF/CH2Cl2 (3:1) in the presence of Na2CO3, which stands as an optimal choice considering the reactivity, peptide solubility, resin swelling property, and operational convenience. The resulting peptide SAL ester is subjected to TFA/H2O (95:5) for global deprotection. No extra scavenger reagents are needed since the extra SAL serves this role well. The reaction proved no epimerization indicated by HPLC analysis.

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3  Development of Serine/Threonine Ligation and Its Applications

3.2.2  N‐Terminal‐Protecting Group for Successive C‐to‐N Ser/Thr Ligations

For the synthesis of peptides involving multiple STLs, the N‐terminal Ser/Thr residues of bifunctional internal fragments have to be properly protected, to avoid the potential side reactions such as self‐cyclization and polymerization. Fmoc is the most straightforward choice. It is removed after ligation by Et2NH in CH2Cl2 [23, 30] or DMSO [16], which renders fast reaction and clean product so that the purification step may be skipped [23]. Azido group is another choice for N‐terminal protection (Scheme 3.8). It is stable under acidic and basic conditions involved in peptide assembly and can be readily removed under reducing conditions; it also displays acceptable hydrophobicity that may not disturb the peptide solubility and purification step. However, it possesses the risk of racemization at certain amino acids, such as racemization‐prone residue serine [31], as is observed in the case of Forteo (Ser1‐Asn16)‐SAL ester as well as hGH‐RH (Ser18‐Met27)‐SAL ester (Scheme 3.9). Moreover, the N‐terminal azido‐serine may also react with the C‐terminal SAL ester through aldol condensation, which destroys the reactive aldehyde for STL. This phenomenon coincides with the reports that the enolate of α‐azido‐ketones can be trapped by electrophiles (such as aldehyde) in the presence of certain bases (such as EtONa or 1,8‐diazabicyclo[5.4.0] undec‐7‐ene, DBU) [32]. This self‐condensation problem is overcome by the expedient dimethyl acetal protection of SAL [33]. Msz (p‐(methylsulfinyl)benzyloxycarbonyl) [34, 35] is another N‐terminal‐ protecting group successfully applied in STL [36]. This safety‐catch type of amino protecting group is stable under both acidic and basic conditions and can be smoothly removed by reductive acidolysis, thus providing a new dimension of orthogonality. It is introduced for the N‐terminal protection of internal fragment since the Fmoc group was found to be partially removed during the activation step toward the Nbz linker. Note that Met residues within the same peptide sequence are likely to be affected upon TFA treatment for global deprotection, where the oxygen exchange between Msz and Met takes place (Scheme 3.10), as is the case in the preparation of hGH‐RH (Ser18‐Met27)‐SAL ester. In this case, Met(O) instead of Met should be introduced during SPPS; the corresponding sulfoxide is concomitantly removed by the reductive cocktail of TMSOTf (1.0 M)–thioanisole (1.0 M)/TFA mixture [37] together with Msz removal.

O

O N3

RCHO

N3

Scheme 3.8  Early reports of azido‐ketone and aldehyde.

O

OH R N3

3.2 ­Serine/Threonine Ligation (STL

O N3

PG

PG

Forteo 1–16 O

O

O O

Na2CO3/THF

PG

N3

O–

O

PG

O

Forteo 1–16

O

O

TFA/H2O

O

PG

PG

Forteo 1–16

N3 O

O O

HO

Scheme 3.9  SAL ester destroyed by N‐terminal azido‐Ser.

3.2.3  Scope and Limitations 3.2.3.1  Effect of Side‐Chain‐Unprotected Lys Residue

Since STL initiates from the imine formation step between the aldehyde of SAL ester and the amino group of the N‐terminal Ser/Thr, the free amino group of internal Lys is likely to compete. To investigate how the internal Lys affects the Ser/Thr ligation, a competitive experiment was designed. Model peptides were prepared as follows: a C‐terminal peptide without any Lys residue (K0) and a series of C‐terminal peptides with a Lys residue at different positions (K2–K5). As a result, peptides K3–K5 reacted similarly to peptide K0, indicating that internal Lys would not disturb the STL pathway. Interestingly, peptide K2 showed faster reaction and higher conversion (Scheme 3.11), leading to the conclusion that Lys at the second position (next to N‐terminal Ser/Thr) may facilitate STL [24]. This effect was also observed in the ligation between oCRH (Ser1‐Met21)‐SAL ester and oCRH (Thr22‐Ala40): the ligation was more efficient (faster reaction and less hydrolysis) when Lys23 (at the second position next to N‐terminal Thr) was side chain free instead of azido protection. To account for this observation, it is likely that the Lys adjacent to the N‐terminal Ser was engaged in the imine formation with the peptide SAL ester, followed by

