DESIGN, SYNTHESIS AND BIOLOGICAL STUDIES OF COUMARIN-BASED PROBES AND TETRAHYDROLIPSTATIN ANALOGS NGAI MUN HONG (MSc., Universiti Teknologi Malaysia) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF CHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2010 To my parents i ACKNOWLEDGEMENTS I would like to express my sincere gratitude and appreciation to my research advisor, Assistant Prof Dr Martin J Lear, for his guidance, support, encouragement and patience throughout the completion of this work Grateful acknowledgements go to Assoc Prof Dr Yao Shao Qin for his advice and guidance My sincere appreciation also extends to Mr Yang Pengyu and Mr Liu Kai from Prof Yao lab for performing the biological assays for THL libraries I wish to extend my gratitude to Assistant Prof Dr Paul MacAry, Ms Song Zhenying from the Department of Microbiology and Dr Song Hongyan for their help in carrying out the bioimaging experiments I would like to thank Ms Cheong Wei Fun and Mr Shareef Mohideen Ismail for their assistance with HRMS I wish to express my indebtedness to all current and former members of the Lear group for their support and friendship I am particularly thankful to Dr Bastien Reux and Mr Oliver Simon for expanding my world view and vocabulary Finally, I wish to thank lab technicians and others who have provided assistance at various occasions ii TABLE OF CONTENTS CHAPTER TITLE PAGE TITLE PAGE DEDICATION i ACKNOWLEDGEMENTS ii TABLE OF CONTENTS iii SUMMARY vi LIST OF TABLES ix LIST OF SCHEMES x LIST OF FIGURES xii LIST OF ABBREVIATIONS xiv LIST OF APPENDICES xvii LIST OF PUBLICATIONS xviii Design and synthesis of coumarin-based fluorescent probe INTRODUCTION References RESULTS AND DISCUSSION 2.1 2.2 2.3 2.4 Development of a coumarin-based fluorescent probe Practical synthesis of N-alkylated maleimides and maleimidocarboxylic NHSesters Synthesis of coumarin probe Proof-of-Principle studies with coumarin probe 2.4.1 Labelling RGDC with coumarin probe 2.20 11 15 18 18 iii 2.5 2.4.2 Antibody-labelling, cell-sorting and cell culture study Conclusion 19 24 References EXPERIMENTAL 29 References 25 42 Synthesis and biological studies of tetrahydrolipstatin analogs INTRODUCTION 43 4.1 43 4.2 4.3 Isolation and biological activities of (-)tetrahydrolipstatin Previous syntheses of (-)-tetrahydrolipstatin 44 4.4 Studies of tetrahydrolipstatin derivatives as fatty acid synthase inhibitors Summary of tetrahydrolipstatin syntheses 61 64 4.5 Objectives 65 References 66 RESULTS AND DISCUSSION 69 5.1 69 Development of a solid-supported strategy to THL analogues 5.1.1 Mechanism of tandem Mukiyama aldol-lactonization (TMAL) 5.2 Synthesis of THL-L analogues 81 5.3 Synthesis of THL-R analogues 85 5.4 Synthesis of THL-T analogues 89 5.5 Biological studies of THL probes 91 5.5.1 78 91 5.5.2 Effects on cell proliferation, phosphorylation of eIf2 and activation of caspase-8 In situ and in vitro proteome profiling 5.5.3 Target identification and validation 95 93 5.6 Conclusion 96 