Science, Faculty of
Chemistry, Department of
DSpace
UBCV
Hwang, Hee-Jong
Doctor of Philosophy - PhD
University of British Columbia
The World Health Organization (WHO) describes antimicrobial resistance (AMR) as one of the three greatest threats to human health. Yet, the current global antibiotic pipeline is scarce, and new resources to combat bacterial pathogens that have become resistant to available antibiotics are few and far between. Therefore, the development of new antibiotics is urgently needed to address the ongoing “antibiotic crisis”. In that regard, thiopeptide antibiotics appear to be good potential sources of new antibacterials. They are potent inhibitors of bacterial protein synthesis and their mode of action is reasonably well understood. Yet, little effort has been made to clinically leverage these natural products. In part, this may be due to the fact that there existed no viable synthetic methods to conduct medicinal chemistry work on these substances. This dissertation presents the first total synthesis of micrococcin P2, a D-series, 26-membered antibiotic, by a synthetic route that incorporates a number of refinements relative to past approaches, and that provides access to large quantities of end product. The synthesis demonstrates economical, concise, efficient, convergent, and diversity-oriented protocols that facilitate medicinal chemistry ventures to a considerable extent. This has enabled an extensive biological evaluation of micrococcin P2 and congeners against Clostridioides difficile, Mycobacterium tuberculosis and non-tuberculous Mycobacterium (NTM). A parallel effort demonstrates the application of the new methodology in a synthetic study toward lactocillin, a metabolite of the human symbiotic bacterium, Lactobacillus gasseri. The work presented herein outlines a route to the most advanced synthetic precursor of lactocillin described to date.
Chemical and Biological Studies on Thiopeptide Antibiotics: Total Synthesis of Micrococcin P2 and Synthetic Efforts towards Lactocillin by Hee-Jong Hwang B. Sc., The University of British Columbia, 2012 M. Sc., The University of British Columbia, 2015 A DISSERTATION SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Chemistry) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) July 2022 ⓒ Hee-Jong Hwang, 2022 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the dissertation entitled: “Chemical and Biological Studies on Thiopeptide Antibiotics: Total Synthesis of Micrococcin P2 and Synthetic Efforts towards Lactocillin” submitted by Hee-Jong Hwang in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Chemistry Examining Committee: Marco A. Ciufolini, Professor, Department of Chemistry, UBC Supervisor Stephen Withers, Professor, Department of Chemistry, UBC Supervisory Committee Member David Perrin, Professor, Department of Chemistry, UBC Supervisory Committee Member Katherine S. Ryan, Associate Professor, Department of Chemistry, UBC University Examiner Yossef Av-Gay, Professor, Department of Medicine, UBC University Examiner Additional Supervisory Committee Members: Jason Hein, Associate Professor, Department of Chemistry, UBC Supervisory Committee Member iii Abstract The World Health Organization (WHO) describes antimicrobial resistance (AMR) as one of the three greatest threats to human health. Yet, the current global antibiotic pipeline is scarce, and new resources to combat bacterial pathogens that have become resistant to available antibiotics are few and far between. Therefore, the development of new antibiotics is urgently needed to address the ongoing “antibiotic crisis”. In that regard, thiopeptide antibiotics appear to be good potential sources of new antibacterials. They are potent inhibitors of bacterial protein synthesis and their mode of action is reasonably well understood. Yet, little effort has been made to clinically leverage these natural products. In part, this may be due to the fact that there existed no viable synthetic methods to conduct medicinal chemistry work on these substances. This dissertation presents the first total synthesis of micrococcin P2, a D-series, 26-membered antibiotic, by a synthetic route that incorporates a number of refinements relative to past approaches, and that provides access to large quantities of end product. The synthesis demonstrates economical, concise, efficient, convergent, and diversity-oriented protocols that facilitate medicinal chemistry ventures to a considerable extent. This has enabled an extensive biological evaluation of micrococcin P2 and congeners against Clostridioides difficile, Mycobacterium tuberculosis and non-tuberculous Mycobacterium (NTM). A parallel effort demonstrates the application of the new methodology in a synthetic study toward lactocillin, a metabolite of the human symbiotic bacterium, Lactobacillus gasseri. The work presented herein outlines a route to the most advanced synthetic precursor of lactocillin described to date. iv Lay Summary Antibiotic resistance is one of the principal public health problems of the 21st century. New antibiotics are urgently needed to treat microbial infections caused by microorganisms that have become resistant to all known antibacterials. In that regard, thiopeptide antibiotics are attractive starting points for the development of new antimicrobial agents. This thesis outlines efficient synthetic avenues to thiopeptides and demonstrates their clinical potential against bacterial pathogens that are designated as serious and urgent threats by the U.S. Centers for Disease Control & Prevention (CDC). v Preface The author, H. -J. Hwang, planned and executed all the chemistry work outlined herein, wrote this dissertation, secured funding from various agencies in S. Korea, and established collaborations with Professor Jin-Hwan Kwak at Handong University, Professor Hyunjoo Pai at Hanyang Medical School for Clostridioides difficile. The other biological evaluation of micrococcin P2 has been carried out at A & J Science by Young-Jin Son. Professor M. A. Ciufolini provided helpful suggestions and thorough editing of this thesis. The research reported in Chapter 3 has been published in: Hwang, H.-J.; Son, Y.-J.; Kim, D.; Lee, J.; Shin, Y.