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Total synthesis of micrococcin P1 Lefranc, David 2008

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 TOTAL SYNTHESIS OF MICROCOCCIN P1  by DAVID LEFRANC Mastère des Sciences de la Matière, École Normale Supérieure de Lyon, 2005  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY  in  THE FACULTY OF GRADUATE STUDIES  (Chemistry)   THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  December 2008 © David Lefranc, 2008 ii Abstract  This thesis describes the total synthesis of the thiopeptide antibiotic micrococcin P1. It unambiguously elucidates its structure, which has been subject to controversy for over thirty years. The centerpiece of the route to the target molecule is a facile one-pot construction of the central thiazole/pyridine cluster developed in our laboratory. This highly- convergent route entails a delicate Michael addition to yield a Hantzsch dihydropyridine intermediate, which undergoes further oxidation to the fully aromatised heterocycle. The synthesis was completed by the coupling of this core with a highly-modified sensitive peptide chain. The modular nature of the synthesis can also accommodate modifications for SAR studies, contributing thereby to the fields of medicinal chemistry, pharmacology, and microbiology. At a purely chemical level, we remain confident that this work will serve as a valuable guide in the elaboration of other members of the thiopeptide family. iii Table of Contents  Abstract .................................................................................................................................. ii Table of Contents .................................................................................................................. iii List of Tables ........................................................................................................................ vii List of Figures ..................................................................................................................... viii List of Schemes ..................................................................................................................... xii List of Abbreviations ............................................................................................................ xv Acknowledgements .............................................................................................................. xix 1. INTRODUCTION ......................................................................................................... 20 1.1 The Thiopeptide Antibiotics................................................................................ 21 1.2 Synthetic Background ......................................................................................... 26 1.2.1 Synthesis of Thiazoline and Thiazole Rings ........................................ 27 1.2.2 Synthesis of Pyridine Rings ................................................................. 33 2. MICROCOCCIN P1: STRUCTURAL AND SYNTHETIC WORK ....................... 44 2.1 Structure Elucidation ........................................................................................... 44 2.2 Synthetic Studies: Shin Synthesis of “Micrococcins” ......................................... 48 2.3 The Ciufolini Synthesis of the Bycroft-Gowland Structure of MP1 ................... 54 2.4 Recent Studies on the Structure of MP1 ............................................................. 63 2.5 The Bagley Synthesis of the Pyridine Core of MP1 ........................................... 65 3. THE TOTAL SYNTHESIS OF MICROCOCCIN P1 .............................................. 68 3.1 Retrosynthetic Analysis....................................................................................... 68 3.2 Synthesis of the Tripeptide Moiety 159 .............................................................. 70 iv 3.3 Synthesis of the Precursors to Pyridine 126 ........................................................ 82 3.4 Exploration of Alternative Routes to Enone 72 .................................................. 90 3.5 Assembly of Pyridine 74 ..................................................................................... 99 3.6 Total Synthesis of Micrococcin P1 ................................................................... 102 4. FERMENTATION ...................................................................................................... 108 4.1 Background Work ............................................................................................. 108 4.2 Culture of the Commercial Bacterium .............................................................. 109 4.3 Isolation from French Raclette Cheese ............................................................. 111 REFERENCES ................................................................................................................... 115 APPENDIX .......................................................................................................................... 125 1.  EXPERIMENTAL PROTOCOLS ................................................................................... 125  1.1. Preparation of (S)–ethyl 2–(1–(tert–butoxycarbonylamino)–2– methylpropyl)thiazole–4–carboxylate (164) ..................................................... 127  1.2. Preparation of (S)–ethyl 2–(1–amino–2–methylpropyl)thiazole–4–carboxylate hydrochloride (165) ........................................................................................... 131  1.3.  Preparation of ethyl 2–((4S,5R)–5–methyl–2–oxooxazolidin–4–yl)thiazole–4– carboxylate (119)............................................................................................... 134  1.4. Preparation of ethyl 2–((S)–1–(2–((1S,2R)–1–(tert–butoxycarbonylamino)–2– hydroxypropyl)thiazole–4–carboxamido)–2–methylpropyl)thiazole–4– carboxylate (226)............................................................................................... 138  1.5.  Preparation of ethyl 2–((S)–1–(2–((1S,2R)–1–amino–2–hydroxypropyl)thiazole– 4–carboxamido)–2–methylpropyl)thiazole–4–carboxylate (160) ..................... 142  1.6.  Preparation of (4S,5R)–3–(tert–butoxycarbonyl)–2,2,5–trimethyloxazolidine–4– carboxylic acid (128) ......................................................................................... 145  v 1.7.  Preparation of (4S,5R)–tert–butyl 4–((1S,2R)–1–(4–((S)–1–(4– (ethoxycarbonyl)thiazol–2–yl)–2–methylpropylcarbamoyl)thiazol–2–yl)–2– hydroxypropylcarbamoyl)–2,2,5–trimethyloxazolidine–3–carboxylate (174) . 148  1.8.  Preparation of (4S,5R)–tert–butyl 4–((Z)–1–(4–((S)–1–(4– (ethoxycarbonyl)thiazol–2–yl)–2–methylpropylcarbamoyl)thiazol–2–yl)prop–1– enylcarbamoyl)–2,2,5–trimethyloxazolidine–3–carboxylate (176) .................. 152  1.9.  Preparation of ethyl 2–((S)–1–(2–((Z)–1–((2S,3R)–2–amino–3– hydroxybutanamido)prop–1–enyl)thiazole–4–carboxamido)–2– methylpropyl)thiazole–4–carboxylate (159) ..................................................... 156  1.10.  Preparation of ethyl 2–(hydroxymethyl)thiazole–4–carboxylate (193) ............ 159  1.11.  Preparation of ethyl 2'–formyl–2,4'–bithiazole–4–carboxylate (117) ............... 162  1.12.  Preparation of ethyl 2'–acryloyl–2,4'–bithiazole–4–carboxylate (72) .............. 166  1.13.  Preparation of 4–((tert–butyldimethylsilyloxy)methyl)–2–methylthiazole (121)  ........................................................................................................................... 170  1.14.  Preparation of (4S,5R)–4–(4–(2–(4–((tert– butyldimethylsilyloxy)methyl)thiazol–2–yl)acetyl)thiazol–2–yl)–5– methyloxazolidin–2–one (71) ........................................................................... 173  1.15.  Preparation of ethyl 2'–(5–(4–((tert–butyldimethylsilyloxy)methyl)thiazol–2– yl)–6–(2–((4S,5R)–5–methyl–2–oxooxazolidin–4–yl)thiazol–4–yl)pyridin–2– yl)–2,4'–bithiazole–4–carboxylate (74) ............................................................. 177  1.16.  Preparation of (4S,5R)–tert–butyl 4–((R)–2–acetoxypropylcarbamoyl)–2,2,5– trimethyloxazolidine–3–carboxylate (220) ....................................................... 181  1.17.  Preparation of (2S,3R)–1–((R)–2–acetoxypropylamino)–3–hydroxy–1– oxobutan–2–aminium chloride (124) ................................................................ 185  1.18.  Preparation of (4S,5R)–tert–butyl 4–(4–(6–(4–((2S,3R)–1–((R)–2– acetoxypropylamino)–3–hydroxy–1–oxobutan–2–ylcarbamoyl)–2,4'–bithiazol– 2'–yl)–3–(4–((tert–butyldimethylsilyloxy)methyl)thiazol–2–yl)pyridin–2– yl)thiazol–2–yl)–5–methyl–2–oxooxazolidine–3–carboxylate (125) ............... 188  vi 1.19.  Preparation of (4S,5R)–tert–butyl 4–(4–(6–(4–((Z)–1–((R)–2– acetoxypropylamino)–1–oxobut–2–en–2–ylcarbamoyl)–2,4'–bithiazol–2'–yl)–3– (4–formylthiazol–2–yl)pyridin–2–yl)thiazol–2–yl)–5–methyl–2– oxooxazolidine–3–carboxylate (223) ................................................................ 192  1.20.  Preparation of micrococcin P1 (1) .................................................................... 196  1.21.  Preparation of 2,4–dibromothiazole (33) .......................................................... 199  1.22.  Preparation of 4–bromo–2–(trimethylsilyl)thiazole (207) ................................ 202  1.23.  Preparation of ethyl 2–aminothiazole–4–carboxylate (202) ............................. 205  1.24.  Preparation of ethyl 2–iodothiazole–4–carboxylate (201) ................................ 208  vii List of Tables Table 1. Reported 1H NMR shifts of thiazoles for natural MP  and synthetic 94 in DMSO-d6 .................................................................................. 63  Table 2. Measured optical rotations of 146 (c = 0.5, H2O, T = 25 °C) .................................. 65  Table 3. Experimental conditions for functionalisation of 33 ............................................... 96  Table 4. Reported 1H NMR shifts for natural MP and synthetic 1 in DMSO-d6 ................. 106  Table 5. Reported optical rotations in 90% EtOH in water. ................................................ 106   viii List of Figures Figure 1. Some thiopeptides ......................................................................................................... 22  Figure 2. Examples of unusual linkages ....................................................................................... 23  Figure 3. Thiazole substitution pattern ......................................................................................... 25  Figure 4. The Walker-Lukacs structural assignment of MP ......................................................... 46  Figure 5. The Bycroft-Gowland structural assignment of MP1 and its later representation ........ 47  Figure 6. The Shin "micrococcins" ............................................................................................... 48  Figure 7. Low-field section of the 1H NMR spectrum of natural MP (top) and synthetic Bycroft-Gowland's MP1 (bottom)................................................................................. 53  Figure 8. Valine-derived thiazole 146 .......................................................................................... 64  Figure 9. The presumed structure of MP1 .................................................................................... 68  Figure 10. Racemate 166 and its (+)-(R)-Mosher derivative ........................................................ 73  Figure 11. Shen's 1H NMR spectrum of 129 contaminated with 172 (arrows) ............................ 76  Figure 12.1H (above) and 13C (below) NMR spectra of 160 ........................................................ 79  Figure 13. Possible chelates of 195 and 196 ................................................................................. 88  Figure 14. 1H NMR spectrum of crude 196 .................................................................................. 88  Figure 15. 1H NMR spectrum of chromatographed 194 ............................................................... 89  Figure 16. 1H NMR spectrum of synthetic (above) and natural (below) MP1, in DMSO-d6 .................................................................... 104  Figure 17. Poor inhibitory activity of the culture broth of S. equorum WS 2733 ...................... 110  Figure 18. Schematic inhibition plate ......................................................................................... 112  Figure 19. Inhibitory activity of "cheese bacteria" against B. cereus ......................................... 113  Figure 20. Inhibitory activity of "cheese bacteria" against MSSA ............................................. 114 Figure 21. 1H NMR spectrum of 164 .......................................................................................... 129 Figure 22. 13C NMR spectrum of 164 ......................................................................................... 129 Figure 23. IR spectrum of 164 .................................................................................................... 130 ix Figure 24. 1H NMR spectrum of 165 .......................................................................................... 132 Figure 25. 13C NMR spectrum of 165 ......................................................................................... 132 Figure 26. IR spectrum of 165 .................................................................................................... 133 Figure 27. 1H NMR spectrum of 119 .......................................................................................... 136 Figure 28. 13C NMR spectrum of 119 ......................................................................................... 136 Figure 29. IR spectrum of 119 .................................................................................................... 137 Figure 30. 1H NMR spectrum of 226 .......................................................................................... 140 Figure 31. 13C NMR spectrum of 226 ......................................................................................... 140 Figure 32. IR spectrum of 226 .................................................................................................... 141 Figure 33. 1H NMR spectrum of 160 .......................................................................................... 143 Figure 34. 13C NMR spectrum of 160 ......................................................................................... 143 Figure 35. IR spectrum of 160 .................................................................................................... 144 Figure 36. 1H NMR spectrum of 128 .......................................................................................... 146 Figure 37. 13C NMR spectrum of 128 ......................................................................................... 146 Figure 38. IR spectrum of 128 .................................................................................................... 147 Figure 39. 1H NMR spectrum of 174 .......................................................................................... 150 Figure 40. 13C NMR spectrum of 174 ......................................................................................... 150 Figure 41. IR spectrum of 174 .................................................................................................... 151 Figure 42. 1H NMR spectrum of 176 .......................................................................................... 154 Figure 43. 13C NMR spectrum of 176 ......................................................................................... 154 Figure 44. IR spectrum of 176 .................................................................................................... 155 Figure 45. 1H NMR spectrum of 159 .......................................................................................... 157 Figure 46. 13C NMR spectrum of 159 ......................................................................................... 157 x Figure 47. IR spectrum of 159 .................................................................................................... 158 Figure 48. 1H NMR spectrum of 193 .......................................................................................... 160 Figure 49. 13C NMR spectrum of 193 ......................................................................................... 160 Figure 50. IR spectrum of 193 .................................................................................................... 161 Figure 51. 1H NMR spectrum of 117 .......................................................................................... 164 Figure 52. 13C NMR spectrum of 117 ......................................................................................... 164 Figure 53. IR spectrum of 117 .................................................................................................... 165 Figure 54. 1H NMR spectrum of 72 ............................................................................................ 168 Figure 55. 13C NMR spectrum of 72 ........................................................................................... 168 Figure 56. IR spectrum of 72 ...................................................................................................... 169 Figure 57. 1H NMR spectrum of 121 .......................................................................................... 171 Figure 58. 13C NMR spectrum of 121 ......................................................................................... 171 Figure 59. IR spectrum of 121 .................................................................................................... 172 Figure 60. 1H NMR spectra of 71 ............................................................................................... 175 Figure 61. 13C NMR spectrum of 71 ........................................................................................... 175 Figure 62. IR spectrum of 71 ...................................................................................................... 176 Figure 63. 1H NMR spectrum of 74 ............................................................................................ 179 Figure 64. 13C NMR spectrum of 74 ........................................................................................... 179 Figure 65. IR spectrum of 74 ...................................................................................................... 180 Figure 66. 1H NMR spectrum of 220 .......................................................................................... 183 Figure 67. 13C NMR spectrum of 220 ......................................................................................... 183 Figure 68. IR spectrum of 220 .................................................................................................... 184 Figure 69. 1H NMR spectrum of 124 .......................................................................................... 186 xi Figure 70. 13C NMR spectrum of 124 ......................................................................................... 186 Figure 71. IR spectrum of 124 .................................................................................................... 187 Figure 72. 1H NMR spectrum of 125 .......................................................................................... 190 Figure 73. 13C NMR spectrum of 125 ....................................................................................... 190 Figure 74. IR spectrum of 125 .................................................................................................... 191 Figure 75. 1H NMR spectrum of 223 .......................................................................................... 194 Figure 76. 13C NMR spectrum of 223 ......................................................................................... 194 Figure 77. IR spectrum of 223 .................................................................................................... 195 Figure 78. 1H NMR of synthetic MP1, 1 .................................................................................... 198 Figure 79. 1H NMR spectrum of 33 ............................................................................................ 200 Figure 80. 13C NMR spectrum of 33 ........................................................................................... 200 Figure 81. IR spectrum of 33 ...................................................................................................... 201 Figure 82. 1H NMR spectrum of 207 .......................................................................................... 203 Figure 83. 13C NMR spectrum of 207 ......................................................................................... 203 Figure 84. IR spectrum of 207 .................................................................................................... 204 Figure 85. 1H NMR spectrum of 202 .......................................................................................... 206 Figure 86. 13C NMR spectrum of 202 ......................................................................................... 206 Figure 87. IR spectrum of 202 .................................................................................................... 207 Figure 88. 1H NMR spectrum of 201 .......................................................................................... 209 Figure 89. 13C NMR spectrum of 201 ......................................................................................... 209 Figure 90. IR spectrum of 201 .................................................................................................... 210 xii List of Schemes Scheme 1. Biosynthesis of thiazoles in thiopeptides .............................................................. 24  Scheme 2. Biosynthesis of pyridine and tetrahydropyridine in thiopeptides ......................... 24  Scheme 3. Scheme of a normal peptide elongation in prokaryotic ribosome ........................ 25  Scheme 4. The Pattenden and the Charette thiazoline and thiazole synthesis ....................... 28  Scheme 5. In situ oxidation of bis-thiazolines ....................................................................... 29  Scheme 6. The Hecht (a) and the Kelly (b) cyclodehydration methods ................................ 29  Scheme 7. The Wipf thiazoline and thiazole synthesis .......................................................... 30  Scheme 8. The Shioiri thiazole synthesis ............................................................................... 