A Study into the Influence of Amyloid-beta Peptide Oxidation on the Rate of Fibril Formation, with a Synthesis of 2-oxo-histidine Hannah Mary Garrett A thesis submitted to THE UNIVERSITY OF LONDON For the degree of DOCTOR OF PHILOSOPHY School of Biological and Chemical Sciences Queen Mary, University of London May 2012 I herewith certify that any material in this thesis that is not my own work has been properly acknowledged. Hannah Garrett 2 I dedicate this thesis to my family and friends but in particular to my late father, Christopher John Wright, who first showed me the wonder of science. 3 ACKNOWLEDGEMENTS Although a PhD thesis only has one name on the title page it is never a solo effort. There are so many people who have advised and supported me over the last four years and without them it is unlikely this thesis would be here. It is impossible to thank everyone who has made this possible but I would like to mention a few of these people. Firstly, I would like thank my two supervisors, Dr Peter Wyatt and Dr John Viles, for giving me the opportunity to undertake this project, for advising me throughout and for being a constant source of suggestions of how to go forward with the project when things didn’t quite go to plan. They have inspired me with their enthusiasm for their work and their breadth and depth of knowledge of their fields. Secondly, I would like to thank the other academics at Queen Mary who have advised and supported me throughout my PhD. Professor Mike Watkinson chaired my panel meetings in a way that ensured I gained the most from them and throughout my PhD has kept me on course. Dr Robin Maytum always brought interesting discussions to my panel meetings and, when I felt like giving up, was incredibly supportive and reminded me of the benefits of a PhD. Professor Vaughan Griffiths kindly replaced Dr Maytum on my panel and brought a new perspective to the meetings, which highlighted important approaches to a PhD that I had not previously considered. Dr Adrian Dobbs, despite not being on my panel, has always encouraged me to increase my knowledge of organic chemistry by teaching me, allowing me to attend several of his group meetings and by being a good friend throughout my PhD. SBCS at Queen Mary is truly blessed in having such a lovely, supportive and friendly community of researchers. Without the friendly faces and comforting chats that I experienced every day in the college, the PhD would have been a much harder process. In addition, the many conversations over cups of tea early in the morning and late in the evenings regarding mechanisms, appropriate reagents and which music was the best to work to, were invaluable. Dr Ana María Blanco Rodríguez has been a good friend and supportive work colleague throughout my PhD. The many evening meals we had together were a good way to unwind after a tough day in the lab. Freda Chio is the most hard working person I have ever met and really inspired me, first as a project student in the Wyatt group and secondly as a fellow PhD student in the Chemistry department, to keep going. I really enjoyed studying the theory of organic chemistry with her and can only slightly apologise for exasperating her when always asking what the correct nomenclature was 4 for every chemical structure I came across. She has been a kind and conscientious friend throughout. Niranjeni Pathumakanthar has the most perseverance I have ever seen in a PhD student. Despite all her hardships she is a lovely, cheerful soul with whom I have enjoyed many lunches and conversations across a fume hood. I wish her all the best with completing her PhD. Dr Farid Uddin has been another true friend throughout my whole PhD. He is both a source of indispensible knowledge of chemistry and has a wicked sense of humour. My PhD would not have been half as enjoyable without his presence in the lab. Dr Rendy Tan, Dr Becky Nadal and Dr Claire Sarrell were all current members of the Wyatt and Viles groups when I joined in 2004. Without them I would never have got up to speed on the many new techniques and knowledge required to do my PhD. Watching them go on to complete their post docs and PhDs was a real inspiration for completing my own project. The other members, past and present, of the Wyatt and Viles groups have made these groups feel like real families; Ahmed Dellali, Jahangir Malik, Zhe Li, Yu Peng, Zhang Yi, Nadine Younan and Helen Stanyon. Finally, but by no means least, my friends and family have been such an encouraging and supportive network throughout my PhD, despite not always understanding the slightly frazzled, PhD-crazed person I had become. My new husband John Garrett has always been a rock throughout my PhD to whom I could turn at any moment for support and love. He even still agreed to marry me after four years of experiencing the PhD stress first-hand and for that I will always admire and love him. My parents have constantly supported me throughout my life and helped me to strive to whatever goal I set myself. They have always taken a keen interest in how my PhD is progressing and my mother even knows the title of my thesis. Rosie and Lucy have been such great sisters throughout my PhD, always with support and able to make me laugh. Finally, my closest friends, Jess, Mary, Zoe, Claire, Lorraine and Amy, have listened to my trials and tribulations and in return provided kind words and fun anecdotes. It is with all this kindness, good will and support that I have completed this PhD and for that I will be eternally grateful. Thank you also to the EPSRC and Queen Mary, University of London for my funding. 5 ABSTRACT The Amyloid Cascade Hypothesis states that fibrillation of the amyloid beta (Aβ) peptide is the primary cause of Alzheimer’s pathology. The trigger for the fibrillation is a subject of much debate, although it is clear, oxidative stress is a key feature of Alzheimer’s aetiology. This thesis explores a possible role of oxidation of Aβ, in particular the effect of histidine and methionine side-chain oxidation, on Aβ fibril growth rates. Within chapters 2 and 3 of this thesis is a discussion of various approaches to chemical synthesis of 2-oxo-histidine with a view to the incorporation of the oxidised amino acids into Aβ peptide using Fmoc approaches. Chapter 2 describes attempted chemical transformation of (protected) L-histidine into L-oxohistidine. Dimethyldioxirane oxidised Boc-His-OMe yielded products containing isopropylidene groups, while oxidation using a Cu(II)/ascorbate generated 2-oxo-histidine but gave very low yields. Within chapter 3, a successful synthesis of protected 2-oxo-histidine is described, via the known imidazolin-2-one-4-carboxylic. Chapter 4 analyses Aβ(1-40) fibrillation kinetics by treating the intact peptide with various oxidants. Contrary to previous reports, hydrogen peroxide alone did not slow fibrillation rates. Cu(II)/Cu(I)- catalysed oxidation increased the likelihood of amorphous aggregation over fibrillation. This thesis shows oxidation of Aβ has a profound influence on fibril growth and that incorporation of a stable oxidised histidine into Aβ is a realisable goal. 6 TABLE OF CONTENTS- Title Page ................................................................................................................... 1 Declaration ................................................................................................................ 2 Dedication .................................................................................................................. 3 Acknowledgments ....................................................................................................... 4 Abstract ...................................................................................................................... 6 Table of contents ........................................................................................................ 7 List of abbreviations ................................................................................................... 13 CHAPTER 1 – INTRODUCTION ......................................................................... 16 1.1. Alzheimer’s Disease (AD) ................................................................................. 17 1.2. The amyloid beta (Aβ) peptide ......................................................................... 18 1.2.1. Evidence for Aβ as the toxic species in AD .................................... 18 1.2.2. Cleavage of the amyloid precursor protein (APP) generates the Aβ peptide. ...................................................................................................... 19 1.2.3. Aβ(1-40) and Aβ(1-42) have different aggregation behaviours and toxicities. ................................................................................................... 21 1.2.4. Aβ structure ..................................................................................... 22 The structure of monomeric Aβ ............................................................ 22 The structure of fibrillar Aβ .................................................................. 23 1.2.5. The kinetic models of Aβ fibrillisation ........................................... 26 1.2.6. Factors that influence Aβ fibrillisation ............................................ 27 1.2.7. Off-pathway Aβ aggregation ........................................................... 28 1.2.8. Toxic species of Aβ ......................................................................... 29 1.3. The Amyloid Cascade Hypothesis