References 97 iv EXPERIMENTAL 101 RESULTS AND DISCUSSION 149 7.1 Introduction to click chemistry 149 7.1.1 Applications of click chemistry 7.2 Synthesis of THL analogs by click chemistry 7.2.1 Mechanism allylation 7.3 Synthesis of azides of Keck asymmetric 151 152 156 158 7.4 Biological evaluation of THL analogs 7.5 Conclusion 162 References 160 163 EXPERIMENTAL 167 References 190 APPENDICES 191 v SUMMARY A coumarin-based fluorescent probe containing a thiol-reacting maleimide group was synthesized A cysteine containing tetrapeptide Arg-Gly-Asp-Cys was successfully labelled with the coumarin probe In addition, an antibody was labelled with the coumarin probe via the selenol-promoted reduction of the native disulfide bond Besides that, one-pot synthesis of maleimido-carboxylic N-hydroxysuccinimide (NHS) esters were accomplished by cyclized maleamic acids with NHS and DCC.……………………………………………………………… O O N H N O fluorophore O O H N HS Biomolecule N O O O linker activating unit Tetrahydrolipstatin (THL) is an FDA-approved anti-obesity drug that selectively inhibits gastrointestinal lipases and fatty acid synthases (FAS) Our study focuses on the synthesis of alkyne-modified THL analogs for identification of new cellular targets Terminal alkyne group was introduced at the left or right side chains, and also at the amino ester group of THL The β-lactone moiety of THL analogs was synthesized by ZnCl2-mediated tandem Mukaiyama aldol-lactonization (TMAL) The vi γ-hydroxy group was introduced via Keck allylation or resolution of the homoallylic alcohol as its (R)-O-acetylmendelic ester OHCHN OHCHN O O O R Mitsunobu O O O O O C6 H 13 C H13 TMAL Keck asymmetric allylation or resolution THL Three THL probes, THL-L, THL-R and THL-T were tested for potential biological activities All three compounds showed antiproliferation activity against HepG2 cells Besides the known FAS, eight new proteins (GAPDH, β-tubulin, Hsp90AB1, ANXA2, RPL7a, RPL14, RPS9 and an unnamed protein) were identified through activity-based proteome profiling approach R O O R NHCHO O NHCHO O O O O O 8 THL-L analogs THL-R analogs H N O O O O O THL-T Another approach for the synthesis of THL analogs was developed by introducing diversity by click chemistry The β-lactone was reacted with eighteen aliphatic and aromatic azides with different electronic properties under click reaction conditions to give THL dialkyne analogs after desilylation Four analogs (7.22k/m/r/s) vii were identified to possess similar anti-proliferative activity against HepG2 cells as compared to THL OHCHN O O O O R N N N structural diversity THL dialkyne analogs NHCHO MeO O S O NH O O NHCHO O O S NH O O O O 7.22m 7.22k NHCHO NHCHO O O Br O N N N N N N O O O O O O2 N N N N 7.22r O O N N N 7.22s viii LIST OF TABLES TABLE NO TITLE PAGE 1.1 Optical properties of Keio-fluors 2.1 13 5.1 Convenient synthesis of N-alkylated maleimides One-pot preparation of maleimido-carboxylic NHS-esters H, 13C, COSY and HMBC data of fluorescence probe 2.20 