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Total synthesis of thiocillin I Aulakh, Virender Singh 2010

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TOTAL SYNTHESIS OF THIOCILLIN I    by  VIRENDER SINGH AULAKH Bachelor of Science, University of British Columbia   A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF   MASTER OF SCIENCE  in   THE FACULTY OF GRADUATE STUDIES  (Chemistry)   THE UNIVERSITY OF BRITISH COLUMBIA  (Vancouver)   October 2010     Virender Singh Aulakh, 2010  ii Abstract   This thesis describes the first total synthesis of Thiocillin I, a thiopeptide antibiotic that is structurally similar to Micrococcin P1. Our group has recently completed the first total synthesis of the latter natural product. In that connection, new methodology had to be devised for the assembly of the central pyridine-thiazole cluster of the molecule. The present work details the development of a considerably more efficient "second generation approach" to that crucial molecular subunit. The new technique relies upon a three- component condensation to form the pyridine core with the full complement of thiazoles already in place.  This transformation is recognized as a variant of the Bohlmann-Rahtz reaction that has heretofore eluded the synthetic community. Minor modification of this chemistry is likely to facilitate the synthesis of several other thiopeptide antibiotics, naturally occurring or otherwise. This presages the advent of technology for the conduct of medicinal chemistry studies of thiopeptide substances and promises to lead to new generations of chemotherapeutic resources.              iii Preface  Work involving the synthesis of intermediates 114, 128, 43, 91 and  150 has been described by Dr. Marco A. Ciufolini and Yong-Chun Shen.26  I used these intermediates as subunits for the synthesis of thiocillin I.  The work described in Chapter 3 has been published.  Aulakh, Virender S. and Ciufolini, Marco A. (2009) An Improved Synthesis of Pyridine-Thiazole Cores of Thiopeptide Antibiotics. Journal of Organic Chemistry, 74: 5750-5753.  I conducted all the experiments and wrote the experimental section.  The remainder of article was written by Marco A. Ciufolini.                     iv Table of contents  Abstract…………………………………………………………………………………..ii  Preface……………………………………………………………………………….......iii  Table of contents…………………………………………………………………...........iv  List of tables………………………………………………………………........................v  List of figures……………………………………………………………........................vii  List of schemes……………………………………………………………………..........xi  List of abbreviations……………………………………………...................................xiii  List of intermediates………………………………………………………………......xvii  Acknowledgements……………………………………………….................................xix  Dedication……………………………………………………………………….............xx  1. Introduction………………………………………………………………............1  1.1 Thiopeptide antibiotics……………………………………………….. 3  1.2  Structural studies of thiocillin I…………………………................... 8  2.  Synthetic studies on thiopeptides Antibiotics…………………………..........10   2.1 Synthesis of pyridine core………………………….….…...………....10   2.2 Synthesis of thiazole heterocycles………………….…..…..…...…….19  3. Total synthesis of thiocillin I…………………………………………….........23   3.1 Synthesis of tripeptide fragment 88 …………………...……....……...24   3.2 Synthesis of pyridine core 127………………...……....………..……..30  3.3 Completion of synthesis thiocillin I ……………...…....………...…....41  4. Conclusion…………………………………………………………………........47  References…………………………………………………………………. ……............49   v Appendix...........................................................................................................................53                                    vi List of tables  Table 1. Comparison of 13C NMR chemical shift data of natural and synthetic thiocillin I ……………………………………………………………..............45                               vii List of figures  Figure 1.  Examples of thiopeptides…………………………………................................3 Figure 2. Pre-thiocillin: the biosynthetic pathway precursor of 1 and 2…………... ..........4 Figure 3. Protein Synthesis………………………………………………………... ..........7 Figure 4. Hydrolytic fragments of thiocillin I ……………………………………............9 Figure 5. Presumed Structure of thiocillin I…………………………..............................23 Figure 6. Retrosynthetic analysis of 1……………………………………………...........24 Figure 7. Retrosynthetic analysis of tripeptide, 88………………………………...........25 Figure 8. Stratagy for preparation of eastern fragment, 89………………………...........30 Figure 9. Retrosynthetic hypothesis for precursor 43 and 126 of fragment 45……........30 Figure 10. Proton spectrum of natural and synthetic thiocillin I………………..............44  Figure 11. Summary of synthesis…………………………………………………..........47  Figure A1. 1H NMR 114…………………………………………………………...........57  Figure A2. 13C NMR 114………………………………………………………..............57  Figure A3. 1H NMR 128……………………………………………...............................59  Figure A4. 13C NMR 128……………………………………………………..................59  Figure A5. 1H NMR 43…………………………………………………………….........62  Figure A6. 1C NMR 43…………………………………………………………….........62  Figure A7. 1H NMR 146…………………………………………………………...........64  Figure A8. 13C NMR 146………………………………………………………..............64  Figure A9. IR spectrum of 146………………………………………………….............65  Figure A10. 1H NMR 41…………………………………………………………...........67  Figure A11. 13C NMR 41…………………………………………………......................67   viii Figure A12. IR spectrum of 41…………………………………………………….........68  Figure A13. 1H NMR 126-a………………………………………………………..........70  Figure A14. 13C NMR 126-a…………………………………………………….............70  Figure A15. IR spectrum of 126-a………………………………………………............71  Figure A16. 1H NMR 126………………………………………………………….........73  Figure A17. 13C NMR 126………………………………………………………............73  Figure A18. IR spectrum of 126…………………………………………………...........74  Figure A19. 1H NMR 129………………………………………………………….........77  Figure A20. 13C NMR 129………………………………………………………............77  Figure A21. 1H NMR 147………………………………………………………….........78  Figure A22. 13C NMR 147…………………………………………………………........78  Figure A23. IR spectrum of 129…………………………………...................................79  Figure A24. IR spectrum of 147…………………………………………………...........79  Figure A25. 1H NMR 131………………………………………………………….........82  Figure A26. 13C NMR 131………………………………………………………............82  Figure A27. IR spectrum of 131…………………………………………………...........83  Figure A28. 1H NMR 135………………………………………………………….........86  Figure A29. 13C NMR 135…………………………………………………………........86  Figure A30. IR spectrum of 135…………………………………...................................87  Figure A31. 1H NMR 134………………………………………………………….........90  Figure A32. 13C NMR 134………………………………………………………............90  Figure A33. IR spectrum of 134………………………………………..………….........91  Figure A34. 1H NMR 145………………………………………………….....................94   ix Figure A35. 13C NMR 145………………………………………………………............94  Figure A36. IR spectrum of 145…………………………………………………...........95  Figure A37. HPLC traces of pyridine cores………………………………………..........96  Figure A38. 1H NMR 137………………………………………………………….........98  Figure A39. 13C NMR 137………………………………………………………............98  Figure A40. IR spectrum of 137…………………………………………………...........99  Figure A41. 1H NMR 139………………………………………………………….......102  Figure A42. 13C NMR 139………………………………………………………..........102  Figure A43. 1H NMR 140………………………………………………………….......103  Figure A44. 13C NMR 140………………………………………………………..........103  Figure A45. IR spectrum 139……………………………………………………..........104  Figure A46. IR spectrum 140……………………………………………………..........104  Figure A47. 1H NMR 107………………………………………………………….......107  Figure A48. 13C NMR 107…………………………………………………………......107  Figure A49. IR spectrum 107……………………………………………………..........108  Figure A50. 1H NMR 110………………………………………………………….......111  Figure A51. 13C NMR 110………………………………………………………..........111  Figure A52. IR spectrum 110…………………………………………….... ..................112  Figure A53. 1H NMR 115………………………………………………………….......115  Figure A54. 13C NMR 115…………………………………………………………......115  Figure A55. IR spectrum 115……………………………………………………..........116  Figure A56. 1H NMR 117………………………………………………………….......119  Figure A57. 13C NMR 117………………………………………………………..........119   x Figure A58. IR spectrum 117………………………………………………………......120  Figure A59. 1H NMR 91…………………………………………….............................122  Figure A60. 13C NMR 91…………………………………………………………........122  Figure A61. 1H NMR 150………………………………………………………….......125  Figure A62. 13C NMR 150………………………………………………………..........125  Figure A63. 1H NMR 152………………………………………………………….......128  Figure A64. 13C NMR 152………………………………………………………..........128  Figure A65. 1H NMR 153………………………………………………………….......131  Figure A66. 13C NMR 153………………………………………………………..........131  Figure A67. 1H NMR 1…………………………………………………………….......135  Figure A68. HMQC 1…………………………………………………………….........135  Figure A69. HMBC 1......................................................................................................136                   xi List of Schemes  Scheme 1.   Biosynthesis of pyridine and tetrahydropyridine units of thiopeptide antibiotics…………………………….............................................................……............5 Scheme 2.   Biosynthesis of thiazole moieties of thiopeptide antibiotics…………...........5 Scheme 3.   Efforts to make aryl stannyl derivatives……................................................11 Scheme 4.   Kelly synthesis of micrococcinic acid……………………….......................12 Scheme 5.   Bach synthesis of pyridine core of GE2270A……………….......................13 Scheme 6.   Shin synthesis of thiocillin I pyridine core…………………………............14 Scheme 7.   Ciufolini’s pyridine core synthesis of micrococcin P1………………..........15 Scheme 8.   Moody’s synthesis of promothiocin A pyridine core………........................17 Scheme 9.   Bagely one-pot protocol of pyridine core synthesis…………………..........17 Scheme 10. Moody’s biomimetic synthesis of pyridne core 60 of 4 ……………...........18 Scheme 11. Nicolaou’s biomemtric synthesis of pyridine core........................................19 Scheme 12. Hantzsch thiazole synthesis…………………………………………...........20 Scheme 13. Racemization of α-amino thiazoles…………………………………...........20 Scheme 14. HMN varient of Hantzsch thiazole synthesis………………........................21 Scheme 15. (a) Hecht and (b) Kelly biomimetric thiazole formation……………...........21 Scheme 16. Shioiri’s synthesis of thiazoles………………………………………..........22 Scheme 17. Attempt to make 103…………………………………………………..........26 Scheme 18. Possible mechanism for formation of 104…………………………….........27 Scheme 19. Synthesis of 110………………………………………….............................28 Scheme 20. Synthesis of compound 94.……………………………………………........29 Scheme 21.  Synthesis of western fragment, 88……………………………………........29  xii Scheme 22. Bagley’s modification of Bohlmann-Rahtz reaction ……..…………..........31 Scheme 23. Keto-enol tautomerism of ketone 43………………………………….........32 Scheme 24. Synthesis of ketone 43………………………………………………...........33 Scheme 25. Synthesis of ynones 131 and 126……………………………………...........34 Scheme 26. Attempted formation of pyridine using Bagley’s conditions.........................34 Scheme 27. Eiden-Herdeis pyridine construction……………………….........................35 Scheme 28. Eiden-Herdeis synthesis of pyridine core reaction……………………........35 Scheme 29. Synthesis of  134 under modified Bagley conditions ……….......................36 Scheme 30. Pyridne formation under modified Bagley conditions ……………….. ........36 Scheme 31. Possible mechanism for the formation of pyridne  140 ……........................37 Scheme 32. Formation of pyridine core of thiocillin I……………………………..........37 Scheme 33. Possible avenue of 131 and 126 via oxidation of 2-mehtyl thiazoles ............................................................................................................................38 Scheme 34. Synthesis of ynones 131 and 126……………………………………...........39 Scheme 35. Synthesis of short side chain, 150………………..…………........................40 Scheme 36. Completion of pyridine core of thiocillin I……………...…………….........41 Scheme 37. Synthesis of thiocillin I………………………………………………..........44           xiii List of abbreviations  ↑↓     reflux  [α]     specific rotation aa-tRNA    amino acid bound transfer ribonucleic acid Ac     acetyl ACN     acetonitrile Aq     aqueous Ar     aryl Bn     benzyl Boc     tert-butyloxycarbonyl BOP-Cl Bis(2-oxo-3oxazolidinyl)phosphonic chloride br broad Bu butyl ° C degrees Celsius cat. Catalytic calcd calculated cm-1 wavenumbers δ chemical shift in parts per million downfield from tetramethylsilane d doublet DBU 1,8-diazabicyclo[5.4.0]undec-7-ene DCC N,N-dicyclohexylcarbodiimide DCM dichloromethane  xiv DDQ 2,3-dichloro-5,6-dicyano-1,4-benzoquinone DIBAL-(H) diisobutylaluminum hydride DMAP 4-N,N-dimethylaminopyridine DMF N,N-dimethylformamide DMP dimethoxypropane DMSO dimethyl sulfoxide DNA deoxyribonucleic acid DPPA diphenylphosphoryl azide EBP ethyl bromopyruvate Ef elongation factor eq equivalent Et ethyl g gram(s) GTP Guanosine-5’-triphosphate h hour(s) HOBt 1-hydroxybenzotriazole HRMS high resolution mass spectrum Hz Hertz (s-1) Im  imidazoyl IR infrared J coupling constant m multiplet M molar (moles per litre); mega  xv Me Methyl min minute(s) mp melting point MP micrococcin P Ms methanesulfonyl MS mass spectrometry MW microwave n normal nBuLi n-butyllithium NBS N-bromosuccinimide NIS N-iodosuccinimide NMR nuclear magnetic resonance PCC pyridinium chlorochromate Ph phenyl ppm parts per million PPTS pyridinium p-toluenesulfonate Pr Propyl PTSA para-toluenesulfonic acid q quartet quant quantitative RNA ribonucleic acid rRNA ribosomal ribonucleic acid RT room temperature  xvi s singlet sat. saturated t tertiary TBAF tetrabutylammonium fluoride TFA trifluoroacetic acid TFAA trifluoroacetic anhydride THF tetrahydrofuran TIPS triisopropylsilyl TLC thin layer chromatography TBDMS (TBS) tert-butyl dimethylsilyl tRNA transfer ribonucleic acid Ts p-toluenesulfonyl                   xvii List of intermediates  Synthesis of ethyl 2–((4S,5R)–5–methyl–2–oxooxazolidin–4–yl) thiazole– 4–carboxylate (114) …………………………………………….