BETAINE ANALOGUES AND RELATED COMPOUNDS FOR BIOMEDICAL APPLICATIONS A thesis presented in fulfilment of the requirement for the degree of Doctor of Philosophy in Chemical and Process Engineering at the University of Canterbury Madhusudan Vasudevamurthy 2006 Department of Chemical and Process Engineering University of Canterbury Christchurch, New Zealand Acknowledgements I owe a debt of gratitude to my supervisor, Dr. Michael Lever for being the force behind this project. He has been my guiding star and a great mentor. His probing questions, enthusiasm to do things right and constantly finding new ways of functioning to resolve a problem was contagious. His humor and never ending discussions were a source of encouragement making him a great friend and confidant. The contribution of my supervisor Dr. Ken Morison is most appreciated. His continual support and guidance during the last couple of years has added immense value to my thesis. His invaluable suggestions, an eagerness to learn and implement has brought about the shape of this thesis. I am deeply indebted to Prof. Peter George and Prof. Steve Chambers for their valuable inputs and suggestions in this thesis. Their willingness to support and accommodate me in their hectic schedule is most appreciated. I am glad of this opportunity to extend my gratitude to Prof. Laurence Weatherley, my previous supervisor, whose encouragement and words of wisdom motivated me to pursue my PhD. His continual support helped me in the founding years of my thesis, opening doors to a wealth of knowledge and a thirst for learning more. His motivation has made me a scientist from an amateur. This acknowledgement would be incomplete without expressing my thanks to Dr. Sandy Slow. Working with her on the Sheep Dialysis Project has helped me develop my scientific skills. Her approach to problems and hard working attitude were very refreshing. I would also like to thank the staff of Sheep Technology Centre, Lincoln University. i Invaluable contribution of Dr. Jeff Upton and his colleagues at Molecular Pathology Department during the PCR project is most appreciated. I am greatly obliged for all the help from Dr. John Lewis and his colleagues at the steroid laboratory during CBG studies. Thanks to Dr. Robert Maclagan for all the theoretical calculations. I would also like to thank Mr. Chris McIntyre for his valuable suggestions for syntheses of solutes and also for being a great friend. I am deeply indebted to Mr. Rewi Thompson for NMR analyses. I wish to record the lasting gratitude that I owe to my loving family who despite all odds and obstacles have supported me to fulfill my ambitions, and be successful in my endeavors. Their constant encouragement and motivation was an essential ingredient for my successful completion and I hope to do them proud someday. I acknowledge with appreciation the love and kindness of my many friends, especially Ekta and Prashu, who have made this foreign city a more comfortable home away from home. I would like to thank all my colleagues at CAPE and CHL for great companionship and making the environment conducive to research. I am thankful to University of Canterbury Scholarship and Canterbury District Health Board for their financial support. ii TABLE OF CONTENTS ACKNOWLEDGEMENTS .............................................................................................. i CHAPTER 1 - INTRODUCTION................................................................................... 1 1.1 GENERAL INTRODUCTION................................................................................. 1 1.2 OBJECTIVES........................................................................................................... 6 1.3 THESIS OVERVIEW............................................................................................... 7 1.4 REFERENCES ......................................................................................................... 8 CHAPTER 2 - PROTEINS AND STABILIZATION MECHANISMS .................... 12 2.1 PROTEINS ............................................................................................................. 12 2.2 MECHANISMS OF PROTEIN STABILIZATION BY COSOLVENTS ............. 14 2.2.1 Preferential Exclusion, Preferential Hydration and Preferential Interaction ... 14 2.2.2 Steric exclusion mechanism............................................................................. 20 2.2.3 Osmophobic Effect .......................................................................................... 21 2.2.4 Protein Stabilization at Low Temperatures (Freeze-thawing and Freeze-drying) ................................................................................................................................... 24 2.3 REFERENCES ....................................................................................................... 30 CHAPTER 3 - SYNTHESES OF COMPENSATORY SOLUTES............................ 35 3.1 INTRODUCTION .................................................................................................. 35 3.2 MATERIALS AND METHODS............................................................................ 37 3.2.1 Ion-exchange chromatography......................................................................... 37 3.2.2 Propio betaine [PB].......................................................................................... 38 3.2.3 Deanol betaine [DB] ........................................................................................ 38 3.2.4 Homodeanol betaine [HDB] ............................................................................ 39 3.2.5 Homoglycerol betaine [HGB].......................................................................... 40 3.2.6 Diethanol homobetaine [DEHB]...................................................................... 40 3.2.7 Triethanol homobetaine [TEHB] ..................................................................... 41 3.2.8 Hydroxypropyl homobetaine [HPHB]............................................................. 42 3.2.9 Dimethylthetin [DMT]..................................................................................... 42 3.2.10 Cyclic Betaines .............................................................................................. 43 3.2.11 Sulfobetaines.................................................................................................. 45 3.2.12 Cyclic-Sulfo betaines..................................................................................... 48 3.3 RESULTS AND DISCUSSION............................................................................. 52 3.4 CONCLUSIONS..................................................................................................... 55 3.5 REFERENCES ....................................................................................................... 55 CHAPTER 4 -PROTEIN STABILIZATION USING SYNTHETIC COMPENSATORY SOLUTES..................................................................................... 57 4.1 Stabilization of Catalytic Activity of Different Model Enzymes ........................... 57 4.1.1 Introduction...................................................................................................... 57 4.1.2 Materials and Methods..................................................................................... 60 4.1.3 Results.............................................................................................................. 63 4.1.4 Discusssion ...................................................................................................... 75 4.2 Assessment of Stabilizing Effect of Compensatory Solutes on the Unfolding of Chymotrypsin Using an HPLC system......................................................................... 81 4.2.1 Introduction...................................................................................................... 81 4.2.2 Materials and Methods..................................................................................... 83 4.2.3 Results.............................................................................................................. 85 4.2.4 Discussion........................................................................................................ 91 4.3 Study of Effect of Compensatory Solutes on Corticosteroid Binding Globulin Using an Enzyme-Linked Immunosorbent assay ......................................................... 94 4.3.1 Introduction...................................................................................................... 94 4.3.2 Materials and Methods..................................................................................... 95 4.3.3 Results.............................................................................................................. 96 4.3.4 Discussion........................................................................................................ 98 4.4 References............................................................................................................... 99 CHAPTER 5 - MEDICAL APPLICATION OF BETAINE ANALOGUES: ADDITION OF GLYCINE BETAINE OR A SYNTHETIC ANALOGUE TO PERITONEAL DIALYSIS FLUID............................................................................. 103 5.1 INTRODUCTION ................................................................................................ 103 5.1.1 Kidney and its function.................................................................................. 103