Electron paramagnetic resonance. Volume 26 978-1-78801-388-8, 1788013883, 978-1-78801-372-7, 978-1-78801-688-9

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Published on 02 November 2018 on https://pubs.rsc.org | doi:10.1039/9781788013888-FP001

Electron Paramagnetic Resonance Volume 26

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A Specialist Periodical Report

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Electron Paramagnetic Resonance Volume 26 A Review of the Recent Literature. Editors Victor Chechik, University of York, UK Damien M. Murphy, Cardiff University, Cardiff, UK Authors Antonio Barbon, University of Padova, Italy Vale ´rie Belle, Aix-Marseille Universite ´, France Julia Cattani, University of Konstanz, Germany Daniel J. Cheney, University of Huddersfield, UK Malte Drescher, University of Konstanz, Germany Andrea Folli, Cardiff University, UK Euge ´ nie Fournier, Aix-Marseille Universite ´, France Nolwenn Le Bretton, University of Strasbourg, France Marle ` ne Martinho, Aix-Marseille Universite ´, France Elisabetta Mileo, Aix-Marseille Universite , ´ France Damien M. Murphy, Cardiff University, UK Emma Richards, Cardiff University, UK Jacob Spencer, Cardiff University, UK Christopher J. Wedge, University of Huddersfield, UK Sabrina Weickert, University of Konstanz, Germany

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ISBN: 978-1-78801-372-7 PDF eISBN: 978-1-78801-388-8 EPUB eISBN: 978-1-78801-688-9 DOI: 10.1039/9781788013888 ISSN: 1464-4622 A catalogue record for this book is available from the British Library r The Royal Society of Chemistry 2019 All rights reserved Apart from fair dealing for the purposes of research for non-commercial purposes or for private study, criticism or review, as permitted under the Copyright, Designs and Patents Act 1988 and the Copyright and Related Rights Regulations 2003, this publication may not be reproduced, stored or transmitted, in any form or by any means, without the prior permission in writing of The Royal Society of Chemistry or the copyright owner, or in the case of reproduction in accordance with the terms of licences issued by the Copyright Licensing Agency in the UK, or in accordance with the terms of the licences issued by the appropriate Reproduction Rights Organization outside the UK. Enquiries concerning reproduction outside the terms stated here should be sent to The Royal Society of Chemistry at the address printed on this page. Whilst this material has been produced with all due care, The Royal Society of Chemistry cannot be held responsible or liable for its accuracy and completeness, nor for any consequences arising from any errors or the use of the information contained in this publication. The publication of advertisements does not constitute any endorsement by The Royal Society of Chemistry or Authors of any products advertised. The views and opinions advanced by contributors do not necessarily reflect those of The Royal Society of Chemistry which shall not be liable for any resulting loss or damage arising as a result of reliance upon this material. The Royal Society of Chemistry is a charity, registered in England and Wales, Number 207890, and a company incorporated in England by Royal Charter (Registered No. RC000524), registered office: Burlington House, Piccadilly, London W1J 0BA, UK, Telephone: þ44 (0) 20 7437 8656. Visit our website at www.rsc.org/books Printed in the United Kingdom by CPI Group (UK) Ltd, Croydon, CR0 4YY, UK

Preface

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DOI: 10.1039/9781788013888-FP005

The applications of Electron Paramagnetic Resonance (EPR) spectroscopy have continued to grow unabated in recent years. The diversity of research fields in which EPR is now finding new use is truly remarkable for a spectroscopic technique which was once considered highly specialised and the exclusive domain of the trained practitioners. With more readily available and cheaper instruments, the traditional continuous wave (CW) technique remains the staple method of choice used by the wider scientific community, with the standard X-band measurements continuing to dominate the field. However, although the use of Pulsed and High Field EPR methods are, by comparison, less widely exploited, the level and sophistication of information provided by these methods on the paramagnetic state is exceptional, unprecedented and unparalleled. This information includes not just the simple detection and quantification of a free radical or paramagnetic ion, but also the detailed insights into the structure, conformation and dynamics of the spin system on length/time scales not easily accessible by other techniques. Furthermore, the application of the EPR method can be extended by use of ancillary spin probes, spin labels or spin traps that provide additional structural and dynamic information on the diamagnetic system or the identity of any transient free radicals. For all these reasons, EPR has and will continue to be the most powerful technique to inform, study and interrogate paramagnetic species in any system. In this volume, we have therefore collected an exciting series of Chapters that we hope will best illustrate and exemplify some of the current application areas of EPR. In Chapter 1, Drescher focusses on intrinsically disordered proteins, demonstrating the unique level of detail and insight that EPR and in-cell EPR can provide on disordered structures. In Chapter 2, Barbon then focusses our attention on the vitally important field of advanced materials, by examining how 2D graphenebased nanomaterials and nanographites can be studied using EPR techniques. Nitroxide spin labels have without doubt become the quintessential paramagnetic probe molecule of choice in EPR spectroscopy over many decades, so Belle illustrates in Chapter 3 how biostructural systems can be studied in this way. In addition to the many application areas of EPR, numerous technical developments are also ongoing in the field. One such example, is the application of light induced hyperpolarization in EPR (and NMR), which has the potential to revolutionize the sensitivity limit and information content of these techniques, and this is covered by Wedge in Chapter 4. Finally, one research field that continues to benefit from EPR is catalysis, since the entire bond-making or bond-breaking catalytic processes frequently involved free radicals, and this area is covered by Murphy in Chapter 5. As always, we hope that both the expert EPR reader and novice practitioner will value these timely reviews, offering a broad perspective on Electron Paramag. Reson., 2019, 26, v–vi | v

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the latest developments in the field. Finally, we would also like to thank all of our reporters for their expert, prompt and efficient cooperation in the production of these Chapters and the staff at the Royal Society of Chemistry for their editorial support and patience.

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Victor Chechik (York) and Damien M. Murphy (Cardiff)

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Author biographies

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Antonio Barbon is staff member at the Department of Chemical Sciences of the University of Padova (IT). He graduated in Chemistry in 1992 and received his PhD in Chemistry at the University of Padova in 1996 working on Twisted Intramolecular Charge Transfer states, under the supervision of Prof. Pierluigi Nordio and Prof. Giorgio Gennari. He spent his post-doc at the Debye Institute at the University of Utrecht (NL) in the group of Dr Ernst van Faassen, working on the characterization by EPR of V(IV)-based catalysts and on defects in polycrystalline Si. In 1998 he returned to Padova, where he continued to work in the field of EPR spectroscopy in the group of Prof. M. Brustolon. There, he was appointed as Researcher in 2002. His research interests are mainly within the characterization of materials by EPR and the study of processes activated by light. He studied the defects of graphene-like materials, both from a structural point of view, but also for the stimulation of toxic effects. He studied the tuning of ISC processes in dyes and the photo-switched electron transfer processes through proteins and membranes. He has been member of the board of the Italian Group of Electron Spin Resonance (GIRSE).

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´rie Belle obtained her PhD in 1995 Vale at the University of Grenoble followed by a postdoctoral stay at the University of ¨rzburg (Germany), working on methodoWu logical aspects of functional and perfusion Magnetic Resonance Imaging. In 1998, she obtained an assistant professor position in Aix-Marseille University and joined the laboratory of Bioenergetics and Protein Engineering. She is now professor at AixMarseille University and teaches general physics to BSc and MSc students. In the past few years, she has focused her research activities on the study of protein dynamics using Site Directed Spin Labeling combined with Electron Paramagnetic Resonance (SDSL-EPR) spectroscopy. She is interested in characterizing structural transitions within proteins mostly arising from protein–protein interaction, in particular in the family of intrinsically disordered proteins. She is also involved in the development of new paramagnetic spin labels aiming at enlarging the potentialities of SDSL-EPR.

Dr Julia Cattani was born in 1984 in Freising. She obtained her Master of Science in Chemistry in 2012 from the University of Regensburg. She joined the research group of Malte Drescher at the University of Konstanz in 2013. In 2017, she completed her PhD on the spectroscopic investigation of the intrinsically disordered protein alpha-synuclein in vitro and in the cell. In 2018, she started working in the industry as a marketing and sales manager for a company in the diagnostic market.

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Daniel Cheney achieved his MChem degree in Chemistry at the University of Warwick (2017). With a strong interest in physical chemistry, he carried out a summer research ´zef Lewandowski on the project with Dr Jo application of NMR relaxation to assessing chromatographic stationary phases, and spent his final year working with Dr Scott Habershon on the application of automated reaction path discovery software to the growth of carbon nanotubes. He is currently undertaking his PhD at the University of Huddersfield with Dr C. J. Wedge, on the development of optically-induced microwave-free Overhauser DNP.

Prof. Dr Malte Drescher received his diploma in Physics in 2001 and his PhD degree in solid-state Physics in 2005 from the University of Karlsruhe, Germany. After a postdoctoral stay with Prof. Edgar Groenen, University of Leiden, The Netherlands, funded by a DFG research fellowship, he started his own independent research group at the University of Konstanz in 2008 as a DFG Emmy-Noether fellow. In 2014 he became a DFG Heisenberg fellow and was appointed as full professor at the University of Konstanz, Germany. Drescher and his group develop and apply electron paramagnetic resonance (EPR) spectroscopy to investigate structure and dynamics of macromolecules. His main research interests include in-cell EPR spectroscopy and EPR spectroscopy on intrinsically disordered proteins. He is principal investigator within the Konstanz Research School Chemical Biology, the collaborative research centres 1214 (Anisotropic Particles as Building Blocks: Tailoring Shape, Interactions and Structures) and 969 (Chemical and Biological Principals of Cellular Proteostasis). Drescher has been awarded an ERC consolidator grant in 2017.

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Andrea Folli is a Research Associate within the EPR group at Cardiff University. Following his Italian Laurea Degree in physical chemistry from the University of Milan (2004), he moved to industry as a junior scientist for Sasol Italy. He returned to academia in 2007 to undertake his postgraduate studies on a Marie Curie Fellowship at the University of Aberdeen (2007–2010). Prior to his current position in Cardiff University, he was the principal investigator on a European FP7 funded project investigating visible light active concretes for air pollution treatment at the Danish Technological Institute (2010– 2015).

´nie Fournier received her BSc degree Euge from the Sciences and Technology Faculty at the University of Lorraine (Nancy, France) in 2013. From there, she moved to Strasbourg, where she obtained in 2015 her MSc degree in analytical chemistry (University of Strasbourg). Since 2015, she has been a PhD student at Aix-Marseille University studying the structural and dynamic behavior of a metalloenzyme, the ACC Oxidase, using Site Directed Spin Labeling combined with EPR spectroscopy. Her research is conducted between two laboratories, the laboratory of Bioenergetics and Protein Engineering (BIP, Marseille) and the Institute of Molecular Sciences of Marseille (iSm2, Marseille).

Nolwenn Le Breton received her PhD in chemistry in 2014 at Aix-Marseille University (France), where she worked on site directed spin labelling combined to EPR spectroscopy. In 2015, she started to work as a postdoctoral research fellow at Queen Mary University of London (United Kingdom) where she focused on the characterisation of a metalloprotein using EPR spectroscopy. She joined the Institut of Chemistry at the University of Strasbourg (France) as a research engineer where she is in charge of the EPR plateform.

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`ne Martinho obtained her PhD in Marle chemistry from the university Paris Sud in 2006. After a post-doctoral position at Carnegie Mellon university (Pittsburgh, PA, USA) in Prof. Eckard Munck’s lab, she moved to Marseille in 2009. She obtained an assistant professor position at the AixMarseille university where she teaches chemistry to BSc and MSc students. Her research work is focused on protein dynamics and protein-protein interactions using site-directed spin labeling and EPR spectroscopy. Her current interests include studying the structure/function relationship into proteins, including IDPs and metalloproteins. In particular, she is interested in using the incorporation of non natural amino acids as targets for nitroxyde probes, as well as the use of the endogenous metal center of metalloproteins for nitroxyde-metal distance measurements.

Elisabetta Mileo obtained her PhD in 2010 at the University of Bologna (Italy). She is a chemist and a spectroscopist with experience on the synthesis and use of nitroxide radicals as spin probes and spin labels in the domain of supramolecular chemistry and Site-Directed Spin Labelling coupled to EPR spectroscopy (SDSL-EPR). After a postdoctoral fellow in the laboratory of P. Tordo and S. Marque (ICR, Marseille, France), she moved to the BIP laboratory (Marseille, France) and she worked on the development of both protocols and methodology to efficiently graft new paramagnetic labels on tyrosine residues on proteins. In 2014, she obtained a position as researcher at CNRS (Marseille, France). Her research activities are focused on the study of structural flexibility and dynamics of chaperone proteins by SDSL-EPR. In particular, she is actively involved in the development of new tools to investigate proteins structural dynamics, protein-protein interaction and the associated structural changes by SDSL-EPR spectroscopy at the molecular level inside cells (in-cell EPR).

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Damien Murphy is Professor of Physical Chemistry at Cardiff University. After obtaining his Chemistry degree from the Dublin Institute of Technology in 1990, he moved to the University of Turin to undertake his PhD in EPR of surface defects on polycrystalline materials. Following successive PDRA appointments at the IST, Lis´ P. et M. Curie, bon (1994) and Universite Paris (1995), he was appointed to a lectureship in Cardiff University, School of Chemistry, where he is currently Head of School. He is a Fellow of the RSC, Fellow of the Learned Society of Wales and a Royal Society Wolfson Research Award holder. His research interests are broadly focused on the applications of advanced EPR methods for catalysis research.

Emma Richards is a University Research Fellow in EPR spectroscopy at Cardiff University (2015) and currently serves as a committee member of the ESR Group of the RSC. Her research involves utilizing pulsed ESR spectroscopy, including ESEEM & HYSCORE, to investigate condensed matter materials of importance in visible-light activated photocatalysis, focusing on electron transfer processes and the role of dopants and surface defects in promoting catalytic activity. Following her UG degree in Natural Sciences at the University of Bath (2004), she completed her PhD on EPR analysis of stable and transient oxygen radicals on polycrystalline TiO2 under the supervision of Prof. Murphy at Cardiff University (2007).

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Jacob Spencer is a current PhD student in the EPR group at Cardiff University, in a collaborative project supported by Johnson Matthey. Originally from Newbury, Berkshire, Jacob completed his MChem (Hons) degree at Cardiff University in 2016. His research interests involve the utilisation of EPR techniques to understand the nature of the catalytic active sites and defect chemistry in functional condensed materials. He is particularly interested in the (in situ and ex situ) characterisation of thin film cathodes for Li-ion battery technologies.

Dr Chris Wedge joined the University of Huddersfield in 2016 as Senior Lecturer in Physical Chemistry. He read Chemistry at Christ Church, University of Oxford, where he received his MChem (2005) and DPhil (2009), his research into radiofrequency magnetic field effects being completed under the supervision of Prof. C. R. Timmel. After postdoctoral work in Oxford with Profs A. Ardavan (2009–2011) and P. J. Hore (2011– 2013), he joined the University of Warwick as an assistant professor and taught course leader of the Integrated Magnetic Resonance Centre for Doctoral Training. His research interests include Spin Chemistry and Electron Paramagnetic Resonance, with a particular focus on optically generated electron and nuclear spin hyperpolarization.

Sabrina Weickert studied Physics at the ¨t (FAU) in Friedrich-Alexander-Universita Erlangen and the Ludwig-Maximilians¨t (LMU) in Munich, where she Universita received her diploma with a focus on Biophysics. She engaged in protein biochemistry while working in the research groups of Prof. Dr Kirsten Jung and Prof. Dr Heinrich Jung at LMU Munich. Sabrina Weickert is currently a PhD candidate in the research group of Prof. Dr Malte Drescher and in the Konstanz Research School Chemical Biology (KoRS-CB) at the University of Konstanz. Her research focuses on the investigation of protein–ligand interactions with methods of electron paramagnetic resonance (EPR) spectroscopy. Electron Paramag. Reson., 2019, 26, vii–xiii | xiii

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CONTENTS

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Cover

Preface

v

Victor Chechik and Damien M. Murphy Author biographies

vii

Intrinsically disordered proteins (IDPs) studied by EPR and in-cell EPR Sabrina Weickert, Julia Cattani and Malte Drescher 1 Introduction 2 Tau 3 a-Synuclein 4 Discussion and outlook References

1

1 9 21 27 29

EPR spectroscopy in the study of 2D graphene-based nanomaterials and nanographites

38

Antonio Barbon 1 Introduction 2 Electronic and magnetic properties of graphene and graphite 3 Adsorption of gases and of metal ions 4 Resolution and analysis of the EPR spectra

38 40 45 48

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5 Nanographites 6 Mono and few-layer graphenes 7 Graphene oxide and reduced graphene oxide 8 Conclusion Acknowledgements References

Nitroxide spin labels: fabulous spy spins for biostructural EPR applications Marle`ne Martinho, Euge´nie Fournier, Nolwenn Le Breton, Elisabetta Mileo and Vale´rie Belle 1 Introduction 2 Nitroxide spin labels used to probe protein dynamics in the liquid state 3 Nitroxide spin labels used to measure distances in biologicals systems 4 Conclusion Acknowledgements References

Applications of light-induced hyperpolarization in EPR and NMR Daniel J. Cheney and Christopher J. Wedge 1 Introduction 2 Electron spin hyperpolarization 3 Optical enhancements in NMR 4 Summary References

Applications of electron paramagnetic resonance spectroscopy for interrogating catalytic systems

50 54 59 61 62 62

66

66 67 78 83 84 84

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Jacob Spencer, Andrea Folli, Emma Richards and Damien M. Murphy 1 Introduction 2 Homogeneous catalytic systems 3 Microporous catalytic systems 4 Photocatalytic catalytic systems 5 Heterogeneous catalytic systems 6 Summary and perspectives Acknowledgements References

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Intrinsically disordered proteins (IDPs) studied by EPR and in-cell EPR Sabrina Weickert, Julia Cattani and Malte Drescher* Published on 02 November 2018 on https://pubs.rsc.org | doi:10.1039/9781788013888-00001

DOI: 10.1039/9781788013888-00001

Intrinsically disordered proteins (IDPs) play important physiological, but also diseaserelated roles. In order to understand the function and malfunction of proteins of this class, electron paramagnetic resonance (EPR) spectroscopy has proven to be a valuable tool, allowing investigation of the protein structural ensembles upon interaction with the environment. This review focuses on the IDPs tau and a-synuclein and gives an overview over recent EPR studies performed with these proteins.

1

Introduction

1.1 IDPs Intrinsically disordered proteins (IDPs) are a class of proteins lacking a stable three-dimensional structure in solution. Nonetheless, these proteins are native and fulfill many important biological functions, among them cell signaling, recognition and regulation.1 IDPs are highly prevalent in humans: Genome analyses predict that around 25% of all human proteins are disordered from end to end, while up to 40% contain unstructured regions.2 Due to their abundance as well as their unique structural and dynamical flexibility, IDPs are key players in many biological pathways, capable of specific interactions with a multitude of binding partners and therefore often binding to or serving as hubs in protein interaction networks.3,4 Due to the same reasons, many members of the IDP family are known to be associated with a variety of human diseases, among them prominently cancers, cardiovascular diseases, diabetes and neurodegenerative diseases.5–7 All of this has made IDPs a research field of tremendous importance and interest since the turn of the century. 1.1.1 The peculiar free energy landscape of IDPs. In the free energy landscape of proteins with a native globular fold there is a pronounced free energy minimum that stabilizes a distinct 3D fold, which often represents the unique functional form of a globular protein (Fig. 1A). The decrease in entropy associated with restrictions of the conformational freedom during folding is compensated by the formation of many intramolecular contacts.8,9 However, the free energy landscape of an IDP looks distinctly different (Fig. 1B): It is characterized by the lack of a global free energy minimum, but shows many local minima instead, which are separated by small energy barriers that allow quick and frequent interconversion between the accessible states. Department of Chemistry and Konstanz Research School Chemical Biology (KoRS-CB), University of Konstanz, 78457 Konstanz, Germany. E-mail: [email protected] Electron Paramag. Reson., 2019, 26, 1–37 | 1  c

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Fig. 1 The free energy landscape for (A) a globular protein and (B) an IDP. (A) In the folding of globular proteins, the increase in free energy is compensated by the formation of intramolecular contacts. Local free energy minima (1, 2) correspond to partially folded intermediates, while the native globular fold is represented by a global free energy minimum. When partially unfolded proteins interact, oligomers, amorphous aggregates or amyloid fibrils may form. (B) Intrinsically disordered proteins do not fold into a compact globular structure in aqueous solution. Upon interaction with binding partners, segments of or the entire IDP may become structured when a reduction in the free energy is accompanying the formation of such a complex (1, 2, 3). For IDPs the formation of oligomers, amorphous aggregates or amyloid fibrils is facilitated in comparison to globular proteins, since no unfolding is necessary prior to the formation of relevant intermolecular contacts. Reproduced from ref. 8 with permission from Elsevier, Copyright 2010.

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Consequently, the conformational ensemble of an IDP in solution is heterogeneous and characterized by a dynamic exchange between many accessible structures. This non-folding behavior is encoded in the amino acid composition of IDPs: These proteins contain less hydrophobic (Ile, Leu, Val) and aromatic (Trp, Tyr, Phe) amino acid residues, but a significantly larger proportion of small and hydrophilic amino acid residues (Arg, Gly, Gln, Ser, Pro, Glu, Lys) and are richer in structurebreaking amino acid residues (Pro, Gly) than typical globular proteins.7,8,10,11 Although IDPs cannot spontaneously fold into a compact globular structure, the presence of interaction partners can alter the IDP free energy landscape in a way that more pronounced energy minima appear: Upon interaction with a partner, IDPs often undergo a structural reorganization that defines a state of clearly reduced free energy, which is thermodynamically stabilized (Fig. 1B). 1.1.2 Linking folding and binding. While some IDPs undergo a folding process as a whole upon interaction with a binding partner, more commonly specific recognition motifs in the disordered protein adopt secondary or tertiary structural elements upon interaction with a binding partner.12–14 As a mechanism of these disorder-to-order transitions, two major models are discussed in the literature – the ‘conformational selection’ model and the ‘induced folding’ model.14 The first model is based on the assumption that the binding partner selects a specific conformation resembling the bound conformation from the wide ensemble of coexisting conformations the IDP adopts when free in solution (Fig. 1B). The latter model is based on the assumption that the IDP binds to its interaction partner in the fully disordered state and folds while bound to the partner, i.e., folding is induced by the partner. In reality, one or the other process may occur, or also some combination of the two models.14 The full dimension of the structural flexibility of IDPs becomes obvious in cases, where one recognition domain of an IDP can adopt various structural folds upon interaction with different binding partners. One prominent example is the C-terminal disordered region of tumor suppressor p53, which can adopt helical, b-strand or irregular structure upon interaction with different partners.14,15 In contrast to a concise disorder-to-order transition, some IDPs may also stay largely disordered and still show fast interconversion between coexisting conformations while in functional complex with a binding partner. Such heterogeneous protein complexes are referred to as ‘fuzzy complexes’.16–18 1.1.3 IDPs in diseases. Similarly to the formation of functional complexes with protein partners, a profound indentation in the free energy landscape of IDPs can be caused by formation of non-functional complexes of IDPs, like oligomers, amorphous aggregates and amyloid fibrils (Fig. 1B).8 Since IDPs are involved in many crucial cellular processes, e.g., signaling and regulation, misfolding of these proteins is often pathogenic.5–7 While some IDPs may exhibit an intrinsic propensity for forming a pathologic conformation, others are misfolding due Electron Paramag. Reson., 2019, 26, 1–37 | 3

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to external factors, e.g., due to disrupted chaperone-interaction, point mutations, interaction with other proteins, toxins and small molecules or impaired post-translational modifications.6 The largest group of misfolding diseases is characterized by the formation of stable, insoluble, highly organized filamentous protein aggregates known as amyloid fibrils, which accumulate in tissues or organs.7,19,20 Prominent among these amyloidogenic diseases are neurodegenerative diseases, where filamentous protein deposits are accumulating in the patient’s brain. Striking examples are the IDPs a-synuclein, which is found as aggregated inclusions in the Lewy bodies in the brain of Parkinson’s disease (PD) patients,21–23 and tau, which is a hallmark of Alzheimer’s disease (AD) in its aggregated form.24–26 1.2 Studying IDPs Their prevalence in humans, their importance in a multitude of biological processes as well as their significance for health and disease makes IDPs a highly relevant and important subject of research. However, their structural and dynamical flexibility as well as the versatility of their shapes and appearances described above makes research on IDPs a challenging matter. Traditionally, protein structures are elucidated using NMR spectroscopy or X-ray crystallography.27,28 However, they are not straightforwardly applicable to obtain a conclusive analysis of the heterogeneous conformational ensemble that is characteristic for IDPs comprising a variety of quickly interconverting structures. Nonetheless, various methods of NMR spectroscopy, e.g., chemical shift dispersion, paramagnetic relaxation enhancement (PRE), and residual dipolar couplings (RDC), are widely applied for the investigation of IDPs.29–31 However, NMR techniques are subjected to limitations when it comes to investigation of large protein complexes, e.g., with lipids or other binding partners. Thus, investigation of IDPs in their relevant macromolecular context using NMR has its boundaries. Many other biophysical methods have been employed to investigate IDPs, among them circular dichroism (CD) spectroscopy,32 fluorescence resonance energy transfer (FRET),33,34 and many more.13,26 Since it is challenging to obtain meaningful experimental results on IDPs, computational approaches like prediction of intrinsic disorder or MD-simulations using experimental restraints, e.g., from PRE, are frequently used in IDP research.13,35 Commonly, the macroscopic morphology of IDPs in the fibril state is judged by transmission electron microscopy (TEM). 1.2.1 Electron paramagnetic resonance (EPR) for studying IDPs. A powerful experimental method that has become increasingly important in IDP research is electron paramagnetic resonance (EPR) spectroscopy. EPR is a versatile spectroscopy technique that allows us to study the dynamical and structural changes of proteins, in particular of IDPs.36 As it can only detect unpaired electrons, EPR spectroscopy is virtually background-free. EPR spectroscopy allows measurements of proteins of any size in arbitrarily complex environments, e.g., in the presence of 4 | Electron Paramag. Reson., 2019, 26, 1–37

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binding partners like proteins, lipids or small molecules, as well as under arbitrary environmental conditions and even in the cell.37,38 1.2.1.1 Site-directed spin labelling. EPR spectroscopy is sensitive to unpaired electrons only. Thus, in order to perform EPR measurements with many proteins, a paramagnetic center has to be introduced as a spin label via site-directed spin labelling (SDSL).39 Depending on the application, a suitable spin label has to be selected from a variety of possibilities. Some examples are shown in Fig. 2. For a recent review, the interested reader is referred to, e.g., Roser et al.40 The spin labels most commonly used for EPR spectroscopy on proteins are nitroxide radicals. Among them, the methanethiosulfonate spin label (MTSL, see Fig. 2A), which is attached to a native or engineered cysteine residue in the protein via disulfide bonding, is most frequently used.41 However, in the reducing environment of a cell, both the N–O-moiety as well as the disulfide bond with the cysteine are susceptible to reduction.41 In order to ensure a stable attachment of the spin label to a cysteine, various linker-moieties have been developed, e.g., the maleimido moiety as used in 3-maleimido-proxyl (see Fig. 2B), which shows enhanced pH stability and is therefore often used for investigation of biological systems.40,41 For cysteine-specific attachment of a spin label, all native cysteine residues in the protein, which are not to be labelled, need to be replaced by non-reactive amino acids (often Ala, Ser) prior to labelling. A different and much more elegant way of introducing a spin label into a protein for in-cell EPR is via genetically encoded spin labelling, where the spin label is introduced as an unnatural amino acid with bio-orthogonal reactivity during the biosynthesis of the protein in vivo, making the technique especially suited for in-cell EPR.42,43 The corresponding unnatural amino acid SLK-1 is shown in Fig. 2C. When using this spin labelling approach, native cysteines present in the protein need not be substituted, implicating a more undisturbed protein than with cysteine-specific spin labelling. This also applies for a proposed spin labelling strategy based on click chemistry targeting unnatural amino acids.44 Approaches for stabilizing the nitroxide radical itself against reduction include the sterical shielding of the unpaired electron.45 A promising approach is the use of pyrrolidine-based nitroxides with ethyl

Fig. 2 Typically used spin labels. (A) (1-oxyl-2,2,5,5-tetramethylpyrroline-3-methyl)methanethiosulfonate (MTSL), (B) 3-Maleimido-2,2,5,5-tetramethyl-1-pyrrolidinyloxy (3-maleimido-proxyl) (C) SLK-1, (D) Gd-4-vinyl-PyMTA. Electron Paramag. Reson., 2019, 26, 1–37 | 5

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groups slowing down the reduction process. As, in general, the stability of nitroxides against reduction is limited, other spin labels have been developed in recent years that pave the way towards in-cell EPR: Recently, a family of Gd31-based spin labels has been introduced, which show superior stability in the cellular environment and exhibit a high sensitivity for EPR spectroscopy.47 Successful in cellula EPR distance measurements at Q- and W-band frequencies (34.5 and 95 GHz, respectively) have been reported with Gd31-chelates like Gd31-PyMTA48 (see Fig. 2D) or Gd31-DOTA-M.49 Moreover, trityl radicals have been shown to be promising spin labels for in-cell EPR due to their favorable reduction characteristics.50 A different approach to spin labelling is the coordination of Cu21-ions by two strategically placed histidine residues. EPR distances determined between two Cu21-ions have been shown to be very narrow and readily relatable to the protein backbone structure.51,52 In the investigation of spin-labelled protein oligomers, e.g., amyloid fibrils, several spin labels from various protein monomers might come into close contact. If contributions from inter-molecular spin–spin interactions are unwanted in the experiment, spin-labelled protein needs to be diluted with diamagnetic protein (Fig. 3). 1.2.1.2 Continuous wave (cw) EPR. Different methods of EPR spectroscopy allow accessing various types of information about the protein under investigation. There are two main experimental EPR techniques: On the one hand, in continuous-wave (cw) EPR spectroscopy the sample is subjected to continuous irradiation with microwave energy. Several excellent reviews describe the applications and developments in cw EPR for the investigation of proteins.39,54–57 Typical cw EPR experiments are performed at X-band microwave frequencies (9 GHz) and deliver information about the following sample parameters: (i) the mobility of the spin label side chain, (ii) the polarity of the spin label microenvironment, (iii) the solvent accessibility of the spin label side chain and (iv) distances to other paramagnetic centers. In the corresponding cw EPR experiments a site scan, i.e., performing the EPR experiment with several samples, where the spin label is attached to

Fig. 3 IDPs (left) can aggregate into ordered b-sheet fibrils (right). Spin dilution of spinlabelled IDP with non-spin-labelled IDP helps to prevent unwanted inter-molecular spin– spin interactions. Reproduced from ref. 53 with permission from Elsevier, Copyright 2017. 6 | Electron Paramag. Reson., 2019, 26, 1–37

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different positions of interest in the protein, helps to create a comprehensive picture of a protein region. For the determination of the spin label mobility (i), the cw EPR experiment is performed at ambient temperature, or particularly at a physiologically relevant temperature. Most commonly, information about the reorientational dynamics of a spin label side chain is extracted from the EPR spectra via spectral shape analysis using full spectral simulations including the determination of the rotational correlation time tcorr using, e.g., the EasySpin software package.58 Cw EPR line shapes in X-band typically resolve spin label dynamics on a 100 ps to 100 ns timescale, which reflect side chain motions, backbone dynamics as well as tertiary contacts.39,59 Changes in the dynamics of a spin label side chain, e.g., due to interaction with a binding partner or conformational transitions of the protein can be detected with cw EPR spectroscopy: Even if the rate of conformational exchange is beyond the EPR timescale, the environment of the spin label is usually subjected to distinct changes, which allow the detection of distinct components in the EPR spectrum reflecting the different conformational states of the protein.39 Often, such EPR spectra contain not only one but several contributions from a superposition of various conformational states of the protein conformational ensemble. Multi-component spectral simulations allow to monitor and to quantify fractions of multiple structural states that coexist or exchange with each other.60 The polarity of the spin label microenvironment (ii) is reflected in the hyperfine-tensor component Azz as well as the g-tensor component gxx. While a polar environment shifts Azz to higher values, gxx is decreased. Azz can easily be obtained from X-band cw EPR spectra recorded in frozen solution samples.61 In room-temperature spectra effects of microenvironmental polarity are reflected in the isotropic hyperfine splitting aiso. The micro-environmental polarity can deliver information about the protein fold, secondary structure elements or embedding of the protein in a lipid layer.54 The accessibility (iii) of the spin label side chain to paramagnetic quenchers can be monitored using cw EPR power saturation curves. While metal ion complexes like NiEDDA and CrOx report on the accessibility of the spin label side chain from the bulk water phase, molecular oxygen reports on the accessibility from a lipid phase.56 In case of interaction of the protein with a lipid phase, accessibility information can be used to judge immersion of labelled protein sites into a lipid layer or exposure to the solvent.62 Periodical patterns in the accessibility also may be interpreted in terms of secondary structural elements like an a-helix. Cw EPR spectroscopy also gives access to (iv) spin–spin distances in the range of 8–25 Å.63 This information manifests itself in spectral broadening due to magnetic dipolar interaction in EPR spectra recorded of frozen solution samples and can be extracted by deconvolution techniques. However, the distance range and thus the applicability of this technique is limited and pulsed EPR spectroscopy provides the means to access much larger distance ranges. Electron Paramag. Reson., 2019, 26, 1–37 | 7

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1.2.1.3 Pulsed EPR. There is a variety of pulsed EPR spectroscopic methods available that provide access to complementary information to cw EPR methods. The most widely applied pulsed EPR technique in the context of proteins and IDPs is pulsed EPR distance measurements using double electron–electron resonance (DEER) spectroscopy, also known as PELDOR (pulsed electron double resonance), which has been reviewed in several excellent articles.64–67 Spin–spin distances between 1.8 and 6 nm in membrane proteins and up to 10 nm in deuterated soluble proteins or even 16 nm in favorable cases are accessible with DEER.65,68 The experiment is commonly performed at cryogenic temperatures (e.g., 50 K) in frozen glassy solution. Since the distance information is frequency-encoded, the result of a DEER experiment provides not only a mean distance, but a precise distance distribution, which allows to understand also non-homogeneous conformational ensembles of proteins and to follow structural transitions upon, e.g., changes in the environmental conditions or interaction with arbitrary binding partners.69 As discussed above, IDPs feature a very broad conformational ensemble, which might even remain fuzzy in the presence of specific binding partners.16–18 In these cases, DEER time traces suffer from a lack of clear dipolar signal modulations and thus, the standard DEER analysis procedures fail.70,71 For such situations, a modulation depth-based data analysis procedure has been established with the IDP osteopontin by Kurzbach et al.: Based on the extraction of an effective modulation depth Deff at a specific dipolar evolution time t it is possible to judge if the average spin–spin distance increases or decreases, since a lower effective modulation depth Deff at the time t corresponds to a larger spin–spin distance (Fig. 4).71,72 Thus, conclusions about transitions in the conformational ensemble of an IDP become possible, even though no clear disorder-to-order transition may take place. The modulation depth of DEER traces is also the source of a different kind of information: Oligomerization of proteins can be monitored using EPR distance measurements with modulation depth calibration73,74 or

Fig. 4 With a modulation depth-based data analysis procedure even modulation-free DEER traces can be interpreted in terms of transitions in the conformational ensemble of an IDP. (A) Basically modulation-free DEER time traces of osteopontin in the presence of varying amounts of urea after background-correction. (B) illustrates, how the effective modulation depth Deff at a specific time t of the dipolar evolution is related to the average spin-spin distance. Reproduced from ref. 71 with permission from American Chemical Society, Copyright 2013. 8 | Electron Paramag. Reson., 2019, 26, 1–37

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monitoring modulation depth vs. flip angle. Thus, it becomes possible to extract useful information about protein–protein interactions. Another pulsed EPR method called electron spin echo envelope modulation (ESEEM) can be used to measure the presence of NMR active nuclei in the environment of a spin label. In the protein context, this is a powerful approach to measure exposure of a protein site to solvent deuterium. 1.2.1.4 In-cell EPR. Ideally, protein structure, conformational transitions, protein function and protein interactions are monitored in the most relevant environment, i.e., inside the living cell. Macromolecular crowding as well as the presence of numerous potential interaction partners may significantly alter the behavior of proteins under investigation compared to an in vitro experimental setup. In general, EPR spectroscopy is suitable for observing proteins work inside the cell and recent research has focused more and more on the development of corresponding experimental protocols.37 Gd31-chelates in combination with microinjection of oocytes of Xenopus laevis48,49 or hypotonic swelling of HeLa cells76 proved to be well suited for intracellular EPR distance measurements. The introduction of MTSL or 3-carboxy-PROXYL into the cell in combination with the oxidizing agent K3Fe(CN)6 also resulted in EPR spectra and DEER traces of good quality.77,78 1.3 Scope of this article: tau and a-synuclein In this article we review the research that has been conducted in the field of IDPs using EPR spectroscopy in the recent years. In particular, we focus on two prominent examples of IDPs, the ‘Parkinson protein’ a-synuclein and tau, a key player in Alzheimer’s disease. Both proteins have been subject of many spectroscopic and especially EPR studies. We will review the latest of these studies and discuss, which developments may become important for future EPR studies on IDPs.