…………………..........55  Synthesis of 4–((tert–butyldimethylsilyloxy)methyl)–2–methyl-thiazole (128) …........58  Synthesis of (4S,5R)–4–(4–(2–(4–((tert butyldimethylsilyloxy) methyl)thiazol–2– yl)acetyl)thiazol–2–yl)–5 methyloxazolidin–2–one (43)...................60  Synthesis of ethyl 2'-methyl-2,4'-bithiazole-4-carboxylate (146)…………………..........63  Synthesis of ethyl 2'-formyl-2,4'-bithiazole-4-carboxylate (41)…………………… ........66  Synthesis of ethyl 2'-(1-hydroxyprop-2-ynyl)-2,4'-bithiazole-4-carboxylate (126-a).......69  Synthesis of ethyl 2'-propioloyl-2,4'-bithiazole-4-carboxylate (126)………………........72  Synthesis of ethyl 2-formylthiazole-4-carboxylate (129)…………………………..........75  Synthesis of ethyl thiazole-4-carboxylate (147)……………………………………........75  Synthesis of ethyl 2-propioloylthiazole-4-carboxylate (131)………………………........80  Synthesis of 1-phenylprop-2-yn-1-one (135)……………………………………….........84  Synthesis of ethyl 2-(5-(4-(acetoxymethyl)thiazol-2-yl)-6-(2-((4S,5R)- 5-methyl-2-oxooxazolidin-4-yl)thiazol-4-yl)pyridin-2-yl)thiazole-4-carboxylate (134)..88  Synthesis of ethyl 2'-(5-(4-(acetoxymethyl)thiazol-2-yl)-6- (2-((4S,5R)-5-methyl-2-oxooxazolidin-4-yl)thiazol-4-yl)pyridin-2-yl)-2,4'-bithiazole-4- carboxylate (145)………….....………………………………………..............................92  Synthesis of ethyl 2-methyl-6-phenylnicotinate (137)……………………………..........97  Synthesis of 2-phenyl-7,8-dihydroquinolin-5(6H)-one (139)………………………......100  Synthesis of phenyl(6-phenylpyridin-3-yl)methanone (140)………………………. ......100  Synthesis of (R)-tert-butyl 1,3-dihydroxy-3-methylbutan-2-ylcarbamate (107)….........105   Synthesis of (S)-methyl 2-(1-(tert-butoxycarbonylamino)-2-hydroxy-2- methylpropyl)thiazole-4-carboxylate (110)………………………….............................109   xviii Synthesis of methyl 2-((R)-1-(2-((1S,2R)-1-(tert-butoxycarbonylamino)-2- hydroxypropyl)thiazole-4-carboxamido)-2-hydroxy-2-methylpropyl)thiazole-4- carboxylate (115)…………………….............................................................................113  Synthesis of (4S,5R)-tert-butyl 4-((Z)-1-(4-((R)-2-hydroxy-1-(4- (methoxycarbonyl)thiazol-2-yl)-2-methylpropylcarbamoyl)thiazol-2-yl) prop-1- enylcarbamoyl)-2,2,5-trimethyloxazolidine-3-carboxylate (117)…………...................117  Synthesis of (4S,5R)-3-(tert-butoxycarbonyl)-2,2,5-trimethyloxazolidine-4-carboxylic acid (91)…………………………………………………………...................................121  Synthesis of (R)-1-((2S,3R)-2-amino-3-hydroxybutanamido) propan-2-yl acetate chloride salt (150)……………………………………………......................................................123  Synthesis of (4S,5R)-tert-butyl 4-(4-(6-(4-((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 (152)………………………………………………........126  Synthesis 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 (153)………...........................................129  Synthesis of thiocillin I (1)……………………………………………………..……....132                   xix Acknowledgements   As a supervisor, mentor and a friend, Professor Dr. Marco A. Ciufolini encouraged and inspired me every step of the way towards understanding the power of synthetic organic chemistry.  The training I have received in graduate school under his guidance is invaluable and has set the foundation to further my career and realize the prospects of this field – thank you.  To my parents and Ting Zhu - thank you for supporting me throughout the past year. Your patience, love and understanding are priceless to me.  Furthermore, I would like to thank Dr. Dan Sorensen, Serge Plamondon and Brian A. Mendelsohn for their efforts in purification of Thiocillin I and its key intermediates.  Also, I would like to thank all the group members of the Ciufolini group, past and present, for their countless discussions and advice.  Their friendship made every moment in the lab an extraordinary experience.           xx              Dedicated to H.H. Sri Satguru Jagjit Singh Ji Maharaj In the loving memory of Jaspal Singh Randhawa  1 1. Introduction The use of antibiotic agents to battle infections caused by bacterial pathogens can be traced back to over 2,500 years ago. For instance, many cultures, including the ancient Egyptians, Greeks and Arabs, developed the use of particular molds to treat infections.1,2 A rational explanation for the effectiveness of such remedies had to await the advent of modern microbiology. Thus, pioneers such as Louis Pasteur and John Tyndall were the first to observe that antagonism between microorganisms provided clues for therapeutic treatments.4 While the work of these scientific giants arguably planted the seeds of modern antibiotic technology, it was Paul Ehrlich, a medicinal scientist in the late 19th century and a pioneer in antibiotic chemotherapy, who discovered the first man-made, structurally well characterized antibiotic: Salvarsan.3 This compound provided the first ever effective treatment against spyrochetal infections (e.g., syphilis); however, it found limited use due to its adverse side affects.  Erlich was also the first to hypothesize that antibiotics could selectively kill bacteria without disturbing the human host.  This visionary proposal found confirmation upon the discovery of penicillin by Sir Alexander Fleming, a veritable turning point in the history of medicine, and a watershed event that spurred intense research on antibiotics. These investigations soon revealed that bacteria develop resistance to those substances. Indeed, antibiotics promote the evolution of resistant bacteria within a population by selectively killing susceptible ones.  As a consequence of the foregoing, and despite the unprecedented success of antibiotic research over the past half century, the World is currently facing a crisis due to  2 the appearance of pathogenic bacteria that are resistant to all known antibiotics. A good example is Staphylococcus aureus. Penicillin and erythromycin, once life saving antibiotics, are now entirely ineffective against resistant strains of that microorganism, which are insensitive even to the action of vancomycin: a drug of last resort. Every year, approximately 500,000 patients in United States hospitals contract life-threatening S. aureus infections, and may die as a consequence.5  As a result of this, there is an urgent need to develop new antibiotic resources, either by isolation from the natural product pool or through chemical modification of existing drugs.                 3 1.1 Thiopeptide antibiotics Thiopeptides are structurally interesting, highly modified peptide natural products produced by bacteria of the genus Streptomyces and Bacillus. Representative thiopeptides are shown in Figure 1.6,7 It is apparent that these compounds incorporate an unusually large number of sulfur atoms in the form of thiazole rings. Indeed, each member of this family exhibits a fully aromatic (cf. 1-3) or partially reduced (4) pyridine core, which is substituted  N N S N S S N N S OH H N O S N HO O HN O NH O HN HO H HO O H N S N NH O Thiocillin I 1 N N S N S S N N S OH H N O S N O HN O NH O HN HO H HO O H N S N NH O Micrococcin P1 2 N N S CO2Me S N N S N S HN O H N O S N HN O NS NH O 4 Amythiamicin D CONHMe N S N S N O H N H HO OHN N S H O H N H HO OH H S N O HN H N S H H N O H N H O N H O NHH O NH H N HOO OH H HH O O R 3 Thiostrepton Figure 1. Examples of thiopeptides  4 with a number of thiazole residues. The resulting pyridine-thiazole cluster is part of a macrocycle that incorporates additional thiazole moieties.  The biosynthesis of thiopeptides is believed to involve a series of post- translational modifications of a large peptide. For instance, thiocillins and micrococcins are assembled from 5, which is termed "pre-thiocillin."47 Only the aminoacids in boldface are incorporated in the ultimate 1-2: the remainder of the peptide chain is excised and lost.  H2N-MSEIKKALNTLEIEDFDAIEMVDVDAMPENEALEIMGASCTTCVCTCSCCTT–COOH 5 Figure 2. Pre-thiocillin: the biosynthetic precursor of 1 and 2.   The genesis of pyridine core is especially interesting. In 1978, Bycroft and Gowland10 proposed that the assembly of this segment could involve an aza Diels-Alder reaction of a pair of dehydroalanine units, as outlined in Scheme 1. The primary adduct 6 may undergo either aromatization to a micrococcin-thiocillin type pyridine such as 7, presumably by sequential dehydration and elimination of R–NH2, or conversion into a thiostrepton-like amino-tetrahydropyridine 8, arguably by sequential dehydration and reduction. Labeling studies by Floss9a,b provided support for the foregoing hypothesis, experimental evidence for which was later adduced by Moody31 and Nicolaou20b.   5 N OH R HN 7 8 N R N R NHCOR' 7 9 –2 H2O tautom. 5 6 N H N R HO HN O R' O N H NH R O HN O R' O N H NH COOH O HN O H2N O OH OH OR' O HN   Scheme 1. Biosynthesis of pyridine and tetrahydropyridine units of  thiopeptide antibiotics   On a different note, the biosynthesis of thiazole moieties is likely to involve the cyclization of N-acyl cysteine segments 10 to thiazolines 11, which subsequently undergo aromatization (Scheme 2).  O N H R3 N H SH O NH R2 O R1 S N O HN R3 NH R2 O R1 S N O HN R3 NH R2 O R1 10 11 12 Scheme 2. Biosynthesis of thiazole moieties of thiopeptide antibiotics  Thiopeptides are strong inhibitors of protein synthesis, and as such they display potent antibiotic activity. They are especially effective against Gram-positive bacteria,7 but they are also significantly active against fungi, cancer cells, and protozoa such as the malarial  6 parasite, P. falciparum. However, their activity against Gram-negative organisms is poor.6  For any substance to be considered for clinical applications, a thorough understanding of the mode of action, beginning with the identification of the target followed by the nature and site of binding, is essential. Extensive research aiming to elucidate such aspects of thiopeptide pharmacology has revealed that these natural products can be distinguished into 2 classes: those which bind to the L11 domain of the 23S ribosomal RNA11 and those that bind to the elongation factor (Ef) that is involved in the elongation cycle of the protein translation.12  Compounds of the first type include MP1 and thiostrepton. Although the precise mechanism of their inhibitory action is not completely understood, it is believed that these thiopeptides bind directly to the rRNA and stabilize its conformation.  This hinders the conformational changes that are required for the elongation step of protein synthesis.14 The overall process is thus blocked.  Substances like GE2270A belong to the second group. Normally in protein synthesis, the only way for an amino acid to be transferred to the ribosome is by the “help” (complex formation) between the amino acid transfer RNA (aa-tRNA) and an enzyme known as the “elongation factor” (Figure 3).  It is believed that substances such as GE2270A inhibits the elongation factor enzyme by complexing to it and as a result prevents the transfer of the amino acid to the ribosome.  Consequently, protein synthesis halts resulting in the death of the organism.    7                 Figure 3.  Protein synthesis    E     P   A E     P   A E     P   A 1 2 3 Transfer of growing peptide chain with formation of new peptide bond Traslocation of growing peptide chain from site A to site P. Deacylated tRNA moves to site E (ready to Exit ribosome) + = aa-tRNA   = Ef•GTP  = Ef•GDP    = tRNA  8 1.2 Structural studies of thiocillin I Knowledge of the precise structure (i.e., constitution and configuration) of a bioactive molecule is also essential for possible clinical applications. In that regard, it is important to note that the structures of most thiopeptide antibiotics are only partially solved, even though the compounds may have been known for several decades.  A case in point is micrococcin P1. This substance was first isolated in 1948; however, its full structure was determined only recently by Ciufolini through total synthesis.8a  In a like manner, the full structures of thiocillin I (the subject of this thesis) and its congeners, thiocillins II and III, remain to be established, yet the compounds have been known since 1976.9 It was rapidly recognized that thiocillin I and MP1 were structurally similar. As reported by Shoji and co-workers,15 acidic hydrolysis of thiocillin I afforded six fragments, shown in Figure 4 as a-f. Five of these, a-e, were identical to those obtained upon hydrolysis of MP1, and correlate with molecular segments A, B, C, D and E.  However, fragment f was a hydroxylated version of the corresponding product obtained from microccin P1. The structural proposals for MP1 promulgated by Walker16- 18 and by Bycroft and Gowland19 thus led Shoji, et al., to postulate (partial) structure 13 for the new antibiotic. It will be seen later that the configuration of segment f is incorrect as assigned.   9 N N S N S S N N S OH H N O S N HO O HN O NH O HN HO H HO O H N S N NH O A B C D E F N N S N S HOOC S N N S HOOC H2N HO H O O COOH COOH S N HO NH2 HO HOOC O S N H2N HOOC a b c d e f 13   Figure 4. Hydrolytic fragments of thiocillin I.            10 2.  Synthetic studies on thiopeptide antibiotics 2.1 Synthesis of pyridine core Over the past two decades, excellent progress has been made toward the total synthesis of thiopeptide antibiotics. Landmark accomplishments in this area include the Moody synthesis of promothiocin A (the first synthesis ever of a thiopeptide natural product)38b, the Nicolaou synthesis of thiostrepton,20a and the Ciufolini synthesis of micrococcin P1.8a  An especially challenging aspect of thiopeptide synthesis is the efficient formation of the central pyridine core with the full complement of thiazole substituents. The pursuit of this objective has relied upon the modification of a preexisting pyridine, either by transition metal mediated coupling reactions or through more classical transformations, or on a de novo construction of the pyridine nucleus via the union of fragments incorporating most — or all — of the requisite thiazoles.  