-J.; Kwon, Y.; Ciufolini, M. A. Diversity-Oriented Routes to Thiopeptide Antibiotics: Total Synthesis and Biological Evaluation of Micrococcin P2. Org. Biomol. Chem. 2022, 20, 1893-1899. H. -J. Hwang and Professor M. A. Ciufolini are the corresponding authors of this paper. Furthermore, an article describing the activity of micrococcin P2 against C. difficile has been accepted for publication in the J. Nat. Prod: Son, Y.-J.; Kim, R.-Y.; Oh, S.-H.; Jung, S.; Ciufolini, M. A.; Hwang, H.-J.; Kwak, J.-H.; Pai, H. Micrococcin P2 targets Clostridioides difficile. H. -J. Hwang is one of the corresponding authors, and Professor M. A. Ciufolini is a co-author of this paper. vi Table of Contents Abstract ......................................................................................................................................... iii Lay Summary ............................................................................................................................... iv Preface .............................................................................................................................................v Table of Contents ......................................................................................................................... vi List of Tables .............................................................................................................................. viii List of Figures ............................................................................................................................... ix List of Schemes ............................................................................................................................. xi List of Abbreviations ................................................................................................................. xiv Acknowledgements ......................................................................................................................xx Dedication .................................................................................................................................. xxii Chapter 1: Introduction ................................................................................................................1 1.1 Background on Antibiotic Discovery ............................................................................. 1 1.2 Economic Incentives for Antibiotic Development ......................................................... 3 1.3 The Thiopeptide Antibiotics ........................................................................................... 5 1.4 Hensens Classfication ..................................................................................................... 8 1.5 Mode of Action ............................................................................................................... 9 1.6 Remarks on Biological Activity and Medicinal Chemistry .......................................... 10 Chapter 2: Previous Syntheses of Thiopeptide Antibiotics ......................................................12 2.1 Background on Thiazole Synthesis ............................................................................... 14 2.2 Background on Pyridine Synthesis - Modification of a Pyridine Nucleus ................... 17 2.3 Background on Pyridine Synthesis - de novo approach................................................ 24 vii 2.4 Objective of the Present Study ...................................................................................... 29 Chapter 3: Total Synthesis of Micrococcin P2 ......................................................................... 32 3.1 Retrosynthetic Considerations ...................................................................................... 32 3.2 Micrococcin P2 : Synthesis of Fragments .................................................................... 38 3.3 Biological Studies of Micrococcin P2 .......................................................................... 50 3.4 Medicinal Chemistry Studies in the Micrococcin P2 Series ......................................... 60 3.5 Remarkable Chemical Property of Micrococcin P2 ..................................................... 61 Chapter 4: Efforts Towards the Total Synthesis of Lactocillin ...............................................68 4.1 Synthetic Background on Lactocillin............................................................................ 69 4.2 Retrosynthetic Analysis of Lactocillin .......................................................................... 75 4.3 Efforts towards the Total Synthesis of Lactocillin ....................................................... 76 Chapter 5: Conclusion .................................................................................................................83 References .....................................................................................................................................86 Appendices ..................................................................................................................................105 A. Experimental Protocol .............................................................................................. 105 B. Synthesis and Characterization of Compounds ........................................................ 106 C. 1H and 13C NMR Spectra, HPLC traces and LC/MS ................................................ 148 D. Experimental Protocol for Biological Evaluations ..................................................... 191 viii List of Tables Table 3.1 Suzuki coupling reactions of 103 .................................................................................. 42 Table 3.2 Activity of MP2 (MIC50 in μg/mL) against various microorganisms .......................... 51 Table 3.3 Rat pharmacokinetic studies of MP2 ............................................................................ 53 Table 3.4 in vitro activity of MP2 against classified 53 C. difficile clinical isolates .................... 54 Table 3.5 Activity of MP2 against M. tuberculosis, including MDR and XDR strains ............... 58 Table 3.6 MP2's' in vitro activity against various NTM bacteria ................................................. 59 Table 3.7 Activity profile of simple aromatic substitution of micrococcin series ........................ 60 ix List of Figures Figure 1.1 Timeline of antibiotic discovery .................................................................................... 