137

138

3  Development of Serine/Threonine Ligation and Its Applications HN–R

HN–R

Reduction

O

O S

O

Oxidation

Msz Stable to acid and base

O O

S Mtz Acid labile

Met

S S HN

O

FmocHN

O

HN

1. Fmoc-SPPS

NH

H2N

2. Activation

O

SARKLLQDI

O

O S

O

O

O

N

N H

N H

Msz-Ser

Rink amide resin

S

H2N

O

S O

SARKLLQDI O

N N H

TFA/H2O

O

HN O

NH2

SARKLLQDI

O

S

O

O

Met(O)

O N N H

O N H

Mtz-Ser

Scheme 3.10  Proposed mechanism of oxygen exchange between Met and Msz.

proximity‐induced imine exchange to the N‐terminal Ser residue via an 11‐membered transition state. On the other hand, how the Lys residue on the N‐terminal peptide affects the ligation has yet to be studied. 3.2.3.2  Effect of the C‐Terminal Amino Acid at Ligation Site

Of the 20 amino acids at the ligation site (X‐Cys) of NCL X‐Cys, the steric property of the amino acid has significantly affected the efficiency of NCL. For instance, the sterically hindered β‐branched amino acids, including Thr, Val, and Ile, resulted in incomplete ligation after 48 h [38]. The ligation with the C‐terminal Pro was also very slow, which is proposed to result from the reduced electrophilicity imposed by the n→π* orbital interaction [39]. Analogously, we aimed to systematically evaluate the compatibility and reactivity of all 20 amino acids at the C‐terminus of the peptide SAL ester. The ligation was carried out between NH2‐SPKALTFG‐CO2H and NH2‐ AEGSQAKFGX‐SAL ester (X represents the 20 amino acids) at a concentration of 1 mM. Within a period of 2 h, Ala, Gly, Ser, Gln, Thr, Phe, and Cys(StBu) appeared to have the fastest conversions (>60%); Val, Leu, Ile, Asn, Phe, Met, and Tyr achieved >30% conversions; the conversion of Arg, Trp, Pro, and His ranging from 7.9% to 28.6% showed to be the slowest. In particular, the ligation

O O

HO O O

HO O

+

O

H2N

HN

HN

H2N O

O N

CHO H2N

O

O

O O

HO N

HN

H2N

Scheme 3.11  Proposed mechanism to explain the accelerated rate of K2 peptide.

140

3  Development of Serine/Threonine Ligation and Its Applications

with C‐Pro and Arg could not be completed after 15 h. Interestingly, the β‐ branched Thr undergoes similar conversion of ligation to the least hindered Gly. In addition, we found that the C‐terminal Asp, Glu, and Lys were not compatible with the current STL condition, due to the instability of the peptide SAL ester with nucleophilic side‐chain functionalities.

3.3 ­Application of STL in Protein Synthesis 3.3.1  Consecutive STL of Peptides/Proteins

A list of marketed peptide drugs was taken as model study, including Teriparatide (Forteo) [23], Corticorelin (oCRH) [23], and Tesamorelin (hGHRH) [36]. Of particular significance, the efficiency of STL was demonstrated in the assembly of a more complex target of biological interest: human erythrocyte acylphosphatase (~11 kDa) [23]. Also, STL is compatible with glycopeptide assembly [30]. 3.3.1.1  Teriparatide (Forteo)