One of the earliest efforts in this area is the Kelly synthesis of micrococcinic acid, 23,21 which is recognized as fragment c arising upon acidic hydrolysis of MP1 or thiocillin I (cf. Scheme 3). Kelly relied upon sequential Stille couplings of preformed thiazole and pyridine units to assemble the desired product. The synthesis began with the union of stannylpyridine 13 with bromothiazole 14.  The emerging 15 was due to  11 N OEtBocHN Me3Sn N S Br a 13 14 N OEtBocHN N S O NHt-Bu O t-BuHN 15 N S SiMe3 Me3Sn 16 X = Br 17 X = SnMe3 b, c N SMe3Si N S O t-BuHN N XBocHN N S O t-BuHN 14 18 19 d N SX N S O t-BuHN 19 20 X = Br 21 X = SnMe3 a (a) 5 mol% (Ph3P)2PdCl2, 55%; (b) Me3SiI, 72%; (c) POBr3, 88%; (d) Br2, 68%; (e) 5 mol% (Ph3P)2PdCl2, (Me3Sn)2 e e  Scheme 3. Efforts to make aryl stannyl derivatives  advance to stannane 17 in preparation for another Stille coupling. However, this transformation proved to be elusive. Likewise, bithiazole 20 was readily constructed through the union of 18 and 14, but subsequent attempts to convert it into stannyl derivative 20 were uniformly unsuccessful. The problem here was that bromides 16 or 20 were rapidly converted into the corresponding homodimers during attempted Pd- mediated stannylation. This provided the inspiration for an alternative approach, in which the exposure of a 1:1 mixture of 16 and 20 to (Me3Sn)2 and a catalytic amount of (Ph3P)2PdCl2 generated the desired 22 in 50% yield. The balance of the product, which   12 (a) 4eq (Me3Sn)2, 11 mol% (Ph3P)2PdCl2, 49%; (b) aq. HNO2, 97%; (c) Tf2O, 58%; (d) 10 mol% (Ph3P)4Pd, 89%; (e) H3O +, 80% 16+20 a NBocHN N S N S N S O t-BuHN NHt-Bu O b, c NTfO N S N S N S O t-BuHN NHt-Bu O N S O Me3Sn 22 23 d, 23 e N N S N S N S O HO OH O N S O 24 25  Scheme 4. Kelly synthesis of micrococcinic acid  had to be discarded, consisted of a mixture of homodimers of the starting bromides (Scheme 4).  Bach determined that the use of Negishi, instead of Stille, coupling reactions22 afforded a partial solution to the foregoing problems, and went on to demonstrate the methodology in a noteworthy synthesis of antibiotic GE-2270A. The assembly of the core unit of this molecule relied on the coupling of trisubstituted pyridine 26 with monothiazole 30 (Scheme 5). It  13 N Br Br Br a,b N BrBr N S O NHBn c N N S O NHBn N S N S MeO O N S AcO PhAcHN N S ZnBr MeO O N S N S NHAc Ph AcO SnMe3 26 30 27 31 29 (a) n-BuLi, THF, then methyl 2-bromothiazole-4-carboxylate, PdCl2(PPh3)2; (b) BnNH2, DIBAL, THF, DCM; (c) 30, PdCl2(PPh3)2, THF, DMA (d) 31, Pd(PPh3)4, dioxane, 80 °C. N Br N S O NHBn N S O MeO d 28  Scheme 5.  Bach synthesis of pyridine core of GE2270A  was hoped that 27 would undergo regioselective coupling at the less hindered C-6 position, but unfortunately, the reaction proceeded with modest selectivity to give a difficultly separable mixture of regioisomeric products (6.5:1 in favor of the desired pyridine). The correct regioisomer advanced to the ultimate 29 upon coupling with stannane 31.  The work of Kelly and Bach underscores the pitfalls of an approach to the pyridine cores of thiopeptides based on transition metal catalyzed coupling reactions. An alternative strategy employed by Shin23 relies instead on more classical transformations, as seen, for instance, in a synthesis of the pyridine core of thiocillin I (Scheme 6). This effort began with the conversion of 1,1,-dimethoxy-2-propanone, 32, into cyanopyridone 33.  14 (a) HCO2Et, NaOEt, THF, 45 °C to rt; (b) NCCH2CONH2, H +, H2O, 60 °C, 23% 2 steps; (c) H2S, DMAP, NEt3, pyridine, 90%; (d) EBP, KHCO3, 0°C, 30 mins; (e) TFAA, pyridine, 0°C, 30min., 53 % steps; (f) Tf2O, DMAP, pyridine, 0°C, 30 min., 93%; (g) ethyl vinyl ether, NEt3, dppp, Pd(OAc)2, toluene, !", 73%; (h) NBS, THF/H2O, RT, 5 min.; (i) KHCO3, DME, RT, overnight; (j) TFAA, pyridine, 0 °C, 30 min.; (k) 2M aq. HCl, THF; (l) phenoxyacetyl cysteine, NEt3, toluene; (m) MnO2, toluene, 9% 3 steps O MeO OMe NH O OMe OMe NC a,b NH O OMe OMe N SEtO O N OMe OMe N S O Br EtO O N S H N O NHBoc TBDPSO S H2N 32 33 34 35 36 35, i c-e f-h N OMe OMe N S N S O EtO HN O 37 OH N S TBDPSO HO NHBoc N N S N S O EtO HN O N S TBDPSO HO NHBoc N S CO2Pac j k-m 38  Scheme 6. The Shin synthesis of the pyridine core of thiocillin I  Addition of H2S to the nitrile afforded a thioamide, which participated in a modified Hantzsch thiazole construction24 to furnish compound 34.  Triflation of the pyridone followed by Heck reaction with ethyl vinyl ether and bromination gave bromoketone 35, which reacted with thioamide 36 to afford 37. Finally, the acetal was released in preparation for a Shioiri thiazole construction.25 Accordingly, the emerging aldehyde was condensed with cysteine and the transient thiazolidine was oxidized (MnO2) to give 38.  15  Syntheses of thiopeptide cores that involve a de novo assembly of the pyridine nucleus have been independently explored by Ciufolini and Moody. The Ciufolini8a,26 construction of the core of micrococcin-like thiopeptides rests on a modified Hantzsch pyridine synthesis that starts with the Michael addition of the bisthiazole ketone 43 to the bis-thiazole enone 42 (Scheme 7).  O NH O N S O N S TBSO HO CN N S OEt O OH O H N S N S OEt O O N S N S OEt O 39 40 41 42 43 42, j O NH O N S O N S TBSO O N S N S OEt O N N S S N N S N S O OEt TBSO O NH O 44 45 (a) H2S, H2O, pyridine, NEt3; (b) EBP, EtOH, !", 35% 2steps; (c) NH3, MeOH; (d) Ac2O, pyridine; (e) Lawesson's reagent, xylenes, !"; (f) EBP, EtOH, !"; (g) PCC, DCM, 41% over 5 steps; (h) 1M H2C=CHMgBr, THF, rt; (i) MnO2, EtOAc, 43% 2 steps; (j) cat. Li2CO3, EtOAc, 92%; (k) NH4OAc, EtOH; (l) DDQ, toluene, 97% 2 steps a,b c-g h,i k,l  Scheme 7.  Ciufolini’s synthesis of the pyridine core of micrococcin P1  This seemingly trivial reaction required considerable experimentation, due to the propensity of 42 to polymerize under a variety of conditions. The desired transformation was ultimately achieved by exposing the reactants to a catalytic amount of powdered lithium carbonate suspended in ethyl acetate, whereupon the 1,5-dicarbonyl adduct 44 was obtained in essentially quantitative yield.  This material was then elaborated to  16 pyridine 45 by treatment with ammonium acetate (formation of an intermediate dihydropyridine), followed by oxidation with DDQ. A weakness of the overall approach is that the preparation of enone 42 required nine steps starting from glyconitrile. The ultimate 45 was thus obtained after a fairly lengthy sequence (12 steps) in a mediocre 9% yield. This setback prompted the development of the more efficient routes that will be discussed later.  Moody and collaborators27 introduced the use of the Bohlmann-Rahtz pyridine synthesis28 for the construction of thiopeptide cores. To illustrate, a key phase of their synthesis of promothiocin A was the assembly of pyridine 52 through a sequence that involved the conversion of ketone 46 into enamine 50-a. This was followed by condensation of the latter with ynone 51 (Scheme 8). The chemistry leading to the formation of 50 is also interesting. Thus, a rhodium catalyzed carbene insertion reaction served to merge 46 and 47 to give rise to 48. Cyclodehydration of the latter to oxazole 49 took place upon treatment with triphenylphosphine and iodine. The ultimate 50 was obtained by ester hydrolysis, activation of the acid with ethyl chloroformate, and reaction of the mixed anhydride with magnesium ethyl malonate. Important work by Bagley later revealed that Bohlmann-Rahtz reactions may be carried out in one pot without isolating the enamine interediate.29 As shown in scheme 9, treatment of a mixture of 53 and 54 with ammonium acetate triggers the following cascade of events: formation of enamine 53-a, Michael addition of the latter to 54, and cyclodehydration of the resultant 55 to pyridine 56. Compound 56 was advanced to dimethyl sulfomycinamate, a component of the thiopeptide antibiotic, sulfomycin I.   17 O H2N Me NHBoc ON2 MeO2C HN MeO2C O NHBoc O N O OEtO2C NHBoc Me O N MeO2C Me N O Me BocHN Me 46 47 48 50 51 52 (a) cat. Rh2(OAc)4, CHCl3, 80 °C, 80%; (b) Ph3P, I2, NEt3, CH2Cl2, 70%; (c) LiOH, MeOH, H2O, 5h, RT; (d) EtO2CCl, NEt3; (e) Mg ethyl malonate, 77% 2 steps; (f) NH4OAc, C6H6-AcOH, 85%; (g) 51, neat, 120 °C, 91% a b g N O O NHBoc Me MeO c-e 49 f N O NH2 EtO2C NHBoc Me 50-a  Scheme 8. Moody’s synthesis of the pyridine core of promothiocin A.  N O O N H O CO2Me OtBu NH4OAc, MeOH !", 5 h SiMe3 MeO2C O Enamine formation Michael addition N CO2Me NH O CO2Me BuOt N O 53 54 55 56 NH2 CO2Me NH O CO2Me BuOt N O O N O NH2 N H O CO2Me OtBu 53-a 54  Scheme 9.  Bagley one-pot protocol of pyridine core synthesis   As described earlier (Scheme 1), the biosynthesis of the pyridine core proceeds via an aza-Diels-Alder reaction of a pair of dehydroaminoacids. Independently, Moody and Nicolaou have devised methodology that successfully duplicates such a remarkable transformation. Thus, the Moody31 biomimetric synthesis of the core of amythiamycin rests upon the reaction of aza-diene 58 with enamide 57 (Scheme 10), which afforded pyridine 60 in 33% yield. Side products arising from the dimerization of 58 was also formed.  In a like fashion, the Nicolaou biomimetic syntheses f the core units of   18 N S BnO2C NHAc N EtO N S CO2Me S N N S BocHN N N S S N N S CO2Me BnO2C N S BocHN a (a) toluene, 120 °C, MW, 33% 57 58 60 N N S S N N S CO2Me BnO2C N S BocHN HN OEt O -HOEt,-NHAc 59  Scheme 10. Moody’s biomimetic synthesis of pyridne core 60 of 4  thiostrepton20, GE2270A, GE2270T and GE2270C130 proceeded through an endo- selective hetero-Diels-Alder dimerization of aza-diene 64, which was obtained from thiazolidine 63 upon treatment with silver carbonate and DBU (Scheme 11). Unfortunately, the reaction occurred without any facial selectivity to give a 1:1 mixture of diasteromeric products. Hydrolysis of the primary adduct afforded the free aminopiperideine 66. This intermediate can either be employed directly in the synthesis of 3, or it can be aromatized to give the pyridine core 67 of GE2270 antibiotics.   19 N S EtO2C N SN NBoc O SH N S OMe O NH2 NBoc O N S OHC S NBoc N S N Boc O N S MeO2C N S N N Boc O NS CO2Et N H2N N S NS S H N BocN O MeO2C CO2Me N N S NS S N BocN O MeO2C CO2Me Towards 3 •TFA (a) KHCO3, MeOH, H2O, 85%; (b) Ag2CO3, BnNH2, DBU, pyridine, -12 °C, then H2O, 64%; (c) DBU, EtOAc, reflux , 50% a b aza-D-A c 61 62 63 66 67 64 N N N S NS S H N BocN O MeO2C CO2Me N S N Boc O N S N Boc O O H H2O hydrolysis 65 62  Scheme 11. The Nicolaou biomimetic approach to the pyridine core  2.2 Synthesis of thiazole heterocycles The construction of the 2,4 disubstituted thiazoles found in thiopeptide antibiotics poses few difficulties. Indeed, one of the earliest techniques for thiazole assembly, the Hantzsch synthesis,24 is very reliable, efficient, and widely used for the such a purpose. The reaction, which was first described in 1889,24 relies on the combination of an α- bromoketone 68 with a thioamide 69. Under "classical" Hantzsch conditions, the components are heated together in a solvent such as ethanol, whereupon the hydrobromic acid liberated during the first step induces dehydration of the intermediate hydroxythiazoline 70, leading directly to thiazole 71 (Scheme 12).  20 O R Br S H2N R' N SR' OH R dehydration N SR' R 68 69 70 71 Scheme 12. The Hantzsch thiazole synthesis   In principle, many enantiopure, α-aminoacid-derived thiazoles of the type found in thiopeptides could be prepared by reaction of the thioamide derivative of an appropriate α-aminoacid with a suitable α-halocarbonyl compound. However, such enantiopure thioamides often afford essentially racemic thiazoles under the original Hantzsch conditions. Available evidence49 indicates that racemization occurs at the stage of the intermediate thiazole 75, which upon protonation by the HBr released into the medium equilibrates with enamine 76, resulting in loss of configuration (Scheme 13). O R' Br S H2N ! 72 73 R NHBoc -HBr N S ! R' NHBoc R -H2O N S ! R' NHBoc R H H Br N S R' NHBoc R H Br H imine-enamine N S R' NHBoc R ± 74 75 76 77  Scheme 13. Racemization of α-amino thiazoles   The foregoing difficulties induced Holzapfel and Meyers to devise a modification of the Hantzsch synthesis that preserves the stereochemical integrity of the starting thioamide.32 The process was subsequently refined by Nicolaou,20 and it is therefore known today as the Holzapfel-Meyers-Nicolaou ("HMN") variant of the Hantzsch thiazole synthesis. The initial reaction of the thioamide with the halocarbonyl compound  21 is thus carried out in the presence of a mild base. This removes the nascent HBr from the medium and affords the 4-hydroxy thiazoline intermediate with no erosion of O R' Br S H2N ! 72 73 R NHBoc N S ! R' OH KHCO3 NHBoc R TFAA/NEt3 N S ! R' O NHBoc R CF3 O H NEt3 N S ! R' NHBoc R 78 79 77 Scheme 14. HMN variant of Hantzsch thiazole synthesis  stereochemical integrity.  The dehydration of the thiazoline with TFAA / Et3N then leads to the thiazole (Scheme 14).  Hecht33 and Kelly34 have studied a biomimetic (cf. Scheme 2) approach to thiazole construction. Treatment of compound 81 with protonic acid or with triflic anhydride promotes cyclization to thiazoline 82, which may then be oxidized to the corresponding thiazole 83 with either MnO2 or NiO2 (Scheme 15). A similar strategy is apparent in what is commonly described as the Shioiri thiazole synthesis, which entails the condensation of a protected α-amino aldehyde 84 with cysteine to give thiazolidine  O N H R NHPG SH CO2Me R S N CO2Me NHPG O N H R NHPG STr CO2Me R S N CO2Me NHPG a b c (a) HCl, CHCl3, 0 °C; (b) TiCl4, (3 eq), DCM, 0 °C or Ph3PO (3 eq) Tf2O (1.5 eq), DCM; (c) MnO2 or NiO2, DCM 80 81 82 83  Scheme 15.  (a) Hecht and (b) Kelly biomimetric thiazole formation.  22 O H R NHPG H2N HS CO2Me R N H S GPHN H CO2Me R N S GPHN CO2Me a b (a) PhH, r.t.; (b) activated MnO2 (20 eq) PhH, 60 °C 84 85 86 87  Scheme 16. The Shioiri thiazole synthesis  86.  Oxidation of the latter is then effected with MnO2 (Scheme 16).                23 3. Total Synthesis of thiocillin I As seen earlier, thiocillin I differs from micrococcin P1 only for the presence of a tertiary hydroxyl group on the isopropyl segment of the valine-derived thiazole. Because both antibiotics derive from pre-thiocillin, it seemed likely that all the other sterocenters have  N S N S N S N S N R O HN HO O H N S N NH O H N N S H N O O N H OH OHO 1 R = H (Micrococcin P1) R = OH (Thiocillin I) Figure 5. Presumed structure of thiocillin I  identical configurations in either molecule. Consequently, we presumed that the structure of thiocillin I was 1 (Figure 5).  