30  Scheme 9. The Hantzsch thiazole synthesis ........................................................................... 31  Scheme 10. The modified-Hantzsch thiazole synthesis ......................................................... 31  Scheme 11. The Moody carbenoid-based thiazole synthesis ................................................. 32  Scheme 12. Synthesis of 2,4-dibromothiazole 33 and the Bach functionalisation thereof .... 33  Scheme 13. The Kelly synthesis of the core of berninamycinic acid .................................... 34  Scheme 14. Attempts to make stannyl derivatives 44 or 45 .................................................. 35  Scheme 15. The Kelly synthesis of micrococcinic acid 48 .................................................... 36  Scheme 16. The Kelly approach to the pyridine synthesis of dimethyl sulfomycinamate .... 37  Scheme 17. The Bach synthesis of the core of ent-GE2270A ............................................... 38  Scheme 18. The Shin synthesis of the pyridine core of thiocilline I ...................................... 39  Scheme 19. The Bohlmann-Rahtz pyridine synthesis applied by Moody ............................. 40  Scheme 20. The Bagley one-pot synthesis of pyridine .......................................................... 40  Scheme 21. The Ciufolini synthesis of the pyridine core of MP1 ......................................... 41  Scheme 22. The Nicolaou synthesis of the core of the GE2270 factors ................................ 42  Scheme 23. The Moody synthesis of the pyridine core of amythiamicin D .......................... 43  Scheme 24. The Shin approach to the pyridine core of MP1 ................................................. 50  xiii Scheme 25. The Shin synthesis of the whole eastern fragment of MP1 ................................ 51  Scheme 26. The Shin synthesis of the peptidic fragment of MP1 ......................................... 52  Scheme 27. The Shin synthesis of isomer 95 of the Walker-Lukacs MP .............................. 53  Scheme 28. Key steps of the Shin synthesis of isomer 96 of the Bycroft-Gowland MP1 ..... 54  Scheme 29. The Ciufolini approach to pyridine 74 ............................................................... 54  Scheme 30. The Ciufolini synthesis of enone 72 ................................................................... 55  Scheme 31. The Ciufolini synthesis of compound 71 ............................................................ 56  Scheme 32. The Stork methodology for Michael addition .................................................... 57  Scheme 33. The Ciufolini synthesis of pyridine 74 ............................................................... 58  Scheme 34. The Ciufolini synthesis of acid 126 .................................................................... 59  Scheme 35. The Ciufolini synthesis of segment 133 ............................................................. 61  Scheme 36. End of the synthesis of the Bycroft-Gowland structure of MP1 ........................ 62  Scheme 37. Synthesis of enamine 150 ................................................................................... 66  Scheme 38. Synthesis of alkynone 154 .................................................................................. 66  Scheme 39. The Bagley synthesis of the pyridine core of MP1 ............................................ 67  Scheme 40. Retrosynthetic analysis, part 1 ............................................................................ 69  Scheme 41. Retrosynthetic analysis, part 2 ............................................................................ 70  Scheme 42. Synthesis of acid 128 and ammonium salt 165 .................................................. 71  Scheme 43. Synthesis of thiazole 119 .................................................................................... 74  Scheme 44. Problems faced during hydrolysis of 119 ........................................................... 75  Scheme 45. Synthesis of amine 160 ....................................................................................... 77  Scheme 46. Synthesis of alcohol 174 ..................................................................................... 79  Scheme 47. Alleged formation of oxazoline during dehydration of threonine ...................... 79  Scheme 48. Synthesis of amine 159 ....................................................................................... 80  Scheme 49. Mechanism of threonine dehydration ................................................................. 81  xiv Scheme 50. Mechanisms of mesylation ................................................................................. 82  Scheme 51. Synthesis of thiazole 121 .................................................................................... 83  Scheme 52. Synthesis of ketone 71 ........................................................................................ 84  Scheme 53. Attempt at obtaining 191 .................................................................................... 84  Scheme 54. Synthesis of aldehyde 117 .................................................................................. 85  Scheme 55. Elaboration of enone 72 from aldehyde 117 ...................................................... 86  Scheme 56. Derivatisation of acrolein ................................................................................... 91  Scheme 57. Retrosynthetic analysis of 72 via 200 ................................................................. 91  Scheme 58. Elaboration of halothiazoles 201 ........................................................................ 92  Scheme 59. Attempt at the functionalisation of 201 .............................................................. 92  Scheme 60. Retrosynthetic analysis of 72 via bis-thiazole 206 ............................................. 93  Scheme 61. Synthesis of thiazole 33 ...................................................................................... 93  Scheme 62. The Dondoni synthesis of 39 .............................................................................. 94  Scheme 63. Products of treatment of dibromothiazole 33 with n-BuLi ................................. 95  Scheme 64. Possible mechanisms for lithiation of dibromothiazole 33 ................................ 97  Scheme 65. Rationale for the formation of thiazole 212 ........................................................ 98  Scheme 66. One-pot double functionalisation of dibromothiazole 33 ................................... 98  Scheme 67. Attempted formation of 218. .............................................................................. 99  Scheme 68. Possible synthesis of bis-thiazole 206 ................................................................ 99  Scheme 69. Synthesis of pyridine 74 ................................................................................... 100  Scheme 70. Synthesis of ammonium salt 124 ...................................................................... 101  Scheme 71. Synthesis of acid 126 ........................................................................................ 102  Scheme 72. Total synthesis of micrococcin P1 1 ................................................................. 103  Scheme 73. Summary scheme of the total synthesis of micrococcin P1 ............................. 107  xv List of Abbreviations [α] aa Ac anh aq Am Ar BHI Bn Boc Bop  BOP-Cl br Bu °C cat calcd cf. cm-1 δ  d dba DBU DCC DCE DCM DDQ DIBAL(H) specific rotation amino acid acetyl anhydrous aqueous amyl aryl brain and heart infusion benzyl tert-butyloxycarbonyl (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate Bis(2-oxo-3-oxazolidinyl)phosphinic chloride broad butyl degrees Celsius catalytic calculated confer (Latin) wavenumber(s) chemical shift in parts per million downfield from tetramethylsilane doublet dibenzylideneacetone 1,8-diazabicyclo[5.4.0]undec-7-ene N,N′-dicyclohexylcarbodiimide 1,2-dichloroethane dichloromethane 2,3-dichloro-5,6-dicyano-1,4-benzoquinone diisobutylaluminum hydride xvi DMA DMAP DME DMF DMP DMSO DNA DPPA dppp EBP ee Ef EI ent Et ESI Fod g GTP GDP h Hex HOBt HRMS Hz i IC50 Im IR J LAH dimethylacetamide 4-N,N-dimethylaminopyridine 1,2-dimethoxyethane N,N-dimethylformamide 2,2-dimethoxypropane dimethyl sulfoxide deoxyribonucleic acid diphenylphosphoryl azide 1,3-bis(diphenylphosphino)propane ethyl bromopyruvate enantiomeric excess elongation factor electron impact enantiomeric ethyl electrospray ionization 6,6,7,7,8,8,8-heptafluoro-2,2-dimethyl-3,5-octanedione gram(s) guanosine 5′-triphosphate guanosine 5′-diphosphate hour(s) hexane 1-hydroxybenzotriazole high resolution mass spectrum Hertz (s-1) iso (as an alkyl group) median inhibitory concentration imidazole infrared coupling constant lithium aluminium hydride xvii m M mRNA Me min mol MOM mp MP MRSA Ms MS MSSA MW n nBuLi NBS NIS NMR p Pac PCC Ph Piv ppm PPTS Pr PTSA q quant RNA multiplet molar (moles per litre); mega messenger ribonucleic acid methyl minute(s) mole(s) methoxymethyl melting point micrococcin P methycillin-resistant Staphylococcus aureus methanesulfonyl mass spectrometry methycillin-sensitive Staphylococcus aureus microwave normal (as an alkyl group) n-butyllithium N-bromosuccinimide N-iodosuccinimide nuclear magnetic resonance para (as a benzene substituent) phenacyl pyridinium chlorochromate phenyl pivaloyl parts per million pyridinium p-toluenesulfonate propyl p-toluenesulfonic acid quartet quantitative ribonucleic acid xviii rRNA RT s sat. sext t TBAF Tf TFA TFAA THF TIPS TLC TBDMS (TBS) TBDPS TMS tRNA Ts UV ribosomal ribonucleic acid room temperature singlet saturated sextuplet tertiary (as an alkyl group) tetrabutylammonium fluoride trifluoromethanesulfonyl trifluoroacetic acid trifluoroacetic anhydride tetrahydrofuran triisopropylsilyl thin layer chromatography tert-butyl dimethyl silyl tert-butyl diphenyl silyl trimethylsilyl (unlike the recommended tetramethylsilane) transfer ribonucleic acid p-toluenesulfonyl ultraviolet xix Acknowledgements   I wish to express my gratitude to my advisor and mentor, Professor Marco Ciufolini. I have admired his dedication to science, commitment to his students’ success and his willingness to share his wisdom. I truly consider myself fortunate to have worked under his direction.  I would also like to thank members of the Ciufolini research group, past and present, for their friendship, insightful discussions and kind advice. Special thanks must go to Dylan, Shawn and Steven for their patience and their help in proofreading this manuscript. 20 1. INTRODUCTION   The discovery of penicillin by Sir Alexander Fleming in the 1920s1 provided humanity with the first effective weapon against the onslaught of bacterial infections and stimulated intense research in the field of antibiotics. As a result, the general level of health and sanitation improved significantly beginning in the 1940’s, at least in North America and the UK.2 In particular, so central was the role of antibiotics in fending off infections resulting from battlefield injury that, as some believe, the Second World War was won more thanks to penicillin than to armaments.3 After the war, the diffusion of antibiotic therapy worldwide, and the discovery of many new such agents, contributed to an unprecedented reduction in mortality due to bacterial disease, both in the infant and the adult populations.  Unfortunately, as early as the 1950s, evidence began to accumulate that pathogenic bacteria were able to develop resistance to antibiotics.4 It is now established that this occurs through genetic mutations.5 By the end of the 1970s many antibiotics had become ineffective. Rather than generating concern, these worrisome observations were ignored by the public (government organisations) and the private (pharmaceutical companies) sectors, and antibiotics research came to a near standstill. The results were only too predictable: one by one, established antibiotics lost their efficacy against pathogenic bacteria and no new resources existed to replace them.  Today, not even a century after Fleming’s discovery, the world faces a veritable antibiotic crisis that threatens to return humanity to the so-called ‘dark age’ of the pre- antibiotic era.6 A prime example of this is the unambiguous identification of vancomycin- resistant strains of Staphylococcus aureus in hospitals in 2002.7 Vancomycin is usually one of the most effective weapons against these organisms, which have been of much concern as 21 the causative agents of numerous, and often deadly, nosocomial infections—infections developed while hospitalised for another illness. Occasionally, such bacteria prove to be sensitive to another known, if uncommon, antibiotic, but generally speaking, vancomycin- resistant S. aureus is extremely difficult to eradicate.7 The prognosis for the unfortunate patient is dire. These developments have rekindled interest in the search for new antibiotics, either through the isolation of new natural products, or the chemical modification of existing drugs,8 or the chemical synthesis of new entities.9  1.1 The Thiopeptide Antibiotics  Amongst antibiotics currently under study, the so-called thiopeptide family, exemplified in Figure 1 by micrococcin P1 (hereafter also referred to as MP1), 1, promothiocin A, 2, thiostrepton, 3, and amithiamycin D, 4, has attracted considerable attention both in the biological and the chemical arenas, as illustrated in a number of excellent reviews.10 Thiopeptides are normally derived from terrestrial organisms such as actinobacteria (mainly Streptomyces spp.); however, some congeners have been isolated from marine sources (e.g., sponges).10a Approximately 80 members of this ever-growing family are known as of this writing. All such compounds are potent inhibitors of protein synthesis and, as such, they display significant antibacterial, antifungal, antiprotozoal, and anticancer activity (vide infra). Finally, a few exhibit gene-inducing activity.  22  Figure 1. Some thiopeptides   As their name suggests, the thiopeptides are rich in sulphur and contain a variety of natural and highly modified amino acids. Most of them include a central heterocyclic cluster composed of a pyridine ring variously decorated with thiazole or oxazole substituents. Reduced forms of the pyridine or of its substituents may be present. Indeed, the precise oxidation state of the nitrogen heterocycles composing the central cluster is a criterion for the classification of thiopeptides.11 This cluster is part of a macrocyclic structure that includes additional heterocycles such as thiazoles, oxazoles, indoles and quinolines at various oxidation states and with diverse substitution patterns, as well as L-amino acids (threonine, isoleucine, etc.) and dehydroamino acids such as dehydroalanine and 23 dehydroaminobutyric acids. Some thiopeptides count three or more macrocyclic subunits which display ether, thioether and thioester linkages (Figure 2).    Figure 2. Examples of unusual linkages   It is noteworthy that many thiopeptides have only partially solved stereostructures, despite the fact that some of them have been known for over 50 years. For instance, glycothiohexide 6 exhibits several asymmetric centres whose configuration is yet to be ascertained. On several of occasions, stereochemical assignments were made through total synthesis.12,13 It should be noted that in all such cases the nitrogen-bearing stereogenic carbons linked to the C-2 position of thiazole residues were found to possess a configuration that corresponds to that of a natural L-amino acid. This is consistent with a biosynthetic pathway that leads to thiazole rings 11 through cyclisation/aromatisation of peptides 7 and 8 (Scheme 1). The same route may be operative in the biosynthetic assembly of analogous oxazoles (cf. 8, 10, 12).14  24   Scheme 1. Biosynthesis of thiazoles in thiopeptides   On the subject of biosynthesis, the sequence leading to the pyridine unit deserves special mention. Bycroft and Gowland proposed in 1978 that the pyridine and tetrahydropyridine core shared by many thiopeptides might result through the fusion of two dehydroalanine residues in an aza-Diels-Alder-like mode.15 Further isotope labelling studies by Floss supported this proposal (Scheme 2).14a,16 Thus, the enamine part of an open macrocycle (13) interacts with the hydroxyaza-diene functionality of another section of the molecule in what may be a concerted mechanism to give aminohydroxytetrahydropyridine 14. At this stage, two different paths are available. In path A, dehydration and elimination of the N-terminus give pyridine 15, whereas in path B, dehydration and reduction lead to tetrahydropyridine 16. It will be seen later that this intriguing proposal provided the inspiration for the noteworthy syntheses of thiostrepton17 and amythiamycin D.18    Scheme 2. Biosynthesis of pyridine and tetrahydropyridine in thiopeptides   25  The manifold bioactivities of these substances appear to be linked to their ability to bind to different zones of the ribosome, thereby disrupting protein synthesis (Scheme 3). Differences in the details of the mechanism of action separate thiopeptides into two categories: those that bind to the L11 binding domain of the 23S ribosomal RNA (a subunit of the 30 S moiety of the ribosome),19 such as MP1 and thiostrepton A, and those that bind to the so-called elongation factor Tu (Ef-Tu) involved in the elongation cycle, like GE2270A.20 Compounds that interact directly with rRNA form a stabilised complex that is    Scheme 3. Scheme of a normal peptide elongation in prokaryotic ribosome Legend: A complex consisting of an appropriate amino acid-carrying tRNA (aa-tRNA, red), elongation factor Tu (Ef-Tu, green) and GTP (teal) binds to a ribosome (consisting of subunits 50S and 30S) which is in the process of elongating a peptide chain (step A). The binding interaction occurs with loss of a phosphotriester linkage and release of an Ef-Tu-GDP complex (purple). The ribosome then triggers the translocation of both peptide and aa-tRNA to bring them together in an active site (step B) where an mRNA promotes the transfer of the amino acid carried by the tRNA to the growing peptide chain. The peptide is released from the deacylated tRNA (step C). This latter tRNA is expulsed from the ribosome concomitantly with the binding of a fresh aa-tRNA-Ef-Tu-GTP unit. The ribosome is thus ready for another cycle.21 unable to undergo the conformational changes necessary to promote the elongation of the nascent peptide chain. As a result, protein synthesis comes to a halt.22 On the other hand, 26 agents that bind to a protein Ef-Tu prevent the formation of a complex between an amino acid-bound-tRNA (aa-tRNA) and GTP-bound Ef-Tu. In this case, protein synthesis is stopped (or strongly inhibited) because the aa-tRNA is unable to bind to the ribosome and deliver the required amino acid that it was carrying.23   As a consequence of the above, thiopeptides exhibit an impressive spectrum of biological properties. They display very strong activity against Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA), a strain that is usually treated with the glycopeptide antibiotic, vancomycin. Some members of the family are active against Gram-negative bacteria,24 others still display antifungal, anticancer or antiprotozoal activity. Significant in the latter connection is the antimalarial action of micrococcin P1, 1, and thiostrepton A, 3, which inhibit the growth of Plasmodium falciparum, at concentrations (IC50’s) of 35 nM and 3.2 nM, respectively, in vitro.25  1.2 Synthetic Background  The molecular complexity of thiopeptide substances, the stereochemical challenge they represent, their reactivity, and their bioactivity have enticed a number of synthetic chemists. Ten thiopeptides have succumbed to total synthesis so far: promothiocin A, 2,12,26 the first such compound ever to be conquered, thiostrepton A, 3,27 amythiamicin A, B, C,28 and D, 4,13,29 GE2270 A,30,31 GE2270 T,31 GE2270 C1,31 siomycin A32 and micrococcin P1, 1, the subject of this thesis. Numerous synthetic studies have also been reported.33  A retrosynthetic analysis of any thiopeptide will reveal a few obvious disconnections: peptide bonds, macrolactone/lactam formation, etc. However, these seemingly straightforward operations represent a serious challenge within the framework of a total 27 synthesis. This is due to the unpredictable reactivity of some functionalities, e.g., dehydroamino acids, during the sequences required to reach the desired target. Sometimes, unanticipated difficulties have forced a re-evaluation of the synthetic strategy. Stereochemical uncertainties add another complication to the synthetic challenge, as illustrated by the convoluted story of MP1 (vide infra). In light of the foregoing, it seems useful to review the chemical strategies that have proven useful in the synthesis of key fragments of thiopeptide architectures. The following section focuses on the methods that were utilised in the synthesis of thiopeptide antibiotics or of some of their fragments.  1.2.1 Synthesis of Thiazoline and Thiazole Rings  The title heterocycles are common features of all thiopeptides, where they are always found as part of a 2,4-disubstituted system such as 17 (Figure 3). Occasionally, 5-methyl substituted analogues of 18 may appear. For example, amythiamycin D, 4, displays 5- methylthiazole ring in its macrocycle.   Figure 3. Thiazole substitution pattern   Thiazolines are customarily assembled by cyclisation of cysteine or serine derivatives. For instance, Pattenden illustrated a method inspired by the work of Shiba34 involving the condensation of amino acid-derived imino-ethers, prepared by reaction of 28 amino acid amides with a Meerwein salt, with cysteine esters.35 The thiazoline formed without erosion of stereochemical integrity. Subsequent oxidation to a thiazole using activated MnO2 also conserved the configuration of the stereogenic carbon. It will be seen shortly that other approaches to like thiazoles may imperil the enantiopurity of the product.    