This document is downloaded from DR‑NTU (https://dr.ntu.edu.sg) Nanyang Technological University, Singapore. Design and synthesis of cysteine‑rich peptides Qiu, Yibo 2014 Qiu, Y. (2016). Design and synthesis of cysteine‑rich peptides. Doctoral thesis, Nanyang Technological University, Singapore. https://hdl.handle.net/10356/62189 https://doi.org/10.32657/10356/62189 Downloaded on 07 Oct 2021 19:28:23 SGT DESIGN AND SYNTHESIS OF CYSTEINE-RICH PEPTIDES Yibo Qiu School of Biological Sciences 2014 DESIGN AND SYNTHESIS OF CYSTEINE-RICH PEPTIDES Yibo Qiu School of Biological Sciences A thesis submitted to the Nanyang Technological University in partial fulfillment of the requirement for the degree of Doctor of Philosophy 2014 Acknowledgements First of all, I would like to express my sincere gratitude to my supervisor Professor Ding Xiang Liu and my co-supervisor Professor James P. Tam for their great ideas and patient guidance throughout my PhD period. In this four-year study, they kept inspiring me with their immense knowledge, great motivation and trained me with a high standard. Their education makes the process an invaluable experience for me. It is my great pleasure to be one of their students and their training will always drive me to become better in my lifetime. My appreciation also goes to Professor Chuan-Fa Liu for his encouragements and technical support during this project. He always offers me practical instructions and professional advices when I need help. I also would like to thank Professor Siu Kwan Sze and Professor Kathy Qian Luo for their help on the mass spectrometry and bioassays. I am grateful to Dr. Xinya Hemu, Dr. Clarence T. T. Wong, Dr. Misako Taichi and Dr. Kien Truc Giang Nguyen. They are friends, mentors and partners. Their intelligence and attitude inspired me a lot and I enjoyed working with them. In addition, I would like to thank my FYP and URECA students for their input in this project. I would like to thank Dr. Ying Liao, Dr. Adrian Guan Ji How, Dr. Quoc Thuc Phuong Nguyen, Dr. Renliang Yang and other SBS friends who helped me during my experiment. Everyone is nice and helpful to me, which makes my stay here a happy and meaningful experience. Last, I would like to thank my parents and my family for their constant support and understanding. i List of Conference Presentations 12th Chinese International Peptide Symposium, Shenyang China, 2012 Oral Presentation, Young Peptide Scientist Award ii List of Publications Qiu, Y., Wong, C. T. T., and Tam, J. P. (2012) Acid-catalyzed N-S acyl shift for preparing peptide thioesters using Fmoc chemistry. in Proceedings of the 12th Chinese International Peptide Symposium. Peptides, Chemistry and Biology. (Liu, K., Cheng, M., and Meng, Q. eds.), Chemical Industry Press, Shenyang, China 1, 22-24 Tam, J. P., Wong, C. T. T., and Qiu, Y. (2012) Engineering of proteinaceous orally active bradykinin peptide antagonists. in Proceedings of the 32nd European Peptide Symposium. Peptide 2012. (Kokotos, G., Constantinou-Kokotou, V., and Matsoukas, J. eds.), John Wiley and Sons, Athens, Greece 18, 130-131 Taichi, M., Hemu, X., Qiu, Y., and Tam, J. P. (2013) A thioethylalkylamido (TEA) thioester surrogate in the synthesis of a cyclic peptide via a tandem acyl shift. Org. Lett. 15, 2620-2623 Hemu, X., Taichi, M., Qiu, Y., Liu, D. X., and Tam, J. P. (2013) Biomimetic synthesis of cyclic peptides using novel thioester surrogates. Pept. Sci. 100, 492-501 Qiu, Y., Hemu, X., Liu, D. X., and Tam, J. P. (2014) Selective bi-directional amide bond cleavage of N-methylcysteinyl peptide. Eur. J. Org. Chem. 2014, 4370-4380 Hemu, X., Qiu, Y., and Tam, J. P. (2014) Peptide macrocyclization through amide-to- amide transpeptidation. Tetrahedron 70, 7707-7713 Nguyen, G. K. T., Wang, S., Qiu, Y., Hemu, X., Lian, Y., and Tam, J. P. (2014) Butelase 1 is an Asx-specific ligase enabling peptide macrocyclization and synthesis. Nat. Chem. Biol. 10, 732-738 iii Table of Contents Acknowledgements ............................................................................................. i List of Conference Presentations ......................................................................ii List of Publications .......................................................................................... iii Table of Contents .............................................................................................. iv List of Figures ................................................................................................. viii List of Tables .................................................................................................. xiii Abbreviations .................................................................................................. xiv Abstract ...........................................................................................................xvii Chapter 1 Introduction...................................................................................... 