2

Tau

2.1 Introduction: tau In its physiological function, the protein tau binds and stabilizes microtubules and has little tendency for aggregation.79 Under pathological conditions however, anomalous aggregates of tau are linked to various neurodegenerative diseases referred to as tauopathies.80,81 This heterogeneous group of dementias and movement disorders also includes Alzheimer’s disease (AD), hallmarked by neurofibrillary tangles (NFTs) of tau filaments that form deposits inside the diseased neurons.25 While most cases of disease are sporadic, tau was also linked to inherited forms of frontotemporal dementia and Parkinsonism caused by mutations in the tau gene MAPT and involving abundant filamentous tau inclusions in the brain.82,83 The mechanisms of tau aggregation in vivo as well as the pathways underlying tau-related neurodegeneration are still enigmatic: Although Electron Paramag. Reson., 2019, 26, 1–37 | 9

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the link between tau and several diseases is well-established, the question, whether NFTs are the primary neurotoxic tau species, is still a matter of much debate.84 The correlation between the appearance of NFTs and disease progression in AD brains was taken as evidence that tau fibrils cause neurodegeneration. However, recent research points in the direction that soluble, pre-filament tau may constitute the most neurotoxic protein species.84,85 Thus, in order to develop effective taudirected therapeutic strategies against tau-based diseases, it is of prime importance to understand the pathomechanisms of tau-mediated neurodegeneration.86 2.1.1 Tau structure and function. Human tau is encoded in the MAPT gene, which is expressed mainly in the axons of the central nervous system (CNS). By alternative splicing, six tau isoforms are generated, which differ by the presence or absence of two N-terminal inserts (N1, N2) and the presence of either three (3R) or four (4R) microtubule binding repeats in the C-terminal half of tau (Fig. 5A).24,87 The longest tau isoform consists of 441 amino acids, and whenever residue numbers are given in this article, they refer to this isoform. The overall amino acid composition of tau is rather hydrophilic, which prevents tau from folding into a globular structure and makes it an IDP.88,89 Tau can be subdivided into two major domains, the C-terminal assembly domain, which has a basic character, and the N-terminal projection domain, which contains a predominantly acidic stretch. Tau is overall basic, but is rather a dipole with two domains of opposite charge.90,91 Despite its generally disordered character, tau was shown to adopt a broad conformational ensemble in solution featuring an overall

Fig. 5 (A) Tau protein domains and alternative splicing in the human CNS. Six isoforms of tau are generated in the human CNS by alternative splicing of the MAPT gene. Distinct amino acid sequences in the N-terminal region of tau are either excluded (0N), or differentially included, giving rise to 1N or 2N tau isoforms. The central region of tau comprises the proline-rich domain (PRD). Alternative splicing in the microtubule binding domain (MTBD) results in 3R or 4R tau isoforms. (B) Tau associates with microtubules through the MTBD. The N and C termini of tau are closely associated when tau is free in the cytoplasm giving rise to the proposed ‘paperclip’ model of tau. Upon binding to microtubules, the terminal regions of tau become separated and the N-terminus of tau projects away from the microtubule surface. Adapted from ref. 24, 10.1007/s00401-0171707-9, under the terms of a CC BY 4.0 license http://creativecommons.org/licenses/by/ 4.0/. 10 | Electron Paramag. Reson., 2019, 26, 1–37

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‘paperclip’ shape, where the C- and N-terminus approach each other and the microtubule binding region (MTBR) (Fig. 5B).34,92 Physiological tau is mainly found in neurons, where it binds to microtubules with the MTBR and flanking regions, while the N-terminal projection domain projects away from the microtubules (Fig. 5B).24 By binding to several tubulin dimers tau stabilizes microtubules, thus regulating microtubule assembly and dynamics as well as axonal transport.93–95 2.1.2 Tau fibrils and disease. Tau inclusions as found in AD patients’ brains consist of paired helical filaments (PHFs) and straight filaments (SFs) formed from all tau isoforms.96–99 The proteaseresistant core of tau filaments is established by the repeat region of tau which folds into b-sheet structures, while the N- and C-terminal regions of tau remain disordered and project away from the core as the so-called ‘fuzzy coat’.100,101 Aggregation was shown to initiate from two short hexapeptide motifs PHF6* (275VQIINK280) and PHF6 (306VQIVYK311) located in repeats R2 and R3 of the MTBR, respectively.102 Only recently, the structure of tau filaments extracted from AD brain was solved with atomic resolution by cryo-electron microscopy by Fitzpatrick and coworkers.99 They showed that the filament core region is C-shaped and consists of intramolecular anti-parallel cross-b structures running perpendicular to the filament axis and that all six tau isoforms are arbitrarily stacked along the filament axis. Interestingly, the fibril core was shown to include repeats R3 and R4 of the MTBR.99 For most studies concerned with the characterization of tau aggregates, filaments are not extracted from brain tissue, but generated in vitro. Tau aggregation can be induced by negatively charged cofactors that compensate the positive charge in the repeat region, like polyanions such as heparin and RNA, or fatty acids.80,103–105 Most commonly, tau fibrils are prepared by the addition of heparin, which has been suggested to induce the formation of AD-like tau filaments.104 Tau is subject to manifold post-translational modifications (PTMs). In particular, phosphorylation at up to 85 potential sites is crucial in regulating the physiological functions of tau, including microtubule binding.106 Hyperphosphorylation reduces microtubule binding and is assumed to drive tau aggregation, since hyperphosphorylated tau from AD brains can self-assemble in vitro.107,108 However, since phosphorylation alone does not seem to be sufficient for aggregation, the issue is still a matter of debate.80 2.2 Tau studied by EPR spectroscopy 2.2.1 Filament architecture investigated by EPR. An important early contribution of EPR spectroscopy to tau research aimed for understanding the architecture of tau filaments along the fiber axis with SDSL cw EPR spectroscopy and line shape analysis using MTSL as a spin label. It was shown that b-strands, which assemble in the repeat region of individual tau molecules, are aligned in parallel with each other such that the same amino acids stack right on top of each other Electron Paramag. Reson., 2019, 26, 1–37 | 11

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Fig. 6 (A) Comparison of X-band cw EPR spectra of tau spin-labelled at position 308 in R3 recorded of tau in the solution state (black) and in heparin-induced fibrils (red dotted). Upon aggregation, the two outer hyperfine lines disappear to give rise to a spinexchanged single-line EPR spectrum indicative of the orbital overlap of several spin labels. (B) Electron micrograph of tau filaments (scale bar ¼ 100 nm). (C) Spectra recorded from diamagnetic dilutions of spin-labelled tau filaments containing 100% (red as in A), 50% (green) or 25% (black) spin-labelled tau molecules. Upon diamagnetic dilution, the hyperfine splitting in the spectra becomes apparent. Adapted from ref. 109 with permission from National Academy of Sciences, USA, Copyright 2004.

along the axis of a heparin-induced tau filament.109 This was implied by very characteristic single-line EPR spectra found for aggregated tau with spin labels attached at positions 301–320 in the repeat R3 (Fig. 6). The loss of the two outer nitroxide hyperfine lines was confirmed to result from spin exchange due to orbital overlap of several spin labels, enabled by the stacking of the same amino acids from different tau molecules along the fibril axis.110 In contrast, EPR spectra recorded of tau, which was spin-labelled outside the filament core (positions 400–404) showed that this region remains structurally dynamic upon aggregation. Independent support for the parallel in-register arrangement of b-strands was provided by quantitative analysis of dipolar broadening in cw EPR spectra of fibrils of singly spin-labelled tau and a-synuclein. The dipolar spin–spin interaction was converted into distance distributions, which are in agreement with the parallel in-register b-sheet arrangement.111 The parallel in-register arrangement of b-strands in the filament core region is not limited to tau, but rather a shared structural characteristic of amyloid fibrils, that has been confirmed (also with methods of EPR spectroscopy) for many other proteins, including a-synuclein and prion proteins.110,112 2.2.2 Polymorphism of Tau fibrils investigated by EPR. Despite all similarities in the general architecture, amyloid fibrils can still appear in different conformations, even when formed from the same protein.113 While the overall arrangement of b-strands running perpendicular to the filament axis is maintained, a large variety is possible in core sizes and b-strand interactions. For example, the introduction of point mutations in repeat R2 of tau was shown to induce local structural changes by EPR spectroscopy, while the overall parallel in-register arrangement of b-strands in the fibril core was unchanged.114 Using diamagnetic dilution experiments and cw SDSL EPR it was shown that tau constructs of 3R tau and 4R tau co-assemble with each other into heterogeneous filaments with a core of parallel, in-register arranged b-strands from soluble tau.115 Implied in this finding is a huge 12 | Electron Paramag. Reson., 2019, 26, 1–37

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number of possible tau fibril conformers: tau may either form 4R fibrils, or 3R fibrils, or 4R/3R heterogeneous fibrils with a variety of ratios between 4R and 3R tau. Different fibril morphologies of tau are diseasespecific: while AD filaments contain 4R and 3R tau, most diseases are characterized by 4R or 3R tau fibrils only.82,116 2.2.2.1 The prion concept for IDPs. In the prion concept, different filament strains characterized by structural polymorphism are thought to be responsible for phenotypic diversity in disease. They spread by mechanisms of intercellular propagation and templated-assisted conversion of monomeric protein onto the filament seeds, while the strainspecific properties are maintained (Fig. 7).25 In the past years, evidence was provided that tau has prion-like properties: Fibril seeds were shown to have the ability to propagate between cells to distant brain regions and assemble wild-type tau into filaments.117,118 Moreover, distinct tau strains were shown to stably reproduce their conformation during propagation with high fidelity defining different tauopathies.82,119 Tau filaments consisting of 3R and 4R tau are characterized by an asymmetric seeding barrier, i.e., 3R tau filaments can recruit 4R tau, while 4R tau filaments cannot recruit 3R tau.120 EPR distance measurements using DEER in combination with SDSL and extensive molecular dynamics simulations were used in order to investigate the molecular basis of the seeding barrier.121 Monomers of 3R and 4R tau fragments containing only the repeat region (known as K19 and K18 tau, respectively) were grown onto seeds formed from 3R or 4R tau and

Fig. 7 Illustration of the mechanism of seeding and growth of amyloid filaments. A pathological filament seed acquires endogenous protein by seeded aggregation and imprints the seed conformation onto the incoming monomers. A fragmentation process produces new seeds, while at the same time growth leads to mature fibrils. Reprinted by permission from Springer Nature, from Sarah K. Fritschi et al. in Proteopathic Seeds and Neurodegenerative Diseases, Edited by Mathias Jucker and Yves Christen, Springer-Verlag Berlin Heidelberg, 2013. Electron Paramag. Reson., 2019, 26, 1–37 | 13

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intramolecular distances were determined between spin labels located in repeat R3 of the fibril core. 4R tau grown onto 4R seeds resulted in distance distributions with several contributions, indicating heterogeneous fibril conformers with at least three distinct conformers. In contrast, when 4R tau and 3R tau were grown onto 3R tau seeds, homogeneous fibrils were indicated by narrow distance distributions. The results suggest conformational variations in R3 of 3R or 4R tau filaments. Moreover, they show a structural plasticity of tau as the initial seeds imprint their conformation onto the recruited tau monomers as evident in the case of 4R monomers which grow into different fibril conformers on different seeds.121 The emergence of structural heterogeneity as well as the conformation-based seeding barrier together with the template-based conversion of monomers are important characteristics of prion-like behavior.121 The influence of single point mutations on the populations of fibril conformers of 4R tau filaments (prepared with truncated K18 tau) was further investigated by DEER spectroscopy.122 Structurally heterogeneous 4R tau seeds were used as initial templates for filament assembly of mutated tau monomers carrying spin labels at positions 311 and 328. Several tau mutations were tested, among them some of well-known relation to diseases (DK280,123 P301S,124 S320F,125 Q336R126). While several of the mutated tau constructs reproduced the intramolecular distance distribution and thus the ratio of fibril conformers of wild-type tau (Fig. 8B), some mutated tau derivatives, in particular some of the disease-related ones, induced large shifts in the populations of fibril conformers (Fig. 8C). As the fibrils are generated by template-assisted growth onto the tau seeds, the proposed mechanism responsible for the change in the fibril conformer populations is seed selection due to changed conformational compatibilities of fibrils formed from pointmutated tau: mutants showing no interference with any of the original conformers will grow onto the wild-type seeds conserving the conformational ensemble. Mutants with incompatibilities to some of the original conformers will change the overall composition of the ensemble, possibly amplifying minor subpopulations (Fig. 8D). The mechanism of seed selection as shown in this study in vitro could provide a plausible explanation of how hereditary mutations might change the initial ensemble of tau conformational composition and lead to the emergence of changed fibril ensembles. The influence of a change in incubation conditions during fibril growth on the conformational ensemble of 3R and 4R tau filaments (prepared with truncated K19 and K18 tau, respectively) was also assessed by means of intramolecular DEER spectroscopy using the spin label positions 311/322 (in 3R tau) and 311/328 (in 4R tau) for determination of intramolecular distances.127 A multicycle seeding scheme was used in order to test the relative stability of individual fibril conformers. While fibrils formed from 3R tau were basically unaltered over 15 cycles of seeding, 4R tau fibril populations changed during consecutive cycles of seeding. Homogeneous 4R fibrils were dominantly formed upon stirring in the first cycle of growth, quiescent growth in the following cycles led to 14 | Electron Paramag. Reson., 2019, 26, 1–37

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Fig. 8 Influence of single point mutations in tau on the conformational ensemble resulting from seed selection. Q-band DEER distance distributions of K18 tau spin-labelled at positions 311 and 328: (A) reproducibility assessed by repetitive experiments using wild-type tau fibrils; (B) mutated tau derivatives with distance distributions similar to wild-type tau; (C) mutated tau derivatives with different distributions compared to wild-type tau. (D) Illustration of how the mechanism of sequence-dependent seed selection reproduces the original ensemble of fibril conformers in the case of full structural compatibility (Mutant 1), causes a switch in the dominant species (Mutant 2) or even amplifies a minor subpopulation of conformers (Mutant 3) in case of restricted compatibility. Adapted from ref. 122 with permission from John Wiley & Sons, Copyright 2014.

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formation of a heterogeneous ensemble of fibril conformers. This change in the fibril structural ensemble is suggested to be a result of the alteration of growth conditions from stirring to quiescent, which could lead to selective amplification of distinct conformers by a mechanism related to the fragilities and growth rates of different fibril conformers: under stirring conditions, fibril conformers with increased fragilities (but decreased growth rates) might have a selective advantage over more stable (faster growing) species due to the production of many new fibril ends. When removing the selective pressure (i.e., stirring), the population ensemble evolves towards the latter conformation. The authors show that fibril conformer populations depend on the subtle balance between fracture and growth.127 Only very recently, Fichou and coworkers questioned the idea that heparin-induced tau filaments are similar to the PHFs and SFs extracted from AD brains: They performed a study comparing intramolecular DEER distance distributions of heparin-induced tau filaments with corresponding simulations based on the AD-filament structure of tau published by Fitzpatrick et al.99,128 They used a truncated version of tau lacking the N-terminal part, but containing all repeats and the C-terminus. As a reference they also measured DEER distances of tau in solution and compared those distances with the corresponding simulations based on a tau solution structural ensemble published previously.129 While Fichou and coworkers find that the DEER distance distributions recorded of tau in solution are in adequate agreement with the published ensemble, larger discrepancies between experiment and simulations appeared in the case of heparin-induced tau filament samples. For all tested doubly spin-labelled tau derivatives they found distance distributions considerably broader than expected from the simulations, indicating that heterogeneous tau fibril conformers are present in heparin-induced tau fibrils, instead of only PHFs and SFs as found in AD-filaments. Moreover, the average inter-spin distances were larger than predicted, suggesting that the key features of AD-filaments, like the C-shape and the cross-b structure, are not the dominant conformations in heparin-induced tau fibrils.128 Upon aggregation with heparin a stretching of intramolecular tau inter-spin distances in repeats R1, R2 and R3 is observed in the distance distributions, consistent with the formation of a cross-b structure. With ESEEM experiments of doubly spin-labelled tau variants the authors determined a homogeneous reduction in the accessibility to deuterium atoms of the solvent across all four repeats of the tau MTBR, suggesting that all four repeats are part of the tightly packed heparin-induced filament. This is in contrast to the AD-filaments, where only R3 and R4 are part of the core region.99 The authors suggest that heparin, which is a fairly extended disordered chain, cannot serve as template for uniform tau aggregates, but rather generates polymorphic tau fibrils.130 However, since tau strains have been shown to be disease-specific, for the meaningful biological analysis of specific tau strains in terms of diseases it might be necessary to employ tau filaments seeded from structurally defined templates, instead of polymorphs resulting from heparin-induced tau aggregation.119 16 | Electron Paramag. Reson., 2019, 26, 1–37

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2.2.3 Early stages of the tau aggregation process investigated with EPR. In the past years some EPR studies have focused on the investigation of very early events in the tau aggregation pathway, monitoring tau conformational states and populations in solution and during earliest stages of aggregation into insoluble fibrils. As there is evidence that primary tau toxicity might be exerted by soluble oligomers formed in the early stages of fibril formation, knowledge about their structure and properties is important for developing tau-based therapeutics. A tau fragment containing 13 residues of the R2 region, including hexapeptide PHF6*, was used to test the hypothesis that subtle changes in the solution conditions, such as changes in buffer type or presence of osmolytes (TMAO vs. urea), influence the aggregation pathways and end products.131 A cysteine-substitution allowed spin labelling at the N-terminal end of the fragment and thus cw EPR measurements in order to access solvent accessibility, packing and mobility of the spin label, as well as the fractions of mobile and fibrillated tau, before and after aggregation initiation. The EPR results together with Overhauser dynamic nuclear polarization (ODNP)-derived surface water diffusivity, ion mobility mass spectrometry (IM-MS), transmission electron microscopy (TEM) and thioflavin T (ThT) fluorescence show that both urea and TMAO strongly influence oligomer formation. While urea breaks apart inter-tau contacts, shifting the tau population towards monomers, and hinders downstream inter-tau fibrillation also in the presence of heparin, TMAO facilitates tau oligomerization already in the absence of heparin. Also the change of buffer type, which is often considered a minor factor in sample preparation, was shown to influence the conformational ensemble of the tau peptide and shifted it to more aggregation-prone populations in comparison to water, which in turn influenced the downstream aggregation of tau peptides after addition of heparin.131 Taken together, the results show that the solution state of tau before initiation of aggregation influences the whole pathway of protein aggregation and the propensity to form fibrils. Using a truncated tau version that comprises all four repeats and the C-terminal region, structural transitions of tau in the early stages of aggregation in solution were probed by ODNP as well as cw EPR line shape analysis as a function of aggregation time.132 Several key positions across R3 and the C-terminus were spin-labelled one at a time for this purpose. Multi-component EPR spectra at different time points after initiation of aggregation consist of (i) a mobile, solvent-exposed component corresponding to monomers or small oligomers of tau, (ii) an immobile component, corresponding to tau immobilized at an inter-tau interface, and in the case of not spindiluted samples (iii) a spin-exchanged single-line component (Fig. 9A,B). The results of this study suggest that as soon as 5 min after initiation of aggregation 40–70% of tau variants carrying a spin label in the R3 region are involved in dynamic and partially structured aggregation intermediates, reflected by immobilization of the spin label side chains. Approximately 5% of all R3 spin-labelled tau are already involved in b-sheet arrangements (Fig. 9C). Electron Paramag. Reson., 2019, 26, 1–37 | 17

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Fig. 9 Early stages of tau aggregation as proposed by Pavlova and coworkers.132 (A–C) EPR line shape analysis of spin dilution X-band cw EPR experiments. Simulated spectra (red) in (A) and (B) are a superposition of a mobile and an immobile component. Without spin dilution (B), a spin-exchanged single-line component is detected, indicative of a bsheet structure. (C) Time-dependent population of each component extracted from EPR spectra labelled at various positions. (D) Electron micrographs display the time-dependent aggregation state of tau. Fibrils are detected starting at 120 min after aggregation initiation (scale bar ¼ 100 nm). (E) Suggested mechanism of tau aggregation including the formation of dynamic soluble oligomers early in the aggregation pathway. Adapted from ref. 132 with permission from National Academy of Sciences, USA.

Importantly, over time, only the fractions of embedded tau change, while spectral simulations show that the EPR parameters of the individual components remain unchanged. At this early time in the aggregation process, no fibrillary assemblies are observable by cryo-TEM (Fig. 9D). This indicates that a major tau population of partially structured intermediates forms within minutes after aggregation initiation, which are on-pathway species towards formation of mature fibrils long before actual fibrils are detected in the sample. After maturation of the fibrils the b-sheet content in the R3 region plateaus at 70–80%. In contrast, the C-terminal spin-labelled residues show no spin-exchanged 18 | Electron Paramag. Reson., 2019, 26, 1–37

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component also after 424 h incubation with heparin, indicating the persistence of disorder in this protein region. Furthermore, the authors find that, once formed, the b-sheet structures do not exchange subunits but form stable entities already in stages of aggregation as early as 5 min. DEER spectroscopy was also used to detect how aggregation initiation acts on the conformational ensemble of soluble tau in a time-dependent manner by monitoring the end-to-end distances of the key fibril-forming hexarepeats PHF6 and PHF6* in freeze-quenched tau samples.133 Eschmann and coworkers analyzed the distances flanking the PHF6 and PHF6* segments in truncated tau fragments containing all four repeats and the C-terminus in their solution state as well as after addition of heparin. In all cases they observe a relatively compact conformational ensemble of tau in solution characterized by an average distance of just above 3 nm (Fig. 10). Upon initiation of aggregation they observe a stretching of the distances spanning both hexarepeats consistent with a conformational extension of these regions to just above 4 nm. Timeresolved DEER experiments show that all distance distributions recorded within 12 h after heparin addition can be described by a 2-state model of conformers, i.e., by a compact and an extended conformation, without the need for intermediates. The compact conformation is attributed to the stable solution ensemble of tau conformers, while the extended conformation is characteristic for a stretched out conformation as can be found in b-sheets of mature tau fibrils. Importantly, already 1 min after addition of heparin roughly 50% of all tau has undergone stretching (Fig. 10D), while 10 min after heparin addition roughly 90% are in the extended conformation (Fig. 10E), which is still a solution state consisting predominantly of monomer and dimer species well before the

Fig. 10 Baseline-corrected time-domain DEER data taken at Ku-band (17.3 GHz) and corresponding intramolecular distance distributions of truncated spin-diluted tau G272C/ S285C before heparin addition (black in all cases) and right after heparin addition ((A) and (D), respectively), as well as 10 min after heparin addition ((B) and (E), respectively), as well as 12 h after heparin addition ((C) and (F), respectively). Reproduced from ref. 53 with permission from Elsevier, Copyright 2017. Electron Paramag. Reson., 2019, 26, 1–37 | 19

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macroscopic appearance of fibrils in the sample. After 1 h the transition in the conformational ensemble towards the compact state is complete. In conclusion, upon initiation of aggregation the population of tau shifts towards an extended state which reflects conformers on the pathway to fibrils, well before b-sheet signatures or fibrils are detectable.133 However, it is not clear whether the formation of the extended b-sheet-like conformation precedes the formation of dynamic aggregation intermediates as suggested by Pavlova and coworkers, or vice versa.132 The results of this study suggest that tau aggregation occurs according to the nucleation conformational transformation model, in which a conformational transformation from a stable tau solution structure to a distinct aggregation-prone structure, as monitored here with EPR spectroscopy, is required to induce the stacking of proteins into fibrils.134,135 2.2.4 Tau and RNA investigated by EPR. In addition to the polyanion heparin, RNA can also induce the aggregation of tau protein.105,136 RNA is a component of every cell, rendering it a highly relevant interaction partner for tau. RNA was shown to drive fibril aggregation and tau fibrils that aggregated in the presence of RNA were shown to adopt a parallel, in-register b-sheet alignment along the fibril axis with characteristic exchange-broadened single-line cw EPR spectra, suggesting that the general fibril architecture is independent of the cofactor used for aggregation induction.137 Also the asymmetric seeding barrier, where 3R tau cannot grow onto 4R tau seeds was shown to be preserved when inducing aggregation with RNA.137 While RNA was found to bind to the tau fibrils formed, heparin replaces RNA on the fibril surface when added to the sample.137 In a recent study, tau was shown to undergo liquid–liquid phase separation (LLPS) in the presence of RNA, forming droplets of high protein density.138 Such protein-rich structures are known as complex coacervates, which are typically formed by two oppositely charged polyelectrolytes in solution, in this case of the polycation tau and the polyanion RNA.139,140 The formation of the droplets is shown to be tunable by salt concentration, tau:RNA ratios and temperature. Despite the molecular crowding inside the droplet, tau was shown to maintain native-like mobility and a locally compact conformation around the PHF6(*) regions as is typical for the tau conformation in dilute solution.138 Thus, a change between the solution state and the coacervate state of tau is reversible and tunable by physiologically viable parameters.138 After prolonged residence in the complex coacervate phase, a low-level b-sheet formation was observed. 2.2.5 Tau in membrane interactions investigated by EPR. Lipid membranes have been shown to enhance tau aggregation in vitro.141–143 It is known that tau interacts with lipid micelles via its MTBR region under the formation of helical structures localized in R1, R3 and R4 of a truncated 3R tau construct containing only the repeat region (K19).144 With SDSL of tau and mobility as well as accessibility analysis of cw EPR spectra, the interaction of tau with POPC/POPS 20 | Electron Paramag. Reson., 2019, 26, 1–37

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vesicles was confirmed and a helical nature of the respective regions in tau was found in the bound state.145 Cw EPR data also imply that the tau amphipathic helical segments interact with the lipid layer periphery and are not deeply immersed into the lipid layer. With DEER distance measurements, distances between the individual helical segments and between the helices and the linker regions were determined. The results suggest that individual helix segments are connected by highly flexible linker regions that allow adaption of the relative orientation of the helices to the spatial constraints posed by the lipid structure.145 The induction of helical structure upon membrane binding has also been shown for other IDPs, e.g., for a-synuclein, and implications of helical segments in the context of protein aggregation are discussed.146 A more detailed discussion of IDP-membrane interactions can be found in the a-synuclein section of this chapter.

3

a-Synuclein

3.1 Introduction: a-synuclein The human a-synuclein (a-syn) consists of 140 amino acids and is highly abundant in the human brain. Its physiological function is not fully understood, but it is enriched in presynaptic nerve terminals and implicated in neurotransmitter release and vesicle trafficking.147–149 a-syn consists of (i) an N-terminal amphipathic region, (ii) a central hydrophobic non-amyloid-b component (NAC) region, and (iii) a highly acidic C-terminal region (Fig. 11). a-syn displays remarkable structural versatility: it is intrinsically disordered but can readily adopt various conformations, e.g., b-sheet structure in aggregates or a-helical structure when bound to lipids.150–153 a-syn is known to pathologically self-assemble into amyloid fibrils and plaques, which are found in Lewy bodies, the pathological hallmark of PD, but it is also associated with other neurodegenerative diseases.25 Like tau fibrils, a-syn fibrils are arranged in a cross-b conformation with b-sheets running perpendicular to the fibril axis. The fibril core ranges approximately from residues 30–100 as indicated by EPR experiments.111,155 As for other amyloid fibrils, a parallel in-register arrangement of a-syn along the fiber axis has been revealed with cw EPR spectroscopy as described in detail for tau.111 Like for other amyloid proteins and tau, a-syn has been shown to have prion-like properties, which allow a-syn-seeds to induce aggregation of endogenous a-syn to form aggregates.156 a-syn was shown to generate polymorphic fibril structures, and two a-syn fibril strains have been

Fig. 11 The primary sequence of a-syn with three functionally distinct regions highlighted in blue, orange and red. Three of the familial PD-related mutations are shown. Adapted from ref. 154, https://doi.org/10.3389/fnmol.2016.00048, under the terms of a CC BY 4.0 license. Electron Paramag. Reson., 2019, 26, 1–37 | 21

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shown to exhibit cell-to-cell transmission and to induce distinct pathologies.157 Various point mutations of a-syn (e.g., A30P, A53T, or E46K) in the N-terminal membrane recognition domain are associated with earlyonset Parkinson’s disease,158–160 indicating the importance of membrane binding for the function of a-syn.154 The N-terminus of a-syn contains seven copies of an 11-residue pseudorepeat, which mediates binding to negatively charged lipids.161,162 Upon membrane binding, a-syn undergoes a structural transformation towards an a-helix,163 which was found by EPR spectroscopy and NMR spectroscopy to be continuous and extended on the surface of unilamellar vesicles164 or to adopt a horseshoe-like conformation of two helices connected by a loop region on SDS micelles,165 while the C-terminal region remains unstructured. As discussed above for tau, in general for amyloid diseases there is a lot of evidence that protein aggregation is the cause for neurodegeneration. Recent results suggest, that the primary neurotoxic species may not be the mature fibril, but prefibrillar oligomers or protofibrils.166 There is evidence that amyloid toxicity may be caused by membrane permeabilization by pore-like early intermediates as shown for a-syn.167 The molecular basis of PD appears to be tightly coupled to the structural diversity of a-syn.168

3.2 a-Syn investigated by EPR spectroscopy 3.2.1 Spin labelling of a-syn. The intrinsically disordered nature of the protein contravenes the typical approach of standard highresolution structural biology methods. EPR spectroscopy in combination with SDSL is a complementary and valuable tool for unraveling the structural ensemble of a-syn. Since human a-syn does not contain any cysteine residues, most commonly site-directed cysteine mutagenesis and cysteine-reactive radicals are used for SDSL. The usual procedure to check whether the spin labelling does affect the functionality of the protein is not applicable, because the function of a-syn is still unknown. Therefore, typical control experiments are CD spectroscopy or measurements of the aggregation kinetics before and after spin labelling.169 Apart from classical spin labelling EPR studies, there are EPR investigations on a-syn, which exploit the fact that a-syn binds Cu21 ions at several positions along the protein: the paramagnetic Cu21 and the resulting EPR signal was employed to analyze the copper-a-syn interaction in various studies.168,170–173 In conclusion, the C-terminal part of a-syn displays low affinity vs. copper, while the N-terminus binds copper with a high affinity of about 0.1 nM with His50 coordinating the bound copper. Dudzik et al. studied the coordination of copper by membranebound a-syn with EPR.174 In this study, copper was found to bind exclusively to the N-terminus (Met1-Asp2, 1eq.) and did not alter the a-helical membrane-bound conformation of a-syn. Drew investigated the Cu21/a-syn binding in solution and found that the dominant copper coordination mode of a-syn is associated with the formation of a-syn 22 | Electron Paramag. Reson., 2019, 26, 1–37

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oligomers, in which Cu ions occupy intermolecular bridging sites of a-syn dimers and trimers.175 3.2.2 a-Syn fibrils investigated by EPR. While early oligomers of a-syn are considered to be neurotoxic,176 fibrils might play a role in spreading the disease from peripheral to central neurons.177 Highresolution information is mainly available for several short fibrilforming peptides,178–181 while fibrillary structures for longer proteins are often less understood.110,182 Structural models of a-syn fibrils were proposed183 based on solid-state NMR and cryo-electron microscopic data. However, long-range distance constraints were missing before the first high-resolution structure of full length a-syn fibrils was published (PDB entry 2N0A).156 Pornsuwan et al. successfully detected several intramolecular long-range distance constrains at the molecular level on the fold of a-syn in fibrils by EPR distance measurements.184 In order to infer the side-chain direction of the spin label within one strand and to conclude on the spatial arrangement of the labels and the strands, they exploited a pair-labelling strategy, i.e., measuring two distances from one labelled residue to two adjacent ones, respectively (Fig. 12). Based on the obtained distance constraints, the authors refrained from proposing a structural model and only concluded that the two inner strands containing residues 54 and 64 might arrange in a more complex fashion than proposed before. Even though a high-resolution structure of full length a-syn is available, polymorphism and coexisting different fibril types need to be taken into account in future studies.157,185–187 3.2.3 a-Syn-membrane interactions investigated with EPR. It is likely, that the physiological function of a-syn, e.g., as protein hub, is connected to a-syn’s membrane binding affinity.188 Additionally, it has been observed that the presence of lipid membranes influences the fibrillation kinetics.189 Depending on the protein : lipid ratio, aggregation is enhanced or reduced. Therefore, a-syn-membrane interactions are studied intensively. Many scientists introduce their conference talks on a-syn by presenting the high-resolution NMR structure 1XQ8 of monomeric, micelle-bound asyn (Fig. 13B) (this often happens even when lipids are not involved at all in the presented research). The 1XQ8 structure was obtained by solution NMR, which is restricted in the size of the complex under study, so that small SDS micelles as membrane mimics were used. NMR structure determination revealed, besides the unbound C-terminal tail, an a-helical region featuring a break, resulting in two anti-parallel a-helices within the N-terminal region, called ‘horseshoe’.165 This model was confirmed by 13 distance constraints obtained by EPR distance measurements between spin-labelled cysteines.190 SDS micelles, however, might be too small to mimic actual organelle membranes. They have typical diameters of 5 nm and therefore may have artificially constrained the protein into the horseshoe structure. Here, the advantage of EPR distance measurements not being restricted in the size Electron Paramag. Reson., 2019, 26, 1–37 | 23

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Fig. 12 (A) 34 GHz four-pulse DEER traces after background subtraction and fit (black) of diamagnetically diluted, doubly labelled a-syn after fibrillation and (B) corresponding normalized distance distributions P(r). Asterisks (*) indicate most probable distances. (C) Scheme of the observed distances between two b-strands containing the spin-labelled residues. The directions of the MTSL side chain within b-strands are also depicted, if known. Reproduced from ref. 184, 10.1002/ange.201304747, under the terms of a CC BYNC 3.0 license http://creativecommons.org/licenses/by-nc/3.0/.

of the complex under study, can be exploited. Subsequent EPR studies were performed using small unilamellar vesicles (SUVs), which can accommodate a-syn in an extended alpha-helical conformation. However, it was found by control experiments using dynamic light scattering, that SUVs are eventually disrupted upon interaction with a-syn, depending on the protein:lipid ratio.191 Large unilamellar vesicles turned out to be stable up to protein : lipid ratios of at least 1 : 200 and were used for subsequent EPR distance measurements unravelling the membrane bound conformation of a-syn. Quantitative binding is found at least for residues 9–69. The helix 24 | Electron Paramag. Reson., 2019, 26, 1–37

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Fig. 13 (A) Model of the extended a-syn a-helix and (B) NMR structure of micelle-bound a-syn.165 Spin labelling positions used by Robotta et al. are indicated in red. Reproduced from ref. 192 with permission from John Wiley & Sons, Copyright 2011.

conformation was found as a mixture of one single, extended helix as well as a horseshoe-like conformation of two helixes with a loop region in between (Fig. 13).192 Kumar et al. studied the a-syn conformation bound to SUVs mimicking the inner mitochondrial and the neuronal plasma membrane.193 They conclude, that their experimental distance constraints obtained by DEER suggest a coexistence of two distinct conformations: firstly, an extended helical conformation and secondly, a broken helix with a larger opening angle between the two helices than the horseshoe-conformation found before on SDS-micelles as well as POPG SUVs and LUVs. This is another hint of the structural variability of a-syn when interacting with membranes. Besides EPR distance measurements, the rotational dynamics at physiological temperatures of spin labels attached at different sites of a-syn can be obtained by EPR spectral shape analysis and can be used to monitor the local degree of membrane binding in the proximity of the labelled site, e.g., as a function of the composition of artificial membranes. The rotational mobility mainly reflects the residual mobility of the spin label. Upon interaction with macromolecular partners, e.g., membranes, reduced mobility is observed. Often, spectral simulations taking two components with different spin label mobilities are used for data analysis. The slow component can be attributed to binding of a-syn to the membrane in the vicinity of the labelled residue, so that the corresponding fraction reflects the local degree of binding of this region of the protein. This approach gives a more differentiated view of the a-syn-membrane interaction than a global binding affinity measurement. Using this approach, Robotta et al. showed that binding of a-syn to membranes is initiated in the N-terminal region.194 At lower surface charge densities, i.e., for membrane compositions with less negatively charged lipids, the binding affinity of regions close to the N-terminus is stronger than that of regions distal from the N-terminus in sequence. This implies, that different binding modes exist for a-syn, i.e., membrane binding involving different stretches of residues (e.g., binding with residues B9–69 or B9–27). Besides the coexistence of different helical conformations, this underlines the extreme conformational flexibility of a-syn. Electron Paramag. Reson., 2019, 26, 1–37 | 25

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Using a similar approach, Kumar et al. focused on the influence of phosphorylation at positions 87 and 129 on the membrane binding behavior of a-syn.195 The serine residues at positions 87 and 129 were exchanged by an aspartic acid one at a time, to mimic phosphorylation at the respective position. Alanine was used as non-phosphorylated reference. The ‘phosphorylation’ at position 87 negatively affects the membrane binding behavior of all investigated labelling positions (27, 56, 63, 69, 76, 90), whereas ‘phosphorylation’ at position 129 has no effect on the binding affinity of a-syn. Also the influence of point mutations A30P and A53T on the membrane binding behavior of a-syn was investigated with EPR spectroscopy.32 Robotta et al. studied ‘wild-type’ a-syn (nevertheless containing spin-labelled cysteines) as well as A30P and A53T mutants labelled at nine different positions (9, 18, 27, 35, 41, 56, 69, 90 and 140) in the presence of LUVs with systematically varied lipid compositions. Their results show that the disease mutation A30P does not only reduce the overall binding affinity of a-syn to LUVs, but is structurally defective and locally affects membrane binding. It is worth mentioning that studies on point mutations using SDSL EPR require (additionally to the point mutation of interested) site-directed mutagenesis and labelling, which also might influence structure and dynamics. Therefore, it is important to directly compare the results of the SDSL-study with and without the point mutation of interest. The a-syn-membrane interaction can also be observed from the perspective of the membrane by using spin-labelled lipids as spin probes instead of spin-labelled a-syn. Following this approach, Pantusa et al. focussed on the influence of a-syn and the disease mutants A30P, A53T and E46K on the lipids of vesicles.196 They used anionic dimyristoylphophatidylglycerol (DMPG) LUVs containing 1 mol% n-PCSL (phosphatidylcholine lipids) with a nitroxide reporter group attached at selected carbon atom position. The spectra reflect the lipid chain order and the membrane dynamics. Their findings suggest that a-syn binds to the membrane without deep penetration into the membrane. A30P showed the smallest influence on the membrane, whereas E46K shows similar influence as wild-type a-syn. 3.2.4 a-Syn interactions with organelles investigated with EPR. The fact that SDSL EPR spectroscopy is virtually background-free, enables studying not only a-syn interactions with artificial membranes, like phospholipid vesicles, but also with isolated organelles, e.g., mitochondria. DEER distance measurements on a-syn were performed in the presence of mitochondria isolated from human HEK 293 cells.197 The DEER data revealed a distance distribution in the presence of mitochondria, which can be described as a superposition of a-helical a-syn, i.e., bound to artificial membranes and intrinsically disordered a-syn in solution. The relative fractions of both components nicely agree with the bound and unbound fractions determined by cw EPR. This result demonstrates how a-syn binds onto isolated mitochondria in a-helical conformation. The binding profiles for two different artificial membrane compositions mimicking the inner and outer mitochondrial 26 | Electron Paramag. Reson., 2019, 26, 1–37

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membrane revealed the binding of a-syn solely to the vesicles mimicking the inner mitochondrial membrane (IMM), which has a higher cardiolipin content than the outer membrane. Taken together, the results suggest that a-syn binds a-helically to the inner mitochondrial membrane. 3.2.5 a-Syn in the cell investigated with EPR. Increasing the level of complexity, spin label EPR spectroscopy can even be performed in the cell. In order to study the intracellular conformation of a-syn, Cattani et al. micro-injected singly spin-labelled, initially monomeric a-syn, including samples with the disease mutants A30P and A53T, into oocytes of Xenopus leavis as model cells. Using intermolecular in-cell DEER experiments intracellular diffusion of a-syn upon microinjection was monitored. To study the (dis-)order of a-syn in the cell, in-cell cw EPR at room temperature was performed.169 Importantly, it was shown by an independent control experiment that the intracellular microviscosity is very similar to aqueous solution. The a-syn in-cell study found no spectral changes compared to spectra measured in aqueous, buffered solution. However, the obtained signal-to-noise ratio, which is limited by the intracellular spin label stability does not allow to exclude a small fraction (up to 20%) of either a-helical or aggregated a-syn in the oocytes. Additionally, there was no difference detectable between the wild-type a-syn and the disease mutations A30P and A53T. The Goldfarb group were the first to perform distance measurements of N-terminally acetylated a-syn doubly spin-labelled with Gd31-DOTA at positions 24/122 and at positions 42/122, which was introduced into A2780 cells via electroporation.198 The obtained distances revealed the intracellular preservation of the disordered structure of a-syn as observed in buffer. However, again it was not possible to exclude a small fraction (up to 20%) of either a-helical or aggregated a-syn in the cell. An intriguing observation in this study was that a-syn seems to interact with some unidentified molecules in cellula as suggested by a drop in NMR signals across the protein.198 This highlights, how the presence of all kinds of possible interaction partners may influence the structure and function of a protein inside a cell.

4 Discussion and outlook Many dysfunctional pathways and contributing factors have been implicated in IDP neurotoxicity, among them hyperphosphorylation, oligomerization, fibrillation, propagation and strain differences.86,199 In order to develop mechanism-based therapeutics against IDP-related diseases, we need to understand the molecular mechanisms underlying their non-functional self-aggregation and characterize toxic and native IDP species as well as their endogenous cofactors regulating relevant structural transformations. EPR spectroscopy has been shown to be a powerful tool for the investigation of IDPs. It was used, e.g., for the elucidation of fibril architecture and fibril polymorphs, the processes of fibril formation, as well as for the analysis of the conformational Electron Paramag. Reson., 2019, 26, 1–37 | 27

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ensemble during interaction with lipid phases. By delivering a wealth of information, EPR spectroscopy has become indispensable in IDP research. Nevertheless, a careful analysis of the studies summarized herein reveals some limitations of the EPR investigations performed. First, usually EPR sensitivity is adequate for studying non-aggregated IDPs at physiological concentrations (some 10 mM). However, many studies on IDP fibrils are conducted with diamagnetic diluted samples. While diamagnetic dilution allows for measuring only intramolecular contributions, it requires very high protein concentrations (e.g., 800 mM)133 in order to reach a sufficient signal-to-noise ratio in the common EPR experiment. Such high protein concentrations are usually nonphysiological and thus may promote a non-physiological shift in the conformation equilibrium of the soluble protein or influence its selfaggregation propensity. High-sensitivity EPR spectroscopy might be the key to reducing molar sample concentrations to the physiologically relevant regime even for diamagnetically diluted samples. Second, many EPR studies have not been performed with the fulllength protein, but with shorter fragments thereof. This is advantageous since it results in greatly accelerated aggregation kinetics,200 while fibril properties are still ‘full-length-like’115,201 and it is a valuable approach for understanding the molecular mechanisms of certain processes. However, since studies have shown that the populations of IDP conformational ensembles and aggregation depend on subtle variations in the sample conditions,131 even small effects accompanying protein truncation might influence the research results downstream, in particular their interpretation in terms of biological processes. Furthermore, IDPs are highly regulated proteins, which are subject to numerous PTMs, e.g., phosphorylation, acetylation, ubiquitination or glycosylation, influencing the IDP energy landscapes.202 Many of these PTMs have been discussed to influence the aggregation behavior of IDPs and thus play important roles in IDP pathology.80,203–205 In vitro experiments are often performed in the absence of PTMs or they investigate the influence of specific modifications. However, it is not clear, if such isolated modifications resemble the physiologically relevant state of an IDP. In order to produce biologically meaningful results in the context of human diseases, it is crucial to further develop in-cell EPR in order to perform EPR experiments with IDPs under the most relevant environmental conditions, i.e., inside the cell. The cellular environment is characterized by molecular crowding and features a huge variety of interaction partners that may modulate the protein structural ensemble and processes of structural reorganization, as well as the oligomerization behavior, resulting fibril conformers and the interaction kinetics with partners like other proteins.206 As a result, in cellula experiments may deliver different findings than the in vitro complement. However, the application of in-cell EPR is still demanding. Apart from the need for spin labels resistant to the reducing intracellular environment, effective ways of inserting the protein of interest into the cell also 28 | Electron Paramag. Reson., 2019, 26, 1–37

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need to be developed. This might be done either by employing transfection techniques like electroporation or by the introduction of alternative in-cell spin labelling strategies, e.g., via unnatural amino acids that allow in vivo expression of spin-labelled protein.42–44 Recent studies showed that the signal-to-noise ratio in in-cell EPR experiments is crucial for the detection of all relevant protein subpopulations: Cattani et al. estimated that up to B20% of a dynamically restricted protein conformation could be present in samples of a-syn in Xenopus leavis oocytes without being detected in the EPR spectra.169 This finding emphasizes the need for suitable labelling strategies as well as for high-sensitivity EPR spectroscopy. Promising spectroscopic approaches for high-sensitivity EPR distance measurements are laser-induced magnetic dipole spectroscopy (Laser-IMD),207 as well as the emerging applications of shaped pulses for DEER spectroscopy, which allow sensitivity enhancement by increasing the excitation bandwidth as well as improving spin dynamics control.208,209 With the development of new spectroscopic techniques, EPR has the potential to further make significant contributions to answering the stillopen questions concerning structure and function of intrinsically disordered proteins.

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EPR spectroscopy in the study of 2D graphene-based nanomaterials and nanographites Antonio Barbon DOI: 10.1039/9781788013888-00038

Graphene-based nanomaterials and nanographites represent 2D and 3D materials, where the transition from one type of materials to the other is without clear boundary. In this type of materials, where the leitmotif is represented by the presence of single or multi-stacked graphene layers, EPR spectroscopy has been fruitfully employed for structural characterization, as well as for the quantification of paramagnetic defects and for the study of magnetic properties. In this type of carbon-based materials, a fundamental role is played by two main actors: (a) conduction electrons, belonging to the extended p-system, and (b) edge states: electrons described by wavefunctions of limited extension associated to zigzag termination of the graphenic layers. A strong interaction exists between conduction and localized electrons, and in the presence of other minor paramagnetic contributions like other types of defects (crystal vacancies), or so-called molecular states (very small graphenic fragments), a vast spectrum of magnetic responses is obtained from the materials, from a ferromagnetic to an antiferromagnetic behavior. In this Chapter, methodological and introductory Sections are followed by a list of examples which highlight the use of EPR in this field.