Scheme 4. The Pattenden and the Charette thiazoline and thiazole synthesis   This method was applied to a limited selection of amino acids (alanine, valine, phenylalanine and isoleucine), but it is likely to be fairly general. In 1998, Charette introduced a modification of the above route, wherein the starting amide is activated as an imidoyl triflate (treatment with Tf2O in the presence of pyridine, Scheme 4). However, the new technique was demonstrated only with amides that lack an α-stereogenic centre. Therefore, it is unclear whether the procedure is applicable to stereochemically labile amides, such as those derived from α-amino acids. It is worthy of note that exposure of bis- thiazolines obtained by this method to the air promotes oxidation of one ring to a fully aromatic thiazole (Scheme 5).36 29   Scheme 5. In situ oxidation of bis-thiazolines   A similar approach involving the cyclodehydration of a cysteine dipeptide finds its inspiration in the biosynthesis of thiazolines and thiazoles. Accordingly, the cyclodehydration of dipeptide 19 or of an (S)-trityl derivative thereof, 20, is achieved under Brønsted acidic conditions,37 or in the presence of TiCl4,38a or with a mixture of Ph3PO/Tf2O.38b Yields of enantiopure thiazolines are thus good to excellent (Scheme 6).    Scheme 6. The Hecht (a) and the Kelly (b) cyclodehydration methods   Suitable activation of the OH group of a serine residue may also induce thiazoline formation. To illustrate, the Wipf thiazoline synthesis calls for cyclisation of a thioamide derivative of serine, 21, in the presence of the Burgess reagent. The use of other dehydration methods (TsCl/Et3N, SOCl2, and Mitsunobu conditions) led to extensive epimerisation of the 30 α-carbon of the thioamide.39 This method requires a protection-deprotection sequence of the serine OH group, but it affords high overall yields and possesses broad scope with respect to the thioamide segment (Scheme 7). Again, thiazolines 22 aromatise easily upon reaction with MnO2. This method has been adapted to the synthesis of oxazoles, thiazines and oxazines.40    Scheme 7. The Wipf thiazoline and thiazole synthesis   A technique for thiazole synthesis related to the foregoing methods was disclosed in 1984 by Shioiri. This involved the condensation of an N-protected α-amino aldehyde with a β-mercapto amine to furnish a thiazolidine, which again was oxidised to a thiazole in moderate to good yield, and with retention of optical integrity, upon reaction with MnO2 (Scheme 8).41 A number of amino acid-derived amino aldehydes were employed successfully in this sequence.    Scheme 8. The Shioiri thiazole synthesis  31  The synthetic community developed renewed interest in the direct construction of thiazoles following the isolations, in the 1980’s, of many bioactive marine natural products containing such heterocycles.42 The Hantzsch synthesis is undoubtedly the method of choice for this purpose. The reaction was discovered in 188743 and entails the condensation of a thioamide with an α-halocarbonyl compound, more commonly in refluxing ethanol (Scheme 9). The method is operationally simple and it affords high yields. However, when applied to     Scheme 9. The Hantzsch thiazole synthesis  the elaboration of thiazoles using amino acid-derived thioamides, it was found to proceed with substantial or complete erosion of optical purity. Extensive investigations revealed that loss of configuration occurs at the stage of thiazoline 25 and that the rate of racemisation is greatly accelerated at higher temperatures (Scheme 10). A method developed by Holzapfel    Scheme 10. The modified-Hantzsch thiazole synthesis  and refined by Meyers44 circumvents these difficulties by conducting the initial reaction between 23 and 24 at low temperature (–15 °C) under gently basic conditions (KHCO3), and by dehydrating the emerging hydroxythiazoline 25 with trifluoroacetic anhydride in the 32 presence of 2,6-lutidine. This so-called modified-Hantzsch reaction preserves the configuration of stereogenic centres.   Finally, Moody developed a general approach to trisubstituted thiazoles and oxazoles that relies on the chemistry of carbenoids.45 The sequence starts with the Rh(II)-catalysed formal insertion of an N-H bond of amide 27 into the carbene produced through deazoniation of methyl diazoacetoacetate 30. The resultant 28 cyclises to thiazole 29 in the presence of Lawesson’s reagent,46 without loss of chirality at the α position (Scheme 11). The method is not applicable to the synthesis of 5-H-thiazoles, due to the difficulties associated with the preparation of diazo compound 31.    Scheme 11. The Moody carbenoid-based thiazole synthesis   Functionalisation of a preformed thiazole is also a useful strategy in thiopeptide synthesis. This normally entails the generation of organometallic derivatives of the heterocycles as a prelude to further reaction with appropriate electrophiles. It should be noted, however, that under certain conditions, anionic thiazole derivatives may prove to be delicate to handle and somewhat unpredictable in their reactivity.47 Our study will also illustrate the unexpected behaviour of such anions depending on reaction conditions. 33  A useful block for the assembly of thiopeptide constructs is 2,4-dibromothiazole 33, readily accessible via the reaction of thiazolidinedione 32 with POBr3 (Scheme 12).48 Many building blocks have been elaborated by successive metal-halogen exchange reactions at the two positions of 33. The greater reactivity of position 2 enables the initial formation of 2- metallo derivatives. Reaction with suitable electrophiles furnishes 4-bromothiazoles, and thence 4-metallothiazoles, which may then be elaborated to a great diversity of useful building blocks.49 Representative examples of this chemistry appear in Scheme 12.49k  N S H N S O O Br Br 32 33 a N S Br Br N S BrMg Brb c N S H2N OTBS Br d N S BocHN OTBS N S Br a) POBr3 (3 equiv.), neat, 110 °C, 61%; b) i-PrMgBr, THF, 0 °C; c) i. (R)-O-TBS-mandelonitrile; ii. NaBH4, EtOH, -78 to 0 °C; d) i. Boc2O, CH2Cl2; ii. ZnCl2, t-BuLi, Et2O, -78 °C. iii. 33, [PdCl2(PPh3)2], THF, rt. 33   Scheme 12. Synthesis of 2,4-dibromothiazole 33 and the Bach functionalisation thereof  1.2.2 Synthesis of Pyridine Rings  Although the synthesis of pyridines has been studied since the late nineteenth century,50 a considerable amount of work has been necessary to obtain the complex pyridine- thiazole clusters found at the core of thiopeptides. Two trends have emerged: the modification of a pre-existing pyridine and the de novo construction of that heterocycle with the full complement of thiazoles. 34  The modification of a preformed pyridine relies on two key technologies: metallation of aromatic rings, either by halogen-metal exchange51 or by directed metallation,52 and palladium-mediated cross-coupling reactions.53 A landmark paper by Kelly in 1984 describing the synthesis of berninamycinic acid illustrated the directed C-3 metallation of a 2,6-bisamidopyridine using BuLi. The dianion thus formed was quenched with two equivalents of N-(methoxymethyl)isothiocyanate and further elaborated to the corresponding bis-thiazole (Scheme 13).54    Scheme 13. The Kelly synthesis of the core of berninamycinic acid   In 1991, Kelly disclosed a synthesis of micrococcinic acid, 48,49d that utilised palladium-catalysed cross coupling reactions for the functionalisation of a pyridine ring (Scheme 14). Micrococcinic acid 48, is a degradation product of micrococcins. In light of our structural studies (vide infra), it is not likely to be a synthetically useful intermediate. However, because this pioneering work is closely related to the object of this thesis, and because it established the first instances of cross-coupling reactions in the thiopeptide field, further detail will be provided in the following section.  The synthesis started with aminopyridone 34, that was transformed in four steps into stannyl derivative 36 (Scheme 14). All four biaryl linkages present in the final product were introduced by palladium-mediated reactions. In this light, couplings of thiazole building 35 block 37 with pyridine 36 and TMS-thiazole 39 produced biaryls 38 and 41 respectively. The ethoxy group in 38 and the trimethylsilyl unit in 41 were modified to give    Scheme 14. Attempts to make stannyl derivatives 44 or 45  bromides 42 and 43. However, subsequent attempts to produce stannanes 44 or 45 by reaction of the bromides with hexabutylditin and Pd(0) afforded instead the homodimers of the respective substrates. Clearly the authors were hoping to accomplish the ultimate formation of 46 by cross-coupling of 42 with 45, or by an alternative 43 with 44. Subtarget 46 was ultimately reached by an ingenious, if moderately efficient, method. Reaction of a 1:1 mixture of bromides 42 and 43 with (Me3Sn)2 and [(Ph3P)2PdCl2] yielded a product 36 which consisted of a statistical mixture of homo- and heterodimers. Accordingly, the desired cross-coupling product accounted for approximately 50% of the product, the remainder being a mixture of undesired homodimers, which were discarded. A fourth palladium- catalysed coupling of triflate 46 with stannyl derivative 47 established the final biaryl linkage. Cleavage of the two tert-butyl-amides furnished micrococcinic acid 48.    Scheme 15. The Kelly synthesis of micrococcinic acid 48   Similar methodology was used in 1995 during the synthesis of dimethyl sulfomycinamate 52.47,55 However, in that case, the authors were unable to create a suitably substituted oxazole for palladium-catalysed Stille coupling, and eventually had to opt for a step-by-step Blümlein-Lewy oxazole synthesis56 (analogous to the Hantzsch thiazole synthesis) (Scheme 16).  37   Scheme 16. The Kelly approach to the pyridine synthesis of dimethyl sulfomycinamate   The problems encountered in the course of the various Stille reactions, namely, the formation of considerable quantities of homodimeric by-products, remained unresolved until Bach introduced the use of Negishi coupling reactions for the assembly of pyridine-thiazole clusters. An instructive example appears in Scheme 17. Thus, two Negishi and one Stille cross-coupling reactions permit a concise and efficient elaboration of 2,3,6-tribromopyridine 53 to compound 57, which is a degradation product of GE2270A.49l,57 This contribution is especially significant in that it also determined the configuration of the 1,2-aminoalcohol segment of 57 and therefore of the natural product. The relative stereochemistry of the desired product derived from the natural material was established to be (S, S)-erythro in the same study, unlike its enantiomer 57, that was synthesised, and which displayed the (R, R)- threo absolute configuration.  38   Scheme 17. The Bach synthesis of the core of ent-GE2270A   This synthesis represented an improvement of the work accomplished by Kelly, but the authors were still faced with a minor complication: Negishi coupling of 54 with 55 proceeded with moderate regioselectivity, leading to an inseparable mixture of 2,3- and 3,6- disubstituted pyridines in a 6.5:1 ratio in favour of the desired 2,6-disubstituted isomer. The isolation of the desired product 57 was accomplished by semipreparative HPLC.   Palladium-mediated reactions are not essential for the assembly of pyridines of the type found in thiopeptides. This is apparent from the work of Shin, who achieved the synthesis of a number of such subunits through the use of more classical chemistry.58 To illustrate (Scheme 18), the core of thiocillin I, 64, was reached through a linear elaboration  39   Scheme 18. The Shin synthesis of the pyridine core of thiocilline I  of pyridone 60. The three thiazole rings were built using the modified-Hantzsch methodology (steps b and d) and the Shioiri thiazole synthesis (step e). The use of Meyers’ conditions in step b is unusual, in that no risk of epimerisation subsisted. The fusion of pyridine 61 and thioamide 62 is clearly the key feature of the synthesis, and it makes for a pleasantly convergent approach.  In his synthesis of promothiocin A,12,26,59 Moody revived a methodology that had not found much use since its discovery in 1957: the Bohlmann-Rahtz pyridine synthesis.60 Thus, heating an ynone with an appropriate enamine such as 65 induces a Michael-type addition that leads to 66. Forceful thermal activation achieves cyclodehydration of 66 to yield pyridine 67 (Scheme 19).  40   Scheme 19. The Bohlmann-Rahtz pyridine synthesis applied by Moody   Subsequently, Bagley studied the Bohlman-Rahtz reaction in greater depth and developed new conditions to conduct the reaction at significantly lower temperatures.61 Thus, exposure of a mixture of a β-ketoester and an ynone, e.g., 68 and 69, to NH4OAc leads directly to a presumed dihydropyridine, which undergoes facile in situ oxidation to 70 when treated with NIS (Scheme 20). It should be noted that in some cases aminedienone intermediates of the type 66 must be isolated and submitted to cyclodehydration / aromatisation in a separate step. The new technique was used in landmark syntheses of dimethyl sulfomycinamate and of the pyridine cores of amythiamicin, cyclothiazomycin, and MP1.62    Scheme 20. The Bagley one-pot synthesis of pyridine   The de novo construction of the pyridine ring often results in a highly convergent approach. For instance, our laboratory reported the synthesis of the complete core of 41 micrococcins, 74, through the union of fragments 71 and 72.63 The initial step involves the heterogeneously-catalysed Michael addition of the enolate of 71 onto enone 72. The resulting 1,5-diketone 73, obtained in virtually quantitative yield, was then treated with ammonium acetate and the resulting dihydropyridine was oxidised with DDQ to give pyridine 74 (Scheme 21). Important aspects of this sequence will be further detailed in the next section.  N S O N S CO2EtO NH O N S N S TBSO O O NH O N S N S TBSO O N SO N S CO2Et O NH O NS N STBSO N S N S CO2Et N a b 71 72 73 74 a) cat. Li2CO3, EtOAc, rt; b) NH4OAc, EtOH, then DDQ in CHCl3.   Scheme 21. The Ciufolini synthesis of the pyridine core of MP1   In 2001, Nicolaou and Moody independently described a remarkable biomimetic (cf. Scheme 2) avenue to thiopeptide cores. Thus, the key step in Nicolaou’s brilliant syntheses of thiostrepton, 3,27 GE2270A, GE2270T and GE2270C1,31 entailed the aza-Diels-Alder dimerisation of 2-azadiene 78 leading to the ultimate formation of tetrahydropyridine 79, which may be optionally oxidised to fully aromatic pyridine 80 (Scheme 22).  42   Scheme 22. The Nicolaou synthesis of the core of the GE2270 factors   Moody’s delightful synthesis of amythiamicin D employed the fusion of enamide 81 with azadiene 82 in an aza-Diels-Alder reaction promoted by microwaves (Scheme 23).13 This route yielded pyridine core 83 in 33% yield. A by-product of this reaction arises from the dimerisation of the diene, akin to the methodology employed by Nicolaou. This observation had been made in numerous studies on the [4 + 2] cycloaddition of 2- azadienes.64  43   Scheme 23. The Moody synthesis of the pyridine core of amythiamicin D   The foregoing review outlines key aspects of the biology and the chemistry of the thiopeptide family of antibiotics. We shall now refocus our attention on the object of this work: the synthesis of micrococcin P1, 1. 44 2.  MICROCOCCIN P1: STRUCTURAL AND SYNTHETIC WORK   The efforts directed toward the structural elucidation and the chemical synthesis of micrococcin P1 constitute a noteworthy, and quite peculiar, story. It is likely that the compound was first isolated in 1948 by Su65 from an unidentified strain of Micrococcus retrieved from a sample of sewage collected from the city of Oxford, UK. That organism may possibly be related to Micrococcus varians.66 No structural studies were carried out on the new substance. In 1955, Fuller67 isolated an antibiotic from a spore-bearing Bacillus pumilus collected in East Africa. The new natural product appeared to be identical to the Su micrococcin. Accordingly, it was later christened “micrococcin P” (“MP”) to underscore its origin from B. pumilus. The material soon proved to be a 7:1 mixture of two compounds: micrococcin P1 (MP1) and micrococcin P2 (MP2).15 Finally, the natural product was more recently isolated from a food-borne strain of Staphylococcus equorum found on the surface of French raclette cheese.68  2.1 Structure Elucidation  The elucidation of the structure of micrococcin occupied Walker, Lukacs and collaborators for over 20 years. In a landmark 1977 publication,69 these scientists summarised results detailed in several earlier papers70 and proposed structure 89 for what they called “micrococcin P”. Salient features of the Walker-Lukacs assignment are as follows (Figure 4): (i) The arrangement of thiazoles around the pyridine nucleus was unequivocally established through an X-ray diffractometric study the bis-4-bromoanilide of micrococcinic acid, 48, a product of acidic degradation of MP1.70c,d,71 45 (ii) Acidic degradation also provided thiazole ketone 86, some L-threonine 84, plus (-)-1-amino-2-propanol (= D-isoalaninol) 87. The lævorotatory property of the latter fragment unequivocally defines its (R)-configuration,70b as shown in the diagram below. (iii) Yet another product of acidic degradation was 85. This valine-derived thiazole was often obtained as the lævorotatory isomer, with specific rotations between – 2° and – 21°, but occasionally it emerged as the racemate.70e The hydrolysis of MP took place in refluxing 20% aqueous hydrochloric acid. One may readily anticipate epimerisation or racemisation of enantiopure 85 under these harsh conditions.70e The absolute configuration of 85 was unknown at that time. Therefore, Walker and collaborators prepared a sample of this material from natural L-valine. They employed techniques of thiazole synthesis that were apt to erode optical purity. Indeed, the resultant emerged once as the racemate and twice as a weakly dextrorotatory isomer with specific rotations between + 2° and + 5°. They thus concluded that natural 85 was the antipode of their synthetic sample and assigned its configuration as corresponding to that of D-valine. (iv) The arrangement of the various hydrolytic fragments within the macrocycle of MP was proposed on the basis of a presumed structural analogy between micrococcin and other thiopeptide antibiotics known at that time. Such a proposal, however, remained unsubstantiated. 46 N S N N S COOH S N S N H2N Et NH2 O N S H2N N S Et HO2C COOH NH2 HO HO2C HO H O O N S N N S H N O S N S N N H O N H O N S NH N S NH O O N H HO O H OH HO H 85 8684 48 87 89   Figure 4. The Walker-Lukacs structural assignment of MP   The Walker-Lukacs assignment was soon revised by Bycroft and Gowland15 on the basis of a thorough study of the kinetics of formation of the various subunits of MP during its hydrolysis. They established by preparative TLC that MP was in fact a mixture of two micrococcins which they called micrococcins P1 and P2. Furthermore, Bycroft and Gowland determined that acidic hydrolysis releases only one threonine unit, plus a product, 2- oxobutyric acid 90, as well as amino acetone 91 that had previously escaped detection. The presence of the latter molecule suggested that MP2 was the C-2 ketone analogue of MP1; i.e., an amino acetone unit was present in lieu of the isolaninol fragment 87. This assumption was further reinforced by the careful reduction of MP2 with NaBH4, which furnished a compound chromatographically identical to MP1.  These workers tacitly accepted other aspects of the earlier structural assignment, in particular, the arrangement of the various subunits in the macrocycle and—seemingly—the 47 alleged D-configuration of the valine-derived thiazole 85. Accordingly, they proposed structures 92 and 93 for MP1 and MP2, respectively (Figure 5). It is apparent from the foregoing discussion that proposed structures 89 and 92 of MP and MP1 are mutually exclusive.    Figure 5. The Bycroft-Gowland structural assignment of MP1 and its later representation   No evidence was available regarding the configuration of the starred carbon atoms in 92, which Bycroft and Gowland, correctly, rendered as undefined. However, since all thiazoles found in thiopeptide antibiotics appear to be derived from natural amino acids, the configuration of those two centres ultimately came to be represented as corresponding to L- threonine (cf. structure 94).   48 2.2 Synthetic Studies: Shin Synthesis of “Micrococcins”  Shin has published extensively on the synthesis of micrococcins. In particular, he achieved the total syntheses of compounds 9572 and 9673 (Figure 6).    Figure 6. The Shin "micrococcins"   These are described as being spectroscopically identical to “micrococcin P” and to “micrococcin P1”, respectively, as if “micrococcin P” and “micrococcin P1” were two distinct natural products. However, we have already seen that this is not so, in that micrococcin P is a 7:1 mixture of micrococcin P1 and micrococcin P2. This implies that the spectra of micrococcin P1, the major component, should be very similar to those of micrococcin P, the mixture. It will be seen later that this is indeed the case. Consequently it is remarkable that, although constitutionally distinct, structure 95 should be spectroscopically identical to a mixture, and structure 96 to its major component. More 49 significantly, general structures 95 and 96 correspond, respectively, to a Walker-Lukacs-like “micrococcin P” and to a Bycroft-Gowland-like “micrococcin P1”. As detailed earlier, these structures are mutually exclusive. Finally, we describe 95 and 96 as “Walker-Lukacs-like” and “Bycroft-Gowland-like” micrococcins because the isoalaninol segment of these synthetic materials possesses the (S)-configuration, instead of the secured (R)-one. Notice that the configuration of the valine-derived thiazole corresponds to that of D-valine, in accord with the Walker-Lukacs assignment. However, the L-threonine-like configuration of the thiazole marked with an arrow in 96 appears to be unsupported by public-domain chemical evidence.   The synthesis of 100 was patterned after an earlier effort toward the pyridine core of thiocilline I (Scheme 18). Accordingly, pyridone 59 was created in two steps from dimethoxyacetone 58 in 23% yield (Scheme 24).58b  50  Scheme 24. The Shin approach to the pyridine core of MP1   The thiazole ring in 61 was introduced via the modified-Hantzsch synthesis, while the α-bromoketone segment resulted upon a Heck reaction of the triflate derivative of the pyridone with ethyl vinyl ether, followed by bromination of the intermediate vinyl ether. Another modified-Hantzsch thiazole synthesis between easily accessible thioamide 98 and 61 gave 99, which advanced to aldehyde 100 upon dehydration of the threonine and deprotection of the acetal functionality. It is worth mentioning that no details are provided concerning the preparation of thiazole 98: Shin and co-workers indicate simply that the compound was made from D-valine according to a procedure described earlier by U. Schmidt that utilised the L-amino acid.74 The latter publication clearly states that the thiazole thus produced exhibits an enantiomeric excess of only about 25%. It is not clear whether Shin utilised the Schmidt procedure verbatim, or whether he implemented a Meyers-type modification thereof. 