Heathcock-Evans anti-aldol reactions 5.2 Attempted TBDPS deprotection 78 5.3 Synthesis of tetrahydrolipstatin derivatives 84 5.4 Synthesis of tetrahydrolipstatin derivatives (THL-R) Synthesis of tetrahydrolipstatin derivatives 87 96 7.1 Proteins identified by pull down and mass spectrometry Optimization of click chemistry 154 7.2 Click chemistry 155 7.3 Synthesis of azides 158 2.2 2.3 5.5 5.6 14 17 74 88 ix l H NMR (500 MHz, CDCl3): δ 8.15 (s, 1H), 7.81 (s, 1H), 7.61 (s, 1H), 7.37 (d, J = 9.5 Hz, 2H), 6.84 (d, J = 9.5 Hz, 2H), 5.82 (d, J = 7.6 Hz, 1H), 5.18 (d, J = 16.4 Hz, 1H), 5.11 (d, J = 16.4 Hz, 1H), 4.52 (m, 1H), 4.29 (m, 1H), 4.00 (q, J = 6.9 Hz, 2H), 3.24 (dt, J = 7.6, 3.2 Hz, 1H), 2.85 (m, 2H), 2.05-2.21 (m, 6H), 1.96 (t, J = 2.6 Hz, 1H), 1.57-1.67 (m, 8H), 1.39 (t, J = 6.9 Hz, 3H), 0.95 (t-like, J = 7.0 Hz, 6H) ESI-MS (m/z) calcd for C32H43N5O7 [M + Na]+ 632.3, found 632.2 HRMS (ESI) (m/z) calcd for C32H43N5O7 [M - H]- 608.3083, found 608.3075 7.21i OHCHN O O O O N N N HN O MeO l H NMR (500 MHz, CDCl3): δ 8.16 (s, 1H), 8.03 (br s, 1H), 7.41 (d, J = 8.8 Hz, 2H), 6.85 (d, J = 8.8 Hz, 2H), 5.91 (br s, 1H), 5.20 (br s, 2H), 5.07 (br s, 1H), 4,52 (br s, 1H), 4.30 (br s, 1H), 3.78 (s, 3H), 3.25 (br s, 1H), 2.86 (br s, 2H), 2.21 (br s, 2H), 2.09 (br s, 2H), 1.96 (t, J = 2.6 Hz, 1H), 1.54-1.64 (m, 24H), 0.95 (t, J = 6.3 Hz, 3H), 0.94 (t, J = 6.3 Hz, 3H) ESI-MS (m/z) calcd for C31H41N5O7 [M + Na]+ 618.3, found 618.2 HRMS (ESI) (m/z) calcd for C31H41N5O7 [M - H]- 594.2927, found 594.2921 176 7.21j OHCHN O O O O N N N HN O l H NMR (500 MHz, CDCl3): δ 8.43 (br s, 1H), 8.12 (s, 1H), 7.92 (d, J = 7.6 Hz, 1H), 7.86 (d, J = 7.6 Hz, 1H), 7.70-7.74 (m, 2H), 7.46-7.55 (m, 3H), 5.63 (d, J = 6.9 Hz, 1H), 5.34 (d, J = 16.4 Hz, 1H), 5.29 (d, J = 16.4 Hz), 5.04 (m, 1H), 4.49 (m, 1H), 4.24 (m, 1H), 3.21 (m, 1H), 2.88 (m, 2H), 2.04-2.22 (m, 6H), 1.95 (t, J = 2.5 Hz, 1H), 1.241.27 (m, 6H), 0.92 (m, 6H) ESI-MS (m/z) calcd for C34H41N5O6 [M + Na]+ 638.3, found 638.2 HRMS (ESI) (m/z) calcd for C34H41N5O6 [M - H]- 614.2978, found 614.2972 7.21k OHCHN O O O O N N N MeO l HN S O O H NMR (500 MHz, CDCl3): δ 8.38 (s, 1H), 7.66 (d, J = 8.2 Hz, 1H), 7.59 (s 1H), 7.03 (d, J = 8.2 Hz, 1H), 5.96 (d, J = 7.6 Hz, 1H), 4.79 (br s, 1H), 4.59 (dt, J = 10.2, 4.5 Hz, 1H), 4.46 (m, 1H), 4.30 (dt, J = 13.2, 4.5 Hz, 1H), 4.18-4.22 (m, 1H), 3.20 (dt, J = 7.6, 3.8 Hz, 1H), 3.00 (m, 1H), 2.86 (m, 2H), 2,79 (m, 1H), 2.63 (t, J = 7.6 Hz, 2H), 2.63 (m, 1H), 2.19 (m, 2H), 2.04 (m, 4H), 1.95 (t, J = 2.5 Hz, 1H), 1.63-1.75 (m, 177 10H), 1.50 (m, 6H), 1.26 (m, 6H), 0.99 (d, J = 6.3 Hz, 3H), 0.97 (d, J = 6.3 Hz, 3H) ESI-MS (m/z) calcd for C33H47N5O8S [M + Na]+ 696.3, found 696.2 HRMS (ESI) (m/z) calcd for C33H47N5O8S [M - H]- 672.3066, found 672.3058 7.21l OHCHN O O O O N N N HN S O O l H NMR (500 MHz, CDCl3): δ 8.38 (s, 1H), 7.68 (d, J = 8.8 Hz, 2H), 7.59 (s, 1H), 7.32 (d, J = 8.8 Hz, 2H), 7.03 (d, J = 8.2 Hz, 1H), 5.98 (d, J = 6.9 Hz, 1H), 4.79 (m, 1H), 4.59 (dt, J = 10.1, 3.8 Hz, 1H), 4.46 (ddd, J = 14.5, 10.7, 