Teriparatide, under the trade name of Forteo, is the human‐made therapeutic form of parathyroid, which increases bone density and bone strength to help prevent fractures. The commercial therapeutic was prepared by Eli Lilly through recombinant sources, where it was well furnished via NCL followed by metal‐free desulfurization [40]. In that strategy, the full‐length hPTH via thiol modified amino acid residues Leu24, Ala39, and Val60 (Scheme 3.12); as an expedient demonstration of STL, the fragment (1–34) was elected as candidate and disconnected at the Asn16‐Ser17 junction. While the fragment (Ser17‐Phe34) was directly released from resin after Fmoc‐SPPS, the synthesis of the peptide (1–16) SAL ester was not straightforward, since the N‐terminal Ser had to be protected. Initial plan with azido H2N

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF

COOH

Forteo (hPTH 1-34)

O PGNH SVSEIQLMHNLGKHLN

HO +

O CHO

H2N

Scheme 3.12  Retrosynthetic plan toward hPTH(1–34).

MERVEWLRKKLQDVHNF

COOH

3.3 ­Application of STL in Protein Synthesi

protection of N‐terminal serine was not successful, as the partial racemization at the azido‐protected serine residue was observed during on‐resin phenolysis; alternative protection with Msz proved to be effective. Gratifyingly, Asn16 at the ligation site did not disturb the STL, contrary to the propensity of the substrates to undergo cyclization to form unproductive C‐terminal succinimides [41]. In fact, the ligation was relatively robust: in pyridine/AcOH (1:1) buffer, even the two parts of ligation fragment were in 1:1 ratio at a concentration of 50 mM, and the reaction completed within 2 h; if the fragment with N‐terminal Ser was in twofold excess, the reaction completed within 0.5 h (concentration of SAL ester at 20 mM). 3.3.1.2 hGH‐RH

The successful synthesis of hGH‐RH via STL largely lies in the well‐managed application of Msz group for N‐terminal Ser protection of the internal fragment (Scheme 3.13). Whereas azido group led to a significant level of epimerization, Fmoc group was partly lost during activation of the Dbz linker; Msz group survived all along the SPPS and on‐resin phenolysis followed by acidolysis and accompanied by Met(O) instead of Met. A careful examination S

HN

O

O

SARKLLQDI

O

O

CHO

H2N

O O

S

HO +

O RQQGESNQERGARARL

hGH-RH (18-27)

NH2

hGH-RH (19–44)

1. Pyridine/AcOH(mol:mol, 1:1), 10 mM, 13h 2. TFA/H2O, 10 min 3. TMSOTf/thioanisole/TFA, 20 min 33% HPLC purification

O

HO

ARKLLQDIMSRQQGESNQERGARARL

H2 N

NH2

1. Pyridine:AcOH(mol:mol, 1:1), 10 mM, 24h AcNH

O

YADAIFTNSYRKVLGQ O

CHO

2. TFA/H2O, 10 min 25% HPLC purification

hGH-RH (1–17) AcNH-YADAIFTNSYRKVLGQLSARKLLQDIMSRQQGESNQERGARARL-NH2

Scheme 3.13  Synthesis of hGH‐RH via STL.

141

142

3  Development of Serine/Threonine Ligation and Its Applications

finally led to clean deprotection of Msz, rendering the fragment for the next ligation. Another concern is N‐terminal acetylation of hGH‐RH, which is clinically applied for the treatment of lipodystrophy in patients with HIV. However, to add an acetyl group on the N‐terminal amine in the presence of the free aromatic amine in the Dbz linker, DAAQ developed by Sutcliffe’s group [42] and applied by Dawson’s group [43] readily dismissed the concern. 3.3.1.3  Human Erythrocyte Acylphosphatase (ACYP1)

Synthesis of the human erythrocyte acylphosphatase (Scheme 3.14) serves as the first illustration of STL in protein synthesis. The human erythrocyte HO FmocHN

ACYP1 (47–68)

HO

O

H N

+

O CHO

ACYP1 (71–98)

H2N

COOH

1. Pyridine/AcOH(mol:mol, 1:1), 25 mM, 10 h 2. TFA/H2O, 10 min 31% HPLC purification Et2NH/CH2Cl2, 1.5 h quant. AcHmb AcNH

ACYP1 (1–44)

O

H N

HO O

+ CHO

H2N

ACYP1 (47–68)

H N

HO O N H

ACYP1 (71–98)

1. Pyridine/AcOH (1:10), 10 mM, 22 h 2. TFA/H2O, 10 min 34% HPLC purification AcHmb AcNH

ACYP1 (1–44)

H N

O HO N H

ACYP1 (47–68)

H N

HO O N H

ACYP1 (71–98)

COOH

1. 10% N2H4 in H2O 2. TFA/H2O/TIPS (95/2.5/2.5, v/v/v) quant.