51  Aldehyde 100 was then coupled to cysteine-derived thiazole 101 in a Shioiri-type reaction and oxidised using MnO2 to give ester 102 in a meagre 28% yield (Scheme 25).    Scheme 25. The Shin synthesis of the whole eastern fragment of MP1   Selective hydrolysis of the phenacyl ester in the presence of the ethyl ester took place in the presence of aqueous 1M LiOH to give the corresponding monoacid, which was coupled to the readily available side chain 103. This intermediate was then hydrolysed to yield fragment 104 with all the desired functionalities in place. The longest linear sequence is 19 steps and the overall yield of 104 along this pathway is 0.4%.  The remainder of the synthesis consisted of the elaboration of the missing dipeptide fragment 109 and its coupling to the acid corresponding to ester 104. The synthesis of the dipeptide started with the coupling of the known protected threonine 105 with N-carboxy 2- amino-2-butenoic acid anhydride 106 followed by ammonolysis to deliver amide 107 (Scheme 26).58c Lawesson reaction gave the corresponding thioamide, which produced thiazole 108 using the modified-Hantzsch conditions. A series of protection/deprotection steps afforded TFA salt 109. 52    Scheme 26. The Shin synthesis of the peptidic fragment of MP1   The final steps featured the coupling of the now available fragments using Bop, after saponification of ester 104. Further global deprotection of the ester, Boc and MOM groups on the seco compound 110, followed by coupling with Bop afforded epimer 95 of the Walker-Lukacs MP1, which was stated to be identical to “micrococcin P” (Scheme 27).   53   Scheme 27. The Shin synthesis of isomer 95 of the Walker-Lukacs MP   A similar approach is apparent in a subsequent synthesis of 96. The diagram below highlights key points of the effort leading to the target molecule, an epimer of the Bycroft- Gowland MP1 that according to Shin and collaborators is identical to MP1 (Scheme 28).  54   Scheme 28. Key steps of the Shin synthesis of isomer 96 of the Bycroft-Gowland MP1  2.3 The Ciufolini Synthesis of the Bycroft-Gowland Structure of MP1  One of the major objectives of this effort was the elaboration of pyridine 74 in a highly convergent fashion through the union of fragments 71 and 72 according to the format of Scheme 29.63 The sequence is recognised as a variant of the Hantzsch pyridine synthesis.75  N S O N S CO2EtO NH O N S N S TBSO O O NBoc O NS N STBSO N S N S CO2Et N 71 72 74   Scheme 29. The Ciufolini approach to pyridine 74 55  The synthesis of enone 72 evolved from glycolonitrile 115 (Scheme 30); treatment with hydrogen sulphide afforded the corresponding thioamide, which underwent a Hantzsch reaction with ethyl bromopyruvate (EBP) in refluxing ethanol. The emerging thiazolyl ester was subject to ammonolysis followed by protection of the free alcohol to furnish amide 116 in 64% yield overall. This compound was advanced to aldehyde 117 in three uneventful steps featuring yet another Hantzsch thiazole synthesis. Addition of vinyl magnesium bromide and oxidation of the allylic alcohol converted 117 to the required 72.    Scheme 30. The Ciufolini synthesis of enone 72   The synthesis of compound 71 started from the known L-threonine derivative 118.76 Ammonolysis followed by chemoselective conversion of the amide into the thioamide set the stage for a Hantzsch thiazole synthesis that gave 119. Condensation of the latter with the anion of readily available methylthiazole 121 (prepared in three steps from commercially available thioacetamide 120) yielded the desired product as a 3:177 mixture of enol (major) and keto tautomers (Scheme 31).  56   Scheme 31. The Ciufolini synthesis of compound 71   The merger of enol 71 and enone 72 turned out to be far from straightforward, requiring extensive experimentation in order to define suitable conditions for each step of the sequence leading to 74. Attempted Michael addition of the enolate of 71 to enone 72 under basic conditions (use of bases such as DBU, Hunig’s base, tBuOK) in different solvents (THF, EtOH, CHCl3 or organic-aqueous mixtures) resulted in decomposition of sensitive enone 72 and recovery of substantially unchanged 71. Bronsted (AcOH) or Lewis (BF3OEt2, Yb(fod)3) acid catalysis provided small quantities of 71, but the desired product was admixed with numerous by-products. The mixtures thus isolated were not synthetically useful. A similar fate awaited attempts to effect the 1,4-addition of silyl ether derivatives of 71 to 72 under electrophilic (Yb(fod)3) conditions.  Solid precedent provided the basis for the suggestion that the loss of 72 was due to Michael polymerisation. Thus, addition of a generic nucleophile to the enone generates a transient enolate that adds to a second molecule of 72 faster that it can be protonated. Such difficulties are certainly not new: several authors have encountered analogous problems and several solutions have been devised over the years. For instance, Stork introduced the use of 57 TMS-enones in such problematic Michael reactions.78 The TMS group stabilises the transient enolate and prevents it from adding to another molecule of enone. Thus, no polymers are formed (Scheme 32).    Scheme 32. The Stork methodology for Michael addition   While a Stork-type modification could have probably cured the foregoing difficulties, an even more practical solution was devised, which relied on an application of heterogeneous catalysis. To wit, a catalytic amount of powdered Li2CO3 suspended in EtOAc converted a mixture of 71 and 72 into 1,5-diketone 73 in nearly quantitative yield. Presumably, the reaction occurs between species adsorbed on the surface of Li2CO3 particles under conditions of extremely low local concentration of 72. This suppresses polymerisation of the latter.  Treatment of 73 with basic agents, including ammonium hydroxide, triggered rapid decomposition, probably via retro-Michael fragmentation and again polymerisation of the nascent 72. Fortunately, a virtually neutral source of ammonia such as NH4OAc yielded dihydropyridine 122 in high yield. A final DDQ oxidation produced the target 74 (Scheme 33).  58   Scheme 33. The Ciufolini synthesis of pyridine 74   Further saponification of the ester and Boc-protection of the oxazolidinone nitrogen yielded acid 123, which was coupled with the threonine derivative 124.79 Notice that the latter fragment incorporates isoalaninol of (R)-configuration (Scheme 34). The silylated alcohol in 125 was deprotected and selectively oxidised to the aldehyde using activated MnO2. The side-chain threonine alcohol was activated using MsCl and Et3N. Subsequent treatment with DBU induced formation of the dehydrobutyric amide segment. It should be noted that no oxazoline 127 was formed in the course of this reaction. Acid 126 was finally obtained through Pinnick oxidation80 of the aldehyde. The order of steps is critical: enamide subunits were found to be extremely sensitive to MnO2, and had to be introduced after oxidation of the thiazolyl alcohol to the aldehyde. Attempted MnO2 oxidation thereof in the presence of dehydroamino acid features resulted in the formation of complex mixtures of poorly characterised products.77 The reasons for this behaviour remain unclear; however, one 59 may assume that the surface of freshly prepared activated MnO2 is acidic; furthermore, it must be properly hydrated for the oxidant to work.81 Consequently, MnO2 may be thought of as a carrier of strong aqueous acid, which may inflict significant hydrolytic damage to dehydro amino acids.  This avenue represents a major improvement of the routes presented earlier, affording acid 126 in 19 steps and in 8% yield along the longest linear sequence.   N S R HN O OMs O R' N S R N O R' O OH ClH3N N H O OAc b O BocN O NS N S N S N SN O HO O H N O N H OAc a) i. LiOH, THF/H2O; iii. Boc2O, DMAP, Et3N, DCM, 95% over 2 step; b) 124, BOP-Cl, Et3N, CH3CN, 86%; c) i. TBAF, THF; ii. MnO2, EtOAc; iii. MsCl, Et3N, DCM; iv. DBU, CHCl3, 68% over 4 steps; v. NaClO2, 2-methyl-2-butene, NaH2PO4, THF, H2O, 80%. 124 126 O H N OH O N H OAc c 125O NBoc O NS N S TBSO N S N S CO2H N 123 74 a O NBoc O NS N S TBSO N S N SN 127   Scheme 34. The Ciufolini synthesis of acid 126   The synthesis of the segment 133 started with Kunieda-type cleavage82 of the oxazolidinone in threonine-derived thiazole 129.79 The latter intermediate was also utilised for the synthesis of the pyridine core (Scheme 35). The D-valine-derived thiazole 130 was 60 prepared according to Meyers.44b Amine 130 emerged in high optical purity, as verified by an NMR study of the Mosher derivative.83 An ominous observation was made at this stage: compound 130 was dextrorotatory, [α]D25 = + 30o ( c= 0.5, CHCl3), even though it was derived from D-valine. Recall that the corresponding acid obtained by Walker upon hydrolysis of micrococcin tended to be laevorotatory, while the acid synthesised from L- valine was dextrorotatory. That which in retrospect proved to be an unwise decision was made at this point, the compound was advanced through the synthesis. Thus, coupling with acid 129 in the presence of DCC, followed by release of the Boc group, furnished dipeptide 131 in high yield. A second DCC-mediated coupling with acid 128 gave alcohol 132, which was advanced to 133 by mesylation of the free threonine alcohol, full deprotection of the threonine subunit and final elimination of the mesylate.   61   Scheme 35. The Ciufolini synthesis of segment 133   The final stage of the synthesis involved coupling of acid 126 with amine 133 in the presence of Bop-Cl to yield 134. Subsequent simultaneous cleavage of the oxazolidinone and the two ester functionalities, followed by deprotection of the N-Boc threonine furnished seco-Bycroft-Gowland MP1, 145. Finally, macrocyclisation using the Yamada reagent, DPPA,84 yielded Bycroft-Gowland micrococcin P1 (Scheme 36) in 23 steps and 5.7% yield from glycolonitrile. 62 O Boc N O N S N S N S N SN O O H N O N H OAc H N N S O NH N S CO2Et N H HO O 126 133 a c Bycroft-Gowland's MP1 94 a) BOP-Cl, Et3N, CH3CN, 90%; b) i. LiOH, THF/H2O; ii. anh HCl, dioxane; c) DPPA, Et3N, DMF, 0 °C, 36h, 65% 3 steps. 134 HO H2N NS N S N S N SN O O H N O N H OH H N N S O NH N S CO2H N H HO O 145 b  Scheme 36. End of the synthesis of the Bycroft-Gowland structure of MP1   Disappointingly, the synthetic material proved to be similar, but definitely not identical, to authentic MP1, as apparent from a comparison of their respective 1H NMR spectra. Especially telling differences were evident in the portion of the 1H NMR spectrum that exhibits the resonances of the thiazole and pyridine hydrogens (Figure 7, Table 1). The optical rotations of synthetic and natural material were not comparable, as measurements were carried out in different solvents ([α]D25 = + 24o (c = 0.13, CHCl3) for 94 vs [α]D25 = + 63.7o (c = 1.19, 90% EtOH) for the natural product).79  63   Figure 7. Low-field section of the 1H NMR spectrum of natural MP (top) and synthetic Bycroft-Gowland's MP1 (bottom) Measured 1H shifts (ppm, natural MP) Measured 1H shifts (ppm, synthetic 94) 8.58 8.43 8.36 8.28 8.19 8.09 8.61 8.46 8.35 8.22 8.16 7.99 Table 1. Reported 1H NMR shifts of thiazoles for natural MP and synthetic 94 in DMSO-d6  2.4 Recent Studies on the Structure of MP1  Ciufolini had thus established that the customary representation of the Bycroft- Gowland structure, 94, is erroneous, but he could not pinpoint the source of error. Indeed, synthetic 94 could have differed from natural MP1 at the level of constitution (recall that the 64 arrangement of the various subunits in the macrocycle was hypothesized based on a presumed structural analogy with other thiopeptides), at that of configuration, or at both.  Extensive spectroscopic studies of the natural product ultimately confirmed the Bycroft-Gowland constitution of MP1,85 indicating that the Ciufolini synthesis had produced a diastereomer of the antibiotic. A likely culprit was the valine-derived thiazole, also because no other known thiopeptide incorporates thiazoles derived from D-amino acids. However, one could not exclude an exceptional case for MP1. Likewise, the configuration of the threonine-derived thiazole, which was left undefined by Bycroft and Gowland, might have corresponded to that of any stereoisomer of the parent amino acid, i.e., L- or D- threonine or allothreonine.  Incisive investigations by Bagley86 resolved stereochemical issues associated with the valine-derived thiazole. Thus, samples of 146 freshly prepared from L-valine using the modified-Hantzsch methodology for the delicate thiazole formation, were unequivocally established to be laevorotatory, just like the analogous product of hydrolysis of micrococcin. Moreover, the specific rotations recorded by Bagley closely match those observed by Walker for the natural material (Figure 8, Table 2). He thus revised the configuration of the valine- derived thiazole of MP1 as the (S)-one, establishing that the fragment originates from natural L-valine. The reason why Walker had obtained a dextrorotatory thiazole from L-valine remains unclear.    Figure 8. Valine-derived thiazole 146 65 Natural 146 Synthetic 146  Bagley Walker  L -derivative D -derivative “L -derivative” – 6.2° – 7.2° – 6.2° + 5.2°  Table 2. Measured optical rotations of 146 (c = 0.5, H2O, T = 25 °C)  2.5 The Bagley Synthesis of the Pyridine Core of MP1  Another significant contribution from the Bagley group is a highly convergent synthesis of the heterocyclic core of MP162c using methodology based on the Bohlmann- Rahtz reaction. The initial plan envisioned the one-pot merger of enamine 150 with alkynone 154. The synthesis of ketoester 149 proceeded uneventfully in seven steps and in very good yield starting from N-Boc-threonine, 147 (Scheme 37). However, conversion of 149 to 150 turned out to be capricious. Treatment with anhydrous ammonia in organic solvents at various temperatures failed to yield the desired enaminoester. In the end, it was found that reaction of 149 with 120 equivalents of ammonium acetate in a mixture of toluene/acetic acid for 10 minutes under microwave irradiation at 150 °C gave the desired product in a reasonable yield (69%). The lack of reactivity of ketoester 149 under usual enamine- formation conditions can partially be explained by two contributing factors: the steric bulk surrounding it, and the fact that the tautomeric enol form of the compound is relatively stable (enough to be seen by 1H NMR and IR spectroscopy). Due to technical constraints related to the large number of equivalents of ammonium acetate required, this reaction could never be tested on more than 100 mg of 149 at a time, but the procedure was highly reproducible.  66 BocHN HO O OH a N Boc O S N O OEt O c N Boc O S N NH2 OEt O 149 150 a) i. DMP, PPTS, THF, reflux, quant; ii. ethylchloroformate, Et3N, 0 °C, then sat. aq. NH3, 78%; iii. POCl3, pyridine, 0 °C, then Et3N, 86%; b) i. (NH4)2S, MeOH, quant; ii. EBP, NaHCO3, DME, rt, then TFAA, pyridine, 0 °C, then Et3N, 87%; iii. ethylchloroformate, Et3N, THF, 0 °C, then potassium ethyl malonate-MeMgBr in THF, 72%; c) NH4OAc (120 equiv.), toluene, AcOH, MW, 150 °C, 69%. b N Boc O CN 148147   Scheme 37. Synthesis of enamine 150   The synthesis of ynone 154 started from dimethoxyacetonitrile 151, which was smoothly transformed into aldehyde 152 (Scheme 38). This intermediate underwent a Grignard reaction with ethynylmagnesium bromide to yield propargyl alcohol 153.    Scheme 38. Synthesis of alkynone 154   Because of the instability of alkynone 154, the alcohol was oxidised just before the Bohlmann-Rahtz coupling, at which point unexpected complications materialised. When an equimolar mixture of precursors 150 and 154 was stirred in ethanol, aminedienone 155 was obtained in only 58% yield, presumably due to the loss of 154 to polymerisation. The yield of 155 reached 81% when an excess (2 equivalents) of 150 were used. Even so, unreacted enamine was recovered from the reaction. Also, cyclodehydration of 155 did not proceed spontaneously, but the addition of a catalytic amount of I2 quickly solved this problem 67 (Scheme 39). Finally, an elegant bidirectional elaboration of the last two thiazoles yielded the desired pyridine core 157.  154150 N Boc O S N NH2 OEt O O N S MeO2C a b N EtO2C N S S N MeO2CNBocO155 156 c N N S S N NBocO 157 N S N S EtO2C EtO2C a) EtOH, 81%; b) I2, MeOH, quant; c) i. LiOH, MeOH, H2O, reflux, quant; ii. ethylchloroformate, Et3N, THF, 0 °C, then sat. aq. NH3, rt, 62%; iii. POCl3, pyridine, 0 °C, 87%; iv. (NH4)2S, Et3N, MeOH, 70%; EBP, KHCO3, DME, then pyridine, TFAA, 0 °C to rt, then Et3N, rt, 96%.   Scheme 39. The Bagley synthesis of the pyridine core of MP1   Again, we are presented here with an interesting synthesis, highly convergent and that illustrates a novel approach to an advanced intermediate en route to MP1. Still, one may anticipate that some experimentation will be necessary to scale up the process in the context of a possible total synthesis, due to the poor stability of the ynone fragment and to technical constraints associated with the microwave-assisted step.  On a different note, the synthesis necessarily leads to a product in which two identical esters (ethyl esters in the case of 157) are present. These have virtually identical reactivity, and it is difficult to differentiate them. An instructive example is found in the Nicolaou synthesis of the GE2270 factors, wherein the saponification of a diester similar to 157 yielded an inseparable mixture of mono-esters, diacid, and starting diester, thus wasting much of the starting material.31 68 3. THE TOTAL SYNTHESIS OF MICROCOCCIN P1   The efforts carried out in our laboratory, the important work of Bagley, and the confusion surrounding the structure of the natural product prompted us to pursue the total synthesis of compound 158 (Figure 9). We hereafter establish that its physical characteristics match those of the natural product. Relative to 94, our target exhibits an L- valine-derived thiazole, in accord with Bagley, and an L-threonine-derived thiazole. This latter decision was made after careful examination of other thiopeptide antibiotics: amino acid-derived thiazoles always derive from natural L-amino acids in the thiopeptide family.    Figure 9. The presumed structure of MP1  3.1 Retrosynthetic Analysis  The present effort relies on key disconnections of MP1 that were dictated by results obtained during the synthesis of 94.  A pair of key disconnections lies in the two amide bonds (a first, then b) that separate the macrocyclic ring into the pyridine core 126 and the tripeptide domain 159 (Scheme 40), providing a highly convergent avenue to the 26- membered ring. The two dehydroamino acid-like features would be created by dehydration of a suitably activated threonine derivative, rather than introduced by employing preformed 69 dehydroamino acid components. In fact, ordinary amino acids are more reactive and better- behaved in coupling reactions than dehydroamino acids, particularly dehydroalanines. A late stage synthesis would then avoid repeated exposure of these sensitive olefins to acids or oxidizing agents. As seen in the previous chapter, the pyridine core of micrococcin, 123, can be regarded as its most interesting and challenging fragment. Following the successful studies by Shen, it would arise from the Michael addition of enolate of 71 onto enone 72, followed by formation of the dihydropyridine under mild conditions and DDQ oxidation thereof.  However, significant work was devoted to improving some problematic steps: for instance, the initial and final steps of the synthesis of enone 72 proved to be more capricious than expected, while a more direct elaboration of the dehydroamino acid fragments was found (vide infra).   Scheme 40. Retrosynthetic analysis, part 1 70  The tripeptide domain, 159, would be assembled from fragments 128 and 160, which would in turn come from thiazoles 129 and 161 (Scheme 41). In this work, the valine- derived thiazole would arise from natural L-valine, as shown below.    Scheme 41. Retrosynthetic analysis, part 2  3.2 Synthesis of the Tripeptide Moiety 159  The synthesis of the building blocks required to construct tripeptide 159 proceeded for the most part as described by Shen.79 Some procedures were improved, while others were modified to deal with side reactions that had previously escaped notice. For example, the known acid 12887 was produced in quantitative yield, as opposed to the 80% originally reported by Shen, by modifying the workup procedure. It was observed that refluxing conditions during the reaction or as means of work-up (evaporation of low-boiling 2,2- dimethoxypropane) produced a yellow to brown solution. This colour is possibly due to the presence of oligomers of 2,2-dimethoxypropane. It was thought that a series of aqueous washes would allow for the isolation of the desired product without contaminants. A first wash of the crude mixture with saturated aqueous NaHCO3 pulls the anion of 128 in the aqueous layer while the organic contaminants in the organic layer are discarded. 71 Acidification of the basic aqueous layer with an aqueous solution of 0.5 M HCl to pH~2 followed by extraction thereof with DCM yields acid 128 quantitative yield (Scheme 42).    Scheme 42. Synthesis of acid 128 and ammonium salt 165   Thiazole 165 was obtained following the modified-Hantzsch reaction in 68% yield over four steps starting from N-Boc L-valine. Ammonolyis of the ethyl ester of 162 constituted a poor route to amide 163. The reaction was very slow even at elevated temperatures (sealed tube), presumably due to the steric hindrance created by the isopropyl and Boc groups around the ester carbonyl. The amidation of 162 was thus achieved through Schotten-Baumann-like reaction88 of the mixed anhydride prepared with ethyl chloroformate with aqueous ammonia solution. Conversion of amide 163 to the corresponding thioamide was performed using Lawesson’s reagent. Flash chromatography of the resulting thioamide afforded material of high purity, but it caused the yield to drop by 50%. Fortunately, the use of crude thioamide in the subsequent thiazole-forming step provided the desired thiazole 164 in good yield: direct reaction of the thioamide with EBP in glyme at –20 °C in the presence 72 of KHCO3 afforded an intermediate alcohol that was not isolated. Removal of the precipitate of potassium salts and addition of TFAA and 2,6-lutidine at –20 °C, followed by warming to 0 °C gave the expected product. The matching measured optical rotation ([a]D18.2 = – 37.9o (c= 1.4, CHCl3)) and literature value44b ([a]D = –37.5o (c= 2.60, CHCl3)) guaranteed the enantiopurity of this compound. Deprotection of the amino group was carried out using 4 M HCl in anhydrous dioxane, providing ammonium salt 165 in quantitative yield. All along this synthesis, anhydrous 4 M HCl in dioxane was preferred to the more commonly used TFA/DCM mixture for practical reasons. The reaction reaches completion within 10 minutes and the workup procedure is simplified, in that it only requires the removal of excess HCl under vacuum. The solution of dioxane is thus concentrated to about half of the initial volume, diluted with anhydrous THF (to decrease the HCl concentration. After another 10 minutes, the volatiles are removed in vacuo and the resulting solid is dried under high vacuum, giving pure ammonium salts in quantitative yield.  