3.8 Hz, 1H), 4.30 (dt, J = 13.9, 4.5 Hz, 1H), 4.20 (dt, J = 8.8, 3.8 Hz, 1H), 3.21 (dt, J = 8.8, 3.8 Hz, 1H), 2.983.04 (m, 1H), 2.97 (sept, J = 6.9 Hz, 1H), 2.86 (m, 2H), 2.77-2.80 (m, 1H), 2.31 (m, 1H), 2.18 (m, 2H), 2.01-2.13 (m, 4H), 1.60-1.78 (m, 10H), 1.49-1.51 (m, 6H), 1.26 (2xd, J = 6.3 Hz, 6H), 0.99 (d, J = 6.3 Hz, 3H), 0.96 (d, J = 6.3 Hz, 3H) ESI-MS (m/z) calcd for C35H51N5O7S [M + H]+ 686.9, found 686.3 HRMS (ESI) (m/z) calcd for C35H51N5O7S [M - H]- 684.3430, found 684.3421 178 7.21m OHCHN O O O O N N N NH S O O l H NMR (500 MHz, CDCl3): δ 8.38 (s, 1H), 7.66 (d, J = 8.2 Hz, 2H), 7.59 (s, 1H), 7.28 (d, J = 8.2 Hz, 2H), 7.03 (d, J = 8.2 Hz, 1H), 5.96 (d, J = 7.6 Hz, 1H), 4.79 (br s, 1H), 4.59 (dt, J = 10.2, 4.5 Hz, 1H), 4.46 (m, 1H), 4.30 (dt, J = 13.2, 4.5 Hz, 1H), 4.18-4.23 (m, 1H), 3.20 (dt, J = 7.6, 3.8 Hz, 1H), 3.00 (m, 1H), 2.86 (m, 2H), 2.79 (m, 1H), 2.63 (t, J = 7.6 Hz, 2H), 2.32 (m, 2H), 2.04 (m, 4H), 1.95 (t, J = 2.5 Hz, 1H), 1.63-1.76 (m, 10H), 1.50 (m, 6H), 1.26 (br s, 6H), 0.99 (d, J = 6.3 Hz, 3H), 0.97 (d, J = 7.0 Hz, 3H) ESI-MS (m/z) calcd for C35H51N5O7S [M + Na]+ 708.3, found 708.2 HRMS (ESI) (m/z) calcd for C35H51N5O7S [M - H]- 684.3430, found 684.3425 7.21n OHCHN O O O O N N N HN S O O l H NMR (500 MHz, CDCl3): δ 8.41 (s, 1H), 8.36 (s, 1H), 7.94 (d, J = 8.2 Hz, 1H), 7.92 (d, J = 8.2 Hz, 1H), 7.89 (d, J = 8.2 Hz, 1H), 7.70 (dd, J = 8.8, 1.9 Hz, 1H), 7.65 (dd, J = 8.8, 1.9 Hz, 1H), 7.60 (dd, J = 8.8, 1.9 Hz, 1H), 7.57 (s, 1H), 7.03 (d, J = 8.2 179 Hz, 1H), 6.16 (d, J = 7.6 Hz, 1H), 4.76 (m, 1H), 4.62 (m, 1H), 4.45 (ddd, J = 13.9, 10.7, 3.8 Hz, 1H), 4.29 (dt, J = 13.9, 4.4 Hz, 1H), 4.15 (dt, J = 8.8, 3.8 Hz, 1H), 3.19 (dt, J = 7.6, 4.4 Hz, 1H), 3.06 (ddd, J = 15.1, 10.7, 5.1 Hz, 1H), 2.84-2.87 (m, 2H), 2.78-2.80 (m, 1H), 2.24-2.30 (m, 1H), 1.98-2.16 (m, 6H), 1.93 (t, J = 2.5 Hz, 1H), 1.68-1.81 (m, 5H), 1.41-1.45 (m, 5H), 1.01 (d, J = 6.3 Hz, 3H), 0.98 (d, J = 6.3 Hz, 1H) ESI-MS (m/z) calcd for C36H47N5O7S [M + Na]+ 716.3, found 716.2 HRMS (ESI) (m/z) calcd for C36H47N5O7S [M - H]- 693.3117, found 693.3105 7.21o OHCHN O O O O N N N l H NMR (500 MHz, CDCl3): δ 8.25 (s, 1H), 7.35 (s, 1H), 6.04 (d, J = 7.6 Hz, 1H), 5.04-5.07 (m, 2H), 4.65 (dt, J = 8.9, 5.1 Hz, 1H), 4.29-4.34 (m, H-4, 3H), 3.74 (t, J = 6.4 Hz, 1H), 3.24 (dt, J = 7.6, 3.8 Hz, 1H), 2.70-2.83 (m, 2H), 2.13-2.21 (m, 2H), 2.04-2.09 (m, 8H), 1.96 (t, J = 1.9 Hz, 1H), 1.67 (s, 3H), 1.61 (s, 3H), 1.33-1.48 (m, 5H), 1.20-1.24 (m, 5H), 0.97 (d, J = 5.7 Hz, 6H), 0.95 (d, J = 6.9 Hz, 3H) ESI-MS (m/z) calcd for C32H50N4O5 [M + Na]+ 593.4, found 593.2 HRMS (ESI) (m/z) calcd for C32H50N4O5 [M - H]- 569.3702, found 569.3699 180 7.21p OHCHN O O O O N N N l H NMR (500 MHz, CDCl3): δ 8.23 (s, 1H), 7.31 (s, 1H), 5.99 (br s, 1H), 5.41 (t, J = 8.0 Hz, 1H), 5.67 (m, 2H), 4.92 (t, J = 7.6 Hz, 2H), 4.66 (m, 1H), 4.30 (dt, J = 8.7, 4.5 Hz, 1H), 3.23-3.27 (m, 1H), 2.71-2.81 (m, 2H), 2.04-2.21 (m, 10H), 1.96 (t, J = 2.5 Hz, 1H), 1.78 (s, 3H), 1.68 (s, 3H), 1.57 (br s, 8H), 0.97 (d, J = 5.1 Hz, 