Our interest in thiocillin I was motivated by a desire to achieve the first synthesis ever of that antibiotic and to ascertain its configuration. Furthermore, we felt that a number of weaknesses affecting our synthesis of micrococcin P1, notably aspects of the assembly of the pyridine-thiazole cluster of the molecule, could be resolved through the development of improved methodology. Indeed, the total synthesis of thiocillin I detailed herein illustrates a considerably more efficient "second generation" construction of the pyridine core. In addition, it incorporates a number of minor improvements that eliminate some protection/deprotection sequences.  24  Our retrosynthetic strategy reflects lessons learned during our work on micrococcin P1 and it is shown in figure 6. One can imagine a disconnection of thiocillin I into 2 major pieces: the "western" tripeptide fragment 88 and the "eastern" pyridine domain, 89.  The two pieces shall be merged to form the 26 membered macrocycle by sequential formation of the "northern" (a) and "southern" (b) amide bonds. This specific order of bond formation is critical for the success of the macrocyclization sequence. Also, such a strategy is convergent and provides for maximum synthetic efficiency.  O H N S N HN O OH HO NH2 N S O OEt N S N N S O H N O N H OAc S N N S Boc N O O H O HO a b Tripeptide Pyridine Domain H 1 88 89 Figure 6.  Retrosynthetic analysis of 1  3.1 Synthesis of tripeptide fragment 88 The tactics used to synthesize the tripeptide segment (Figure 7) follow principles established during previous work from this laboratory.8a,26   25 O OH O NBoc OH S HN O N S OH O OMe H2N 91 92 HO NHBoc N S O OH OH N S OMe O H2N 94 95 O H N S N N H O OH HO NH2 N S O OEt 88 93 O H N S N N H O OH N S O OEt N O Boc HO 90 N S O OEt O NBoc O Figure 7.  Retrosynthetic analysis of tripeptide 88  Accordingly, the construction of 88 involves the coupling of a molecule of the known (L)-threonine derivative 918a with 92, which in turn results upon the union of fragments 94 and 95. Component 95 differs from the corresponding subunit of micrococcin P1 in that it contains a tertiary OH group. Thus, while the analogous micrococcin thiazole was prepared from (L)-valine, compound 95 may be obtained starting with 3-hydroxyvaline or an equivalent thereof.  A convenient avenue to L-hydroxyvaline was devised in our group during work on the synthesis of luzopeptins.8b The method was utilized verbatim in the present context as well. Thus, commercial (D)-serine methyl ester hydrochloride was N- and O- protected to afford 97 (Scheme 17).  26 O OMe NH2 HO O OMe NHBoc THPO BocN O O HO BocN O O HO O BocN O O H2N O BocN O O N S O EtO NBoc OH N S O EtO HN O O N S O EtO (a) Boc2O, DCM, NaHCO3, H2O, 40 °C, 12 h;  (b) DHP, PPTS, DCM, rt, 12 h, 97% 2 steps; (c) MeMgBr (4 eq), THF, 0!45 °C, 45 min. (d) NaH, THF, 55 °C, 3 h, then Boc2O; (e) cat. PTSA, MeOH, 4 h, 70% 3 steps; (f) (COCl)2, DMSO, then NEt3 (g) NaClO2, t-BuOH, H2O, 0 °C, 90% 2 steps; (h) Ethyl chloroformate, pyridne, 0!RT; (i) NH3, MeOH, RT, 12 h, 70% 2 steps; (j) Lawesson's reagent, benzene, 12 h, "#; (k) EBP, EtOH, "#, 2 h, 30 % 2 steps; (l) Cs2CO3, EtOH, RT, 12 h 96 97 99 100 101 102 103 104 a,b d,e f,g h,i j,k l c OH NHBoc THPO 98 l  Scheme 17.  Attempt to make 103  Treatment with methylmagnesium bromide gave tertiary alcohol 98, which upon treatment of sodium hydride and Boc2O gave the protected oxazolidine 99.  At this point, the primary alcohol was deprotected  and oxidized to carboxylic acid 100 , which was then transformed into amide 101.  This material was treated with Lawesson’s reagent and then with EBP to yield the thiazole 102.  Our intention was now to release the oxazolidine to give compound 103. However, to our surprise only 104 was produced upon treatment of 102 methanolic Cs2CO3 (Kunieda conditions).41a Whereas this result seems to reflect a preferential release of the BOC unit from the substrate, an alternative explanation envisions that the Kunieda oxazolone cleavage occurs normally. However, an Ingold gem-alkyl effect,41b strongly favors recyclization of the desired 106 to 104.   27 BocN O O N S O EtO HN OH N S O EtO HN O O N S O EtO 102 105 104 Cs2CO3 MeOH CsOMe O OtBu CsOMe HN OCs N S O EtO O OtBu 106 Scheme 18.  Possible mechanisms for the formation of 104  In any event, no cure was found to palliate this difficulty: careful monitoring of the reaction, changes in temperature, amounts of reagents and concentration were fruitless.  This setback induced us to utilize a Shioiri oxazole synthesis35 for the construction of L-hydroxyvaline. A similar approach was utilized by Shin during his own synthetic studies toward thiocillin I.23 The new strategy required only a minor modification of the sequence shown in Scheme 18. Accordingly, the THP group in synthetic intermediate 98 was released under acidic conditions to furnish diol 107 in excellent yield (Scheme 19). Parekh-Doering oxidation42 furnished the delicate aldehyde 108, which was immediately condensed with cysteine to form a mixture of diastereomers of thiazolidine 109. Finally, the latter advanced to the desired 110 upon oxidation with manganese dioxide. It is important to note that the quality of manganese dioxide is very crucial to the oxidation of thiazolidines.  Reagent purchased from either Sigma-Adrich or Alfa-Aesar is of average quality (90%) and does not oxidize the thiazolidine. The same is true for freshly prepared Attenburrow MnO2.36 In accord with Shioiri,37 only 99% manganese dioxide purchased from Wako Pure Chemicals (Japan) proved to be competent in this step. We were then able to oxidize 109 to 110 in up to 34% yield.    28 OH NHBoc OH OH BocHN SN OMe O c (a) cat. PTSA, MeOH, 4 h, 99%; (b) SO3•pyridine , DMSO, NEt3, DCM 0 °C ! RT; (c) Cysteine, NaHCO3, MeOH, H2O (1:1), RT, 6 h; (d) MnO2, ACN, "#, 6 h, 30% 3 steps. 107 110 98 OH NHBoc OTHP a b OH NHBoc OHC 108 d OH BocHN SHN OMe O 109 H  Scheme 19.  Synthesis of 110.   At this juncture we turned to the synthesis of the known8a compound 94 (Scheme 20). Commercial (L)-threonine was transformed into 94 in 67% overall yield by sequential treatment with triphosgene and Fischer esterification with methanolic thionyl chloride. Aminolysis of the ester followed by treatment with Lawesson’s reagent in refluxing benzene gave thioamide 113. The use of benzene, boiling point = 80 °C, as the solvent in this step promotes smooth, selective conversion of the primary amide into the thioamide, without interference from the oxazolidinone. By contrast, the use of higher boiling solvents, especially the customary xylene8a results in formation of variable amounts of the undesired thiono-oxazolidinone, which interferes with the subsequent Hantzsch thiazole construction. Treatment of crude 113 with EBP gave compound 114 in good yield. Finally, the oxazolidinone was activated with Boc2O and then released with LiOH to give 94.   29 HO NH2 OH O OMe O O NH O O NH O NS O EtO OH NHBoc NS O HO a,b c-d f,g (a) Triphosgene, NaOH, dioxane, 0 °C!RT; (b) SOCl2 (3 eq), MeOH, "#, 12 h, 67%  2 steps; (c) NH3(g), MeOH, RT, 6 h; (d) Lawesson's reagent, benzene, #", 2 h; (e) EBP, EtOH, #", 1 h, 53% 3 steps; (f) Boc2O, DMAP, NEt3, DCM, 1 h; (g) LiOH (6 eq), THF:H2O (1:1) , 3 h, 97% 2 steps 111 112 114 94 NH2 S O NH O 113 e  Scheme 20.  Synthesis of compound 94  The assembly of the complete western fragment started with the coupling of 94 and 95 with EDCI, whereupon compound 115 emerged in 65% yield (Scheme 21). Further treatment with TFA followed by coupling with 91 gave compound 116 in excellent yield.  OH NHBoc S N MeO O a,b HO NHBoc N S O H N OH N S OMe O c,d HO HN N S O H N OH N S OMe O O O NBoc 116 e HN N S O H N OH N S OMe O O O NBoc 117 110 115 (a) TFA:DCM (1:4), RT, 2 h; (b) EDCI, HOBt, 94, DCM, RT, 12 h, 65% 2 steps; (c) TFA:DCM (1:4), RT, 2 h; (d)  EDCI, HOBt, 91, DCM, RT, 12 h; (e) MsCl, NEt3, then DBU, 32% 3 steps Scheme 21.  Synthesis of the western fragment   30 Reaction of the latter with MsCl and DBU triggered elimination of the OH group, thereby providing a form of the western fragment, 117, which is suitable for the conduct of end- game operations. We note that the dehydroaminoacid-like motif present in 117 was obtained exclusively as the Z-isomer, which is favored both on kinetic and thermodynamic grounds.  Now, we shall turn to the synthesis of the central pyridine core.  3.2 Synthesis of the eastern fragment 89 The eastern fragment of thiocillin I, 89, is identical to the corresponding subunit of micrococcin P1. A key step in the preparation of 89 is the union of the complete pyridine core, 118, with threonine amide 119 (Figure 8). Segment 118 is by far the most interesting and challenging subgoal of the synthesis. The route to this material that we utilized during our work on micrococcin P1 requires 12 steps from glycolonitrile and it  N S N N S O H N O N H OAc S N N S Boc N O O H O HO H 89 N N S S N O NH O TBSO O H2N OH N H S N N S OH O 118 119 OAc  Figure 8. Strategy for the preparation of the eastern fragment, 89.  proceeds in only 9% overall yield (Scheme 7). The following aspects of synthesis are especially troublesome. First, glycolonitrile is expensive and it tends to decompose, to  31 formaldehyde and HCN, during the reaction with H2S leading to the corresponding thioamide. The yield of thiazole 40 is therefore unsatisfactory. Second, the addition of vinylmagnesium bromide to aldehyde 41 proceeds with formation of a number of byproducts that complicate the isolation and the purification of the desired vinylcarbinol, the yield of which is rarely greater than 50%. The problem may be alleviated by the use of vinyllithium in lieu of the Grignard reagent, but the preparation of vinyllithium is impractical. While the sequence becomes extremely reliable after the foregoing roadblocks, it clearly holds much room for improvement, and indeed, a considerable portion of the research described in this Thesis aimed to establish a more efficient approach.  In that connection, we became aware of the work of Bagley,38 whose variant of the Bohlmann-Rahtz reaction permits the creation of a pyridine through the condensation of a readily enolizable ketone, 120, and an ynone, 121, in the presence of ammonium acetate in refluxing ethanol (Scheme 22). The presumed mechanism of the reaction is as shown before in Scheme 9, and yields are generally excellent. It is important to note that substituent R2 in 120 is usually a carbethoxy group, which favors the formation of the R1 R2 O R3 O NH4OAc EtOH reflux R1 R2 NH2 R3 O R1 R2 NH2 R3 O ±H+ N R2 R1 R3 -H2O 120 121 122 123 124  Scheme 22.  The Bagley modification of the Bohlmann-Rahtz reaction   32 intermediate enamine 122, and that emerges at position C-3 of the final pyridine 124. The elaboration of 124 into a thiopeptide-type core may then continue with the conversion of R2 into a thioamide followed by a Hantzsch thiazole synthesis.  Ketone 43, one of the building blocks in the Ciufolini synthesis of micrococcin P1 (cf. Scheme 7), is readily enolizable, and that indeed it exists primarily as the enol tautomer, 125 (ca. 3:1; Scheme 23). O N S O N H O S N COOEt 43 OH N S O N H O S N COOEt 125 Scheme 23.  Keto-enol tautomerism in ketone 43.  The carbethoxythiazole subunit acts as an electron withdrawing group in a manner similar to a COOR substituent by (i) acidifying the methylene group in 43, thereby promoting enolization, and by (ii) stabilizing the enol form through both conjugation and hydrogen bonding. If ketone 43 were to provide the corresponding enamine efficiently under Bagley conditions, then segment 89, in the form of ester 45, could arise via a three- component condensation of 43, 45 and ammonium acetate (Figure 9).  N N S S N O NH O TBSO S N N S OEt O N S S N O NH O TBSO O S N N S OEt O O 45 43 126 NH4OAc [ ? ]  Figure 9.  Retrosynthetic hypothesis for precursor 43 and 126 of fragment 45  33 To test this hypothesis, we proceeded to synthesize 43 using an established route (Scheme 23),8a,26 which starts with DIBAL reduction of commercial 2-methyl-4- carbethoxythiazole, 127, followed by TBS protection of the alcohol to give 128. Deprotonation of the latter with n-butyllithium occurs exclusively at the 2-methyl position,8a,26 and the resulting organometallic (3 equivalents) condenses efficiently with compound 114 (Scheme 24, 1 equivalent) to furnish the desired 43. The extra 2 equivalents of the anion of 128 are necessary to deprotonate the oxazolidinone NH and the emerging 43, which as seen earlier is quite acidic. These proton transfer steps regenerate 128, which is subsequently recovered (column chromatography) and reused.  N S CO2Et N Sa,b 127 128 (a) DiBAL-H, DCM, -78 °C!RT, 2 hr; (b) TBS-Cl, imidazole, DCM, RT, 12 hr, 85% 2 steps; (c) n-BuLi, THF, -78 °C, 0.33 eq of 114, - 78 °C ! -40 °C, 1 hr, 75%. OTBS O NH O N S O N S TBSO 43 c  Scheme 24.  Synthesis of ketone 43   Parallel work (Scheme 25) produced ynones 131 and 126 from compound 40 and 41, which were prepared as detailed earlier in Scheme 7. Alcohol 40 was thus oxidized to an aldehyde in preparation for addition of alkynylmagnesium bromide. Contrary to the case of vinyl Grignard reagents, the alkyne addition reaction proceeded extremely  34 cleanly. The resultant propargyl alcohol 130 then underwent smooth Dess-Martin oxidation43 to 131. Bis-thiazole 41 was advanced to 126 in a like fashion. N S CO2Et a 40 HO N SOHC CO2Et 129 N S CO2Et 41 S N OHC b,c b c N S CO2Et 130 HO N S CO2Et 131 O N S CO2Et 126-a S N HO (a) PCC, DCM, 70%, RT (b) ethynyl magnesium bromide, THF, RT (75% for 126-a) (c) Dess-Martin reagent, DCM, RT (96% for 126, 85% 2 steps for 131) N S CO2Et S N O c 126  Scheme 25.  Synthesis of ynones 131 and 126   Initial attempts to promote pyridine formation from 43 and 131 under Bagley conditions failed to yield 132 (Scheme 26). Interestingly, Eiden and Herdeis39 had shown that 1,5 diketones of the type 133 react with ammonium acetate in refluxing acetic acid to afford pyridines (Scheme 27).  