AcNH

ACYP1 (1–44)

H N

O HO N H

ACYP1 (47–68)

H N

HO O N H

ACYP1 (71–98)

COOH

Folding Human erythrocyte acylphosphatas e

Scheme 3.14  Total synthesis of human erythrocyte acylphosphatase via STL.

COOH

3.3 ­Application of STL in Protein Synthesi

acylphosphatase is composed of 98 amino acid residues and an acetylated NH2 terminus. Because it incorporates 19 of the 20 proteinogenic amino acids found in nature except Cys, NCL cannot be directly utilized for its total synthesis. On the other hand, the well distribution of Ser/Thr residues provides opportunities for the application of Ser/Thr ligation. Taking advantage of the Gly residues, the peptide sequence was disconnected into three fragments: (1–45), (46–69), (70–98). Thus, the full length was achieved throughout serine ligation followed by threonine ligation, furnishing the native form of the acylphosphatase. After successful folding [44], the assay was carried out by incubating the enzyme at 25 °C with benzoylphosphate as the substrate in 0.1 M acetate buffer, pH 5.3; UV‐absorption analysis in the region of 260–300 nm confirmed the synthetic acylphosphatase did exhibit the reported hydrolytic activity toward benzylphosphate [45], which suggests that STL can be used to synthesize complex bioactive enzymes. 3.3.1.4  MUC1 Glycopeptides

Convergent syntheses of MUC1 glycopeptides (40‐mer and 80‐mer) via STL proved that STL is well tolerated with peptidoglycans. MUC1 glycoprotein is expressed by a variety of normal and malignant epithelial cells, cancer‐associated MUC1 being different from MUC1 on normal cells; variable level of glycosylation of MUC1 during tumor progression makes it a tumor‐specific antigen and thus a valid target for immunotherapy [46]. Of particular significance is the chemical synthesis of MUC1 fragments with different lengths of the 20‐mer MUC1 tandem repeat displaying varying degrees of glycosylation, as is ingeniously addressed [47–50]. In this context, Ser/Thr ligation has been successfully used to ligate the repeat unit to form up to an 80‐mer MUC1 fragment [30] (Scheme 3.15). 3.3.2  STL‐Mediated Peptide Cyclization 3.3.2.1  STL in Head‐to‐Tail Tetrapeptide Cyclization

Head‐to‐tail cyclic peptides represent an attractive class of drug‐like molecules in the therapeutic industry, exhibiting remarkable biological activities with extraordinary potency and oral bioavailability owing to their compacted structures and high stability against enzymatic degradation and physical denaturation. However, efficient synthetic approach toward such molecules is far from mature, considering the great demand of the development their medicinal chemistry. The standard dehydration‐reagent‐mediated reactions of linear peptide precursor failed to meet this requirement, mainly due to slow cyclization in comparison to intermolecular peptide coupling reaction, thus accompanied with competing dimerization and oligomerization. Epimerization at non‐Gly/Pro is also an often‐met problem. Cyclic tetrapeptide bears the top synthetic

143

144

3  Development of Serine/Threonine Ligation and Its Applications Glycan

Glycan

HO FmocHN

O

H N

MUC1 tandem repeat

1. Pyridine/AcOH (1:1, mol:mol), 4h 2. TFA/H2O, 10 min 3. Et2NH/CH2Cl2 (1:2, v:v) Iterate twice

O CHO

O = Glycan OAcOAc O AcO AcHN O Glycan

HO H2N

O

Glycan

MUC1 tandem repeat

Glycan

HO

N H

Glycan

Fmoc H N

N H

Glycan H N

O

H N

Glycan

H2N

O

O

H N

O

OH

OH

O

1. Pyridine/AcOH(1:1,mol:mol), 4h

O

2. TFA/H2O, 10 min

Glycan

MUC1 tandem repeat

H N

O

O

Glycan

HO N H

Glycan H N

O

Glycan

HO N H

MUC1 tandem repeat

H N

O

O HO N H

OH

O

Glycan

Glycan

Glycan

MUC1 tandem repeat

5% Hydrazine (for Ac- and Fmoc- removal)

