DESIGN, SYNTHESIS, AND EVALUATION OF IRREVERSIBLE PEPTIDYL INHIBITORS FOR CLAN CA AND CLAN CD CYSTEINE PROTEASES A Dissertation Presented to The Academic Faculty By Marion Gabriele Götz In Partial Fulfillment Of the Requirements for the Degree Doctor of Philosophy in Chemistry Georgia Institute of Technology May 2004 Copyright © Marion Götz DESIGN, SYNTHESIS, AND EVALUATION OF IRREVERSIBLE PEPTIDYL INHIBITORS FOR CLAN CA AND CLAN CD CYSTEINE PROTEASES Approved by Dr. James C. Powers (Thesis Advisor) Dr. Donald Doyle Dr. Nicholas Hud Dr. Niren Murthy Dr. Suzanne Shuker Date Approved January 16, 2004 DEDICATION To my parents, Dagmar and Helmut Götz. To my grandparents, Margot and Johann Walch. To Colin Heyes, my future husband. iii ACKNOWLEDGEMENTS I would like to express my sincerest gratitude to my advisor, Professor James C. Powers. I have the highest respect for him as a scientist and as a person. His kindness, his incredible patience, his humor, and what is most, his integrity have helped me grow both professionally and personally. He has been a remarkable teacher to me with his never-ending supply of advice, may it be on the most difficult research problems or on how to dress for a wintry hike. I will remain forever grateful for being accepted into his group and for the preparation he has provided me with for my future scientific endeavors. I would like to thank my Ph.D. thesis committee members Professor Donald Doyle, Professor Nicholas Hud, Professor Niren Murthy, and Professor Suzy Shuker for their insights and interest in my research, Dr. Leslie Gelbaum and Dave Bostwick for their assistance with spectroscopy, the School of Chemistry and Biochemistry, and my collaborators Conor Caffrey and Professor Jim McKerrow at the University of California, San Francisco. My wonderful group members, past and present, have brightened my time as a graduate student. Özlem Doğan Ekici, Sylvia Shadinger, Amy Campbell, Brian Rukamp, Juliana Asgian, Karen Ellis, Karrie Rukamp, and Temam Juhar have been my family for four years with their friendship and support. I thank Zhao Zhao Li for sharing her insights in organic chemistry with me and for her guidance. I am grateful to Bryan White, Samer Salim, Julie Ha, Liang Zhang, Josh Sasine, Beth Brewster, Veronica DeSilva, and my friends in Germany, Tanja Kuklinsky and Peter Tranitz, for spending time with me on this rollercoaster ride. Especially my iv friendship with Özlem Doğan Ekici will always remain with me. She has been the kindest friend through times of trouble without judgment or pride. My caring sister, Andrea Orr, has always believed in me. Even though our lives are very different, she always manages to put herself in my shoes and to find words of wisdom for any situation presented by life. I will always be grateful to my dear grandparents, Margot and Johann Walch, who have been like parents to me. They have played an important role in my education and growth as an individual and always treated me with understanding and warmth. I thank my loving parents, Dagmar and Helmut Götz, for believing in me and showing their continuous support across the Atlantic Ocean. They have given me direction, and taught me how to respect and appreciate life. Finally, I would like to thank my future husband, Colin Heyes, who has brought meaning and balance to my life, and always placed my well-being before his. Thank you for teaching me how to see the good in people and how to focus on what really matters. v TABLE OF CONTENTS Dedication………………………………………………………………….….iii Acknowledgements……………………………………………………………iv List of Tables………………………………………………………………….ix List of Figures……………………………………………………………….…x Abbreviations…………………………………………………………………xii Summary…………………………………………………………………….xvii Chapter 1: Background………………………………………………………...1 Proteases………………………………………………………………..1 Substrate Specificity……………………………………………….…...4 Inhibitor Design…………………………………………………….…..8 Irreversible Kinetics…………………………………………………….9 References……………………………………………………………...13 Chapter 2: Design and Evaluation of Inhibitors for Dipeptidyl Peptidase I…..15 Introduction………………………………………………………….…15 Chemistry………………………………………………………………19 Results and Discussion………………………………………………...21 Summary and Perspectives…………………………………………….29 Experimental…………………………………………………………...30 References……………………………………………………………..36 Chapter 3: Peptidyl Allyl Sulfones: A New Class of Inhibitors for Clan CA Proteases……………………………………………..39 vi Introduction…………………………………………………………….39 Inhibitor Design………………………………………………………..42 Results and Discussion………………………………………………...43 Conclusions…………………………………………………………….50 Experimental…………………………………………………………...51 References……………………………………………………………...60 Chapter 4: Design, Synthesis, and Evaluation of Potent and Selective Inhibitors of S. mansoni Legumain………….