O NH O N S O N S TBSO O N S CO2Et NH4OAc EtOH reflux (Bagley conds.) O NH O N S N N S N S CO2Et TBSO 43 131 132  Scheme 26.  Attempted formation of pyridine using Bagley’s conditions  35  Ph O O OH NPh OH NH4OAc AcOH reflux 97% 133 134 Scheme 27.  Eiden-Herdeis pyridine construction   Given that the formation of ynone 131 was much more efficient than that of enone 42 (Scheme 7), we saw an advantage in building the pyridine core, 45 in an Eiden-Herdeis mode. We thus deliberately induced the conjugate addition of 43 to 131 by exposing an equimolar solution of the reactants in ethyl acetate to an equimolar amount of DBU, and then performed the Eiden-Herdeis step. A 45% overall yield of 134 was thus realized. Notice the exchange of OH protecting groups (TBS --> Ac) incurred during this step. Evidently, the hot acetic acid present in the medium promoted both the release of the TBS group and Fischer esterification of the liberated alcohol.   43  +  131 O NH O N S N N S N S CO2Et AcO 134 O NH O N S O N S N S CO2Et TBSO O 133 NH4OAc AcOH reflux 45% DBU  Scheme 28.  Eiden-Herdeis synthesis of the pyridine core, 134. Reasoning that the formation of an enamine such as 122 was likely to be an acid- catalyzed process, we wondered whether the direct combination of 43, 131, and NH4OAc  36 O NH O N S O N S TBSO O N S CO2Et NH4OAc AcOH reflux, 12 h 63% O NH O N S N N S N S CO2Et AcO 43 131 134  Scheme 29. Synthesis of 134 under modified Bagley conditions   might become possible by operating in an acidic medium. To our pleasant surprise, this proved to be the case: the same reaction that had failed in refluxing ethanol (Bagley conditions) proceeded in 63% yield in refluxing acetic acid (Scheme 29).40 Furthermore, the new procedure worked well with other substrates. For instance, ynone 135 and ethyl acetoacetate combined under the above conditions to afford pyridine 137 in 85% yield, while pyridine 139 was obtained in 62% yield from the analogous condensation of 135 with 138 (Scheme 30).  The latter reaction also delivered byproduct 140 (22% yield), the genesis of which may be rationalized by invoking 1,4-addition of ammonia to 135, followed by conjugate addition of the resultant enamine 141 to a second molecule of 135 and final cyclization – dehydration of adduct 140 (Scheme 31).40 O CO2Et O O OPh NPh CO2Et N O Ph N Ph OPh 135 136 137 138 62% 22% 139 140 + OPh 135 + NH4OAc AcOH reflux NH4OAc AcOH reflux 85%  Scheme 30. Pyridine formation under modified Bagley conditions  37  Of course, the most significant aspect of the new procedure is that it enabled the three-component synthesis of the micrococcin-thiocillin pyridine core in useful yield.  N Ph OPh 140 OPh 135 O Ph NH2 NH3    HOAc OPh OPh NH O Ph [± H+ ] N Ph OPh HO [± H+ ] NH Ph OPh HO [± H+ ] – H2O [± H+ ] 141 144 135 142 143 NH4  OAc [H+ ] [H+ ]  Scheme 31. Possible mechanism for the formation of pyridine 140. O NH O N S O N S TBSO O N S N S OEt O O NH N S N O N S AcO N S N S EtO2C a (a) NH4OAc, AcOH, reflux, 12 hr, 52% 12643 145  Scheme 32.  Formation of pyridine core of thiocillin I Thus, the reaction of 43 with ynone 126 afforded 125 in 52% yield (Scheme 32). Once again, the transformation occurred with desilylation / acetylation of the primary OH group.  The new avenue to 145 was shorter than the original route (Scheme 7) by two steps and it circumvented the difficulties encountered in the addition of vinylmagnesium bromide to 41. Still, it relied on the troublesome preparation of 41 from glycolonitrile. To realize the full potential of the methodology, it was essential to establish a more direct  38 and efficient access to the ynone.  One possibility envisioned the oxidative conversion of readily available 2-methyl thiazoles into the corresponding aldehydes, e.g. with selenium dioxide,40 as a prelude to the now familiar addition of ethynylmagnesium bromide and oxidation (Scheme 33). Interestingly, this transformation was not documented in the N S CO2Et N S S N O H CO2Et 127 41 MgBr N S S N O CO2Et 126 N S COOEt O [ O ] [ ? ] N S S N CO2Et 146 1. 2. [ O ] [ O ] [ ? ] MgBr1. 2. [ O ] OHC S N CO2Et 129 131  Scheme 33. Possible avenue to 131 and 126 via oxidation of 2 methylthiazoles.  literature at the onset of our investigations.  2-methyloxazoles such as 127 and 146 resisted the action of SeO2 under the customary conditions (refluxing ethanol or dioxane). Fortunately, the transformation occurred as expected in refluxing acetic acid (Scheme 34). When the oxidation of 127 was carried out at a substrate concentration greater than 1M, significant quantities of thiazole 147 were obtained as a byproduct.  This may possibly be due to overoxidation of the aldehyde to the corresponding carboxylic acid and in situ decarboxylation. The problem may be contained by performing the reaction at a concentration of ca. 0.5M.  The desired product 129 can then be isolated in 55% yield, together with 21% of 147.  Interestingly, the oxidation of bithiazole 146 proceeded with formation of no such decarboxylated byproduct. Indeed, no byproducts at all were  39 N S CO2Et N S COOEt N S N S CO2Et S NH2 a b,c + N S S N O H CO2Et O H 127 129 148 147 41 e,g N S S N O CO2Et 126 (a) SeO2, AcOH, reflux, 12 hr, 55%; (b) NH4OH, 70 °C, 2hr; (c) Lawesson's reagent, toluene, reflux, 2 hr; (d) EBP, EtOH, reflux, 1hr, 80% 3 steps; (e) Ethynyl magnesium bromide, THF, RT, 75%; (f) IBX, DMSO, 35 °C, 12 hr, 85% over 2 steps; (g) Dess-Martin, DCM, 2 hr, 96%. N S COOEt O 131 d N S S N CO2Et a e,f 146  Scheme 34. Route to ynones 131 and 126 by oxidation of 2-methylthiazoles  apparent in the crude product mixture, although a portion of the starting material was clearly lost in the course of the reaction. Secondary products could possibly have been entrained — and lost — in the dark precipitate of selenium-containing matter that forms during this step. Finally, addition of ethynylmagnesium bromide to the aldehydes, followed by oxidation with IBX (for monothiazole 129) or Dess-Martin periodinane (for bithiazole 41) gave the ynones in good yield. In this manner, fragment 145 of thiocillins and micrococcins could be accessed in 7 steps and in 16% overall yield from commercial thiazole 127: a significant improvement over the previous route, which required 12 steps and proceeded in 9% yield (Scheme 7).  The elaboration of compound 145 to the complete pyridine domain, 89, requires the attachment of the short side chain fragment, 150. This small subunit was easily prepared starting with an EDCI coupling of commercial (R)-isoalaninol, 149, and N-Boc-  40 threonine acetonide, 91 (Scheme 35).  The secondary alcohol was then acetylated and the acetonide and Boc protecting groups were simultaneously cleaved with HCl in dioxane.  O NBoc O OH H2N OH+ a-c (a) EDCI, HOBt, NEt3, DCM, 12 hr; (b) Ac2O, pyridine, 0 °C!RT; (c) 4M HCl, dioxane, 30 min, 95% 3 steps O N H OAc NH2•HCl HO 91 149 150  Scheme 35.  Synthesis of short side chain, 150   At this point, we were ready to merge fragment 145 and 150 and to complete the pyridine domain of the natural product. To that end, we elected first to replace the acetyl unit in 145 with a TBS protecting group (Scheme 36). This blocking group switch facilitated the handling of synthetic intermediates and it allowed us to utilize the same end-game sequence developed earlier for micrococcin P1 without reoptimization of individual steps. The subsequent hydrolysis of the ester in 45 was followed by Boc protection of the oxazolidine moiety. The actual coupling of the two fragments was carried out using BOP-Cl to yield 152 in 20% over 5 steps. Finally, treatment of 152 with MsCl and then DBU introduced the dehydroaminoacid motif (cf. Scheme 29). Smooth deprotection of the alcohol with TBAF followed by oxidation with Dess-Martin periodane furnished the pyridine core of thiocillin I in excellent yields.  41 145 N S N S N S N O NH O TBSO N S COOEt N S N S N S N O NBoc O N S O H N N H O OAc (a) K2CO3, DCM:EtOH (1:1), 48 hr; (b) TBS-Cl, DMF, RT, 12 hr; (c) LiOH, THF:H2O (1: 1), RT, 4 hr (d) Boc2O, NEt3, DCM; (e) BOP-Cl, 150, NEt3, ACN, rt, 6 hr, 20% over 5 steps; (f) MsCl, NEt3, DCM, 2 hr, then DBU, 3 hrs; (g) TBAF, THF, 3 hr; (h) Dess-Martin, DCM, RT, 5 hrs, 84% 3 steps a, b TBSO OH 45 151 c, d N S N S N S N O N O TBSO N S CO2H Boc 152 e N S N S N S N O NBoc O N S O H N N H O OAc O H 153 f-h  Scheme 36. Completion of pyridine core of thiocillin I.  3.3  Completion of synthesis: Thiocillin I The final sequence leading to thiocillin I (Scheme 37) began with the deblocking of tripepetide 117 with TFA to obtain the corresponding primary amine 154 as the TFA salt. In parallel, aldehyde 153 was oxidized to carboxylic acid 89 with sodium chlorite. Fragments 154 and 89 were then merged to yield 155 using BOP-Cl as a coupling agent. Reaction of the emerging 155 with aqueous LiOH caused saponification of the ethyl ester, release of the side chain acetate, and Kunieda cleavage of the activated oxazolidine. This was followed by treatment with TFA to provide mono-seco thiocillin I, 156, setting  42 the stage for macrocyclization.  This final step was carried out by the use of DPPA8a as the condensing agent to yield fully synthetic thiocillin I.  Synthetic 1 was obtained in 12% yield after column chromatography from the sequence of Scheme 29. The purity of this material, ca. 90% as estimated by 1H NMR, was insufficient for full characterization. Further purification was therefore attempted by HPLC. This effort was carried out at MerckFrosst Canada, Ltd., by Dr. Dan Sorensen, whom we thank warmly for his assistance in this matter. Unfortunately, significant losses were incurred during these operations, due in part to decomposition of the antibiotic during elution with aqueous mixtures containing TFA, and in part to degradation of the sample during prolonged storage. In any event, the sample of synthetic thiocillin I ultimately obtained upon HPLC purification (32% H2O (0.1%TFA) and 67% MeCN (0.1% TFA); pressure: 171 bar; Column Temperature: 40 °C) consisted of between 50 and 80 µg (estimated by 1H NMR) of 1. A solution of this material in 200 µL of DMSO- d6 was utilized for characterization, which was also carried out at Merck Frosst Canada, Ltd., through the courtesy of Dr. Dan Sorensen. Synthetic 1 thus purified was spectroscopically (1H and 13C NMR) identical to the natural product, a sample of which was kindly provided by the Shionogi Co.  Figure 10 shows the proton NMR spectra of both natural and synthetic thiocillin I, and Table 1 provides a tabulation of carbon-13 NMR chemical shifts for the two substances. It should be noted that the amount of material recovered through HPLC purification was insufficient to record a 13C spectrum in the direct observation mode: the data in Table 1 were obtained through a 2D inverse detection experiment (HSQC- direct proton carbon correlation and HMBC-long range proton-carbon correlation). In a like manner, a reliable optical rotation could not be  43 117 N S N S N N S S N HO O HN HO O H N S N NH O O NBoc O MeO O (a) TFA, MeOH, 3hr, quant; (b) NaClO2, 2-methyl-2-butene, NaH2PO4, THF, H2O, 4 hr; (c) BOP-Cl, 154, NEt3, ACN; (d) LiOH, THF:H2O (1:1); (e) TFA, DCM, 4 hr; (f) DPPA, NEt3, DMF, 5 hr, 12 % over 5 steps N S H N O O N H OAc N S N S N S N O NBoc O N S O H N N H O OAc O H 153 a b HN N S O HN O O NBoc OH N S COOMe 154 N S N S N S N O NBoc O N S O H N N H O OAc O HO 89 HN N S O HN O OH NH2 • TFA OH N S COOEt + c d, e 155 N S N S N N S S N HO O HN HO O H N S N NH O NH2 • TFA O OH N S H N O O N H OH OH 156 f 1  Scheme 37.  Synthesis of thiocillin I  measured, although an [α]D25 value between + 100 and + 160° was obtained from the foregoing sample, which, once again, consisted of between 50 and 80 µg of purified 1 in  44 200 µL of DMSO-d6 {literature: [α]D24.5 = 97.8 ± 0.8° (c = 2.028, 90% aq. EtOH)}.48 a) b)  Figure 10. (a) Synthetic thiocillin I  (b) natural thiocillin I   45 Table 1.  Comparison of 13C NMR data of natural and synthetic thiocillin I Measured 13C shifts (ppm, natural Thiocillin I) Measured 13C shifts (ppm, synthetic Thiocillin I) Difference (ppm) 170.45 170.85 0.4 168.56 168.80 0.24 168.36 168.30 -0.06 167.90 167.88 -0.02 166.80 166.84 0.04 164.37 164.72 0.35 164.31 164.72 0.26 161.38 161.48 0.10 160.47 161.17 0.70 159.77 159.86 0.09 159.75 159.87 0.12 159.02 159.87 0.85 153.02 153.33 0.31 151.13 150.91 0.22 150.43 150.50 0.07 147.88 149.77 -0.11 149.73 149.42 -0.31 149.43 149.09 -0.34 148.77 148.79 0.02 148.33 148.59 0.26 140.94 140.73 -0.21 130.61 130.47 -0.14 129.34 129.36 0.02 128.89 128.76 -0.13 128.44 128.67 0.23 127.97 127.98 0.01 125.87 125.41 -0.46 125.60 125.31 -0.29  46 Table 1.  Comparison of 13C NMR data of natural and synthetic thiocillin I Measured 13C shifts (ppm, natural Thiocillin I) Measured 13C shifts (ppm, synthetic Thiocillin I) Difference (ppm) 125.45 125.29 -0.16 124.72 124.43 -0.29 121.72 121.47 -0.25 120.77 120.62 -0.15 118.64 118.41 -0.23 71.37 71.26 -0.11 68.32 67.96 -0.36 67.03 66.67 -0.36 65.11 64.87 -0.24 57.21 56.97 -0.24 56.74 56.38 -0.36 56.64 56.29 -0.35 46.86 46.52 -0.34 27.48 27.22 -0.26 25.83 25.51 -0.32 21.07 20.71 -0.36 21.00 20.67 -0.33 20.25 19.91 -0.34 13.70 13.38 -0.32 13.56 13.24 -0.32       47 4. Conclusion The total synthesis of thiocillin I detailed herein is convergent and it affords the final product in a 4% yield over 20 steps starting from thiazole 119 (Figure 8). Furthermore, it provides an unequivocal structural proof for the natural product. At a chemical level, this effort demonstrates a "second generation" approach to the construction of a key segment  HO NHBoc O OH O O NH2 HO NH2 OH N S O O HO NH2 O OH O NH O N S CO2Et NBocO CO2H Tripeptide NHO O N S O N S OTBS S N N S CO2Et O Pyridine core NBocO O OH O N H ClH3N OH OAc Complete pyridine core Thiocillin I 157 91 91 150 96 95 111 114 N S O EtO N S O EtO 127 127 43 126 153 117 145   Figure 11. Summary of synthesis   48 of thiopeptide architectures, which is significantly more efficient that other known routes, and that should be applicable to the synthesis (and structural elucidation) of related compounds such as thiocillins II-III and YM-266983.  More importantly, thanks to the new technology, the exploration of the structure- activity relationship (SAR) and the medicinal chemistry of thiopeptide substances becomes a realistic possibility. 