1 Figure 1.2 The dwindling global antibiotic development ............................................................... 2 Figure 1.3 Chemical structure of plazomicin and eravacycline ...................................................... 4 Figure 1.4 Representative D-Series thiopeptide antibiotics ............................................................ 6 Figure 1.5 Examples of Hensen's Group A and B thiopeptides ...................................................... 8 Figure 1.6 Examples of Hensen's Group C and E thiopeptides ...................................................... 9 Figure 1.7 Binding mode of 26-membered thiopeptide in a cleft between L11 and ribosome .... 10 Figure 2.1 Representative thiopeptide obtained by total synthesis ............................................... 13 Figure 2.2 Substitution pattern of thiazoles found in thiopeptide antibiotics ............................... 14 Figure 2.3 Structure of micrococcinic acid ................................................................................... 18 Figure 3.1 Hypothetical boronic acid 141..................................................................................... 43 Figure 3.2 1H NMR of synthetic MP2 .......................................................................................... 48 Figure 3.3 13C NMR of synthetic MP2 ......................................................................................... 48 Figure 3.4 1H NMR spectral comparison of reduced MP2 vs MP1 (DMSO-d6) .......................... 49 Figure 3.5 1H NMR spectrum reduced MP2, containing 1:1 mixture of MP1 and epi-MP1 (152), in CDCl3 (400 MHz) ..................................................................................................................... 49 Figure 3.6 Cellular toxicity assay of MP2 .................................................................................... 52 Figure 3.7 Twenty-four time-kill curves for vancomycin and MP2. ............................................ 55 Figure 3.8 In vivo mouse CDI-IBD efficacy of MP2.................................................................... 57 Figure 3.9 MP2 immune action against cytokines IL-1β and IL-6. Dexamethasone is used as a control ........................................................................................................................................... 58 x Figure 3.10 The substitution of second thiazole ring to 150, 151................................................. 61 Figure 3.11 Acid 153: the precursor of all clinically interesting MP2 analogues. ....................... 62 Figure 3.12 Chemical structure of eribulin ................................................................................... 63 Figure 4.1 The chemical structure of lactocillin ........................................................................... 68 Figure 4.2 The Siegel advanced intermediate for lactocillin ........................................................ 69 xi List of Schemes Scheme 1.1 Key steps in the biosynthesis of the pyridine nucleus of thiopeptide antibiotics ........ 7 Scheme 2.1 The Hantzsch thiazole synthesis ............................................................................... 14 Scheme 2.2 Racemization of α-amino thiazoles in the course of Hantzsch reaction ................. 15 Scheme 2.3 The Holzapfel-Meyers-Nicolaou (HMN) modification of Hantzsch thiazole synthesis....................................................................................................................................................... 15 Scheme 2.4 Example of the HMN modification of the Hantzsch thiazole synthesis ................... 16 Scheme 2.5 The Shioiri thiazole synthesis ................................................................................... 16 Scheme 2.6 The White-Siegel thiazole synthesis ......................................................................... 17 Scheme 2.7 Heterodimer formation during attempted stannylation of 42 and 43 ...................... 19 Scheme 2.8 Kelly’s synthesis of 48 by Stille coupling ................................................................. 20 Scheme 2.9 Bach synthesis of the heterocyclic core of ent-GE2270A......................................... 21 Scheme 2.10 Synthesis of the heterocyclic core of baringolin ..................................................... 22 Scheme 2.11 The Siegel synthesis of the MP1 core ..................................................................... 23 Scheme 2.12 The Bohlmann-Rahtz pyridine synthesis .............................................................. 24 Scheme 2.13 The Moody synthesis of the pyridine core of promothiocin A ............................... 25 Scheme 2.14 Bagley’s route to dimethyl sulfomycinamate.......................................................... 25 Scheme 2.15 The Ciufolini synthesis of heterocyclic cluster of thiocillin I ................................. 26 Scheme 2.16 Assembly of the MP1 core via a modified Hantzsch pyridine synthesis ................ 27 Scheme 2.17 Moody’s biomimetic route to the core of amithiamycin ....................................... 28 Scheme 2.18 Nicolaou’s biomimetic approach to the core of thiostrepton .................................. 29 Scheme 2.19 A hypothetical diversity-oriented synthesis of thiopeptides ................................... 30 xii Scheme 3.1 Retrosynthetic analysis of micrococcin P2 ............................................................... 33 Scheme 3.2 The Ciufolini synthesis of ester 115 .......................................................................... 34 Scheme 3.3 Requirement for excess organolithium species 116 to prepare ketone 117 .............. 34 Scheme 3.4 The preparation of ketone 89 .................................................................................... 35 Scheme 3.5 Synthetic logic for pyridine 108 .............................................................................. 36 Scheme 3.6 Selenium dioxide oxidation of 2-methylthiazoles en route to ynones ...................... 37 Scheme 3.7 Plan for the preparation of ynone 111 ....................................................................... 37 Scheme 3.8 Preparation of nitrile 121........................................................................................... 38 Scheme 3.9 Preparation of ketone 110 ......................................................................................... 