1

Introduction

This Chapter focuses on the use of the EPR techniques for the characterization of graphene-based 2D nanomaterials and nanographites. These structures form a rather vast class of materials that play a prima donna role in the literature during last twenty years. A strong impulse was given to the research in this area as a Nobel prize was awarded to Geim and Novoselov in 2010 for their work on graphene.1,2 Graphene-based materials have shown numerous different properties which spawn a considerable number of technological applications, including photovoltaics,3–5 electronics and spintronics,6,7 fibers and composites,8 drug delivery,9 energy storage,10 catalysis and photocatalysis;4,11–13 here EPR also has been given a contribution. The selection of nanomaterials presented here is based on the presence of single or multi-stacked graphene nanosheets in the material. Given the diversity of structures belonging to this category of materials, the choice was not a simple one. Though a 2D material, nanosheets have a relatively high tendency to stack and to form platelets; as the number of layers increases, their properties approach those of nanographites, 3D materials. As mentioned earlier, the transition from graphene nanoparticles to nanographites is continuous rather than discrete, with no clear boundaries between the different structures. Geim set to 10 the number of stacked layers that exhibit a real 2D character,14 but this Department of Chemical Sciences, University of Padova, Via Marzolo 1, Padova, 35131, Italy. E-mail: [email protected] 38 | Electron Paramag. Reson., 2019, 26, 38–65  c

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threshold depends on the spectroscopy technique used to measure properties. Nanographites were included in this discussion because the transition from few-layer graphene materials to nanographites is blurred and also because these structures are present in many syntheses, moreover, most of the materials discussed here have characteristics that can be considered as intermediate between those of graphene and nanographites. Finally, graphene oxide (GO) and reduced-GO (RGO) were added to the discussion because of their importance for practical applications. Many of their properties are determined by the nanographenic fraction that is preserved within the material even when containing a high percentage of non-carbon atoms (proton and oxygen atoms). Graphene is the parent structure of all 2D carbon-based materials; ideally, it is an infinitely-extended flat structure made of carbon atoms that are all sp2 hybridized and organized as condensed hexagons. Graphene-like materials can be produced with a variety of techniques,15 that lead to the formation of particles with hetero-disperse dimension, shape and topology. This accounts for the high demand for spectroscopic tools able to provide information on the structure and the properties of these materials, as well as for the detection of the presence of defects, such as discontinuities in the structure, vacancies, imperfect stacking of layers, alteration of the sp2 structure, that are responsible for a dramatic change of the material properties.16 So far, Raman spectroscopy has been the most used technique for their characterization.17,18 EPR does not offer the spatial selectivity of Raman, as the samples normally consist of a quantity of material of the order of mg, whereas Raman is able to focus even on the single nanometric-size flake. This limitation for EPR can be also considered an advantage, as EPR gives information on the whole sample. Also, Raman is currently used for the determination of the dimension of the crystallites (the fraction of nanoparticle that preserve high order) based on the ID/IG parameter (the ratio between the Raman intensities of the bands attributed to defects, ID, and that typical of graphenic breathing mode, IG).19 However, this parameters was recently found to be related to the electron spin relaxation T1,20 showing once more the potential of the EPR spectroscopy for the study of graphene-based systems. A drawback of the EPR spectroscopy is that the cw-EPR spectra produced by these materials are composed of only a few lines. They do not exhibit sufficiently resolved hyperfine interactions, and only sometimes they appear to be inhomogeneously broadened because of g-anisotropy. The literature reported here shows that more work is required for this class of materials which possess a large variability in terms of dimension, shape or disorder. Currently, spectroscopic data are mostly used to confirm the presence of the few sample structures for which the properties are well established. So far, the properties and the origin of the EPR signals have been interpreted based on a general model describing the features of the nanoparticles. It is, in fact, assumed that the nanoparticles have distinct regions that are sketched in Fig. 1: (a) a core region, having the properties of bulk materials, like the infinitely extended graphene or graphite crystals, (b) a crown portion of the nanoparticle, close to the termination Electron Paramag. Reson., 2019, 26, 38–65 | 39

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Fig. 1 Schematic model of a bulk graphitic/graphenic flake (core region, of size La) surrounded by a crown region, inferring the material specific properties, distinct from those of bulk material. The ultimate edge contains all imperfections of the structure like chemical disorder, reacted edges and small graphene fragments.

of the particle, where most, but not all, of the imperfections of the ordered hexagonal motif are found, (c) an ultimate edge, characterized by a structural and possibly chemical disruption and reacted or reconstructed edges of the graphenic structure, also with the formation of ultra-small nanoparticles that resemble more the molecular states than extended graphenic states. Additionally, for the interpretation of the EPR data and to complete the entire picture, we have to consider the presence of defects within the core region (vacancies, reorganizations. . .). In sufficient quantity, such defects may modify bulk core properties. Nevertheless, only vacancies have been discussed by EPR; and their effect has been treated like that produced by a termination of the flake, in fact, it has been shown, not only by EPR,21 that such internal core defects are able to scatter electrons, like edges do.22 In this Chapter the following two Sections will be devoted to general electronic and magnetic properties of the ideal and real structures giving origin to the EPR signals, and to the spectral effects generated by adsorption of gases or metal ions on these signals. A further Section will display the methods used for interpretation of the EPR spectra. Finally, the results obtained from the study of different types of materials will be given in the remaining Sections. To conclude this Introduction, the reader should be warned that, even if a big effort has been done for the classification of the structures present in graphene-based materials,23 a consolidated nomenclature in the scientific arena has not yet been reached making it difficult to assign a particular structure to one of the categories.

2 Electronic and magnetic properties of graphene and graphite sp2 hybridized carbon atoms can form planar structures in which the pz electrons form delocalized p networks. In graphene the p density extends all over the plane and it is the result of the summation of the densities of the very delocalized single-electron wavefunctions. The energies of these functions are grouped in two bands, whose structure, for the ideal graphene, has been theoretically studied by Wallace24 and Coulson25 at the 40 | Electron Paramag. Reson., 2019, 26, 38–65

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Fig. 2 Sketch of the Density Of States and population of the states at 0 K for (a) the ideal graphene and (b) non-ideal graphene.

Fig. 3 Temperature dependence of the spin susceptibility (red squares, rising at low temperature) of few-layer graphene obtained by scotch-tape method. The susceptibility has been measured from the EPR intensity. Blue squares represent the spin susceptibility after the subtraction of a Curie contribution. The inset shows a sketch of the conical band-structure model for graphene in the vicinity of the neutrality point. Reprinted with permission from ref. 27, Copyright r 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

time when graphene was not experimentally known. Graphene is a unique zero-bandgap semiconductor, with a Density Of States (DOS) distribution sketched in Fig. 2a, thus, at 0 K, only the lower Valence Band is completely filled, and the higher energy Conduction Band is completely empty. Conduction and, partially, magnetic properties derive from thermic excitation of electrons from the valence band to the conduction band. The thermal population of the states was used by McClure et al. for a theoretical estimation of the magnetization, for both graphene and graphite, as a function of the temperature.26 The temperature dependence of magnetization is linear with positive slope,27 in stark contrast with the standard Curie paramagnetic behavior (see Fig. 3), where the Electron Paramag. Reson., 2019, 26, 38–65 | 41

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magnetization is inversely proportional to temperature; also, a non-zero intercept is present because of a temperature-independent Pauli contribution. The stacking of layers does not change substantially the behavior of the material, and for a high-quality graphite crystal, the experimental verification of the linear dependence has been conducted by Wagoner.28 In non-ideal graphene-like materials, contributions of paramagnetic states, eventually coupled with either ferromagnetic or antiferromagnetic interactions, are due to the presence of different type of defects (sketched in Fig. 2b as the semi-occupied states at the Fermi level). These contributions emerge in particular at the lowest temperatures often resulting in a rise of the magnetization (see Fig. 3); this argument will be expanded in the next Section. 2.1 Defects, edges and their role for the magnetic properties In this Section most of the discussion will focus on the presence of edges and different types of defects, and their impact on graphene properties. In nanographites (stacked layers of graphene), apart from some details, the properties of the materials can simply be considered as the sum of the properties of the single layers (see K. Wakabayashi in ref. 29) and will not be presented separately. Besides the change of the magnetic properties induced into graphenebased materials by the inclusion of adatoms in the structures,8 the magnetic properties of nano-sized graphenes and graphites are significantly perturbed by the presence of interruptions of the crystal structure, in particular by zigzag edges.30–33 The termination of the basic planar crystal structure can topologically be distinguished in two forms, reported in Fig. 4a, that are classified as zigzag (vertical, left termination) and armchair edges (horizontal, bottom termination). Zigzag edges are able to localize electrons in the so called localized states, in fact, associated to these motif, there are wavefunctions that have specific characteristics: (a) they are not extended, rather they can be consider pseudo-molecular orbitals as they are a combination of a limited number

Fig. 4 (a) termination of graphenic planes with different topology: Zigzag (blue, left vertical termination) and arm-chair (red bottom horizontal termination). (b) electron density of the edge unpaired electron (represented by circles) calculated with a simple Hu ¨ ckel method. 42 | Electron Paramag. Reson., 2019, 26, 38–65

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of pz atomic orbitals of the carbon atom next to the edge; (b) these orbitals are energetically located at the Fermi level, and they are singly occupied like those represented in Fig. 2b. An example of this type of orbitals is given in Fig. 4b, where the orange circles represent the electron ¨ckel density; this localization effect is observed already by a simple Hu calculation. We can observe that the localization of the unpaired electron is along the longest zigzag edge and that no electron localization is found on the armchair terminations. In general, three or four zigzag carbon sites are sufficient to develop an edge state.34 EPR is able to detect the presence of unpaired electrons that are found in fragments with an odd-number of electrons, like for the case just presented, but, unexpectedly, unpaired electrons can even be found in structures with an even number of electrons under particular circumstances: in the presence of either low-lying open shell states35 or of a peculiar topology, in particular of the edges, as discussed below. Open shell states in molecules with an even number of electrons are typically found for excited states, but for a series of polyacenes DFT calculation has shown that the energy splitting between the lowest-energy triplet and the (close-shell) singlet ground state progressively reduces when the number of fused rings increases; for acenes longer than eight rings there is even an inversion of the order of the states as displayed in Fig. 5, i.e. the ground state of the molecule is a paramagnetic open shell triplet state.36,37 This can explain both the high reactivity of longer acenes and the difficulty in obtaining some of them by chemical synthesis.38 For our purpose, it accounts for a possible presence of open-shell ground state, where agglomeration of the material can prevent the reaction and extends the state lifetime.

Fig. 5 Singlet-Triplet energy separation of acenes with increasing lengths according to DFT calculations (B3LYP/cc-pVTZ for singlets and UB3LYP/cc-pVTZ for triplets). The ground state is predicted to be a triplet for acenes containing more than 8 fused rings. Reproduced courtesy of M. Tommasini.36 Electron Paramag. Reson., 2019, 26, 38–65 | 43

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The topology also plays a fundamental role in the stabilization of openshell states. An interesting discussion on these aspects can be found in ´-like structures for some graphenic ref. 39: the inability to write Kekule fragments, with all electrons paired into double bonds, is responsible for the formation of open-shell ground states. One of the important criteria in examining whether molecules forming extended p-systems have nonbonding states (paramagnetic) is Lieb’s theorem:40 the calculation starts with the starring of carbon atoms so that, whenever possible, every starred atoms is linked only to unstarred atoms. Finally, the number of nonbonding states is obtained as the difference between the number of starred and unstarred carbon atoms.40 For example, for triangulenes, formed by m-rings on the edges, the number of unpaired electrons can be deduces from the expected maximum multiplicity spin S ¼ 12(m  1).41 Regrettably, apart from some particular cases, there is no general theory for the prediction of the number of unpaired electrons in complex structures.42 Another aspect of particular relevance for the magnetic response of the materials containing unpaired electrons, is the question of whether these electrons are magnetically independent or correlated. Emblematic is the case of graphene oxide reported in ref. 43 and discussed further in Section 7, where similarly prepared samples exhibited antiferromagnetic, paramagnetic or ferromagnetic behavior, suggesting the co-presence of the different types of interactions between the electrons within the material. Because conduction electrons give a paramagnetic Pauli contribution, these properties are mainly governed by defects, and in particular by edge states. Different interaction mechanisms between edge electrons are invoked, depending on their localization as they can be on the same edge, on different edges of the same flake, or on edges of different flakes. A number of papers report that edge-state spins are strongly coupled to each other through ferromagnetic intra-zigzag edge interactions.29,33,44,45 The direct exchange interaction J0 is estimated to be of the order of 103 K, more than enough to spin-polarize the interacting unpaired edge electrons. Moreover, in a nanographene sheet with zigzag edge regions separated by the presence of armchair edges, an antiferromagnetic interzigzag edge coupling is active, having a strength J1 of ca. (101–102) J0.45 Such a high value is accounted for by the presence of conduction electrons, which act as mediators of magnetic interactions, as discussed further. Finally, interaction between electron belonging to different sheets in stacked graphene layers is antiferromagnetic in nature and its strength (generally weaker than J1) depends on the details of the stacking structure of the sheets and on the geometry of the periphery of each sheet in the stack.46,47 Conduction electrons are strongly interacting with localized electrons, in particular with edge electrons, in a similar way as in metals (Kondo effect).48 Kondo effect accounts for an anomalous component of the resistivity as a result of spin-flip scattering of the conduction electrons coupled anti-ferromagnetically with localized magnetic moments. This interaction has implications for the relaxation properties of the spin system, as it will be shown in Section 6, but mobile electron spins can 44 | Electron Paramag. Reson., 2019, 26, 38–65

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also act as mediators of magnetic interaction between electrons in different edge states if they retain a coherent state for a time sufficiently long to allow the interaction with edge electrons of different terminations. The so-called Ruderman–Kittel–Kasuya–Yoshida (RKKY) coupling45,49,50 is a mechanism able to explain, in graphite or in graphene-like materials, the coupling of electrons in nearby edges with both ferromagnetic or antiferromagnetic interactions. Saremi49 reported that RKKY coupling between magnetic impurities is ferromagnetic only if the edges belong to the same sublattice and antiferromagnetic for edges in different sublattices. So far we have considered the contribution to magnetization deriving from unpaired electrons in p-orbitals, but we expect that additional unpaired electrons can derive from the presence of dangling bonds at the terminations or at the vacancies. Few papers deal with the presence of unpaired electrons in dangling bonds, as their presence is thought in general not to be very relevant for the ability of 2D structures to reconstruct and saturate vacancies bonds.16,51 Graphitic materials produced from diamonds exhibit the same EPR defect as that found in nanodiamond ( g ¼ 2.0027, LWpp ¼ 0.85 mT), that is attributed to electrons in the sp3 orbital.52 Unfortunately, the overlap of the signals, and, likely, their low concentration prevents a clear-cut determination of these species in structures other than those produced from diamonds. T. Enoki in ref. 47 discusses the differences between edge electrons and dangling bonds. In this case, the identification of dangling bonds required other techniques like NEXAFS. To conclude this Section, a remark is devoted to the existence of other possible structures, with relatively small dimensions: small graphene fragments or islands inside the graphene structure where the p-system is very limited in extension because of the presence of carbon vacancies or crystal faults. The description of such p-electrons is rather close to that of molecules, being described in terms of a limited numbers of pz. These states, then, can be referred to as molecular states.53 It’s worth noting that these states have few chances to interact with delocalized electrons as they can be considered isolated islands, moreover, they are expected to have also low interactions with other paramagnetic centres, like for edges, because RKKY mechanisms are excluded. Consequently, they are characterized by long T1 and T2 values, in particular at low temperatures. As such, they can be conveniently studied by pulse techniques for the determination of spin relaxation properties, and for the resolution of eventual hyperfine interactions.

3

Adsorption of gases and of metal ions

Gases inside the sample tube containing graphene-like and nanographite materials are responsible for several effects. First of all, admission of gases into the EPR tube favour heat exchange with the environment, and this fact is particularly important when working at relatively high powers (102 mW in cw-EPR) as the sample can undergo heating problems. Both intensity variation and g-shift phenomena have been observed in this Electron Paramag. Reson., 2019, 26, 38–65 | 45

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case, that are simply explained by the temperature dependence of both the g-values and the intensity (see the discussion in Section 2). The presence of gases inside the tube can then attenuate heating problems of the sample by improving of heat exchange with the environment.54 Besides this physical effect, oxygen is adsorbed at the surface of nanosized graphene and graphite materials. Interaction can be both physical18,55 or chemical in nature.56 A careful study of the effect of oxygen on the linewidth has been conducted by J. Joly et al.46 who considered the effects of pressure and temperature (see Fig. 6). In a fresh sample it has been observed that below 100 K the effect is very small, as compared to the temperature range 100–200 K, due to condensation of oxygen molecules on the surface of the flakes. In the condensed form, oxygen is less efficient in inducing spin relaxation because the first layer of condensed molecules can undergo a partial charge transfer from the flakes (chemisorption) and turn into a diamagnetic state, thus unable to induce spin relaxations. On rising of the temperature, as chemisorption is a process partially reversible, a progressively increasing quantity of adsorbed oxygen can be desorbed and reform free molecular oxygen, resulting in significant linewidth broadening. Another effect of the interaction with oxygen may be observed in ballmilled graphites. A non-reversible chemisorption of oxygen has been observed from the significant quenching of the EPR intensity (nearly an order of magnitude) upon oxygen exposure of a freshly prepared sample (see Fig. 7).51 The milling of the graphite produced a quantity of reactive sites (possibly characterized by dangling bonds) with paramagnetic character. These sites are spontaneously able to reorganize with time as discussed in Section 2.1, and further in Section 5.1, but in the fresh-prepared

Fig. 6 Temperature dependence of the EPR line-width of nanographites (ACF) under oxygen pressures 0 (vacuum), 0.1, 1, 5, 10 and 20 Pa. Reprinted with permission from ref. 46, Copyright 2009 Elsevier Ltd. 46 | Electron Paramag. Reson., 2019, 26, 38–65

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Fig. 7 CW-EPR spectra at 100 K measured on ball-milled graphite samples (circles): (a) as-prepared, (b) aged in an Ar atmosphere for several months, and (c) immediately after exposure to air; please note the intensity in (c) is multiplied by a factor of 20. Solid red line is a fit, while dotted lines represent the individual components used to fit the spectra. Reprinted with permission from ref. 51, Copyright 2011 American Physical Society.

sample, where the concentration of these centers is high, a large part can by quenched by chemical reaction with the oxygen. A peculiar and apparently opposite effect upon oxygen exposure has been observed in RGO: in the initial purified sample, kept under vacuum, no EPR signals were observed, whereas, a signal appeared upon exposure to oxygen.57 In this case, initially, the electrons are very mobile, they diffuse through the sample and are not observed by EPR; upon adsorption of guest molecules at the surface of graphene layers, potential barriers for electron hopping are created which reduce the charge mobility and allow for their observation. With respect to oxygen, gas molecules of HNO3 can also intercalate within the graphene sheets in graphite, generating superficial layers of intercalated graphite on top of buried bulk graphite. The resulting materials have specific characteristics typical of metals sprayed over another metal substrate58 with altered g-values with respect to those of graphite: g8 ¼ 2.0019  0.0002, and g> ¼ 2.0030  0.0002. The boundary contact between the intercalated volumes and the non-intercalated volumes are assumed to be non-conductive.58 A significant interaction with the surface of the layers is established also with metal ions. Of particular interest is the interaction of Mn, extensively used for the production of graphene oxide. EPR, in Electron Paramag. Reson., 2019, 26, 38–65 | 47

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combination with NMR, has proven Mn to be anchored to the graphene oxide planes.59,60 Broad bands are then observed, which can dominate the EPR spectrum. The observation of well-resolved hyperfine Mn21 structure indicated that ions exist in magnetically diluted paramagnetic complexes rather than in magnetically concentrated Mn salts, thus graphenic layers offer the possibility to provide ion dispersion in a solid matrix without the need of a solvent.

4 Resolution and analysis of the EPR spectra The cw-EPR spectra of this type of carbon-based materials, as stated in the Introduction, are relatively simple, and composed of one or few lines. Due to the heterogeneity of the samples, the spectra may include several overlapping signals15,61 and the first concern, when approaching these samples, is relative to the separation of the different contributions. Often the lines are simple Lorentzians but they might occur with different lineshapes and in some cases they are inhomogeneously broadened because of g-anisotropy. The simulation of the cw-EPR spectra generally allows for the determination of two or three major contributions at most, and requires sometimes a particular care in order to correctly determine the spectroscopic parameters, like the g-factors, in particular when the spectrum presents bands with Dysonian lineshapes. This type of lineshape can be found also in nanopowders composed of particles whose spatial dimension is smaller than the skin depth,62 but allows for electric contact among them.54,63–65 Under these circumstances, the skin depth depends on the macroscopic conductivity of the overall sample and, in turn, the conductivity depends on the electric contact between the particles. This parameter is not a fixed characteristic of the material, and depends on the state of the sample: electrical contacts are improved by rising the compactness of the sample64 and they are reduced by dispersion of the nanoparticles in an inert matrix. 54 Consequently, different preparations of the sample (compression of the powder, dispersion in an inert matrix. . .) or the use of tubes with different sizes can result in EPR spectra with different lineshapes. As anticipated in the introduction, the EPR spectra of these materials are characterized by the presence of bands attributed to electrons having, in general, quite different mobility. Moreover, different mobility is associated to a proper lineshape function fi0 ½B  B0 ðOÞ that in general can be Lorentzian, Gaussian or Dysonian (eqn 1).66 In Fig. 8 a clear experimental example of cw-EPR spectra where all three types of contributions are simultaneously present (Table 1 reports the detail of the various contributions). We focus now the attention on the problem of the simulation of the component with Dysonian lineshape. The problem arises because of the heterogeneity of samples prevents the use of a single standard formula for it. On the other hand, inaccurate parameters are obtained if regular Lorentzian or Gaussian lineshapes are used instead.54 According 48 | Electron Paramag. Reson., 2019, 26, 38–65

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Fig. 8 cw-EPR spectra of the sample nanographite produced by ball-milling (6 h) of a pure-carbon graphite: solid line is the experimental spectrum and dashed line the simulation. The EPR spectra have been simulated as a sum of three contributions: one Dysonian, one Gaussian and one Lorentzian, with relative weights that depend on the temperature. Reprinted with permission from ref. 21, Copyright Springer-Verlag 2011.

Table 1 Fitting parameters of the cw-EPR spectra of fig. 8. LW is the homogeneous line width for the different components indicated in the table, present with different weight (wt%). For the g tensor, only the average value could be determined. Reprinted by permission from Springer, A. Barbon, M. Brustolon, Appl Magn Reson (2012) 42:197–210 r Springer (2012) https://doi.org/10.1007/s00723-011-0285-6.21

Dysonian

Gaussian

Lorentzian

T/K

w%

LW/mT

hgi

280 70 7 280 70 7 280 70 7

50 46 41 50 42 32 o5 12 27

7.0 9.0 5.7 10.0 8.5 8.0 0.3 0.24 0.24

2.01355 2.0101 2.0071 2.0184 2.0288 2.0004 2.00334 2.00344 2.00404

to Joshi et al.,67 the homogeneous Dysonian lineshape can be fitted by using a phenomenological expression that, in derivative form, reads as:21 f 0 ðB  B0 ðOÞÞ /

a 8½B  B0 ðOÞ  fG þ a½B  B0 ðOÞg  4½B  B0 ðOÞ2 þ G2 f4½B  B0 ðOÞ2 þ G2 g2 (1)

where G is the homogeneous linewidth, and B0(O) the resonance field, B0 ðOÞ ¼ hn exp =½ geff ðOÞb, which depends on the effective g-value for the particular orientation O of the magnetic field in the g-tensor frame. For each contribution, characterized by a proper lineshape, the overall spectrum is then calculated as a powder spectrum by integration Electron Paramag. Reson., 2019, 26, 38–65 | 49

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over all orientations. Finally, all contributions are summed up with a weight wi: 15 0

I ðBÞ /

X

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i

ð 4p wi 0

0

fi ½B  B0 ðOÞdO

(2)

the i index runs over all resolved species contributing to the spectrum. As stated earlier, a careful simulation of the spectra, like that shown in Fig. 8, typically allows to disentangle the presence of two-three components. Other minor contributions might be overlooked because of their relatively low intensities, but some of them can be revealed by using other methods, like, for instance, Echo-Detected EPR (ED-EPR)68 and Fourier Transform-EPR (FT-EPR) techniques.61,69 Both ED-EPR and FTEPR suppress the intensity of the species which dominate the cw-EPR spectrum, normally with a fast spin relaxation times, and thus they act like a filter that allows the observation of (minor) species with slow relaxation times. As the spectra of these species are not very broad, one has to consider the acquisitions of the spectra with high spectral resolution. Although in solid state phases echo techniques are usually most informative,68 in this specific case, with limited or absent hyperfine interactions, the use of FT-EPR is convenient.70 With this technique, the spectral resolution is directly related to the reverse of the acquisition time, consequently, long Free Induction Decays (FIDs) have to be recorded.71 Please note that these systems are ideal for acquisition of dead-free FIDs: by using an echo experiment, the FID is obtained from the echo time profile, as the echo is formed by two symmetrically disposed FID’s. ED-EPR spectra are obtained from the measurement of the 2p echo intensity as a function of the magnetic field. For improving the spectral resolution and to avoid distortions, selective pulses are required together with long integration times of the echo intensity. Moreover, the variation of the delay between the pulses can reveal also contributions with different relaxation properties; a spectrum profile variation upon change of the delay is a clear indication of the presence of more than one species. After these initial Sections devoted to the general presentation of the EPR of the materials considered in this Chapter, a presentation of the results obtained by analysis of the EPR spectra are given for the different categories of materials.

5

Nanographites

Nanographites generally share properties with their parent material, graphite. From an EPR point of view, the most relevant one is the presence of a large g-tensor anisotropy.28,72 This property is attributed to the stacking of layers, to inter-layer interactions and to the mixing of 3d orbitals with p-bonds, that makes the tensor temperature dependent.26,72 Interaction between layers is required as McClure has found that the calculated average gz-shift in graphites vanishes in the absence of 50 | Electron Paramag. Reson., 2019, 26, 38–65

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interaction due to electrons and holes having the same density of states but opposite g-shifts.26 A further confirmation of this requirement was given by an experimental study where hydrostatic pressure was used to modulate the interplane-distance, and consequently the interaction between layers.73 The study has revealed clearly a sensitivity of the gz component to compression, having the larger deviation from ge with respect to the in-plane components. Given that in nanographites all the above mentioned effects due to the stacking of the layers are also present, the similarity of the properties between graphite and nanographites is not so general. In the coming Sections it will be shown that nanographites exhibit the larger variability of g-values, both in terms of average values and in terms of g-anisotropy. Anisotropies have been reported to be as high as Dg ¼ 0.02, reminiscent of bulk graphite,21,74 or close to zero, with components (isotropic) below ge.75,76 This variability depends mostly on extrinsic factors, like the sample conductivity, but also on intrinsic factors, like the dimensions of the particles. Conductivity of the samples might have impact on the measured g-values: the ability of electrons to diffuse through the sample can reduce the measured anisotropy.21,77 For instance, a fast motion through a large number of randomly oriented particles produces a partial or a total averaging of the principal components of the g-tensor,77 moreover, this may result in the observation of EPR bands with an isotropic value and an unexpectedly high g-values.21,69 Disorder, or different order in the stacking of the layers, also has an effect on the measured principal values: different ordering of the layers from the standard AB stacking (hexagonal graphite), like in the case of ABC stacking (rhombohedral graphite), can give a gz component as high as 2.24 at room temperature.78 The remaining part of this Section will present the EPR studies conducted on nanographites prepared by using different methods, and showing the specificity of nanographites with respect to graphite. They will show that often, but not always, the conduction electrons are characterized by broad signals due to their g-anisotropy or to their short T1 values. Alongside these signals, narrow lines are present that can be related to the defectivity of the sample. 5.1 Ball milled nanographites Nanographites obtained by ball milling are often characterized by the presence of two types of well-separated EPR bands: (a) broad components, with linewidths up to some hundreds of Gauss, and (b) narrow bands.21,51 The milling of the samples has the effect of enhancing the narrow component while the broad components tend to reduce; nanographites produced by prolonged milling might lack this broad components.20,21,69 The narrow component is in general attributed to different types of defects (localized spins), whose concentration rises with prolongation of the milling. Typically, the number of spins are of the order of 1017–1020 spin per g,21,69 the higher value was obtained in a sample after Electron Paramag. Reson., 2019, 26, 38–65 | 51

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80 h milling. The narrow linewidth is indicative of a strong exchangenarrowing limit between the mobile electrons and the localized electrons in the different types of defects (homogeneously broadened Lorentzian bands).51 This type of interaction has been experimentally investigated in detail for graphene,79 and a further discussion can be found in the next Section. In the ball-milled nanographites, the presence of mobile electrons has been determined not only from the appearance of the broad component, but also on the basis of both antiferromagnetic exchange interactions51 and Pauli contributions at room temperature determined for the observed single component with narrow linewidth. A rather high value of wCW/wPE0.7 has been determined in prolonged ball milled graphites and analyzed in Fig. 9. This implies that the contribution of itinerant electrons can be substantial also in absence of a visible broad band. Likely, prolonged milling produces a quantity of very small pieces of graphite where, due to the limited extension of the layers, mobile electrons have a markedly different behavior with respect to those of graphite, and get closer to a behavior typical of nanoribbons (see further). For ball-milled nanographites, eventually exposed to oxygen and showing a single narrow band, the EPR intensities as a function of the temperature are reported in Fig. 9. A paramagnetic Curie-like behavior is obtained with the sample freshly prepared in inert atmosphere, in which dangling bonds have not yet reacted with gases, like oxygen, or vapors, like water. The quenching of these sites either with aging of the sample or by reaction with oxygen, makes edge states dominate the EPR spectrum; edge states exhibit antiferromagnetic exchange coupling character.51

Fig. 9 Temperature dependence of the EPR spin susceptibilities for as-prepared ballmilled nanographite (circles), aged in inert atmosphere (squares), and air exposed (triangles). Solid lines represent fits to a sum of Pauli temperature-independent and Curie–Weiss spin susceptibilities with different antiferromagnetic exchange interaction. Inset: Temperature dependence of the inverse EPR spin susceptibilities for the same samples. Reprinted with permission from ref. 51, Copyright 2011 American Physical Society. 52 | Electron Paramag. Reson., 2019, 26, 38–65

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Fig. 10 Linear correlation of Raman intensity ratios (ID/IG), measured with different excitation wavelengths, vs. spin lattice relaxation time (T1) revealed by EPR on nanographites obtained by ball milling for different times. Ball milling produces a shortening of T1. Adapted with permission from ref. 20, Copyright r 2011 Elsevier B.V.

The antiferromagnetic character is, unexpectedly, found also for nanographites showing a broad components with Dysonian lineshape, in stark contrast with the typical trends found in graphites.21 This divergent behavior, specific of nanographites, accounts once more for a strong correlation between conduction electrons and localized electrons in highly defective materials, which makes both types of electrons belong to the same bath.21 It is interesting to mention the analysis conducted on the linewidth of the mobile electrons in nanographites within the model of Elliott–Yafet. In the model, defects are considered centers able to induce relaxation of mobile electrons by spin/orbit mechanisms where possible relaxation centers are edges, or other types of defects like sp3 bonding.80,81 Extrinsic spin–orbit fields, mainly due to ripples, in principle can also participate81 but they have been excluded: for nanographites, the correlation between the density of defects (whose value was obtained from the Raman spectroscopy ID/IG ratio) and the T1 value (obtained from the homogeneous EPR linewidth) displayed in Fig. 10, supports the initial hypothesis of the Elliott–Yafet mechanism as dominant for the spin relaxation for conduction electrons.20 5.2 Nanographites from nanodiamonds EPR spectroscopy was used to reveal a structural phase transition of nanodiamonds (detonation-synthetized with size of 4–5 nm) to hollow, onion-like, polyhedral nanographite structures.52 This transition, characterized by an increasing sp2/sp3 ratio upon heat-treatment, produces at first quasi-spherical followed by polyhedral nanographites. Initially the spectrum is characterized by a line with LWpp ¼ 0.85 mT and g-value of 2.00265, attributed to dangling C–C sp3 bonds, and located in the sp3 core or within the surface layer of the nanodiamond (spin concentration around 1019 spins per g). Upon the heating of the sample at high Electron Paramag. Reson., 2019, 26, 38–65 | 53

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temperature, the graphitization process starts. The room temperature cwEPR spectra report a decrease of the narrow signal (insensitive to oxygen), while a broad signal (gB2, LWpp ¼ 10.9 mT) starts to develop and at the same time to narrow with the prolongation of the treatment. For 120 min thermal treatment, only this last component is observed with g ¼ 2.0013– 2.0014, LWpp ¼ 1.14–2.33 mT. Andersson et al. in similarly prepared systems could observe the broad component only by cooling the sample below 170 K.76 Interesting is the observation obtained by B. L. V. Prasad et al. who estimated, on average, a single free electron in each nanographite particle.75

6

Mono and few-layer graphenes

EPR has been applied to the study of single-layer graphene in a quantity of the order of 101–102 mg. Fig. 11 reports the cw-EPR spectrum of single-layer graphene obtained by the scotch-tape method.27 The spectrum is essentially composed of a single Lorentzian line, with a g-value varying from 2.0040 ca. at temperatures close to liquid helium temperature, to 2.0045 at room temperature.27 The linewidth, in the same temperature interval, varies from ca. 9 to 6 G. The intensity variation of the line is reported in Fig. 3 and it is consistent with the variation expected from conduction electrons: a decrease of the temperature cause a depletion of the conduction band. A small additional contribution from paramagnetic states, observed from the mild rise of the intensity at the lowest temperatures, accounts for a low defectivity of the sample.

Fig. 11 cw-EPR spectrum of graphene obtained by Scotch-tape method at 150 K. Reprinted with permission from ref. 27, Copyright r 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. 54 | Electron Paramag. Reson., 2019, 26, 38–65

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Beside the scotch-tape method, chemical-vapor deposition (CVD) has been used for the preparation of high quality single layer graphene sheets.82 However, observation of EPR signals is possible only after the samples have been kept under high vacuum for several days.83 Apart from the similar intensities, the g-value (g ¼ 2.00245  0.00005) differs substantially from that of the samples produced by the scotch-tape methods, moreover, the variation of the intensity with the temperature has a Curie– Weiss dependence, and it is characterized by an antiferromagnetic coupling. The observed line in CVD-prepared samples is attributed consequently to the presence of localized electrons: edges or vacancies. However, for large flakes, vacancies are expected to dominate over edges not only for a geometrical reason, but also because the intensity has been observed to vary with time, in a fashion coherent with a reorganization of highly energetic defects, like vacancies.83 Vacancies and internal defects, created by irradiation, have been studied by S. Just et al.84 A single line appears upon Ar1 bombardment, composed by a mixture of Gaussian and Lorentzian lineshapes, and with g B 2.002. This mixing indicates that both broadening due to exchange narrowing (Lorentzian) and unresolved inhomogeneities of the local g-factor or dipole–dipole interactions (Gaussian) contribute to the linewidth. On top of this, antiferromagnetic or ferromagnetic interactions have been found in samples irradiated with different Ar1 fluence, whereas inhomogeneities have been revealed from the slightly different g-value observed by orienting the sample with the plane parallel or perpendicular to the magnetic field. EPR was used on CVD-produced graphene also to determine the spin relaxation time of conducting electrons and to help resolve the busillis of a short experimental relaxation times, when theory suggested long spin lifetimes.79,85 Augustyniak-Jabłokow et al. recorded single-Lorentzian line spectra around room temperatures with g-factor of 2.00245  0.00005. On cooling of the sample down to 9 K the g-factor did not change, whereas the linewidth LWpp ¼ 0.6 G of the Lorentzian line decreased linearly with the lowering of the temperature. The analysis of the temperature dependence of the relaxation times was conducted by means of a set of Bloch–Hasegawa equations in the form proposed by Barnes. Within the frame of a strong exchange coupling limit, the localized spin angular momenta are allowed to be transferred to the conduction electrons, and subsequently transferred back before a spin/lattice relaxation event. From the solution of the equations, they obtained a linear dependence of the linewidth with temperature:79 1 1 1 ¼ þ const T ¼ a þ bT Teff TsL Tel

(3)

where the spin relaxation rate 1/Teff is a combination of the spin–lattice relaxation time TsL and the conduction electrons spin relaxation Tel. This result is rather close to that obtained from the Korringa model for metals, which retains the linear dependence on the temperature.86 AugustyniakJabłokow et al. have finally concluded that the interaction between Electron Paramag. Reson., 2019, 26, 38–65 | 55

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mobile and localized electrons can be claimed as the main source of spin relaxation, given that the Kondo mechanism was proven to be effective in graphene.87 In other words, due to this interaction, electrons can easily pass from the localized bands to the conduction one and back thus further generating the exchange narrowing of the signal and the averaging of g-tensor anisotropy.79 ´ra et al. for graphene as An analogous result was reached by B. Do obtained from Li-intercalation of a high pure graphite.85 They concluded that the main source of spin relaxation for conduction electrons is an intrinsic spin–orbit coupling, ruling out other possible mechanisms, namely Bychkov–Rashba, and ripple spin–orbit coupling-induced spin relaxation.85 Here ‘intrinsic’ is intrinsically associated to ‘edges’. 6.1 Nanoribbons Under the label nanoribbons falls a family of graphenic structures characterized by small dimensions. The magnetic properties of these structures are dominated by the presence of localized edge states in the zigzag-shaped edge regions, together with conduction electrons;88 the materials can exhibit an interesting interplay between magnetism and electron transport similar to what found in traditional s-d metal magnets.88 Nanoribbons produced by CVD were studied by V. L. J. Joly et al.88 The CVD synthesis was followed by a thermal treatment of the material at high temperature to favor self-healing of lattice faults and the coalescence of edges, with the aim of controlling edge formation. EPR spectra of samples annealed at different temperatures show that a thermally activated graphitization process starts already for a treatment at 1500 1C, a relatively inadequate temperature, much lower than 2000 1C at which graphitization is normally induced. The EPR spectra attributed to the nanoribbons are composed by a narrow Lorentzian line whose intensity decreases with the rising of the annealing temperatures above 1500 1C. For the sample annealed at 1500 1C, the temperature dependence of both the linewidth (linearly dependent on the temperature) and the g-value (varying between g ¼ 2.003 at 4 K and g ¼ 2.005 at 300 K) made the authors attribute the line to the overlap of the signals of edge states and conduction electrons in the strong exchange limit as detailed in the following. Firstly, according to the Korringa model,45 or eqn (4), a proportionality is expected between T1 and the temperature. The line width, determined by the relaxation between the edge-state spins and the conduction carriers, is determined by the inverse of the relaxation time Tedge-p that can be expressed as:   1 4p 2 ¼ DðEF Þ2 kB T (4) LW / J Tedgep h edgep  where D(EF) is the density of states at the Fermi level EF and Jedge-p is the exchange interaction between the edge-state spin and the conduction p electrons.47 Secondly, in the strong interaction limit, a single Lorentzian band is observed, whose g-value is expressed in terms of a combination of the individual g-values weighted by their relative 56 | Electron Paramag. Reson., 2019, 26, 38–65

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intensities: g ¼ (gnwn þ gcwc)/(wn þ wc), where the labels n and c stand for edges and conduction electrons, and wi the relative magnetic susceptibilities. An alternative method of production of nanoribbons is by chemical unzipping of carbon nanotubes.89–91 An acid treatment of multi wall carbon nanotubes (MWCNT) with sulfuric acid, followed by oxidation with KMnO4 can form nanoribbons up to 4 mm long, with widths of 100– 500 nm and thicknesses of 1–20 graphene layers. Successive different types of reduction processes can partially restore the disruption of the p-system obtained during the oxidation processes. The EPR signals of the materials are characterized by high spin concentration (1018 spin per g) and almost temperature independent g-values of 2.0029–2.0032.89 Both the g-invariance and the Curie-type rise of the intensity with the lowering of the temperature made the authors attribute the signals to dangling bond or, in the most oxidized samples, to organic radicals related to the presence of –OH, –O, –CO and –CQO groups.89 Ferromagnetic interactions, found also in edge states, were obtained by unzipping of MWCNT in reducing conditions by using potassium like for the chemical exfoliated graphites (see next Section).92 The corresponding cw-EPR spectrum exhibits a single Lorentzian line with g ¼ 2.0025, nevertheless, HYSCORE68 measurements showed the presence of hyperfine interactions with protons of the order of 25 MHz, attributed to protons on the edges of the nanoribbon. Structures are formed with localized wave functions, extended over a limited number of carbon atoms. Exchange interactions of varying strengths among these structures are thought to be sufficient to induce ferromagnetic ordering.92 Subsequent high-field EPR studies confirmed that the lineshape is not fully Lorentzian,90 and has Gaussian contributions.

6.2 Chemically exfoliated graphites Within the methods considered for the production of graphene materials in a scalable way, chemical exfoliation of graphites has been investigated. ´rkus Exfoliation can be obtained in different ways, for instance, B.G. Ma et al. used intercalation of graphite with potassium, in combination with post-processing treatments: ultrasound treatment, shear mixing, and magnetic stirring.77 The produced materials have been characterized by EPR, and displayed the presence of a broad signal attributed to conduction electrons in graphitic/graphenic structures, with variable linewidth, and a narrow line, attributed to localized electrons. The process allowed obtaining one sample with the linewidth of the broad component close to that of a single-layer graphene. F. Tampieri et al.,15 with similar exfoliation methods and the use of either potassium or strong acids, followed by post treatments based on microwave thermal treatment and/or sonication, produced materials with a wide range of characteristics, from expanded graphites, with typical graphitic properties, to graphenes with a restricted number of layers. For this last type of samples, a careful analysis of the single-band EPR spectrum, (see Fig. 12), showed that the spectrum at high temperature Electron Paramag. Reson., 2019, 26, 38–65 | 57

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Fig. 12 Intensity variation of the two components of the cw-EPR spectrum (shown in the inset) of few-layers graphene obtained by exfoliation methods. Adapted from ref. 15 with permission from The Royal Society of Chemistry.

was an overlap of two main components and dominated by the signal due to mobile electrons residing in few-layer graphenes. Interpretation of the spectra required a series of spectral deconvolutions at various temperatures. It was found, in fact, that the dominant contribution to the spectrum at high temperature has both g-factor (g ¼ 2.0027) and linewidth (10 G) close to that of single-layer graphene.27 Moreover, the decrease in intensity with the temperature in the range 300–150 K appears consistent with a signal from electrons that were thermally promoted to the conduction band. Finally, minor contributions from molecular-type states have been obtained by using pulse techniques. Alongside the presence of conduction and localized electrons, smalldimension fragments of the order of the molecular size, as consequence of chemical expansion, have been detected by pulse-EPR. Such p-systems, formed by a limited number of carbon atoms, are better described by using a molecular approach. For example, these systems have a long spin relaxation time because of the lack of conduction electrons in direct contact,79 moreover, hyperfine interactions are not washed out. Echo detection is then the proper method to selectively determine these contributions. The spectra show that the small contribution evidenced by the use of ED-EPR68 (see Fig. 13(a)) is inhomogeneously broadened because of hyperfine interactions with protons as revealed by 2pulse-ESEEM: a rather intense proton band, with respect to that of 13C band, was observed (see Fig. 13b). Clearly it turns out that during the preparation process, likely because of the highly reducing environment, small graphene fragments have reacted and the edges have been passivated with protons. The width of the proton bands obtained by ESEEM or ENDOR have been used to have an estimate of the dimension of these fragments. It is possible, in fact, to compare the larger values of the dominant isotropic hyperfine interactions (amax) with those estimated for reference systems, like coronene (C24, amax ¼ 1.5 G) and circumcoronene (C54, amax ¼ 0.6 G).61 58 | Electron Paramag. Reson., 2019, 26, 38–65

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Fig. 13 (a) 2p Echo-detected EPR of a chemically expanded graphite; interpulse delay t ¼ 200 ns. Two species are present with g-values typical of organic radicals. (b) 2p-ESEEM spectrum of the same sample, showing the presence of coupled C and H atoms. Reproduced from ref. 15 with permission from The Royal Society of Chemistry.