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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  54 except THF, Et2O (both freshly distilled from Na/benzophenone under argon) and CH2Cl2 (freshly distilled from CaH2 under argon). Commercial n-BuLi was titrated against N-benzylbenzamide in THF at –78°C until persistence of a light blue colour. Flash 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.                     55 Ethyl-2–((4S,5R)–5–methyl–2–oxooxazolidin–4–yl)thiazole–4–carboxylate (114)       L-threonine (5.0g, 42.0 mmol) was dissolved in 125 mL of aqueous solution of sodium hydroxide and cooled to 0º C. Triphosgene (13.7g, 46.2 mmol) was dissolved in 70 mL of dioxane and added slowly to the aqueous solution and left to stir overnight to room temperature.  The reaction was concentrated to give a slurry of white precipitate.  This was re-suspended in 40 mL of methanol and cooled to 0º C.  Thionyl chloride (9.13 mL, 126 mmol) was added slowly and then the reaction mixture was heated to reflux for 12 hours.  The solution was then concentrated and the crude was subjected to 40 mL of saturated NaHCO3 solution.  The oxazolidine ester was then extracted with EtOAc (3 x 40 mL) and dried with Na2SO4. Concentration under vacuum gave a yellow oil (4.12 g, 67%).  The crude product was then re-dissolved in 40 mL of methanol and was saturated with NH3 gas.  The mixture was stirred overnight at room temperature before it was concentrated to give the amide as thick yellow oil.  The resulting amide was suspended in 35 mL of benzene with Lawesson’s reagent (5.4 g, 13.0 mmol) and refluxed at 100° C for 2 hours.  The solution was then cooled to r.t. and concentrated to give the thioamide. This was then dissolved in 40 mL of ethanol and EBP (3.62 mL, 25.9 mmol).  The solution was refluxed for 1 hour at 90º C.  The crude was then subjected to 30 mL of saturated NaHCO3 and extracted with EtOAc (3 x 30 mL).  The organic layer was dried with Na2SO4 and concentrated under vacuum. The residue was purified by O NH O N S O EtO  56 chromatography (1:1 EtOAc/ Hexanes) to give 3.5 g (52% over 3 steps) of the known 114 [Ciufolini, M.A.; Shen, Y.-C. J. Org. Chem. 1997, 62, 3804].  1H NMR: 8.20 (s, 1H), 6.10 (br s, 1H), 4.97 (d, 1H, J = 5.9), 4.67   (p, 1H, J= 6.1), 4.43 (q, 2H, J = 7.1), 1.63 (d, 3H, J =   6.2), 1.41 (t, 3H, J = 7.1)  13C NMR: 170.9, 160.9, 157.8, 148.0, 128.0, 80.0, 61.8, 60.9, 19.9,   14.3  MS:  257.2 [M + H+], 279.1 [M + Na+]  HRMS: calcd for C10H12N2O4SNa+: 279.0416    found:    279.0415            57    Figure A1. 1H NMR of 114   Figure A2. 13C NMR of 114       58 4–((tert–butyldimethylsilyloxy)methyl)–2–methylthiazole (128)        Ethyl 2-methylthiazole-4-carboxylate (1.41 g, 8.24 mmol) was dissolved in 20 mL of DCM and cooled in -78° C.  DIBAL-H (3M in hexanes, 18.0 mL, 18.1 mmol) was added dropwise and stirred for 45 minutes at -78° C.  The reaction was then warmed to r.t. for 1 hour before it was quenched with 30 mL of saturated Rochelle salt solution and stirred for 1 hour. The organic layers were separated and extracted twice with 20 mL of DCM. The organic layer was dried with Na2SO4 and concentrated under vacuum to give the alcohol as orange oil.  The crude was then dissolved in 15 mL of DCM with imidazole (1.00 g, 14.7 mmol) and TBS-Cl (1.24g, 8.22 mmol) and stirred overnight at r.t. Washing of the organic layer with 10 mL saturated solution of NaHCO3 followed by another wash with 10 mL of brine, drying over Na2SO4 and concentrating under vacuum gave a deep red liquid.  The crude was subjected to chromatography (5% EtOAc/ Hexanes) to give 1.7 g (85% over 2 steps) of the known 128 [Lefranc, D; Ciufolini, M.A. Angew. Chem. Int. Ed. 2009, 48, 4198] as a pale pink liquid. 1H NMR: 7.00 (s, 1H), 4.82 (s, 2H), 2.69 (s, 3H), 0.94 (s, 9H), 0.11   (s, 6H)  13C NMR: 166.0, 156.8, 112.9m 62.3, 25.9, 19.1, 18.4, -5.4  MS:  244.4 [M+H+], 266.4 [M+Na+]  HRMS: calcd for C11H21NOSSiNa+: 266.1011    found:    266.101  N S TBSO  59 Figure A3. 1H NMR of 128    Figure A4. 13C NMR of 128  60 (4S,5R)–4–(4–(2–(4–((tert-butyldimethylsilyloxy) methyl) thiazol–2– yl)acetyl)thiazol– 2–yl)–5 methyloxazolidin–2–one (43)                TBS-thiazole 128 (4.51g, 18.6 mmol) was dissolved in 13 mL of THF and cooled to -78º C.  A solution of butyllithium (1.3 M in hexanes, 14.3 mL, 18.6 mmol) was added dropwise to give a deep maroon color solution.  The reaction mixture was stirred for 10 minutes before a solution of 95 (1.59 g, 6.20 mmol) dissolved in 13 mL of THF, was added dropwise.  The reaction was then allowed to stir for one hour before it was quenched with 30 mL of H2O.  The reaction was then acidified to pH 3 with 1 M HCl and diluted with 30 mL of THF.  The organic layer was separated and extracted with EtOAc (2 x 15 mL), dried with Na2SO4 and concentrated under vacuum.  The crude was subjected to chromatography (30% EtOAc/Hexanes) to give 2.10 g (75%) of the known 43 [Lefranc, D; Ciufolini, M. A. Angew. Chem. Int. Ed. 2009, 48, 4198] as a yellow solid. Proton and 13C NMR spectra of this material indicated the presence of both the keto (major) and the enol form in a ca. 3:1 ratio.  1H NMR: 8.29 & 7.76(s, 1H), 7.17 & 6.99 (2t, 1H, J = 1.1), 6.71   (s,1H),5.98 & 5.81 (s, 1H), 4.90-4.65 (m, 5H), 1.65 (d,   3H, J = 6.0), 0.96 & 0.94 (2s, 9H), 0.15 & 0.11 (2s, 6H)  O NH O N S O N S OTBS  61 13C NMR: 188.9, 170.7, 169.9, 167.8, 161.7, 158.9, 156.8, 155.2,   154.5,154.1, 151.8, 127.4, 118.1, 114.7, 110.5, 93.5,   79.9, 79.8, 62.2, 61.7, 61.2, 61.0, 44.0, 25.9, 19.9, 18.4,   –5.3, –5.4  MS:  454.2 [M+H+], 476.2 [M+Na+]  HRMS: calcd for C19H27N3O4S2SiNa+:  476.1110  found:      476.1117                        62 Figure A5. 1H NMR of 43  Figure A6. 13C NMR of 43  63 Ethyl-2'-methyl-2,4'-bithiazole-4-carboxylate (146)     Ethyl 2-methylthiazole-4-carboxylate (8.7 g, 51.1 mmol) was dissolved in aq. conc. NH4OH solution (40 mL) and the solution was heated to 70 °C for 2 h. The solution was cooled to rt and extracted with EtOAc (3 x 30 mL). The combined extracts were evaporated and the residue of 2-methylthiazole-4-carboxamide was directly treated with the Lawesson’s reagent (10.3 g, 25.6 mmol) in refluxing toluene (40 mL) for 2 h. Upon cooling to 0 °C, the thioamide precipitated as a brown solid. The toluene was decanted and the thioamide was treated with ethyl bromopyruvate (10.0 g, 51.1 mmol) in refluxing ethanol (40 mL) for 2 hours. The cooled reaction mixture was evaporated and the residue was suspended in EtOAc (10 mL). Neutralization with aq sat NaHCO3 sol (40 mL) caused the precipitation of a white solid, which was filtered and washed with small amounts of cold hexane to give 10.6 g (80% over 3 steps) of the known 146 (Huang, L.; Quada, J. C., Jr.; Lown, J. W. Heterocyclic Commun. 1995, 1, 335) as a white solid. 1H NMR 8.17 (s, 1H), 8.01 (s, 1H), 4.45 (q, 2H, J = 7.14), 2.78 (s,   3H), 1.43(t, 3H, J = 7.14)   13C NMR 166.8, 163.4, 161.5, 148.0, 147.9, 127.6, 117.0, 61.5,   19.2, 14.4  MS:  255.2 [M+H+]  HRMS: calcd for C10H10N2S2O2Na+: 277.0081    found:    277.0075  m.p.:  95-97 °C  IR:  3434, 3127, 3117, 2974, 1705 N S S N COOEt  64 Figure A7. 1H NMR of 146   Figure A8. 13C NMR of 146   65  Figure A9. IR spectrum of 146                       66 Ethyl-2'-formyl-2,4'-bithiazole-4-carboxylate (41)      A solution of compound 146 (4.9 g, 19.2 mmol) and SeO 2  (6.4 g, 57.8 mmol) in AcOH (40 mL) was refluxed for 12 h. The mixture was filtered through Celite to remove a dark precipitate and the filtrate was evaporated. The residue was treated with aqueous saturated NaHCO3 solution (30 mL) and extracted with EtOAc. The combined extracts were dried (Na2SO4) and evaporated to give 2.8 g (55%) of the known aldehyde (Lefranc, D; Ciufolini, M.A. Angew. Chem. Int. Ed. 2009, 48, 4198).  1H NMR 10.06 (d, 1H, J = 1.0), 8.55 (d, 1H, J = 1.1), 8.26 (s, 1H),   4.47 (q, 2H, J = 7.1), 1.45 (t, 3H, J = 7.1)  13C NMR 183.2, 165.9, 161.9, 161.1, 151.1, 148.2, 128.5, 123.9,   61.7, 14.3  MS:  301.2 [M+MeOH+H]+  HRMS: calcd for C10H8N2O3S2Na+: 290.9874    found:    290.9865  m.p.: 156-157 °C (recrystallized from 1:1 EtOAc/Hexanes)  IR:  3127, 3098, 2839, 1721, 1695       N S OHC S N COOEt  67  Figure A10. 1H NMR of 41     Figure A11. 13C NMR of 41       68  Figure A12. IR spectrum of 41                      69 Ethyl-2'-(1-hydroxyprop-2-ynyl)-2,4'-bithiazole-4-carboxylate (126-a)      A solution of 41 (3.5 g, 13.0 mmol) in THF (8 mL) was added dropwise to a commercial 0.5 M solution of ethynylmagnesium bromide in THF (57.6 mL, 28.8 mmol) at rt. The mixture was stirred for 30 min, then it was quenched with aqueous saturated NH4Cl solution (30 mL). The organic layer was separated and the aqueous phase was extracted with EtOAc (2 × 30 mL). The combined extracts were dried (Na 2 SO 4 ) and evaporated. Flash chromatographic purification of the residue (40% EtOAc/hexanes) gave 126-a (2.9 g, 75%) as a white solid.  1H NMR (DMSO-d6):  8.54 (s, 1H), 8.35 (s, 1H), 7.08 (d, 1H, J = 6.0),     5.73 (dd, 1H, J= 6.0, 2.2), 4.32 (q, 2H, J = 7.1),     3.68 (d, 1H, J = 2.2), 1.31 (t, 3H, J = 7.1)  13C NMR (DMSO-d6): 174.0, 162.8, 161.1, 147.8, 147.4, 130.0, 119.4,     83.16, 77.3, 61.3, 60.8, 14.7  MS:    295.1 [M+H+]  HRMS:   calcd for C12H11N2O3S2+:  295.0211      found:     295.0191  m.p.:    145-148 °C  IR:    3247, 3111, 2116, 1709  N S S N COOEt HO  70 Figure A13. 1H NMR of 126-a     Figure A14. 13C NMR of 126-a    71  Figure A15. IR spectrum of 126-a                       72 Ethyl-2'-propioloyl-2,4'-bithiazole-4-carboxylate (126)        Dess−Martin periodinane (735 mg, 1.8 mmol) was added in small portions to a suspension of alcohol 126-a (435 mg, 1.5 mmol) in CH 2 Cl 2  (5 mL) at rt and with good stirring. The solution became clear after 10 min. After 2 h of stirring, the reaction was complete (TLC), whereupon it was diluted with 10 mL each of aqueous saturated NaHCO3 and aqueous saturated Na2S2O3 solutions. The organic layer was separated and further washed with aqueous saturated NaHCO3 solution (10 mL), then it was dried (Na 2 SO 4 ) and concentrated to give 126 as an orange solid (415 mg, 96%). This reactive material was best utilized in crude form, because purification induced unacceptable loss of product.  1H NMR: 8.54 (s, 1H), 8.26 (s, 1H), 4.46 (q, 2H, J = 7.0 Hz), 3.66   (s, 1H), 1.44 (t, 3H, J = 7.0 Hz)  13C NMR: 168.6, 165.7, 161.9, 161.2, 151.1, 148.2, 128.6, 124.7,   84.0, 79.1, 61.7, 14.3  MS:  315.1 [M+Na+]  HRMS: calcd for C12H9N2O3S2+:  293.0055    found:     293.0067  m.p. 107-109 °C (a sample purified by flash chromatography 40% EtOAc/Hexanes)  IR:  3214, 3122, 2996, 2098, 1725, 1640   N S S N COOEt O  73                  Figure A16. 1H NMR of crude 126     Figure A17. 13C NMR of crude 126      74     Figure A18. IR spectrum of 126                     75 Ethyl-2-formylthiazole-4-carboxylate (129) and Ethyl thiazole-4-carboxylate (147)       A solution of Ethyl 2-methylthiazole-4-carboxylate (4.0 g, 23.6 mmol) and SeO 2  (7.8 g, 70.8 mmol) in AcOH (95 mL) was refluxed for 12 h, then it was evaporated. The residue was neutralized with aqueous saturated NaHCO 3  solution (30 mL) and extracted with EtOAc. The combined extracts were dried (Na 2 SO 4 ) and evaporated. Flash chromatographic purification of the residue (30% EtOAc/ hexanes) afforded 2.4 g (55%) of the known 129 (Patterson, A.W.; Peltier, H.M.; Ellman, J.A. J. Org. Chem. 2008, 73, 4362) as a white solid and 780 mg (21%) of the known 147 (Borgen, G.; Grohowitz, S. Acta Chem. Scand. 1966, 20(9), 2593) as a pale yellow solid.  1H NMR (129): 10.08 (d, 1H, J = 1.3 Hz), 8.52 (d, 1H, J = 1.3 Hz), 4.50    (q, 2H, J =7.1 Hz), 1.45 (t, 3H, J = 7.1 Hz)  13C NMR (129): 183.6, 166.1, 160.6, 149.6, 133.0, 62.1, 14.3   1H NMR (147): 8.86 (d, 1H, J = 1.7), 8.26 (d, 1H, J = 1.7), 4.45 (q, 2H, J    = 7.1),1.43 (t, 3H, J = 7.1)  13C NMR (147): 161.3, 153.4, 148.2, 127.2, 61.6, 14.3 MS (129):  207.9 [M+Na+]  MS (147):  180.2 [M+Na+]  HRMS (129):  calcd for C7H8NO3S+:  186.0225     found:    186.0224  HRMS (147):  calcd for C6H7NO2Na+: 180.0095     found:    180.0098 N S OHC COOEt N S COOEt  76  m.p. (129):  65-66 °C [lit . 65 °C – 67 °C51]  m.p. (147):  49-50 °C  [lit . 52 °C – 53 °C52]  IR (129):  3371, 3105, 2986, 2940, 2863  IR (147):  3085, 2986, 1712                              77 Figure A19. 1H NMR of 129        Figure A20. 13C NMR of 129   78 Figure A21. 1H NMR of 147    Figure A22. 13C NMR of 147  79  Figure A23. IR spectrum of 129    Figure A24. IR spectrum of 147   80 Ethyl-2-propioloylthiazole-4-carboxylate (131)       A solution of 129 (1.7 g, 9.0 mmol) in THF (5 mL) was added dropwise with good stirring to a commercial 0.5 M solution of ethynylmagnesium bromide (27.0 mL, 13.5 mmol) at rt. The mixture was stirred for 30 min at rt, then it was cautiously quenched with aqueous saturated NH4Cl solution (20 mL). The organic layer was separated and the aqueous phase was extracted with EtOAc (2 × 20 mL). The combined extracts were dried (Na2SO4) and concentrated. The crude product, a thick oil, was directly added to a solution of IBX (2.5 g, 18.0 mmol) in DMSO (10 mL) and heated at 35 °C for 12 h. The cooled mixture was diluted with EtOAc (30 mL) and water (40 mL) and stirred vigorously for 10 min, then it was filtered over Celite. The organic phase was separated and the aqueous layer was extracted with ether (3 × 30 mL). The combined extracts were sequentially washed with saturated aqueous NaHCO3 (30 mL) and saturated aqueous NaCl (30 mL) solution, dried (Na 2 SO 4 ), and concentrated to give 1.6 g (85% over 2 steps) of the known (Bagley, M.C.; Xiong, X. Org. Lett. 2004, 6, 3401) 131 as a dark solid. Despite the color, this material was of sufficiently good quality to be used in the next step without purification. A sample purified by flash column chromatography (20% EtOAc/hex) was obtained as a white solid.  1H NMR: 8.50 (s, 1H), 4.48 (q, 2H, J = 7.2 Hz), 3.68 (s, 1H), 1.44 (t, 3H, J= 7.2 Hz)  N S COOEt O  81 13C NMR:  169.1, 166.0, 160.5, 149.6, 133.5, 84.8, 78.9, 62.1, 14.3  MS:   210.2 [M+H+], 232.1 [M+Na+]  HRMS:  calcd for C9H8NO3+: 210.0225     found:   210.0175  m.p.: 114-116 °C (sample purified by flash chromatography, 20% EtOAc/ Hexanes)  IR: 3212, 2101, 1718, 1638                        82  Figure A25. 