39 Scheme 3.10 Preparation of ynone 111 ........................................................................................ 39 Scheme 3.11 Synthesis of pyridine core 108 ................................................................................ 40 Scheme 3.12 Preparation of fragment 107 by the literature method .......................................... 41 Scheme 3.13 Union of 108 and 107 and macrocyclization sequence to afford 103 ..................... 41 Scheme 3.14 Successful borylation of 103 ................................................................................... 44 Scheme 3.15 Preparation of thiazolyl coupling partners of 147 and 148 ..................................... 45 Scheme 3.16 Total synthesis of MP1 and MP2 ............................................................................ 46 Scheme 3.17 Sodium borohydride reduction of MP2, resulting in 4 and 152 .............................. 47 Scheme 3.18 MP2 analogue synthesis via Suzuki coupling ......................................................... 60 Scheme 3.19 Synthesis of eravacycline (2) .................................................................................. 63 Scheme 3.20 Nozaki-Hiyama-Kishi reaction applied to the synthesis of eribulin ....................... 64 Scheme 3.21 Mechanistic hypothesis for the highly chemoselective hydrolysis of MP2 ............ 66 Scheme 3.22 Bioengineering sequences leading to the formation of MP2 using B. subtilis ........ 67 Scheme 4.1 Siegel’s retrosynthetic logic for lactocillin ............................................................... 70 xiii Scheme 4.2 Construction of pyridine core of lactocillin by Siegel .............................................. 71 Scheme 4.3 Preparation of nitrile 172........................................................................................... 71 Scheme 4.4 Synthetic efforts to afford 175 .................................................................................. 72 Scheme 4.5 Successful coupling of 169 with 175 and dehydration to yield 177 ......................... 72 Scheme 4.6 Preparation of enantiopure stannylthiazole 186 ...................................................... 74 Scheme 4.7 Stille coupling of 177 and 186 to afford 163 ............................................................ 75 Scheme 4.8 Retrosynthetic analysis of lactocillin ........................................................................ 76 Scheme 4.9 Synthesis of serine derived cyanoketone 196 ........................................................... 76 Scheme 4.10 Synthesis of the pyridine core of lactocillin ............................................................ 77 Scheme 4.11 Synthesis of sensitive thiazoline fragment 203 ....................................................... 78 Scheme 4.12 Successful formation of the lactocillin macrocycle ................................................ 80 Scheme 4.13 Late-stage Suzuki coupling to install the tail fragment ........................................... 81 Scheme 4.14 Anticipated completion of lactocillin methyl ester ................................................. 82 xiv List of Abbreviations [α] specific rotation Ac acetyl ACN acetonitrile Alloc allyloxycarbonyl AMK amikacin AMR Antimicrobial Resistance APA aminopenicillanic acid aq aqueous Ar argon AUC Area Under the Curve br broad Boc tert-butyloxycarbonyl Bn benzyl group BQL below the quantification limit Bu butyl oC degrees Celsius CARB-X Combating Antibiotic-Resistant Bacteria cald calculated cat. catalytic CDC Centers for Disease Control and Prevention CDI C. difficile infection CIP ciprofloxacin xv Cl clearance cm-1 wavenumbers CMC Chemistry, Manufacturing & Control COVID-19 SARS-CoV-2 Variant Coronavirus δ chemical shift (parts per million down from tetramethylsilane) d doublet dba tris(dibenzylideneacetone) DBU 1,8-diazabicyclo[5.4.0]undec-7-ene DCC N,N’-dicyclohexylcarbodiimide DCE dichloroethane DCM dichloromethane DDQ 2,3-dichloro-5,6-dicyano-1,4-benzoquinone Dex dexamethasone DIBAL-H diisobutylaluminum hydride DIPEA diisopropylethylamine DMAP 4-N,N’-dimethylaminopyridine DME dimethoxyethane DMF N,N’-dimethylformamide DMSO dimethyl sulfoxide DPPA diphenylphosphoryl azide dppf 1,1’-bis(diphenylphosphino)ferrocene DSS dextran sulfate sodium EBP ethyl bromopyruvate xvi EDCI 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide Ef-G elongation factor G Ef-Tu thermo-unstable elongation factor epi epimer equiv equivalents Et ethyl FDA Food and Drug Administration g gram(s) GAIN Generating Antibiotic Incentives Now Act GARDP Global Antibiotic Research & Development Partnership h hour(s) HEK human embryonic kidney cells HMN Holzapfel-Meyers-Nicolaou modification HOBt 1-hydroxybenzotriazole hydrate HPLC high performance liquid chromatography HRMS high resolution mass spectrometry Hz Hertz (s-1) i iso (as an alkyl group) IBD inflammatory bowel disease IL interleukin INH isoniazid IPA isopropyl alcohol IR Infrared xvii J coupling constant LC-MS liquid chromatography-mass spectrometry LIN linezolid LOD limit of detection LPS lipopolysaccharides m multiplet M molar (moles per liter) MAC Mycobacterium avium complex MDR multi-drug resistant Me methyl MIC minimal inhibitory concentration min minute(s) MLST multilocus sequence typing MOM methoxymethyl mp melting point MP micrococcin P MPLC medium pressure liquid chromatography Ms methanesulfonyl MRSA methicillin-resistant Staphylococcus aureus MRT mean residence time MW microwave MXF moxifloxacin n normal (as an alkyl group) xviii NBS N-bromosuccinimide n-BuLi n-butyllithium NMM N-methylmorpholine NMR nuclear magnetic resonance NTM non-tuberculous Mycobacterium p para (as a benzene substituent) PAMPA parallel artificial membrane permeability assay PCR polymerase chain reaction Pg unspecified protecting group Ph phenyl ppm parts per million PPTS pyridinium p-toluenesulfonate py pyridine q quartet quant quantitative RIF rifampicin Rf retention factor RNA ribonucleic acid RT ribotype r.t room temperature s singlet SAR structure-activity relationship sat. saturated xix SEM trimethylsilylethoxymethyl SH-SY5Y human neuroblastoma cell line t triplet t tertiary (as an alkyl group) T1/2 half-life TASF tris(dimethylamino)sulfonium difluorotrimethylsilicate TBAF tetra-n-butylammonium fluoride TBDPS tert-butyldiphenylsilyl TBS tert-butyldimethylsilyl TES triethylsilane Tf