MUC1 tandem repeat

H N

Glycan

MUC1 tandem repeat

2

Glycan

O

O

Glycan

HO N H

O

HO

H N

O

HO O

O

CHO

MUC1 tandem repeat

H N

MUC1 tandem repeat

O

HO

Glycan

H2N

Glycan

MUC1 tandem repeat

Glycan

MUC1 tandem repeat

FmocHN

Glycan

HO O

H N

Glycan

HO

Glycan = OH OH O HO AcHN O Glycan

MUC1 tandem repeat

H N

O OH

O

3

Scheme 3.15  Synthesis of native 80‐mer MUC1 section.

difficulty attributable to the rigid 12‐membered ring backbone with constrained geometry. A general strategy of the synthesis of such molecules relies on either external conformational assistance [51, 52] or the introduction of internal turn‐inducing residues, such as glycine, proline [53, 54], d‐amino acids, tertiaryamide, or pseudoproline [55]. STL‐mediated peptide cyclization has been successfully used for head‐to‐tail tetrapeptide cyclization with high reaction efficiency, chemoselectivity, and product integrity. To name a few examples, cyclo‐(TLLA), cyclo‐(TVVA), and cyclo‐(SYIA) were obtained with high monomer‐to‐dimer ratio (9:1, 9:1, 99:1, respectively). Taking cyclo‐(SAAA) as an example (Scheme 3.16), molecular modeling study has revealed the underlying mechanism [25]. Note that the direct amide bond formation in the cyclization of both unsubstituted tetrapeptide SAAA (Scheme 3.16c) and its SAL equivalent (Scheme 3.16d) gives substantially higher values. This well explains the previous observation that the head‐to‐tail tetrapeptide cyclization is difficult to realize using

3.3 ­Application of STL in Protein Synthesi

O CHO OH

NH2

(a) ∆G = 7.0

O

O

NH O

(b) ∆G = 6.7

O

O OH

NH2

(c) ∆G = 14.3

OH

OH

NH2

O OHC

O

(d) ∆G = 14.8

Scheme 3.16  Estimated activation energies (kcal mol−1) for cyclization of (a) SAL ester tetrapeptide SAAA forming a Schiff base; (b) N,O‐benzylidene structure; (c) direct lactamization of tetrapeptide SAAA; and (d) direct aminolysis of the tetrapeptide SAAA‐ SAL ester.

a conventional approach and indicates that the high energy barrier in the cyclization can be bypassed by an imine‐induced ring‐closing/ring‐contraction strategy using tetrapeptide SAL esters. 3.3.2.2  STL in Head‐to‐Tail Cyclization of Peptides of Various Sizes

Apart from the synthesis of cyclic tetrapeptides, STL‐mediated peptide cyclization strategy has been well executed in the synthesis of different types of cyclic peptides with various ring sizes. For instance, in the synthesis of kynurenine‐containing peptides cyclomontanin B (Scheme 3.17), Kyn was generated from Trp via on‐resin ozonolysis, which concomitantly released SAL ester from the resin [56]. Other cyclic peptides with small‐to‐medium sizes achieved by intramolecular STL include yunnanin C and its analogs [57], anticancer stylopeptide 1, phakellistatin 4, and anti‐inflammatory cyclosquamosin D. Additionally, Liu et al. have synthesized a Thr‐containing cyclic heptapeptide with antimalarial activity, mahafacyclin B [26]. 3.3.2.3  Total Synthesis of Daptomycin via Serine‐Ligation‐Mediated Peptide Cyclization

Daptomycin, under the trade name Cubicin, represents a new generation of antibiotics with a novel mode of action and unique potency against a series of otherwise antibiotic‐resistant Gram‐positive pathogens, including methicillin‐resistant Staphylococcus aureus (MRSA), vancomycin‐resistant enterococci (VRE), and vancomycin‐resistant S. aureus [58]. For the development of next‐generation daptomycin‐based antibiotics, preparation of a library of various daptomycin analogs will be necessary to establish its structure–function relationship [58]. To this end, a limited number of daptomycin analogs have been obtained via chemoenzymatic combinatorial biosynthesis [59, 60]. An alternative and effective approach would be chemical synthesis.