……………………65 Introduction……………………………………………………………65 Chapter 4.1: Aza-Peptide Epoxides: Potent and Selective Inhibitors of S. mansoni Legumain….…………………………..69 Inhibitor Design………...……………………………………………...69 Chemistry……………………………………………………………....70 Results and Discussion………………………………………………...74 Summary………………………………………………………………78 Chapter 4.2: Aza-Peptide Michael Acceptors: Potent and Selective Inhibitors of S. mansoni Legumain……………………………...80 Inhibitor Design……………………………………………………….80 Chemistry………………………………………………….…………..82 Results and Discussion………………………………………………..87 Summary………………………………………………………………96 Conclusions and Perspectives…………………………………………98 Experimental………………………………………………………….100 vii References…………………………………………………………….144 Vita……………………………………………………………………148 viii LIST OF TABLES Table 2.1. Inhibition of Intracellular DPPI in RNK-16 Cells by Dipeptide Vinyl Sulfones……..…………………………...………………...18 Table 2.2. Inhibition of Bovine Spleen DPPI by Dipeptide Vinyl Sulfones….…...……23 Table 2.3. Inhibition of Various Cysteine Proteases by Dipeptide Vinyl Sulfones……..25 Table 2.4. Inhibition of DPPI by Dipeptidyl Vinyl Sulfones with a Charged P1 Amino Acid……………………………………………………..26 Table 2.6. Stability of Dipeptide Acyloxymethyl Ketones and Vinyl Sulfones………...28 Table 3.1. Irreversible Inhibitors for Calpain…………………….………………..……40 Table 3.2. Inhibition of Various Cysteine Proteases by Dipeptidyl Allyl Sulfones…….48 Table 4.1.1. Inhibition of Legumains by Aza-Peptide Epoxides………………………..75 Table 4.2.1. S. mansoni Legumain Inhibition with Aza-Peptide Michael Acceptors…...88 Table 4.2.3. Summary of Aza-Peptide Epoxides and Aza-Peptide Michael Acceptors...98 ix LIST OF FIGURES Figure 1.1. Nomenclature for Protease Subsite Specificity……………………..………2 Figure 1.2. Classification of Cysteine Proteases into Families and Clans……..……..…3 Figure 2.1. Processing of Progranzymes by DPPI to form Active Granzymes...………16 Figure 2.2. General Preparation of the Dipeptidyl Vinyl Sulfone…...…………………20 Figure 2.3. DPPI Mechanism of Inhibition by Dipeptidyl Vinyl Sulfones...…………..27 Figure 3.1. Structure of the Cysteine Protease Inhibitor E-64………...……………..…41 Figure 3.2. Hypothesis for a New Functional Group…...………………………………43 Figure 3.3. Preparation of Cbz-protected Vinyl Sulfones...…………………………….44 Figure 3.4. Possible Constitutional Isomers of the Vinyl Sulfone Starting Material…..45 Figure 3.5. Preparation of the Peptidyl Oxazoline...……………………………………46 Figure 3.6. Isomerization of Dipeptidyl Vinyl Sulfones to Allyl Sulfones...…………..47 Figure 3.7. Proposed Enzyme Mechanisms of Inhibition...…………………………….50 Figure 4.1. Schistosome Life-Cycle……...……………………………………………..66 Figure 4.1. Previously Reported Legumain Inhibitors………...………………………..68 Figure 4.1.1. Design of Aza-Peptide Epoxide Inhibitors………….……………………..70 Figure 4.1.2. Synthesis of Enantiomerically Pure Epoxysuccinate Esters….…………...70 Figure 4.1.3. Synthesis of the Epoxide Moiety…………………………………………..73 Figure 4.1.4. Synthesis of Aza-Peptide Epoxide Inhibitors……………………………...74 Figure 4.2.1. Aza-Peptidyl Michael Acceptor Design Derived from Aza-Peptidyl Epoxides……………………………………………………………………81 Figure 4.2.2. Preparation of Fumarate Precursors……………………………………….83 x Figure 4.2.3. Coupling of Fumarate and Acryloyl Precursors to Peptidyl Hydrazides…85 Figure 4.2.4. Preparation of a Biotinylated Michael Acceptor Inhibitor………………..86 Figure 4.2.5. Mechanism of Thioalkylation of Aza-Peptidyl Michael Acceptor Inhibitors with DTT………………………………………………………..93 Figure 4.2.6. Proposed Future Modification of the Aza-Peptide Michael Acceptor……99 xi ABBREVIATIONS AA amino acid residue AAsn aza-asparagine AAsp aza-aspartic acid Ac acetyl Adp adipic acid Ala alanine AMC 7-amino-4-methylcoumarin Arg arginine Asn asparagine AS allyl sulfone Asp aspartic acid Boc tert-butoxycarbonyl Brij polyoxyethylenelaurylether Bzl benzyl, CH2Ph 13C NMR carbon nuclear magnetic resonance Cbz benzyloxycarbonyl CHAPS 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate Cys cysteine DEAD diethyl azodicarboxylate DCC 1,3-dicyclohexylcarbodiimide DCU 1,3-dicyclohexylurea xii DMAP 4-dimethylaminopyridine DMF N,N-dimethylformamide DMSO dimethylsulfoxide DMSO-d6 dimthylsulfoxide-d6 deuterated DPPI dipeptidyl peptidase I DTT dithiothreitol E-64 L-trans-epoxysuccinyl-leucylamido(4-guanidino)butane EDC 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride EDTA ethylenediaminetetraacetic acid EP epoxide Eps epoxysuccinyl ESI-MS electrospray ionization mass spectrometry Et3N triethylamine EtOAc ethyl acetate FAB fast atom bombardment Fla fluoresceine amine FMK fluoromethyl ketone FT-IR Fourier transform infrared spectroscopy Fum fumarate Gln glutamine Glu glutamic acid Gly glycine HEPES N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid xiii His histidine HOBt N-hydroxybenzotriazole