trifluoromethanesulfonyl TFA trifluoroacetic acid TFAA trifluoroacetic anhydride THF tetrahydrofuran TLC Thin Layer Chromatography TMS trimethylsilyl Trt triphenylmethane μ micro Van vancomycin VRE vancomycin-resistant Enterococci Vss volume of distribution WHO World Health Organization XDR extensively drug resistant xx Acknowledgements First and foremost, I must express my deepest gratitude to my supervisor, Professor Marco A. Ciufolini. His immense knowledge in science, sharp wisdom about life, and dedication to excellence have been very inspirational. My life has been transformed by his guidance and support. He has taught me core values of life, let alone a high standard of professionalism. He is and always will be remembered as the greatest mentor, and I feel very fortunate to have chances to work with him. I will always remind myself to share his undying legacy – to leave a positive impact on someone’s life. I thank my committee members, Professor Stephen Withers and Professor David Perrin, for their support throughout the years and their thorough review of this manuscript. I highly admire their ground-breaking scientific discoveries. My sincere gratitude goes to my university examiners, Professor Katherine Ryan and Professor Yossef Av-Gay. It was a humbling experience to work with fantastic Ciufolini group members – mostly in the past as of this moment. Since my undergrad, this very vibrant group occupies a special place in my heart. Night group meetings, inheriting valuable laboratory skills, and the moments of our shared joys will never be forgotten. When we all get to meet, I hope we could laugh over the times we spent in Ciufolini group. Thank you - Dr. Josh Zaifman, Dr. Matthew Smith, Dr. Takahito Kasahara, Dr. Sanjia Xu, Dr. Leanne Racicot, Dr. Marco Paladino, Dr. Nikita Jain, Dimitrios Panagopolous, Xueqing Wang, Mai Lam Ferguson, Doris Tang and countless undergraduates. To be honest, it feels rather uneasy to wrap up as the last graduate student of this group. I must acknowledge the Department of Chemistry, and UBC for the support I am deeply xxi grateful for. This is my true alma mater, who taught me to appreciate the real meaning of “Tuum Est”. Through many precious moments and lessons, I will find ways to contribute to society, and the world. I would have to express gratitude to South Korean collaborators, especially the scientists at A & J Science. The replenishment of broken antibiotic pipeline is urgently needed and I hope our science will help patients who are in need of new antibiotics. Let us hope that our passion and rigor for science are to be remembered and cherished. My special gratitude goes to my parents. Words cannot simply express how thankful I am to have parents who are loving and supportive. Thank you so much for believing in me and for being the best role model I could ever ask for. My brother deserves my deep gratitude for being the best brother in the world and his support in so many ways. My final dedication goes to my wife, Yeji Lee, for all the support and love she has brought in my life. Thank you for believing in me. You are true sunshine in my life and I look forward to spending our many predestined days with you. xxii Dedication To my parents 1 Chapter 1. Introduction 1.1 Background on Antibiotic Discovery The discovery of penicillin by Sir Alexander Fleming in 1928 changed the world of medicine, in that previously intractable bacterial infections became easily treatable. This event revolutionized the management of infectious disease. The advent of antibiotics not only saved millions of lives, but it also stimulated extensive research that led to the discovery of new antibacterial agents, such as streptomycin1, aminoglycosides2, tetracyclines3, beta-lactams4, macrolides5, and so on. Figure 1.1 provides a timeline of antibiotic discovery and shows that the rate at which new antibacterials were introduced peaked in the 1950’s and 1960’s. Indeed, global pharmaceutical companies were engaged in a highly competitive race from the 1940’s to the mid- Figure 1.1. Timeline of antibiotic discovery (reproduced from: Current opinion in Microbiology, 2019, 51, 72-80) 2 1960’s to expand antibiotic arsenals, because of the lucrative rate of economic return of such medicines7. The two decades 1950-1970 are thus known as the ‘golden era’8 of antibiotics research. Soon after the introduction of early antibiotics, it was observed that bacterial pathogens can develop resistance against such agents. It is now established that this occurs through horizontal9 and vertical10 gene transfer. Antibiotic resistance remained a manageable difficulty so long as new anti-infectives reached the market at an appropriate pace, so that infections caused by pathogens that had become resistant to an extensively prescribed agent, e.g. penicillin, could be treated with a new one. Paradoxically, antibiotic research abated greatly during the 1970’s, and very few new such compounds have been discovered and developed since. The last important class of anti-infectives, the oxazolidinones6, entered medical practice almost 20 years ago. Today, only a handful of new antibacterials are under development (Figure 1.2). Figure 1.2. The dwindling global antibiotic development (reproduced from: Clinical Infectious Disease, 2011, 52, S397-S428) 3 The next chapter briefly addresses why antibiotic development came to a near halt. Regardless, the dwindling antibiotic pipeline threatens to return humankind to the “dark age”11 of pre-antibiotic times. The UK-Government-commissioned O’Neill report12 projects that 10 million people will die from drug resistant infections by 205013 if no immediate action is taken. Even so, the antimicrobial resistance (AMR) problem is still being largely ignored, or at least not taken seriously. Yet, history provides a very clear picture of what an unprepared world may face if an epidemy caused by an untreatable pathogen were to break out. Suffice it to mention the plagues that befell the Old World from the XIV to the XVIII century and that decimated its population, or the ongoing coronavirus pandemic (SARS-CoV-2 Variant: COVID-19)14, which has already caused more than 5.9 million deaths worldwide as of February 2022. The author hopes that the world will never have to “lock the stable door after the horse is stolen”. 