Development and Evaluation of Novel Hydrocarbon Stapled Peptidomimetics with Future Application to Bionic Proteins David James Yeo Submitted in accordance with the requirements for the degree of Doctor of Philosophy University of Leeds School of Chemistry June 2014 ii iii Intellectual Property and Publication Statements The candidate confirms that the work submitted is his own, except where work has formed part of jointly-authored publications has been included. The contribution of the candidate and the other authors to this work has been explicitly indicated below. The candidate confirms that appropriate credit has been given within the thesis where reference has been made to the work of others. The work reported in Chapter 3a formed the basis of a research article published in September 2013: ‘Monosubstituted alkenyl amino acids for peptide “stapling” David J. Yeo; Stuart L. Warriner and Andrew J. Wilson, Chemical Communications, 2013, 49, 9131-9133. The contributions of the authors were as follows: DJY (the candidate), SLW and AJW designed the research, DJY (the candidate) performed the research and drafted the manuscript, SLW and AJW edited the manuscript into its present form (see attached copy). This copy has been supplied on the understanding that it is copyright material and that no quotation from the thesis may be published without proper acknowledgement. © 2014 The University of Leeds and David James Yeo iv Acknowledgements Firstly, I would like to thank my formidable panel of supervisors, Dr. Stuart Warriner, Prof. Sheena Radford and Prof. Andy Wilson who have ensured that my project has been an intellectual challenge but were malleable enough to recognise when the project needed to evolve. Moreover, for the advice and balanced guidance throughout my PhD that'll be retained during my career development. I also want to extend my gratitude to Dr. Julie Fisher, who was instrumental in helping me turn things around at the 21 month nadir. Also, Sri, who has feigned interest at the appropriate moments to keep me motivated. This project has been a collaborative effort and would not have been anywhere near as successful without the help of several people from Chemistry and Astbury. The technical staff, Tanya Marinko-Covell (MS), Simon Barrett (NMR), Iain Manfield (ITC), Chris Empson (FAA) and particularly Martin Huscroft (HPLC) have been wonderfully tolerant of me over the last four years. A special thanks to Nasir Khan (CD) who gave me the first hope with the circular dichroism results of the BID peptide series and to Lynette Steele and Jane Levesley at LIMM for the cell work. Dr. Jen Miles, Dr. Alice Bartlett, Hannah Kyle and Dr. Thomas Edwards have also been helpful in either providing me with protein for testing and for teaching me how to express it myself, especially Jen and Ed, whose crystal trials have been of the upmost importance. Right then. Sentiments. You can all assume how much you've meant to me. Job satisfaction is partly down to the description, but mostly down to the environment. The people who I've worked with, from supervisors to placement students have been a drive of my PhD when things have been at their worst. The Radford and Edwards groups have been supportive into my forays into biology and biophysics and have been wonderful people to interact with. The first six months of the PhD have been the most memorable, thanks to the people in 1.32 and the inorganic corridor. Similarly, the people in the Warriner/Turnbull/Webb groups who've cheerfully welcomed my presence in their labs, in addition to the Marsden group who helped clear the blue air during the upscaling attempts. To keep me away from descending into v chemistry madness, the footballers, volleyballers, squash players and grappler have been dear to me. The Wilson Group. So many thanks to the group members who welcomed me when I first started and got me settled into my first forays into scientific research. Then, to the 2013/2014 Wilson group. I feel that I'm leaving the group at the wrong time. You are all wonderful, wonderful people who should never doubt their scientific abilities or personalities. The sweetness of the submission of this thesis will never be balanced by the bitterness of leaving you all. Jayapal for an unrepressed spirit; Irène for all of your craziness and massages; Sarah – thank you for the Staircase of Rant and calming me down; Jen for keeping me properly sane; George Burslem, who then undermined that proper sanity; Hannah for being a football and gin outlet; Kérya for the insults; Ludwig it's a crying shame that you didn't join the group earlier - I'll never forget Old Trafford; Silvia for teaching me that sometimes blunt is best; Kelly for teaching me that unreserved loveliness is best mixed with bluntness; Anna - the Lab Mum. Phil Winkworth, it seems appropriate that the first person I met in my PhD programme is at the end of the acknowledgments. Valeria. Nothing ever ever changes. Finally, I want to acknowledge the support and (enthusiastically feigned in some cases) understanding of my families, the Parker family, the Scotland clan a arbennig o Yeo tylwyth; a Mam a Tad. Diolch yn fawr. The last word, to my fiancée Charlotte, who has put up with being a Chemistry- and Thesis-widow for long enough. Our bright future together starts here. This thesis is dedicated to the memory of my grandfathers, Neville Yeo and Peter Scotland - who inspired and nurtured my scientific interest from the very beginning. Their passing happened far too soon. In five years, I hope to reopen this thesis and realise that whatever my next step was after the PhD was both my best decision and my worst mistake. vi Abstract Hydrocarbon stapling is a method of constraining a short polypeptide through the incorporation of alkenyl alanine unnatural amino acids,1, 2 which are metathesised during peptide synthesis to afford a covalent crosslink on one face of the peptide. The purpose of 'stapling' is to improve the therapeutic properties of a peptide by improving transport properties and resistance to proteolysis.2, 3 Stapling also reduces the conformational plasticity of a peptide, which in turn, should improve the potency of the crosslinked peptide with its binding partner. Alkenyl glycine ('monosubstituted') derivatives of amino acids have been previously synthesised, but overlooked for peptide stapling experiments.4 This project investigated the utility of monosubstituted amino acids as surrogates for hydrocarbon stapling with a therapeutically relevant family of proteins, the apoptosis regulator Bcl-2 family and p53 proteins.5, 6 This led onto an investigation of the foundations of hydrocarbon stapling, to assess the extent that hydrocarbon stapling modulates potency through various biophysical and structural experiments. Hydrocarbon stapled peptides are classified as peptidomimetics within the field of foldamers,7, 8 which aims to construct scaffolds from synthetic oligomers which can replicate the three dimensional topography and functionality of native proteins.7, 8 Since the de novo design of synthetic proteins is out of reach, regions of proteins with distinct architecture have been replaced with mimetics to afford semi-synthetic proteins in a technique described as 'protein prosthesis'.9, 10 The project also aimed to chemically synthesise a protein with four distinct helical regions, colicin immunity protein Im7,11 with the view to replace one of the helical regions with a stapled peptide or an oligobenzamide helix mimetic12 to afford a semi-synthetic protein to add to the growing field of secondary and tertiary structure mimetics. vii Table of Contents Intellectual Property and Publication Statements............................................. iii Acknowledgments .......................................................................................................... iv Abstract .............................................................................................................................. vi Table of Contents .......................................................................................................... vii List of Tables ....................................................................................................................xii List of Schemes .............................................................................................................. xiii List of Figures ................................................................................................................ xiii List of Abbreviations ................................................................................................. xviii Chapter 1 ............................................................................................................................. 1 Introduction ....................................................................................................................... 1 1.1 Protein Prosthesis: Replacing Sections of Native Proteins ..................... 2 1.1.1 Proteins - A definition ................................................................................. 2 1.1.2 Protein Synthesis - Biological Machinery ............................................ 2 1.1.3 Modification of proteins - a mutagenesis approach ........................ 4 1.1.4 Modified Proteins Through Chemical Synthesis ............................... 6 1.1.4.1 Solid Phase Peptide Synthesis (SPPS) Methods ..................... 6 1.1.4.2 Native Chemical Ligation Approach to Protein Synthesis .........................................................................................................