7

Graphene oxide and reduced graphene oxide

Graphene Oxide (GO) is produced mostly by the Hammer method, which uses KMnO4 as a strong oxidant to partially disrupt the layers, and introduces heteroatoms. Beside this, other methods are available.43,93 GO consists then of graphene flakes which are decorated with oxidized hydrophilic organic groups15,57,94,95 like alcohols or ketones. Paramagnetic defects, like vacancies and dangling bonds, are generated within the flakes and at the edges, and they are observed by EPR.95 Reduction processes saturate these defects and decrease the oxygen content (typically from 10–30% to few %) by chemical reaction.57 During the synthesis a careful purification of the materials is required to remove Mn21 ions, as the latter avidly adsorb onto the graphenic planes (see Section 3). The material produced in this way is called Reduced Graphene Oxide (RGO). Compared with the other nanographene-based materials, RGO has a relatively high content of carbon atoms hybridized sp3. A commercially available RGO (Single Layer Graphene, ACS) has been studied by F. Tampieri et al.15,61 A combination of techniques (cw-EPR and pulse EPR) and the analysis of the temperature-dependence of the contributions to the spectra, see Fig. 14, allowed the identification of two main different components to the cw-EPR spectra at each temperature, all characterized by a Lorentzian lineshape. The nature of these major components in the cw-EPR spectra is different at high and at low Electron Paramag. Reson., 2019, 26, 38–65 | 59

Published on 02 November 2018 on https://pubs.rsc.org |

60 | Electron Paramag. Reson., 2019, 26, 38–65 Fig. 14 (a) cw-EPR spectra at different temperature of a commercial sample of RGO (Single Layer Graphene, ACS). The inset compares the integrated cw-EPR and the ED-EPR spectra at 20 K. (b) temperature dependence of the intensity of the two components of the cw-EPR spectra adapted from ref. 15 with permission from The Royal Society of Chemistry.

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temperatures. At high temperature there are species with relatively high g-values of 2.0031 and 2.0032 (components ‘1’ and ‘2’, respectively) which get smaller on the lowering of the temperature, in addition, also the intensities decreases. This behavior is close to that of quasi-ideal graphene (see Section 6) and is associated to conduction electrons.27 Below about 80 K, additional Curie-type contributions start to be dominant. The lines get narrower, and the g-values increase. These contributions have been attributed to localized (edge) states. Finally, ED-EPR spectra allowed the determination of further (narrow) contributions to the cw-EPR at low temperature (see Inset in Fig. 14a), characterized by a small intensity and Gaussian lineshape, thus inhomogeneously broadened. Indeed the ENDOR spectrum displayed hyperfine interactions with 13C and 1H.61 The presence of the 13C band and the strength of the proton hyperfine interaction as shown in the previous Section, allowed to estimate the spatial extension of the p-system of about 25 carbon atoms. ´ iric´ et al., exhibited a single RGO materials, differently prepared by L. C Lorentzian line in the entire studied temperature range (4–295 K) whose linewidth monotonically increases with the temperature as a result of a strong correlation between mobile electrons and localized electrons in the system. The magnetization of this sample has a more complex behavior: at low temperature the EPR intensities are dominated by species with Curie-like behavior.43 At high temperature, instead, the EPR spectra are dominated by conduction electrons, with the typical linear proportionality of the intensity on the temperature. Here it was possible to distinguish samples prepared in different ways: samples whose reduction process was conducted at higher temperature had a larger conduction electrons contributions, indicating a more efficient restoration of the p-system in this case. The GO materials used for the preparation of the RGO just described were separately studied.43 In this case the effect of the dimension of the graphenic flakes on the magnetic properties was considered. For the scope, GO with different size of the graphenic flakes were prepared: largesize (average surface B2500 mm2), intermediate-size (B1 mm2), and smallsize (B500 nm2). A very different response was obtained from these systems. Large-size flakes exhibit a metallic behavior while intermediate-size flakes showed an almost standard Curie-like behavior. An anomalous Curie law, with the susceptibility dropping faster than 1/T, was observed instead for the small-size flakes. The departure from the typical Curie law might result either from the increasing of the interaction between localized moments on decreasing temperature or from the thermal excitation of weakly localized spins to the conduction band. In this latter case, the intensity of the Pauli contribution to the susceptibility would be too low to be observable. Clearly these measurements confirm the strong effect of the particle size on the material properties.

8 Conclusion EPR has been applied to a variety of carbon-based 2D materials and to nanographites to obtain different type of information. The interpretation Electron Paramag. Reson., 2019, 26, 38–65 | 61

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of the cw-EPR spectra is normally conducted based on the presence of two major contributions deriving from conduction electrons, belonging to the extended p-system, and from edge states, electrons described by wavefunctions of limited extension associated with zigzag termination of the graphenic layers. The presence of both conduction and localized electrons play an important role in the magnetic response of the materials as revealed by EPR. Antiferromagnetic of ferromagnetic interactions of different strength between edge electrons localized in different edges have been found. Also, different interaction strength between edge states and conduction electrons has been observed, leading to the formation of either separate or overlapping EPR lines for the two types of spin centers. The interactions are responsible for efficient spin relaxations, falling often into the strong-exchange coupling limit, so that single Lorentzian lines are found for edge states in graphenic-like materials. The stacking of several layers is characterized by a broadening of the mobile electrons EPR band because in graphitic-like particles the interaction between layers favors the increase of the g-tensor, and this represent normally a fingerprint of the presence of such structures inside the materials. Finally, also different types of defects have been studied and characterized, like vacancies, s-bonds or small graphenic fragments. To conclude, this Chapter has shown how EPR can be applied to the study of carbon-based nanomaterials. All these data are precious for a careful structural analysis of the materials, besides the determination of the magnetic response, and they can also be exploited for the study of composite materials, often developed for technological applications, where nanocarbon materials, and in particular 2D graphenic structures, are used for their interesting properties.

Acknowledgements I would like to thank Prof. M. Tommasini (Politecnico di Milano, IT) for providing Fig. 5, and Prof. M. Brustolon (University of Padova, IT) and Dr Ernst van Faassen (University of Leiden, NL) for advice and suggestions during writing of the Chapter.

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Nitroxide spin labels: fabulous spy spins for biostructural EPR applications Published on 02 November 2018 on https://pubs.rsc.org | doi:10.1039/9781788013888-00066

a a Marle ` ne Martinho, Euge ´nie Fournier, a Nolwenn Le Breton,b Elisabetta Mileoa and Vale ´ rie Belle*

DOI: 10.1039/9781788013888-00066

Characterizing proteins in action requires appropriate biophysical techniques sensitive to protein motions. One of the technique dedicated to monitor protein dynamics is SiteDirected Spin Labelling combined with EPR spectroscopy (SDSL-EPR). The main purpose of this chapter is to describe and illustrate the different strategies based on the use of nitroxide spin labels either as reporters or as a means to measure inter-label distances. The complementarity of these different approaches to answer biological questions will be addressed. The objective is also to give non-specialist readers an overview of the recent developments in the field of SDSL-EPR dedicated to the study of protein dynamics. A particular emphasis will be devoted to describe the design and application of new nitroxide spin labels that allow overcoming the limitations of the classical ones.

1

Introduction

The view that we have in mind when we talk about biological macromolecules such as proteins is often related to their beautiful 3D crystallographic structures that give a general impression of static objects. But proteins are dynamic entities that need to move to accomplish their function. This dynamics allows proteins to change their conformation and to adapt their structure to the presence of other molecules (ligands, partner protein, cofactors. . .) or to variations of their environment (pH, temperature. . .). This dynamic behaviour can be on a small scale such as atomic fluctuations for some of them or, on the contrary, on large structural rearrangements for others.1 Characterizing this dynamics is a complex task that requires the use of appropriate techniques. In parallel with the development of methods leading to structure resolution of proteins, there is an increasing need to develop biophysical techniques also able to describe structural flexibility as this dynamical aspect is closely related to protein function. Among the various techniques able to give access to dynamical properties of biomolecules is Site-Directed Spin Labelling combined with Electron Paramagnetic Resonance (SDSL-EPR). The general principle consists in grafting an EPR-active label at a selected site of the macromolecule under interest followed by its observation using an appropriate EPR technique or by combining different EPR strategies. This chapter is dedicated to the use of nitroxide radicals that are the most used spin labels for applications in biostructural EPR, in particular in the field of protein dynamics. It is divided into two parts a

BIP Laboratory, Aix Marseille Univ and CNRS UMR 7281, 31 chemin J. Aiguier CS 70071, 13402 Marseille Cedex 09, France. E-mail: [email protected] b POMAM Laboratory, University of Strasbourg and CNRS UMR 7177, 4 rue Blaise Pascal CS 90032, 67081 Strasbourg, France 66 | Electron Paramag. Reson., 2019, 26, 66–88  c

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corresponding to the two main approaches used in SDSL-EPR. The first approach consists of probing the micro-environment of the spin label in the liquid state by continuous wave (cw) EPR spectroscopy. This approach has been pioneered by W. Hubbell and co-workers in the 90’s.2 Taking a model protein (lysozyme T4), this group demonstrated the correlation between well-defined structural elements (loop, alpha-helix, buried sites) and the EPR spectral shapes of a nitroxide spin label grafted successively at different positions. This key demonstration has led to the success of this technique that is now recognized in the community working on structural dynamics of biological macromolecules. The second approach is based on the measurement of inter-label distances using pulsed EPR techniques. Among the different techniques available, those based on double resonance sequences are the most commonly used. First introduced by Y. D. Tsetkov and coworkers in 1984 called PELDOR for Pulsed Electronic Double Resonance,3 a dead-time free version of the pulse sequence has been introduced by G. Jeschke and coworkers and is referred to as Double Electron–Electron Resonance or DEER.4 In the last decade, the use of such pulsed EPR techniques has rapidly gained interest in the scientific community and has contributed to generate increasing needs of pulsed EPR equipment in laboratories. Other approaches such as the measurement of solvent accessibility or polarity of labelled sites have also been the focus of many studies but are beyond the scope of this chapter. Interested the readers are referred to the abundant literature on these topics.5–9 Finally, the aim of this chapter is to show the complementarity of two approaches mentioned above to answer key biological questions and to underline the importance of combining information coming from both of them. Each section of this chapter will be illustrated by examples and the recent developments in the synthesis of new spin labels to enlarge the potential of SDSL-EPR will also be highlighted. Additionally, the objective is also to give some practical and experimental considerations all along the chapter. For simplicity reason, all the examples given in this chapter are taken from studies done by our group. However, we would like to recommend the reading of excellent reviews made by scientists working in the same field.10–17 All these reviews have been a continuous source of inspiration for the writing of this chapter.

2 Nitroxide spin labels used to probe protein dynamics in the liquid state The power of SDSL-EPR in the liquid state is based on the extreme sensitivity of the EPR spectral shape of a nitroxide radical related to its mobility and we will first describe the physical principles underlying this sensitivity. A second part is devoted to the description of a classical SDSLEPR experiment and we will illustrate how this technique can be powerful to map a local induced folding in the case of an intrinsically disordered protein. Finally, in a third part, we will discuss the limitations of the current nitroxide spin labels that stimulate researchers to design new nitroxide spin labels for specific applications. Electron Paramag. Reson., 2019, 26, 66–88 | 67

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2.1 EPR spectral shape of a nitroxide radical: effect of mobility Nitroxide are stable radicals characterized by a general form: R1R2–N–O . The NO chemical group is enclosed in either a six-membered piperidine or a five-membered pyrrolidine ring (see examples of pyrrolidine rings in Fig. 1A). The stability of the molecule is ensured by the steric screening of the four adjacent methyl groups. The paramagnetism of such radical is the result of the occupancy of the unpaired electron in the 2pz-orbital that is a p-orbital delocalized over the two atoms N and O. The consequence of this chemical property is that nitroxide radicals are anisotropic paramagnetic centres characterized by the interaction between its electronic spin S ¼ 12 and the nuclear spin I ¼ 1 arising from the magnetism of the 14 N nucleus. If the tumbling dynamics of the system is high, as is the case for free radicals in non-viscous solution at room temperature, the magnetic parameters are averaged and the cw EPR spectrum only reveals giso and Aiso. Let’s describe the EPR spectrum for such a simple example as depicted in the energy diagram (Fig. 1B). Considering the electronic spin S ¼ 12, the two possible orientations of the spin determine two energy states (ms ¼ þ 12; ms ¼  12) which are degenerate in the absence of an external magnetic field B. In the presence of B, the Zeeman interaction induces a splitting of the two energy states, with an energy difference DE between the two states proportional to the value of B. Taking into account the hyperfine interaction between the electronic spin and the

Fig. 1 EPR of nitroxides: A Commonly used five-membered pyrrolidine ring nitroxides. B Energy diagram for a spin system (S ¼ 1/2 and I ¼ 1) in the isotropic regime, the 3 transitions are indicated by arrows and give three lines. C EPR spectral shape modifications as a function of the mobility of the spin label described by its rotational correlation time tc. The spectra have been simulated using EasySpin for different values of tc: 0.01 ns (spectrum a), 1 ns (spectrum b), 10 ns (spectrum c) and 100 ns (spectrum d).

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nuclear N spin (I ¼ 1), each level is subdivided into three levels corresponding to each possible value of mI (mI ¼ 1, mI ¼ 0, mI ¼ þ 1). A cw EPR experiment is based on the application of a continuous electromagnetic radiation at a fixed frequency n (typically n ¼ 9.8 GHz at X-band). Recording an EPR spectrum is done by varying continuously the external magnetic field. The resonance condition is obtained when the energy hn (h Planck constant 6.626.1034 m2 kg s1) matches exactly with DE. This leads to the absorption of the electromagnetic wave that gives the EPR line. The selection rules for spin transitions impose Dms ¼ 1 and DmI ¼ 0. For our isotropic system (S ¼ 1/2; I ¼ 1), these rules give 2I þ 1 ¼ 3 transitions. The resonance conditions are thus fulfilled for three different values of the external magnetic field, called resonant fields BmI, given by the equation: BmI ¼ (hn  AisomI)/gisob, with b the Bohr electron magneton (b ¼ 9.274.1024 J T1). As giso is close to 2, the EPR spectrum is centred at around B ¼ 340 mT with a spectrometer operating at X-band. The EPR spectrum is composed of three equidistant lines separated by the quantity DB ¼ Aiso/gisob, typically of 1.5 mT. Note that, in order to improve sensitivity, the external magnetic field is modulated in amplitude, this leads to the acquisition of the signal as the first derivative of the absorption lines. The centre of a line is thus taken as it crosses zero. Now, if the tumbling dynamics of nitroxide radicals in solution is reduced, for example by reducing the temperature or by using a viscosity agent, the magnetic parameters are no longer averaged. We have to define xyz-axes with respect to the molecule: z-axis is taken perpendicular to the plane of the nitroxide, x-axis along the NO bond and y-axis is deduced from the previous two. The magnetic axes are approximated to be parallel to these geometric axes. The Zeeman interaction is slightly anisotropic with gx4gy4gz and typical values of (2.0085, 2.0065, 2.0027).8 On the contrary, the magnetic hyperfine interaction between the electron spin and the 14N nucleus is highly anisotropic and nearly axial with AxBAyoAz with typical values of (0.5 mT, 0.5 mT, 3.5 mT). At X-band the EPR spectral shape of a nitroxide is thus dominated by the hyperfine interaction. The EPR spectral shape is very sensitive to the rotational motion of the molecule (Fig. 1C). This mobility is described by a dynamic parameter: the rotational correlation time tc that expresses the molecular re-orientation of the molecule with respect to the external magnetic field. As seen previously, when the radical is highly mobile, magnetic anisotropies are fully averaged and the spectrum displays three narrow lines (Fig. 1C, spectrum a). As the motion becomes progressively slower, magnetic anisotropies are partially averaged and this results in a differential broadening of lines in the spectrum, while line positions remain constant. This is the so-called rapid regime of mobility, valid until tc around 1 ns (spectrum b). Decreasing the mobility, averaging becomes less and less efficient, leading to spectral shape distortions in this intermediate motional regime (spectrum c). The limit is obtained for an immobilized spin label as it is the case in a frozen solution (spectrum d) where the anisotropy is fully revealed. Electron Paramag. Reson., 2019, 26, 66–88 | 69

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2.2 Nitroxide spin labelling In the previous section, we described how EPR spectra of nitroxide radicals are highly sensitive to molecular motion. This is exactly the property we will take advantage of in SDSL-EPR applications in the liquid state. Until now, we only discussed the EPR-sensitive part of the molecule corresponding to the NO bond environment. To be qualified as a spin label, the nitroxide molecule has to be functionalized to be able to be grafted at a specific position of a protein. SDSL consists of manipulating the biological system of interest so that it contains reactive amino acid residues well-controlled both in numbers (usually one or two) and positions. Most of the commercial spin labels are designed to target cysteine residues because chemical modifications of cysteines are well-mastered and highly specific. The technique thus requires the construction, expression and purification of cysteine variants. Cysteine residues can be modified with a variety of reagents, the most commonly used being either methanethiosulfonate or maleimide derivatives (Fig. 2A). The most frequently used nitroxide spin label is the MTSL (1-oxyl-2,2,5,5-tetramethylpyrroline-3-methyl) methanethiosulfonate 1 leading to the formation of a disulphide bridge between the side-chain of the cysteine and the label (Fig. 2A, side-chain R1).18 Its relatively small size (comparable to natural tryptophan or phenylalanine side chains) together with its flexibility around the different chemical bonds minimise perturbations of the biological system under interest. Other commercial spin labels are available such as the 3-maleimido-2,2,5,5-tetramethyl-pyrrolidinyloxy referred to as ‘‘Proxyl’’ 2 depicted in Fig. 2A (side-chain R2). The maleimide-functionalized spin labels have the advantage of forming a thio-ether bond with the protein side-chain preventing the label release in reducing environments. These labels are however more sterically demanding and slightly more rigid compared to MTSL and they can react with amines at high pH.18 In practice, prior to the labelling reaction, a first step of cysteine reduction is often required to prepare the redox state of this residue and ensure a good labelling yield (see the illustration of a typical labelling protocol in Fig. 2B). After each incubation time (typically one hour), gel filtration (desalting columns for example) is needed to remove (i) the excess of reducing agents and (ii) the excess of spin labels. A final step of concentration is often needed to have sufficiently concentrated samples to allow the recording of EPR spectra in typically several minutes. The labelling yield can be determined by dividing the spin concentration (obtained by comparison with a standard solution of known concentration) over the protein concentration (determined for example by the measurement of OD280 nm). Labelling efficiency can also be checked by global mass spectrometry analyses (mass increment of 186 Da for R1 side-chain). Importantly, covalent modification of proteins with extrinsic labels presents the risk of damaging protein function. Labels can be innocuous at certain sites while not at others and this point is difficult to predict. It is thus crucial to perform control experiments to check the non-invasiveness of the label with respect to protein structural and/or functional properties.

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Fig. 2 Spin Labelling. (A) Labelling reaction scheme for cysteine residues with two classical spin labels: MTSL 1 and Proxyl 2. (B) Classical protocol for spin labelling involving two steps: cysteine reduction and labelling reaction.

Let’s give an example that nicely illustrates the power of SDSL-EPR in the liquid state to reveal the local folding of an intrinsically disordered region of a protein. Intrinsically Disordered Proteins (IDPs) or Regions (IDRs) are the most highly dynamical biological systems that lack a well-defined 3D structure under physiological conditions while being associated to key functions such as regulation, molecular assembly, signalisation.19–22 The following example concerns the nucleoproteins (N) from the Measles (MeV) virus. Multiple copies of N are structurally organized to encapsidate the viral genome and these macromolecular

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assemblies play a crucial role in the replicative complex of the virus.19 MeV nucleoprotein has been extensively studied by means of complementary biophysical techniques.23 Structurally speaking, MeV N consists of two regions: a N-terminal globular one (aa 1–400) and a C-terminal domain NTAIL (aa 401–525) that is fully disordered. This disordered part plays an essential role in the transcription and replication of the virus via an interaction with the phosphoprotein P of the viral polymerase complex. Our study was focused on the interaction between MeV NTAIL and the C-terminal part (X Domain) of the phosphoprotein PXD (aa 459–507 of P) composed of 3 a-helices.24 The aim of the study was to precisely localize the regional folding that MeV NTAIL undergoes in the presence of PXD. We targeted 14 sites for MTSL spin-labelling within NTAIL, 12 of which being localised in the region (aa 488–525) that was known to be specifically involved in the interaction with MeV PXD.24 Among these labeled positions, Fig. 3 displays for three of them (positions 407, 491 and 496) the room temperature EPR spectra recorded in the absence (panel A) and in the presence of PXD (panel B).25 In the absence of the partner protein, the EPR spectra are very similar whatever the position. All spectra are composed of three narrow lines indicating a high mobility of the labels, consistent with the disordered character of NTAIL alone in solution. Behind the term ‘‘mobility’’ we can distinguish three types of motions: the internal dynamics of the spin-label side-chain, the local backbone fluctuations (local flexibility) and the global motion of the entire protein.26 The EPR spectra reflect all these contributions, in particular in our example in which flexibility is present at the different levels. Note that for proteins of molecular weight higher than 50 kDa, the contribution of its global motion is negligible whereas for smaller proteins, this contribution can be cancelled by using sucrose as a viscosity

Fig. 3 Illustration of EPR spectral shape modifications consecutive to an induced folding. NTAIL is an intrinsically disordered region labelled with MTSL at 3 positions (407, 491, 496). X-band room temperature EPR spectra of NTAIL alone (A) and in the presence of its partner protein PXD (B). Spectral modifications are observed for positions 491 (see arrow) and 496 and reflect the local a-helical induced folding caused by PXD binding. 72 | Electron Paramag. Reson., 2019, 26, 66–88

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agent. Even if the contribution of the different motion types is not easy to determine, the main point of this kind of experiments is to detect spectral changes induced by an event (the addition of a partner protein in our case) and to interpret them as structural modifications. Let’s go back to the example of NTAIL to illustrate this particular point. For the label grafted at position 407, the EPR spectrum remains the same between the two conditions: free NTAIL (panel A) and NTAIL bound to PXD (panel B). This indicates that this region is not involved in the interaction with the partner protein. On the contrary, at position 491, in the presence of the partner protein, a broad shape component is observed, indicating a very restricted environment of the spin label in the bound form (see arrow on panel B). Note that a proportion of unbound NTAIL is also detected and contributes to the signal (approximately 25%). At position 496, a spectral shape modification is also observed revealing a structural change in the environment of the label. These observations allowed us to confirm the structural model of a chimera construct between MeV PXD and a small NTAIL region (aa 486–504) in which the amino acid side chain 491 points towards PXD whereas the amino acid side chain 496 is solvent-exposed.28 Extending the studies to the 14 labeled sites, we were able to map the region involved in the interaction restricted to the 488–502 region and to attribute the spectral modifications to the formation of an a-helix using complementary circular dichroism (CD) analyses.25 Interestingly, the mobility of this latter region was found to be slightly but significantly restrained even in the absence of the partner protein, a behavior that could indicate the existence of a pre-structuration of this region. This observation has been further confirmed combining SDSL-EPR data and modeling of local rotation conformational space29 as well as by NMR studies and modeling of this region as a dynamic equilibrium between a completely unfolded state and different partially helical conformations.30 Using the same strategy based on multiple individual labelling sites, we studied the dynamics of NTAIL from other viruses (Hendra and Nipah) and mapped the induced folding that NTAIL undergoes in presence of PXD.31 This study validated previously proposed structural models obtained by homology modelling.32 Other IDPs have been studied and dynamically characterized by SDSL-EPR.33–35 This approach has been also successfully applied to reveal various structural states and higherorder organizations of flexible proteins involved in neurodegenerative diseases such as a-synuclein,36 amyloid-b peptide37 and tau.38 SDSL-EPR is applicable to all possible varieties of proteins whatever the size and complexity of the system from soluble globular protein39 to membrane proteins.40,41

2.3 Limitations and design of new nitroxide spin labels In the previous section, we examined an example that illustrates the property of nitroxide spin labels of being perfect reporters or ‘‘spy spins’’ of their local environment and the power of such molecules to answer biological questions. Nevertheless, the technique has still some limitations. In the following, we will present three limitations of conventional Electron Paramag. Reson., 2019, 26, 66–88 | 73

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nitroxide spin labels applications and propose, for each of them, new nitroxide-based spin labels to overcome these limitations. 2.3.1 Diversification of the spectral shapes. One limitation comes from the poor diversity of EPR spectral shapes of nitroxide labels, so that EPR spectra from different nitroxide labels can strongly overlap. The similarity of these spectral shapes precludes the simultaneous investigation of two different regions of a protein or two interacting proteins. This is a situation that can be encountered in allosteric mechanism in which ligand binding at one site influences binding at another site through a propagated structural change within the protein.42,43 The idea was thus to design a new nitroxide with different magnetic properties able to provide a different spectral shape. A new spin label based on a maleimide-functionalized b-phosphorylated nitroxide referred to as phosphorylated Proxyl ‘‘PP’’ has been synthesized (nitroxide spin label 3, Fig. 4). PP can be grafted on cysteine residues as the maleimide Proxyl does (Fig. 4). Moreover the presence of a phosphorous atom near the NO bond leads to a supplementary high magnetic coupling between the electron spin and the nuclear spin of 31P (I ¼ 1/2). This new label gives a well-resolved 6-line spectrum composed of a doublet of triplets.44 Taking MeV NTAIL as a model protein, our first objective was to demonstrate the ability of PP to be a good reporter. Four grafting sites on NTAIL were judiciously chosen within and outside the induced folding region. EPR spectral modifications induced by PXD were recorded and compared to the classical Proxyl grafted at the same positions. As an example, Fig. 4A shows the EPR spectra obtained for Proxyl (spectrum a) and PP (spectrum b) grafted at position 491. In the case of protein-protein interaction, EPR spectra are often composite with at least one spectral shape corresponding to the unbound and one to the bound form. To determine the proportion of both forms, spectral simulations are needed. Simulations also provide the determination of the rotational correlation tc which constitutes a dynamic indicator. Different packages are nowadays available for EPR spectra simulation, the most commonly used being EasySpin,45 a toolbox supported by MATLAB. More recently, a graphical user interface of EasySpin, called SimLabel, has been developed in order to provide a simulation tool for non-expert users of MATLAB.46 In the example of Fig. 4, the EPR spectra were simulated using ROKY software47 because, at the time of this study, it was not possible to simulate spectra in the intermediate regime of motion with two hyperfine couplings using EasySpin. Taken together the results demonstrated that PP is able to monitor from subtle to larger structural transitions, as efficiently as the classical spin label 2.44 As a proof of principle, the second objective was to show that combining Proxyl and PP opens the way to study two protein sites simultaneously. Fig. 4B shows the EPR spectrum resulting from a mixture of NTAIL labeled with PP at position 407 and PXD labeled with Proxyl at position 496. The composite spectrum allows distinguishing clearly the 3-line spectrum from the 6-line ones and simulation of the whole spectral shape was possible (unpublished results). 74 | Electron Paramag. Reson., 2019, 26, 66–88

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Fig. 4 Spectral shape diversification. A X-band room temperature EPR spectra of Proxyl 2 (upper spectrum a) and Phosphorylated Proxyl 3 (upper spectrum b) grafted at the same position of NTAIL (491) in the presence of its partner protein PXD. The spectra have been simulated using ROKY software (lower spectrum) and reveal two components corresponding to the unbound and bound forms of the labelled protein. B Composite EPR spectrum obtained after mixing NTAIL labelled with 3 and PXD with 2. The different components can be identified and the whole spectrum simulated (lower spectrum).

With a similar idea, other authors proposed to modify the nitroxide spectral shape using isotopically modified 15N (I ¼ 1/2) nitroxide spin label for application in inter-label measurements.48 2.3.2 Diversification of the grafting sites. Another limitation of conventional nitroxide spin labels concerns the fact that only cysteines are the target of the labels. This strategy becomes unsuitable when sulfhydryl groups play important roles either in the function and/or in structural elements (like in active sites or disulfide bridges). Two different approaches can be set up. The first one consists in targeting another amino acid. Following this idea, we used two nitroxide spin labels able to react with the phenol group of a tyrosine amino acid via a three-components Mannich type reaction.49,50 Best results were obtained with the newly synthesized Electron Paramag. Reson., 2019, 26, 66–88 | 75

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isoindoline-based nitroxide: the 5-amino-1,1,3,3-tetramethyl-isoindolin2-yloxyl, referred to as ‘‘Nox’’ (nitroxide spin label 4, Fig. 5A).50 We applied this new labelling strategy to a small chloroplastic protein called CP12 (80 amino acids) from the green alga C. reinhardtii with the aim of studying its association with the glyceraldehyde-3-phosphate dehydrogenase (GAPDH). This association is a key step towards the formation of a ternary supramolecular complex involved in the regulation of the Calvin cycle in many photosynthetic organisms.51 CP12 contains four cysteine residues engaged in two disulfide bridges in its oxidized state and presents some a-helical structural elements modeled from each side of the N-terminal disulfide bridge, whereas the C-terminal part appears

Fig. 5 Grating site diversification. (A) Tyrosine spin labelling using nitroxide 4 and illustration of its use on the CP12 protein (unique Tyrosine at position 78). (B) Targeting the unnatural amino acid p-acetyl-L-phenylalanine (p-AcF) with HO-4120 nitroxide 5 and illustration of its use on the ACCO enzyme (position 192, spheres). 76 | Electron Paramag. Reson., 2019, 26, 66–88

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mainly disordered (Fig. 5A). This C-terminal region has been demonstrated to be mainly responsible for the redox regulation of GAPDH.53 This region contains a single tyrosine located at position 78, almost at the end of the protein. This tyrosine, highly conserved in CP12s from different organisms, is particularly interesting as it is close to residues that play a crucial role in the activity modulation of GAPDH either in the binary complex54 or in the ternary complex with PRK.55 The new isoindoline-based nitroxide 4 (Fig. 5A) was used to selectively target this unique tyrosine. The labelling conditions were harsher compared to a classical labelling protocol: it necessitates a rather long incubation period (around 16 hours) in the presence of formaldehyde. Typical labelling yield of 30% was obtained under these conditions. The small spectral modification observed in the presence of GAPDH led us to conclude that this region is in the vicinity of the interaction site without being directly involved in it.56 This finding was in good agreement with the partial view of the CP12/GAPDH complexes from other organisms obtained by crystallographic and NMR studies in which only the 20 last amino acids of CP12 were detected.57,58 More recently, this strategy has been combined with conventional cysteine labelling on the protein PTBP1 to measure inter-label distances.59 The second strategy to avoid cysteine labelling is an orthogonal labelling strategy aiming at inserting an unnatural amino acid (uaa), bearing a chemical function that is not present in the common 20 natural amino acids. The first method proposed in the literature was based on the insertion of the genetically encoded uaa p-acetyl-Lphenylalanine (p-AcPhe) bearing a ketone function.60 This uaa is able to react specifically with a hydrolylamine-functionalized reagent (nitroxide spin label 5, HO-4120) to generate a ketoxime-linked nitroxide side chain called K1 (Fig. 5B). Note that the EPR spectra of the resulting K1 mutants were shown to reflect higher mobilities compared to classical MTSL-labelled mutants due to the higher flexibility of K1 compared to R1 side-chain.60 However, the reaction is rather slow and requires the use of acidic pH that limit the application of this method in its simple form. Several catalysts have been proposed in the literature to work at neutral pH and improve labelling efficiency.61 Fig. 5B illustrates the application of this strategy on the 1-Amino-Cycloproprane-1-Carboxylic Oxidase (ACCO) which is a non-heme iron(II) containing enzyme responsible for the production of ethylene in plants.62,63 The need to develop such strategy for this particular biological system came from the existence of four cysteines, one of which leading to the loss of enzyme activity after mutation in serine. The position of the uaa was chosen in the C-terminal region (position 292), a part of the protein that is supposed to be flexible and involved in the enzymatic function (unpublished results). More recently, another approach has been proposed based on azide-alkyne click chemistry to provide fast and highly selective labelling reactions.64 2.3.3 Towards in-cell EPR. All the experiments presented until now were performed on purified proteins in adequate buffers and, in the Electron Paramag. Reson., 2019, 26, 66–88 | 77

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case of protein-protein interaction, the only two partners were present. What about protein structural dynamics and protein–protein interaction in cell? This is a very challenging question that currently generates great interests and efforts in the scientific community.65–68 In living cells, proteins are subjected to an extreme ‘‘macromolecular crowding’’ and can meet and possibly interact with many other molecules such as proteins, nucleic acids, ligands, . . . In-cell EPR has become in the last years an explorative and highly attracting field of interests. Although several studies demonstrated the feasibility of incell EPR experiments,69–76 the major limitation lies in the short persistence of currently available nitroxide-based spin labels because of the reductive conditions of the cellular medium. This leads in a few minutes to the EPR-silent hydroxylamine form that dramatically limits the potential of this approach.77–79 To overcome this limitation, we designed and synthesized a new nitroxide called M-TETPO (nitroxide spin label 6, Fig. 6) characterized by a high resistance of the nitroxide moiety to bioreduction and of the linker to cleavage. The resistance to bioreduction is ensured by the presence of gem-diethyl groups that protect the NO group whereas the maleimide function brings a noncleavable thio-ether bond.80 We took the chaperone protein NarJ from E. coli as a model protein that we previously studied with classical MTSL spin labels.81 The resistance to reduction (in a 20-fold molar excess ascorbic acid) on the labelled NarJ (position 119) was checked by comparing the drop of the EPR signal as a function of time between the conventional Proxyl and the new shielded nitroxide (Fig. 6A). A further crucial point was to evaluate the ability of the M-TETPO 6 to be a good reporter on its micro-environment, an essential property for its use in the liquid state cw EPR. For this, three different sites characteristic of different structural environments were chosen on NarJ on the basis of our previous study.81 For each label site, similar spectral shapes were observed with Proxyl 2 and M-TETPO 6, thus demonstrating that the new label is as sensitive to its structural environment as the classical one (Fig. 6B).80 The ability of M-TETPO grafted on NarJ to be used for in-cell applications has been demonstrated after microinjection of a labelled sample into Xenopous laevis oocytes.80 Alternative approaches based on the use of Gd31-based tags and trityl radicals have been proposed for in-cell EPR investigations.71,72,82 However, despite their resistance to bioreduction, Gd31-based tags require the use of high frequency (W-band, 95 GHz) and are not applicable for liquid state EPR.

3 Nitroxide spin labels used to measure distances in biologicals systems In the previous section, we described the ability of a single nitroxide spin label to report on its local environment in the liquid state. The same labels can also be used in pairs in order to measure the distance between them. For biological systems, the possibility of measuring distances 78 | Electron Paramag. Reson., 2019, 26, 66–88

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Fig. 6 Bioresistant nitroxide for in-cell applications. (A) Kinetic of reduction (20-fold molar excess of ascorbic acid) of the chaperone NarJ labeled either with Proxyl 2 or with the M-TETPO nitroxide 6 at position 119. The peak-to-peak amplitude of the central line is measured (B) EPR spectral shapes given by 2 and 6 grafted at three different positions of NarJ (119, 149 and 207). The similar shapes observed demonstrate that M-TETPO is a good reporter of its local environment.

between paramagnetic species is extremely attractive. Several techniques have been developed, in particular based on pulsed EPR methods that will be the focus of the present section. Electron Paramag. Reson., 2019, 26, 66–88 | 79

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3.1 Measuring nitroxide–nitroxide distances The method of choice to measure distances between two nitroxides grafted on biological systems is the 4-pulse Double Electron–Electron Resonance (DEER) experiment.4 The range of measurable distances by DEER is typically between 1.5 nm and 8.0 nm, a range that perfectly matches the large conformational transitions encountered in proteins. This sequence is dedicated to measure distances between two weaklycoupled spins by means of dipolar interaction measurement. It is classically used on frozen solution due to too short phase memory time Tm of conventional nitroxides. A qualitative description of the pulse sequence is the following: it is based on the use of two frequencies that excite selectively two different spin populations A and B (Fig. 7A). These two frequencies are chosen on the basis of the Echo Field Sweep signal corresponding to the absorption spectrum of all nitroxides present in the sample (Fig. 7B and C, left). Spins A, excited by the frequency nobs, are the spins that are observed through the measurement of a refocused echo obtained after a (p/2  p–p) sequence. The inter-pulse delays are constant so that the position of the echo remains the same along the sequence. These delays are adjusted according to the relaxation properties of the system. Magnetization of the spin B population is inverted by a p-pulse applied at frequency npump that leads to the modification of the magnetic field experienced by spins A. The position of this pulse varies along the sequence according to the evolution time t. This leads to a modulation of the refocused echo as a function of t that gives the so-called DEER trace. This modulation contains the information relative to the interspin distance r as the dipolar coupling frequency is inversely proportional to r3. The most used software for calculating distribution distance profiles from DEER traces is the DeerAnalysis program developed by G. Jeschke and coworkers.83 An example of DEER measurement is given in Fig. 7B on the ACCO enzyme (see y 2.3.2) on which two nitroxides were introduced at position 60 and 292 respectively. In folded proteins, in which the label positions are well-defined, the modulation of the echo in DEER traces is in general clearly visible. On the contrary, in the case of IDPs characterized by an ensemble of interconverting conformations, a non-modulated DEER trace is generally observed leading to a broad inter-spin distance distribution. A recent study has shown that measuring the effective modulation depth Deff of the DEER traces as a function of a denaturing agent leads to information on the structural behavior of an IDP, revealing in this example cooperative transition events between compact and expanded conformations.84 In a last example, by combining both approaches: label dynamics monitored by cw EPR and DEER measurements, we were able to demonstrate that the dimerization interface of the IF1 peptide from yeast involved both folded and unfolded regions.85 Analyses of label dynamics by cw EPR at different positions of the peptide allowed demonstrating that the C-terminal part (position 54) was a disordered region (Fig. 8A). Interestingly DEER measurement between the two sites in the dimeric form showed a well-defined distance centred at 2.7 nm that was not expected for disordered segments (Fig. 8B). Thanks to the 80 | Electron Paramag. Reson., 2019, 26, 66–88

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Fig. 7 Inter-label distance measurements: (A) 4-pulse DEER pulse sequence based on the application of two frequencies: the ‘‘observed’’ and the ‘‘pump’’ frequency. The delays t1 and t2 are fixed and adjusted to detect a refocussed echo. t is the evolution time of the inversion pump pulse incremented at each step of the sequence. (B) X-band 60K echo field sweep (left) of the nitroxide bi-labelled ACCO enzyme (positions 60 and 292) with the indication of the chosen frequencies. Background corrected DEER trace (middle) and the resulting distance distribution (right) calculated by DeerAnalysis are shown. (C) Echo field sweep (left) of the nitroxide mono-labelled ACCO enzyme reconstituted with Cu(II) in its active site with the indication of the chosen frequencies. Corrected DEER trace (middle) and the resulting distance distribution (right) calculated by DeerAnalysis are shown. This example illustrates that nitroxides as well as metal centres can be used on the same biological system to combine measurements of either NO–NO or NO-metal distances.

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Fig. 8 Complementary strategies. (A) cw EPR spectrum of MTSL 1 grafted at position 54 of the IF1 peptide. The spectrum reveals the high dynamics of the corresponding region. (B) Background corrected DEER trace of the dimeric form of IF1 shows a narrow interlabel distribution centred at 2.7 nm. (C) Structural model of IF1 dimer calculated from a set of inter-label distance measurements.

combination of the two approaches we concluded that the C-terminal region is folded back to the central a-helical region of the peptide and that its high dynamics is spatially restrained to this particular region (Fig. 8C). This example shows how important it is to take into account data coming from both cw and pulsed DEER EPR experiments. Concerning practical considerations, great advantages in terms of sensitivity improvement of using Q-band (35 GHz) instead of X-band (9.8 GHz) have been demonstrated.86,87 Sample preparation is also a crucial point to improve sensitivity. It has been shown that deuterated solvents extend transversal relaxation time and allows longer evolution time and thus longer distance range measurement. It is also important to dilute the sample with a certain proportion of a cryoprotectant (often glycerol) to avoid excluding volume effects and provide a homogeneous distribution of the proteins.88,89 3.2 Measuring nitroxide-metal distances Interestingly the application of DEER is not restricted to nitroxides but can also be used with naturally occurring paramagnetic species such as metals that constitute perfect endogenous labels. DEER has been used to 82 | Electron Paramag. Reson., 2019, 26, 66–88

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90,91

measure metal-metal distances and metal-nitroxide distances as well.92–94 This list is not exhaustive but only provides different examples described in the literature. The ACCO enzyme is also an appropriate system to measure distance between metal and radical. Fig. 7C shows the result of Cu(II)–NO (position 292) distance measurement on this system. Even if Cu(II) is not the natural ion in this non heme Fe(II) enzyme, it has been shown to occupy the same position in the active site.95 Cu(II) has the advantage of having a spin S ¼ 1/2 and moderate relaxation rates. The example presented in Fig. 7 illustrates the different strategies that can be set up on the same biological system by measuring either NO-NO distances or NO-metal distances. In the context of metal-nitroxide distance measurements, DEER is not the most appropriate technique because of the large difference between the two paramagnetic species both in anisotropy and in relaxation properties. An alternative to DEER is the so-called RIDME (Relaxation Induced Dipolar Modulation Enhancement) sequence first introduced by Kulik96 and later proposed in its dead-time free version by M. Huber and coworkers.97 This method requires the application of a single frequency in a sequence based on the detection of a stimulated echo. Here the flip of spins B is not induced by a pump pulse as in the DEER sequence but is left to the spontaneous longitudinal relaxation of B spins, avoiding the problem of the limited excitation bandwidth. It is dedicated to determination of the interaction between low g-anisotropy centre (nitroxide) and high g-anisotropy centre (metal) and is suitable for species of different relaxation properties (fast-relaxing metal centre and slow-relaxing nitroxide). 3.3 Other labels for interspin distance measurements As for liquid state SDSL-EPR, new nitroxide spin labels have also been proposed to improve their use in DEER measurements. Classical experiments are performed under cryogenic conditions (typically 60 K) which raised questions about potential differences between physiological and frozen states. Moreover, the use of cryoprotectant may alter distance distribution or conformational equilibrium. New nitroxide spin labels with spirocyclohexyl substituents have been synthesized and were shown to have longer Tm.98,99 The use of this label allowed DEER measurements at room temperature with a model protein embedded in a glassy trehalose matrix.100 Other spin labels are also used for distance measurements by pulsed techniques such as Gd(III) tags101–104 or trityl radicals.82,105,106

4 Conclusion As a conclusion, we would like to point out the complementarity of the two approaches presented in this chapter: nitroxide dynamics probed by cw EPR and inter-label distance measurement by pulsed EPR. One approach is not better compared to the other. A careful examination of data coming from both approaches can be crucial to understand the structural behaviour of proteins under various conditions. The progress in the field of SDSL-EPR requires many skills from different scientific Electron Paramag. Reson., 2019, 26, 66–88 | 83

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disciplines. We need biologists to have well-controlled/high quality samples and ask relevant biological questions. We need organic chemists able to design and synthesize new spin labels, to enlarge the panoply of the existing ones. We need spectroscopists/physicists to develop new methods and tools to acquire and analyse data in the best possible conditions. The combination of these skills and the sharing of knowledge between the different communities are the key to continue the progress and make SDSL-EPR always more competitive in the field of biostructural applications. Finally, combining different biostructural techniques, in particular NMR and EPR, is probably also an efficient strategy to improve the fine understanding of dynamic behaviour in proteins. For example, nitroxide spin labels are also used in Paramagnetic Enhancement Resonance (PRE) experiments in which the paramagnetic species is used to accelerate the relaxation of the NMR-detected nuclei.107 This technique has the ability to reveal long-range contacts and is in particular useful for the study of IDPs.108 Whereas PRE is blind near the label site due to too fast relaxing effect, EPR can precisely report on this particular site, obviating the complementarity of the two techniques. Although very few examples exist in the literature combining PRE and EPR,84,109,110 we are convinced that this joint strategy is going to develop in the next future.

