MECHANISM–BASED INHIBITORS FOR COPPER AMINE OXIDASES: SYNTHESIS, MECHANISM, AND ENZYMOLOGY By BO ZHONG Submitted in partial fulfillment of the requirements For the degree of Doctor of Philosophy Thesis Adviser: Dr. Lawrence M. Sayre, Dr Irene Lee Department of Chemistry CASE WESTERN RESERVE UNIVERSITY January, 2010 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of ______________________________________________________ candidate for the ________________________________degree *. (signed)_______________________________________________ (chair of the committee) ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ (date) _______________________ *We also certify that written approval has been obtained for any proprietary material contained therein. Table of Contents Table of Contents ............................................................................................................... I List of Tables .................................................................................................................... V List of Figures .................................................................................................................. VI List of Schemes ................................................................................................................ XI Acknowledgements ...................................................................................................... XIV List of Abbreviations ..................................................................................................... XV Abstract ........................................................................................................................ XVII Chapter 1. Introduction ................................................................................................... 1 1.1 General introduction to copper amine oxidases (CAOs) .......................................... 2 1.2 Mechanism for CAOs-catalyzed turnover................................................................. 3 1.3 General introduction to mechanism-based inhibitors................................................ 8 1.4 Kinetics for mechanism-based inhibition .................................................................. 9 1.5 References ............................................................................................................... 13 Chapter 2. Inactivation of two different copper amine oxidases by allenic amines 15 2.1 Introduction ............................................................................................................. 16 I 2.2 Results ..................................................................................................................... 18 2.2.1 Synthesis............................................................................................................... 18 2.2.2 Inactivation of bovine plasma amine oxidase by allenic amines ........................ 20 2.2.3 Inactivation of Arthrobacter globiformis amine oxidase (AGAO) by allenic amines............................................................................................................................ 27 2.2.4 Structure of AGAO-inhibitor rac-2 complex ....................................................... 29 2.3 Discussion and conclusion ...................................................................................... 30 2.4 Experimental procedures ......................................................................................... 38 2.5 References ............................................................................................................... 47 Chapter 3. Inactivation of bovine plasma amine oxidase (BPAO) by acetic acid-3- amino-propenyl ester ...................................................................................................... 49 3.1 Introduction ............................................................................................................. 50 3.2 Results and discussion ............................................................................................. 53 3.2.1 Synthesis............................................................................................................... 53 3.2.2 Enzyme assay of inhibition of BPAO by (E)- and (Z)-acetic acid-3-amino- propenyl ester (1 and 2) ................................................................................................. 54 3.2.3 Metabolism of acetic acid (E)-3-amino-propenyl ester (1) by BPAO ................. 56 3.2.4 Enzyme assay of inhibition of BPAO by aldehyde product 3-(acetyloxy)-(E)-2- propenal and its hydrolytic product MA ....................................................................... 59 II 3.2.5 Evaluation of 3-amino-propionaldehyde and its analogs as inhibitors and substrates of BPAO ....................................................................................................... 60 3.3 Mechanistic conclusion ........................................................................................... 72 3.4 Experimental procedures ......................................................................................... 76 3.5 References ............................................................................................................... 85 Chapter 4. Kinetic analysis of propargylamine derivatives as both substrates and inhibitors for bovine plasma amine oxidase (BPAO) and Arthrobacter globiformis amine oxidase (AGAO) ................................................................................................... 87 4.1 Introduction ............................................................................................................. 88 4.2 Results and discussion ............................................................................................. 92 4.2.1 Steady-state kinetic parameters for AGAO/BPAO-catalyzed oxidation of propargylamine derivatives ........................................................................................... 92 4.2.2 Kinetic parameters for inactivation of AGAO/BPAO by propargylamine derivatives ..................................................................................................................... 94 4.2.3 Inactivation of substrate-reduced BPAO by α, β-unsaturated aldehydes ............. 99 4.2.4 Anaerobic inactivation of BPAO by propargylamine derivatives ..................... 102 4.3 Conclusions ........................................................................................................... 104 4.4 Experimental procedures ....................................................................................... 105 4.5 References ............................................................................................................. 111 III Chapter 5. Investigate structural basis of inactivation of bovine plasma amine oxidase (BPAO) by 5-aryl-3-pentynamines ................................................................ 114 5.1 Introduction ........................................................................................................... 115 5.2 Results ................................................................................................................... 116 5.2.1 Synthesis............................................................................................................. 116 5.2.2 Irreversible inactivation of BPAO by 5-aryl-3-pentynamines (1-3) .................. 117 5.2.3 Mechanism of inactivation of BPAO by 5-aryl-3-pentynamines....................... 121 5.2.3.1 Covalent adduct between BPAO and 5-aryl-3-pentynamines......................... 121 5.2.3.2 Immunochemical analysis of inactivation of enzyme BPAO by inhibitor 3 .. 126 5.2.3.3 Attempt to resolve the structural basis of inactivation of BPAO by inhibitor 2 using enzymatic digestion and LC/MS ....................................................................... 127 5.3 Discussion ............................................................................................................. 130 5. 4 Conclusions .......................................................................................................... 134 5. 5 Experimental procedures ...................................................................................... 135 5.6 References ............................................................................................................. 144 Appendix ........................................................................................................................ 146 Bibliography .................................................................................................................. 152 IV List of Tables Chapter 2 ......................................................................................................................... 15 Table 2.1 The inhibitory potencies and reversibility of inhibition on BPAO by allene compounds ........................................................................................................................ 21 Table 2.2 The inhibitory potencies and reversibility of inhibition on AGAO by allene compounds ........................................................................................................................ 28 Chapter 4 ........................................................................................................................