145

OH

O

O

Boc-SPPS

N H

BocHN OtBu

O

H N

N O

O

H N

N H

N H

O O

O

H N

O

O

O

N H

NHXan N OHC

O

O N H NH N

O OO

H N O

N H OH

HN

1. TMSOTf/TFA/thioanisole

O O

2. O3, –78 C, CH2Cl2/TFA (95/5), next Me2S

NH2 1. Pyridine/AcOH(1:2) 2. TFA/H2O 3. TrisHCl, pH = 8, 4 h

O H2N

O H2N

H N

N

OH

Cyclomontanin B

Scheme 3.17  Synthetic scheme of cyclomontanin B.

O

O N H

H N O O NH2

O N H

H N O O H2N

O O O

3.3 ­Application of STL in Protein Synthesi

Daptomycin molecule contains a 31‐membered ring made up of 10 amino acids and a linear 3‐amino‐acid side chain modified with an n‐decanoyl lipid at the N‐terminus (Scheme 3.18). Within the sequence, there are two nonproteinogenic amino acids, kynurenine (Kyn) and 3‐methyl glutamic acid (3‐ mGlu), along with d‐Asn, d‐Ser, and d‐Ala. The esterification of Kyn to the Thr residue was found to be very difficult and has been realized via ozonolysis of Trp after the esterification of Trp to the side chain of Thr. An additional challenge was the synthesis of 3‐mGlu [61]. The total synthesis, thereby, was achieved via peptide synthesis in solid phase in combination with solution phase; the critical peptide macrocyclization was achieved by Ser‐ligation‐ mediated peptide cyclization. Based on this strategy, around 100 daptomycin has been synthesized for further screening (to be reported). H2N O NH CONH2

O C9H19

NH O

N H

H N O

O

O N H

CO2H

NH

O

N H

O N H O

Kyn

NH2

O

H N

O HO2C HO2C O

HO H N O

N H HN

O O NH

N H

O

CO2H mGlu

Later on, with the Kyn and 3‐mGlu accessible and the orthogonal protecting group [57], an on‐resin total synthesis strategy was also reported to prepare daptomycin and several analogs [62]. Thus, preparation of daptomycin is not a formidable task any more. However, to date, the mode of action of daptomycin remains far from clear, albeit a number of proposed mechanisms give putative explanation such as formation of oligomeric pores in bacterial membranes [63, 64] or others [58]. In this sense, STL‐based total synthesis offers an attractive alternative to the generation of such analogs in a readily accessible manner. 3.3.3  Thiol SAL Ester‐Mediated Aminolysis in Peptide Cyclization

Given the success of STL in peptide linear assembly and head‐to‐tail cyclization, its thiol SAL ester counterpart, interestingly, did not proceed through the same pathway. Initially, Fmoc‐Ala thiol SAL ester indeed reacted with Ser‐Phe dipeptide in pyridine/AcOH (1:1, mol:mol) buffer, followed by acidolysis to

147

1. Pyridine/AcOH(1:1, mol:mol), 4 h NH2 O

O OHC

Kyn

O

2. TFA/H2O, 10 min

NH2 O

H N

Kyn H2N COOH

OH D-Ser

3-mGlu

Scheme 3.18  Ring closure of daptomycin was achieved by STL.