6H) ESI-MS (m/z) calcd for C32H48N4O5 [M - H]- 567.4, found 567.4 HRMS (ESI) (m/z) calcd for C32H50N4O5 [M - H]- 567.3546, found 567.3548 7.21q OHCHN O O OH O O N N N l H NMR (500 MHz, CDCl3): δ 8.25 (s, 1H), 8.02 (s, 1H), 7.42 (dd, J = 8.3, 1.3 Hz, 1H), 7.28 (t, J = 7.8 Hz, 1H), 7.17 (d, J = 8.3 Hz, 1H), 6.99 (t, J = 7.8 Hz, 1H), 5.96 (br s, 1H), 5.06-5.11 (m, 1H), 4.61 (dt, J = 9.5, 4.4 Hz, 1H), 4.31 (dt, J = 8.2, 4.4 Hz, 1H), 3.25 (dt, J = 7.6, 4.4 Hz, 1H), 2.87-2.95 (m, 2H), 2.12-2.23 (m, 5H), 2.09 (dt, J = 10.8, 3.8 Hz, 1H), 1.95 (t, J = 2.5 Hz, 1H), 1.69-1.83 (m, 4H), 1.53-1.62 (m, 7H), 0.98 (d, J = 6.3 Hz, 3H), 0.97 (d, J = 6.3 Hz, 3H) ESI-MS (m/z) calcd for C28H36N4O6 [M + H]+ 525.3, found 525.2 HRMS (ESI) (m/z) calcd for C28H36N4O6 [M - H]523.2556, found 523.2546 181 7.21r OHCHN O O Br O O N N N l H NMR (500 MHz, CDCl3): δ 8.24 (s, 1H), 7.87 (s, 1H), 7.64 9s, 4H), 5.93 (d, J = 7.6 Hz, 1H), 5.05-5.09 (m, 1H), 4.61 (dt, J = 8.8, 3.8 Hz, 1H), 4.31 (dt, J = 8.8, 4.5 Hz, 1H), 3.25 (dt, J = 7.6, 4.4 Hz, 1H), 2.81-2.92 (m, 2H), 2.07-2.20 (m, 6H), 1.95 (t, J = 2.5 Hz, 1H), 1.70-1.72 (m, 4H), 1.53-1.60 (m, 5H), 0.98 (d, J = 6.3 Hz, 3H), 0.97 (d, J = 6.3 Hz, 3H) ESI-MS (m/z) calcd for C28H35BrN4O5 [M]- 587.2, found 587.2 HRMS (ESI) (m/z) calcd for C28H35BrN4O5 [M - H]- 585.1713, found 585.1713 7.21s OHCHN O O O 2N l O O N N N H NMR (500 MHz, CDCl3): δ 8.40 (d, J = 8.9 Hz, 2H), 8.26 (s, 1H), 8.09 (s, 1H), 8.01 (d, J = 8.9 Hz, 2H), 5.92 (br s, 1H), 5.01-5.06 (m, 1H), 4.55-4.60 (m, 1H), 4.30 (dt, J = 8.2, 3.8 Hz, 1H), 3.24 (dt, J = 7.6, 4.4 Hz, 1H), 2.86-2.96 (m, 2H), 2.12-2.21 (m, 5H), 2.08 (dt, J = 11.4, 3.8 Hz, 1H), 1.95 (t, J = 2.5 Hz, 1H), 1.71-1.82 (m, 4H), 1.52-1.60 (m, 5H), 0.99 (d, J = 6.3 Hz, 3H), 0.97 (d, J = 6.3 Hz, 3H) l3C NMR (125 MHz, CDCl3): δ 712.1, 170.3, 161.2, 125.5, 120.3, 83.8, 74.7, 71.2, 68.8, 57.0, 50.0, 40.4, 39.3, 33.4, 27.9, 27.0, 25.7, 25.0, 22.9, 21.5, 21.0, 18.1 ESI-MS (m/z) calcd for 182 C28H35N5O7 [M - H]- 552.3, found 552.4 HRMS (ESI) (m/z) calcd for C28H35N5O7 [M - H]- 552.2457, found 552.2455 Synthesis of (S)- and (R)-Mosher esters for determination of ee and absolute configuration3 CF3 OMe O F3 C (S) O O DCC, DMAP, CH2 Cl2 , rt 8.1S MeO OMe OH (R) OH CF3 OH O OMe CF3 O O DCC, DMAP, CH2 Cl2 , rt 7.15 8.1R Scheme 8.1: Synthesis of (S)- and (R)-Mosher esters (S)-((R)-oct-1-en-7-yn-4-yl) 3,3,3-trifluoro-2-methoxy-2-phenylpropanoate (8.1S): DCC (21 mg, 0.102 mmol) and DMAP (2 mg, 0.015 mmol) was added to a mixture of (S)-Mosher acid (22 mg, 0.095 mmol) and homoallylic alcohol 7.15 (9 mg, 0.073 mmol) in CH2Cl2 The reaction mixture was stirred at rt overnight The precipitate was filtered through a pad of celite, the filtrate was evaporated under reduced pressure Purification by flash chromatography on SiO2 (95:5, hexanes:EtOAc) to provide the (S)-Mosher ester 8.1S (21.2 mg, 85%) as a colourless oil lH NMR (500 MHz, CDCl3): δ 7.53-7.54 (m, 2H), 7.40-7.41 (m, 3H), 5.61-5.63 (m 1H), 5.25-5.30 (m, 1H), 5.07 (br d, J = 3.8 Hz, 1H), 5.04 (s, 1H), 3.54 (s, 3H), 2.41 (br t, J = 6.3 Hz, 2H), 2.22-2.29 (m, 1H), 2.19 (ddd, J = 17.1, 7.6, 2.6 Hz, 1H), 2.00 (t, J = 2.5 Hz, 1H), 1.83-1.91 (m, 2H) (R)-((R)-oct-1-en-7-yn-4-yl) 3,3,3-trifluoro-2-methoxy-2-phenylpropanoate (8.1R): The title compound was prepared from (R)-Mosher acid following the same procedure that was used for the synthesis of 8.1S; yield 54%; pale yellow oil lH NMR 183 (500 MHz, CDCl3): δ 7.54-7.55 (m, 2H), 7.39-7.41 (m, 3H), 5.72-5.81 (m, 1H), 5.30 (m, 1H), 5.14 (dd, J = 6.3, 1.3 Hz, 1H), 5.11 (br s, 1H), 3.57 (d, J = 1.3 Hz, 3H), 2.47 (t, J = 6.3 Hz, 2H), 2.09-2.15 (m, 1H), 2.01 (ddd, J = 15.1, 7.6, 2.5 Hz, 1H), 1.96 (t, J = 2.5 Hz, 1H), 1.80-1.84 (m, 2H) General procedure for the synthesis of azide from alkyl bromide N3 1-azidooctane (z1):4 NaN3 (505 mg, 7.77 mmol) was added to a solution of 1bromooctane (1 g, 5.18 mmol) in DMF (2 mL) The reaction mixture was stirred at rt for h Water (20 mL) was added to the reaction mixture and the aqueous layer was extracted with EtOAc (3 x 20 mL) The combined organic phase was washed with brine (30 mL), dried over anhydrous Na2SO4 and evaporated in vacuo to give azide z1 (793 mg, 99%) as a colourless oil lH NMR (300 MHz, CDCl3): δ 3.25 (t, J = 6.9 Hz, 2H), 1.60 (quin, J = 6.7 Hz, 2H), 1.28 (br s, 10H), 0.80 (t, J = 6.4 Hz, 3H) l3C NMR (75 MHz, CDCl3): δ 51.5, 31.7, 29.13, 29.10, 28.8, 26.7, 22.6, 14.0 l3C NMR (125 MHz, CDCl3): δ 72.6, 70.60, 70.57, 70.55, 70.49, 70.44, 70.2, 70.0, 61.7, 50.6 N3 1-azidohexane (z2):5 The title compound was prepared from hexyl bromide following the same procedure that was used for the synthesis of z1; yield 89%; colouless oil lH NMR (300 MHz, CDCl3): δ 3.26 (t, J = 6.9 Hz, 2H), 1.55-1.64 (m, 2H), 1.26-1.39 (m, 6H), 0.90 (t, J = 6.6 Hz, 3H) l3C NMR (75 MHz, CDCl3): δ 51.5, 31.3, 28.8, 26.4, 22.5, 13.9 l3C NMR (125 MHz, CDCl3): δ 138.0, 128.4, 127.74, 127.72, 73.3, 70.7, 69.4, 69.2, 69.0, 37.7 184 N3 (3-azidopropyl)benzene (z5):6 The title compound was prepared from (3bromopropyl)benzene following the same procedure that was used for the synthesis of z1; yield 99%; colourless oil lH NMR (300 MHz, CDCl3): δ 7.31 (m, 2H), 7.21 (m, 3H), 3.29 (t, J = 6.9 Hz, 2H), 2.72 (t, J = 7.4 Hz, 2H), 1.92 (pent, J = 6.9 Hz, 2H) l3C NMR (75 MHz, CDCl3): δ 128.5, 128.4, 126.1, 50.6, 32.7, 30.4 N3 Citronellyl azide (z15):7 The title compound was prepared from citonellyl bromide following the same procedure that was used for the synthesis of z1; yield 75%; pale yellow oil.lH NMR (500 MHz, CDCl3): δ 5.09 (t, J = 7.0 Hz, 1H), 3.23-3.34 (m, 2H), 1.94-2.06 (m, 2H), 1.69 (s, 3H), 1.61-1.66 (m, 4H), 1.61 (s, 3H), 1.54 (m, 1H), 1.311.45 (m, 2H), 1.17-1.22 (m, 1H), 0.91 (d, J = 6.3 Hz, 3H) l3 C NMR (125 MHz, CDCl3): δ 131.5, 124.4, 49.5, 36.9, 35.6, 30.0, 25.7, 25.4, 19.2, 17.6 N3 Geranyl azide (z16):8 The title compound was prepared from geranyl bromide following the same procedure that was used for the synthesis of z1; yield 65%; pale yellow oil lH NMR (500 MHz, CDCl3): δ 5.34 (m, 1H), 