Acknowledgements The authors are grateful to the EPR facilities available at the national TGE RPE facilities (IR CNRS 3443). We would like to thank our collaborators and their teams: S. Longhi (AFMB, CNRS-AMU UMR 7257), B. Gontero (BIP, CNRS-AMU UMR 7281), S.R.A Marque and O. Ouari (ICR, CNRSAMU UMR 7273), A. Magalon (LCB, CNRS-AMU UMR 7283), J. Simaan (iSm2, CNRS-AMU 7313) and F. Haraux (I2BC, CNRS-UPS 9198). E. ´ Fournier is grateful to the Excellence Initiative of Aix-Marseille Universite (A*MIDEX) for her PhD fellowship.

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Applications of light-induced hyperpolarization in EPR and NMR Daniel J. Cheney and Christopher J. Wedge* Published on 02 November 2018 on https://pubs.rsc.org | doi:10.1039/9781788013888-00089

DOI: 10.1039/9781788013888-00089

Magnetic resonance methods are widely used to provide atomic level information on the structure and dynamics of chemical and biochemical systems, but often suffer from poor sensitivity. This review examines how optical excitation can provide increased electron spin-polarization, and how this can be used to increase sensitivity and/or information content in both Nuclear Magnetic Resonance (NMR) and Electron Paramagnetic Resonance (EPR) spectroscopy.

1

Introduction

Nuclear Magnetic Resonance (NMR) spectroscopy is applied ubiquitously in the chemical sciences and increasingly in a biochemical context to probe for example the structure and dynamics of biological macromolecules. However, both routine exploitation and further developments of NMR’s applications are limited by low intrinsic sensitivity.1 Signal intensity depends upon the net absorption or emission of radiation and hence in turn on the population difference across an allowed spectroscopic transition, given under equilibrium conditions by the Boltzmann distribution. While for a typical optical transition the energy separation of the states involved is often large compared to the thermal energy kBT, in magnetic resonance the energy gaps are small and hence the final state (B) of the transition has almost equal population to the initial state (A). The tiny fraction of the total spins that actually contribute to the observable signal may be expressed as the polarization, P:   nA  nB hgB0  P¼ ¼ tanh nA þ nB 2kB T where nA,B are the populations of states A and B, g the gyromagnetic ratio and B0 the applied magnetic field. For example, due to the weak nuclear Zeeman interaction, the room temperature polarization of protons at 23.5 T (1 GHz) is only about 0.008%.2 Whilst this is sufficient for 1H NMR of materials available in large quantities, studies on low-concentration biological molecules, or less abundant isotopes, may potentially be rendered impossible. Polarization may be enhanced by brute force methods, such as increasing the magnetic field strength or cooling the sample to cryogenic temperatures, Fig. 1. However, high-resolution NMR is dependent upon the rapid tumbling of molecules that occurs only in the solution phase, and the properties of low-temperature superconductors used for conventional NMR magnets means that fields in excess of Department of Chemistry, University of Huddersfield, Queensgate, Huddersfield, HD1 3DH, UK. E-mail: [email protected] Electron Paramag. Reson., 2019, 26, 89–129 | 89  c

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Fig. 1 Boltzmann electron and nuclear spin polarization at different resonant frequencies (and magnetic fields). Optical excitation can potentially enable close to 100% electron polarization even at high temperature.

25.9 T (1.1 GHz) are unlikely to be reached with this technology.1 Furthermore, even at the highest stable magnetic fields so far obtained, the observed nuclear polarization is still no higher than B0.1% at 20 K.3 While small sensitivity gains are possible by optimizing the coil design, or by reducing sources of noise,1,2 the most promising technique for overcoming the intrinsic sensitivity limitation is hyperpolarization: the generation of non-Boltzmann spin-polarization. There are a number of mechanisms that may be exploited to achieve hyperpolarization. These include para-hydrogen induced polarization (PHIP),4,5 which uses the nuclear symmetry properties of the hydrogen molecule, and optical pumping, which uses circularly-polarized light to generate large polarizations of noble gas nuclei.6,7 But by far the most studied technique is dynamic nuclear polarization (DNP). First theorized in 1953 by Overhuaser,8 and experimentally verified later that same year by Carver and Slichter,9 DNP involves transfer of polarization from electron to nuclear spins. This relies on the fact that the Boltzmann polarization for electrons is much greater than that of nuclei, due to the larger gyromagnetic ratio (Fig. 1). Electron Paramagnetic Resonance (EPR), the older cousin of NMR, is more selective, by detecting only paramagnetic species such as radicals or certain metal centres. Whilst this specificity has resulted in less widespread use of EPR, the method has found increasing importance in recent years, with applications ranging from biological docking problems intractable by NMR10,11 to quantum information.12 The higher polarization of electron spins make EPR intrinsically more sensitive than NMR, yet further sensitivity enhancements would still be welcome and are achievable through electron spin hyperpolarization, as can be generated by photochemical and photophysical processes. Such hyperpolarized electron spins not only produce alterations in EPR signal intensities, but can be used to generate nuclear hyperpolarization.13 90 | Electron Paramag. Reson., 2019, 26, 89–129

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While early work lead to pessimism about the possibilities to exploit DNP at high magnetic fields (41 T), recently the field has seen a renaissance and DNP-NMR instrumentation based on solid-state microwave irradiation is now a commercial reality.14,15 Such microwave DNP approaches based upon thermally polarized electron spins have been reviewed extensively elsewhere.16–19 Here the focus will be on lightenhanced electron and nuclear spin hyperpolarization. We begin by introducing the three main mechanisms for generating chemically induced dynamic electron polarization (CIDEP), discuss applications of these mechanisms in EPR, and finally examine how they may be applied to nuclear spin hyperpolarization.

2

Electron spin hyperpolarization

CIDEP (and chemically induced dynamic nuclear polarization, CIDNP) were discovered in the 1960s, beginning with the radical pair mechanism (RPM).20,21 As Forbes et al. also highlight initially they were explained by a ‘‘dynamic’’ Overhauser type process, although the now well-established theories tell us this is not the case.13 The term ‘‘chemically induced’’ is also a misnomer as chemical reaction, while sometimes occurring, is not necessary for the generation of spin polarization. 2.1 Time-resolved electron paramagnetic resonance Before discussing the various CIDEP mechanisms, it is instructive to discuss EPR methods for detection of such polarization. The detection method must be ‘‘time resolved’’, because CIDEP typically has a lifetime of the order of microseconds or less. Conventional time-resolved EPR (TR-EPR) uses continuous-wave microwave irradiation, but whereas EPR experiments without high temporal resolution apply magnetic field modulation and phase sensitive detection to significantly boost detection sensitivity,22 in TR-EPR the signal is sampled directly from the microwave bridge.23 This means TR-EPR signals do not possess the characteristic firstderivative shape associated with field-modulated spectra, and instead the sign of the signal directly represents enhanced absorption or emission. The TR-EPR experiment uses a pulsed laser to initiate photochemical reactions, and a boxcar integrator or fast digital oscilloscope to detect the subsequent transient EPR signal. A pulse delay generator controls the experimental timing, often using a fast photodiode to detect laser pulses for synchronisation and jitter reduction. Both dark (before the laser pulse) and off-resonance signals are used for baseline correction, the latter accounting for any heating effect of the laser on the sample or the microwave resonator.24,25 TR-EPR is unable to detect the signal arising from a Boltzmann distribution of electron spins, and is thus insensitive to the thermal polarization, by detecting only hyperpolarized spins. It is also worth noting that the decay of TR-EPR signals is similarly governed not by the lifetime of the transient species, but by spin-relaxation leading to loss of the hyperpolarization necessary for detection. A time-resolution of around 10 ns can be achieved, with higher resolution not in-fact desirable as this is already comparable to the time required for the Electron Paramag. Reson., 2019, 26, 89–129 | 91

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hyperfine interaction to evolve, and at X-band (B0.3 T), is close to the minimum time of B0.1 ns needed for any EPR signal to appear.13,26 It is now increasingly common for pulsed EPR spectrometers to be available, allowing Fourier transform or spin-echo detected EPR experiments.27 Such instruments are often capable of continuous-wave TR-EPR as described above using in-built fast digitizers for signal detection, but also allow for what have become known as ‘‘delay after flash’’ (DAF) experiments.25 As in other pulsed EPR experiments, DAF uses a sequence of nanosecond microwave pulses to generate a free induction decay or electron spin echo, which is monitored with a fast digitizer, but with one or more additional laser pulses added to the sequence. For monitoring kinetics, the time delay between the laser flash and detection sequence can be varied to provide similar information to TR-EPR, but for photostable samples producing sufficiently long-lived (typically triplet) states, the full suite of advanced pulsed EPR techniques may be applied. Whilst detection sensitivity and bandwidth can present significant challenges in pulsed EPR methods,28 they do offer a considerable advantages over continuous-wave TR-EPR in that Boltzmann signals may be acquired. While in many TR-EPR experiments the laser pulse generates the paramagnetic species so there may be no dark signal, the point is particularly pertinent to recent investigations of the radical triplet pair mechanism (RTPM). This mechanism can hyperpolarize persistent radicals, and measurement of the thermal signal enables quantification of the level of hyperpolarization generated (vide infra). Detailed descriptions of the instrumentation and experimental considerations of both approaches are given elsewhere.13,24,25 2.2 Hyperpolarization mechanisms 2.2.1 Triplet mechanism. The triplet mechanism (TM) was first observed in the form of a number of anomalous EPR line patterns.29 As shown in Fig. 2, the TM occurs when intersystem crossing (ISC) to the triplet state follows initial formation of an excited singlet by optical excitation. A key finding is that in chromophores that do not possess spherical symmetry, ISC is spin-selective: each of the triplet sublevels, labelled TX, TY, and TZ, are populated at different rates due to anisotropy in the spin–orbit coupling. Not only is this polarization evident in the characteristic EPR signal of the triplet itself,24,25,30 but if the triplet undergoes subsequent photochemical reaction to form radicals before

Fig. 2 The triplet mechanism, showing generation of two emissively polarized radicals; see text for details. Image inspired by ref. 13. 92 | Electron Paramag. Reson., 2019, 26, 89–129

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spin–relaxation produces a Boltzmann distribution over the triplet states, then the radicals gain net electronic polarization. In the 1970s, a number of groups presented detailed theories for how this spinselectivity arises including Wong et al.,31 followed by Atkins and Evans,32 and Pedersen and Freed.33 Briefly, the triplet sublevels are selectively populated and are thus ‘‘polarized’’, in the absence of an external magnetic field. The molecule, however, possesses no net magnetization until evolution of the zero-field states under the influence of the applied field.34 For a concise description of the necessary transformation of the triplet states from the molecular to the laboratory frame, the reader is referred to the recent review by Forbes et al.13 As noted there, the physical reason for the unequal population of the laboratory frame triplet states (T1, T0, T), is the uneven distribution of the molecular triplet states (TX, TY, TZ) about the zero-field energy. Steiner et al. showed that as well as selective triplet population, some molecules may undergo spin-selective depopulation of the triplet sublevels.35 This is known as depopulation-type (d-type) TM, or sometimes reversed TM, to distinguish it from the selective population-type (p-type) TM described above. The reversed TM occurs much more rarely than p-type TM, since it requires ISC to the ground state to be fast enough to compete with radical formation. The reversed TM has also been observed by Savitsky and Paul, for 2-cyano-2-propyl radicals formed from AIBN.36,37 A theory for reversed TM has been proposed by Serebrennikov and Minaev.38 2.2.2 Radical pair mechanism. The RPM was the first CIDEP and CIDNP mechanism to be discovered, and has been known since 1963 for CIDEP20 and 1967 for CIDNP.21 Theories for the RPM were first proposed by Closs,39 and by Kaptein and Oesterhoff,40 although the diffusive excursion and re-encounter critical to the operation of the RPM, as we understand it today, was first postulated by Adrian in his ‘‘grazing encounter’’ model for radical pairs (RPs).41 A geminate RP (g-pair) is a pair of radicals formed simultaneously from photochemical dissociation and may exist in either a singlet (S) or a triplet (T) state, depending on the spin multiplicity of the precursor excited state. The g-pair may recombine, or the two radicals may diffuse apart, with an RP formed from a random encounter of two such radicals termed a free pair (f-pair).42 The spin Hamiltonian for a radical pair is given by:   X X X 1 ^ ðSN  g N  B0 Þ þ ðaiN SN  I iN Þ HRP ¼  J ðr Þ 2SA  SB þ þ mB 2 N ¼ A;B N ¼ A;B i in which aiN is the isotropic hyperfine coupling between electron SN and the ith nuclear spin IiN of radical N, and the isotropic exchange interaction J(r) is given by: J ðr Þ ¼

1 ðES  ET Þ D J0 expðlr Þ 2 Electron Paramag. Reson., 2019, 26, 89–129 | 93

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Usually, J0 is negative, meaning that the triplet levels lie above the singlet levels. The eigenstates in this system are given by:

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1 7Si ¼ pffiffiffi ð7aA bB i  7bA aB iÞ 2 7T1i ¼ 7aAaBi 1 7T0 i ¼ pffiffiffi ð7aA bB i þ 7bA aB iÞ 2 7T_i ¼ 7bAbBi The only difference between the S and T0 states is their phase, so at long inter-radical separations r the radical pair oscillates between these states. Interconversion is driven by the difference QAB ¼ 12 ðoA  oB Þ in the Larmor precessional frequencies of the two unpaired electrons arising from small differences in their g-values and hyperfine couplings:43 oN ¼

gN mB B0 X þ aiN I i h  i

While the S and T0 states become degenerate at large separations (negligible J) that allowing mixing to occur, the T  states are energetically isolated owing to the substantial Zeeman splitting, although S–T  mixing can become important at low magnetic fields.42 The RPM therefore operates as follows: a RP is formed in a pure state, and separates leading to S–T0 mixing (once J is negligible). If the radicals re-encounter within a few nanoseconds, J becomes large and the system must once again become either a singlet or a triplet state. This induces spin polarization, although the polarization cannot be observed until the radicals have permanently diffused apart.41,42 There are two types of polarization that may be formed from the RPM: net and multiplet (Fig. 3).43 Net polarization occurs when the two radicals have different g-factors, but little or no hyperfine couplings. This effect is seen as one radical in absorption (A) and the other in emission (E). It should be noted that if the two radical spectra overlap exactly (i.e., the g-factors are equal), then the A and E lines are co-incident and cancel out, with only a weak signal observed due to unequal magnitudes of the oppositely phased signals (Fig. 3(b)). Multiplet polarization is seen when the radicals have similar g-factors, but strong hyperfine couplings. This manifests as the low- and high-field lines having different phases (A/E or E/A pattern). The intensity (and phase) of the CIDEP lines of radicals A and B are given by:43 PA ¼

X

Pij

PB ¼ 

B

94 | Electron Paramag. Reson., 2019, 26, 89–129

X A

Pij

Published on 02 November 2018 on https://pubs.rsc.org | Electron Paramag. Reson., 2019, 26, 89–129 | 95

Fig. 3 CIDEP patterns arising from RPM polarization: (a) Net polarization, where Dg exceeds the linewidth, (b) Net polarization, where Dg ¼ 0 leading to cancellation, and (c) Multiplet polarization, where Dg ¼ 0 with significant hyperfine. The example shows a pair of identical radicals split by six equivalent protons – convolution of the expected 1 : 6 : 15 : 20 : 15 : 6 : 1 hyperfine septet with the transition dependent CIDEP efficiency causes the central line to be absent and gives the A/E polarization pattern a ‘‘sine wave’’ shape (see ref. 13 for data inspiring this example).

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in which the summations are over all nuclear spin states, and 

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Pij ¼ p  sgnðQAB J0 ðrS ð0Þ  rT ð0ÞÞ

 31=3 7QAB t71=2 ld

An important consequence of this is that CIDEP intensity (which superimposes the use binomial pattern for EPR intensity) decreases near the centre of the spectrum as QAB becomes small.40 In certain cases, such as the formation of an identical pair of radicals, the central line in the spectrum is not visible, as there is no g-factor difference and the sum of nuclear spin quantum numbers is zero (Fig. 3(c)).13 As can be seen from this relation, the polarization pattern observed depends on the initial spin multiplicity (S or T) and the sign of J (usually negative). Kaptein developed simple sign rules based on these parameters for the prediction of CIDNP phases (vide infra) which have been extended to CIDEP.42,44 Direct observation of the spin-correlated radical pair (SCRP), the geminate radical pair that has not yet recombined or permanently diffused apart, is of particular interest. The term ‘‘spin-correlated’’ refers to the entanglement, or non-separability of the spin system.45 The spindensity operator of a SCRP cannot be represented simply as the direct product of the spin density operators of the constituent radicals. The practical implication is that the phase relationship between the two electron spins results in populations of the electron-nuclear spin states that are significantly different from those formed from an f-pair.46 As previously noted, if the EPR lines of the two radicals overlap, they cancel out and no net polarization is seen (Fig. 3(b)). However, in the case of the SCRP, inter-radical interactions (normally J) remove the degeneracy of the transitions, leading to a spectrum of lines of alternating phase called an anti-phase splitting (APS) pattern (Fig. 4).47 The doublet splitting is 27 J7 and the polarization is maximised when 7 J7 and 7QAB7 are comparable.45 The direct observation of SCRPs requires that their lifetimes are long enough on the TR-EPR timescale. This can be achieved by confining

Fig. 4 Anti-phase splitting lineshape of a spin-correlated radical pair. Non-zero exchange interaction during detection causes the oppositely polarized components to be offset, preventing cancellation. 96 | Electron Paramag. Reson., 2019, 26, 89–129

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48,49

the RP in nanostructures, such as carbon nanotubes or micelles, as in the widely studied photoreduction of benzophenone in micelles of the anionic surfactant sodium dodecyl sulfate (SDS).46,50 The APS intensity formed from SDS-anthraquinone pairs has been controlled by changing the charge of the anthraquninone. More highly charged anthraquinones are more hydrophilic, so tended to have a greater affinity for bulk water than the micelles, leading to reduced APS intensity and predomination of the RPM.51 However, in the case of cetyltrimethylammoniam chloride (CTAC), the opposite trend was true, due to the anthraquinone’s strong electrostatic attraction to the surfactant’s cationic head group. In non-ionic Brij-based micelles, the TM can also be observed, when the relaxation rate of the sensitizer triplet is slow enough.52 ¨ttler et al. intro2.2.3 Radical triplet pair mechanism. In 1990, Bla duced a new mechanism to explain the CIDEP observed when a triplet molecule and a radical encounter, dubbed the radical triplet pair mechanism.53 This involves a collision between a doublet radical and an excited triplet to form a radical–triplet encounter complex, which can exist in either quartet or doublet states. It was already well-known that free radicals act as excellent quenchers for excited triplet states, as such encounter complexes offer a spin-allowed pathway for the return of a triplet state to a singlet ground state, known as enhanced intersystem crossing (EISC).54 To demonstrate how this EISC generates spin polarization, we consider the spin Hamiltonian for the radical-triplet pair given as:56,57 ^ ðr Þ ¼ gmB B0 ðSTZ þ SRz Þ  1 J ðr Þð1 þ 4ST  SR Þ þ VZFS H 3 The through-space exchange interaction between radical SR and Triplet ST takes the usual form: J(r) ¼ 2RT0  4RT0D J0 exp{ g(rd) } where J0o0 is the interaction strength at d, the distance of closest approach, and r is the intermolecular distance. Crucially, the zero-field splitting (ZFS) term of the triplet in the molecular frame of reference, assuming DcE, is given by:   1 VZFS ¼ D S2Tz  S2T 3 and does not commute with the exchange coupled elements of the RT Hamiltonian.13 At separations where J(r) is negligible, the non-interacting pair states formed from the triplet (7T1i, 7T0i, 7Ti) and radical (7ai, 7bi) are Electron Paramag. Reson., 2019, 26, 89–129 | 97

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appropriate, whereas at rEd, the eigenstates are quartet and doublet states: rffiffiffi rffiffiffi 1 2 | Tþ biþ | T0 ai | Q þ1 i ¼ 3 3 2

rffiffiffi rffiffiffi 1 2 | T aiþ | T0 bi | Q 1 i ¼ 3 3 2

| Qþ3 i ¼ | Tþ ai

| Q3 i ¼ | T bi

2

rffiffiffi rffiffiffi 2 1 | Tþ bi | T0 ai | D þ1 i ¼ 3 3 2

2

rffiffiffi rffiffiffi 2 1 | T ai | T0 bi | D 1 i ¼ 3 3 2

As seen in Fig. 5, there are three level crossings in this system at distances determined by the relative magnitude of the electron Zeeman and RT exchange interactions. Due to the presence of the ZFS perturbation to the spin Hamiltonian, these become level anti-crossings (LACs),58 leading to state mixing. With notable similarity to the movement between strong and weak exchange regions of the RPM, polarization is generated as follows: the RT pair encounters and doublet states undergo spin-allowed quenching (EISC) to lower-lying 2RS0 states (radical-singlet pair), as quenching of quartet states remains spin-forbidden. Doublet quenching alone equally depopulates 7ai and 7bi radical states, but state mixing at the LACs means 7Q3/2i and 7Q1/2i are also partially depleted. The nonmixed states 7Q13/2i and 7Q11/2i which correlate with 7T1ai and 7T0ai, retain their initial populations, leading to excess 7ai spin population manifest as emissive CIDEP.55,59–61 The RTPM may also operate via the interaction between an excited singlet state and a radical.62 Such a regime is referred to as doublet precursor RTPM (DP-RTPM), while RTPM from triplet states is called quartet precursor (QP-RTPM). The former dominates when ISC is inefficient. In this regime, the energy levels, as well as their LACs, are the same as in the QP-RTPM, except the radical-singlet states are above the RT pair states. Upon encounter 2RS0 undergoes efficient quenching, populating 2 RT0 via internal conversion and during separation the LACs drive formation of an excess of 7bi radical states giving rise to absorptive CIDEP. The preceding discussion assumed that Jo0, which is true for most RT pairs including all triplet–TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl) systems. However, Kawai has reported examples of systems in which J40.63,64 In this situation, the doublet states of the radical-triplet pair lie above the quartet states, meaning that QP-RTPM now produces a net absorptive CIDEP and DP-RTPM net emissive CIDEP. The existence of an unusual positive J is due to a charge transfer (CT) interaction, which is significant if the energy gap between the 2Dye–1R1 (CT) state and the 3 Dye–2R (RT) state is small (with due consideration for solvent reorganization and the coulombic interaction energies).65 Similar considerations of the energy gap between the RT state and the CT or 1Dye–2R* state, can be made in relation to the plausible triplet-quenching mechanism.66 Thus far we have only considered net RTPM polarization; however, as for the RPM, many systems exhibit multiplet CIDEP, that is intensity variations over the different hyperfine lines. For example, the 4-oxo-TEMPO 98 | Electron Paramag. Reson., 2019, 26, 89–129

Published on 02 November 2018 on https://pubs.rsc.org | Electron Paramag. Reson., 2019, 26, 89–129 | 99

Fig. 5 Energy diagrams for quartet precursor RTPM, encounter and quenching (left), and separation (right), after ref. 55. Open (filled) circles represent initial (final) populations.

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Fig. 6 Multiplet RTPM seen for TEMPONE in benzene with the triplet formed from (a) phenazine and (b) acetone. Reproduced from ref. 67 with permission from American Chemical Society, Copyright 1991.

(TEMPONE) system shown in Fig. 6(a) shows stronger emissive CIDEP towards lower field, as a result of an E/A (E for mI ¼ þ1 and A for mI ¼ 1) multiplet pattern superimposed on net emissive CIDEP; this is termed an E*/A pattern. The inversion of polarity for mI ¼ 1 in Fig. 6(b) due to the absorptive multiplet CIDEP exceeding the net emissive CIDEP is uncommon.67 The multiplet effect arises as the hyperfine coupling is a further perturbation to the RTPM Hamiltonian leading to additional 7Q11/2i27D11/2i and 7Q1/2i27D11/2i state mixing. Importantly, multiplet CIDEP can only be explained by the RTPM but not the ESPT mechanism introduced below.55 The RTPM is also supported by the direct observation of quartet and doublet states.68,69 We now consider the kinetics of the processes involved in the RTPM: hn

ISC

S0 ! S1 ! T1 kq

ðoptical excitation followed by intersystem crossingÞ

T1 þ R ! S0 þ R*

ðelectron spin polarization transferÞ

100 | Electron Paramag. Reson., 2019, 26, 89–129

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R* ! R

ðspin-lattice relaxationÞ

kt

T1 ! S0

ðtriplet decayÞ ktt

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T1 þ T1 ! 2S0

ðtriplet-triplet annihilationÞ

The rate of build-up of magnetization is given by the Bloch and kinetic equations:53,70 dMy My ¼  o1 Mz  kq My ½T dt T2 Mz  Peq ½R dMz ¼ þ ðPn  Pm Þkq ½R½T þ o1 My dt T1 d½T ¼  kq ½R½T  kt ½T  2ktt ½T2 dt where Pn, Pm and Peq are the net, multiplet and equilibrium polarizations, respectively. The first equation, in which o1 is the microwave field strength, is only necessary if the continuous-wave rather that pulsed TR-EPR method is applied. Numerical solution of the coupled differential equations allows fitting of the RTPM kinetics. While in the case of continuous-wave TR-EPR absolute detection sensitivity is hard to calibrate so Mz and hence Pn,m cannot be obtained quantitatively, using pulsed detection the thermal radical signal may also be observed. This allows the ratio Pn/Peq to be determined, with specialist pulse sequences also being used to remove any ESPT contribution.70 Shushin and coworkers have carried out extensive theoretical analysis into the factors affecting the magnitude of RTPM, proposing formulae for two limiting cases.59–61 If 7 J074o0 (the strong exchange limit), then:   p D2zfs o0 d 1 4 Pn ¼  þ 45 o20 gDr 1 þ x2 4 þ x2 where x ¼ 1/o0tc, and Dr is the diffusion coefficient. In the weak exchange limit, 7 J07oo0, then: ! 8 D2zfs J0 d 1 4 Pn ¼  þ 45 o20 gDr ð1 þ x2 Þ2 ð4 þ x2 Þ2 An important distinction is that in the latter regime the LACs are inaccessible, occurring at rod, hence CIDEP creation is inefficient.55,57 In 1986 Immamura et al. had also considered the CIDEP arising from radical–triplet interactions, proposing that triplet polarization from differential population is transferred to the radical.71 This mechanism, known as electron spin polarization transfer (ESPT), predominates in solids. The RTPM is more commonly used to explain CIDEP generation in solution, where radical quenching competes ineffectively with rapid relaxation of the polarized triplet, which also means the triplet itself is Electron Paramag. Reson., 2019, 26, 89–129 | 101

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not observed. There is, however, experimental evidence for the occurrence of ESPT in porphyrin-TEMPO systems in viscous solutions; under such conditions averaging of the porphyrin ZFS by rotational motion is ineffective giving rise to the slow spin–lattice relaxation of the triplet necessary for ESPT.72,73 2.3 Applications The development of TR-EPR and the theory of Spin Chemistry have been intimately associated with the investigation of the electron transfer processes in photosynthetic reaction centres.74 In the last two decades, since the proposal that its magnetically sensitive radical-pair reactions may be responsible for the avian magnetic compass sense,75 the focus of attention has been on the flavoprotein cryptochrome (and the photolyases).26,76 In both cases, which are well reviewed elsewhere,24,26,77 the biological systems intrinsically contain the necessary components for the photochemical generation of spin hyperpolarization. Here, instead, the focus will be on less obvious candidates, those systems in which utilization of electron hyperpolarization is dependent on addition of some extrinsic radical and/or chromophore. 2.3.1 Singlet oxygen. The Kawai group have extensively studied the use of CIDEP for lifetime measurements of singlet oxygen. This key species is relevant to studies in a variety of areas including cardiovascular disorders,78 photosynthesis79 and photodynamic therapy.80 TREPR investigations of RTPM generated polarization in the anthracene/ TEMPO system observed the expected net emissive signal ( Jo0) when samples were deoxygenated. However, when open to the air, an extremely large and much longer lived (tB25 ms) net absorptive polarization was seen.81 Molecular oxygen (3Sg ) is well known to undergo spin-allowed quenching of triplet states to generate singlet oxygen (1Dg), which is itself known to be effectively quenched by nitroxide radicals.82 The observed absorptive polarization arises as this second quenching step occurs via a DP-RTPM, as confirmed by experiments in which the TR-EPR decay rate was shown to have a linear dependence on the concentration of singlet oxygen quencher b-carotene.81 A number of other triplet sensitizers have been shown to generate absorptive CIDEP due to nitroxide quenching of singlet oxygen, including porphyrins in aerated toluene solutions,73 along with benzophenone, naphthalene and 9,10-acenaphthenequinone in benzene.83 Even trace amounts of oxygen remaining after argon bubbling are sufficient to permit absorptive polarization to be observed, highlighting the importance of rigorous deoxygenation in TR-EPR experiments. Only net CIDEP is generated by singlet oxygen as the large ZFS of triplet oxygen is dominant over much smaller hyperfine interactions. The ZFS is also large compared to the Zeeman energy (at X-band) such that the quartet and doublet states can be considered to mix over a wider range of separations than the conventional level crossing region, leading to accelerated doublet-quartet mixing.84 The absolute magnitude of the 102 | Electron Paramag. Reson., 2019, 26, 89–129

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CIDEP is therefore large, with the measured value of Pn ¼ (340  40)Peq being among the highest reported polarizations generated by the RTPM and implying quenching via EISC in the strong exchange limit. The unusually large polarization permits observation of extremely strong TREPR signals for TEMPO in benzene despite a low quenching rate Pand fast radical relaxation due to Heisenberg spin exchange with O2 (3 g ). The slow quenching by TEMPO leads to the CIDEP decay rate being invariant under radical concentration.85 Using the usual Bloch equation approach to model the CIDEP time profile, the lifetime of singlet oxygen can be determined in good agreement with literature values under the same conditions. TR-EPR has therefore been proposed as an alternative detection strategy for singlet oxygen, in place of time-resolved measurements of singlet oxygen phosphorescence at 1270 nm which suffer from low sensitivity and interference from other luminescence sources.81,84 Singlet oxygen lifetimes depend on the relative occurrence of C–H and C–D bonds in the solvent. The Turro group extracted lifetimes from the CIDEP of anthracene and TEMPONE in aerated d6-benzene (h6-benzene) of 88  10 ms (27  5 ms) mirroring the trend measured by phosphorescence in a pure solvent of 681 ms (30 ms), to further verify participation of singlet oxygen in the CIDEP generation.86 Phosphorescence measurements confirmed that the lifetime in d6-benzene is significantly reduced in the presence of the radical and dye. The spin polarization efficiency per quenching event of three structurally similar nitroxides increases in the order TEMPONEoTEMPOoTEMPOL (4-hydroxy-TEMPO), which is the opposite to the trend in singlet oxygen quenching rate constants determined by phosphorescence measurements.86 It was suggested that this implies that quenching and polarization are independent processes. To obtain this interesting result simultaneous detection of CIDEP from a mixture of two radicals was performed, one 15N labelled to enable signals to be independently resolved. The equal decay rates and intensities of the integrated signals despite the differences in the independently measured quenching rates, required different values for polarization efficiency in the Bloch kinetic analysis. It was also noted that solutions should be air-saturated and not oxygen-saturated as excess oxygen concentrations broaden the EPR spectra sufficiently that CIDEP cannot be observed. 2.3.2 Masers. A number of groups have exploited the polarization inversion afforded by CIDEP mechanisms to design room temperature masers. Blank and Levanon proposed the use of the RTPM to generate maser action, specifically from the interaction between a trityl radical and the triplet state of etioporphyrin.87,88 However, no maser oscillation was seen for this system and the calculated optical illumination power of B10 W necessary for continuous-wave operation was deemed incompatible with the necessity to avoid photodegradation and excessive sample heating. More recently, Oxborrow et al. reported a functional solid-state maser at room temperature.89 This made use of the triplet mechanism in p-terphenyl doped with pentacene, contained in a high Q-factor sapphire Electron Paramag. Reson., 2019, 26, 89–129 | 103

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ring, and microwave emission at 1.45 GHz was achieved. A major disadvantage was that its threshold optical illumination power was 230 W and its peak power was 1.4 kW. It also operated in burst mode, whereas continuous maser operation would be more desirable for most applications. Replacing the sapphire ring with strontium titanate to optimize the Purcell factor of the cavity was found to reduce the threshold optical power to 2 W and the peak optical power to 70 W.90 In addition the visible-to-microwave conversion efficiency has been measured as a function of excitation energy, using nanosecond laser pulses, in order to improve the design of the maser.91 Computational studies identified diaza-substituted pentacene derivatives as potential candidates for generating continuous wave maser action,92 but others proposed nitrogenvacancy centres in diamond (which will be discussed later in terms of nuclear hyperpolarization) as appropriate systems due to their extremely long spin lifetimes. Numerical simulations estimated such a maser would require pumping at less than 10 W,93 and such a continuous-wave maser was recently demonstrated with a threshold pump power of 138 mW.94 2.3.3 Polymers. As reviewed elsewhere,13 Forbes and co-workers have carried out extensive TR-EPR based studies into the photodegradation of acrylic polymers, both confirming the nature of the radicals involved and using CIDEP to probe conformational dynamics. In these studies, the polymer radicals are spin polarized due to the TM, which is important because the concentration of radicals formed is so small that a Boltzmann distribution of electron spins is undetectable. In fact, the strong TM effect has been suggested to be dependent on slow, anisotropic rotation of the polymer radicals, leading to more selective triplet sublevel population.96 While these systems have spinpolarization due to an intrinsic chromophore, here we focus on a more recent development, in which addition of an extrinsic radical enables probing of long-range polymer motions using the RTPM.95 If a persistent nitroxide radical is incorporated into a polymer chain, it can interact with a photoexcited ester (triplet state) to produce spin polarization, which can be easily recognized in the TR-EPR spectrum as a 3-line hyperfine pattern (Fig. 7). The competition between and relative intensity of RTPM and TM CIDEP provides information about the flexibility of the polymer chain. This competition is sensitive to the level of nitroxide incorporated within the polymer, which will alter the distance distribution between the excited side-chains and randomly incorporate radical. Incorporation of the radical into the PMMA chain resulted in a smaller RTPM signal than that of a separated PMMA chain and TEMPO radical, verifying that the polymer chain restricts access of the radical to the excited triplet. Other effects such as temperature, solvent and steric bulk to the side chain were investigated by this method.95 2.3.4 Biological structure I: RTPM. Having obtained evidence that RTPM polarization may be observed in covalently linked chromophorenitroxide systems,97 the Corvaja group proposed the use of intramolecular 104 | Electron Paramag. Reson., 2019, 26, 89–129

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Fig. 7 TR-EPR spectra of nitroxide-containing copolymer as a function of nitroxide incorporation (left, 120 1C) and as a function of temperature (right, 1 mol%). At low loading, transitions of the TM polarized main chain polymeric radical of PMMA are dominant: these are denoted by asterisks in the 1.0 mol% 120 1C spectra. Adapted from ref. 95 with permission from American Chemical Society, Copyright 2014.

Fig. 8 Structures of the spin label and chromophores integrated into peptide structures.

RTPM interactions to study peptide conformation in solution.98 Their work exploited the interactions between the photoexciteable amino acids Bin, Bpa or Trp and the nitroxide spin label 2,2,6,6-tetramethyl-N-oxyl-4amino-4-carboxylic acid (TOAC) (Fig. 8).98–100 As discussed above, RTPM polarization requires passage through the LAC region, which is at a radical-triplet separation determined by the exchange interaction J(r). In the intermolecular case this is achieved by free translational diffusion, however in the intramolecular case the residual motion of the structure must permit the appropriate approach distance to be obtained and sufficient residence time for polarization generation. Information on the RT separation is therefore accessible, with a proof of principle study testing Electron Paramag. Reson., 2019, 26, 89–129 | 105

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this by placing the labels at an appropriate separation (B6 Å) on a 310helix, allowing polarization to be observed via TR-EPR.98 A full understanding of the distance dependence of the exchange interaction is necessary to obtain quantitative measurements, whereas this first study simply noted that observation of superimposed net and multiplet polarization was consistent only with J values expected for a helical rather than unfolded conformation. Further studies on a series of peptides in which the radical-chromophore separation and relative orientation was varied, modelled the time evolution of the CIDEP signal using modified Bloch equations.99,100 Triplet quenching rates were lower for greater separations but the effect was insufficient to provide quantitative distance information, possibly due to flexibility of the chromophoric amino acid Bpa. A cyclic peptide showed no spin polarization, despite having the same number of bonds between Bin and TOAC as a linear peptide in which polarization was observed, implying through-space rather than through-bond interactions dominate. Signal intensities were larger for the intramolecular systems than in tests with a freely diffusing chromophore, and appeared to vary with separation of the two units in the linked systems, but in the continuous-wave TR-EPR method used intensities could not be precisely obtained.100 It would be interesting to revisit these systems using the pulsed approach to TR-EPR to obtain quantitatively the variation in polarization efficiency. Kawai et al. proposed the application of the RTPM to study protein folding, probing the solvent accessibility of Trp residues by monitoring of CIDEP intensity in the presence of a nitroxide radical.101 As Trp absorbs at longer wavelengths than other amino acids, selective photoexcitation at 280 nm to generate triplet Trp was possible. Quenching of the triplet state requires close approach (passage through the relevant LAC region) of the freely diffusing radical which is possible only if Trp is solvent exposed (c.f. photo-CIDNP described below, in which Trp is not excited but itself reacts with a freely diffusing triplet dye). A proof-of-concept study verified generation of RTPM polarization between N-acetyl-Trp and TEMPO, with strong emissive polarization observed (Pn ¼ 30 Peq), before application of the method to the small peptide a-lactalbumin. The CIDEP intensity was shown to have a pH dependence (Fig. 9) which mirrored pre-existing fluorescence intensity data and could be interpreted in terms of known conformational transitions. Full kinetic modelling of CIDEP intensity does, however, require independent optical measurements to obtain relevant parameters such as quenching rate constants and triplet quantum yield. The method has more recently been applied to the water-soluble chlorophyll protein WSCP.102 In this case the chlorophyll triplet was quenched and nitroxides of various charge states utilized. Observation of CIDEP only with neutral and cationic radicals was in agreement with a negative surface potential around the pore through which the chlorophyll was solvent exposed. Further possibilities exist to utilize this method not only with biomolecules containing endogenous chromophores, but also through labelling with dyes of high triplet yield and photostability.

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Fig. 9 Effect of pH on CIDEP time profiles of TEMPO with a-lactalbumin. Reproduced from ref. 101 with permission from Springer Vienna, Copyright 2010.