HO COOH 3-mGlu

NH O D-Ser

3.3 ­Application of STL in Protein Synthesi

yield Fmoc‐Ala‐Ser‐Phe‐OMe tripeptide. However, when a model heptapeptide (Ac‐SPKMVQG thiol SAL ester) was reacted with a hexapeptide (NH2‐ SFAVGA‐CO2H) with N‐terminal Ser under the same conditions mentioned earlier, surprisingly, no ligated product was observed, whereas the starting material remained intact. Noticing the precedent examples of peptide aminolysis using hemiaminal‐mediated acyl transfer by Kemp and Ito [65, 66] (Scheme 3.19), which was limited to simple substrate (1–2 amino acid, for instance), the utility of peptide thiol SAL esters for peptide condensation was thus investigated. The optimal condition for peptide thiol SAL ester‐mediated peptide aminolysis involves using 1–3 equiv. of DIEA as a base in DMSO. However, significant epimerization at the reacting C‐terminal amino acid was detected, similar to other aminolysis‐mediated peptide condensation [67], thus limiting the condensation at Gly or Pro site; Gly generally gave faster reaction compared to Pro. Scope studies on the reacting N‐terminal amino acids showed that Phe, Gly, Leu, Ala, Val, Pro, Ser, Asp, Glu were compatible. A comparison study of the aminolysis rate between a peptide thiol SAL ester and peptide thiophenyl ester [68] was carried out; the slower reaction of the latter confirmed that the hemiaminal‐formation‐mediated reaction helped expedite the peptide aminolysis. This strategy was then applied to peptide cyclization and a series of natural cyclic peptides with variable ring sizes (7–11 amino acid residues) (Table 3.1), and various activities were synthesized. The reaction was rapid and clean: all the substrates were consumed within 4–8 h (see the following discussion), giving monocyclic product as the major or even sole product [69]. 3.3.4  A Fluorogenic Probe for Recognizing 5‐OH‐Lys Inspired by STL

It is noted that lysine 5‐hydroxylation emerged as a new posttranslational modification. The hydroxylation of the lysine residue, as an important genre of posttranslational modifications, plays a crucial role in stabilizing intra‐ and intermolecular cross‐links of collagen proteins; for instance, Jmjd6 has been shown to catalyze lysyl‐5‐hydroxylation of many proteins associated with RNA metabolism, processing, and splicing [70]. The key chemoselectivity of Ser/Thr ligation is realized by taking advantage of the 1,2‐hydroxyl amine bifunctionality of N‐terminal Ser or Thr. Since 5‐hydroxylysine possesses 1,2‐hydroxyl amino group, it is conceived that Ser/Thr ligation can be used to detect or profile 5‐hydroxylysine‐containing proteins. To the end, a model study with the probe (ortho‐aldehyde 4‐methylumbelliferone trimethyl acetate) gave positive results. After the electron‐withdrawing aldehyde group was masked as N,O‐benzylidene acetal via STL pathway, facile O‐to‐N acyl transfer liberated the aromatic hydroxyl group, leading to fluorescence regeneration via a fluorescence turn‐on mechanism. The

149

Kemp and Vellaccio [65] O

O O

CHO

H2NBn

O

Bn HN O

Bn N OH OH

OH

OH CHO

O N H

Bn +

Ito and coworkers [66]

S

FmocHN O

+ NO2

OtBu

H2N

DIEA N–Methylmaleimide DMF-H2O

O

H N

FmocHN

OtBu

O

O

O O

O

S

Me N O

+

1,4-Addition

NO2 O

S

Me N O

NO2 OH

Scheme 3.19  Previous examples using a hemiaminal‐mediated acyl transfer strategy [65,66].

3.3 ­Application of STL in Protein Synthesi

Table 3.1  Synthesis of natural cyclic peptides via peptide thiol SAL ester-mediated cyclization. Entry

Name

Sequence

Ring size M: D

Yielda (%)

1

Phakellistatin‐13

cyclo‐[FGPTLWP]

7

8:1

41

2

Cyclosquamosin D

cyclo‐[GVVSYYPG]

8

99:1

49

3

Dichotomin G

cyclo‐[LPSTFPPIP]

9

9:1

50

4

Antamanide

cyclo‐[AFFPPFFVPP]

10

99:1

48

5

Stelladelin D

cyclo‐[VPSPYFPAAIG]

11

99:1

45

M, monocyclic product; D, dimerized product. a)  Isolated by HPLC.

fluorescence intensity increased over time and reached the maximum at 2 h, with the maximum λem value being observed at 425 nm. Furthermore, it was proved effective on peptide level. About 1.2‐fold excess of the probe incubated with the peptide at 5 mM concentration enabled complete labeling within 2 h, as verified by LC–MS as well as fluorescent analysis. To make it more convincing, the reaction mixture was subjected to TFA treatment; acidolytic cleavage removed the N,O‐benzylidene acetal and released the product with trimethyl acetate, further confirming the reaction in STL pathway. The detection retains high sensitivity, even in the presence of hydroxylysine‐containing peptide as little as