5.10 (m, 1H), 3.77 (t, J = 7.6 Hz, 1H), 2.11 (m, 4H), 1.71 (s, 3H), 1.70 (s, 3H), 1.61 (s, 3H) 185 General procedure for the synthesis of azide from alcohol OH MsCl, Et 3N, CH2 Cl2 8.2 OMs 8.3 NaN3 , DMF, 80 °C N3 z6 Scheme 8.2: Synthesis of azide z6 (5-azidopentyl)benzene (z6): Methanesulfonyl chloride (696 mg, 6.08 mmol) was added to a solution of alcohol 8.2 (500 mg, 3.04 mmol) in CH2Cl2 (5 mL) cooled at °C, followed by Et3N (922 mg, 9.12 mmol) The reaction mixture was warmed to rt and stirred for h The reaction mixture was diluted with CH2Cl2 (10 mL) and extracted with H2O (2 x 10 mL), brine (20 mL), dried over anhydrous Na2SO4 and the solvent was evaporated in vacuo to give a colourless oil The product was used for next step without further purification lH NMR (300 MHz, CDCl3): δ 7.28 (m, 2H), 7.18 (m, 3H), 4.22 (t, J = 6.6 Hz, 2H), 2.98 (s, 3H), 2.63 (t, J = 7.4 Hz, 2H), 1.73-1.83 (m, 2H), 1.62-1.72 (m, 2H), 1.41-1.50 (m, 2H) l3C NMR (75 MHz, CDCl3): δ 128.4, 128.3, 125.8, 69.9, 37.4, 35.6, 30.8, 29.0, 25.0 NaN3 (293 mg, 4.5 mmol) was added to a solution of crude 8.3 in DMF (5 mL) The reaction mixture was heated at 80 °C for h The reaction mixture was cooled to rt and diluted with water (20 mL) and the organic phase was extracted with EtOAc (3 x 10 mL) The combined organic phase was washed with brine (20 mL), dried over anhydrous Na2SO4 and the solvent was evaporated in vacuo The residue was chromatographed on silica gel (hexanes/EtOAc, 1:0 to 98:2) to give azide z6 (560 mg, 186 97%, steps) as a colourless oil lH NMR (300 MHz, CDCl3): δ 7.26-7.28 (m, 2H), 7.17-7.21 (m, 3H), 3.26 (t, J = 6.9 Hz, 2H), 2.63 (t, J = 7.4 Hz, 2H), 1.59-1.72 (m, 4H), 1.38-1.48 (m, 2H) l3C NMR (75 MHz, CDCl3): δ 128.4, 128.3, 125.7, 51.4, 35.8, 31.0, 28.7, 26.3 BnO O OH MsCl, Et3 N, CH 2Cl2 BnO NaN 3, DMF O 8.4 OMs BnO O N3 z3 8.5 Scheme 8.3: Synthesis of azide z3 ((2-(2-azidoethoxy)ethoxy)methyl)benzene (z3): The mesylate was prepared from ethyleneglycol 8.4 following the same procedure that was used for synthesis of mesylate 8.3; colourless oil lH NMR (500 MHz, CDCl3): δ 7.29-7.35 (m, 5H), 4.55 (s, 2H), 3.76-3.78 (m, 2H), 3.69-3.70 (m, 2H), 3.63-3.65 (m, 2H), 3.02 (s, 3H) The azide was prepared from mesylate 8.5 following the same procedure that was used for synthesis of azide z6; colourless oil lH NMR (500 MHz, CDCl3): δ 7.287.35 (m, 5H), 4.58 (s, 2H), 3.64-3.70 (m, 6H), 3.40 (t, J = 5.1 Hz, 2H) l3C NMR (75 MHz, CDCl3): δ 138.2, 128.4, 127.7, 127.6, 73.3, 70.8, 70.1, 69.5, 50.7 HO O 8.6 OTs NaN 3, DMF HO O N3 z4 Scheme 8.4: Synthesis of hexaethyleneglycol azide z4 17-azido-3,6,9,12,15-pentaoxaheptadecan-1-ol (z4):9 The title compound was prepared from monotosylate 8.6 following the same procedure that was used for the synthesis of z3; yield 99%; colouless oil lH NMR (300 MHz, CDCl3): δ 3.64-3.72 (m, 187 20 H), 3.58-3.60 (m, 2H), 3.37 (t, J = 5.3 Hz, 2H) l3C NMR (75 MHz, CDCl3): δ 72.6, 70.60, 70.57, 70.55, 70.49, 70.44, 70.2, 70.0, 61.7, 50.6 General procedure for the synthesis