1 1. Cysteine-rich peptides ...................................................................................... 1 2. Circular peptides .............................................................................................. 7 3. Cyclotides ........................................................................................................ 9 3.1 Discovery of cyclotides ............................................................................. 9 3.2 Structure and classification of cyclotides .................................................. 9 3.3 Biosynthesis of cyclotides ....................................................................... 12 3.4 Biological activities of cyclotides ........................................................... 18 4. CRPs as peptide biologics .............................................................................. 20 5. Amide-to-amide chemoenzymatic synthesis of cyclic peptides .................... 31 5.1 Intein-mediated synthesis of cyclic peptides ........................................... 31 5.2 Sortase-mediated synthesis of cyclic peptides ........................................ 35 6. Amide-to-amide chemical synthesis of cyclic peptides ................................. 37 6.1 Solid-phase peptide synthesis ................................................................. 38 6.2 Chemical ligation .................................................................................... 40 6.3 The thia zip assisted cyclization .............................................................. 48 6.4 Preparation of thioesters for cysteine thioester ligation .......................... 50 7. Oxidative folding of CRPs ............................................................................. 60 8. Aim ................................................................................................................ 66 Chapter 2 Material and methods.................................................................... 67 1. Chemical Synthesis of Cyclic CRPs .............................................................. 67 1.1 General information ................................................................................ 67 1.2 MeCys-mediated Nα-thioester formation in aqueous conditions ............ 68 iv 1.3 MeCys-mediated synthesis of cyclic peptide SFTI-1 ............................ 70 1.4 MeCys-mediated Cα-thioester formation in strongly acidic conditions .. 71 1.5 β-thiolactone formation ........................................................................... 76 1.6 TEBA-mediated Nα-thioester formation in aqueous conditions ............ 77 1.7 TEBA-mediated synthesis of cyclic peptide SFTI-1 ............................. 78 1.8 Synthesis of kalata B1 via a hydrazide linker ......................................... 79 2. Oxidative folding of CRPs ............................................................................. 81 2.1 Oxidative folding of kB1 in organic conditions ...................................... 81 2.2 1H NMR characterization ........................................................................ 81 2.3 Synthesis of ET-1 .................................................................................... 81 2.4 Oxidative folding of ET-1 in organic solvent. ........................................ 82 3. Butelase-mediated synthesis of cyclic peptides ............................................. 82 3.1 Synthesis of peptide library XXXGIR-OH ............................................. 82 3.2 Optimization of butelase-mediated ligation reaction .............................. 83 3.3 Ligation of KALVINHV and XXXGIR mediated by butelase 1 ............ 83 3.4 Synthesis of C-terminal substrate library KALVXNHV ........................ 84 3.5 Butelase 1 mediated ligation of KALVXXHV and HIGGIR ................. 84 4. Design and engineering of cysteine-rich peptides ......................................... 85 4.1 Synthesis of SFBK, BKSF ...................................................................... 85 4.2 Synthesis of SFBK-L, BKSF-L, DALK ................................................. 86 4.3 Heat and pH stability assay ..................................................................... 87 4.4 Pepsin and trypsin stability assay ............................................................ 88 4.5 Serum stability assay ............................................................................... 88 4.6 Trypsin inhibition assay .......................................................................... 88 4.7 Cytotoxicity ............................................................................................