While a strongly acidic treatment of allegedly stereochemically labile thiazole 164 was a source of some trepidation, it seemed well established that the racemisation during the Hantzsch synthesis occurs at the stage of the thiazoline.44b Once the thiazole is in place, the molecule becomes more configurationally stable. Thus, we hoped that our thiazole would withstand the harsh acidic conditions of the N-deprotection step. Indeed, it was readily established that no erosion of enantiopurity had taken place during deblocking. To wit, coupling of the product amine with Mosher’s acid83 and careful analysis of the 19F NMR spectra of the resultant amide revealed the presence of only one diastereomer. It is worthy of note that the racemic 166 utilised in the preparation of Mosher amide 167 was obtained from the traditional Hantzch procedure, which proceeded with almost complete loss of configuration as established by 19F NMR spectroscopy (Figure 10). Furthermore, the 73 subsequent coupling of 165 with 129 yielded 160 as a single diastereomer, thus insuring the optical integrity of both fragments (vide infra).    Figure 10. Racemate 166 and its (+)-(R)-Mosher derivative   The synthesis of acid 129 started from L-threonine. Formation of the oxazolidinone ester 118 was performed by treatment of the free amino acid with triphosgene in dioxane63,89 and subsequent esterification. The latter reaction was found to proceed spontaneously when a methanolic solution of acid 168 was allowed to stand overnight in the presence of a catalytic amount of DMAP at room temperature. It is not clear whether esterification occurred under conditions of autocatalysis or whether it was promoted by traces of acidic contaminants or acyl chloride in 168. Ammonolysis of the ester occurred smoothly upon dissolution at room temperature into a methanolic solution of anhydrous NH3(g) in the presence of a catalytic amount of DMAP. Its behaviour thus differed significantly from that of 162, which, as detailed earlier, resisted ammonolysis under such conditions. Selective thionation of amide 169 in the presence of the oxazolidinone was accomplished with Lawesson’s reagent in refluxing benzene. The use of higher-boiling solvents, such as toluene or the customary xylenes, promoted variable degrees of thionation of the oxazolidinone as well. Treatment of the resultant 170 with EBP in refluxing ethanol yielded ester 119, a 74 common building block to the tripeptide moiety and the pyridine core, in a very satisfactory yield of 75% from L-threonine (Scheme 43).    Scheme 43. Synthesis of thiazole 119   A complication that had previously escaped notice materialised at the stage of the conversion of 119 into 129. This transformation required the prior activation of the oxazolidinone nitrogen as a Boc derivative, in accord with Kunieda.82 While this initial step proceeded efficiently upon reaction of 119 with Boc2O and DMAP, the subsequent simultaneous hydrolysis of the oxazolidinone and ester functionalities (treatment with aqueous LiOH in THF) yielded an inseparable 9:1 mixture of acids 129 (desired, major product) and 172 in 80% yield (Scheme 44). The ratio of acids was determined by integration of 1H signals from the corresponding NMR spectrum of the mixture.  75   Scheme 44. Problems faced during hydrolysis of 119   The same reaction had previously been carried out in our laboratory by other workers, who appear to have overlooked the formation of 172. To illustrate, the spectrum of 129 that appears in the dissertation of Y. C. Shen (Rice University, 1998) clearly shows signals arising from 172, even though its presence is mentioned neither in the discussion nor in a later publication (Figure 11).79  76   Figure 11. Shen's 1H NMR spectrum of 129 contaminated with 172 (arrows)   The extreme polarity of compounds 129 and 172 disallowed separation by flash chromatography; moreover, all attempts to purify 129 by recrystallisation failed. In an effort to minimise formation of 172, we attempted first to cleave the oxazolidinone ring according to the original Kunieda procedure, and then to saponify the ester. Indeed, the conditions required for oxazolidinone cleavage (catalytic Cs2CO3 in methanol) are mild, selective, and induce no loss of optical purity in the product.82b It was hoped that this would limit or suppress the formation of the elimination product.  Our expectations were partially realised: scrutiny of the NMR spectra of the crude product of oxazolidinone cleavage revealed a diminished extent of formation of 172. However, the overall yield of desired 129 after the subsequent saponification of the ester was comparable to that of the one-step sequence. Therefore, we saw no advantage in 77 implementing the 2-step route to 129, and the mixture of acids resulting from the simultaneous double hydrolysis was used as such in the following coupling reaction.   The last stages of the synthesis of tripeptide 159 involved the coupling of the three fragments and dehydration of the threonine-derived thiazole. Thus, condensation of the mixture of acids 129 and 172 with thiazole amine 165 in the presence of DCC and HOBt, followed by deprotection of the amine using anhydrous HCl in dioxane, yielded dipeptide 160 in 75% (Scheme 45).    Scheme 45. Synthesis of amine 160   It is noteworthy that no epimerisation of potentially labile nitrogen-bearing stereogenic carbons occurred under the acidic conditions required for Boc release. This is borne out of the fact that product 160 was obtained as a single diastereomer within the limits of 300 MHz 1H and 75 MHz 13C NMR spectroscopy (Figure 12).  78     Figure 12.1H (above) and 13C (below) NMR spectra of 160   A second coupling using DCC between amine 160 and acid 128 yielded tripeptide 174 in 79% yield (Scheme 46). Past schemes for the advancement of an epimer of 174 to the epimer of 159 relied on a four-step sequence involving mesylation of the free OH group, TFA release of the Boc, acetonide hydrolysis with aqueous HCl, and DBU elimination of the mesylate. This modus operandi was presumably dictated by an anticipated sensitivity of the dehydroamino butyramide segment to the acidic conditions required for the deprotection of the threonine segment: it thus seemed unwise to introduce the dehydroamino acid feature prior to acidic hydrolysis.  OH NH2 N S O HN N S CO2Et 160 79   Scheme 46. Synthesis of alcohol 174   Furthermore, it was indicated that the elimination of the mesylate succeeded only in the presence of a fully deblocked threonine segment, because oxazoline formation occurred if that amino acid residue was blocked (Scheme 47).77  OH H N R O N BocO OMsH N R O N BocO MsCl N O N Boc O R   Scheme 47. Alleged formation of oxazoline during dehydration of threonine   The literature provides ample precedent for the sensitivity of dehydro alanines, which are customarily introduced at the last step of a synthesis.26,27,32 However, no evidence could be garnered in support of a hypothetical sensitivity of dehydro butyramides to anhydrous protonic acids or to dilute aqueous acidic solutions. This led us to surmise that it may be possible to convert 174 into 159 in only two steps, provided that conditions could be found for the simultaneous activation-elimination of the OH group and the subsequent deblocking of the terminal threonine segment in the presence of the dehydro butyramide.  80  Fortunately, only a modest amount of effort was necessary to translate the foregoing hypothesis into practice. Reaction of 174 with MsCl (1.0 equivalent) and Et3N (1.3 equivalents) in DCM at room temperature resulted in rapid conversion to a mixture of mesylate 175 and traces of dehydroamino acid 176 (Scheme 48).    Scheme 48. Synthesis of amine 159   Contrary to claims presented in an earlier publication from our laboratory,79 some mesylate elimination was already taking place in the presence of a fully protected threonine unit. Introduction of additional triethylamine caused no further elimination. However, addition of excess DBU promoted complete, if slow (12 hours) conversion of the mesylate to 176. The dehydro butyramide unit was thereby installed in a single step and in 93% chromatographed yield. Subsequent treatment of 176 with anhydrous 4M HCl in dioxane triggered rapid release of the Boc group. Vacuum was then applied to remove excess HCl, 81 and finally the solution was diluted with THF and water. The residual acidity still present in the medium sufficed to promote hydrolysis of the acetonide with no adverse effect upon the dehydroamino acid. Amine 159 was obtained in quantitative yield after a basic aqueous wash.  It was interesting to observe some elimination product during the mesylation step, even though excess Et3N had no effect on 175. Evidently, the elimination of the mesylate from 176 cannot be attributed to its interaction with Et3N. This raises a question: what is the mechanism of the observed elimination reaction?  It is commonly accepted that the mechanism of mesylation in the presence of Et3N involves dehydrochlorination of MsCl (Scheme 49). An alcohol then attacks the resultant sulfene 177, and a presumed transient dipolar intermediate 179 thus formed advances to the ultimate 180 through a series of proton transfer reactions.90 We speculate that as a result of the activation provided by the thiazole ring to the starred proton in 178, an intermediate of the type 179 partitions between a reaction pathway involving proton transfer events (path a), and leading to 180, and one involving a 5-centre intramolecular proton transfer (path b), and that leads to 181 which displays the wrong geometry for the double-bond. A possible in situ isomerisation of 181 may occur to give the desired and observed geometry of the olefin.    Scheme 49. Mechanism of threonine dehydration 82  Some support for the foregoing hypothesis derives from an observation made earlier in this group.91 Thus, mesylation of salicylaldehydes 182 in the presence of Et3N produced variable amounts of cyclic sulfonate 184 in addition to the expected 183. Remarkably, base treatment of 183 (NaH, t-BuOK) failed to produce additional 184, none of which was observed when the mesylation reaction was carried out in the presence of pyridine. This is consistent with the accepted mechanism of pyridine-mediated mesylation reactions, which are believed to proceed via intermediate 187.92    Scheme 50. Mechanisms of mesylation  3.3 Synthesis of the Precursors to Pyridine 126  As shown earlier in Scheme 40, the pyridine-thiazole cluster of MP1 arose through the merger of ketone 71 and enone 72. Substance 71 was formed by the addition of the anion 83 of 121 onto ester 119, which thus served as a building block for two key fragments of the natural product. 2-Methylthiazole 121 was prepared following Shen’s method: thioacetamide, 120, and EBP were reacted in refluxing EtOH to yield ester 189. This ester was reduced to the corresponding alcohol using two equivalents of DIBAL. Protection of the OH group (TBSCl in the presence of imidazole) gave 121 in 91% yield (Scheme 51).    Scheme 51. Synthesis of thiazole 121   The union of the two fragments entails treatment of 119 with three equivalents of the anion of 121. The first equivalent serves to deprotonate the nitrogen atom of the oxazolidinone, the second to perform the addition onto the ester, and the third to deprotonate the acidic product ketone (Scheme 52). At the end of the reaction, the desired ketone 71 was isolated as a 1:3 mixture of the keto (minor) and enol (major) tautomers, while excess 121 was recovered by flash chromatography. The desired product was accompanied by a small amount of alcohol 190, which results from double addition of the anion of 121 to 119. Compound 190 was isolated in 7% chromatographed yield from a reaction carried out on a 2-gram scale. This contaminant had escaped detection during past work carried out in our laboratory.77  84   Scheme 52. Synthesis of ketone 71   Later in the synthesis, the oxazolidinone in 119 must be cleaved by a Kunieda-type reaction: this requires activation of the oxazolidinone as the N-Boc derivative. To decrease the number of equivalents of 121 utilised in the synthesis of 71 and to shorten the longest linear sequence leading to the ultimate goal, we attempted condensation of the anion of 121 with the N-Boc derivative of 119, ester 171 (Scheme 53). However, such attempts proved to be unsuccessful, affording only intractable mixtures of products that seemed to contain none of the desired 191. Presumably, the N-Boc derivative of the oxazolidinone is sufficiently reactive to condense with the anion of 121.    Scheme 53. Attempt at obtaining 191  85  Unlike the avenue to 71, the established sequence leading to 72 (Scheme 30)63 was in need of significant improvement. A difficulty was encountered during the very first step. Thioamide 192, required for the preparation of thiazole 193, is extremely polar and water soluble, complicating purification by column chromatography or aqueous extractions. The procedure devised originally63 entails reaction of a commercial 55% aqueous solution of glycolonitrile with gaseous H2S in the presence of pyridine and triethylamine, followed by evaporation of the reaction mixture to a thick oil. This leaves a residue of crude 192 which may be used directly in a Hantzsch thiazole synthesis. Because the formation of the thiazole ring is consistently quite efficient on a variety of thioamides, one may surmise that the yield of thiazole reflects the yield of thioamide obtained in the previous step (Scheme 54).    Scheme 54. Synthesis of aldehyde 117   The above procedure proved to be capricious, furnishing thiazole 193 in a variable 10-40% yield. Attempted thionation of glycolonitrile by addition of Na2S in the presence of organic or inorganic bases (for example Et3N, pyridine, pH 4, 7 or 10 phosphate buffers, KHCO3) to the commercial aqueous solution of substrate apparently produced none of the required thioamide. In fact, utilisation of the residue obtained upon evaporation of the above solution in a Hantzsch reaction yielded none of the thiazole 193. This was all the more 86 disappointing in light of Bagley’s reported thionation of dimethoxyacetonitrile 151 in quantitative yield with ammonium sulphide in anhydrous methanol.61 Ultimately, an attempt of the sequence with 250 mmol of glycolonitrile afforded enough alcohol 193 (35% yield) to continue the synthesis. The next five steps proceeded uneventfully to afford aldehyde 117 in a very satisfactory 41% overall yield from 193, after a few minor modifications in the route devised by Shen. Namely, after the Lawesson reaction, the solid in suspension in the crude mixture was filtered off and after concentration under vacuum and dissolution in CHCl3, a basic wash (saturated aqueous NaHCO3) allowed for the removal of most of the by-product of the Lawesson reagent. Aldehyde 117 is a nicely crystalline solid, (m.p. 156-157 oC), with a very long shelf life (> 3 years).  The final steps in the synthesis of enone 72 featured the addition of vinyl magnesium bromide to aldehyde 117 and oxidation of the resultant alcohol (Scheme 55).    Scheme 55. Elaboration of enone 72 from aldehyde 117   The Grignard reaction soon proved to be problematic when run on scales greater than 30 mg. The original procedure called for careful addition of the organomagnesium species into a cooled (–78 °C) solution of the aldehyde in anhydrous THF. However, even dilute (0.01 M) THF solutions of the aldehyde turned into a slurry when cooled to –78 °C. Such dilute solutions, however, remained homogeneous at temperatures higher than –40 °C. 87 Addition of vinylmagnesium bromide to a homogeneous solution in the range of –40 °C to room temperature resulted in incomplete conversion. A typical crude reaction mixture contained about 40% of the desired alcohol, 30% of starting aldehyde, and 30% of a mixture of at least four unidentified by-products. Mass spectrometry suggested that some of these had resulted from double or triple addition of the Grignard reagent to the substrate. The use of more than about 1.5 equivalents of Grignard reagent did not solve the problem, in that it caused formation of an inseparable mixture of polyalkylated compounds. Also, reduction of the aldehyde occurred and the corresponding alcohol was isolated as a major product when the reaction was run under refluxing conditions.  Running the reaction in anhydrous Et2O as a solvent, instead of THF, was troublesome on account of the poor solubility of 117 in the new solvent. Adding 117 to the Grignard reagent at temperatures between –20 and +25 °C caused the yield of 194 to drop, probably because under these conditions the aldehyde introduced into the reaction found itself in the presence of excess Grignard reagent. This favours the occurrence of multiple addition events.  To complicate matters, perfectly homogeneous solutions of crude alcohol 194 in, e.g., CDCl3, produced 1H NMR spectra that were unusually broad. Also, the compound appeared as a streak on analytical thin-layer chromatographic plates, regardless of the solvent system utilised as the eluant (e.g.: mixtures of DCM, MeOH, EtOAc, hexanes, AcOH). We suspect that this was due to the fact that 194 had coordinated metallic species, probably Mg2+ ions, to form complexes of general structure 195 (Figure 13).  88   Figure 13. Possible chelates of 195 and 196   Aquo- or hydroxo ligands around the metal might promote proton exchange reactions at a rate comparable to the sampling speed of the NMR spectrometer, resulting in severe line broadening (Figure 14). In a like vein, sequestration of the Grignard reagent by chelation of the magnesium centre by the two nitrogen atoms of the bis-thiazole, forming compounds such as 196, may partially explain the unexpected behaviour of 117 in this seemingly straightforward reaction.    Figure 14. 1H NMR spectrum of crude 196  N S R N S CO2Et Mg Br 196 89 Finally, chromatography of crude 194 did afford a pure sample exhibiting the anticipated spectral characteristics, but the yields ranged from 20-40% (Figure 15).    Figure 15. 1H NMR spectrum of chromatographed 194   It ultimately transpired that crude alcohol 194 could be directly oxidized to 72. The enone could then be purified with no significant losses. Reproducible yields of 72 in the 30- 40% range from 89 were thus realised by refluxing a solution of 1.2 equivalents of the Grignard reagent and 1.0 equivalent of aldehyde in anhydrous THF, and by subjecting the resulting crude alcohol to oxidation with MnO2 (Scheme 55, route a).  The search for an even better alternative led us to investigate the addition of the more reactive vinyl lithium to the aldehyde. Several sources of vinyl lithium were examined, as this reagent is not commercially available. Vinyllithium prepared by transmetallation of tetravinyltin or tributylvinyltin with n-BuLi gave the desired alcohol in yields that varied between 10 and 50%. While the crude 194 was still contaminated with aldehyde and with some products of multiple addition, it displayed none of the foregoing problems with broad N S HO N S CO2Et 194 90 spectra and streaky TLC traces. Finally, a quantum improvement was realised by preparing vinyllithium by halogen-metal exchange reaction of vinyl bomide with nBuLi. The yield of chromatographically purified 194 was 65%. The subsequent MnO2 oxidation of 194 to enone 72 occurred in quantitative yield (Scheme 55, route b). We note that the observed differences in the reactivity of “vinyllithium” prepared by different methods are consistent with the work of Seyferth, who as early as 1961 determined that reagent prepared from vinyltin species by a transmetallation reaction behaves differently from that prepared from vinyl bromide by the direct method or by halogen-metal exchange.93  3.4 Exploration of Alternative Routes to Enone 72  Parallel work focused on shorter routes to 72 that would circumvent the problems with the thionation of glycolonitrile and with the vinylmetallic addition step. One attractive possibility consisted in the linear elaboration of the two thiazoles starting from a compound already displaying the vinyl and OH groups found in 194. To this end, acrolein 197 was converted into the known TMS-protected cyanohydrine 198 by reaction with TMSCN.94 A technical modification of the literature procedure consisted in suppressing all additives: mixing one equivalent each of reactants furnished the desired product, a colourless oil which is indefinitely stable under argon at -18 °C, in quantitative yield. This intermediate was then subjected to various conditions in pursuit of the formation of the corresponding thioamide 199 (Scheme 56). However, the use of various sources of sulphide ion on 198 (H2S(gas), anhydrous and nonahydrate Na2S), in the presence of a wide range of bases (e.g., pyridine, Et3N, phosphate buffer, DMAP…), different solvent systems (H2O, MeOH, pyridine, THF and various mixtures thereof) and Lewis acid catalysis (BF3OEt2), resulted, at best, in the 91 desilylation of the alcohol. More frequently, the solution darkened and oligomers of 198 were obtained, as apparent from mass spectroscopy analysis.    Scheme 56. Derivatisation of acrolein   A second approach aimed to utilise the linear elaboration of the thiazoles in the hope of forming a suitable organometallic species at a later stage following a methodology developed by Knochel.49e-g Accordingly, intermediate 200 would be obtained from 201 using the now familiar linear sequence (Scheme 57).  