1.2 Economic Incentives for Antibiotic Development Economic considerations provided the driving force both for the dawning of the ‘golden era’ of antibiotic research and for its demise8. While the antibiotic business was quite profitable, top pharmaceutical companies soon realized that better rates of return were possible by focusing on chronic illnesses such as cancer and metabolic disease. Patients suffering from these conditions would have to take medications for years, whereas antibiotics are typically prescribed for two weeks. In this light, it is not surprising that the current global antibiotic pipeline is nearly dry compared to that for cancer and metabolic diseases.15 In 2012, the U.S government enacted the GAIN16 (Generating Antibiotic Incentives Now Act) to incentivize the development of new antibacterials. In a like vein, non-profit organizations 4 created to address the global antimicrobial resistance problems, such as CARB-X17 (Combating Antibiotic-Resistant Bacteria) in the U.S and GARDP18 (Global Antibiotic Research & Development Partnership) in Europe have garnered support from policy makers.19 Small, innovative biotech companies have responded favorably to such incentives. Indeed, antibiotic development is currently taking place mostly at small biotechs, which are more readily inclined to avail themselves of the foregoing incentives than large pharmas22. The ensuing research efforts have produced two noteworthy medicaments: Acahogen’s Plazomicin (1)20, approved for human use in 2016, and Tetraphase’s Eravacycline (2)21, approved in 2018 (Figure 1.3). Unfortunately, these drugs failed to generate adequate profits. Acahogen filed for bankruptcy in 2018 and Tetraphase was acquired by La Jolla Pharmaceuticals in 2020. The financial concern that such unfortunate events have engendered among global antibiotics companies has been exacerbated by Novartis’ recent exit from the field. Yet, the specter of antibiotic resistance remains quite real: someone must absolutely take the initiative to tackle antimicrobial resistance. On a more positive note, the present state of affairs may have rekindled interest in the search for new antibacterial resources.23,24,25,26 Figure 1.3. Chemical structure of plazomicin and eravacycline 5 1.3 The Thiopeptide Antibiotics The so-called thiopeptides are a largely unexploited family of potent antibacterial agents that seem to be good starting points for the development of new antibiotics.27,28 Two aspects of their biochemistry render them attractive. First, they possess an interesting mode of action29,30,31: they strongly inhibit bacterial protein synthesis by tight binding to ribosomes. Second, thiopeptides are nontoxic to mammalian cells at antibacterial concentrations32. This translates into a large selectivity index (SI; the difference between efficacious and toxic doses), a favorable property that should be exploited. On the other hand, their complex chemical structures, exemplified in Figure 1.4 by micrococcin P2 (3, MP2)34,141, micrococcin P1 (4, MP1)34,35, thiocillin (5) 36, and lactocillin (6) 37, greatly complicate the conduct of medicinal chemistry research33. As apparent from Figure 1.4, thiopeptides are sulfur-rich, highly modified macrocyclic peptide natural products. 6 Figure 1.4. Representative D-Series thiopeptide antibiotics They are primarily isolated from Streptomyces and Bacillus species.38,39,40 Thiopeptides are characterized by a heterocyclic core consisting of a pyridine, which may be present in partially or completely reduced form, decorated with various thiazole and/or oxazole residues41,42. The first thiopeptide to be discovered is “micrococcin P”, now known to be a mixture of MP1 and MP2, which was described in 1948. Since then, approximately 100 members of the family have been reported.42 The biosynthesis of these natural products is believed to involve a series of post-translational modifications of a large peptide43,44. This hypothesis, first promulgated in 197843, was 7 later confirmed thanks to the work of Walsh and others45,46,47,48. The biosynthetic assembly of the pyridine is especially interesting. To illustrate, thiocillin I, 5, is now accepted to emanate from 14 C-terminal residues of a 52-residue peptide. This peptide, rendered with the simplified structure 7 in Scheme 1.1, incorporates two crucial serine segments. The latter are first dehydrated, then one of the resulting dehydroalanines undergoes amide tautomerism, resulting in formation of azadiene 8. Scheme 1.1. Key steps in the biosynthesis of the pyridine nucleus of thiopeptide antibiotics Such an event sets the stage for a hetero Diels-Alder reaction, presumably orchestrated by a Diels-Alderase enzyme. The emerging 9 may undergo various fates, giving rise to diverse thiopeptides that differ for the oxidation state of the pyridine nucleus. For instance, sequential dehydration and elimination of R-CONH2 produce the fully aromatic pyridine 10 found in micrococcins and thiocillins, while sequential dehydration and reduction lead to the piperidine nucleus of thiopeptin and congeners, or the tetrahydropyridine core of thiostrepton and related substances. The structures of these compounds are shown in the next section. 8 1.4 Hensens Classification The so-called Hensens classification49 assigns thiopeptides to five different groups, A-E, depending on the oxidation state of the pyridine nucleus. Group A includes compounds exhibiting a piperidine core, i.e., a fully reduced pyridine, as apparent in; e.g., thiopeptin A3a50, 11, while group B is characterized by a tetrahydropyridine core; e.g., thiostrepton A51,52,53, 12 (Figure 1.5). Figure 1.5. Examples of Hensen’s Group A and B thiopeptides Group C, the rarest in the thiopeptide family, includes substances that display an imidazopiperidine core. Only one member of this family, Sch 4083233, 13, is known to date (Figure 1.6). Group D encompasses substances that exhibit a 2,3,6-trisubstituted pyridine, such as the natural products depicted in Figure 1.4. Finally, compounds in group E contain the most highly oxidized pyridine in the form of a 3-hydroxypyridine, as seen in nosiheptide32,54,55, 14. 9 Figure 1.6. Examples of Hensen’s Group C and E thiopeptides 1.5 Mode of Action As indicated earlier, thiopeptides are potent inhibitors of bacterial protein synthesis by virtue of their tight binding to particular regions of the ribosome29,30,31. Their precise mode of action differs slightly depending on the size of the macrocycle. Those with 26-membered macrocycles bind to a cleft between the 23S ribosomal RNA subunit and L11 Domain56,57. This results in the blockage of the binding region of elongation factor G (Ef-G), which inhibits translocation of the growing peptide (Figure 1.7). 