1H NMR of 131    Figure A26. 13C NMR of 131   83   Figure A27.  IR spectrum of 131                               84 1-phenylprop-2-yn-1-one (135)      A solution of benzaldehyde (1.0 g, 9.2 mmol) in THF (3mL) was added dropwise to a commercial 0.5 M solution of ethynylmagnesium bromide (28.3 mL, 14.1 mmol) in THF at rt. The mixture was stirred for 10 min at 0°C, then it was quenched with aq sat NH4Cl sol (10 mL). The organic layer was separated and the aqueous phase was extracted with EtOAc (2 x 10 mL).  The combined extracts were evaporated to give the propargyl alcohol as yellowish oil. Without purification, this material was added to a solution of IBX (5.3 g, 18.8 mmol) in DMSO and the solution was heated to 35 °C for 12 h. The cooled reaction mixture was diluted with EtOAc (20 mL) and water (30 mL) and stirred vigorously for 10 min, then it was filtered over celite. The organic layer was separated and the aqueous phase was extracted with ether (3 x 20 mL). The combined extracts were sequentially washed with aq sat NaHCO3 (20 mL) and NaCl (20 mL) solutions, dried (Na2SO4) and concentrated to give 1.2 g (95% over 2 steps) of the known (Bowden, K.; Heilbron, I. M.; Jones, E. R. H.; Weedon, B. C. L. J. Chem. Soc. 1946, 39 ) 135 as a white solid. 1H NMR  8.16 (m, 2H), 7.65 (m, 1H), 7.51 (m, 2H), 3.44 (s, 1H)  13C NMR  177.4, 136.1, 134.5, 129.7, 128.7, 80.7, 80.3  MS:   131.1 [M+H+], 153.0 [M+Na+]  HRMS:  cacld for C9H7O+:  131.0497     found:    131.0495  O  85 m.p.:   46-47 °C [lit. 49 °C -50 °C 50]  IR:   3231, 2092, 1639                                 86  Figure A28. 1H NMR of 135     Figure A29. 13C NMR of 135  87  Figure A30. IR spectrum of 135                              88 Ethyl-2-(5-(4-(acetoxymethyl)thiazol-2-yl)-6-(2-((4S,5R)-5-methyl-2-oxooxazolidin-4- yl)thiazol-4-yl)pyridin-2-yl)thiazole-4-carboxylate (134)         A solution of ketone 43 (256 mg, 564 µmol), ynone 131 (118 mg, 564 µmol), and NH 4 OAc (65 mg, 846 µmol) in AcOH (5 mL) was refluxed for 8 hr, then it was concentrated, neutralized (aqueous saturated NaHCO 3  solution), and extracted with EtOAc (2 × 20 mL). The combined extracts were dried (Na 2 SO 4 ) and evaporated. Flash chromatographic purification of the residue (50% EtOAc/hexanes) afforded 134 (203 mg, 63%) as a light orange solid.  1H NMR 8.42 (d, 1H, J = 8.1), 8.32 (s, 1H), 8.19 (d, 1H, J = 8.1),   8.01 (s, 1H), 7.39 (s, 1H), 5.59 (s, br, 1H), 5.22 (s, 2H),   4.65 (dd, 1H, J = 6.3, 1.2), 4.54 (p, 1H, J = 6.2), 4.48 (q,   2H, J = 7.1), 2.13 (s, 3H), 1.48 (d, 3H, J = 6.3), 1.46 (t,   3H, J = 7.1)  13C NMR 170.8, 168.7, 168.5, 165.3, 161.3, 158.0, 154.1, 151.6,   150.5 (2 overlapping peaks), 148.6, 140.0, 130.1, 129.7,   121.5, 119.23, 119.2, 79.6, 61.7, 61.7, 60.8, 20.9, 19.9,   14.4  MS:  572 [M+H+], 594 [M+Na+]  HRMS: calcd for C24H22N5O6S3+:  572.0732  found:     572.0727  O NH O N S N N S AcO N S OEt O  89 [α] D  :  +21.1° (c 0.99, acetone)  m.p.:  95-98 °C  IR:  3312, 3106, 2980, 1730, 1729, 1728                     90  Figure A31. 1H NMR of 134  Figure A32. 13C NMR of 134      91 Figure A33.  NMR spectrum of 134                              92 Ethyl-2'-(5-(4-(acetoxymethyl)thiazol-2-yl)-6-(2-((4S,5R)-5-methyl-2-oxooxazolidin-4- yl)thiazol-4-yl)pyridin-2-yl)-2,4'-bithiazole-4-carboxylate (145)  O N H O N N S AcO S N N S O OEt N S    A solution of ketone 43 (380 mg, 832 µmol), ynone 126 (248 mg, 832 µmol), and NH 4 OAc (97 mg, 1.2 mmol) in AcOH (5 mL) was refluxed for 12 h, then it was concentrated, neutralized (aq. sat. NaHCO 3  sol.), and extracted with EtOAc (2 × 25 mL). The combined extracts were dried (Na 2 SO 4 ) and evaporated. Flash chromatographic purification of the residue (70% EtOAc/hexanes) afforded 145 (285 mg, 52%) as a pale yellow solid.  1H NMR: 8.34 (d, 1H, J = 8.0 Hz), 8.32 (s, 1H), 8.24 (s, 1H), 8.20   (d, 1H, J = 8.0), 8.03 (s, 1H), 7.40 (s, 1H), 5.78 (s, br,   1H), 5.23 (s, 2H), 4.65 (dd, 1H, J = 6.0, 1.1), 4.54 (p, 1H,   J = 6.1), 4.47 (q, 2H, J = 7.2), 2.13 (s, 3H), 1.48 (d, 3H, J   = 6.2), 1.45 (t, 3H, J = 7.2)  13C NMR: 170.8, 168.4, 168.3, 165.4, 163.1, 161.4, 157.7, 154.2,   151.6, 150.7, 150.6, 149.9, 148.1, 140.0, 129.6, 127.9,   121.5, 120.5,119.1, 118.8, 79.6, 61.8, 61.6, 60.8, 21.0,   19.9, 14.4  MS:  655 [M+H+], 677 [M+Na+]  HRMS: calcd for C27H23N6O6S4+:  655.0562    found:     655.0558  [α]D:  +5.5° (c 0.91, CHCl 3 )   93 m.p.:  210-211 °C  IR:  3279, 3124, 3108, 1780, 1733, 1716                         94 Figure A34. 1H NMR of 145     Figure A35. 13C NMR of 145        95  Figure A36. IR spectrum of 145                              96 HPLC Traces of Pyridines 134 and 145. Column: Agilent ZORBAX Bonus-RP, 3.5 µm, 4.6 mm x 150 mm Solvent:  A = MeOH, B = H2O, 50 mmol (NH4)SO4, pH = 5.5 Detection:  UV, 254 nm    Time (min) Flow (mL/min) %A %B Gradient: 1 0.00 1.00 5.0 95.0  2 10.00 1.00 100.0 0.0  3 25.00 1.00 100.0 0.0  4 31.00 1.00 5.0 95.0  Pyridine 134.   Pyridine 145.  Figure A37. HPLC traces of pyridine cores A U 0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 Minutes 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 O NH O N S N N S AcO N S OEt O A U 0.000 0.002 0.004 0.006 0.008 0.010 0.012 0.014 0.016 0.018 0.020 0.022 0.024 0.026 Minutes 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 O NH O N S N N S AcO N S N S O OEt  97 Ethyl 2-methyl-6-phenylnicotinate (137)      A solution of ethyl acetoacetate (229 µL, 1.8 mmol), alkynone 22 (234 mg, 1.8 mmol), and NH4OAc (208 mg, 2.7 mmol) in AcOH (5 mL) was refluxed for 12 h, then it was cooled to rt and evaporated. The residue was treated with aq sat NaHCO3 sol (10 mL) and extracted with EtOAc (2 x 20 mL). The combined extracts were dried (Na2SO4) and evaporated and the residue was purified by chromatography (10% EtOAc/ hexanes) to give 369 mg (85%) of the known 11644 as a white solid.  1H NMR: 8.28 (d, 1H, J = 8.3), 8.10-8.03 (m, 2H), 7.64 (d, 1H, J =   8.3), 7.54-7.41 (m, 3H), 4.41 (q, 2H, J = 7.1), 2.92 (s,   3H), 1.43 (t, 3H, J = 7.1)  13C NMR: 166.5, 159.1, 158.9, 139.3, 138.4, 129.6, 128.8, 1127.3,   123.6, 117.3, 61.1, 25.3, 14.3  MS:  242.1 [M+H+]  HRMS: calc for C15H16NO2+:  242.1181    found:    242.1182  m.p.  44-45 °C  IR:  2987, 1714   N O EtO  98  Figure A38. 1H NMR of 137     Figure A39. 13C NMR of 137           99  Figure A40. IR spectrum of 137                       100 2-phenyl-7,8-dihydroquinolin-5(6H)-one(139) and phenyl(6-phenylpyridin-3- yl)methanone (140)          A solution of 1,3-cyclohexanedione (276 mg, 2.5 mmol), alkynone 135 (321 mg, 2.5 mmol), and NH4OAc (285 mg, 3.7 mmol) in AcOH (7 mL) was refluxed for 8 hours, then it was cooled to rt and evaporated. The residue was neutralized with aq sat NaHCO3 sol (10 mL) and extracted with EtOAc (2 x 20 mL).  The combined extracts were dried (Na2SO4) and evaporated and the residue was purified by chromatography (10% EtOAc/hexanes) to give 341 mg (62%) of the known 139 as a white solid, plus 139 mg (22 %) and 140 as a pale yellow solid.  1H NMR (139): 8.34 (d, 1H, J = 8.2), 8.10-8.03 (m, 2H), 7.72 (d, 1H, J =    8.2), 7.56-7.44 (m, 3H), 3.23 (t, 2H, J = 6.3), 2.73 (t, 2H,    J = 6.3), 2.24(p, 2H, J = 6.3)  13C NMR (139):  197.9, 163.8, 160.8, 138.4, 135.8, 130.0, 128.9, 127.5,    126.5, 119.0, 38.6, 32.9, 22.0  1H NMR (140): 9.08 (dd, 1H, J = 2.4, 0.6), 8.22 (dd, 1H, J = 8.3, 2.2),    8.15 (m,2H), 7.88 (m, 3H), 7.65 (tt, 1H, J = 7.41, 2.31),    7.54 (m, 5H)  13C NMR (140): 194.7, 160.4, 151.2, 138.2, 137.0, 133.0, 131.2, 130.01,    129.97,129.0, 128.6, 127.4, 119.9  MS (139):  224.3 [M+H+]  MS (140):  260.3 [M+H+]  HRMS (139):  calcd for C15H14NO+:  224.1075 N O O N  101     found:    224.1073  HRMS (140):  calcd for C18H14NO+:  260.1075     found:    260.1082  m.p. (139):  129-130 °C  m.p. (140):  83-85 °C  IR (139):  2952, 1674  IR (140):  3050, 1639                         102    Figure A41. 1H NMR of 139 Figure A42. 13C NMR of 139    103 Figure A43. 1H NMR of 140   Figure A44. 13C NMR of 140   104 Figure A45. IR spectrum of 139  Figure A46. IR spectrum of 140  105 (R)-tert-butyl 1,3-dihydroxy-3-methylbutan-2-ylcarbamate (107)       D-Serine methyl ester hydrochloride (4.14 g, 26.6 mmol) was suspended in 15 mL of DCM. NaHCO3 (2.23g, 26.6 mmol) dissolved in 10 mL of H2O was added.  Boc2O (6.38 g, 29.3 mmol) was dissolved in 15 mL of DCM and added to the mixture.  NaCl (4.63 g, 79.8 mmol) was added and the mixture was heated at 50° C for 12 hours.  The solution was then cooled to r.t. and the organic layer was separated.  The product was extracted with DCM (2 x 30 mL) and the combined extracts were dried (Na2SO4) and concentrated under vacuum. The crude N-Boc serine was dissolved in 30 mL of DCM. Neat DHP (4.82 mL, 53.2 mmol) was added, followed by PPTS (0.668 g, 2.66 mmol). The mixture was stirred overnight, then it was washed with 20 mL of sat. NaHCO3 sol. and concentrated to give the protected alcohol.  The crude was dissolved in 20 mL of THF and then added to a solution of 3M methyl magnesium bromide (35.4 mL, 106 mmol) in 10 mL of THF under argon at 0º C. After 5 minutes, the reaction mixture was heated to 45° C for 1 hour.  Upon cooling to r.t., the reaction was quenched by the slow addition of 20 mL of sat. NH4Cl sol., followed by 30 mL of water; then it was extracted with EtOAc (3 x 35 mL).  The combined extracts were dried (Na2SO4) and concentrated in vacuum. The crude residue was redissolved in MeOH (40 mL) containing PTSA (0.5 g, 2.7 mmol) and the solution was stirred for 3 hours. The mixture was then evaporated and the residue redissolved in 40 mL of EtOAc.  The organic layer was washed with 10 mL of saturated NaHCO3 solution, dried (Na2SO4) and evaporated in vacuo.  The residue was then HO NHBoc OH  106 purified by flash chromatography (30% EtOAc/ Hexanes) to give the title compound 107 (4.80 g, 82% over 4 steps) as a colorless oil, which solidified upon standing.  1H NMR: 5.38 (br d, 1H), 4.04 (m, 1H), 3.81 (m, 1H), 3.47 (br m,   1H), 2.61 (s, 1H), 2.53 (m, 1H), 1.46 (s, 9H), 1.36 (s,   3H), 1.26 (s, 3H)  13C NMR: 156.5, 79.6, 73.6, 63.1, 57.8, 28.3, 27.4, 27.1  MS:  242.3 [M + Na+]  HRMS: calcd for C10H21NO4Na+:  242.1368    found:     242.1366  [α]D:  -17.3° (c = 1, CHCl3)  m.p.:  61-63 °C  IR:  3418, 3272, 2977, 2937, 1709          107 Figure A47. 1H NMR of 107    Figure A48. 13C NMR of 107       108  Figure A49. IR spectrum of 107                             109 (S)-methyl-2-(1-(tert-butoxycarbonylamino)-2-hydroxy-2-methylpropyl)thiazole-4- carboxylate (110)   NHBoc OH S NMeO O    Sulfur trioxide-pyridine complex (5.30 g, 33.2 mmol) was dissolved in 20 mL of warm DMSO and added to a solution of compound 107 (2.43 g, 11.1 mmol) in 30 mL of DCM. The reaction was allowed to stir for 45 minutes before it was quenched with 30 mL of saturated solution of NaHCO3.  The volatiles were removed in vacuum and the product was extracted with EtOAc (3 x 20 mL).  The organic layer was then washed with 15 mL of 1M NaHSO4, 15 mL of brine, 15 mL of H2O and then dried with Na2SO4 and concentrated to give the sensitive aldehyde, which was immediately dissolved in 20 mL of MeOH and treated with a solution of L-cysteine methyl ester hydrochloride (2.85 g, 16.6 mmol) in 10 mL of H2O.  After stirrin for 2 hours the volatiles were evaporated and the product was extracted with EtOAc (3 x 30 mL).  The organic layer was dried with Na2SO4 and concentrated in vacuum to give the thiazolidine as a 1:1 mixture of diastereomers.  The crude thiazolidine was dissolved in 30 mL of MeCN containing oven-dried "chemical" MnO2 (38.6 g, 444 mmol, purchased from Wako Pure Chemicals, 99.5% purity).  The reaction mixture was heated to 60° C for 24 hrs, then it was filtered over Celite® and concentrated in vacuum.  The residue was purified by flash chromatography (5% acetone/DCM) to give the title compound 110 as a white solid (1.10 g, 30% over 3 steps).  1H NMR: 5.38 (br s, 1H), 4.04 (m, 1H), 3.81 (m, 1H), 3.47 (br,   1H), 2.61 (s,1H), 1.46 (s, 9H), 1.36 (s, 3H), 1.26   (s,3H)  110  13C NMR: 156.5, 79.6, 73.6, 63.1, 57.8, 28.3, 27.4, 27.1  MS:  242.3 [M+Na+]   HRMS: calcd for C10H21NO4Na+: 242.1368    found:    242.1366  [α]D:  +24.5° (c = 0.2, CHCl3)  m.p.:  47-50 °C  IR:  3386, 2978, 1708                   111 Figure of A50. 1H NMR of 110   Figure A51. 13C NMR of 110        112 Figure A52. IR spectrum of 110                              113 Methyl-2-((R)-1-(2-((1S,2R)-1-(tert-butoxycarbonylamino)-2-hydroxypropyl)thiazole-4- carboxamido)-2-hydroxy-2-methylpropyl)thiazole-4-carboxylate (115)             A solution of ester 114 (1.00 g, 3.90 mmol), Boc2O (1.02 g, 4.68 mmol), triethylamine (0.54 mL, 3.90 mmol), and a spatula tip of DMAP in 10 mL of DCM was stirred at room temperature for 2 hours, then it was concentrated under vacuum. The residue was purified by flash chromatography (10% EtOAc/Hexanes) and the Boc-protected oxazolidinone thus obtained was dissolved in 7 mL of 1:1 mixture of THF and H2O and treated with LiOH monohydrate (980 mg, 23.3 mmol).  The mixture was stirred for 2 hours and then acidified to pH 3 using 1M HCl. The solution was extracted with EtOAc (4 x 10 mL). The combined extracts were dried with Na2SO4 and concentrated in vacuum to give carboxylic acid 94 (1.12 g, 95% over 2 steps) as a light yellow solid.  A solution of hydroxyvaline-derived thiazole 110 (711 mg, 2.15 mmol) in 5 mL of a 4:1 mixture mL of DCM and TFA was stirred for 1 hr under argon.  The reaction was then concentrated under vacuum. The residue was taken up with more DCM (5 mL) and the solution again concentrated in vacuum to promote complete removal of TFA. The residue of TFA salt of amine 92 thus obtained (quantitative yield) was dissolved in 5 mL of DCM and added to a solution of acid 94 (0.651g, 2.15 mmol), HOBt (0.349 g, 2.58 mmol) and triethylamine (0.95 mL, 6.88 mmol) in 7 mL of DCM.  The mixture was stirred for 2 minutes, then N S OMe O HN HO O S N BocHN OH  114 EDCI (0.495 g, 2.58 mmol) was added and stirring was continued overnight.  The reaction was quenched with 10 mL of aq. sat. NH4Cl sol. and the organic layer was separated and concentated under vacuum. The residue was redissolved in 30 mL of EtOAc and was sequentially washed with 10 mL of aq. sat. NaHCO3 sol., 10 mL of H2O and 10 mL of brine, then dried with Na2SO4 and concentrated under vacuum to give a brown solid.  