2.3.5 Biological structure II: PELDOR. Since the start of the millennium, Pulsed Electron Double Resonance (PELDOR),107 also known as Double Electron-Electron Resonance (DEER), has become a widely used tool in biostructural studies. Using the size of the dipolar interaction between two paramagnetic centres, PELDOR measures interspin distance distributions, typically in the 1.8 to 6 nm range,108 although in favourable cases protein deuteration has almost doubled the upper limit.108,109 The original PELDOR sequence consisted of three pulses: a 2-pulse echo with constant inter-pulse delay as an observer sequence at frequency (oprobe) and an inversion pulse at a second microwave frequency (opump) so as to excite a different spin ensemble. The requirement that microwave pulses cannot overlap on commercial pulsed-EPR spectrometers results in a dead-time, hence the 4-pulse variant is more commonly used. In this approach, the less-intense refocused echo is observed, permitting overlap of the pump pulse with the primary echo, essentially removing the deadtime at the expense of signal intensity.108 The basis of both sequences, which are shown in Fig. 10, is that the pulse sequence applied to the observer spin refocuses inhomogeneous broadening of the EPR line, but inversion by the pump pulse of other dipolar coupled spins shifts the frequency of the detected spin by odip. This arises as inversion of the pump spin inverts its contribution to the local magnetic field experienced by the observer spin. The frequency shift of the observer spins is not refocused, hence monitoring echo intensity as a function of the time of the pump pulse leads to a modulation of the decaying time-domain signal. Fourier Transformation leads to a Pake pattern from which the modulation frequency odip /

ð3cos2 y  1Þ r3

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108 | Electron Paramag. Reson., 2019, 26, 89–129 Fig. 10 Pulse sequences for conventional and photogenerated triplet PELDOR variants.103–106

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may be obtained, where r is the interspin distance, and y the angle between the external magnetic field and the interspin vector. Sophisticated data analysis methods permit extraction of spin–spin distance distributions,108 with attention now focussing on extraction of angular information in orientation-selective PELDOR.110 Ideally the spin labels should be spectrally well separated, with the excitation bandwidth27 of each pulse small enough to selectively excite one spin, but large enough to achieve a deep modulation and high detection sensitivity. While site directed spin labelling of proteins with nitroxides is a well-developed approach,111 the broadening of their EPR spectra by anisotropic interactions is not ideal from a sensitivity perspective and high flexibility often negates the possibility of obtaining orientation data. A number of alternative spin-labels based on paramagnetic metal centres have therefore been developed.112 In addition to metal–metal distance measurements, these may be combined with a nitroxide to provide a spectroscopically distinct pair of spin labels, in the so-called orthogonal labelling approach. However, the broad spectra of many metal centres whilst providing good orientation selectivity, at best lead to low sensitivity due to fractional excitation and at worst exceed the accessible excitation bandwidth range for a two-frequency experiment.110 Short relaxation times also limit applications of metal centres. Di Valentin and co-workers recently demonstrated the application of spin polarization to PELDOR by pumping a nitroxide radical and observing a photoexcited porphyrin triplet state, Fig. 10(c). The porphyrin moiety offers high triplet quantum yield, high natural abundance in biomolecular systems, desirable relaxation behaviour and good photostability. The measured radical-triplet separation in a series of seven small synthetic a-helical peptides containing TOAC and covalently linked at the N-terminus to free-base porphyrin, was in excellent agreement with that obtained from a structural model for distances in the 1.7 to 6 nm range.103,104 At 20 K a signal-to-noise ratio comparable to nitroxide-nitroxide PELDOR was obtained with a 100-fold reduction in number of scans.103 High sensitivity arises from the triplet’s intrinsic spin polarization due to selective ISC, which is only partially negated by the high spectral width associated with anisotropy of the triplet ZFS tensor. The orthogonal nature of the spin labels permitted the experiment to be optimised, achieving maximum inversion efficiency by application of the pump pulse at the maximum of the nitroxide spectrum and detection of the triplet via the most intense emissive transition. Inserting Cu(II) into the free-base porphyrin enabled comparison of the light-induced method to a conventional DEER measurement on the Cu(II)-nitroxide system, with excellent agreement in the measured distance (Fig. 11).104 This validates treatment of the triplet state via the point dipole approximation despite the significant delocalisation of spin density over the porphyrin moiety. The phase and spin relaxation rates of the triplet are comparable to those of a nitroxide under usual cryogenic PELDOR measurement conditions, whereas those of Cu(II) are significantly shorter. It should be noted that Electron Paramag. Reson., 2019, 26, 89–129 | 109

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Fig. 11 Four-pulse PELDOR trace after background correction (left) and corresponding Fourier transform (right) of porphyrin-peptide-nitroxide system. Data recorded at 20 K under photoexcitation for free-base porphyrin (3TPP-NO ) and without photoexcitation for the copper analogue Cu(II)TPP-NO . Adapted from ref. 104 with permission from John Wiley and Sons, Copyright r 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

the Pake pattern does not show any effects of orientation selection. This can be attributed to the fact that at the observer position nearly all possible orientations with the tetrapyrrole plane parallel to the external field are detected. However, in theory the ZFS anisotropy of a triplet state could permit orientation selection, with triplet polarization counteracting the reduced sensitivity of such measurements and applications utilizing photoselection with polarized light might also be imagined. Lightinduced PELDOR has been successfully extended to use of the triplet state of endogenous chromophores as a spin probe in the peridininchlorophyll a-protein,105 with other future possible targets including heme groups following Zn(II) substitution and flavins.103 Inspired by Di Valentin’s work, a variant technique has been developed, dubbed laser-induced magnetic dipole (LaserIMD) spectroscopy.106 This replaces the pump pulse with a triplet generating laser flash, whilst observing a nitroxide spin-label, in what may be considered a variant of 3-pulse PELDOR (Fig. 10(d)). The method recovers the signal intensity advantage of the 3-pulse method but with zero deadtime as laser and microwave pulse overlap is possible. As this variant requires only a single microwave frequency, cavity tuning can be optimised for detection sensitivity, rather than being compromised to gain increased bandwidth. The method observes the Boltzmann polarized nitroxide radical so does not gain a sensitivity advantage from the triplet hyperpolarization. As all chromophore orientations may be excited however, the authors claim that the lack of orientation selection of the pump pulse may increase sensitivity and effectively unlimited excitation bandwidth should reduce the lower limit of distances than can be measured. This does however make assumptions regarding the fraction of molecules in the triplet state, which is limited by the triplet quantum yield. LaserIMD was demonstrated using the same peptide used for demonstration of light-induced PELDOR, and was also successful in 110 | Electron Paramag. Reson., 2019, 26, 89–129

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recovering an interspin distance close to the expected value, though with no sensitivity advantage over conventional nitroxide–nitroxide PELDOR. LaserIMD has also been applied to an endogenous chromophore, the heme group in spin-labelled cytochrome C, recovering an interspin distance close to that estimated from theory. Iron porphyrins show very short excited state lifetimes113 and no triplet spin-echo could be observed. The authors argue that this is due to short phase memory only, which LaserIMD is not restricted by since the triplet is the pumped rather than observed species.106 Both laser-induced-PELDOR and LaserIMD utilize the interaction of a polarized triplet and a nitroxide radical, but in neither case has any radical polarization been reported. The RTPM requires that the radicaltriplet pairs are able to obtain a separation close to the LAC region. It is possible that the rigid tertiary structure of the peptides either does not provide the appropriate separation, or permits insufficient motion for J modulation, such that radical polarization is not generated. Intramolecular RTPM polarization has been reported in a series of flexibly linked systems114 and more rigid helical peptides,98–100 but in both cases in liquid solution at or close to room temperature. PELDOR methods by necessity use cryogenic temperatures to achieve the necessary spin relaxation times. While Yamauchi investigated radical-triplet interactions at low temperatures the focus was direct observation of quartet and doublet states in strongly coupled systems.115,116 It would be interesting to investigate further whether under appropriate conditions radical polarization can be observed in weakly coupled systems used for lightinduced PELDOR or LaserIMD, either at cryogenic temperatures or by using radicals that permit measurements in a rigid matrix at room temperature.117

3

Optical enhancements in NMR

There are a variety of ways that optical excitation may be used to tackle the sensitivity problem in NMR, as illustrated by the following two examples. Dissolution DNP generates a large nuclear hyperpolarization through microwave pumping of radicals in the solid-state under cryogenic conditions, followed by rapid warming, faster than nuclear relaxation times, to enable solution-state NMR detection at room temperature.15,118 The radicals necessary for DNP generation reduce nuclear relaxation times causing loss of hyperpolarization and line broadening hence must be separated from the sample. Removing the radical rapidly during warming is challenging but has been achieved by dissolution of the solid in an immiscible solvent. Recent studies have instead utilized UV-generated radicals able to mediate polarization buildup at low temperature, yet which are unstable at higher temperatures and disappear during the warming step.119,120 The nuclear relaxation induced by paramagnetic species has also been harnessed to reduce the often long relaxation delays in NMR experiments, hence increasing the repetition rate, in the Photo-Induced Reversible Acceleration of T1 Relaxation (PIRAT) method.121 Two colour illumination of a spin-crossover Electron Paramag. Reson., 2019, 26, 89–129 | 111

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compound switches between a paramagnetic state that rapidly relaxes the nuclei back to thermal equilibrium and a diamagnetic form that does not broaden the NMR spectrum during signal acquisition. While both techniques show the exciting possibilities afforded by the use of optical excitation in NMR, they do not in fact utilize spin hyperpolarization, which will be our focus for the remainder of this chapter.

3.1 Optical nuclear polarization Under suitable conditions photoexcited triplet states in solids are able to generate extremely large nuclear spin polarizations, as first reported in 1967 by Hausser and co-workers.122 This Optical Nuclear Polarization (ONP) involves initial generation of triplet polarization through differences in the ISC rates to or from the different triplet sub-levels, just as in the TM described above. The hyperfine interaction couples the nuclear spins to the triplet states, allowing a partial polarization transfer to local nuclei, which by spin diffusion through the dipolar coupled network of spins may be distributed to the bulk. We begin our discussion of ONP by considering organic chromophores doped into molecular crystals, before moving on to the more recent emergence of colour centres in diamond as a hyperpolarization source. In addition to the ZFS of the triplet state, under an applied magnetic field, the T  sub-levels are split by the electron Zeeman interaction. Depending on the molecular orientation relative to the applied magnetic field, one of these levels will cross T0, at a field strength also dependent on the size on the ZFS. The additional contribution of the hyperfine interactions causes these crossings to become LACs and the resultant nuclear-spin dependent state mixing generates nuclear hyperpolarization through a coherent process. Actually the LACs come in pairs, each with a different width and opposite sign, leading to broad and a narrow features in the ONP field-dependence (Fig. 12).123 Orienting the crystal correctly is of paramount importance because any misalignment introduces a transverse field component which, if in excess of the hyperfine perturbation, significantly decreases the polarization generated. To ensure polarization transfer before decay or spin-lattice relaxation of the triplet state, microwave pumping is commonly applied to drive electron-nuclear polarization transfer, in the techniques NOVEL (nuclear orientation via electron spin-locking)124 and ISE (integrated solid effect) as shown in Fig. 13.125,126 The former, as the name implies, transfers the electron spin magnetization present after a laser pulse to the transverse plane whereupon a continuous-wave spin-locking pulse is applied. By choosing an appropriate microwave field strength the Hartmann–Hahn condition is achieved, that is the rotating frame precession frequency is matched to that of the nuclei leading to efficient polarization transfer. The latter method applies continuous microwaves after the laser pulse, initially off-resonance but with an adiabatic magnetic field sweep which at two points matches the electron nutation and nuclear larmor frequencies leading to polarization transfer. NOVEL and ISE are discussed 112 | Electron Paramag. Reson., 2019, 26, 89–129

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Fig. 12 LACs and corresponding ONP spectra in a molecular triplet when the applied magnetic field is parallel to the molecular z-axis (left), and y-axis (right). Reproduced from ref. 3 with permission from Taylor & Francis (http://www.tandfonline.com), Copyright 2017.

Fig. 13 Pulse sequences used to drive electron-nuclear polarization transfer in ONP.

in more detail in a series of papers by Henstra and Wenckebach;127–129 here we simply note that ISE is in general more efficient.3 As the ZFS significantly broadens the powder EPR spectra of the triplet state, most ONP studies have taken place in a small number of suitable target molecules in a single crystal host, commonly pentacene in p-terphenyl or naphthalene. Using the latter system and by crushing the sample, Takeda et al. were able to show that polarization can still be Electron Paramag. Reson., 2019, 26, 89–129 | 113

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observed in polycrystalline materials by restricting the ISE field sweep width to a narrow part of the EPR spectrum.130 The measured 1H NMR signal enhancement of 3160 (at 13.6 MHz/319 mT, 100 K) is lower than for a single crystal, not only due to excitation of only a fraction of the spectrum, but also due to reduced spin–lattice relaxation time after pulverization of the crystals and laser scattering. ONP has also been observed in glassy matrices at low temperature, using benzophenone and o-terphenyl as hosts for pentacene.131 In 2014 Tateishi et al. reported a 1H spin polarization of 34% at room temperature, giving a signal enhancement of 2.5105 in a 0.40 T field.132 Building upon previous studies that had independently investigated effects of proton concentration in the host matrix133 and by deuteration of the guest,134 this high polarization was achieved using pentacene in a p-terphenyl matrix, with full deuteration of the guest molecule and partial deuteration of the host. The former increases the electronic relaxation time, whilst the latter selectively targets the contribution from pendulum motion of the central benzene ring of the terphenyl to the nuclear spin lattice relaxation time.132 Despite the high polarization obtained, and demonstration of cross-polarization to enhance 13C spin polarization of the host matrix, a significant challenge in the field of ONP remains in developing applications able to utilize this impressive spin polarization beyond fundamental physics experiments. Recently the possibility of utilizing optical polarization of colour centres in diamond for hyperpolarization has generated significant attention, with specific focus on the negatively charged nitrogen vacancy centre labelled NV. The physics of this centre is reviewed in detail by Doherty et al.135 Here we note only that competition between radiative and non-radiative decay pathways upon green-light illumination overpopulates the mS ¼ 0 sub-level of the triplet ground state of this S ¼ 1 defect, with 95% polarization achieve within a few microseconds. With a large axial ZFS of 2.88 GHz, this gives well separated strongly polarized absorptive and emissive EPR transitions.136 The spectrum is however exquisitely sensitive to the precise alignment of the field parallel to the h111i axis connecting the nitrogen and adjacent vacancy and it must be remembered that the defect is able to occupy four symmetry-related orientations within the diamond lattice. Early work on nuclear polarization of NV was concerned with qubit initialization and by applying a 50 mT static field to reach a LAC of the excited state, 98% polarization of 15N was demonstrated at a single centre through optically detected magnetic resonance.137 The large optical polarization is however transferred via the hyperfine interaction to 13C nuclei surrounding the defect and subsequently throughout the diamond lattice via spin diffusion; bulk 13C NMR enhancements of B500 were achieved by optical pumping at B50 mT and rapid shuttling to 4.7 T for measurement.138 Polarization transfer may also be achieved by using the microwave driven approaches of NOVEL and ISE as introduced above, to reach the Hartmann–Hahn matching condition between electron and nuclear frequencies.139–141 To enable polarization transfer out of the diamond to some substrates of interest, nanodiamonds are of particular 114 | Electron Paramag. Reson., 2019, 26, 89–129

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interest with suggested use in enhancing magnetic resonance imaging (MRI) sensitivity.142 With a randomly oriented ensemble of colour-centres however the aforementioned sensitivity to alignment becomes particularly problematic. The ISE and NOVEL methods are therefore modified for application to nanodiamonds, a quasi-adiabatic sweep of the microwave frequency enabling polarization transfer for a range of orientations. A further challenge with diamond hyperpolarization schemes is transfer of polarization to a substrate whose NMR signal is of interest: however a method to polarize liquids using nanodiamonds immobilised in a hydrogel inside a flow channel has been proposed, theoretically capable of enhancing water polarization to 4700 times the thermal level at 0.35 T.143 Although much attention has focussed on the NV centre, the diamond lattice may contain numerous other defects that can be optically polarized including the similar silicon vacancy SiV0. 144 An especially interesting report noted that optical pumping of a diamond containing the 15Ns0 and 15 N3V0 defects in close proximity (o3 nm separation) enhanced the 15N signal to 2000 times the thermal equilibrium signal.145 Interestingly both are S ¼ 12 defects so the usual mechanisms of solid-state polarization cannot be at play, and the mechanism is insensitive to the applied magnetic field up to B20 T.146 Polarization transfer to 13C occurs without any need for microwave driving, giving 13C NMR enhancements at 7.04 T of 200 at room temperature and 500 at 240 K which are comparable to those achieved by microwave pumping of NV at low fields. 3.2 Chemically induced dynamic nuclear polarization In 1967, Bargon and Fischer, and Ward and Lawler, independently reported unusual emissive lines in NMR spectra of radical reactions,21,147 explained shortly afterwards by the development of the radical pair mechanism.39,148 While CIDNP may arise from radical reactions in the dark, here we consider the common case that the polarization arises from a photochemically generated RP, so-called photo-CIDNP. The radical pair mechanism (RPM) has already been described in detail above. The key point for generation of CIDNP is consideration of the radical recombination process, singlet encounters of the geminate pair allowing spin-allowed electron transfer and recombination, whereas triplet pairs cannot recombine so separate and eventually return to the ground state through f-pair collisions (termed the ‘‘escape’’ route). As the rate of S–T0 interconversion depends on the nuclear spin states of the radical pair, the recombination and escape products while chemically identical have different nuclear spin state populations. This leads to oppositely polarized NMR transitions, which do not cancel out because the second-order f-pair recombination is much slower than geminate recombination. The electron spin of the freely diffusing radicals therefore relaxes their nuclear spin states, allowing the nuclear hyperpolarization of the geminate recombination products to be observed. These processes are summarised in Fig. 14, for the common application of CIDNP to the study of protein structure, which utilizes radical pair formation between a triplet dye, typically a Flavin, and solvent accessible aromatic amino acid residues Trp, Tyr and His.149 Electron Paramag. Reson., 2019, 26, 89–129 | 115

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Fig. 14 CIDNP generation through quenching of a triplet state by an aromatic amino acid. Reproduced from ref. 149 with permission from Elsevier, Copyright 2004.

It is important to note that the radical pairs exist in a solvent ‘‘cage’’ which allows two radicals to collide with each other several times. The cage lifetime depends on the distance of closest approach and relative diffusion coefficient, and for maximal CIDNP should be close to the inverse of the S–T0 interconversion rate. With too short a lifetime, the radicals diffuse apart before any CIDNP has developed, and with too long a lifetime all RPs eventually undergo cage recombination, irrespective of spin state.3 Considering the larmor precessional frequencies of the two radicals in the simple case of coupling to only a single spin-1/2 nucleus leads to the conclusion that net hyperpolarization via S–T0 mixing is most efficient when DgmBB0 ¼ 7a7/2. Extending the consideration to the case of two nuclei, it instantly becomes apparent that the interconversion rate depends on whether the magnetization of the two nuclear spins is parallel or anti-parallel.3 This multiplet polarization will occur even when Dg ¼ 0 (which leads to no net CIDNP) and results in intensity variation of lines within the same NMR multiplet. Kaptein developed a set of simple rules for determining the polarity of the polarization.44 The sign of net polarization is given by: Gne ¼ mE  sgn(Dg)sgn(ai) where m ¼ þ1 denotes RP formation from a triplet precursor and m ¼ 1 for a singlet precursor. Similarly, E ¼ þ1 denotes geminate products and E ¼ 1 escape products. The overall sign of G indicates whether a line is in absorption (positive) or emission (negative). Similar rules apply to the multiplet case and other CIDNP mechanisms, such as those arising from LACs of the S and T  states at low-field or due to non-zero exchange interaction.3,150

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As mentioned above, a key application of photo-CIDNP has been in probing the surface structure of proteins. As radical pair formation is necessary for generation of spin polarization, the method does not provide a generalised sensitivity enhancement over the entire spectrum, but is specific for the three aromatic amino acids tyrosine, tryptophan, histidine (Fig. 15) and rarely methionine.149 These residues react with a photosensitizer (typically 2,2 0 -dipyridyl or a flavin) via electron transfer (Trp, Tyr) or hydrogen atom transfer (His), only if the amino acid is solvent exposed, meaning CIDNP is sensitive to the protein’s tertiary structure. NMR acquisition occurs after the end of the illumination period such that the radicals have recombined and there is no paramagnetic broadening of the spectrum and, as the photoreaction is cyclic, the final NMR spectrum still represents the intact protein. CIDNP acquisition involves recording the difference between the ‘‘light’’ spectra collected after laser illumination and ‘‘dark’’ spectra showing Boltzmann polarization. Although this subtraction simplifies CIDNP spectra, showing only spin-polarized NMR transitions, it does result in a signal to noise pffiffiffi ratio penalty of 2. Experimental considerations and applications to protein studies have been reviewed extensively;149,151,152 here we highlight only two biological examples and some most recent developments. Use of NMR as a method to characterize unfolded states of proteins is hampered by poor spectral resolution in the unfolded state. Photo-CIDNP is able to avoid this problem, probing solvent accessibility (of Trp, Tyr and His) by photochemical reaction in an unfolded or partially folded state, followed by refolding which, if faster than nuclear spin-lattice relaxation, permits acquisition of resolved NMR spectra in the native state. The ‘pulse-labelling’ approach is enabled by a device that homogenously mixes the solution to permit NMR acquisition only 50 ms after injection of a refolding buffer into a sample within the NMR spectrometer.154 Hore and co-workers extended this approach by using the Nuclear Overhauser Effect to transfer polarization from the hyperpolarized CIDNP-active side chains to other nearby residues before the refolding step, providing additional information on the structure of the unfolded state. Tests on the mini-protein TC5b found that hydrophobic collapse resulted in residual structure of the unfolded state, contributing to a very short refolding time of just 4 ms.155 Whilst detection of only three CIDNP-active residues simplifies photoCIDNP spectra of proteins compared to conventional NMR, assignment can still be complicated if more than one residue of the same type is enhanced, or cross-polarization has transferred polarization to nuclei close to those involved in the primary CIDNP step.152 To solve the problem of overcrowding it is possible to apply 2D NMR methods, with CIDNP variants of COSY and NOESY first demonstrated by Kaptein and co-workers.156,157 Photo-CIDNP was extended to heteronuclear experiments, with 2D 15N–1H HSQC introduced by Lyon et al. to discriminate between individual Trp sidechains in unfolded states of proteins (1H resolution alone would be sufficient in the native state).158 Whilst attractive, owing to the strong 15N CIDNP observed and since the dark

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118 | Electron Paramag. Reson., 2019, 26, 89–129 Fig. 15 1H NMR and CIDNP spectra at 600 MHz of N-acetyl derivatives of (A) tryptophan, (B) tyrosine, and (C) histidine. Reproduced from ref. 149 with permission from Elsevier, Copyright 2004.

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15

N NMR spectrum is so weak that subtraction is unnecessary, applicability to this single moiety only is a major limiting factor. With 13C photo-CIDNP already known, Cavagnero and co-workers were motivated by the impressive enhancements of this heteronuclear method to develop a 1H detected 13C photo-CIDNP constant time reverse INEPT method (13C-PRINT).159 This method is applicable to a number of 13C–1H bond pairs in aromatic amino acid residues, and with a 16-fold enhancement in signal-to-noise per unit time compared to a reference non-CIDNP experiment (1H–13C SE-HSQC), time-savings of 256-fold are made for this 2D experiment. Despite hyperpolarization, sensitivity is an issue in CIDNP as only a fraction of molecules are photoexcited and photostability prevents indefinite signal averaging. Goez et al. developed novel multi-flash pulse sequences to tackle this issue,160 whereas more recently Cavagnero and co-workers introduced a tri-enzyme system to directly tackle photodegradation.161 This system consists of glucose oxidase, catalase and nitrate reductase; the former two act as oxygen scavengers, preventing generation of the sample degrading singlet-oxygen by oxygen quenching of the photogenerated triplet, while nitrate reductase specifically oxidises any photoreduced dye back to the to the initial state in the absence of oxygen. The tri-enzyme system not only allows longer averaging of spectra without photodegradation, but after identification of fluorescein as a superior chromophore to flavin in the low-concentration regime, has enabled photo-CIDNP experiments with as little as 1 mM substrate. These experiments showed significant time savings over higher-field nonCIDNP experiments (Fig. 16).153 An interesting application of CIDNP is the indirect detection of shortlived radicals not easily detectable by EPR. This is possible, because nuclear polarization generated in a few microseconds, survives for several seconds. In most cases, the CIDNP intensity of each spin-possessing nucleus is proportional to its hyperfine coupling constant (HFCC). Using this relationship to create a calibration plot for the known HFCC for one member of the radical pair, the values and signs of couplings for the unknown radical (Fig. 17) and g-factor difference relative to the known radical can be calculated.3,162

Fig. 16 NMR of 1 mM Trp. 1H–13C SE-HSQC at 900 MHz (21.2 T) in a cryoprobe requires nearly 2000 times more scans to match the signal-to-noise of 1D 13C PRINT at 600 MHz (14.1 T) in a room temperature probe. Adapted from ref. 153 with permission from American Chemical Society, Copyright 2016. Electron Paramag. Reson., 2019, 26, 89–129 | 119

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120 | Electron Paramag. Reson., 2019, 26, 89–129 Fig. 17 Left: CIDNP spectrum and radical structures for hydrogen transfer from L-Tyr to triplet-excited tetracarboxybenzophenone (TCBP). Right: Correlation between CIDNP intensities and HFCC of known L-Tyr radical (circles) used to determine HFCC of TCBP ketyl radical (squares). Adapted from ref. 162 with permission from the PCCP Owner Societies.

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Finally, we note that CIDNP can occur in the solid state, being observed both at high-field via magic angle spinning NMR163 and at the earth’s magnetic field.164 The phenomenon involves the subtle interplay of a number of different mechanisms, which have resulted in nearly all observations being confined to photosynthetic reaction centres.165 This topic will not be discussed further here; the interested reader is referred to the review by Matysik and co-workers who have worked extensively in this field.166 3.3 Light-induced overhauser DNPy Until recently Overhauser DNP studies have generally used Boltzmann electron spin systems,19,167,168 although the potential advantages of hyperpolarized electron spins have been discussed, particularly with reference to shuttle DNP.17 In the shuttle DNP approach, nuclear hyperpolarization is generated by microwave-driven cross-relaxation of the electron-nuclear spin system, which occurs most efficiently at lower magnetic fields, with subsequent rapid transfer of the sample to a higher magnetic field necessary for high-resolution NMR detection. Enhancements after accounting for total measurement time of up to a factor of 4 have been achieved,169 but the transfer imposes a Boltzmann penalty equivalent to the field ratio so further improvement necessitates use of hyperpolarized electron spins. Consider the four-level system comprised of a coupled electron and nuclear spin, as shown in Fig. 18. Typically, the electron Zeeman transitions are saturated by microwave pumping and polarization transfer occurs via a cross-relaxation process due to a difference in the rate of zero- and double-quantum transitions (W0 and W2).8 Considering the

Fig. 18 Energy levels of an electron spin coupled to an I ¼ 1/2 nucleus. Solid arrows indicate the allowed NMR (Wn) and EPR (We) transitions, while dotted arrows indicate the zero (W0) and double (W2) quantum transitions. y

Unlike CIDEP and CIDNP whose names are a misnomer, the results discussed in this section are truly a ‘‘dynamic’’ nuclear polarization in the sense of operating via an Overhauser mechanism. Electron Paramag. Reson., 2019, 26, 89–129 | 121

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time-dependence of the nuclear polarization, and solving to obtain the steady-state solution, gives the enhancement factor, e, as:

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  g  hIz i  I0 e¼ ¼  z fs S : I0 gI This is the well-known Overhuaser equation,167 in which gS/gI is the ratio of the electron and nuclear gyromagnetic ratios. The coupling factor z indicates the efficiency of the cross-relaxation process and varies from þ0.5 for a purely dipolar coupling to 1 for a purely scalar interaction, but has conventionally been thought to become small at fields of over 1 T. The leakage factor f represents the paramagnetic contribution to the overall nuclear relaxation and is given by f ¼ (T10  T1)/T1, where T10 is the spin-lattice relaxation time in the absence of radicals, and as such has a maximum value of 1. The saturation factor s ¼ (S0  hSzi)/S0 also has a maximum value of 1 in microwave pumped DNP, giving a maximum enhancement for protons of ge/gHB660. Utilizing hyperpolarized electron spins should permit 7s741, leading to eventual NMR enhancements of 104 or higher. The first observation of optically generated bulk-DNP in the liquid state was recently reported using continuous-pumping from a 1 W laser diode.170 Using the rose-bengal/TEMPO system, previously highlighted as generating an electronic hyperpolarization of 150 times, the thermal polarization via the RTPM,66 gave a four-fold enhancement of the 1H NMR signal of the aqueous solvent (Fig. 19(a)). Whereas pulsed laser illumination is conventionally used to obtain high transient electronic hyperpolarization necessary for direct EPR detection, use of a high-power continuous-wave laser was key to the detection of nuclear hyperpolarization.170 This may be understood by considering that the DNP build-up timescale is determined by the nuclear relaxation rate as recently experimentally verified.171

Fig. 19 (a) 1H NMR of water in the presence of rose-bengal and TEMPO, and (b) enhancement factor as a function of TEMPO concentration. Adapted from ref. 170 with permission from the Royal Society of Chemistry. 122 | Electron Paramag. Reson., 2019, 26, 89–129

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Considering the Overhauser equation, the variation in enhancement factor as a function of radical concentration was rationalised; the coupling factor is independent of the radical concentration, whereas the leakage factor increases with the radical concentration (and solvent deuteration). Observation of a peak enhancement at 1 mM with reduction at higher concentrations, Fig. 19(b), therefore implies that the saturation factor decreases as the radical concentration increases. The fraction of radicals polarized must decrease because of insufficient available triplet states. The saturation factor at high radical concentrations (for optimal leakage factor) may be increased by raising the triplet concentration, however, increased dye concentrations would lead to a decrease in optical penetration depth and higher laser powers may result in excessive sample heating and photodegradation. The reliance on intermolecular quenching of the triplet state may be partially responsible for the low saturation factor. Flexibly linked radical-dye systems produce higher electron polarization than unlinked systems,114 and could enable higher saturation factors and hence larger NMR enhancements if applied to light-induced DNP. The Bennati group have applied the light-induced DNP method using chemically linked fullerene nitroxide derivatives as polarizers. DNP enhancement factors of only r0.5 were obtained, the positive NMR enhancement indicating an overall negative saturation factor in this case.171 In these rigid systems, the exchange interaction is positive hence the quartet state of the RT pair lies below the doublet.97 Both states are directly observed in the TR-EPR spectra, distinguishable by their g-factors and show a mixture of absorptive and emissive polarization. The TR-EPR signal intensity depends on both the electronic polarization and the fraction of molecules excited to the triplet state. Determining the latter by comparison to a 3C60 standard indicated a polarization ratio of Pn/Peq ¼ 30, which may provide an explanation as to the lower DNP enhancements reported compared to the rose-bengal/TEMPO system (Pn/Peq ¼ 150).

4 Summary This chapter has explored the interplay between photochemistry and magnetic resonance, how light-induced primary electron hyperpolarization may be used to increase the sensitivity or information content of both EPR and NMR. A varied range of applications have been considered, yet optical hyperpolarization is not a panacea for the sensitivity problem in magnetic resonance. Despite strong sensitivity enhancements, many of the methods described have so far been shown to be applicable only to a limited range of systems. There is also an (almost) universal challenge; many of the techniques described substitute the sensitivity problem for that of photodegradation and indeed some even lack sensitivity due to insufficient concentrations of the relevant photoexcited triplet state. Increasing both the quantum yield of triplet formation and the fraction of chromophores excited, without increasing photodegradation (as excessive optical illumination power is bound to do) would significantly Electron Paramag. Reson., 2019, 26, 89–129 | 123

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widen the appeal of both LaserIMD and light-induced Overhauser DNP. In this regard, the advances described in photo-CIDNP through use of the tri-enzyme system offer hope for liquid state methods. A renaissance in high-field DNP was heralded nearly a decade ago and it seems safe to conclude that optical methods will continue to contribute to the flourishing field of hyperpolarization. Further advances will require improved understanding of spin physics, progress in chemical synthesis and hardware developments.

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Applications of electron paramagnetic resonance spectroscopy for interrogating catalytic systems Jacob Spencer, Andrea Folli, Emma Richards and Damien M. Murphy* DOI: 10.1039/9781788013888-00130

Species bearing unpaired electrons, including paramagnetic redox metal centres, surface defect centres, reactive oxygen species, adsorbed radical anions, are often involved in catalytic reactions. These species can be readily and thoroughly interrogated using Electron Paramagnetic Resonance (EPR) spectroscopy, providing information on the identity, chemical composition and even the dynamics of the centres themselves, thereby helping to elucidate the involvement of the radicals in the reaction cycles. This review will summarise and highlight the applications of EPR in heterogeneous, homogeneous, photocatalytic and microporous materials, all of which are of vital importance to the field of catalysis.

1

Introduction

Electron Paramagnetic Resonance (EPR) spectroscopy, which includes continuous wave (CW) and pulsed methods, is frequently used in the study of catalytically active systems. The role of EPR in these studies varies considerably from, on the one hand, a comprehensive description of the electronic states of the paramagnetic active site in the catalytic cycle to, on the other hand, a simple analytical confirmation that a radical centre is present. Another extremely valuable contribution that can be provided by EPR, and is certainly growing again in recent years, is to employ spin traps that can capture transient reactive oxygen (ROS) species in solution, and thereby identify the nature of the ROS by analysis of the resulting spin adduct spectra. In some cases, EPR can also indirectly provide value insights into diamagnetic catalytic systems, by employing suitable paramagnetic spin probes that can access the structural aspects of an oxide surface to revealing dynamic aspects of the system, or by employing probe transition metal ions. In all cases the paramagnetic species, which are directly or indirectly studied by EPR, may include surface defects on metal oxide surfaces, transition metal complexes in solution or supported on surfaces, anchored radical anions or cations, or ROS at the solid–liquid interface, which all provide valuable information concerning the catalytic reaction. In all of the above cases, a characteristic EPR signal with a unique set of spin Hamiltonian parameters may be detected. These parameters are then used to unambiguously identify the radical species or paramagnetic centres involved in the reaction cycle. However, as one may expect, the behaviour of these paramagnetic centres and radical species, including School of Chemistry, Cardiff University, Main Building, Park Place, Cardiff CF10 3AT, UK. E-mail: murphydm@cardiff.ac.uk 130 | Electron Paramag. Reson., 2019, 26, 130–170  c

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their reactivity, stability, dynamics or structure, may vary considerably from one catalytic system to another. Inevitably, the spin Hamiltonian parameters may in turn vary considerably from one catalytic reaction to another, and therefore one must be extremely careful in the analysis of the experimental spectra. This variation in spin Hamiltonian parameters, as a function of the catalytic system under investigation, makes EPR such a powerful diagnostic technique when interrogating catalytic reactions, because one is not simply confirming the presence of a sought after paramagnetic centre, by using EPR as an analytical or finger printing method. It is also possible to investigate the subtle changes in the properties of the radicals from one catalytic site to another, through analysis of the changes to the EPR spectrum. Unfortunately, this also means that EPR signals can often be mis-interpreted in catalytic studies. One must therefore always pay due care to the interpretation of the EPR spectra, which will change depending on the numerous reaction conditions, such as solvent, support, temperatures, etc., and indeed measurement conditions, such as power, modulation amplitude, resonator settings, etc. A final point to note, is that the accurate analysis of the EPR spectrum requires an accurate simulation to extract the spin Hamiltonian parameters, and in some cases these parameters can be readily verified and compared to DFT derived values. Traditionally, EPR has been widely used in the broad field of catalysis, from homogeneous to heterogeneous to enzymatic catalysis. There are numerous and outstanding review articles devoted to this topic. In the current chapter, we will focus on the literature in the past 4–5 years pertaining to the applications of EPR for studies of homogeneous catalytic systems (primarily first role transition metal ions), catalysis by microporous materials, systems of photocatalytic interest and finally heterogeneous catalysis. Most of our attention will be devoted to CW EPR, as this traditional method is still the most widely used and readily accessible method of choice in the catalysis field.

2

Homogeneous catalytic systems

Over recent years, the number of research groups utilising EPR spectroscopy and hyperfine methodologies to investigate radicals and paramagnetic species in homogeneous catalysis has increased. It is now well recognised that important structural and electronic properties concerning the catalyst itself can be obtained from the EPR and hyperfine data, under a range of experimental conditions (liquid phase, frozen solution and single crystals). This allows for a full investigation of interacting substrates, transient intermediates (that may be on- or offcycle) and identification of reaction products, thus gaining access to a deep understanding of reaction mechanisms that can be used to guide rational catalyst design. 2.1 Iron There are a growing number of EPR investigations that describe iron coordination complexes, driven by their capacity to facilitate atom- and Electron Paramag. Reson., 2019, 26, 130–170 | 131

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group-transfer catalysis to unreactive C–H bonds and olefinic functionalities, and also as mimics of low-valent iron centres in metalloenzymes.1–11 The availability of multiple oxidation and spin states of iron centres requires a complete characterisation with a combination ¨ssbauer and of multi-frequency EPR measurements, zero-field 57Fe Mo SQUID magnetometry to relate activity to metal oxidation state, metal spin state, and electronic structure. Ferric imido complexes, which are useful as group transfer catalysts, are known to span electronic configurations of S ¼ 1/2-5/2, with the high-spin configuration proposed to provide a driving force for substrate functionalization. Whilst isolated S ¼ 1/29 and S ¼ 3/2 complexes10,11 have been reported, experimental evidence for the putative high-spin 5/2 configuration was lacking. Betley et al.,4 prepared a series of Fe(III) imido complexes that facilitate nitrene transfer including C–H bond functionalization. The single quadrupole doublet (d: 0.62 mm s1, ¨ssbauer spectrum of an |DEQ |: 0.86 mm s1) identified in the 57Fe Mo (ArL)Fe complex (where ArL ¼ 5-mesityl-1,9-(2,4,6-Ph3C6H2)-dipyrrin) and a measured meff ¼ 1.7mB at 75 K via SQUID magnetometry were consistent with a low-spin Fe(I) centre. The EPR spectrum for this complex was characterised by a rhombic g-tensor with geff ¼ 2.24, 2.04 and 1.98. Addition of adamantyl azide resulted in the formation of the target iron–imido complex (ArL)Fe-(NAd), which displayed a single broad ¨ssbauer spectrum, representative of an Fe(III)–  NAr transition in the Mo system. No EPR spectrum of this complex could be observed at T450 K. However at 4 K, a rhombic signal with geff ¼ 8.62, 5.35, 3.10 was easily resolved. The spectrum was successfully simulated using the parameters S ¼ 5/2, g ¼ 2 and |E/D| ¼ 0.145, in which each intra-doublet transition was treated as an effective S ¼ 1/2 system, in accordance with the weakfield limit. A single-point DFT calculation confirmed the S ¼ 5/2 groundstate assignment, and calculated spin density plots revealed 78% iron and 16% imido nitrogen atom contributions to the total spin. The highspin Fe(III) imido complexes reported can facilitate nitrene transfer including C–H bond functionalization. Furthermore, of the possible spin states S ¼ 1/2–5/2 of ferric imido complexes, the sextet configuration is the only electronic structure that permits both H-atom abstraction and the subsequent recombination reaction to occur. In addition to their utility in catalysis, rare linear two-coordinate 15 valence electron cyclopentadienyl iron(I) complexes have recently been reported to exhibit single-molecule magnet (SMM) behaviour due to, it is thought, their unquenched orbital momenta.5 The X-band EPR data recorded at 30 K of [CpArFe(IiPr2Me2)] (where IiPr2Me2 ¼ 1,3-diisopropyl4,5-dimethylimidazol-2-ylidene, see Scheme 1) revealed that the Fe(I) complex was nearly EPR silent, as expected for an S ¼ 3/2 system with a negative zero field splitting parameter D. However, at very low temperature (8 K) and high microwave power (200 mW), evidence of the formally forbidden DmS ¼ 3 transition within the |msi ¼ |  3/2i Kramer’s doublet of the S ¼ 3/2 (Do0, E/DB0) species was observed as a relatively sharp low-field peak at geff ¼ 7.76. At X-band frequencies, the zero-field splitting is large relative to the microwave energy, thus geff should depend only on 132 | Electron Paramag. Reson., 2019, 26, 130–170

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Scheme 1 Structure of earth-abundant organometallic complexes utilised in homogeneous catalytic reactions.

the rhombicity parameter E/D and the real gz-value of the S ¼ 3/2 species, giving an expected value of geffB6. The deviation of the measured geff from this was an indication of a z-contribution to the g-tensor, which was confirmed by simulation using the following parameters: S ¼ 3/2, gx ¼ gy ¼ 2.18, gz ¼ 2.55, D ¼ 33.4 cm1, E/D ¼ 0.001. Thus, the authors proposed that the basis for the SMM behaviour was the unusual electronic structure, where extensive metal–ligand interactions and 3d–4s mixing arising from a bent structure produce a nearly orbitally degenerate ground state, giving rise to slow relaxation of the magnetization in the presence and even in the absence of an external magnetic field. 2.2 Cobalt EPR spectroscopy has been used to study the utility of cobalt catalysts in a range of reactions including cyclopropanation of electron-deficient alkenes, ring-closing C–H amination of aliphatic azides and C–C coupling reactions for the synthesis of fused 6-membered ring-systems.12–14 Recently, de Bruin et al.,15 employed [Co(Corrole)] complexes in the Electron Paramag. Reson., 2019, 26, 130–170 | 133

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synthesis of N-containing heterocycles via C–H amination, which proceeded through an activated nitrene precursor. Using CoCp2 as a chemical reductant, [CoIII(Br2Cor)(PPh3)] (Scheme 1) could be reduced to give a species with an anisotropic EPR spectrum, characteristic of cobalt(II) metalloradical formation. The EPR spectra of the complexes revealed cobalt hyperfine couplings on the g1 and g2 components (g1 ¼ 3.56, A(59Co)1 ¼ 740 MHz; g2 ¼ 2.22, A(59Co)2 ¼ 260 MHz), but no hyperfine was observed on the high-field feature (g3 ¼ 1.85, Table 1). The absence of resolvable 31P hyperfine coupling indicated PPh3 dissociation during the one-electron reduction step. DFT calculations revealed a dxy orbital ground state leading to different spin–orbit interactions than in the closely related [CoII(Por)] complexes,16–18 as reflected by changes in the spin Hamiltonian parameters (see Table 1). Soper et al.,19 have recently explored low-coordinate Co complexes supported by redox-non-innocent tridentate [OCO] pincer-type bis(phenolate) N-heterocyclic carbene (NHC) ligands for base-metalmediated organometallic coupling catalysis. Previous EPR investigations of analogous Ir[OCO] complexes had been unable to provide a definitive assignment to metal-centred or ligand-centred radical in oxidized materials.20 Measured solution magnetic moments (meff) for a series of Cobalt-[OCO] complexes (see Scheme 1) were in the range 1.88– 1.90. These are consistent with square-planar low spin d7 Co(II), which are well-known to have significant contributions from the angular momentum term, typically leading to higher than spin-only values of meff. The corresponding X-band EPR spectrum displayed a rhombic signal with well-resolved cobalt hyperfine coupling (Table 1), characteristic of a metal-centred metalloradical of S ¼ 1/2 system. Chemical oxidation resulted in the formation of a paramagnetic [(OCO)Co(THF)2][BPh4] complex, with ueff ¼ 2.88 consistent with a spin-only S ¼ 1 centre. The X-band EPR (20 K) spectrum of this complex gave no signal in the range of 0–600 mT, consistent with the authors assignment to a metal-based triplet system with large zero-field splitting parameter D, originating from either an intermediate-spin Co(III) centre or a ferromagnetically coupled low-spin Co(II) and ligand radical.