10 Figure 1.7 Binding mode of 26-membered thiopeptides in a cleft between L11 and ribosome57 Those with 29-membered macrocycles bind strongly to the thermo-unstable elongation factor known as Ef-Tu factor58. This prevents the transfer of aminoacyl residues to the nascent peptide chain. In either case, bacterial protein synthesis is severely impaired. The molecular target of 35-membered macrocycles is unknown27. 1.6 Remarks on Biological Activity and Medicinal Chemistry The potent antimicrobial activity of thiopeptide is largely confined to Gram-positive pathogens27,28,32, including species such as methicillin-resistant Staphylococcus aureus (MRSA)59, drug-resistant Streptococcus pneumoniae60, vancomycin-resistant Enterococci (VRE)61, all of which are designated as “serious threats” by the U.S Centers for Disease Control and Prevention (CDC)26. Remarkably, a number of thiopeptides also exhibit elevated activity against 11 Mycobacterium tuberculosis62. The question of whether this organism is Gram-positive or Gram-negative remains unsettled, although genetic analysis suggests a closer relationship with Gram-negative species115. Furthermore, our own studies have revealed a clinically useful activity of micrococcin P2 against Clostridioides difficile (C. difficile)63 and non-tuberculous Mycobacterium (NTM)64. This will be discussed in Chapter 3. It seems likely that converting a native thiopeptide into a clinical candidate will require appropriate chemical modifications, as apparent, e.g., from Novartis’ effort toward LFF57199. In turn, this requires knowledge of the structure-activity relationship (SAR) of the original compound. However, the complex structure of thiopeptides and the presence of multiple reactive sites hamper the conduct of traditional medicinal chemistry work. Indeed, SAR studies would be best carried out using fully synthetic materials. This imposes a requirement for economical, concise, efficient, convergent, and diversity-oriented synthetic routes. These elements add extra challenges to a synthetic effort and require meticulous synthetic planning, tenacity, and courage to reach the desired objective. 12 Chapter 2. Previous Syntheses of Thiopeptide Antibiotics Thiopeptides have garnered significant attention from the synthetic community since the late 1990’s by virtue of their intriguing chemical structure and interesting biological activity. Even so, only nine total syntheses of these substances have been achieved as of this writing, arguably because of the chemical difficulties associated with the assembly of their complex structure. The members of the class that have yielded to total synthesis are promothiocin A (15)65, in 1998, amythiamicin D (16)66, and thiostrepton A (12)51,52, in 2005, siomycin A67 in 2008, GE2270A (17)68, in 2007, micrococcin P1 (4) 35,69,70, in 2009, thiocillin (5) 36, in 2011, baringolin (18)71, in 2013, and nosiheptide (14)54, in 2016 (Figure 2.1). An analysis of successful synthetic strategies clearly indicates that the key to an efficient synthesis is the rapid assembly of their pyridine-thiazole cluster. However, the preparation of this complex heterocyclic core has always been a challenging task. Interestingly, each total synthesis showcases new techniques for the construction of these crucial subunits, which imposes upon the operator the double challenge of preparing and handling stereochemically labile thiazoles, as well as creating highly substituted pyridine nuclei. The discussion that follows will first address the issues encountered in the synthesis of the thiazole moieties. Subsequently, it will illustrate the methods that have been employed to create the pyridine cores. 13 Figure 2.1. Representative thiopeptides obtained by total synthesis 14 2.1 Background on Thiazole Synthesis Thiopeptides commonly feature 2,4-disubstituted thiazoles (19, Figure 2.2), but some of them harbor 5-methyl substituted analogues, 20. Figure 2.2. Substitution pattern of thiazoles found in thiopeptide antibiotics There are countless methods to prepare thiazoles74. In the context of thiopeptide synthesis, these heterocycles have been obtained by Hantzsch75, Shioiri76,77, and White-Siegel78,79 reactions. The Hantzsch thiazole synthesis was first reported in 1887, and it entails the condensation of a thioamide, 21, with an α-halocarbonyl compound, 22, usually in refluxing ethanol. This is a high-yielding protocol and is easy to implement (Scheme 2.1). However, thioamides that bear a Scheme 2.1. The Hantzsch thiazole synthesis stereocenter at the α-position to the thiocarbonyl group, such as amino acid-derived thioamides, suffer substantial or complete racemization during the reaction. The problem occurs at the stage of thiazole 25, which upon protonation by HBr released into the medium equilibrates with enamine 26, resulting in loss of configuration80 (Scheme 2.2). 15 Scheme 2.2. Racemization of α-amino thiazoles in the course of Hantzsch reaction Fortunately, this limitation can be circumvented by condensing an enantioenriched thioamide 24 with α-halocarbonyl compound 22 at low temperature, and under gently basic conditions. The emerging hydroxythiazole 28 can be carefully dehydrated by the action of Scheme 2.3. The Holzapfel-Meyers-Nicolaou (HMN) modification of Hantzsch thiazole synthesis 16 trifluoroacetic anhydride (TFAA) in the presence of pyridine and triethylamine (Scheme 2.3). This procedure is commonly described as the Holzapfel-Meyers-Nicolaou (HMN) modification81,82 of the Hantzsch thiazole synthesis, and the method has been widely utilized in the synthetic arena, especially in the thiopeptide field. For instance, we have relied upon this technique during our efforts towards nosiheptide55 (Scheme 2.4). Scheme 2.4. Example of the HMN Modification of the Hantzsch thiazole synthesis The Shioiri thiazole synthesis76,77 entails the MnO2 oxidation of thiazolidines obtained by condensation of N-protected, α-aminoaldehydes derived from amino acids with cysteine derivatives (Scheme 2.5). This operation is largely free from stereochemical erosion, and in some cases it may be more efficient compared to the