72 N S RMg N S CO2Et S N CO2Et I 200 201   Scheme 57. Retrosynthetic analysis of 72 via 200   Iodothiazole 201 was unknown when we first considered it a possible intermediate (2006), unlike its 2-bromo analogue.47 The compound was prepared from aminothiazole, 202 which in turn was made by Hantzsch reaction of thiourea and EBP. Iodination of 202 under traditional Sandmeyer conditions (NaNO2, aq. HCl, then KI)95 formed an essentially 1:1 mixture of desired 201 plus the reduced thiazole 203 (ca. 60% yield), which were difficult to separate. Happily, 201 resulted in 60% yield upon treatment of 202 with isoamyl nitrite in 92 acetonitrile at 0 °C in the presence of diiodomethane. A recent paper by Bach describes the synthesis of 201 by the same method.30  a) EBP, EtOH, reflux, quant; b) CH2I2, CH3CN, then i-AmONO, 0 °C, 60%. H2N S NH2 a 202 203 b N S I O OEtN S H2N O OEt 201 N S H O OEt   Scheme 58. Elaboration of halothiazoles 201   The elaboration of the second thiazole required ammonolyis of 201 and Lawesson thionation of the resultant amide (Scheme 59). These two steps proceeded with abnormally low recovery of material (less than 20%). The crude product thus retrieved was not fully characterised, but it was directly reacted with EBP. This gave a complex mixture containing only trace amounts of the desired bis-thiazole 205 (detected by 1H NMR and mass spectroscopy). The precise reasons for this failure were not investigated, and indeed the route was soon abandoned. Perhaps, the iodo substituent underwent aromatic nucleophilic displacement in the presence of agents such as NH3 or thioamide 204, to the detriment of overall efficiency.    Scheme 59. Attempt at the functionalisation of 201  93  A modification of the above strategy envisioned a Kelly-type49d coupling of 201 and 207 and reaction of the resultant with an appropriate acyl chloride, aldehyde or ketene, according to Dondoni’s methodology49a-c (Scheme 60).96 The coupling of thiazole fragments 201 and 207 required conversion of either unit to an organometallic species. Since iodothiazole 124 was already available, we concentrated on the preparation of the known trimethylsilylthiazole 207. Seeing the sometimes poor behaviour of stannylated compounds faced by Kelly, we decided that another metal could be installed to effect the subsequent cross-coupling reaction.    Scheme 60. Retrosynthetic analysis of 72 via bis-thiazole 206   To this end, halothiazole 33 was prepared as shown in Scheme 61. Thiazolidinedione 32 was treated with neat POBr3 at 110 °C to produce dibromothiazole 33 in 63% chromatographed yield. Some tribromothiazole (5-10% yield) was also obtained as a by- product.    Scheme 61. Synthesis of thiazole 33  94  Dondoni detailed the following procedure for the preparation of stannylthiazole 39.49c A solution of bromothiazole 33 in Et2O was added over 1 h to a stirred solution of n-BuLi in Et2O at –78 °C, and the mixture was kept at that temperature for another hour. An Et2O solution of an electrophile, e.g., TMSCl, was introduced over 15 minutes and the mixture was again stirred at –78 °C for another hour before a standard aqueous workup. Exactly the same procedure was recommended for the conversion of 207 into 39, except that the intervening anion was intercepted with Me3SnCl (Scheme 62).    Scheme 62. The Dondoni synthesis of 39   The Dondoni chemistry could be duplicated without problems for the synthesis of 207. However, some unexpected results arose upon slight modifications of his procedure. Replacing diethyl ether with THF in the first step of the sequence afforded a mixture of 209 (E = TMS) and 210 (Scheme 63). Furthermore, a mixture of compound 211 and starting 33 emerged from one run of the same step in THF in which the electrophile was accidentally introduced immediately after the end of the addition of 33 to n-BuLi, instead of allowing the mixture first to stir for the prescribed hour. More interestingly, running the reaction in THF with addition of n-BuLi to 33 (opposite order of addition relative to Dondoni) promoted formation of 212 in high yield. The nature of regioisomer 212 was established by 95 protonolysis to the known 213.49c All these reactions were duplicated numerous times using different electrophiles (E), as detailed below.    Scheme 63. Products of treatment of dibromothiazole 33 with n-BuLi   Table 3 provides a summary of such experiments. All reactions were carried out in flame-dried flasks under argon atmosphere. In column 4, “I” stands for “inverse addition” of the brominated species to n-BuLi, and “D” refers to the “direct” mode of addition of n-BuLi to the brominated species. A “slow addition” means that the substrate was added over 1 h whereas “fast addition” means it was added dropwise over 5-10 minutes. Three electrophiles were used: TMSCl, Me3SnCl and Bu3SnCl. Yields of the announced products or mixtures varied very little from an entry to another and ranged from 75 to 85% chromatographed yields. The electrophiles are hereafter referred to as “E+” (respectively “E’+”) when under their chlorinated form, and “E” (respectively “E’”) after exchange. After the addition of the electrophile, the reaction was stirred for 1 h at –78 °C and quenched by addition of a saturated aqueous NaHCO3 solution according to Dondoni’s procedure. The procedures were reproduced at least twice for entries 1-5 with at least two electrophiles.  96 Entry Solvent Equiv. of n-BuLi Mode of addition of n-BuLi Duration of Mt/X exchange Mode of addition of E+ Product 1 Et2O 1.1 I, slow 1 h slow 209 2 THF 1.0 I, slow 1 h slow 209 and 210 3 THF 1.2 I, slow - fast 211 and 33 4 THF 1.4 D, fast 30 min fast 212 5 THF 0.9 D, fast 30 min fast 212 6 THF 2.0 I, slow 1 h slow E+, slow E’+ complex mixture 7 Et2O 2.0 I, slow 1 h slow E+, slow E’+ under study Table 3. Experimental conditions for functionalisation of 33   It appears that the major factor affecting the outcome of the reaction, besides the solvent, is the mode of addition and the amount of time that the solution is allowed to stir before addition of the electrophile. The precise amount of n-BuLi utilised in the reaction has little effect on the outcome, as illustrated by entries 4 and 5.  Entries 1, 2 and 3 allow us to infer a possible mechanism for this reaction (Scheme 64). Slow addition of substrate on an n-BuLi solution promotes rapid formation of mono- anion 214 by halogen-metal exchange. However, the nascent 214 reacts with excess n-BuLi present in the medium, again by halogen-metal exchange, to form dianion 215. Once all the n-BuLi has been neutralised—probably after addition of 0.5 equivalents of dibromothiazole– the solution contains roughly 0.5 equivalents of dianion. Continued addition of dibromothiazole induces halogen-metal exchange between the more reactive anionic position of 215 (C-4) and the more reactive electrophilic one 33 (C-2). This reaction may be slow, 97 rationalising the requirement for a 1 h equilibration after the end of the addition of the starting dibromothiazole. Addition of the electrophile produces the various products isolated. When the mixture is not allowed to equilibrate, and the electrophile is added immediately, 211 is formed, which corresponds to the reaction of the most reactive site of the dianion with the electrophile. It is of note that no material was isolated that possessed two TMS groups.    Scheme 64. Possible mechanisms for lithiation of dibromothiazole 33   When the reaction is carried out in THF a parasite reaction takes place during the equilibration phase: the dianion reacts with by-product bromobutane to give 2-butylthiazole 210.  The formation of product 212 shown in entries 4 and 5 is not as easily explained. However, a rationale involving intermolecular rearrangement of the mono-anion of the thiazole could be supported by observations made by others in their work with thiazoles. Dondoni documented the deprotonation of 2-TMS-thiazole and established that it favours position C-5 of the ring.49c,96 The rearrangement of thiazole anions was touched upon by Kelly in his endeavours towards the synthesis of dimethyl sulfomycinamate.47 Thus, in the 98 absence of excess n-BuLi anion 214 could rearrange to 216, which, upon treatment with an appropriate electrophile E+ would yield product 212 (Scheme 65).    Scheme 65. Rationale for the formation of thiazole 212   According to the mechanisms proposed above, treatment of 33 with two equivalents of n-BuLi should give a solution of dianion 215 which would undergo bis-functionalisation in one pot with two different electrophiles to give 217 (Scheme 66).    Scheme 66. One-pot double functionalisation of dibromothiazole 33   When this reaction was attempted in THF (entry 6), a mixture of various thiazoles was obtained that could not be fully separated. The reaction carried out in Et2O (entry 7) is under study in our laboratory at the time of redaction of this document. Various electrophiles could be employed and give access to a wide range of compounds in one step, including, but not limited to stannyl, zinc, silyl or borate derivatives, dry ice, aldehydes or even acyl chlorides. When acrolein was used as an electrophile during a reaction following entry 6 (Table 3), a product containing the allylic alcohol at position 2 was observed in the mixture of products. 99  When 212 was obtained with Bu3Sn as “E”, the treatment of this compound with various Pd species (e.g., [Pd(OAc)2], [PdCl2(PPh3)2], [Pd2(dba)3], [Pd(PPh3)4]) were attempted. Various solvents (DMF, dioxane, benzene) and reaction temperatures were also considered. It was hoped that trimer 218 would form (Scheme 67). Unfortunately, the starting material was recovered unchanged from several such attempts.    Scheme 67. Attempted formation of 218.   As mentioned earlier, the alternative coupling of bromo-derivative 207 with a suitably substituted organometallic species such as 219 could also lead to compound 206 (Scheme 68).49b Efforts toward the synthesis of fragment 206 by the use of this technique are ongoing in our laboratory.  N S TMS Br 207 N S CO2Me BrZn 219 N S TMS N S CO2Et 206   Scheme 68. Possible synthesis of bis-thiazole 206  3.5 Assembly of Pyridine 74  The union of fragments 71 and 72 was realised according to the originally-designed route. Michael adduct 73 was formed in high yield upon reaction of an equimolar solution of 71 and 72 in EtOAc under the catalytic influence of suspended, powdered Li2CO3 (Scheme 100 69). Treatment of an ethanolic solution of 73, a fragile, base-sensitive intermediate, with NH4OAc, a neutral source of ammonia, followed by DDQ oxidation of the presumed dihydropyridine 122, afforded pyridine 74. When the reaction was carried out on scales of 10-20 mg, 5-10 equivalents of NH4OAc sufficed to induce conversion of 73 into 122 at a reasonable rate (2-3h, monitored by 1H NRM spectroscopy). On larger scales (>200 mg), a greater excess of the salt (up to 20 equivalents) was necessary. Perhaps as a result of secondary reactions between excess NH4OAc and the DDQ utilised in the subsequent oxidation step, the yield of 74 drops substantially (as low as 44% after chromatography) under these conditions, unless excess salt is removed. This is readily accomplished by addition of brine to the ethanolic solution and extraction of the mixture with EtOAc. This solution is dried over MgSO4, concentrated and redissolved in toluene. It is then titrated with a solution of DDQ in toluene (as DDQ proved to be insoluble in chloroform, the solvent originally used) until a sole light blue spot is visible under UV light.   Scheme 69. Synthesis of pyridine 74 101   Pyridine 74 was intended to undergo coupling with the isoalaninol-containing fragment 124. This intermediate was prepared as shown in Scheme 70. Coupling of (R)-1- amino-2-propanol with bis-protected threonine 128 using DCC and acetylation of the free alcohol (Ac2O, pyridine, DMAP) yielded amide 220 in 84% yield. Release of the Boc and the acetonide protecting groups using the same method detailed earlier for segment 159 (4 M anh HCl in dioxane, followed by dilution of the solution with THF, then H2O) yielded ammonium salt 124 in quantitative yield.    Scheme 70. Synthesis of ammonium salt 124   Pyridine 74 was advanced to acid 123 by ester hydrolysis and protection of the oxazolidinone using Boc2O. Coupling with ammonium 124 afforded amide 125 in 77% yield over three steps. One-pot dehydration of the threonine subunit with MsCl and Et3N followed by DBU gave olefin 221. Finally, acid 126 was reached by cleavage of the silyl protecting group in 125, Dess-Martin97 oxidation of alcohol 222 to the corresponding aldehyde, followed by Pinnick oxidation80 (Scheme 71).  102   Scheme 71. Synthesis of acid 126   One-step oxidation of the primary alcohol after deprotection of the TBS-protected alcohol was attempted using a mixture of IBX and 2-hydroxypyridine, HOBt or N- hydroxysuccinimide.98 These conditions afforded inseparable mixtures of the desired acid plus an overoxidised byproduct, which we believe to be the pyridine N-oxide derivative of 126 on the basis of its 1H NMR and mass spectra.  3.6 Total Synthesis of Micrococcin P1  The final sequence of the synthesis started with the coupling of acid 126 with amine 159 in the presence of BOP-Cl. Exposure of the resulting 224 to the action of aqueous LiOH 103 in THF promoted the simultaneous saponification of the two ester functionalities (the side- chain acetyl alcohol and the macrocyclic ethyl ester) and the cleavage of the oxazolidinone. Release of the N-Boc with 4 M anhydrous HCl in dioxane afforded monoseco intermediate 225. Yamada macrolactamisation84 gave totally synthetic micrococcin P1, 1, in 29% yield over five steps (Scheme 72).    Scheme 72. Total synthesis of micrococcin P1 1 104  This compound was spectroscopically and polarimetrically identical to the natural product, an authentic sample of which was kindly provided by Prof. E. Cundliffe, of the University of Exeter, U.K. Relevant spectra and data are shown in Figure 16, Table 4 and Table 5 below.     Figure 16. 1H NMR spectrum of synthetic (above) and natural (below) MP1, in DMSO-d6      105 Measured 1H shifts (ppm, natural MP) Measured 1H shifts (ppm, synthetic 94) 9.53 a (br s, 1H) 9.48 a (br s, 1H) 8.59 (s, 1H) 8.46 (d, 1H) 8.46 (s, 1H) 8.38 a (1H) 8.37 (s, 1H) 8.35 (d, 1H) 8.30 (s, 1H) 8.22 a (1H) 8.21 (s, 1H) 8.12 (s, 1H) 7.88 a (br t, 1H) 7.86 a (br d, 1H) 6.52 (q, 1H) 6.48 (q, 1H) 5.42 b (d, 1H) 5.15 (dd, 1H) 5.09 (dd, 1H) 4.80 b (br d, 1H) 4.71 (dd, 1H) 4.67 b (d, 1H) 4.39 (m, 1H) 4.03 (m, 1H) 3.72 (m, 1H) 3.09 (m, 2H) 2.51 (m, 1H) 9.50 (br s, 1H) 9.50 (br s, 1H) 8.59 (s, 1H) 8.45 (d, 1H) 8.45 (s, 1H) 8.38 (s, 1H) 8.37 (s, 1H) 8.34 (d, 1H) 8.29 (s, 1H) 8.21 (s, 1H) 8.20 (s, 1H) 8.11 (s, 1H) 7.89 (br t, 1H) 7.85 (br t, 1H) 6.51 (q, 1H) 6.46 (q, 1H) 5.41 (d, 1H) 5.13 (m, 1H) 5.07 (m, 1H) 4.77 (br d, 1H) 4.68 (dd, 1H) 4.63 (d, 1H) 4.38 (m, 1H) 4.01 (m, 1H) 3.70 (m, 1H) 3.08 (m, 1H) 2.52 (m, 1H) 106 Measured 1H shifts (ppm, natural MP) Measured 1H shifts (ppm, synthetic 94) 1.76 (d, 3H) 1.72 (d, 3H) 1.39 (d, 3H) 1.04 (d, 3H) 1.03 (d, 3H) 0.98 (d, 3H) 0.87 (d, 3H) 1.75 (d, 3H) 1.69 (d, 3H) 1.37 (d, 3H) 1.03 (d, 3H) 1.01 (d, 3H) 0.97 (d, 3H) 0.86 (d, 3H) aNH proton.  bOH proton.  Table 4. Reported 1H NMR shifts for natural MP and synthetic 1 in DMSO-d6  Natural 1 Synthetic 1 + 63.7 ° (c = 1.19, T = 21 °C) 99 + 68.9 ° (c = 0.45, T = 19.7 °C) Table 5. Reported optical rotations in 90% EtOH in water.   To the best of our knowledge, this effort constitutes the first total synthesis of the thiopeptide antibiotic, micrococcin P1. The synthesis is convergent and it yields MP1 in 1.0% yield along the longest linear sequence, which comports 21 steps from glycolonitrile (Scheme 73). The work also established the absolute stereostructure of the natural product in an unambiguous manner. Hopefully, this will clear the considerable degree of confusion that has accumulated in the micrococcin field over the past 30 years as a result of seemingly contradictory claims and counterclaims.  107   Scheme 73. Summary scheme of the total synthesis of micrococcin P1   The modular nature of the synthesis can also accommodate modifications for SAR studies, contributing thereby to the fields of medicinal chemistry, pharmacology, and microbiology. At a purely chemical level, we remain confident that this work will serve as a valuable guide in the elaboration of other members of the thiopeptide family.  108 4. FERMENTATION   As indicated earlier, a sample of authentic micrococcin P was secured through the courtesy of Prof. E. Cundliffe. This did not dissuade us from attempting to ferment additional antibiotic for the purpose of comparison with our synthetic material and for other studies. This chapter summarises the efforts to isolate the natural product following the procedure designed by Scherer.68  The work detailed herein could not have been accomplished without the expert assistance of Dr. Elena Polishchuk and the staff of the Biological Services Laboratory of the Department of Chemistry of the University of British Columbia, and of Dr. David Williams from the group of Prof. Raymond Andersen, also of this Department. We express our heartfelt gratitude to our colleagues for their insightful discussions and invaluable help.  4.1 Background Work  As mentioned in the second chapter of this work, three different bacteria have been reported to produce MP or MP1: a strain of Micrococcus, a strain of Bacillus pumilus and Staphylococcus equorum WS 2733. The first organism had been found in sewage water from Oxford, England, the second one was isolated from soil collected in East Africa, and the last one was reported to grow on French raclette cheese. Anticipating that, if need be, it would be easier to get raclette cheese than to collect East African soil or British sewage water, we opted for the caseous approach. To avoid additional isolation steps, we started our study by the direct culture of the bacterium identified by Scherer.   109 4.2 Culture of the Commercial Bacterium  We were delighted to find that the micrococcin-producing strain of S. equorum studied by Scherer is commercially available from American Type Culture Collection (ATCC). An inoculum of the commercial bacteria was incubated for 24 hours at 30 °C in a chicken brain and heart infusion (BHI) broth. The rapid appearance of cloudiness in the broth confirmed that this bacterium is non-fastidious.  In order to test for activity, paper discs were dipped in the broth and placed on agar gel cultures of six strains of Gram-positive Bacillus cereus and Enterococcus cloacae. Some inhibitory activity was detected against two strains of B. cereus and very weak activity against one strain of E. cloacae (Figure 17). In the other cases, no activity was detected, and occasionally overgrowth of the host bacterium was observed. No activity was observed even when the broth was tested against against methicillin-sensitive S. aureus (MSSA), although this organism is known to be quite sensitive to MP. It is worthy of note that MSSA is an attenuated form of the highly pathogenic methicillin resistant S. aureus (MRSA).  110   Figure 17. Poor inhibitory activity of the culture broth of S. equorum WS 2733   The lack of activity might have been due to the inappropriate timing of collection of the broth. Indeed, many organisms produce their secondary metabolites (= natural products) after variable periods of induction. However, the same weak activity was detected after 1, 3, 5, 7 and 14 days of incubation. This was rather disappointing, considering the report by Scherer: 217 out of 226 strains of Gram-positive bacteria tested were sensitive to the action of a culture containing S. equorum.  Parallel work aimed to detect, and hopefully isolate, natural micrococcin P from fermentation broth collected at each of the above time intervals. A 300 mL portion of broth was centrifuged at 12,000 g for 20 minutes at 4 °C. The supernatant was collected and extracted with 250 mL portions of DCM (3 times), EtOAc (3 times) and 1-butanol (3 times). The extracts were combined, dried over MgSO4 and concentrated under vacuum. Each time, TLC, MS, and 1H NMR analysis of the residue failed to indicate the presence of MP1. The 111 pellets of biomass obtained upon centrifugation were suspended in methanol and the suspension was sonicated to destroy the cells and release their contents. Filtration and evaporation left a residue that was redissolved in water and extracted following the same procedure as earlier. Again, no MP was detected. These setbacks induced us to start from the cheese itself, i.e., the reported source of S. equorum.  4.3 Isolation from French Raclette Cheese  Kilogram samples of authentic French Raclette cheese were obtained from reputable Fromagers located in the cities of Paris100 and Lyon.101 Obviously, there is very little chance that these samples had came from the same cheese factory as the one in which the cheese studied by Scherer was produced. Therefore, it is likely that the bacteria found by Scherer on his cheese and the ones that inhabited the rinds of ours may differ slightly. Nonetheless, we carried on with our study.  Only the rind of the cheese was required for the experiments described below. Accordingly, the exterior of our samples was scraped carefully and the rind shavings were collected with utmost care and retained. The bulk of the cheese was not useful for our work, but it was utilised to initiate and delight a number of group members unfamiliar with the finer points of French cuisine to the joys of raclette preparation and consumption.102  A few cm² of scraped cheese rind were added to a BHI broth and the resultant was allowed to ferment for 2 days. Paper disks soaked in this broth exhibited weak, but definite, inhibitory activity E cloacae, B cereus, and MSSA. It seemed reasonable that the cheese rind would host several species of bacteria. Therefore, it was likely that more than one bacterium might have been responsible for the observed antimicrobial activity. In order to isolate the possible different species of such bacteria, a minute amount of one of the inhibition rings 112 was collected from an agar plate (Figure 18) and inoculated into a virgin plate. By spreading the inoculum over a large surface, it is often possible to grown distinct colonies of individual species of bacteria.    Figure 18. Schematic inhibition plate   Five colonies were thus isolated that were initially thought to have slightly different phenotypes. Each colony was transferred to BHI broth and allowed to ferment for 2 days. Each broth was tested in the usual fashion and found to possess inhibitory activity identical to the original mixture. Although the five colonies were not submitted to gene sequencing, their identical bioactivity suggests that they may well originate from a single strain. Regardless, the bioactivity was still unsatisfactory. Following an observation recorded by Walker,69 we doped the fermentation broth with an excess (10% by weight!) of cysteine, a key building block of thiopeptide architectures, to stimulate the production of antibiotic. 