2.3 Nickel The chemistry of paramagnetic Ni(I) in homogeneous catalytic cycles and enzymatic processes is now well-established.21–25 New insights into the involvement of the low oxidation state in both stoichiometric and catalytic transformations has been achieved due to the increased number of isolated Ni(I) species that have been prepared and characterized.26 Our group has contributed to this field, by utilising N-heterocyclic carbene (NHC) ligands to prepare highly reactive two- and three-coordinate Ni(I) species.27–29 In all cases, the starting point for our chemistry was the three-coordinate species [Ni(6-Mes)(PPh3)Br], (where 6-Mes ¼ 1,3-bis(2,4,6trimethylphenyl)-3,4,5,6-tetrahydropyrimidin-2-ylidene). Treatment with TlPF6 (a bromide abstracting agent) in THF yielded the three-coordinate cationic THF complex labelled [Ni(6-Mes)(PPh3)(THF)][PF6], and further 134 | Electron Paramag. Reson., 2019, 26, 130–170

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Table 1 Spin Hamiltonian parameters for various organometallic complexes of catalytic relevance. a

g values

A values/MHz

Electron Paramag. Reson., 2019, 26, 130–170 | 135

Complex

g1

g2

g3

giso

Nuc

A1

A2

A3

aiso

Ref

[CoII(Mes2Cor)] Co[OCO] Co(TPP) [Ni(6-Mes) (PPh3)Br] [Ni(6-Mes) (PPh3)(THF)][PF6] [Ni(6-Mes) (PPh3)(CO)][PF6] CpNi(6-mes)

3.56 2.500 2.930 2.050

2.22 2.105 2.930 2.265

1.85 2.061 1.925 2.365

2.54 2.222 2.595 2.227

2.210

2.490

2.242

740 170 780 184 6 292

260 o30 780 194  27 210

0 45 420 250 70 419

333 81.7 660 209 12 307

15 19 17 27

2.025

Co Co Co P Br P

2.035

2.121

2.185

2.114

P

21

29

48

33

27

2.052

2.204

2.324

2.193

H

2.052

2.154

2.313

2.170

H

CpNi(6-mesDAC)

2.032 (2.054) 2.050 2.026

2.095 (2.076) 2.064 2.180

2.262 (2.262) 2.208 2.272

2.130 (2.131) 2.107 2.159

H

9.1 3.4 6.4 3.4 8.6 6.8 30.6 48.8 — —

10.9 6.8 11.2 6.8 13.3 7.6 27.8 — 34.3 36.9

5.4 0.4 4.6 0.4 6.3 1.8 29.6 —

30

CpNi(7-mes)

3.7 2.3 3.9 2.3 3.0 4.7 30.4 33.2 — —

36 31

—

31

101 326 134 60(60) 36.5(36.5)

101 326 134 60(60) 36.5(36.5)

51 227 106 600(582) 30(30)

84 293 125

32 32

[LNiI]1[A] [(tBuN4) NiIII(PhF)Br]1 [(tBuN4) NiIII(PhF)Cl]1 (dppf)NiI-Ph (dppf)NiI-mes

2.028

2.175

2.282

2.161

N Br N N

2.12 2.06

2.12 2.08

2.29 2.27

2.14 2.14

P P

Cu(Phen)Cl/DBAD/NMI

2.055(2.052)

2.055(2.052)

2.21(2.23)

2.107(2.111)

Cu N

a

For Euler angle rotations of g/A frames with respect to the molecular axis, the reader should refer to the original references.

27

30 30

44

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exposure of this solution to 1 atm of CO afforded an unusual example of a cationic Ni(I)–CO complex. The CW X-band EPR spectra of these complexes were characterised by a rhombic g-tensor, with one component (g1) close to the free spin value, indicating a considerable 3dz2 character in the SOMO. The coordination geometry around the metal centre, indicating deviation from ideal D3h symmetry, is reflected by the d-orbital distribution and profile of the g-tensor associated with the paramagnetic centre. In particular, an increase in the dx2y2 contribution to the SOMO corresponds to a shift in the g2 value away from g3 towards g1, and indeed for T-shape complexes g2 is closer to g1 than to g3, highlighting a geometry induced shifting of the g-tensor. An almost entirely isotropic 31 P superhyperfine interaction is observed for the neutral and cationic complexes, with a much smaller HFC in the case of the CO complex as a result of significantly less spin density on the 31P nucleus. We have furthermore prepared a series of paramagnetic twocoordinate nickel N-heterocyclic diamino/diamidocarbene complexes (6-MesDAC, Scheme 1) via potassium graphite reduction of the Ni(II) three-coordinate precursor.30 The structure of these three complexes was investigated by EPR/ENDOR spectroscopy, revealing rhombic g-tensors with considerable anisotropy (Table 1), as expected for low symmetry Ni(I) centres. The electronic perturbation from the presence of the DAC ligand resulted in a smaller deviation from ge, and variable-temperature EPR measurements also revealed the presence of a second Ni(I) centre at temperatures of 470 K, consistent with a dynamic equilibrium of the nickel between two sites identified in the X-ray structure, which in turn is manifested on the EPR time scale. The 1H hyperfine tensors for the Cp ring protons were also evaluated through simulation of the angular selective ENDOR measurements, revealing similar A(1H) tensors for the three complexes. Of interest to us and others is the use of nickel complexes in catalytic organometallic transformations, such as Negishi, Kumada, and Suzuki cross-coupling reactions. Whilst Pd-catalyzed cross-coupling is well understood to proceed via two-electron transfer oxidative addition and transmetallation, the mechanisms of Ni-catalyzed reactions are not clearly defined due to the ability of Ni to undergo both one and twoelectron redox events. Mirica et al.,31 recently provided strong evidence for the involvement of Ni(III) species in the cross-coupling mechanism, through the identification of a series of Ni(III) complexes via EPR spectroscopy. The six-coordinate organometallic Ni(III) complexes [(tBuN4)Ni(III)(PhF)X]1, (X ¼ Br, Cl; Scheme 1) were produced via oxidation of the Ni(II) precursor with [Fc1]PF6, and displayed distorted octahedral geometry as characterized via XRD. The EPR spectra revealed rhombic signals with giso ¼ 2.160–2.162 (Table 1), in addition to superhyperfine coupling to the two axial N donors observed in the gz direction and Br superhyperfine coupling along the gy and gz directions. These spin Hamiltonian parameters strongly suggest the presence of a Jahn–Teller distorted octahedral d7 Ni(III) centre. Supporting DFT calculations indicate a major contribution (74%) from the 3dz2 and minor population (22%) of the 3dx2y2 Ni orbitals supporting the observation of 79,81Br 136 | Electron Paramag. Reson., 2019, 26, 130–170

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superhyperfine coupling to the equatorial Br ligand. The [(tBuN4)Ni(III)(PhF)X]1 complexes then undergo C–halide bond formation via a reductive elimination, which is proposed to yield an unstable NiI species that undergoes rapid disproportionation to Ni0 and the [(tBuN4)NiII(halide)(MeCN)]1 species, giving strong evidence for Ni(III)(aryl)halide species to act as key intermediates in oxidatively induced C–heteroatom bond formation from Ni(II) precursors. Further identification of an [(tBuN4)Ni(III)(PhF)13CH3]1 intermediate, characterized by a rhombic EPR spectrum with broad signals in the x and z directions arising due to 13C-superhyperfine coupling of the methyl group, confirmed the presence of a detectable Ni(III)(aryl)alkyl intermediate. This was observed to be active in subsequent C–C reductive elimination, suggesting the viable activity of Ni(III) in cross-coupling reactions. The role of Ni(I)-aryl species, in cross-coupling reactions was also recently investigated by Hazari et al.32,33 Reaction of isolated (dppf)Ni(I)Cl with a series of aryl-Grignard reagents resulted in the appearance of paramagnetic complexes, assigned to (dppf)Ni(I)(aryl). Increased steric bulk on the aryl group led to increased stability of the Ni(I)-aryl species, but caused a distortion of the molecule and lowering of the symmetry at the metal centre. This was reflected in the EPR spectra, which were accurately modelled as either axial or rhombic species, with superhyperfine coupling to increasingly inequivalent phosphorous nuclei (Table 1). The Ni(I)-aryl species were observed to decompose in the presence of boronic acid, providing an explanation for why they have not previously been observed in-situ in Suzuki–Miyaura reactions, and are suggested to be off-cycle species that undergo decomposition to generate catalytically active Ni(0) and Ni(II) complexes. The authors also reported the identification of identical Ni(I)-aryl complexes in cross-coupling reactions involving naphthyl sulfamate substrates. Catalytic systems based on nickel a-diimine complexes activated with organoaluminum compounds (i.e., Brookhart-type catalysts) are increasingly being explored for ethylene polymerization, producing branched polyethylenes with unique properties via a proposed ‘‘chain-walking’’ mechanism.34 A detailed understanding of the mechanism of their catalytic action is required to overcome the rapid deactivation of Ni-based catalysts at high temperatures (80–100 1C), thus leading to the rational design of thermally stable and active catalysts. Talsi et al.,35 and Gurinovich et al.,36 have independently used EPR to examine the redox processes that can take place both at the metal centre, and on the redoxactive diimine ligand, which demonstrate a tendency to accept electrons and become radical anions. In similar fashion to the Cr(I/III) system described in detail previously by us,37–39 the role of the organoaluminium activator has been actively investigated with some evidence to suggest that it serves only as an alkyl donor that combines with nickel to form an agostic cationic alkyl complex. Talsi et al.,35 investigated the nature of the Ni(I/II) species formed throughout the reaction of LNiIIBr2 pre-catalyst (L ¼ 1,4-bis-2,4,6-dimethyl-phenyl-2,3-dimethyl-1,4-diazabuta-1,3-diene), with a high excess (4100 eq. Al) of MAO (methylaluminoxane) activator. Gradual heating of the LNiIIBr2 pre-catalyst with MAO or B(C6F5)3/AlMe3 Electron Paramag. Reson., 2019, 26, 130–170 | 137

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led to disappearance of the diamagnetic Ni(II) species (observed via in situ NMR) and emergence of an almost axially anisotropic EPR spectrum (g1 ¼ 2.050, g2 ¼ 2.061, g3 ¼ 2.208), with partially resolved hyperfine splitting from two equivalent nitrogen atoms with A> ¼ 1.06 mT superimposed on the g1,2 features. These signals arose from reduction to the corresponding Ni(I) complexes in which the a-diimine ligand remained coordinated to the Ni centre after its reduction from Ni(II) to Ni(I). The authors demonstrated that either the heterobinuclear ion pair [LNi(II)(mMe)2AlMe2]1[MeMAO] or agostic ion pair [LNi(II)–tBu]1[MeMMAO] are formed at the initial stages of activation. Both ion pairs are thermally unstable, converting to Ni(I) species at T420 1C. The Ni(II)-alkyl ion pair species are proposed as the precursors of the active sites of olefin polymerization catalysts, whilst the role of Ni(I) is yet to be fully elucidated. In a parallel investigation, Gurinovich et al.,35 studied Ni(COD)2 and Ni(COD)DPP-DAB activation using boron trifluoride etherate BF3.OEt2 to determine whether activation of zero-valent nickel complexes by nonalkyl activators is possible. The EPR spectrum of a red-brown solution of Ni(COD)2/MAO/DPP-DAB revealed two distinct anisotropic signals, characterized by the g-values of g1 ¼ 2.037, g2 ¼ 2.054, g3 ¼ 2.368 assigned to [Ni(COD)2]1, and g1 ¼ 2.024, g2 ¼ 2.043, g3 ¼ 2.238, assigned to [Ni(COD)(COD 0 )]1. An isotropic radical signal (gisoB2.00) related to a (DPP-DAB) AlR2 complex was also observed. No EPR evidence for radical diimine coordinated to the nickel was observed. However, activation of a preformed [Ni(COD)(DPP-DAB)] with MAO resulted in an EPR signal (g1 ¼ 2.040; g2 ¼ 2.065; g3 ¼ 2.280), originating from complete oxidation to [Ni(COD)(DPP-DAB)]1. These results indicated that in systems using MAO (i.e. where the activator cannot serve as oxidizing agent), formation of Ni(I) complexes took place in presence of diimine ligand only. As Ni(0) diimine complexes exist in the form of charge-transfer complexes, the mechanism is thus proposed to proceed via coordination of diimine to Ni(0), resulting in electron transfer from nickel to ligand; the intermediate complex Ni þ L  interacts with MAO, giving a Ni(I) olefin complex and radical anionic complex L AlR2. Similar results were obtained using non-ligand Lewis acids such as boron trifluoride etherate (BF3OEt2). The investigated systems were active in ethylene polymerization (Table 1), wherein comparison of the catalytic activity of systems containing Ni(I) complexes revealed a positive correlation between the relative quantity of Ni(I) complex and the consumption rate of ethylene. In the presence of diimine Ni(I) complex, the catalytic activity for ethylene polymerization was reported to be rather higher.

2.4 Copper Nitrogen-ligated copper complexes in the presence of an organic nitroxyl co-catalyst, e.g. TEMPO or ABNO, have shown efficacy in chemoselective oxidation of primary alcohols and dehydrogenation of secondary alcohols respectively, mimicking the cooperative redox chemistry directing galactose oxidase.40,41 The oxidation state of the copper metal centre, and redox activity of the nitroxyl co-catalyst remains strongly debated,42 with 138 | Electron Paramag. Reson., 2019, 26, 130–170

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some experimental evidence suggesting that replacing Cu(II) with a Cu(I) source leads to a significant rate enhancement. To explore this further, Stahl et al.,43 undertook a thorough mechanistic study of (bpy)Cu(I)/ TEMPO/NMI catalyzed aerobic alcohol oxidation (bpy ¼ NMI ¼ N-methylimidazole). The possibility that Cu(II) oxidizes the nitroxyl co-catalyst to TEMPO1 was ruled out as no visible changes were observed in the relative signal intensities of the metal-based EPR signal and the organic radical. The authors concluded that Cu(II) and TEMPO act in concert as one-electron oxidants to achieve the net two-electron alcoholto-aldehyde oxidation reaction. Replacing the TEMPO co-catalyst with azodicarboxylates, provides broader synthetic scope, and yielded similar mechanistic details as probed by the EPR signal of different (phen)Cu(II) species, characterized by slight differences in the nitrogen superhyperfine coupling indicative of changes in electron spin density at the metal centre (Table 1). More recently, it has been suggested that the oxidation can proceed in the absence of the expensive nitroxide co-catalyst.44 Thus, Stahl et al.,45 have also recently reported a mechanistic investigation of aerobic alcohol oxidation employing a nitroxide-free [Cu(MeCN)4]PF6/DBED/DMAP catalyst system (DBED ¼ N,N 0 -di-tert-butylethylenediamine; DMAP ¼ p(N,N-dimethylamino)pyridine). The EPR spectrum of the pre-catalyst in the absence of alcohol substrate was composed of an axial signal (gx ¼2.03, gy ¼ 2.07, gz ¼ 2.26, and Az ¼ 535 MHz). Upon addition of alcohol, a second organic radical signal (g ¼ 2.002) with hyperfine coupling to a 14N nucleus (A ¼ 85 MHz) was observed. The authors reported a decrease in the signal intensity of the organic radical, with a corresponding increase in Cu(II) signal during the course of the reaction. The EPR data thus revealed that the catalyst undergoes an in situ oxidative self-processing step in which Cu/O2 mediates oxygenation of the DBED diamine ligand to afford a nitroxyl radical that promotes efficient alcohol oxidation in cooperation with Cu.

3

Microporous catalytic systems

Microporous materials present an incredibly important research topic for modern day catalytic processes, molecular separations, and technologies for energy and health.46 The range of porous-type materials, and variations thereof, are continually growing as more applications and tailored designs become apparent. One of the limitations at the moment is the ability to improve upon the industrially established materials,46 but their inexpensive preparations, coupled with superior surface areas, modification, tuneability and porosities make them a highly attractive tool for improving upon current protocols. Once again, EPR techniques play an important role in the characterisation of these microporous materials in catalytic applications. 3.1 Zeolites and zeotype materials Zeolites, and more generally the related ‘zeotype’ materials, are a class of crystalline microporous aluminosilicates, primarily consisting of AlO4 and SiO4 tetrahedra. A massive variation and arrangement of subunits Electron Paramag. Reson., 2019, 26, 130–170 | 139

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provide a degree of tuneability, with regards to the physical and chemical properties of the materials, only paralleled by other related microporous materials. They have had a huge impact on modern science and technology, in particular, playing a key role in modern sorption47 and catalytic processes.48 The global annual demand has been reported to be several thousand metric tons.49 Zeolites can be catalytically active via the inclusion of redox-active dopant ions within or anchored to their framework, adopting multiple low symmetry sites that are often highly sensitive to experimental and synthetic conditions. EPR spectroscopy, coupled with the related hyperfine techniques, have been successfully used in the characterisation of zeolites for over 40 years. Many recent investigations of the local environment of redox-active metal centres,50–55 radical intermediate species and spin probes,56–58 have been readily evaluated with significant implications for understanding catalytic pathways and with complementary spectroscopic techniques. Recent technological advances have seen an increased use of in situ spectroscopic investigations, for the quantitative real-time59 and even spatial60 (EPR-imaging) analysis of catalytic processes, although the experimental conditions required for true operando studies remain difficult to achieve. A recent paper by Godiksen et al.,61 presented a CW EPR investigation of Cu(II) sites in Cu–CHA zeolites for the selective catalytic reduction of nitrogen oxide using ammonia (NH3-SCR). Although several highly active zeolites have been identified and are subject to further research, the authors indicate that little is known regarding the identity, speciation and activity of Cu(II) species within the catalytic process. In addition, the EPR results were of considerable interest when compared to alternative spectroscopic methods, such as X-ray diffraction, IR and UV–Vis spectroscopies. Conventional frequency CW EPR (X-band) provides ready accessibility to the magnetic parameters of the Cu(II) ions inherent to their electronic structure, and by extension, local coordination environment. Cu(II) has been well characterised in the literature and therefore allows for a straightforward comparison between the experimental parameters, and hence coordination geometries, owing to the early work of Peisach and Blumberg.62 In the fully hydrated state, Godiksen et al.,61 showed that the Cu–CHA sample presented two distinct Cu(II) environments. The first constituted a broad isotropic signal with the absence of a hyperfine splitting, attributed to a hydrated Cu(II) centre in which free movement or rotation is relatively unhindered. The second species was identified as an axially symmetric signal with a weakly resolved 4-line hyperfine pattern, from interaction with the Cu nucleus (63,65Cu, I ¼ 3/2), assumed to be a restricted Cu(II) environment in which the framework oxygen is involved within the coordination sphere. Upon dehydration, under a flow of O2/He at 400 1C, Godiksen et al.,61 reported that the EPR spectrum changed significantly, showing a complex anisotropic signal because of speciation within the zeolite framework. Simulation of the signal revealed three distinct isolated Cu(II) species, with the spin Hamiltonian parameters, labelled ‘A1’ (g8 ¼ 2.325, A8 ¼ 487 MHz), ‘A2’ (g8 ¼ 2.358, A8 ¼ 464 MHz) 140 | Electron Paramag. Reson., 2019, 26, 130–170

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Scheme 2 Structures attributed to the A1/A2 sites in Cu–CHA zeolites (after61).

and ‘B’ (g8 ¼ 2.388, A8 ¼ 530 MHz) respectively, all with axial symmetry and observed orders of magnitude of g84g> and A84A>, indicative of Cu(II) in a distorted octahedral or square planar geometry, with a dx2y2 ground state. Both ‘A1’ and ‘A2’ were attributed to Cu(II) residing within a 6-membered ring site with two aluminium T-sites (Scheme 2). This was supported by the spin Hamiltonian parameters corresponding to a tetragonal 4O coordination mode, as well as similar behaviours upon dehydration, and comparison with alternative zeolites not containing the same ring sizes. The final simulation component, site ‘B’, was reported by the authors61 to be comparable to species observed in earlier works63,64 within hydrated zeolites measured at cryogenic temperatures, that possessed 5- or 6-coordinate sites. This site was then assigned to a tetragonal square pyramidal geometry coordination, possibly containing traces of bound water within the first coordination sphere. The local geometry and electronic structure of the redox centres in zeolite materials is a point of considerable interest due to their active nature. Maurelli et al.,65 investigated the nature of catalytically active V41 sites, introduced within microporous aluminophosphates (AlPOs), for use as size- and shape-selective oxidation catalysts for processes such as alkane ammoxidation and alkene epoxidation. Although it is well known that these catalysts exhibit high selectivities (495%), the local structure of the vanadium units was not sufficiently explored in previous research. This was, in part, due to its ready ability to exist in variable oxidation states, coordinations and occupations within the AlPO framework. Due to its coordinative sensitivity, EPR techniques proved particularly useful for the description of vanadium in its þ4 oxidation state (3d1, S ¼ 1/2) to rationalise its distribution, and catalytic activity, with respect to the synthesis and experimental conditions. Vanadium in its þ5 oxidation state is diamagnetic (S ¼ 0), and therefore cannot be investigated by means of EPR or related techniques. With this in mind, Maurelli et al.,65 performed a combined multifrequency (X-band, 9.75 GHz, and Q-band, 34 GHz) CW EPR, hyperfine sub-level correlation spectroscopy (HYSCORE) and DFT investigation to afford a microscopic description of the local metal sites within VAPO-5 prepared via a hydrothermal synthesis. Beginning with the CW EPR experiments, measurements of the VAPO-5 sample indicated the presence Electron Paramag. Reson., 2019, 26, 130–170 | 141

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of two distinct vanadyl sites, with well resolved 8-line hyperfine patterns, typical of an S ¼ 1/2 system interacting with the dipole moment of V nuclei (51V, I ¼ 7/2, 99.76% abundance). The resolution and narrow linewidths observed indicated that V was present in an isolated site, in contrast to a tetrahedral clustered or polymeric V(IV) species. This was further supported by empirical correlations presented in previous works by Lohse et al.,66 in which the isotropic spin-Hamiltonian parameters may be used to readily distinguish between tetrahedral V(IV) and vanadyl units, and also conclusions presented by previous IR investigations with probe molecules.67 In order to yield further information regarding the local coordination environment of the identified vanadyl species, Maurelli et al.,65 employed X- and Q-band HYSCORE experiments to probe the spin density distribution over neighbouring magnetically active nuclei (1H, I ¼ 1/2, 27 Al, I ¼ 5/2 and 31P, I ¼ 1/2). The HYSCORE data revealed a series of distinct 27 Al (aiso ¼ þ 4.5 MHz and þ2 MHz for each species) and 31P (aiso ¼ þ 18 MHz and þ6.7 MHz for each species) cross-peaks with relatively large hyperfine parameters, consistent with V-O-P and V-O-Al type structural units. Further, the presence of a 1H signal, unaltered after dehydration, confirmed the presence of an equatorial proton in the first coordination shell of the vanadyl species not attributed to coordinated water, strongly suggesting a charge compensation effect from Al(III) framework substitution. When compared with DFT-derived spin Hamiltonian parameters, two possible models were then presented by the authors for vanadyl, in isomorphously substituted Al(III) sites, and another as extraframework vanadyl species grafted onto the surface of the AlPO matrix. The use of mesoporous silica (SBA-15) and titanium silicalite (TS-1) for supporting or incorporation of framework substituted paramagnetic ions, also continues to attract considerable attention for investigating the magnetic properties using advanced EPR techniques.68–70 In particular, these techniques can give unprecedented insights into the local topology surrounding the paramagnetic ions, and this becomes even more relevant with studying adsorbate interactions. Dinse et al.,68 provided an excellent account, using pulsed 2D EPR and ENDOR, on the structure of the paramagnetic V(IV) centres in SBA-15 as revealed through the full set of g-matrix and vanadium hyperfine parameters. The H2 reduced SBA-15 supported VOx catalysts are used for oxidative dehydrogenation of alkanes. Accurate g and A values for the as-prepared samples, extracted by both CW and field-swept echo (FSE) EPR, were reported with typical values of g1 ¼ 1.987, g2 ¼ 1.990, g3 ¼ 1.944, A1 ¼ 194.5, A2 ¼ 215.8 and A3 ¼ 549.3 MHz. Angular selective vanadium and proton ENDOR were both employed to obtain information on the surrounding environment, and this information is nicely complimented by the HYSCORE measurements. An iron grafted Fe/SBA-15 catalyst, used for the direct arylation of biphenyl methane and benzene, was also investigated by EPR.69 As discussed earlier, for earth abundant homogeneous catalysts such as iron, considerable efforts are also devoted to anchoring these ions into mesoporous materials. Controlling the nature and distribution of the iron centres in such materials is however challenging, with a distribution of species forming inside the (meso- or micro-) porous network, 142 | Electron Paramag. Reson., 2019, 26, 130–170

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including small iron clusters, isolated iron centres and iron oligomers. In such cases, techniques like EPR are vital in order to better understand the speciation of the metal centres. Typically, such EPR spectra are very broad, and therefore it is difficult to extract a great deal on information on the spin system. In the Fe/SBA-15 case, a broad spectrum was also observed with a g-value of 2.14, which was assigned to polynuclear oxyhydroxy Fe(III) arrays. The absence of signals at g ¼ 2.2–2.5 indicates the sample is free from any iron oxide species. Morra et al.,70 also examined the paramagnetic properties of the open shell Ti(III) centres in TS-1, using EPR and HYSCORE. The Ti(III) centres were generated by reaction of triethylaluminium with the Ti(IV) centres within the TS-1 framework. The CW EPR spectra of the isolated Ti(III) centres produced a well resolved rhombic signal with g1 ¼ 1.922, g2 ¼ 1.939, g3 ¼ 1.989. However, of greater diagnostic value was the observation of 29Si hyperfine coupling through the HYSCORE measurements, giving A1 ¼ 6.8, A2 ¼ 7.1, A3 ¼ 10.0 MHz. This hyperfine tensor was then decomposed in the usual way, to obtain the anisotropic and isotopic components of the hyperfine interaction to determine the Si 3s orbital character. The accessibility of the framework Ti(III) ions was demonstrated by ammonia adsorption studies, where a clear preference for Ti(III) ions in TS-1 with five-fold coordination was found, compared to the expected case of tetrahedral Ti(III) ions in the framework of porous aluminophosphate molecular sieves.

3.2 Metal-organic frameworks (MOFs) Another growing and significant class of microporous materials in catalysis are MOFs. Although zeolites, are more suited to extreme temperature conditions due to their inorganic nature, the inclusion of organic components in MOFs presents an almost unlimited arrangement of sub-units, topologies, porosities and functions.71 In fact, the precise control of the MOF assembly allows designs almost completely tailored to its application, with typically large surface areas, and densities of catalytic sites.72 In contrast to zeolites, MOFs typically contain redox-active metal centres embedded within the framework, which can be an issue for EPR spectroscopy, due to dominating electron spin-exchange interactions that encompass signal features relating to more sensitive, and informative interactions. However, this is not significant in every case, and routes to magnetic dilution, such as co-metal syntheses, and post-synthesis modification are readily available. Although research into MOFs has only become significant within the last 20 years, a number of recent studies demonstrating the power of EPR spectroscopy to characterise the redox centres,65,73–77 and adsorption properties via spin probe studies,56,78,79 have been reported. Hua and coworkers80 recently investigated an example of an electroactive MOF in terms of redox and spectral properties of the tris-(p-tetrazolylphenyl)amine (H3TTPA) ligand in solution, and by means of a triarylamine probe, generated in contact with the frameworks [Mn3(TTPA)2(MeOH)6]n and [Mn3(TTPA)2(DMF)6]n. The triarylamine Electron Paramag. Reson., 2019, 26, 130–170 | 143

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probe is known to undergo a one-electron oxidation to a triarylamine radical cation state via chemical or electrochemical oxidation,81 and is well characterised in the literature, providing a good basis for its use as a probe in the characterisation of MOFs and other related materials. In this case, in situ electrochemical EPR spectroscopy was employed to characterise the identity and electronic structure of electrochemically generated radicals. Understanding the nature, and behaviour of electroactive MOFs provides a fundamental basis for its application in functional electronic or optical devices.80 Hua et al.,80 initially reported measurements of the H3TTPA ligand in electrolyte solution [(n-C4H9)4N]PF6/CH2Cl2] with an applied potential. Upon application of a 1.7 V potential, a weak EPR signal was observed at g ¼ 2.0068 with a weakly resolved hyperfine structure indicating an interaction with two inequivalent nitrogens (14N, I ¼ 1, 99.6% abundance) with values of aN1 ¼ 12.6 MHz and aN2 ¼ 10.4 MHz respectively, sup¨ckel calculations. The higher hyperfine value was attributed ported by Hu to the nitrogen atom at the centre of the triarylamine radical, whilst the latter was attributed to a nitrogen atom attached to a proton within the tetrazole ring. Concerning the measurements of the [Mn3(TTPA)2(MeOH)6]n framework in the same electrolyte solution, Hua et al.,80 reported that upon increasing the applied potential from 1.4 V to 1.65 V, a signal centred at g ¼ 2.005 was observed corresponding to the triarylamine radical. The anisotropic nature of the signal indicated that the radical was present in the solid state, i.e. adsorbed to the framework. The authors reported that, upon generation of the triarylamine radical Mn(II) leaching into the solution was observed in the form of a 6-line hyperfine pattern, attributed to the interaction with a Mn(II) nucleus in solution (55Mn(II), d5, S ¼ 1/2, I ¼ 5/2) with the spin Hamiltonian parameters of g ¼ 2.001 and aMn ¼ 270 MHz. In the case of the [Mn3(TTPA)2(DMF)6]n framework in the same electrolyte, the authors reported a similar anisotropic signal at g ¼ 2.007 at a potential of 1.5 V, again expected to be a triarylamine radical cation species attached to the framework. Leaching of Mn(II) occurred after the potential was increased to 1.9 V with similar parameters reported as in the previous case (g ¼ 2.001, aMn ¼ 280 MHz). The observation of Mn(II) leaching is an important factor to note in improving the stability of electroactive MOFs for functional applications.80 Whilst the work of Hua et al.,80 demonstrated the use of radical probes for framework characterisation, another interesting example concerns the characterisation of defective metal sites embedded within the ¨nder et al.,82 involved a single crystal EPR framework. The work of Friedla investigation of mononuclear Cu(II) defect species in the well-known HKUST-1, containing Cu2(COO)4 paddlewheel units. In the conventional Cu2 units, super-exchange interactions between the neighbouring electron spins, on respective Cu(II) units, dominate the hyperfine interaction, and appear as a broad isotropic signal due to the S ¼ 1 excited state at ambient temperatures. This not only causes difficulty in accessing a meaningful description of these environments, but may also envelope less intense but potentially important paramagnetic species around free 144 | Electron Paramag. Reson., 2019, 26, 130–170

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´el temspin values. At sufficiently low temperatures (i.e. below the Ne perature) the Cu2 units are antiferromagnetically coupled and the resulting EPR signal is lost in the S ¼ 0 ground state. A dielectric resonator was employed to aid sensitivity enhancement which was estimated as a factor of 8.6. ¨nder et al.,82 reported an initial X-band powder EPR spectrum Friedla (7 K) in which a complex signal was observed, consisting of multiple distinct environments. The experimental spectrum showed an anisotropic signal, characteristic of an S ¼ 1/2 system interacting with the nuclear magnetic moments of Cu (63,65Cu, I ¼ 3/2). As expected, no signal attributed to the S ¼ 1 system was observed at this temperature. Deconvolution of the parallel low-field component of the signal indicated the presence of two species, denoted ‘A’ and ‘B’, with well resolved parallel hyperfine patterns and principle tensor values: for ‘A’, g[2.055 2.317], ACu[18 173] 104 cm1 and for ‘B’ g[2.044 2.359], ACu[18 153] 104 cm1. The observed tensor values for ‘A’ and ‘B’ indicated a square pyramidal oxygen coordination environment, similar to values reported in a previous paper.83 To obtain further geometric information about the Cu(II) defective ¨nder et al.,82 also performed single crystal EPR measspecies, Friedla urements in order to demonstrate the angular dependence of the Cu(II) signal components observed in the powder spectrum. The series of spectra indicated a clear, 2-fold angular dependence, with angles of the z a,b a,b principle axis ya,b a ¼  201, yb ¼ 901 and yc ¼  701 of the two tensor orientations assigned to species ‘A’ and ‘B’. This linked to the crystal structure of the framework with space group I212121, and perpendicular screw axes along three dimensions, resulting in two magnetically inequivalent, symmetry related sites. The outcome of the single crystal experiments concluded that the two species are located at well-defined mononuclear sites within the framework, rather than an impurity phase or extra-framework site. The authors proposed that these observed species are the formation of defective Cu/Cu paddlewheel units with a missing cupric ion, and rule out the possibility of Cu(I) oxidation to Cu(II), which is present as a distorted tetrahedral coordination to 4 nitrogens in the centre of the paddlewheel unit. It was suggested that approximately 10% of the paddlewheel units were defective in this manner, which agreed with complementary XRD analysis.

3.3 Porous carbon materials Porous carbon materials, in particular activated carbons, are another industrially important class of frameworks used as heterogeneous catalyst supports, as water desalination systems, for photochemical and sensing applications.84 The molecular design of the systems, with respect to the various morphologies of carbon materials along with framework modification by incorporation of heteroatoms, leads to efficient tailoring of the system in terms of porosity, electrochemical response, surface polarity and wettability.84 The application of EPR spectroscopy is less well known in this class of materials, however a number of interesting uses Electron Paramag. Reson., 2019, 26, 130–170 | 145

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have been reported recently to probe the nature of incorporated metal sites,85 defect chemistry86 and radical generation,87–91 in steady-state and in situ applications. Due to the inherent sensitivity of the technique, EPR is a useful tool in identifying structural features and radical intermediates, not usually detectable by other means. A study by Wang et al.92 investigated nano zero-valent iron, encapsulated in porous carbon spheres (Fe0/Fe3C@CS) for the activation of peroxymonosulfate (PMS) for phenol degradation. A remarkable catalytic activity, and stability, was reported by the authors, due to subsequent encapsulation and magnetic separation. This class of catalyst acts via an advanced oxidation process, employing oxidising substances such as sulfate (SO4 ) and hydroxyl OH ) radicals, or non-radicals such as singlet O2 to degrade pollutants. Although structural components within forms of carbon are not paramagnetic, important features of the degradation mechanism, involving the radicals, listed vide infra, were investigated using EPR spectroscopy and radical trapping methods, which is a subject of much debate among recent literature on porous carbon materials for oxidative degradation.85,87,89,93,94 It is generally accepted that reactive species, such as hydroxyl and sulfate radicals, are the dominating active species when attacking organic pollutants. Such organic radicals, however, are typically short-lived in solution, and spin-trapping agents are therefore necessary to extend the radical half-life beyond operational time-frames for an EPR spectrometer. In the work presented by Wang et al.,92 5,5-dimethylpyrroline oxide (DMPO) was utilised to indicate the presence of expected radical intermediate species in the catalytic mechanism, as well as classical radical quenching tests. In the absence of DMPO, no signals were resolved within the EPR spectrum, indicating that hydroxyl and sulfate radicals were not present, or at least at a concentration below the EPR detection limit. Upon the addition of DMPO (80 mM), and phenol (20 ppm) to a PMS solution, the authors reported signals associated with the oxidised form of DMPO, where the Hb-attached carbon position is oxidised to a carbonyl group. As the catalyst was added to the reaction mixture of PMS (6.5 mM), DMPO (80 mM) and phenol (20 ppm), hyperfine patterns associated with the DMPO- OH and DMPO-SO4  adducts were reported by the authors with variable signal intensities through the course of the reaction. Within the first minute, both adducts were resolved with hyperfine parameters of aN ¼ aH ¼ 41.7 MHz for DMPO- OH and aN ¼ 37 MHz, aH ¼ 28 MHz, aH ¼ 4 MHz and aH ¼ 2.1 MHz, respectively. The EPR signal associated with the DMPO-SO4  adduct was considerably less intense than the former case. After 4 minutes, this signal intensity decreased, which could have suggested consumption of the sulfate radical species in the oxidation process. However, upon extension to 9 minutes, a larger relative signal intensity was observed for the DMPO- OH signal, indicating an excess of trapped radical adducts in solution. Although the presented study does not categorically prove the activity of OH or SO4  within the catalytic process, it does confirm the presence of such species generated during the course of the reaction. Quenching experiments, in 146 | Electron Paramag. Reson., 2019, 26, 130–170

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comparison with EPR data, confirmed that  OH was selectively generated versus SO4  radicals after PMS activation. Another exciting class of porous carbon materials are porous Si : C and SiO2 : C layers, which have had much attention, as support materials for commercial catalysts,95 and also as a replacement for graphite anodes in new Li-ion battery technologies96 among a wide range of applications. SiC materials exhibit high thermal conductivity, resistance towards oxidation, high mechanical strength and chemical inertness,97 making it a promising support material, although achieving a high surface area has been a limitation. Savchenko and coworkers86 performed a CW EPR study investigating the nature of paramagnetic carbon-based surface defects in various silicon-carbide materials (por-Si, por-Si : C and por-SiO2 : C), in which the respective spin Hamiltonian parameters had not been previously reported. Using this data, their aim was to correlate the nature of these defects to various technological treatments during preparation and application.86 In the initial por-Si layer, an irregular isotropic lineshape was observed near free spin, which was attributed to the presence of two distinct environments, previously reported in the literature.98 A signal at g ¼ 2.0043 (DHpp ¼ 0.58 mT) was attributed by the authors to Si dangling bonds (Si-DB), localised in nanocrystalline Si, whereas the other signal at g ¼ 2.0073 (DHpp ¼ 0.43 mT) is associated to a Pb0 interface defect, assigned to a dangling bond existing at the Si/SiO2 interface with structure Si  Si3. After carbonisation at 850 1C of the por-Si : layer, the two signals for Pb0 and Si-DB were observed at g ¼ 2.0065 (DHpp ¼ 0.57 mT) and g ¼ 2.0045 (DHpp ¼ 0.51 mT), respectively, with some evidence of amorphous phase growth, and differences in strain localisation within the material due to the shift in g. Upon carbonisation of the por-Si layer, a third EPR signal was reported by the authors86 at g ¼ 2.0035 (DHpp ¼ 0.45 mT), which was attributed to a carbon related defect (CRD) with a nearby O heteroatom. All three types of paramagnetic centres indicated vide infra were reported for the por-SiO2 : C layers, with two of small intensity; Pb0 g¼2.0077 (DHpp ¼ 0.9 mT) and Si-DB g ¼ 2.0049 (DHpp ¼ 0.5 mT); and a larger signal attributed to the CRD g ¼ 2.0030 (DHpp ¼ 0.8 mT). The authors suggested86 that the decrease in g-value could be due to the appearance of sp2 coordinated C clusters formed after oxidation, which were also suggested in STEM work. The main contribution to the EPR signals for por-Si : C and por-SiO2 : C arose from the CRD and C clusters, while the Si related defects were in low concentration.