Purification by flash chromatography (70% EtOAc/ Hexanes) gave 115 as a white solid (0.720 g, 65% over 3 steps).  1H NMR: 8.22 (d, 1H, J = 9.1), 8.13 (s, 1H), 8.05 (s, 1H), 5.70 (d,   1H, J =  8.7), 5.34 (d, 1H, J = 8.7), 4.89 (d, 1H, J = 9.3),   4.63 (m, 1H), 3.92 (s, 3H), 1.46 (s, 9H), 1.36 (s, 3H),   1.33 (d, 3H, J = 6.4), 1.28 (s, 3H)  13C NMR: 173.2, 169.1, 161.5, 160.8, 155.9, 149.0, 146.2, 128.0,   124.5, 80.4,72.7, 68.5, 57.5, 57.3, 52.4, 28.3, 27.8, 26.4,   19.6  MS:  537.3 [M + Na+]  HRMS: calcd for C21H30N4O7S2Na+  537.1454    found:     537.1464  [α]D:  -33.0° (c = 0.2, CHCl3)  m.p.:  73-75 °C  IR:  3387, 3116, 2987, 1731, 1695             115     Figure A53. 1H NMR of 115       Figure A54. 13C NMR 115    116     Figure A55. IR spectrum of 115                         117 (4S,5R)-tert-butyl-4-((Z)-1-(4-((R)-2-hydroxy-1-(4-(methoxycarbonyl)thiazol-2-yl)-2- methylpropylcarbamoyl)thiazol-2-yl)prop-1-enylcarbamoyl)-2,2,5-trimethyloxazolidine- 3-carboxylate (117)               Compound 115 (0.720 g, 1.49 mmol) is dissolved in 10 mL of DCM and TFA (4:1) and stirred for one hour. The reaction was then concentrated under vacuum (repeated twice for complete removal of TFA) and re-dissolved in 5mL of DCM. This was then added to a solution of N-Boc acetonide threonine 91 (0.387g, 1.49 mmol), HOBt (0.246g, 1.82mmol) and triethylamine (0.923 mL, 1.82 mmol).  After the mixture was allowed to stir for 2 minutes, EDCI (0.350 g, 1.82mmol) was added and stirred overnight.  The reaction was quenched with 10 mL of saturated solution of NH4Cl and the organic layers were separated.  DCM was stripped under vacuum and the crude was redissolved in 30 mL of EtOAc.  The organic layer was washed once with 10 mL of saturated NaHCO3, 10 mL of H2O and 10 mL of brine.  The organic layer was dried with Na2SO4 and concentrated under vacuum to give a brown solid.  The crude brown solid was then redissolved in 4mL of DCM with triethylamine (0.61 mL, 4.47 mmol) and MsCl (0.14 mL, 1.79 mmol) under argon for 2 hours.  Then DBU (0.67 mL, 4.47 mmol) was added to the reaction mixture and stirred for 4 hours.  Once the reaction was complete, it was quenched by the addition of 10 mL of saturated NH4Cl solution.  The organic layer was N S OMe O HN HO O S N HN O NBoc O  118 separated and further extracted 3 times with DCM (3 x 10 mL).  The organic layer was combined, dried with Na2SO4 and concentrated under vacuum.  The crude was then subjected to chromatography to give a white solid (0.30g, 32% over 3 steps)  1H NMR: 8.19 (d, 1H, J = 8.6 Hz), 8.12 (s, 1H), 7.99 (s, 1H), 6.57   (br s, 1H), 5.34 (d, 1H, J = 9.4 Hz), 4.36 (m, 1H), 4.29   (br s, 1H), 4.02 (d, 1H, J = 9.4), 3.90 (s, 3H), 1.86 (d, 3H,   J = 6.7), 1.64 (s, 3H), 1.62 (s, 3H), 1.47 (d, 3H, J = 6.0),   1.42 (br s, 9H), 1.35 (s, 3H), 1.26 (s, 3H)  13C NMR:       169.2, 168.3, 167.0, 161.5, 160.6, 152.4, 149.1, 146.3, 127.9,127.6, 123.8, 95.1, 81.3, 74.3, 72.6, 67.7, 57.4, 52.3, 28.3, 27.7, 26.7, 25.7, 19.2, 14.4  MS:                 660.3 [M+H+]  HRMS:           calcd for C28H39N5O8S2Na+:   660.2138              found:      660.2131  [α]D:               -22.4 ° (c = 1, CHCl3)  m.p.:               122-124 °C  IR:            3387, 3360, 2979, br 1677         119  Figure A56. 1H NMR of 117     Figure A57. 13C NMR of 117     120  Figure A58. IR spectrum of 117                             121 (4S,5R)-3-(tert-butoxycarbonyl)-2,2,5-trimethyloxazolidine-4-carboxylic acid (91)       A solution of N-Boc-L-theronine (2 g, 9.1 mmol) in 15 mL DCM containing 2, 2- dimethoxypropane (22.3 mL, 0.182 mmol) and PTSA (0.15g, 0.91mmol) was stirred at r.t. for 2 hours.  The mixture was washed with aq. sat. NaHCO3 sol. (3 x 20 mL).  The aqueous layer was re-acidified to pH 3 with 1M HCl and back-extracted with EtOAc (3 x 30 mL).  The combined organic phases were dried (Na2SO4) and concentrated under vacuum to give 91 (Lefranc, D; Ciufolini, M.A. Angew. Chem. Int. Ed. 2009, 48, 4198) as a white solid (2.31 g, 98%).   1H NMR (CD3OD):  4.17 (p, 1H, 6.2), 3.83 (d, 1H, J = 7.9), 1.59 (s,    3H), 1.43 (s, 9H), 1.38 (d, 3H, J = 5.9)  13C NMR (CD3OD): 171.5, 172.0, 151.2, 151.5, 94.6, 94.4, 80.8, 80.4,    74.0, 73.7, 66.3, 66.1, 27.4, 27.3, 27.0, 25.6, 23.9,    23.0, 18.2, 18.0  MS:   282.3 [M + Na+]  HRMS:  calcd for C12H21NO5Na+: 282.1317     found:     282.1319       O OH O NBoc  122 Figure A59. 1H NMR of 91  Figure A60. 13C NMR of 91      123 (R)-1-((2S,3R)-2-amino-3-hydroxybutanamido)propan-2-yl acetate chloride salt (150)  O N H NH3 OAc OH Cl   A solution of acetonide 91 (1.00 g, 3.85 mmol), (R)-isoalaninol (0.303 mL, 3.85 mmol), HOBt (0.572 g, 4.23 mmol), EDCI (0.81g, 4.23 mmol) and Et3N (1.17 mL, 8.47 mmol) in 10 mL DCM was stirred overnight under Ar, then it was treated with 10 mL of aq. sat. NH4Cl solution. The organic phase was separated and washed sequentially with 10 mL of aq. sat. NaHCO3 sol. and 10 mL of brine, then it was dried (Na2SO4) and concentrated. The white solid residue was dissolved in 20 mL of pyridine, cooled to 0 °C, treated with Ac2O (0.74 mL, 7.70 mmol, added dropwise) and stirred at r.t. for 6 hours.  The mixture was concentrated and the residue was redissolved in 30 mL of EtOAc, washed with aq. sat. NaHCO3 (2 x 50 mL), aq. sat. NH4Cl sol. (2 x 50 mL) and brine (2 x 50 mL).  The organic layer was then dried with Na2SO4 and concentrated to give acetate.  This crude material was then dissolved in 5 mL of commercial 4M HCl in dioxane and stirred for 1 hour.  The excess HCl was then removed under water aspirator vacuum. The solution was then concentrated in vacuum to give the known 150 (Lefranc, D; Ciufolini, M.A. Angew. Chem. Int. Ed. 2009, 48, 4198) as a white solid (0.932g, 95% over 3 steps).  1H NMR (CD3OD): 8.55 (br s, 1H), 4.96 (br, 1H), 3.98 (br, 1H), 3.62    (br, 2H), 3.35 (br,2H), 3.19 (br, 1H), 2.00 (s, 3H),    1.22 (br m, 6H)  13C NMR (CD3OD): 171.0, 167.4, 69.2, 66.0, 59.1, 43.3, 20.1, 19.0,    16.6  MS:   219.3 [M+H+]   124 HRMS:  calcd for C9H18N2O4Na+:  241.1164     found:     241.1157                          125 Figure A61. 1H NMR of 150     Figure A62. 13C NMR of 150         126 (4S,5R)-tert-butyl-4-(4-(6-(4-((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 (152)   O N O N N S TBSO S N N S O NH N S OH O N H OAc Boc   A solution of pyridine 145 (0.559 g, 0.854 mmol) in 15 mL of 1:1 DCM and EtOH containing suspended K2CO3 (23.6 g, 171 mmol) was stirred overnight, then it was filtered and concentrated to give the liberated alcohol.  A solution of this crude material in 5 mL of DMF was treated with TBS-Cl (0.128 g, 0.854 mmol) and imidazole (0.117g, 1.71 mmol) and stirred at r.t for 24 hrs.  The mixture was then diluted with 15 mL of EtOAc, washed with aq. sat. NH4Cl sol. (3 x 5 mL), and concentrated under vacuum. The residue was purified by flash chromatography to give the protected alcohol (0.388 g) as a yellow solid.  A solution of the latter in 10 mL of 1:1 THF - water containing LiOH⋅H2O (0.067g, 1.6 mmol) was stirred for 2 hours, then it was acidified to pH = 3 and extracted with EtOAc (3 x 10 mL). The combined extracts were dried (Na2SO4) and concentrated to give the expected carboxylic acid 151.  A solution of this crude substance in 4 mL of DCM containing NEt3 (0.15mL, 1.07 mmol), a spatula tip of DMAP, and Boc2O (0.290 g, 1.33mmol) was stirred under Ar for 3 hours, then it was treated with 5 mL of H2O and acidified to pH = 3 with 1M HCl solution.  The mixture was extracted with EtOAc (3 x 10 mL) and the combined extracts were dried (Na2SO4) and concentrated to give the Boc protected oxazolidinone. A solution of this crude substance  127 and 76 (0.133 g, 0.533 mmol) in 3 mL of MeCN containing NEt3 (0.23 mL, 1.70 mmol) and BOP-Cl (0.131g, 0.533 mmol) was stirred for 3 hours and then quenched with 3 mL of saturated NH4Cl solution and EtOAc (10 mL) and the layers were separated.  The product was extracted futher with EtOAc (2 x 5 mL).  The residue was purified by flash chromatography (EtOAc/Hexanes) to give the known 152 (Lefranc, D; Ciufolini, M.A. Angew. Chem. Int. Ed. 2009, 48, 4198) as a white solid (0.167g, 20% over 5 steps).   1H NMR 8.34 (d, 1H, J = 8.3), 8.29-8.21 (m, 2H), 8.19 (s, 1H),   8.01 (s, 1H), 7.31 (s, 1H), 7.09 (t, 1H, J = 5.9), 5.16-4.96   (m, 2H), 4.89 (s, 1H), 4.65-4.44 (m, 3H), 3.78 (br s, 1H),   3.61-3.45 (m, 1H), 3.45-3.22 (m, 1H), 2.04 (s, 3H), 1.49   (s, 9H), 1.26 (d, 3H, J = 6.4), 1.23 (d, 3H, J = 6.4), 0.96   (s, 9H), 0.14 (s, 6H)  13C NMR 171.5, 170.8, 170.8, 168.8, 166.8, 164.5, 162.8, 162.2,   157.7, 153.5, 150.8, 150.4, 149.8, 149.8, 148.9, 140.0,   130.1, 124.5, 121.8, 120.3, 118.8, 75.6, 69.5, 66.3, 62.3,   62.2, 56.6, 43.7, 27.9, 25.9, 21.2, 20.2, 18.4, 18.3, 17.6,   –5.3  MS:                 1021.8 [M+Na+]  HRMS:            calcd for C43H54N8O10S4SiNa+:  1021.2513                          found:      1021.2502               128     Figure A63. 1H NMR of 152    Figure A64. 13C NMR of 152  129 (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 (153)         A solution of 152 (0.167 g, 0.166 mmol), NEt3 (0.06 mL, 0.415 mmol) and MsCl (0.03 mL, 0.332 mmol) in 5 ml DCM were stirred at rt for 1 h under Ar, then neat DBU (0.050 mL, 0.332 mmol) was added.  The mixture was stirred for 1 more hour, then it was quenched with 3 mL of aq. sat. NH4Cl solution. The organic layer was separated, dried (Na2SO4) and concentrated to give dehydroaminoacid derivative.  A solution of this crude substance in commercial in 0.1M TBAF in THF (0.82 mL, 0.830 mmol) was stirred for 3 h, then it was diluted with 3 mL of EtOAc and 3mL of aq. sat. NH4Cl solution.  The organic layer was separated, dried with Na2SO4 and concentrated.  The crude alcohol thus obtained was dissolved in 3 mL of DCM containing Dess-Martin periodinane (0.147g, 0.349 mmol) and stirred for 2 hours.  The reaction was quenched with 3 mL of aq. sat. Na2S2O4 sol. and extracted with EtOAc (3 x 10 mL).  The combined extracts were dried (Na2SO4) and evaporated under vacuum. The residue was purified by flash chromatography (2% MeOH in EtOAc) to give the known aldehyde 153 (Lefranc, D; Ciufolini, M.A. Angew. Chem. Int. Ed. 2009, 48, 4198) (0.120 g, 84% over 3 steps).  O N O N N S O S N N S O NH N S O N H OAc Boc H  130 1H NMR 10.05 (s, 1H), 8.73 (br s, 1H), 8.37 (s, 1H), 8.37 (overlap-   ping d, 1H,  J = 8.0), 8.25 (s, 1H), 8.24 (s, 1H), 8.22 (s,   1H), 8.19 (d, 1H, J =8.0), 6.62 (q, 1H, J = 7.2), 6.57 (t,   1H, J = 5.2), 5.10-4.98 (m, 1H), 4.94 (d, 1H, J = 3.8),   4.51-4.40 (m, 1H), 3.62-3.52 (m, 1H), 3.50-3.38 (m, 1H),   2.03 (s, 3H), 1.87 (d, 3H, J = 7.2), 1.45 (s, 9H), 1.43   (overlapping d, 3H), 1.28 (d, 3H, J = 6.3)  13C NMR 184.2, 171.1, 168.6, 166.4, 166.2, 164.9, 162.6, 159.4,   154.7, 153.4, 151.0, 150.6, 150.2, 150.0, 148.7, 140.5,   131.0, 131.0, 129.4, 128.5, 125.0, 122.3, 120.2, 118.6,   85.0, 75.3, 69.9, 61.8, 44.5, 27.9, 21.2, 20.4, 17.7, 14.1  MS:  887.5 [M+Na+]  HRMS: calcd for C37H36N8O9S4Na+:  887.1386    found:     887.1370                   131 Figure A65. 1H NMR of 153   Figure A66. 13C NMR of 153        132 Thiocillin I (1)                  Aldehyde 153 (0.120g, 0.133 mmol), NaClO2 (24.2 mg, mmol), 2-methylbutene (0.66mL, 1.33 mmol, 2M in THF) and NaH2PO4 (33 mg, 0.275 mmol) are dissolved in 1mL of 1:1 mixture THF and H2O for 2 hours before being acidified to pH = 2 with 1M HCl solution. The carboxylic acid was extracted twice with 3 mL DCM, dried with Na- 2SO4 and concentrated under vacuum.  This crude material, NEt3 (0.05 mL, 0.360 mmol), the unprotected tripeptide 88 (which was prepared by exposing 99 (0.170g, 0.266 mmol) to TFA:MeOH for 3 hours and then concentrating to give the trifluoro acetate salt) and BOP-Cl (0.070 g, 0.274 mmol) were stirred in 2 mL of acetonitrile under Ar overnight.  It was diluted with 5 mL of DCM and successively washed with saturated solutions of NH4Cl, NaHCO3 and NaCl.  The organic layer was then dried with Na2SO4 and concentrated under vacuum.  The crude was then dissolved in 2 mL of 1:1 mixture of THF/H2O, LiOH (0.033g, 7.86 mmol) and stirred at r.t. for 2 hours before it was acidified to pH = 3 with 1M HCl.  The compound was then extracted with DCM (3 x 5 mL), dried with Na2SO4 and concentrated under vacuum to give the crude carboxylic acid.  This was N S N N S N S S NO N H O O H N OH S N O HN O N S N H O HN OH OH OH N H  133 then dissolved in TFA/DCM (1:4) and stirred for 2 hours at r.t. before it was concentrated under vacuum to give the trifluoroacetate salt.  The crude material was then dissolved in 1 mL of DMF, DPPA (0.029 mL, 0.134 mmol) and NEt3 (0.055 mL, 0.396 mmol) and stirred for 2 hours at r.t. before being diluted with 5 mL of DCM.  The organic layer was successively washed with saturated solutions of NH4Cl, NaHCO3, NaCl and dried with Na2SO4 and concentrated under vacuum.  The crude was then purified (MeOH/EtOAc) to give Thiocilline I (1) (20 mg, 12 % over 5 steps).  1H NMR (DMSO-d6, 600 MHz): 9.70 (s, 1H), 9.50 (s, 1H), 8.60 (s,      1H), 8.47 (s, 1H), 8.41 (d, 1H), 8.37      (s, 1H), 8.35, (d, 1H), 8.30 (d, 1H),      8.26 (s, 1H), 8.00 (s, 1H), 7.90 (br t,      1H), 7.58 (d, 1H), 6.50 (2 overlapping      q, 1H + 1H), 5.48 (d, 1H), 5.04 (dd,      1H), 4.70 (dd, 1H), 4.50 (br s, 1H),      3.95 (t, 1H), 3.69 (q, 1H), 3.07 (m,      2H), 1.70 (d, 3H), 1.73 (d, 3H), 1.37      (d, 3H), 1.25 (s, 3H), 1.22 (s, 3H),      1.02 (d, 6H)  13C NMR (DMSO-d6, 600 MHz): 170.85, 168.80, 168.30, 167.88,      166.84, 164.72, 164.72, 161.48,      161.17, 159.86, 159.87, 159.87,      153.33, 150.91, 150.50, 149.77,      149.42, 149.09, 148.79, 148.59,      140.73, 130.47, 129.36, 128.76,      128.67, 127.98, 125.41, 125.31,      125.29, 124.43, 121.47, 120.62,      118.41, 71.26, 67.96, 66.67, 64.87,      56.97, 56.38, 56.29, 46.52, 27.22,      25.51, 20.71, 20.67, 19.91, 13.38,      13.24  MS:     1160.3 [M + H]  HRMS:    calcd for C48H49N13O10S6Na+: 1182.1947   134      found:    1182.1974  [α]D:     +100 °                          135 Figure A67. 1H NMR of Thiocillin I (1)  Figure A68.  HMQC of 1 (13C data tabulated on page 43)   136 Figure A69.  HBMC of 1 (13C data tabulated on page 43) 

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