113  Both the commercial and the cheese rind bacteria underwent the same treatment, which had no effect on the former. However, we were delighted to find that activity was significantly enhanced in the broth containing cheese rind (Figure 19).    Figure 19. Inhibitory activity of "cheese bacteria" against B. cereus   Another encouraging result arose from a test against MSSA: excellent activity was detected for all five colonies (Figure 20).  114   Figure 20. Inhibitory activity of "cheese bacteria" against MSSA   Each of these fermentations was then submitted to the same extractive treatment mentioned earlier. We were once again disappointed to find no evidence for the presence of MP1 either in the extracts or in the pellets.   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Multiplicities are reported as “s” (singlet), “d” (doublet), “t” (triplet), “q” (quartet), “quin” (quintuplet), “sex” (sextuplet), “dd” (doublet of doublets), “ddd” (doublet of doublets of doublets), “m” (multiplet), “app” (apparent) and “br” (broad). Infrared (IR) spectra (cm–1) were recorded on a Perkin–Elmer model 1710 Fourier transform spectrophotometer from films deposited on NaCl plates while optical rotations were measured on a Jasco P–1010 polarimeter at the sodium D line (589 nm). Unless otherwise stated, low–resolution mass spectra (m/z) were obtained in the electrospray (ESI) mode on a Waters Micromass ZQ mass spectrometer. High–resolution mass spectra (m/z) were recorded in the electrospray (ESI) mode on a Micromass LCT mass spectrometer by the UBC Mass Spectrometry laboratory. Low– and high–resolution mass spectra obtained in electron impact (EI) mode were recorded on MASPEC II System mass spectrometer by the UBC Mass Spectrometry laboratory. Melting points (uncorrected) were measured on a Mel–Temp apparatus.  All reagents and solvents were commercial products and used without further purification except THF, Et2O (both freshly distilled from Na/benzophenone under argon) and CH2Cl2 (freshly distilled from CaH2 under argon). Commercial n-BuLi was titrated againt N-benzylbenzamide in THF at –78°C until persistence of a light blue colour. Flash 126  chromatography was performed on Silicycle 230-400 mesh silica gel. Analytic and preparative TLC was carried out with Merck silica gel 60 plates with fluorescent indicator. Spots were visualised with UV light. All reactions were performed under dry argon in flame– or oven–dried flasks equipped with Teflon™ stirbars. All flasks were fitted with rubber septa for the introduction of substrates, reagents and solvents via syringe. Solvents, pure liquid reagents or reagents in solution, and solids were added in one portion, unless otherwise stated.  127  1.1. Preparation of (S)–ethyl 2–(1–(tert–butoxycarbonylamino)–2– methylpropyl)thiazole–4–carboxylate (164) N S NHBoc O OEt   Triethylamine (3.9 mL, 27.6 mmol, 1.2 equiv) was added to a cold solution (0°C) of N–Boc–L–valine (5.0 g, 23.0 mmol, 1.0 equiv) in DCM (100 mL) before ethyl chloroformate (2.4 mL, 25.3 mmol, 1.1 equiv) was introduced dropwise over 5 minutes. The solution was stirred at 0°C for 1h before being warmed up to room temperature. After 1h at this temperature, ammonium hydroxide (4.6 mL, 15 M in water, 69 mmol, 3.0 equiv) was added and stirred for 30 minutes. The solution was diluted with DCM (50 mL) and washed with deionised water (3 × 80 mL), dried over MgSO4 and concentrated, to yield the desired amide as a white solid (4.8 g, 22.0 mmol, 96%). This resulting amide was suspended in benzene (100 mL) and Lawesson’s reagent (4.5 g, 11.0 mmol, 0.5 equiv) was added. The mixture was heated to reflux for 1h before being diluted with ethyl acetate (100 mL) and washed successively with sat. aq. NH4Cl, sat. aq. NaHCO3, and brine (80 mL each). The organic layer was then dried over MgSO4 and concentrated to yield the corresponding thioamide (5.0 g, 22.3 mmol, 97%) as an off–white, foamy solid. A cold solution (–40 °C) of the crude residue in DME (90 mL) under argon was treated with solid KHCO3 (8.5 g, 85.4 mmol, 4.0 equiv) and EBP (3.3 mL, 23.5 mmol, 1.1 equiv). The solution was warmed up to –20 °C and stirred for under argon overnight. Solids were then filtered and the filtrate was cooled back down to –20 °C before trifluoroacetic anhydride (10.1 mL, 75.9 mmol, 3.3 equiv) and 2,6– lutidine (17.6 mL, 161 mmol, 7.0 equiv) were added carefully under argon. After 1h at 0 °C, the mixture was covered with ethyl acetate (90 mL) and successively washed with sat. aq. 128  NaHCO3 and brine (100 mL each), dried over MgSO4 and concentrated in vacuo. The crude mixture was purified by flash column chromatography (EtOAc:Hex, 30:70) to furnish thiazole 164 (4.7 g, 14.3 mmol, 62% overall) as a white solid.  m.p.   116-117 °C (lit. 116-117 °C: Downing, S. V.; Aguilar, E.; Meyers, A. I. J.   Org. Chem. 1999, 64, 826). 1H NMR: 8.03 (s, 1H), 5.31 (br d, 1H, J = 9.0), 4.85 (br dd, 1H, J = 8.4, 5.4), 4.35 (q,   2H, J = 7.2), 2.39 (m, 1H), 1.39 (s, 9H), 1.34 (t, 3H, J = 7.2), 0.92 (d, 3H, J =   6.9), 0.85 (d, 3H, J = 6.6).  13C NMR:  173.4, 161.3, 155.4, 145.4, 126.8, 80.0, 61.4, 58.0, 33.2, 28.3, 19.4, 17.2,   14.3.  IR:   2976, 2933, 1716, 1502. MS:   351 [M + Na+]. HRMS: calcd for C15H24N2O4NaS+: 351.1354, found 351.1364. [α]D18.2 = – 37.9° (c = 1.4, CHCl3) (lit. [α]D23 = – 37.1° (c = 0.5, CHCl3): ibid.).  129   Figure 21. 1H NMR spectrum of 164   Figure 22. 13C NMR spectrum of 164  130   Figure 23. IR spectrum of 164  131  1.2. Preparation of (S)–ethyl 2–(1–amino–2–methylpropyl)thiazole–4–carboxylate hydrochloride (165)   N–Boc–protected thiazole 164 (4.7 g, 14.3 mmol, 1.0 equiv) was treated with a 4 M solution of anhydrous HCl in dioxane (10 mL, 40.0 mmol, 2.8 equiv) for 20 minutes. The excess HCl was removed using a suction aspirator and the solvent was then evaporated in vacuo, giving amine hydrochloride 165 in quantitative yield (3.8 g, 14.3 mmol) as a white foam.  1H NMR:  9.26 (br s, 3H), 8.17 (s, 1H), 4.69 (d, 1H, J = 6.6), 4.30 (q, 2H, J = 7.2), 2.54   (app sex, 1H, J = 6.6), 1.30 (t, 3H, J = 7.2), 1.13 (d, 3H, J = 6.6), 0.93 (d, 3H,   J = 6.6).  13C NMR:  165.7, 161.5, 146.2, 129.4, 61.7, 58.7, 32.7, 19.1, 18.6, 14.2. IR:   3445, 2876, 1728, 1480. MS:   229 [M + H+]. HRMS:  calcd for C10H17N2O2S+: 229.1011, found 229.1012. [α]D20.3 = – 10.5° (c = 2.0, in MeOH).  132   Figure 24. 1H NMR spectrum of 165   Figure 25. 13C NMR spectrum of 165  133   Figure 26. IR spectrum of 165  134  1.3. Preparation of ethyl 2–((4S,5R)–5–methyl–2–oxooxazolidin–4–yl)thiazole–4– carboxylate (119)    L–Threonine (5.0 g, 42.0 mmol, 1.0 equiv) was dissolved in a 1 M aqueous solution of NaOH (125 mL, 125 mmol, 3.0 equiv) to which was added slowly, at 0 °C, a solution of triphosgene (13.7 g, 46.2 mmol, 1.1 equiv) in dioxane (100 mL). The reaction was then stirred at room temperature for 6h before being concentrated. The crude mixture was then dissolved in methanol (300 mL) and stirred overnight. The solution was concentrated in vacuo to approximately 100 mL of solvent, and was saturated with dry ammonia after DMAP (50 mg, 0.41 mmol, 0.01 equiv) was added. The mixture was stirred at room temperature for 12h before being concentrated. The residue and Lawesson’s reagent (8.5 g, 21.0 mmol, 0.5 equiv) suspended in benzene (125 mL) were heated to reflux for 2h. Concentration of the crude yielded the thioamide intermediate, which was directly reacted with EBP (5.9 mL, 42.0 mmol, 1.0 equiv) in refluxing ethanol (90 mL). After 30 minutes, the mixture was concentrated and the residue was dissolved in CHCl3 (100 mL), washed with sat. aq. NaHCO3 and brine (80 mL each), dried over MgSO4 and concentrated. The crude mixture was purified by flash column chromatography (EtOAc:Hex, 50:50) and thiazole-ester 119 (8.1 g, 31.6 mmol, 75% overall) was obtained as a pure white solid.  m.p.   133-134 °C (lit. 133-134 °C: Ciufolini, M. A.; Shen, Y.-C. J. Org. Chem.   1997, 62, 3804). 135  1H NMR:  8.18 (s, 1H), 6.97 (br s, 1H), 4.99 (s, 1H, J = 6.0), 4.65 (app q, 1H, J = 6.0,   6.3), 4.40 (quin, 2H, J =7.2), 1.62 (d, 3H, J = 6.3), 1.39 (t, 3H, J = 7.2).  13C NMR:  171.4, 160.9, 168.5, 147.9, 128.0, 80.0, 61.7, 61.0, 19.9, 14.3. IR:   3244, 2979, 1762, 1223. MS:   279 [M + Na+]. HRMS: calcd for C10H12N2O4NaS+: 279.0415, found 279.0407.  136   Figure 27. 1H NMR spectrum of 119   Figure 28. 13C NMR spectrum of 119  137   Figure 29. IR spectrum of 119  138  1.4. Preparation of ethyl 2–((S)–1–(2–((1S,2R)–1–(tert–butoxycarbonylamino)–2– hydroxypropyl)thiazole–4–carboxamido)–2–methylpropyl)thiazole–4–carboxylate (226)  HO BocHN N S O HN O OEtN S   Boc2O (2.9 g, 13.2 mmol, 1.2 equiv) was added under argon to a stirred solution of triethylamine (1.7 mL, 12.0 mmol, 1.1 equiv) and oxazolidinone 119 (2.8 g, 11.0 mmol, 1.0 equiv) in DCM (50 mL) at room temperature. A spatula tip of DMAP was added as a catalyst. The reaction was stirred for 45 minutes before the solvent was removed in vacuo, and the crude purified by flash chromatography (EtOAc:Hex, 40:60). The resulting compound was dissolved in 1:1 mixture of H2O and THF (100 mL). Lithium hydroxide monohydrate (2.8 g, 67.0 mmol, 6.0 equiv) was added and the reaction was stirred overnight before being diluted with EtOAc (50 mL). A buffered solution of sodium phosphate monobasic in H2O (pH~2) was then added at 0 °C until pH~3 was reached. The organic layer was separated and the aqueous layer was washed with EtOAc (3 × 100 mL). The combined organic layers were dried over MgSO4 and concentrated to yield 129 as an inseparable mixture of acids. This resulting acid (616 mg, ca. 2.0 mmol, 1.0 equiv) and amine hydrochloride 165 (465 mg, 2.0 mmol, 1.0 equiv) were dissolved in DCM (8 mL) under argon. HOBt (290 mg, 2.11 mmol, 1.1 equiv) and DCC (420 mg, 2.0 mmol, 1.0 equiv) were added and the mixture was stirred for 2h at room temperature. The solution was then cooled down to 0 °C and stirred for 15 minutes before being filtered over Celite. The 139  resulting solution was then successively washed with a 1 M solution of HCl in water (5 mL), a 5% solution of NaHCO3 in water (2 × 10 mL), and brine (5 mL), before being dried over MgSO4 and concentrated in vacuo. The crude mixture was purified by flash chromatography (EtOAc:Hex, 50:50) to yield the title compound 226 (860 mg, 1.7 mmol, 84%) as a colourless oil.  1H NMR:  8.07 (s, 1H), 8.04 (s, 1H), 7.95 (d, 1H, J = 9.0), 5.69 (br d, 1H, J = 9.0), 5.30   (dd, 1H, J = 9.0, 6.9), 4.91 (br d, 1H, J = 9.0), 4.63 (br s, 1H), 4.40 (q, 2H, J   = 7.2), 2.85 (d, 1H, J = 3.0), 2.56 (app sex, 1H, J = 6.9, 6.6), 1.46 (s, 9H),   1.39 (t, 3H, J = 7.2), 1.33 (d, 3H, J = 6.6), 1.02 (d, 3H, J = 6.9), 0.98 (d, 3H, J   = 6.6).  13C NMR:  173.1, 171.4, 161.2, 160.7, 155.9, 149.2, 147.3, 127.0, 124.4, 80.4, 68.7, 61.4,   57.6, 56.5, 33.2, 28.3, 19.7, 18.1, 14.3.  IR:   3390, 3119, 2975, 1715, 1681, 1538. MS:   513 [M + H+]. HRMS:  calcd for C22H32N4O6NaS2+: 535.1661, found 535.1655. [α]D20.5 = – 6.3° (c = 4.3, in MeOH).  140   Figure 30. 1H NMR spectrum of 226   Figure 31. 13C NMR spectrum of 226  141   Figure 32. IR spectrum of 226  142  1.5. Preparation of ethyl 2–((S)–1–(2–((1S,2R)–1–amino–2–hydroxypropyl)thiazole– 4–carboxamido)–2–methylpropyl)thiazole–4–carboxylate (160)   N–Boc protected amine 226 (655 mg, 1.2 mmol, 1.0 equiv) was treated with a 4 M solution of anhydrous HCl in dioxane (2 mL, 8.0 mmol, 6.7 equiv) for 20 minutes. The excess HCl was removed using a suction aspirator and the solvent was then evaporated in vacuo. The resulting solid was dissolved in DCM (10 mL) and sat. aq. NaHCO3 (5 mL) and stirred for 10 minutes before the organic layer was separated and the aqueous layer washed with DCM (3 × 5 mL). The organic layers were then dried over MgSO4 and concentrated to yield amine 160 (497 mg, 1.2 mmol, 100%) as a white foam.  1H NMR:  8.07 (s, 1H), 8.06 (s, 1H), 7.96 (d, 1H, J = 9.3), 5.31 (dd, 1H, J = 9.3, 6.9),   4.40 (q, 2H, J = 7.2), 4.21 (app quin, 1H, J = 6.3, 5.1), 4.01 (d, 1H, J = 4.8),   2.60 (app sex, 1H, J = 6.9, 6.6), 2.25 (br s, 2H), 1.38 (t, 3H, J = 7.2), 1.26 (d,   3H, J = 6.3), 1.03 (d, 3H, J = 6.9), 0.99 (d, 3H, J = 6.6).  13C NMR:  175.5, 171.5, 161.2, 160.9, 149.2, 147.3, 127.0, 124.2, 69.8, 61.4, 59.4, 56.4,   33.2, 19.7, 18.1, 14.3.  IR:   3384, 2968, 1719, 1654, 1540. MS:   413 [M + H+]. HRMS:  calcd for C17H24N4O4NaS2+: 435.1137, found 435.1150. [α]D20.9 = – 3.2° (c = 1.7, in MeOH).  143   Figure 33. 1H NMR spectrum of 160   Figure 34. 13C NMR spectrum of 160  144   Figure 35. IR spectrum of 160  145  1.6. Preparation of (4S,5R)–3–(tert–butoxycarbonyl)–2,2,5–trimethyloxazolidine–4– carboxylic acid (128)   N–Boc–L–threonine (1.0 g, 4.6 mmol, 1.0 equiv) was dissolved in DCM (25 mL). 2,2–dimethoxypropane (15.2 mL, 91.0 mmol, 20.0 equiv) and a spatula tip of PTSA were added and the solution was stirred for 2h under argon. The organic layer was then washed with sat. aq. NaHCO3 (3 × 25 mL) and then discarded. The aqueous layer was acidified with a buffered solution of sodium phosphate monobasic in H2O (pH~2) until pH~3 was reached, and subsequently extracted with EtOAc (5 × 25 mL). The organic layer was dried over MgSO4 and concentrated to yield acid 128 (1.16 g, 4.5 mmol, 98% yield).  m.p.   89-90 °C. 1H NMR  5.08 (br s, 1H), 4.19 (app m, 1H), 3.91 & 3.85 (2d, 1H, J = 7.5), 1.61 (s, 3H), (MeOD): 1.57 (s, 3H), 1.51 & 1.45 (s, 9H), 1.45 & 1.40 (2d, 3H, J = 6.0).  13C NMR  172.5 & 171.9, 152.2 & 151.6, 94.6 & 94.4, 80.8 & 80.4, 74.0 & 73.7, 66.3 & (MeOD): 66.1, 27.3 & 27.1, 26.9 & 25.5, 23.8 & 22.9, 18.0 & 17.8.  IR:   3500 (br), 3195 (br), 2981, 2937, 1712, 1394. MS:   258 [M–H–]. HRMS: calcd for C12H20NO5–: 258.1341, found 258.1340. [α]D19.3 = – 38.6° (c = 1.1, in MeOH).  146   Figure 36. 1H NMR spectrum of 128   Figure 37. 13C NMR spectrum of 128  147   Figure 38. IR spectrum of 128  148  1.7. Preparation of (4S,5R)–tert–butyl 4–((1S,2R)–1–(4–((S)–1–(4– (ethoxycarbonyl)thiazol–2–yl)–2–methylpropylcarbamoyl)thiazol–2–yl)–2– hydroxypropylcarbamoyl)–2,2,5–trimethyloxazolidine–3–carboxylate (174)   Amine 160 (423 mg, 1.0 mmol, 1.0 equiv) and acid 128 (266 mg, 1.0 mmol, 1.0 equiv) were dissolved in DCM (4 mL). DCC (212 mg, 1.0 mmol, 1.0 equiv) was added and the solution was stirred under argon at room temperature for 1h. The mixture was cooled down to 0 °C and stirred for 15 minutes before being filtered over Celite. The resulting solution was then washed with a 1M aqueous HCl solution (5 mL), a 5% solution of NaHCO3 in water (2 × 10 mL), and brine (5 mL), before being dried over MgSO4 and concentrated. The crude mixture was purified by flash chromatography (EtOAc:Hex, 70:30) to yield tripeptide 174 (532 mg, 0.81 mmol, 81%) as a white solid.  m.p.:   123-124 °C. 1H NMR:  8.09 (s, 1H), 8.08 (s, 1H), 7.97 (d, 1H, J = 9.3), 7.12 (br d, 1H, J = 5.7), 5.32   (dd, 1H, J = 9.3, 6.9), 5.26 (dd, 1H, J = 9.0, 3.0), 4.62 (m, 1H), 4.41 (q, 2H, J   = 7.2), 4.36 (m, 1H), 3.94 (d, 1H, J = 7.2), 3.25 (d, 1H, J = 3.6), 2.56 (m, 1H),   1.87 (s, 3H), 1.63 (s, 3H), 1.61 (s, 3H), 1.41 & 1.39 (s, 9H), 1.40 (t, 3H, J =   7.2), 1.32 (d, 3H, J = 6.3), 1.04 (d, 3H, J = 6.9), 1.00 (d, 3H, J = 6.9).  13C NMR:  171.2, 170.4, 170.3, 161.2, 160.5, 149.3, 149.2, 127.0, 124.4, 94.9, 81.3, 77.2,   69.1, 67.5, 61.5, 56.5, 56.0, 33.4, 28.3, 27.5, 25.6, 24.9, 19.7, 19.3, 18.2, 14.3.  IR:   3367, 3237, 3082, 2977, 2934, 1728, 1684, 1660, 1403. 149  MS:   676 [M + Na+]. HRMS:  calcd for C29H43N5O8NaS2+: 676.2451, found 676.2450. α]D18.6 = – 57.5° (c = 0.8, in CHCl3).  150   Figure 39. 1H NMR spectrum of 174   Figure 40. 13C NMR spectrum of 174  151   Figure 41. IR spectrum of 174  152  1.8. Preparation of (4S,5R)–tert–butyl 4–((Z)–1–(4–((S)–1–(4– (ethoxycarbonyl)thiazol–2–yl)–2–methylpropylcarbamoyl)thiazol–2–yl)prop–1– enylcarbamoyl)–2,2,5–trimethyloxazolidine–3–carboxylate (176)   Alcohol 174 (220 mg, 0.34 mmol, 1.0 equiv), Et3N (0.12 mL, 0.84 mmol, 2.5 equiv), and MsCl (0.14 mL, 1.0 mmol, 3.0 equiv) were dissolved in DCM (3.5 mL) under argon. The solution was stirred for 1.5h and DBU (1.0 mL, 6.7 mmol, 20.0 equiv) was added to the mixture. After stirring overnight at room temperature, the reaction was quenched by addition of sat. aq. NH4Cl (5 mL) and the aqueous layer was further extracted with DCM (5 mL). The organic layers were combined, dried over MgSO4 and concentrated in vacuo. The crude mixture was purified by flash chromatography (EtOAc:Hex, 80:20) to yield olefin 176 (199 mg, 0.31 mmol, 93%) as a white solid.  m.p.:   134-136 °C. 1H NMR:  8.07 (s, 1H), 8.01 (s, 1H), 7.91 (d, 1H, J = 9.0), 7.79 (br s, 1H), 6.63 (app br   s, 1H), 5.31 (dd, 1H, J = 9.3, 7.2), 4.40 (q, 2H, J = 7.2), 4.40 (overlapped m,   1H), 4.05 (d, 1H, J = 7.2), 2.62 (app sex, 1H, J = 6.9, 6.6), 1.87 (d, 3H, J =   6.9), 1.65 (s, 3H), 1.63 (s, 3H), 1.46 (d, 3H, J = 6.3), 1.44 (s, 9H), 1.39 (t, 3H,   J = 7.2), 1.02 (d, 3H, J = 6.9, 0.98 (d, 3H, J = 6.6).  13C NMR:  171.7, 171.6, 168.4, 167.0, 161.3, 160.7, 149.4, 147.4, 127.6, 127.0, 123.7,   123.6, 95.2, 81.3, 77.2, 67.6, 61.4, 56.6, 32.9, 31.5, 28.3, 25.9, 19.7, 19.4,   18.2, 18.1, 14.4.  IR:   3226, 3082, 2972, 1732, 1699, 1674, 1650, 1531. 153  MS:   658 [M + Na+]. HRMS:  calcd for C29H41N5O7NaS2+: 658.2345, found 658.2354. [α]D20.7 = + 38.6° (c = 2.0, in MeOH).  154   Figure 42. 1H NMR spectrum of 176   Figure 43. 13C NMR spectrum of 176  155   Figure 44. IR spectrum of 176  156  1.9. Preparation of ethyl 2–((S)–1–(2–((Z)–1–((2S,3R)–2–amino–3– hydroxybutanamido)prop–1–enyl)thiazole–4–carboxamido)–2–methylpropyl)thiazole– 4–carboxylate (159)   Acetonide 176 (150 mg, 0.24 mmol, 1.0 equiv) was dissolved a 4 M solution of anhydrous HCl in dioxane (1.0 mL, 4.0 mmol, 16.7 equiv) of for 20 minutes. The excess HCl was evaporated using a suction aspirator and the solvent was then removed in vacuo. The resulting solid was then dissolved in a 1:1 mixture of H2O and THF (1 mL). After 5 minutes, the solution was diluted with DCM (5 mL) and sat. aq.NaHCO3 (5 mL). The organic layer was separated, dried over MgSO4 and concentrated to yield amine 159 (117 mg, 0.24 mmol, 100%) as white foam.  1H NMR:  9.19 (br s, 1H), 8.47 (br d, 1H, J = 9.3), 8.05 (s, 1H), 7.95 (s, 1H), 6.46 (q,   1H, J = 7.2), 5.34 (m, 1H), 4.48 (m, 1H), 4.39 (q, 2H, J = 7.2), 3.49 (d, 1H, J   = 1.2), 2.42 (app sex, 1H, J = 6.9, 6.6), 1.88 (d, 3H, J = 6.9), 1.39 (t, 3H, J =   7.2), 1.34 (d, 3H, J = 6.6), 1.02 (d, 3H, J = 6.9), 0.95 (d, 3H, J = 6.6).  13C NMR:  170.8, 170.8, 166.4, 161.1, 160.5, 149.0, 148.9, 147.2, 127.2, 126.8, 123.1,   68.7, 61.5, 59.9, 55.9, 55.8, 34.1, 19.4, 18.4, 14.4, 14.4.  IR:   3390, 3300, 2969, 2933, 1727, 1668, 1537, 1215. MS:   496 [M + H+]. HRMS:  calcd for C21H30N5O5S2+: 496.1688, found 496.1677. [α]D20.4 = + 55.0° (c = 1.6, in MeOH). 157   Figure 45. 1H NMR spectrum of 159   Figure 46. 13C NMR spectrum of 159  158   Figure 47. IR spectrum of 159 159  1.10. Preparation of ethyl 2–(hydroxymethyl)thiazole–4–carboxylate (193)   H2S gas, generated from Na2S·9H2O (180.0 g, 0.75 mol, 3 equiv) and conc. HCl (63 mL, 0.75 mmol, 3.0 equiv) was passed through an aqueous solution of glycolonitrile (25.0 mL, 250 mmol, 1.0 equiv, 55% in H2O) in pyridine (25 mL) and Et3N (15 mL). After being stirred at room temperature for 3h, the mixture was concentrated in vacuo. The residue was taken in ethanol (400 mL), and EBP (35 mL, 250 mmol, 1.0 equiv) and conc. H2SO4 (4 mL) were added. The solution was then heated to reflux for 30 minutes before being cooled down to room temperature and concentrated in vacuo. The crude mixture was dissolved in EtOAc (250 mL) and washed with sat. aq. NaHCO3 (3 × 200 mL), dried over MgSO4 and concentrated. The resulting oil was purified by flash chromatography (EtOAc:Hex, 50:50) to yield alcohol 193 (16.4 g, 87.7 mmol, 35%) as a white solid.  m.p.   76-77 °C. 1H NMR:  8.11 (s, 1H), 4.98 (d, 1H, J = 3.3), 4.37 (q, 2H, J = 7.2), 4.21 (br s, 1H), 1.37   (t, 3H, 7.2).  13C NMR:  173.5, 161.4, 146.8, 127.5, 62.0, 61.6, 14.3. IR:   3380, 3129, 1725, 1511. MS:   188 [M + H+]. HRMS: calcd for C7H9NO3NaS+: 210.0201, found 210.0197.   160   Figure 48. 1H NMR spectrum of 193   Figure 49. 13C NMR spectrum of 193  161   Figure 50. IR spectrum of 193 162  1.11. Preparation of ethyl 2'–formyl–2,4'–bithiazole–4–carboxylate (117)   A solution of ester 193 (1.91 g, 10.2 mmol, 1.0 equiv) in MeOH (80 mL) saturated with dry NH3(g) was stirred at room temperature overnight. The volatiles were removed before the residue was reacted with Ac2O (2.0 mL, 20.4 mmol, 2.0 equiv) and a catalytic amount of DMAP in pyridine (20 mL) at room temperature for 2h. The crude mixture was concentrated, dissolved in DCM (50 mL), and successively washed with sat. aq. NH4Cl and sat. aq. NaHCO3 (30 mL each). The organic layer was dried over MgSO4 and concentrated in vacuo. A suspension of the crude acetylated alcohol and Lawesson’s reagent (2.0 g, 5.0 mmol, 0.5 equiv) in xylenes (45 mL) was heated to reflux for 1h before being cooled down to room temperature and filtered over Celite. The organic layer was concentrated and then dissolved in CHCl3 (80 mL) and washed sat. aq. NaHCO3 (2 × 30 mL). The organic layer was dried over MgSO4 and concentrated in vacuo. The intermediate thioamide was reacted with EBP (1.5 mL, 10.2 mmol, 1.0 equiv) in refluxing ethanol (50 mL) for 25 minutes. This solution was then treated with K2CO3 in small portions until no more gas CO2 evolved, and the solvent evaporated. The residue dissolved in chloroform (50 mL) was washed with sat. aq. NaHCO3 (50 mL), dried over MgSO4 and concentrated in vacuo. Finally, to a stirring solution of the resulting alcohol in DCM (15 mL) was added a suspension of PCC (4.5 g, 20.4 mmol, 2.0 equiv), Celite (4 g) and MgSO4 (4 g) in DCM (15 mL). After being stirred for 2h under argon at room temperature, the solution was filtered and concentrated in vacuo. The crude residue was purified by flash chromatography (EtOAc:Hex, 30:70) to yield aldehyde 117 as an off–white solid (1.1 g, 4.2 mmol, 41% over 5 steps). 163  m.p.   156-157 °C (recrystallised from EtOAc/heptanes) (lit. 134-136 °C, Ciufolini,   M. A.; Shen, Y. C. J. Org. Chem. 1997, 62, 3804). 1H NMR:  10.04 (d, 1H, J = 1.2), 8.55 (d, 1H, J = 1.2), 8.25 (s, 1H), 4.46 (q, 2H, J =   7.2), 1.43 (t, 3H, J = 7.2).  13C NMR:  183.2, 165.9, 161.9, 161.2, 151.1, 148.3, 128.5, 124.0, 61.7, 14.3. IR:   3127, 3099, 1723, 1702, 1211. MS:   301 [M + MeOH + H+]. HRMS:  calcd for C10H8N2O3NaS2+: 290.9874, found 290.9865.  164   Figure 51. 1H NMR spectrum of 117   Figure 52. 