4 Photocatalytic catalytic systems 4.1 Earth abundant transition metals as dopants in TiO2 photocatalysis In this section, we look at recent trends in Earth-abundant transition metal (EAM) doped TiO2 photocatalysts where the electron trapping events and the reduced state of the oxide can be investigated by EPR spectroscopy. Electron Paramag. Reson., 2019, 26, 130–170 | 147

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Metal oxide doping with elements foreign to the crystal lattice has long been an area of great scientific interest, starting from materials science to physics and chemistry. When semiconductor metal oxides are used as photocatalysts (TiO2 still largely dominates this area of research), small amounts of dopants can play a pivotal role in stabilising phases that are otherwise thermodynamically unstable, altering the electronic configuration and energetics of the host lattice, and therefore governing the magnetic, optical as well as chemical and catalytic properties of the host metal oxide.99–110 After almost half a century from the first observation of the Fujishima–Honda effect,111 TiO2 remains the most widely used semiconductor metal oxide photocatalyst, owing to its photoelectric conversion efficiency, good photocatalytic activity, photoand chemical stability, non-toxicity, very high abundance (Ti) and global production (in terms of Ti mining and TiO2 processing).112–116 Nevertheless, many drawbacks also exist, specifically, UV photosensitisation (band gap43 eV), weak separation efficiency of the photogenerated electron–hole pairs, and their high recombination rates. To overcome these limitations, doping strategies such as self-doping,117–119 non-metal doping,120–125 transitional metal doping,99–104,108–110,126–178 and rare-earth metal doping,179,180 have been largely explored in the last three decades, to optimise the optical properties for visible light harvesting, to improve the kinetics of charge separation for reducing the massive recombination events, to enhancing interface and surface characteristics. As far as transition metals for doping and surface decoration are concerned, despite a persistent focus on platinum group metals (PGM), mostly due to their great performances as co-catalysts, their scarcity, constantly high demand, low global production rates and nonsustainable profile, have forced researchers into looking at replacements amongst the Earth abundant metals, including V, Nb, Cr, W, that will be discussed here. The partially filled d-orbitals of these guest elements creates a series of new energy levels below the conduction band edge of the host semiconductor that can be accessible. Often, the result is a red shift of the band-gap transition and a modulation of the semiconductor Fermi level. Furthermore, these new energy levels can alter the charge carrier equilibrium concentration by serving as electron–hole trapping centres, potentially suppressing charge carrier recombination (if both electron and hole are separately trapped) or enhancing charge carrier recombination (if both electron and hole are trapped by the same centre or, in the absence of charge scavengers, one of the two charge carriers is trapped whilst the other is not). Electron trapping generally occurs at a much faster rate than hole trapping, and in most of the cases the net result is a reduction of the oxidation state of the metal ions in the TiO2 crystal lattice. Therefore, a careful analysis of the electron trapping centres and reduced metal ions in doped TiO2 becomes imperative when designing high performance photocatalysts and semiconductor sensitizers. EPR spectroscopy has proven to be a technique of supreme elegance, sensitivity and wealth of information for such specific investigations. 148 | Electron Paramag. Reson., 2019, 26, 130–170

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4.2 V-group metal doping: V- and Nb-doped TiO2 TiO2 semiconductors doped with V-group metals, mostly V(V) and Nb(V) ions,100,104,108–110 have been synthesised and tested for their photocatalytic performances, although a greater interest has focused on their use as dielectric semiconductors, conductive oxides for thin film coatings as well as pigments for high-applied electric fields and non-linear resistivity applications. V-doping strategies have been adopted for both rutile and anatase polymorphs. A common feature was the detection of reduced V(IV) ([Ar]3d1, S ¼ 1/2) in TiO2 that was doped with V(V) ([Ar]3d0). Energetically, the existence of V(IV) in TiO2 doped with V(V) is interpreted as a 3d electron at energy ca. 1.0 eV below the conduction band edge, for the V-doped anatase case109,131 (the edge of the conduction band is also situated at a lower energy compared to un-doped TiO2; a scenario occurring when a severe distortion of the octahedral geometry is caused by small doping ions like V(V)109). Crystallographically, the formation of V(IV) can be explained by ion substitution (for Ti(IV) [Ar]3d0) and electronic compensation of the extra positive charge carried by V(V), followed ¨ger–Vink notation: by electron trapping; or in the Kro 2TiO2

V2 O5 ! 2VTi þ 4OxO þ 2e0 þ 12 O2 VTi þ e0 ! VXTi

ðelectron trapping at a substitutional siteÞ

þ e0 ! V V i i

ðelectron trapping at an interstitial siteÞ

In the above equations V represents vanadium, not a crystal vacancy ¨ger–Vink notation). Kokorin et al.,132 (also an upper-case V in the Kro studied V-doped rutile TiO2 at X-, Q- and W-band frequencies, reporting the presence of two distinct V(IV) signals upon doping with V(V). The first signal was characterised by g and A tensors (hyperfine arising from the coupling of the unpaired electron to the I ¼ 7/2 51V nucleus), with principal values (averaged amongst X-, Q- and W-bands) of gx ¼ 1.915, gy ¼ 1.913, gz ¼ 1.956 and Ax ¼ 91.1 MHz, Ay ¼ 128.9 MHz, Az ¼ 425.4 MHz, which was assigned to lattice V(IV) at Ti(IV) sites, given that they match perfectly previous reports for V(IV) centres in rutile single crystals133 (g and A tensor frames are collinear where x is along the crystal [10], y is along the crystal [001] which is the c-axis and z is along the crystal ¯0]). The second signal with the spin Hamiltonian parameters of [11 gx ¼ 1.984, gy ¼ 1.990, gz ¼ 1.938 and Ax ¼ 137.2 MHz, Ay ¼ 180.1 MHz, Az ¼ 321.6 MHz, was attributed to interstitial V(IV), with total spin g1 of the interstitial V(IV) centres approximately ten times smaller than the amount of substitutional V(IV) centres. The comparison of the multifrequency analysis with older works based on similar polycrystalline powders, as well as single crystals,134–138 allowed the authors to establish that the V(IV) ion location in the TiO2 lattice influences the magnitude and the shape of the g tensor, with gx, gy4gz for interstitial V(IV) centres and gx, gyogz for substitutional V(IV) centres. On the contrary, only the magnitude of the A tensor is affected, as in both the cases Ax, AyoAz. In addition, these authors found that the concentration of paramagnetic V(IV) species is maximum at 0.5 at.% of V doping and slightly decreases Electron Paramag. Reson., 2019, 26, 130–170 | 149

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41

afterwards with the portion of paramagnetic V species not exceeding 0.5% of the total V at 5.0 at.% of V doping (i.e. most of the V is in the diamagnetic V(V) state), although the absolute difference is very much contained in the whole range of 2–5 at.%. This considerably differed from the case of doping TiO2 directly with isovalent V(IV) (precursor used was VO2 rather than V2O5),134 where, as it would stand to reason, the amount of V(IV) in spin/g was found to increase linearly with the total V doping used in the synthetic procedure. Interestingly though, quantification by EPR spectroscopy revealed that for total loadingo0.5 at.%, the V(IV) ions were isolated in the rutile lattice, whilst at loadings 40.5 at.% a new microphase with the mixed composition {TiO2—VO2} and noticeably narrower band gap than the initial TiO2 was formed. The maximum of isolated V(IV) ions in the lattice was again corresponding to a total V loading of 0.5 at.%, in perfect agreement with the results by Kokorin et al.132 Chang and Liu109 have recently investigated a series of surface doped anatase TiO2 nanoparticles and used EPR, in connection to other spectroscopies (i.e. XPS and UV–vis), to study the paramagnetic species formed upon electron trapping and their energetics. In the case of V(V) doping, a signal with g tensor principal components equal to g> ¼ gxEgy ¼ 1.962 and g8 ¼ gz ¼ 1.938 was recorded, and characterised by a hyperfine to the I ¼ 7/2 51V nucleus with A tensor principal components equal to AxEAy ¼ 145.7 MHz and AzE462.4 MHz. This signal was attributed to reduced V(IV) (also independently confirmed by XPS). Despite a clear evidence of reduced vanadium species, no signal associated with reduced titanium species was evident, i.e. Ti(III) ([Ar]3d1, S ¼ 1/2), was detected prior to irradiating the sample. Upon light irradiation and generation of electron–hole pairs, no significant increase in the V(IV) signal was recorded, however a new, very narrow isotropic EPR resonance appeared at g ¼ 2.003. Chang and Liu109 described this as a trapped hole, i.e. O radical. However it is difficult to reconcile this isotropic resonance with what has been extensively reported for trapped holes. The O radicals in TiO2 were widely shown to exhibit an anisotropic EPR profile with axial symmetry,139–142 bearing typical g-values of g> ¼ 2.026 and g8 ¼ 2.002 for Evonik P25139 (i.e. a 75% anatase and 25% rutile mixed polymorphs used as a benchmark in semiconductor photocatalysis), and with 2.020rg>r2.043 and g8 ¼ 2.002 for rutile TiO2.141 It is most likely that what these authors observed at free spin, upon irradiation, is the sharp line of a medium-polarised conduction electron spin resonance, commonly observed in reduced or photo-irradiated anatase TiO2 samples.141,143,144 Similarly to V(V), Nb(V) ([Kr]4d0) replaces Ti(iv) in the titania lattice aliovalently, leading to a change in the stoichiometry of the composite in analogy to what reported for V(V). Contrary to V(V) though, the Nb(V) substitution is isomorphic, given that Nb(V) has a very similar size to Ti(IV). Interestingly, the valence induced electron trapping, at least for the anatase polymorph, seem to generate mostly Ti(III) ([Ar]3d1, S ¼ 1/2) rather than Nb(IV) ([Kr]4d1, S ¼ 1/2).100,145,146 Giamello and co-workers146 and Folli and co-workers100 independently showed that Ti(III) formed as a 150 | Electron Paramag. Reson., 2019, 26, 130–170

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result of valence induction when Nb(V) aliovalently replaces Ti(IV) are structurally very different from Ti(III) formed as a consequence of chemical or chemo-/thermal reduction of pure TiO2. Whilst the latter tend to be localised surface species, the former seem to be highly delocalised bulk species with optical absorptions deep in the infrared146 and responsible for the remarkable electronic conductivity of Nb-doped TiO2147 (effectively the excess electrons are shared by more than one lattice Ti). These bulk delocalised Ti(III) centres exhibited an anisotropic EPR profile at X-band and 77 K characterised by axially symmetric g-values equal to g> ¼ 1.988 and g8 ¼ 1.957.100 The amount of delocalised Ti(III) could also be augmented by photo-injection of extra conduction electrons.100,146 For these reasons, besides the use of the material as a photocatalyst, Nb-doped TiO2 composites are attracting a significant amount of industrial interest as transparent conductive oxides (TCOs),145,148,149 and transparent electrodes for solar cells and GaNbased LEDs.150,151 A clear Nb(IV) signal including well resolved hyperfine coupling of the unpaired 4d electron to the 100% abundant 93Nb nucleus was observed at 4.2 K in Nb-doped rutile single crystals152,153 and in Nb-doped TiO2 powders containing both anatase and rutile polymorphs.154 In rutile single crystals, Zimmermann152 found the spin Hamiltonian parameters were gx ¼ 1.973, gy ¼ 1.981, gz ¼ 1.948 and Ax ¼ 5.0 MHz, Ay ¼ 23.8 MHz, Az ¼ 7.0 MHz (g and A tensor frames are ¯0], y is along the crystal [110] and collinear where x is along the crystal [11 z is along the crystal [001] which is the c-axis; Zimmermann152 used a reference system which is rotated compared to Chang’s for V(IV)133). Owing the very fast relaxation of this paramagnetic species,152 the signal quickly broadens beyond detection at higher temperature (477 K), with hyperfine structure already unresolved at 20 K.152,153 However, De Trizio et al.,145 at liquid helium temperature could not detect Nb(IV) at all in Nb-doped colloidal anatase nanocrystals. Furthermore, Folli and co-workers100 could not observe Nb(IV) in (Nb, N)-codoped anatase samples. The absence of detectable Nb(IV) signals in anatase TiO2 has yet to be fully understood, and it is still matter of debate in the field.

4.3 VI-group metal doping: Cr- and W-doped TiO2 As one of the Earth abundant transition metals, Cr has also been investigated as a dopant for TiO2 photocatalysis. Chromium exists in several oxidation states, but the most stable ones, together with the metal form Cr(0), are the trivalent Cr(III) ([Ar]3d3 S ¼ 3/2), and the hexavalent Cr(VI) ([Ar]3d0) systems. Given that Cr(VI) is hemotoxic, genotoxic, and carcinogenic,155 whilst Cr(III) is, to a greater extent, less toxic, poorly soluble in water and also an essential trace mineral in the human diet,156 it is clear why most of the catalysis research has focused on doping TiO2 with Cr(III) (also much more interesting than Cr(VI) from an EPR perspective). As far as photocatalysis is concerned, Cr(III) doping was shown to narrow the band gap of TiO2 and therefore allow visible light photoresponse.157–161 In addition, increase of the photocatalytic activity towards the degradation of model pollutants, like 2,4-dichlorephenol Electron Paramag. Reson., 2019, 26, 130–170 | 151

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under visible light, was also reported, with an optimal doping level corresponding to ca. 1 at.% of Cr(III). Cr(III) substitutes for a Ti(IV) ion, decreasing the n-type character of TiO2 (it acts as a p-type dopant) and leads to the formation of oxygen vacancies: 2TiO2

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Cr2 O3 ! 2Cr0Ti þ 3OxO þ V O The g-values of g> ¼ 1.9730 and g8 ¼ 1.9718 with the zero-field splitting parameter D ¼ 1118.2 MHz for high spin Cr(III) in anatase octahedral clusters, have been recently calculated using a complete high-order perturbation formula based on the cluster approach, involving both crystal field and charge transfer mechanisms.163,164 These values were found to be in perfect agreement with experimental values measured some time ago by Grunin and coworkers.165 Cho166 studied the substitution of Cr(III) in rutile, suggesting a strong paramagnetic behaviour and a weak ferromagnetic contribution (even at room temperature) with every small magnetic moment per doping ion. They reported an X-band CW EPR spectrum that could be interpreted with g ¼ 1.97, D ¼ 16488.5 MHz and E ¼ 8094.3 MHz for Cr(III) (matching the spin Hamiltonian parameters measured in 1959 by Gerritsen et al.,167 for rutile single crystal), plus a signal potentially arising from the presence of Cr(V) in interstitial positions. A series of papers appeared in catalysis-specialised journals162,168–170 where EPR spectroscopy was used to improve the fundamental understanding of the structural location of the Cr dopants and draw a clearer picture of the structure/function relationship in Cr(III) doped anatase and anatase, rutile mixed polymorphs TiO2 photocatalysts. Their X-band CW EPR spectra could be mostly simulated with a Cr(III) centre having g, D (and potentially E) values as reported above, plus an additional broad line for Cr loadings higher than 1 at.%, corresponding to Cr(III) in segregated Cr2O3 clusters. Nevertheless, these authors identified the low-field and centre-field resonances of a unique Cr(III) signal with fine structure (consequence of zero-field interaction) as two distinct signals, called d and g respectively, suggesting that they arise from Cr(III) in two different crystallographic environments. It is important to highlight this point, because this rather unusual and controversial interpretation of the Cr(III) spectrum continuously appears in the catalysis-specialised literature, usually through multiple cross-references that can be traced back to (at least) the mid-1990’s. W is known to easily generate stable mixed valence (5þ and 6þ ) oxides, i.e. tungsten bronzes, upon reduction of WO3, and potentially form even lower oxidation states with increased metallic character. TiO2– WO3 mixed oxides and TiO2–WO3 heterojunction films have attracted a lot of interest as visible light photocatalysts which are also capable of efficiently storing photo-generated electrons. This would enable the systems to sustain photocatalytic activity in the darkness.171–173 Recently, the same rationale has been tested on W(VI) doped TiO2, to probe whether the benefits of the mixed oxide systems and heterojunctions could be retained in lattices that, from a crystallographic point of view, are anatase but containing W(VI) point defects.99,101,174,175 When introduced as a 152 | Electron Paramag. Reson., 2019, 26, 130–170

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Fig. 1 Experimental (a) and simulated (b) X-band CW EPR spectrum of substitutional W(V) in rutile octahedra of Ti0.999W0.001O2 (88% anatase and 12% rutile). The spectrum was recorded at 50 K.

dopant in TiO2, W(VI) ([Xe]4f 14) aliovalently and isomorphically substitutes for Ti41: 1 TiO2  WO3 ! WTi þ 2OxO þ 2e0 þ O2 2 increasing the n-type character of the semiconducting metal oxide. In mixed polymorphs anatase/rutile nanoparticles at low W loadings, a clear anisotropic EPR signal of W(V) ([Xe]4f 145d1) is evident (Figure 1). The diagonalized g-tensor has principal components of gx ¼ 1.473, gy ¼ 1.443, gz ¼ 1.594. The unpaired d electron also couples to the 183W nucleus (natural abundance 14.31%) with A-tensor principal values of |Ax | ¼ 122.4 MHz, |Ay | ¼ 191.1 MHz, |Az | ¼ 277.5 MHz. The very low g-values for W(V) compared to the transition metal ions previously discussed here are due to its very high spin–orbit coupling, equal to 2700 cm1 (i.e. 80.94 THz).176 The presence of W(V) is the result of the trapping of valence-induced electrons, which are necessary to balance the extra positive charges carried by W(VI) in comparison to Ti(IV), in a very similar fashion to what is already seen for V(V) and Nb(V). Interestingly, the g- and A-values reported above match exactly the ones reported some time ago by Chang133 for W(V) in rutile single crystal (gx ¼ 1.473, gy ¼ 1.443, gz ¼ 1.594 and |Ax | ¼ 122.4 MHz, |Ay | ¼ 191.1 MHz, |Az | ¼ 277.5 MHz; g and A-tensor frames are collinear where x is along the crystal [110], y is along the crystal c-axis and z is along the crystal ¯0]). This would indicate at the fact that in mixed polymorph powder [11 nanoparticles, substitutional W(V) might prevalently form in rutile octahedra rather than anatase octahedra. Indeed, for similar W loadings in anatase only nanoparticles, we observed no evidence for such W(V) ion. This result is peculiar, even more so as it reflects exactly what is observed Electron Paramag. Reson., 2019, 26, 130–170 | 153

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for the case of isomorphic substitutional Nb(V) and its reduction to Nb(IV) through electron trapping. Robust evidence and reasoning explaining this phenomenon have yet to be provided. For W loadings greater than 5 at.%, non-stoichiometric W clusters can form on the surface of anatase TiO2 nanoparticles. Folli et al.,174 have shown that at these high loadings, reduced W states are mostly associated with these clusters. Two types of W(V) centres in different crystallographic environments can indeed be formed: one associated with unsaturated W(V) species in WxOy clusters strongly anchored to the surface of the host crystal, with g>¼ 1.85 and g8 ¼ 1.50; the second one associated with W(V) in tungsten bronze-like superstructures, weakly bound to the surface of the host crystal, with g>¼ 1.80 and g8 ¼ 1.64. No hyperfine coupling to the 183W nucleus was detected in this case,174 most likely due to the very low signal intensity and low S/N ratio. Folli and coworkers have highlighted that photo-generated electron storage is indeed possible in W(VI) doped TiO2 nanoparticles, unveiling a correlation between the mount of electrons stored and W loading in the nanoparticles.174 Indeed, EPR showed that substitutional W(V) in TiO2 octahedra and W(V) in WxOy clusters and bronze-like superstructures tend to increase in concentration upon irradiation.174,177 This would suggest that photo-generated electrons can be trapped exactly like valence-induced electrons. Furthermore, they observed that photo-induced, reduced W species may be responsible for accessing redox processes that are kinetically unfeasible with undoped TiO2. A typical example is the electrochemical and photoelectrochemical two-electron reduction of molecular oxygen,174 where W can reproduce the benefits of PGM cocatalysts.174,178

5

Heterogeneous catalytic systems

In general terms, as heterogeneous catalysis remains such a large and globally important field, it is of little surprise that a large number of catalysis research papers include EPR techniques for analysis of the surface centres involved in the catalytic reactions. In recent years, a number of excellent specific reviews have appeared on the use of EPR to investigate heterogeneous catalysis, which demonstrate and highlight the wealth of information that can be extracted.181–184 Morra et al.,181,182 summarise some of their own results on Ti-based heterogeneous catalytic materials, particularly isomorphously substituted Ti in open framework materials, including tetrahedrally coordinated Ti ions in silicalite TS-1, TiAlPO-5 and ETS-10,181 but also on systems relevant to Ziegler–Natta catalysts and TiO2 based photocatalysts.182 These review articles illustrate nicely how a thorough and comprehensive understanding, at the molecular level, of the structure–property relationships between catalytically active sites and the surrounding matrix, as well as the interaction with selective adsorbates, can be obtained through pulsed hyperfine EPR methods, such as HYSCORE. Sobanska et al.,183 present a detailed description of the diagnostic features of the superoxide radical anion (O2 ) on catalytic surfaces and how the g- and A-tensor can be used to interpret the molecular framework of the radical. This radical is 154 | Electron Paramag. Reson., 2019, 26, 130–170

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commonly involved in heterogeneous oxidation reactions, and unfortunately is also commonly mis-assigned in the analysis of EPR spectra in the literature. However, in this review, Sobanska et al.,183 provide an excellent account on the electronic nature of the g-tensor, for both electrostatic and covalent adducts, and on the analysis of the 17O hyperfine A-tensor. Any practitioner of EPR employing the technique to study heterogeneous oxidation reactions, where O2  anions will most certainly be involved, should refer to this work,183 and the references therein. Finally, in their review, Manck and Sarkar184 have summarised, through some selected examples, the use of EPR to unravel reaction mechanism of relevance to catalytic bond action reactions.

5.1 Supported metal complexes The functionalization of surfaces with transition metal complexes, or simply anchoring homogeneous catalysts onto heterogeneous supports, has remained a very popular means of generating novel heterogeneous catalysts. In many cases, organic ligands capable of strongly binding redox active metal ions are employed, such as Schiff base ligands185 or phthalocyanines.186 Anbarasu et al.,185 used EPR as an analytical tool to demonstrate the formation of the Cu(II)-Schiff base complex on a silica surface. The EPR spectrum obtained was poorly resolved, so only limited diagnostic information could be extracted about the catalyst. Huang et al.,186 also studied the generation of reactive oxygen species by EPR using an anchored Co(II)-phthalocyanine complex on activated carbon fibres, where the focus of their study was primarily the chemistry of the ROS. Bilis et al.,187 provided an in-depth catalytic study of non-heme Fe based catalysts covalently grafted onto a silica support for catalytic oxidation of cyclohexene. These heterogeneous anchored catalysts proved to be very resilient and displayed improved oxidative stability compared to the homogeneous analogues. In addition to the high spin (S ¼ 5/2) Fe(III) centres, characterised by a broad peak at g ¼ 9.2 and with higher field peaks at g1 ¼ 4.63, g2 ¼ 4.21, g3 ¼ 3.80, EPR also revealed the presence of a low spin (S ¼ 1/2) Fe intermediate, in the presence of H2O2, and tentatively assigned to a Fe(III)–OOH hydroperoxide centre. This centre was characterised by the principal g-values of g1 ¼ 2.02, g2 ¼ 1.96, g3 ¼ 1.86 and the high spin to low spin transition only occurred in the presence of hydrogen peroxide. The involvement of free radicals was demonstrated by complimentary spin trapping studies using DMPO and TEMPO. This excellent paper illustrates nicely the wealth and depth or information that EPR can provide in catalysis studies. Barman et al.,188 recently prepared a series of vanadium-based catalysts for the oxidative dehydrogenation of propane. The catalysts were prepared on silica using surface organometallic chemistry to deliver a m2–oxo-bridged, bimetallic [V2O4(acac)2] complex precursor which, following activation, leads to a well-defined and isolated monovanadate VOx species. The well resolved EPR signals of the catalyst revealed the spin Hamiltonian parameters of g8 ¼ 1.94, g> ¼ 1.98, A8 ¼ 17.4103 cm1, A> ¼ 6.8103 cm1, and revealing that the first coordination sphere is completed by singly Electron Paramag. Reson., 2019, 26, 130–170 | 155

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bound oxygen atoms. Finally, the anchoring or grafting of homogeneous species onto heterogeneous supports is not limited to transition metal complexes. Organic radicals can also be covalently grafted onto supports to enhance or generate new catalytic functionalities. Shakir et al.,189 grafted 4-amino-TEMPO onto graphene oxide through the amide bond. The resulting composite materials were successfully used as a catalyst for the selective oxidation of some alcohols in very mild conditions. In this work, the EPR signals were very broad, but nevertheless demonstrated the presence of the grafted radicals on the graphene support. Piotrowski et al.,190 also used TEMPO to functionalize C60 fullerenes deposited on gold surfaces for the catalytic oxidation of selected alcohols. A redox based mechanism was proposed for this C60TEMPO10@Au catalytic system for the oxidation, under mild conditions, of primary and secondary alcohols to the corresponding aldehyde and ketone analogues. Crucially during the catalytic cycle, it was found that the TEMPO moiety was continuously regenerated in situ in the presence of a primary oxidant. The EPR signals displayed the expected triplet profile with g ¼ 2.006 and aN ¼ 14.98– 15.45 G superimposed on a broad background resonance caused by the nitroxide radicals interacting with other.

5.2 Ethylene polymerisation Without question the area of ethylene polymerisation continues to attract significant attention, with considerable efforts devoted to probing the nature of the active sites and the mechanism.191–196 Whilst most of this work pertains to heterogeneous systems,191–195 the homogeneous analogues, for example based on chromium complexes,196 continue to be extremely important (although the homogeneous systems will not be considered here). This is particularly pertinent to the Philips ethylene polymerization catalysts, which after 60 years of application and whilst accounting for 50% of the world’s annual high density polyethylene, still has many unknowns associated with the mechanism. Brown et al.,191 recently focussed on probing the polymerisation step which is initiated without using an alkylating co-catalyst. It is believed that the Cr/SiO2 catalyst bears Cr(II) as the lowest oxidation state of chromium accessed, and this is required to activate ethylene to form an organo-Cr active site. Cr(II) is not visible by EPR spectroscopy at conventional frequencies, but it can be detected using very high frequencies. In their work, the authors recorded a signal at 105.6 GHz showing four distinct features located far from the free spin region, indicating the presence of a nonKramers (integer spin number) species with a large zero-field splitting. The spin Hamiltonian parameters extracted from the spectrum gave the parameters S ¼ 2, D ¼ 2.06 cm1, E ¼ 0, giso ¼ 1.99. Higher frequency measurements were also performed at 212 and 317 GHz, confirming the reported spin Hamiltonian parameters. Additional features in the spectra were assigned to the Kramers form of chromium, namely Cr(V) and Cr(III), which is not unexpected in such a complex catalytic system. In another mechanistic study of the Philips catalyst, Delley et al.,192 examined the elementary steps in ethylene polymerisation on the 156 | Electron Paramag. Reson., 2019, 26, 130–170

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isolated Cr(III) sites on the silicate surface. The X-band EPR spectra displayed a broad (ca. 600 G) symmetric signal at g ¼ 4.6 along with a dispersive signal at g ¼ 2.0 (ca. 220 G wide) and consistent with an axial g-tensor. The broad absorptive signal at lower magnetic field consisted of two broad and overlapping signals that are consistent with the presence of two different Cr environments on the catalyst surface. In combination with other techniques, the authors were thus able to demonstrate that 60% of the tri-coordinated Cr(III) sites can initiate the ethylene polymerisation step.192 The group of Chiesa et al.,193–195 have also undertaken detailed investigations into olefin polymerisation, but based on Ziegler–Natta type catalytic systems. They studied the fourth-generation Ziegler–Natta type catalyst TiCl4/MgCl2/phthalate following activation with triethylaluminum which leads to the formation of Ti(III) centres. These paramagnetic centres were then probed using CW and pulsed EPR methods. Two dominant Ti(III) sites were identified from analysis of the X- and W-band EPR spectra with g-values of g1 ¼ 1.93, g2 ¼ 1.88, g3 ¼ 1.84 for site 1 (76% abundant) and g1 ¼ 1.96, g2 ¼ 1.94, g3 ¼ 1.89 for site 2 (23% abundant). Q-band HYSCORE spectra furthermore revealed the presence of 35,37Cl signals from which the authors were able to report the hyperfine and nuclear quadrupole tensors. Based on their analysis of the data, and with complimentary DFT calculations, the authors concluded that the dominant Ti(III) active centres were located the MgCl2(110) surfaces. Finally, the accessibility and reactivity of these centres was also demonstrated by exposure of the catalyst to molecular oxygen, leading to the formation of the superoxide radical anion (O2) with rhombic g-values of g1 ¼ 2.003, g2 ¼ 2.010, g3 ¼ 2.021.193 In a series of follow up studies, Chiesa et al.,194,195 also examined the catalytic synergy between Ti(III) and Al(III) sites on a chlorinated Al2O3 surface,194 and also on the related Al2O3/TiClx catalyst.195 The authors developed a unique step-by-step approach to synthesize and characterize a bifunctional heterogeneous catalyst composed of isolated Ti(III) and Al(III) centres on the chlorinated alumina surface. It was found that the two sites, in close proximity, acted in a concerted fashion to synergistically boost the conversion of ethylene into branched polyethylene. The Ti(III) centres were identified by CW X-band EPR while the local environment around the centre was probed by Q-band HYSCORE spectroscopy which revealed clear modulations with the 27Al nucleus. The large isotropic hyperfine interaction offered confirmation for the presence of Ti(III)–Cl–Al linkages, with a spin density of 0.15–0.60% from the Ti(III) unpaired electron into the 3s Al orbital. By grafting TiCl4 onto a transitional alumina surface, the authors also studied the Ti–Al synergies in the Al2O3/TiClx catalyst.195 A detailed characterisation of the Ti(III) sites was obtained by CW and pulsed EPR, providing a detailed view on the electronic and geometrical structure of the paramagnetic centres after activation with triethylaluminum. Two different Ti(III) sites were once again observed with the g-values of g1 ¼ 1.97, g2 ¼ 1.94, g3 ¼ 1.90 for site 1 (35% abundant) and g1 ¼ 1.95, g2 ¼ 1.92, g3 ¼ 1.88 for site 2 (65% abundant). HYSCORE also revealed the presence of 27Al interactions, indicating that Electron Paramag. Reson., 2019, 26, 130–170 | 157

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the reduced Ti centres and their interactions with nearby Al(III) ions may be a common feature in these types of catalysts.

5.3 Heterogeneous free radical generation In studies of advanced oxidation processes or Fenton-like chemistry, involving heterogeneous catalysts suspended in the liquid phase, inevitably short lived reactive oxygen species (ROS) are involved, such as OH , O2 , OOH , RO , etc. In systems where persulfate is involved, SO4  species may also be involved at the solid–liquid interface. In all cases, spin trapping methods combined with EPR are frequently necessary to capture and identify these transient species, in order to better understand the reaction mechanism. In the past few years, a large number of investigations have been reported that use spin trapping EPR methods in heterogeneous catalytic systems to identify the transient radicals. These catalysts are based on perovskites,197,198 iron nanoparticles,199 nanodiamonds,200,201 graphene,202,203 carbon,204,205 manganese oxides,208 substituted phosphotungstates,209 iron oxides210 and CuMgFe layered oxides.211 A number of authors have demonstrated clearly how SO4  and OH radicals could be trapped using the popular 5,5-dimethyl-1-pyrroline (DMPO) trapping agent.197,210 In particular, Su et al.,197 developed a novel double perovskite material, labelled PrBaCo2O51d, for the heterogeneous generation of free radicals from peroxymonosulfate for the oxidative degradation of organic wastes in the aqueous phase. They demonstrated that these transient radicals played important roles in the complex catalytic oxidation cycle. Although the spin adduct spectra are reasonably clear, no simulations were provided to confirm the assignments and thereby extract the isotropic hyperfine parameters for the trapped radicals. Extended work by the same group198 also investigated the transient hydroxyl and sulfate radicals involved in a closely related perovskite catalyst used for the effective peroxymonosulfate (PMS) activation forming the radicals. The reported spin adduct EPR spectra were incredibly well resolved and the authors provided detailed hyperfine parameters for the two adducts (DMPO-OH: aH ¼ 14.8, aN ¼ 14.8 G and DMPO-SO4: aH ¼ 0.78, aH ¼ 1.48, aH ¼ 9.6, aN ¼ 13.2 G). Unfortunately, no simulations of the spin adduct signals were provided. A very detailed catalytic study of persulfate activation, using various oxyanions (such as peroxymonosulfate) in the presence of iron nanoparticles as the reducing agent and single-walled carbon nanotubes as the electron transfer mediators, was investigated by Yun et al.199 It was found that the oxidative degradation of organic substrates was achievable through one-electron reduction of oxyanions on the nanoparticle surfaces, with electron transfer mediation from the organics to the oxyanions on the carbon nanotubes. The EPR spectra evidenced the presence of DMPO-OH and DMPO-SO4 spin adducts, which helped to elucidate the radical mechanism, although no spin adduct hyperfine parameters are given. Duan et al.,200–204 also investigated the radical generation using nanodiamonds,200,201 nanocarbons,202 a-MnO2203 or graphene,204 all as metal free catalysts. The motivation for this work was to develop new 158 | Electron Paramag. Reson., 2019, 26, 130–170

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metal-free based catalysts for advanced oxidation processes. Persulfate activation over annealed nanodiamonds was investigated and EPR spectroscopy provided valuable information on the relative abundances of the DMPO-OH and DMPO-SO4 spin adducts as a function of the reaction conditions, which were initialized from oxidizing water molecules on the nanodiamond surfaces.200,201 The authors have extended their work to include N-doped graphene nanomaterials which was capable of degrading phenol solutions by metal-free catalytic activation of peroxymonosulfate (PMS). The role of the trapped SO4  and OH radicals in the cycle was confirmed by EPR, although the experimental EPR spectra were not simulated. A very detailed spin trapping EPR study of graphene oxide as a metalfree catalysts for light assisted Fenton-like chemistry was described by Espinosa et al.205 The focus of their work was to investigate the catalytic activity of reduced graphene oxide in the presence of sunlight for phenol degradation. The formation and participation of hydroxyl radicals was confirmed by EPR using the phenyl a-tert-butyl nitrone (PBN) spin trap. In this case, well resolved and simulated EPR spectra of the spin adducts were reported yielding the associated spin Hamiltonian parameters for PBN-OH (aH ¼ 2.7 G, aN ¼ 15.5 G) and a second minor PBN-tert-butyl aminoxyl adduct (aH ¼ 13.9 G, aN ¼ 14.58 G). The role of the radical mechanism was confirmed using DMSO, since it reacts with OH radicals and thus quenches the reaction. The reaction mechanism is carefully articulated and explained throughout in their work.205 Spin trapping EPR was used more extensively to investigate the radical based mechanism in the oxidation of cyclohexane.206,207,209 In their work, Liu et al.,206 provided detailed simulations and hyperfine parameters for all of the observed spin adducts in liquid phase oxidation over a bimetallic Au–Pd/MgO catalysts. The observed radicals included a di-tertbutyl-nitroxide derivative formed by DMPO oxidation (aN ¼ 14.31 G), a DMPO–O–C6H11 spin adduct (aN ¼ 13.37, aH(b) ¼ 6.01, aH(g) ¼ 1.90 G), a DMPO–OO–C6H11 adduct (aN ¼ 14.49, aH ¼ 10.64 G) and a carbon centred adduct (aN ¼ 15.74, aH ¼ 25.61 G) possibly attributed to an DMPO– C(OH)R2 adduct. The relative contributions of each adduct was determined by simulation of the spectra, so the role of the radicals in the reaction mechanism was easier to quantify, assign and determine. Yang et al.,207 also studied cyclohexane oxidation using multi-walled carbon nanotubes. An EPR signal at g ¼ 2.0037 was observed in the nanotubes and assigned to an O2  intermediate. This species has a well-defined rhombic g-tensor,183 so the observation of a single peak or g-value is insufficient to unambiguously make an assignment to a superoxide radical. Similar to the work of Liu et al.,206 the authors207 also identified  spin adducts associated with C6H11OO , C6H11O or C6H11 radicals. Finally, Song et al.,209 used a transition-metal-substituted phosphotungstate with hydrogen peroxide in their investigation of cyclohexane oxidation. In their case, spin adducts of DMPO-OH and DMPO-OOH were reported. However, the spin adduct EPR spectra are incredibly broad, and in the absence of any simulations to extract the spin Hamiltonian parameters, it is difficult to rationalise these assignments. Electron Paramag. Reson., 2019, 26, 130–170 | 159

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The Fenton-like free radical chemistry of various heterogeneous catalysts was also explored using DMPO over ZnMn2O4 nanorods or Au–Fe3O4 nanocomposites. DMPO-OH208,210 or DMPO-OOH208 adducts were reported, but in the absence of spectral simulations and hyperfine parameters, these assignments cannot be confirmed.

5.4 Supported metal oxides and nanoparticles Traditionally, EPR techniques have been widely used to directly characterise heterogeneous surfaces, particularly metal oxides. In the past few years, this trend and application has continued with considerable emphasis on supported Au nanoparticles,212–215 mixed metal oxides,216,217 cobalt oxides,219 and supported nickel,220 palladium,221 or lithium222 based systems. In their review, Ong et al.,212 describe how different techniques, including EPR, can be used to study the ligand shell of coated Au nanoparticles. In these systems, EPR can be used to study the thiolated nitroxide free radicals, from which valuable information on molecular conformation and lateral mobility can be determined. The incorporation of Au nanoparticles into a porous coordination polymer, was also investigated by Agarwal and Gupta. Very broad EPR signals with g-values in the range of 2.020–1.852 and 2.042–1.785 were detected and assigned to the presence of nanoparticles with differing sizes. The presence of what appears to be Mn(II) signal in their Ag/AgCl@Mn-PCP system is also evident in their spectra. The oxidation of CO over Au/TiO2 catalysts was investigated in a combined TAP and EPR study. The EPR spectra revealed well resolved signals associated with Ti(III) centres, as characterised by the g-values of g> ¼ 1.978 and g8 ¼ 1.964 in an inert atmosphere. After subsequent exposure to CO, the intensity of the signal increased, with slight changes observed to the g-values (g> ¼ 1.976 and g8 ¼ 1.958); this change was ascribed to the alteration in the distorted local environment. The role of the lattice oxygen in the chemistry is discussed in detail. Notably, a symmetric signal at g ¼ 2.004 was assigned to an F centre. This assignment is not discussed in detail; it is not clear how such a centre can be present on TiO2 and it appears close to the medium polarised conduction electron signal observed in TiO2 with a very similar profile. Oxygen activation on CeO2/Al2O3 supported Au was also examined using 18 O/16O isotopic exchange and EPR. Despite the growing use of supported Au for activating a host of simple molecule, the primary or initial oxygen activation is still under debate. The authors identified several O2  species with different g-values, which they were able to assign to the CeO2, Au/CeO2, CeO2/Al2O3 and Au/CeO2/Al2O3 surfaces respectively (the parameters were slightly different in each case). Giamello et al.,216,217 have reported the structural and reversible electron transfer properties of the mixed oxides ZrO2–TiO2216 and CeO2– TiO2,217 since the redox properties of these reducible oxides are important in many catalytic processes. Traces of a Zr(III) signal (with g> ¼ 1.960 and g8 ¼ 1.978) can be detected in the as-prepared sample. In the reduced sample, well defined signals attributed to Ti(III) centres are detected. 160 | Electron Paramag. Reson., 2019, 26, 130–170

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Simulation of the spectra reveals three Ti(III) environments with g-values of g> ¼ 1.905 and g8 ¼ 1.978 for surface and subsurface species, g> ¼ 1.929 and g8 ¼ 1.967 in t-ZrO2 and g>¼ 1.918 and g8¼ 1.958 in mZrO2. Subsequent exposure of the reduced sample to molecular oxygen results in the formation of the surface adsorbed superoxide anion O2  with the g-values of gzz ¼ 2.035–2.032, gyy ¼ 2.010 and gxx ¼ 2.003. A more detailed investigation of the reversible nature of the electron transfer process through the intermediary of the superoxide anion is also described.217 Exposure of the activated mixed phase oxide to molecular oxygen results in the formation of O2  as characterized by the g-values of gzz ¼ 2.0256, gyy ¼ 2.0206 and gxx ¼ 2.0126 and indicative of O2  stabilisation at Ce(IV) sites. By comparison, formation of O2  on bare CeO2 only occurs if the sample is partly reduced (leading to Ce(III) centres) and the O2  centre is not reversibly formed. Further information on the identity of the reversibly formed radical on the mixed-phase oxide was obtained through 17O isotopic labelling studies where the 17O2  tensor is reported. The redox properties of another mixed-phase oxide system, namely CuO–CeO2, was also investigated by in situ EPR.218 A better understanding of the redox properties of such systems is extremely value in catalytic chemistry, and EPR is therefore the ideal technique to monitor the changes in the oxidation states of the transition metal ions. The authors were able to demonstrate conclusively that two redox mechanisms are operative, including a synergetic step involving the redox pair Ce(IV)/ Ce(III) during oxidation of Cu(0)/Cu(I) species to Cu(II) and a direct step that bypasses the Ce(IV)/Ce(III) redox pair.218 The EPR spectra revealed the presence of isolated monomers, isolated dimers (exhibiting fine structure, with g> ¼ 2.035, g8 ¼ 2.216, A> ¼ 1.3 mT, A8 ¼ 9.1 mT and D ¼ 69.1 mT) and amorphous clusters of Cu(II) ions. The behaviour of these signals under reductive and reoxidation conditions was then monitored by EPR, enabling the authors to suggest the proposed mechanism. This is an excellent contribution, demonstrating the wealth of information that advanced EPR techniques can provide to heterogeneous, mixed-oxide systems. An operando EPR study was reported using EPR over supported Ni catalysts for butane oligomerization at 353 K and up to 16 bar. Single Ni(I)/Ni(II) redox couples were identified as the active sites. The EPR spectra revealed well resolved axial signals associated with two different Ni(I) centres on the support, with g> ¼ 2.069, g8¼ 2.536 and g>¼ 2.051, g8 ¼ 2.443. After exposure of the catalyst to butene, two new signals emerged in the spectra and were assigned to a NiI p-complex with 1-butene (g> ¼ 2.012, g8 ¼ 2.219) and to a NiI–(CH2)3CH3 complex (g> ¼ 2.090, g8 ¼ 2.233) formed by insertion of 1-butene into the Ni–H bond. A new signal was also observed at g> ¼ 1.970 and this was accounted for due to the interaction of the Ni d-electrons with the p-electrons of the olefin ligands. Vedyagin et al.,221 examined CO oxidation over a Pd/ZrO2 catalyst using a range of analytical and spectroscopic techniques including EPR. The spin probe approach was taken in this study, using 1,3,5-trinnitrobenzene (TNB), to characterise the active sites where a Electron Paramag. Reson., 2019, 26, 130–170 | 161

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good correlation was found between the abundance of surface radical centres, corresponding to the donor sites of the support, and the catalytic behaviour during light-off tests. The signals of the radical anions are easily identified by their g- and A-values corresponding to gav ¼ 2.005– 2.004 and Aav ¼ 31.0–26.5 G. This work offers a good example of how diamagnetic oxide based catalysts can still be studied by EPR when using the appropriate spin probe molecules. Finally, a catalyst that remains of high interest for the oxidative coupling of methane is Li doped MgO (Li/MgO).222 Although the catalyst suffers from a well-known stability issue, due to strong and fast loss of Li, nevertheless the system remains of general interest due to the detailed insights it continues to provide on the mechanism. In a very detailed and comprehensive study, Simon et al.,222 used EPR/NMR and DFT to unravel the local topology in a Gd–Li/MgO and Fe–Li/MgO doped system. CW and FSE EPR at X-band and 239.2 GHz, as well as transient nutation EPR at Q-band were all utilised. No change was observed in the Fe(III) EPR pattern when co-doped with Li, indicating that no local correlation of Fe(III) and Li(I) as next neighbours occurs. Analysis of the EPR spectra of Gd–Li/MgO samples provided clear evidence for the anticipated existence of nearest-neighbour correlated Gd–Li pairs. This correlation was confidently assigned based on the observation that Li co-doping leads to a change in the EPR pattern, which in turn is accounted for by assuming a change in the local symmetry of Gd(III).

6

Summary and perspectives

There is little doubt that catalysis is essential for solving many of the global challenges facing society, from clean energy and the environment, to developing chemicals with less waste, to enhancing the quality of life). In all these and many other cases, we must develop better, cheaper and more-sustainable catalysts. To develop such new catalysts, we will continue to rely on better analytical techniques that can interrogate the catalytic reaction at faster time scales, detected and characterise the reactive intermediates and ideally to determine the structure of the active sites at relevant conditions. Since most catalytic reactions involve bond making/breaking processes, electron transfer reactions, or oxygen transfer steps, then free radicals or unpaired electrons are typically involved. EPR spectroscopy then becomes a powerful technique to untangle and study the details of the reaction mechanism. Here, we have illustrated how homogeneous, heterogeneous, microporous and photocatalytic systems, have all benefited from the utilisation of EPR, through a detailed a comprehensive analysis of the spectra and the spin Hamiltonian. In all cases, great care and attention must be devoted to the simulation of the experimental data; only then can a confident and reliable assignment of the paramagnetic centre or free radical be achieved.

Acknowledgements The authors would like to thank the EPSRC for funding (EP/P019951). 162 | Electron Paramag. Reson., 2019, 26, 130–170

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