University of Alberta Chemical Synthesis and Biological Evaluation of Unusual Sulfur-Containing Peptides and Efforts Toward Chemical Synthesis of Crocin by Zedu Huang A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Chemistry ©Zedu Huang Fall, 2012 Edmonton, Alberta Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission. Abstract In order to minimize side reactions when preparing peptides containing a C- terminal cysteine residue, a method of protection of the carboxyl of the C-terminal cysteine as a cyclic ortho ester was developed. N-Fmoc-S-StBu cysteine cyclic ortho esters 31 (L) and 32 (D) were synthesized with excellent enantiomeric purity. This moiety was incorporated onto a solid support via a disulfide linkage, and model tripeptides (Gly- Phe-Cys) and the analgesic peptide, crotalphine, were synthesized with excellent diastereomeric purity. In the second chapter of this thesis, a structure-activity relationship (SAR) study of the crotalphine was developed. Eleven analogs were prepared to elucidate the essential structural features for the analgesic activity and identify an active analog with increased metabolic stability. The biological evaluation of these analogs is ongoing in the laboratory of our collaborators. The Vederas group discovered a new family of Class II bacteriocins, such as thuricin-β, featuring sulfur to α-carbon bridges. In the third chapter, efforts toward asymmetric synthesis of a bis-amino acid containing a thioether bridge were attempted. This bis-amino acid could be used as a standard to confirm the stereochemistry at the α- carbon of the modified residues using chiral GC-MS. Bis-amino acid derivative (126) was synthesized as a single diastereomer using chiral tricycloiminolactone 129 and efforts toward the removal of the auxiliary are ongoing. In the second part of the third chapter, a SAR study of thuricin-β was performed. The cysteine residues of thuricin-β were replaced by glycine, serine or alanine. All of the synthetic analogs were inactive against Bacillus firmus. In the future, cyclic analog 163, will be synthesized and evaluated for antimicrobial activity. Crocin is a major component of saffron and shows activity against various diseases. The last project of this thesis was towards chemical synthesis and biological evaluation of crocin and its analogs. The polyene acid 198 was synthesized in 10 steps from geranyl acetate. Attempts to attach this acid to β-D-glucose were unsuccessful. In the future, polyene ester 214 will be coupled to β-D-glucose or β-gentibiose. When the glycosylated products are obtained, they will be subjected to cross metathesis conditions to form crocin and its analogs. Acknowledgements I would like to thank my research supervisor, Professor John C. Vederas for his constant support, encouragement and understanding. As an excellent teacher and mentor, he has provided a stimulating learning environment. I have been inspired by his enthusiastic attitude towards science and his commitment to promoting independent thought. The time spent in his group has undoubtedly prepared me for the journey ahead. I would like to thank Drs. Jennifer Chaytor and David Dietrich for their careful proofreading of this manuscript. I am thankful to all the past and present members of the Vederas group who have contributed to a positive and enjoyable work environment. I would especially like to thank Dr. Darren Derksen for his collaboration in the cyclic ortho ester and crotalphine projects, Dr. David Dietrich for his collaboration in the crocin project, and Christopher Lohans for his collaboration in the tridecaptin A project. I would also like to thank Stephen Cochrane for his work on a collaborative research project not described in this thesis. I would like to thank my collaborators outside the Vederas group, Dr. M. Joanne Lemieux, Dr. Yara Cury, and Dr. Cory Brooks. Their excellent collaboration and support had enormous impacts on my research projects. I also want to thank all of the staff members from the service labs of the Department of Chemistry at the University of Alberta. Dr. Randy Whittal, Dr. Angie Morales-Izquierdo, Jing Zheng, Bela Reiz and Wayne Moffat all provided tremendous help. Lastly, but most importantly, thank you to my parents and sisters for their unconditional love and support. Whenever I felt frustrated or depressed, they were always ready to listen to me and cheer me up. Without these overseas conversations, I could not finish my PhD studies. Thanks to all of you for always believing in me and helping me realize my goals. Table of Contents Chapter 1 : Preparation and Use of Cysteine Cyclic Ortho Esters for Solid-Phase Peptide Synthesis ................................................... 1 1.1 Introduction .................................................................................................... 1 1.1.1 Fmoc-based solid phase peptide synthesis ........................................................ 1 1.1.2 Naturally occurring peptides containing C-terminal cysteine ........................... 6 1.1.3 Fmoc-based SPPS of peptides containing C-terminal cysteine ........................ 7 1.1.3.1 Epimerization and piperidine adduct formation during Fmoc-based SPPS 7 1.1.3.2 Established methods to prevent or reduce undesired side reactions during Fmoc-based SPPS of peptides containing a C-terminal cysteine residue ............. 10 1.1.3.2.1 Use of trityl resin ............................................................................... 10 1.1.3.2.2 Side-chain anchoring strategy ............................................................ 11 1.1.4 Serine cyclic ortho esters ................................................................................. 12 1.2 Project objectives: Fmoc-based SPPS of peptides containing C-terminal cysteine residues without epimerization or piperidine adduct formation by the protection of the carboxyl group as a cyclic ortho ester .................................... 15 1.3 Results and discussion .................................................................................. 17 1.3.1 Synthesis and chiral HPLC analyses of cysteine cyclic ortho esters ............... 17 1.3.2 Synthesis and evaluation of epimerization of model tripeptides ..................... 21 1.3.3 Synthesis and evaluation of epimerization of natural crotalphine (2) and its D- Cys1 diastereomer (53) ............................................................................................. 26 1.3.4 Synthesis and evaluation of epimerization of all-D crotalphine ...................... 33 1.4 Conclusions and future directions ................................................................ 36 Chapter 2 : Structure-Activity Relationship Study of Crotalphine and Efforts Toward Racemic Crystallization of Crotalphine ........ 38 2.1 Introduction .................................................................................................. 38 2.1.1 Crotalphine and pain relief .............................................................................. 38 2.1.2 Structure-activity relationship study of crotalphine ........................................ 40 2.1.3 Racemic crystallization of peptides ................................................................. 44 2.2 Project objectives: Structure-activity relationship study of crotalphine and efforts toward the racemic crystallization of crotalphine ................................... 46 2.3 Results and discussion .................................................................................. 47 2.3.1 Synthesis of crotalphine analogs ...................................................................... 47 2.3.2 Biological evaluation of crotalphine analogs ................................................... 50 2.3.3 Racemic crystallization study of crotalphine ................................................... 50 2.4 Conclusions and future directions ................................................................ 51 Chapter 3 : Study of New Bacteriocins Featuring Sulfur to α- Carbon Bridges .............................................................................. 53 3.1 Introduction .................................................................................................. 53 3.1.1 New members of Class II bacteriocins featuring sulfur to α-carbon bridges . 53 3.1.2 Structural elucidation of peptides containing sulfur to α-carbon bridges ........ 56 3.1.3 Mechanism of formation of sulfur to α-carbon bridges ................................... 57 3.1.4 Chemical synthesis of sulfur to α-carbon bridges ........................................... 60 3.1.5 Structure-activity relationship study of bacteriocins featuring sulfur to α- carbon bridges ........................................................................................................... 60