13C NMR spectrum of 117  165    Figure 53. IR spectrum of 117 166  1.12. Preparation of ethyl 2'–acryloyl–2,4'–bithiazole–4–carboxylate (72)   To a solution of vinylbromide (1.5 mL, 1 M in THF, 1.5 mmol, 1.0 equiv) in THF (10 mL) under argon, cooled down to –78 °C, was added via syringe a solution of n-BuLi in hexane (1.0 mL, 1.6 mmol, 1.6 M in hexane, 1.1 equiv). After being stirred at –78 °C for 45 minutes, the solution was transferred slowly via cannula to a solution of aldehyde 117 (389 mg, 1.5 mmol, 1.0 equiv) in THF (20 mL) at –78 °C. The mixture was stirred at –78 °C for an hour before being warmed up to room temperature, quenched by addition of sat. aq. NH4Cl (15 mL), and diluted with Et2O (30 mL). The organic layer was extracted, washed with brine (30 mL), dried over MgSO4 and concentrated in vacuo. The crude was purified by flash chromatography (EtOAc:Hex, 20:80) to yield alcohol intermediate 194 (320 mg, 1.1 mmol, 72%). This allylic alcohol was dissolved in EtOAc (5 mL) and activated MnO2 (650 mg, 7.5 mmol, 5.0 equiv) was added. The mixture was stirred 5h at room temperature before being filtered over Celite. The solid was washed with EtOAc (3 × 20 mL) and the organic layer was concentrated in vacuo. Enone 72 was obtained as a white solid (309 mg, 1.1 mmol, 70% over 2 steps).  m.p.   158-159 °C (lit. 129-130 °C, Ciufolini, M. A.; Shen, Y. C. J. Org. Chem.   1997, 62, 3804) 1H NMR:  8.48 (s, 1H), 8.23 (s, 1H), 7.58 (dd, 1H, J = 17.4, 10.5), 6.78 (dd, 1H, J =   17.4, 1.4), 6.08 (dd, 1H, J = 10.5, 1.4), 4.45 (q, 3H, J = 7.2), 1.43 (t, 3H, J =   7.2).  167  13C NMR:  181.3, 167.5, 162.3, 161.2, 150.2, 148.2, 132.3, 130.6, 128.3, 124.3, 61.7,   14.3. IR:   3131, 3090, 1723, 1669, 1209. MS:   327 [M + MeOH + H+]. HRMS:  calcd for C13H14N2O4NaS2+: 349.0293, found 349.0286 ([M + MeOH +   Na+]).  168   Figure 54. 1H NMR spectrum of 72   Figure 55. 13C NMR spectrum of 72  169   Figure 56. IR spectrum of 72 170  1.13. Preparation of 4–((tert–butyldimethylsilyloxy)methyl)–2–methylthiazole (121)   A mixture of thioacetamide (1.0 g, 13.3 mmol, 1.0 equiv) and EBP (1.9 mL, 13.3 mmol, 1.0 equiv) in ethanol (50 mL) was heated to reflux for 30 minutes before being concentrated under vacuum. The white solid was dissolved in THF (50 mL) and cooled down to –78 °C under argon before DIBAL (27.0 mL, 1 M in hexane, 27.0 mmol, 2.0 equiv) was slowly added via syringe. After being stirred for 1h at –78 °C, the mixture was warmed up to room temperature and a sat. aq. Rochelle salt solution (80 mL) was added. After stirring at room temperature, the organic layer was separated. The aqueous layer was extracted with DCM (3 × 40 mL). The organic layers were gathered and washed with brine (40 mL), dried over MgSO4 and concentrated. The resulting crude alcohol was dissolved in DMF (25 mL) under argon and TBSCl (2.0 g, 13.3 mmol, 1.0 equiv) and imidazole (1.6 g, 23.4 mmol, 1.8 equiv) were added. The mixture was stirred for 1h at room temperature before sat. aq. NaHCO3 (30 mL) was added. The organic layer was diluted with DCM (50 mL) and washed with brine (4 × 40 mL), before being dried over MgSO4, and concentrated in vacuo to yield thiazole 121 as a pale yellow oil (2.96 g, 12.1 mmol, 91% over 3 steps).  1H NMR:  7.00 (s, 1H), 4.82 (s, 2H), 2.69 (s, 3H), 0.94 (s, 9H), 0.11 (s, 6H). 13C NMR:  165.8, 156.8, 112.9, 62.3, 25.9, 19.1, 18.4, –5.4. IR:   2955, 2930, 2886, 2857, 1534, 839. MS:   244 [M + H+]. HRMS:  calcd. for C11H21NONaSiS+: 266.1011, found 266.1012. 171   Figure 57. 1H NMR spectrum of 121   Figure 58. 13C NMR spectrum of 121  172   Figure 59. IR spectrum of 121 173  1.14. Preparation of (4S,5R)–4–(4–(2–(4–((tert–butyldimethylsilyloxy)methyl)thiazol– 2–yl)acetyl)thiazol–2–yl)–5–methyloxazolidin–2–one (71)   A cold (–78 °C), colourless solution of thiazole 121 (3.80 g, 15.6 mmol, 3.0 equiv) in THF (10 mL) turned red upon slow addition n-BuLi (of 12.0 mL, 1.6 M in hexane, 15.6 mmol, 3.0 equiv) via syringe. After being stirred at –78 °C for 10 minutes, a solution of ester 119 (1.33 g, 5.2 mmol, 1.0 equiv) in THF (10 mL) was introduced via syringe. The mixture was stirred at –78 °C for 30 minutes before being warmed up to –40 °C for 20 minutes. The reaction was quenched by slow addition of H2O (25 mL) and acidified to pH~6 with a 0.5 M aq. HCl solution. The solution was diluted with EtOAc (30 mL), extracted and the organic layer was washed with brine (20 mL), dried over MgSO4 and concentrated. The crude mixture was then purified by flash chromatography (EtOAc:Hex, 50:50) to produce ketone 71 as a light yellow foamy solid (1.92 g, 4.2 mmol, 82%).  m.p.  45-51 °C. 1H NMR:  8.27 & 7.74 (s, 1H), 7.16 & 6.98 (2 t, 1H, J = 1.2), 6.88 & 6.63 (s, 1H), 6.70   (s, 1H), 4.89–4.65 (m, 5H), 1.63 (d, 3H, J = 6.3), 0.96 & 0.94 & 0.92 (3 s,   9H), 0.14 & 0.10 (2 s, 6H).  13C NMR:  188.9, 170.4, 169.6, 167.8, 161.6, 158.5, 157.0, 155.3, 154.5, 154.2, 152.0,   127.2, 118.1, 114.7, 110.5, 79.9, 79.8, 62.2, 61.7, 61.2, 61.0, 44.1, 25.9, 19.9,   18.4, –5.3, –5.4.  IR:   3289, 2953, 2929, 2856, 1755, 1632, 1092. MS:   454 [M + H+]. 174  HRMS:  calcd. for C19H27N3O4NaSiS2+: 476.1110, found 476.1108 . 175   Figure 60. 1H NMR spectra of 71   Figure 61. 13C NMR spectrum of 71  176   Figure 62. IR spectrum of 71 177  1.15. Preparation of ethyl 2'–(5–(4–((tert–butyldimethylsilyloxy)methyl)thiazol–2– yl)–6–(2–((4S,5R)–5–methyl–2–oxooxazolidin–4–yl)thiazol–4–yl)pyridin–2–yl)–2,4'– bithiazole–4–carboxylate (74)   A mixture of ketone 71 (360 mg, 0.79 mmol, 1.0 equiv), enone 72 (232 mg, 0.79 mmol, 1.0 equiv)and Li2CO3 (16 mg, 0.2 mmol, 0.3 equiv) in EtOAc (8 mL) was stirred at room temperature for 2h. The reaction was quenched by careful acidification to pH~4 with 0.01 HCl in H2O. The organic phase was separated, dried over MgSO4 and concentrated to yield diketone 73. This crude material was dissolved in EtOH (5 mL) to which was added NH4OAc (910 mg, 11.8 mmol, 15 equiv) and stirred at room temperature overnight before being diluted with EtOAc (15 mL), and washed with H2O (2 × 10 mL) and brine (10 mL). The aqueous layer was extracted with EtOAc (2 × 15 mL) and the organic layers were collected, dried over MgSO4 and concentrated in vacuo. The dihydropyridine thus formed was redissolved in toluene (5 mL) and titrated with a 1 M solution of DDQ in toluene until only a blue fluorescent stop was apparent by TLC under UV lamp. The mixture was then diluted with toluene (10 mL) washed with sat. Aq. NaHCO3 (2 × 10 mL) and with brine (10 mL), before being dried over MgSO4, concentrated in vacuo and purified by flash chromatography (EtOAc:Hex, 45:55) to afford pyridine 74 (512 mg, 0.70 mmol, 89% over 3 steps).  178  m.p.  96-97ºC (lit. 97-99 °C, Ciufolini, M. A.; Shen, Y. C. J. Org. Chem. 1997, 62,   3804). 1H NMR:  8.32 (d, 1H, J = 8.4), 8.31 (s, 1H), 8.22 (d, 1H, J = 8.1), 8.22 (s, 1H), 7.95 (s,   1H), 7.33 (t, 1H J = 1.2), 6.23 (br s, 1H), 4.88 (br s, 2H), 4.68 (dd, 1H, J =   6.0, 1.2), 4.53 (overlapped m, 1H), 4.46 (q, 2H, J = 7.2), 1.47 (d, 3H, J = 6.3),   1.46 (t, 3H, J = 7.2), 0.96 (s, 9H), 0.14 (s, 6H).  13C NMR:  168.6, 168.5, 164.7, 163.2, 161.4, 158.1, 157.5, 154.2, 150.5, 150.4, 149.8,   148.0, 139.8, 130.0, 127.9, 121.4, 120.5, 118.9, 116.4, 79.8, 62.2, 61.6, 60.9,   25.9, 19.9, 18.4, 14.4, –5.3.  IR:   3118, 2954, 2929, 2856, 1766, 840. MS:   727 [M + H+]. HRMS:  calcd. for C31H34N6O5NaSiS4+: 749.1141, found 749.1154.  179   Figure 63. 1H NMR spectrum of 74   Figure 64. 13C NMR spectrum of 74  180   Figure 65. IR spectrum of 74 181  1.16. Preparation of (4S,5R)–tert–butyl 4–((R)–2–acetoxypropylcarbamoyl)–2,2,5– trimethyloxazolidine–3–carboxylate (220)   A mixture of acid 128 (5.4 g, 20.8 mmol, 1.0 equiv), (R)–isoalaninol (2.0 mL, 25.2 mmol, 1.2 equiv) and DCC (4.5 g, 20.8 mmol, 1.0 equiv) in DCM (90 mL) were stirred overnight under argon at room temperature. The mixture was then cooled in an ice bath (0 °C) and stirred for 15 minutes before being filtered over Celite. The filtrate was washed with a 0.1 M aq. HCl solution (3 × 40) and sat. aq. NaHCO3 (2 × 20), before being dried over MgSO4 and concentrated. The crude mixture and a spatula tip of DMAP were dissolved in pyridine (100 mL) and Ac2O (4.0 mL, 42.4 mmol, 2 equiv) was added dropwise to the solution via syringe, at 0 °C. The solution was allowed to warm up to room temperature and was stirred for 2h under argon. The mixture was then concentrated and the crude oil was redissolved in DCM (100 mL), washed with sat. aq. NaHCO3 (2 × 100 mL). The organic layer was dried over MgSO4 and concentrated. It was further purified by flash chromatography (EtOAc:Hex, 80:20) to afford N–Boc–threonine derivative 220 as a white solid (6.4 g, 17.4 mmol, 85% yield from acid 128).  m.p.   116-118 °C. 1H NMR:  6.36 (br s, 1H), 4.97 (m, 1H), 4.20 (app br s, 1H), 3.76 (d, 1H, J = 7.5), 3.51   (ddd, 1H, J = 14.1, 6.0, 3.6), 3.32 (ddd, 1H, J = 12.9, 7.2, 6.0), 2.03 (s, 3H),   1.61 (s, 3H), 1.56 (s, 3H), 1.43 (s, 9H), 1.36 (d, 3H, J = 6.0), 1.23 (d, 3H, J =   6.6).  13C NMR:  170.7, 170.0, 170.0, 94.8, 80.9, 74.1, 69.9, 67.6, 44.0, 28.3, 25.4, 21.2, 19.1,   17.6. 182  IR:   3327, 2981, 2936, 1740, 1707, 1386. MS:   381 [M + Na+]. HRMS:  calcd for C17H30N2O6Na+: 381.2002, found: 381.2004. [α]D19.6 = – 49.3° (c = 0.8, in MeOH).  183   Figure 66. 1H NMR spectrum of 220   Figure 67. 13C NMR spectrum of 220 184   Figure 68. IR spectrum of 220   185  1.17. Preparation of (2S,3R)–1–((R)–2–acetoxypropylamino)–3–hydroxy–1– oxobutan–2–aminium chloride (124)   A mixture of acetonide 220 (1.1 g, 3.0 mmol, 1.0 equiv) and a 4 M solution of HCl in dioxane (5 mL, 20.0 mmol, 6.5 equiv) was stirred under argon for 20 minutes after which the excess HCl was removed using a water aspirator. The solution was concentrated to a volume of approximately 2 mL and THF (6 mL) and H2O (2 mL) were added successively. After being stirred for 15 minutes, the solution was concentrated to afford amine hydrochloride 124 as a white solid (648 mg, 3.0 mmol, 100%).  m.p.  119-121 °C.84 1H NMR  5.00 (app sex, 1H, J = 6.6, 3.9), 4.02 (app quin, 1H, J = 6.3), 3.73 (d, 1H, J = (MeOD): 6.6), 3.64 (dd, 1H, J = 14.1, 3.9), 3.21 (dd, 1H, J = 14.1, 6.9), 2.04 (s, 3H),   1.29 (d, 3H, J = 6.3), 1.25 (d, 3H, J = 6.6).  13C NMR 171.1, 167.5, 69.2, 66.0, 43.1, 19.9, 18.8, 16.4. (MeOD):  IR:   3377, 2980, 2937, 1735, 1671, 1251. MS:   219 [M + H+]. HRMS:  calcd for C9H18N2O4Na+: 241.1164, found: 241.1156. [α]D18.8 = – 13.5° (c = 2.0, in MeOH).84  186   Figure 69. 1H NMR spectrum of 124   Figure 70. 13C NMR spectrum of 124  187   Figure 71. IR spectrum of 124 188  1.18. Preparation of (4S,5R)–tert–butyl 4–(4–(6–(4–((2S,3R)–1–((R)–2– acetoxypropylamino)–3–hydroxy–1–oxobutan–2–ylcarbamoyl)–2,4'–bithiazol–2'–yl)– 3–(4–((tert–butyldimethylsilyloxy)methyl)thiazol–2–yl)pyridin–2–yl)thiazol–2–yl)–5– methyl–2–oxooxazolidine–3–carboxylate (125)    After being stirred 3h at room temperature, a mixture of ester 74 (236 mg, 0.34 mmol, 1.0 equiv) and lithium hydroxide monohydrate (28 mg, 0.67 mmol, 2.0 equiv) in a 1:1 mixture of H2O:THF (2 mL) was cooled to 0 °C and carefully acidified with a buffered solution of NaHSO4 until pH~2.5 was reached. The solution was diluted with EtOAc (5 mL) and the organic layer was dried over MgSO4 and concentrated. The crude acid in DCM (3 mL) was stirred under argon for 20 minutes with Et3N (0.24 mL, 1.7 mmol, 5.0 equiv), a spatula tip of DMAP and Boc2O (184 mg, 0.85 mmol, 2.5 equiv). It was quenched with H2O (0.5 mL) after which the mixture was cooled down in an ice bath and acidified with a buffered solution of NaHSO4 to pH~2.5 and EtOAc (5 mL) was added. The organic layer was dried over MgSO4 and concentrated in vacuo. The crude acid, amine hydrochloride 124 (86 mg, 0.34 mmol, 1.0 equiv), Bop-Cl (86 mg, 0.34 mmol, 1.0 equiv) and Et3N (0.1 mL, 0.67 mmol, 2.0 equiv) in acetonitrile (1.5 mL) were stirred for 2h under argon. The mixture was quenched with sat. aq. NaHCO3 (2 mL) and diluted with DCM (3 mL). The organic layer was separated and washed successively with sat. aq. NH4Cl and brine (5 mL each), 189  before being dried over MgSO4 and concentrated in vacuo to afford alcohol 125 (260 mg, 0.26 mmol, 77%).  m.p.  123-124 °C. 1H NMR:  8.29 (d, 1H, J = 8.1), 8.29 (br t, 1H, J = 7.5), 8.20 (d, 1H, J = 8.1), 8.19 (s,   1H), 8.13 (s, 1H), 7.98 (s, 1H), 7.29 (s, 1H), 7.25 (br t, 1H, J = 6.3), 5.02 (d,   1H, J = 4.2), 5.02 (overlapped m, 1H), 4.87 (s, 2H), 4.59-4.47 (m, 3H), 4.13   (d, 1H, J = 2.7), 3.54 (ddd, 1H, J = 14.1, 6.0, 3.6), 3.31 (ddd, 1H, J = 14.1,   7.8, 6.0), 1.46 (s, 9H), 1.45 (d, 3H, J = 6.6), 1.24 (d, 3H, J = 5.7), 1.21 (d, 3H,   J = 6.3), 0.94 (s, 9H), 0.12 (s, 6H).  13C NMR: 171.0, 170.8, 170.8, 168.8, 166.8, 164.5, 162.7, 161.9, 157.7, 153.5, 150.8,   150.3, 149.9, 149.7, 148.9, 139.9, 130.1, 124.4, 121.8, 120.3, 118.8, 116.0,   84.9, 75.6, 69.5, 66.6, 62.3, 62.2, 57.0, 43.7, 27.9, 25.9, 21.2, 20.2, 18.4, 18.3,   17.6, –5.3.  IR:   3296, 3116, 2979, 2954, 2857, 1835, 1731, 1646, 1539. MS:   999 [M + H+]. HRMS:  calcd for C43H54N8O10NaSiS4+: 1021.2513, found: 1021.2534.  190   Figure 72. 1H NMR spectrum of 125   Figure 73. 13C NMR spectrum of 125  191   Figure 74. IR spectrum of 125 192  1.19. Preparation of (4S,5R)–tert–butyl 4–(4–(6–(4–((Z)–1–((R)–2– acetoxypropylamino)–1–oxobut–2–en–2–ylcarbamoyl)–2,4'–bithiazol–2'–yl)–3–(4– formylthiazol–2–yl)pyridin–2–yl)thiazol–2–yl)–5–methyl–2–oxooxazolidine–3– carboxylate (223) O NBoc O NS N S O N S N S O N H N H N O OAc H   A mixture of alcohol 125 (45 mg, 45 µmol, 1 equiv), Et3N (16 µL, 0.11 mmol, 2.5 equiv) and MsCl (7 µL, 90 µmol, 2.0 equiv) in DCM (0.3 mL) was stirred at 0 °C under argon for 10 minutes after which no alcohol was visible by TLC or MS analyses and DBU (13 µL, 90 µmol, 2.0 equiv) were added. The mixture was further stirred for 30 minutes at room temperature under argon before being diluted with DCM (2 mL), and sat. aq. NH4Cl (1 mL) was added. The organic layer was separated, dried and concentrated to yield crude alkene intermediate 221 (44 mg, 45 µmol, 100%). A solution of this intermediate (37 mg, 38 µmol, 1.0 equiv) and a 0.1 M solution of TBAF in THF (0.95 mL, 95 µL, 2.5 equiv) in THF (0.2 mL) was stirred for 10 minutes under argon before being diluted with EtOAc (1 mL) and sat. aq. NH4Cl (1 mL). The organic layer was extracted, dried over MgSO4 and concentrated in vacuo. The crude mixture was purified by flash chromatography (MeOH:EtOAc, 4:96) to yield alcohol 222 (32 mg, 37 µmol, 100%). A solution of this alcohol intermediate, Dess-Martin periodinane (19 mg, 41 µmol, 1.1 equiv), NaHCO3 (31 mg, 0.37 mmol, 10 equiv) in DCM (0.1 mL) was stirred under argon at room temperature for 1h. This solution was diluted with DCM (2 mL) before being washed successively with sat. aq. NH4Cl and brine (1 mL each). The organic layer was then dried over MgSO4, 193  concentrated in vacuo and purified by flash chromatography (MeOH:EtOAc, 2:98) and yielded aldehyde 223 (28 mg, 32 µmol, 88% over 3 steps).  m.p.  144-146 °C. 1H NMR:  10.00 (s, 1H), 8.78 (br s, 1H), 8.35 (s, 1H), 8.30 (d, 1H, J = 8.1), 8.20 (s, 1H),   8.19 (s, 1H ), 8.17 (s, 1H), 8.14 (d, 1H, J = 8.1), 6.74 (br t, 1H, J = 5.7), 6.58   (q, 1H, J = 6.9), 5.06-4.97 (m, 1H), 4.92 (d, 1H, J = 3.9), 4.42 (dq, 1H, J =   6.3, 3.9), 3.52 (ddd, 1H, J = 14.1, 5.6, 3.9), 3.45-3.36 (m, 1h), 1.99 (s, 3H),   1.82 (d, 3H, J = 6.9), 1.41 (s, 9H), 1.40 (overlapped d, 3H), 1.23 (d, 3H, J =   6.3).  13C NMR 171.0, 168.5, 166.4, 166.3, 165.0, 162.5, 159.4, 154.7, 153.4, 150.9, 150.6,   150.1, 150.0, 149.9, 148.7, 140.5, 135.1, 132.8, 131.1, 130.9, 129.5, 129.3,   128.5, 125.9, 125.0, 122.3, 120.3, 118.6, 84.9, 75.3, 69.9, 61.8, 44.4, 27.9,   21.2, 20.3, 17.7, 14.0.  IR:   3560, 3343, 3125, 2959, 2925, 2854, 1809, 1716, 1259. MS:   887 [M + Na+]. HRMS:  calcd for C37H36N8O9NaS4+: 887.1386, found: 887.1371  194   Figure 75. 1H NMR spectrum of 223   Figure 76. 13C NMR spectrum of 223   195   Figure 77. IR spectrum of 223  196  1.20. Preparation of micrococcin P1 (1)   A mixture of aldehyde 223 (21 mg, 24 µmol, 1.0 equiv), NaClO2 (4.4 mg, 48 µmol, 2.0 equiv), 2–methylbutene (0.12 mL, 2 M in THF, 0.24 mmol, 10.0 equiv) and NaH2PO4 (6 mg) in a 1:1 mixture of THF/H2O (0.4 mL) was stirred at room temperature for 2h before being acidified to pH~2.5 with a buffered solution of NaHSO4 (pH~1.5). DCM (3 mL) was added and the organic layer was dried over MgSO4 and concentrated to yield crude acid 126 (18 mg, 20 µmol, 84%). This crude material, amine 159 (20 mg, 40 µmol, 2.0 equiv), BopCl (10 mg, 40 µmol, 2 equiv) and Et3N (6 µL, 40 µmol, 2.0 equiv) was stirred under argon at room temperature for 2h in acetonitrile (1.5 mL). The solution was then diluted with DCM (1 mL), washed successively with sat. aq. NH4Cl, sat. aq. NaHCO3 and brine (1 mL each), before being dried over MgSO4 and concentrated in vacuo. The crude mixture was purified by flash chromatography (MeOH:DCM, 7:93) to afford seco compound 224 (23 mg, 17 µmol, 70% over 2 steps). A mixture of ester 224 (23 mg, 17 µmol, 1.0 equiv) and lithium hydroxide monohydrate (3.6 mg, 85 µmol, 5 equiv) in a 1:1 mixture of H2O:THF (0.6 mL) was stirred at room temperature for 4h before being acidified to pH~2.5 with a buffer solution of NaHSO4. DCM (2 mL) was added and the organic layer was dried over MgSO4 and concentrated in vacuo. The crude acid was dissolved in a 4 M solution of HCl in dioxane (0.8 mL) and stirred at room temperature for 15 minutes before the excess HCl was removed using a water aspirator. The solution was further concentrated to yield intermediate amine hydrochloride 225. The crude material was then dissolved in DMF (0.2 mL) and DPPA (3.2 197  µL, 15 µmol, 1.0 equiv) and Et3N (2 µL, 4.5 µmol, 3.0 equiv) were added. The solution was stirred for 2h under argon at room temperature before being diluted with DCM (2 mL) and H2O (2 mL). The organic layer was washed with sat. aq. NH4Cl and sat. aq. NaHCO3 (2 mL each) before being dried over MgSO4 and concentrated in vacuo. The crude mixture was purified by preparative scale TLC (MeOH:EtOAc, 5:95) to yield micrococcin P1 1 (8 mg, , 41% over 3 steps).  1H NMR:  9.50 (overlapped s, 1H), 9.50 (br d, 1H, J = 6.0), 8.59 (s, 1H), 8.45 (d, 1H, J =   8.1), 8.45 (s, 1H), 8.38 (overlapped br d, 1H), 8.37 (s, 1H), 8.34 (d, 1H, J =   8.1), 8.29 (s, 1H), 8.21 (overlapped d, 1H), 8.20 (s, 1H), 8.11 (s, 1H), 7.89 (br   t, 1H, J = 8.0), 7.85 (br d, 1H, J = 11.1), 6.51 (q, 1H, J = 6.8), 6.46 (q, 1H, J =   6.9), 5.41 (d, 1H, J = 6.3), 5.13 (app t, 1H, J = 9.1), 5.07 (dd, 1H, J = 8.8,   4.4), 4.77 (br d, 1H, J = 4.4), 4.68 (dd, 1H, J = 8.0, 3.6), 4.63 (d, 1H, J = 4.5),   4.41-4.34 (m, 1H), 4.04-3.97 (m, 1H), 3.73-3.67 (m, 1H), 3.12-3.03 (m, 2H),   2.52 (overlapped m, 1H), 1.75 (d, 3H, J = 6.6), 1.69 (d, 3H, J = 6.8), 1.37 (d,   3H, J = 6.6), 1.03 (d, 3H, J = 7.8), 1.01 (d, 3H, J = 6.3), 0.97 (d, 3H, J = 6.5),   0.86 (d, 3H, J = 6.7).  MS:   1144 [M + H+]. HRMS:  calcd for C48H39N13O9NaS6+: 1166.1998, found: 1166.2030 [α]D19.7 = + 68.9° (c = 0.45, in 10 % H2O in EtOH).  198   Figure 78. 1H NMR of synthetic MP1, 1  199  1.21. Preparation of 2,4–dibromothiazole (33) Br N S Br   A mixture of commercial 2,4–thiazolidinedione (17.0 g, 145 mmol, 1.0 equiv) and phosphorus oxybromide (125.0 g, 435 mmol, 3.0 equiv) was heated at 110 ºC for 3 h. The resulting dark solid was cooled in an ice bath and 4 M NaOH in water was added until pH 8 was reached. The mixture was extracted with ether (3 × 200 mL). The combined ethereal extracts were washed with brine, dried and concentrated to give a brown solid. The crude compound was purified by flash column chromatography (Hex:diethyl ether, 100:0 to 99:1) to give thiazole 33 as a white solid (21.5 g, 88.9 mmol, 61%).  m.p.  77-78 ºC (lit. m.p. 82 ºC). 1H NMR:  7.21 (s, 1H). 13C NMR:  136.3, 124.2, 120.9. IR:   3117, 2360, 1455, 1022, 744. EI–MS:  243 [M+]. EI–HRMS: calcd for C3HNS79Br81Br+ = 242.8176, found 242.8173. 200   Figure 79. 1H NMR spectrum of 33   Figure 80. 13C NMR spectrum of 33 201   Figure 81. IR spectrum of 33 202  1.22. Preparation of 4–bromo–2–(trimethylsilyl)thiazole (207) TMS N S Br   To a stirred solution of n-butyllithium (6.5 mL, 1.6 M in hexanes, 10.5 mmol, 1.1 equiv) in diethyl ether (30 mL), cooled at –78 ºC, was added dropwise over 1h a solution of 2,4–dibromothiazole, 33, (2.3 g, 9.5 mmol, 1.0 equiv) in diethyl ether (20 mL). The mixture was then stirred at –78 ºC for 1h, and a solution of trimethylsilyl chloride (1.3 mL, 10.5 mmol, 1.1 equiv) in diethyl ether (15 mL) was added dropwise over 15 min. After 1h at –78 ºC, the reaction was brought to room temperature and washed with sat. aq. NaHCO3 (80 mL). The aqueous layer was extracted with diethyl ether (2 × 80 mL). The combined organic layers were dried and concentrated in vacuo. The crude residue was purified by flash column chromatography (Hex:diethyl ether, 98:2) to give thiazole 207 as a clear colourless liquid (1.7 g, 7.2 mmol, 76%).  1H NMR:  7.36 (s, 1H), 0.38 (s, 9H). 13C NMR:  176.2, 128.1, 119.2, –1.3. IR:   3121, 2960, 1142, 1253, 844. EI–MS:  235 [M+]. EI–HRMS:  calcd for C6H10NS79Br Si [M]+ = 234.9487, found 234.9487. 203   Figure 82. 1H NMR spectrum of 207   Figure 83. 13C NMR spectrum of 207 204   Figure 84. IR spectrum of 207  205  1.23. Preparation of ethyl 2–aminothiazole–4–carboxylate (202) H2N N S O OEt   A mixture of thiourea (1.0 g, 13.3 mmol, 1.0 equiv) and EBP (1.9 mL, 13.3 mmol, 1.0 equiv) in ethanol (50 mL) was heated to reflux for 30 minutes before being concentrated under vacuum. The crude mixture was dissolved in DCM (50 mL), washed with sat. aq. NaHCO3 (50 mL) before being dried and concentrated to yield pure 202 as a white solid (2.3 g, 13.3 mmol, 100%).  m.p.   154-156 ºC. 1H NMR 7.45 (s, 1H), 4.31 (q, 2H, J = 7.2), 1.36 (t, 3H, J = 7.2). (MeOD):  13C NMR 169.6, 161.4, 141.9, 116.7, 60.7, 13.2. (MeOD):  IR:   3127, 1696, 1338, 1239. MS:   173 [M+H+]. HRMS:  calcd for C6H9N2O2S+ = 173.0385, found 173.0387. 206   Figure 85. 1H NMR spectrum of 202   Figure 86. 13C NMR spectrum of 202 207   Figure 87. IR spectrum of 202 208  1.24. Preparation of ethyl 2–iodothiazole–4–carboxylate (201) I N S O OEt   To a stirred solution of amine 202 (1.4 g, 4.9 mmol, 1.0 equiv) and CH2I2 (0.40 mL, 4.9 mmol, 1.0 equiv) in acetonitrile (50 mL) was added dropwise at 0ºC isoamyl nitrite (0.73 mL, 5.5 mmol, 1.1 equiv). The solution was stirred for 3h at room temperature. The mixture was concentrated in vacuo and the residue was dissolved in EtOAc (50 mL), washed with sat. aq. NaHCO3 (50 mL) and saturated brine (50 mL), dried over MgSO4 and concentrated. The crude product was purified by flash chromatography (EtOAc:Hex, 90:10) to yield 201 (1.1 g, 3.8 mmol, 78%) as a colourless solid.  m.p.   84-86 ºC. 1H NMR:  8.12 (s, 1H), 4.40 (q, 2H, J = 7.2), 1.38 (t, 3H, J = 7.2). 13C NMR:  160.0, 149.5, 133.5, 101.4, 62.0, 14.5. IR:   3130, 2980, 1714, 1235, 989. MS:   306 [M+Na+]. HRMS:  calcd for C6H6NO2NaSI+ = 305.9062, found 305.9055.  209   Figure 88. 1H NMR spectrum of 201   Figure 89 13C NMR spectrum of 201  210   Figure 90. IR spectrum of 201 

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