of aromatic azide 2-azidophenol (z17):10 Concentreated HCl (2.3 mL) was added to a suspension of 2aminophenol (1.09 g, 10 mmol) in water (25 mL) cooled at °C NaNO2 (690 mg, 10 mmol) in water (3 mL) was added to the reaction mixture After 10 min, NaN3 (780 mL, 12 mmol) was added portionwise The reaction mixture was warmed to rt and stirred for h The reaction mixture was extracted with EtOAc (3 x 20 mL) The combined organic phase was washed with brine (30 mL), dried over anhydrous Na2SO4 and the solvent was evaporated in vacuo to give a colourless oil (1.283 g, 95%) The product was used without further purification N3 OH l H NMR (300 MHz, CDCl3): δ 7.09 (dd, J = 7.4, 1.3 Hz, 1H), 7.04 (dd, J = 7.4, 1.3 Hz, 1H), 6.91-6.97 (m, 2H), 5.33 (br s, 1H) N3 Br 1-azido-4-bromobenzene (z18):11 The title compound was prepared from pbromoaminobenzene following the same procedure that was used for the synthesis of 188 z17; yield 87%; pale yellow oil lH NMR (500 MHz, CDCl3): δ 7.46 (d, J = 8.6 Hz, 2H), 6.91 (d, J = 8.2 Hz, 2H) N3 NO 1-azido-4-nitrobenzene (z19):12 The title compound was prepared from pnitroaminobenzene following the same procedure that was used for the synthesis of z17; yield 97%; pale yellow oil lH NMR (500 MHz, CDCl3): δ 8.25 (d, J = 8.8 Hz, 2H), 7.14 (d, J = 8.8 hz, 2H) 189 References Rubinshtein, M.; James, C R.; Young, J L.; Ma, Y J.; Kobayashi, Y.; Gianneschi, N C.; Yang, J Org Lett., 2010, 12, 3560-3563 White, J D.; Kim, T.-S.; Nambu, M J Am Chem Soc 1995, 117, 5612-5613 Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H J Am Chem Soc 1991, 113, 4092-4096 Ju, Y H.; Kumar, D.; Varma, R S J Org Chem 2006, 71, 6697-6700 Masuda, Y.; Hoshi, M.; Arase, A Bull Chem Soc Jpn 1984, 57, 1026-1030 Lepore, S D.; Mondal, D.; Li, S Y.; Bhunia, A K Angew Chem Int Ed 2008, 47, 7511-7514 Bosanac, T.; Wilcox, C S Org Lett 2004, 6, 2321-2324 Hagiwara, H.; Sasaki, H.; Hoshi, T.; Suzuki, T Synlett 2009, 643-647 Muller, M K.; Brunsveld, L Angew Chem Int Ed 2009, 48, 2921-2924 10 Pirali, T.; Gatti, S.; Di Brisco, R.; Tacchi, S.; Zaninetti, R.; Brunelli, E.; Massarotti, A.; Sorba, G.; Canonico, P L.; Moro, L.; Genazzani, A A.; Tron, G C.; Billington, R A ChemMedchem 2007, 2, 437-440 11 Shi, F.; Waldo, J P.; Chen, Y.; Larock, R C Org Lett 2008, 10, 2409-2412 12 Barral, K.; Moorhouse, A D.; Moses, J E Org Lett 2007, 9, 1809-1811 190 ... NBD-sarcosine (1. 14) and 7-dimethylamino coumarin- 4-acetic acid (1. 15).9 R O R O MeO O O O H O O O O O O O O O 1. 12 R = OAc 1. 16 R = 1. 14 1. 17 R = 1. 15 O2 N 1. 13 R = OAc 1. 18 R = 1. 14 1. 19 R = 1. 15 N N... F Eur Pat 19 98, EP08479 91 13 Paterson, M J.; Jovanovic, A Arkivoc, 2 010 , 10 , 11 -16 14 Nielsen, O.; Burchardt, O Synthesis, 19 91, 819 -8 21 15 Dey, B B J Chem Soc 19 15, 10 7, 16 06 -16 51 16 (a) Pederson,... enantiosynthesis of THL 54 4 .11 Kumaraswamy’s synthesis of THL 56 4 .12 Synthesis of β-hydroxy aldehyde 4. 71 57 4 .13 Davies’ synthesis of THL 57 4 .14 Raghavan’ synthesis of THL 59 4 .15 Hanson’ synthesis of