Holzapfel-Meyers-Nicolaou80,81,82 reaction. Scheme 2.5. The Shioiri thiazole synthesis 17 A more concise route towards thiazole has been developed by Siegel70,78 and collaborators on the basis of the White thiazole synthesis79. Siegel and his group have employed this method for the total synthesis of MP1 (4), which they achieved in 202070 (Scheme 2.6). The White-Siegel method has proven to be advantageous in the context of thiopeptide synthesis and we have employed it extensively in the present study (Chapter 3). Scheme 2.6. The White-Siegel thiazole synthesis 2.2 Background on Pyridine Synthesis – Modification of a Pyridine Nucleus Two chemical trends are apparent from the various solutions that have been proposed over the years for the elaboration of the pyridine-thiazole core cluster of thiopeptide antibiotics: the modification of a pre-existing pyridine via metal-mediated coupling reactions72,73, and the de novo assembly of the pyridine nucleus with the full complement of thiazoles35,36,65,66. The present section focuses on the modification of a pyridine nucleus, whereas methods for the construction of the pyridine ring will be discussed in Chapter 2.3. The modification of a pre-existing pyridine through metal-mediated coupling reactions is a key strategic idea in the earliest recorded study in the thiopeptide field: the 1991 Kelly synthesis of micrococcinic acid, 41 (Figure 2.3), a product of acidic hydrolysis of MP1.72 18 Figure 2.3. Structure of micrococcinic acid Kelly’s approach centered on Stille coupling83 of a pyridine with an appropriate thiazole. One heterocycle would participate in the reaction as a bromo derivative; the other, as a stannyl organometallic. The latter, in turn, would be obtained from a corresponding bromide through a palladium-catalyzed stannylation. Unfortunately, serious complications plagued this strategy. The conversion of bromopyridine 42 or bromothiazole 43 to the corresponding organotin derivatives failed, resulting instead in formation of homodimers 45 and 47, respectively. Evidently, the rate of Stille coupling of a palladated derivative of either heterocycle with an initially formed stannyl derivative of the same heterocycle is faster than the reaction with hexamethyldistannane (Scheme 2.7). 19 Scheme 2.7. Heterodimer formation during attempted stannylation of 42 and 43 A moderately successful remedy emerged in the form of the reaction of a 1:1 mixture of 42 and 43 with (Me3Sn)2 in the presence of a catalytic amount of (Ph3P)2PdCl2, resulting in formation of the desired product 48 in 49% yield, plus heterodimers 45 and 47 (Scheme 2.8). 20 Scheme 2.8. Kelly’s synthesis of 48 by Stille coupling Bach’s 2005 synthesis of GE2270A68,73 resolved some – but not all – of the foregoing difficulties. To illustrate, lithium-halogen exchange of pyridine 49 occurred with the anticipated C-3 regioselectivity, enabling in situ generation of 3-pyridylzinc species 50. An ensuing Negishi coupling with bromothiazole 51 occurred in high yield. However, a subsequent Negishi coupling of the resulting 54 with 2-thiazolylzinc species 52 suffered from moderate C-6 regioselectivity (6.5:1) and produced considerable quantities of homodimers. Fortunately, the final Stille coupling reaction of 57 and 55 occurred in a satisfactory manner to furnish the desired 58 in 61% yield. (Scheme 2.9). The regioselectivity and homodimerization issues outlined above remain unresolved as of this writing. Indeed, later efforts in the thiopeptide field circumvented the problem by avoiding the use of a polyhalogenated pyridine in palladium-mediated reactions. For instance, the more recent 2013 synthesis of baringolin, 1871, employed a 2-chloro-6-methoxy pyridine, in lieu of a 2,6-dihalopyridine, as a substrate for Stille coupling. Thus, the carboxylic acid in commercial pyridine 59 was elaborated to an oxazole through a modified Wipf protocol84. 21 Scheme 2.9. Bach synthesis of the heterocyclic core of ent-GE2270A Stille coupling of the resulting 60 with 61 occurred smoothly to produce 62 in 88% yield. The methoxy group was subsequently converted into a triflate to enable Negishi coupling85 with 64, leading to the desired heterocyclic core 65 in a quantitative yield (Scheme 2.10). 22 Scheme 2.10. Synthesis of the heterocyclic core of baringolin The very recent (2020) Siegel synthesis of MP170 adopts similar methodology. Commercial 2-chloro-3-cyanopyridine, 66, was transformed into 67 through the White-Siegel thiazole synthesis illustrated in Chapter 2.1. A 6-cyano group was then introduced via a Polonowski-type reaction of pyridine N-oxide 68. The emerging 69 underwent smooth Stille coupling with 70 to afford the desired pyridine 71, which was advanced to the heterocyclic cluster 73 through a second White-Siegel thiazole synthesis (Scheme 2.11). 23 Scheme 2.11. The Siegel synthesis of the MP1 core The foregoing serves only to outline the methodology that has been employed for the elaboration of a preformed pyridine to the heterocyclic cluster of a thiopeptide. Significant additional examples are found, e.g., in the work of Shin116 and Arndt54. 24 2.3 Background on Pyridine Synthesis – de novo approach It is apparent from the foregoing that the construction of a pyridine bearing as many as possible – ideally, all – of the requisite heterocyclic substituents would be desirable in terms of efficiency, economy, conciseness, and convergence. Indeed, the three major players in the thiopeptide area, C. J. Moody, K. C. Nicolaou, and M. A. Ciufolini, have all focused on a de novo assembly of the heterocyclic cluster, and in the process, they developed noteworthy solutions. In that connection, Moody introduced a strategy relying upon a Bohlmann-Rahtz pyridine synthesis86. This transformation entails the cyclocondensation of a vinylogous amide 74 (R2 = alkyl) or carbamate (75, R2 = alkoxy) with an ynone 76. An initial Michael addition forms adduct 77 or 78 which can be isolated. Upon heating, 77 or 78 undergoes E-Z isomerization and cyclodehydration to pyridine 79 or 80 (Scheme 2.12). Scheme 2.12. The Bohlmann-Rahtz pyridine synthesis It is interesting to review how the Bohlmann-Rahtz approach evolved over the years, starting with Moody’s synthesis of promothiocin A65, 15, which also constitutes the first synthesis of a naturally occurring thiopeptide. Thus, pyridine 84 was prepared through the union of ynone 82 and enamine 81 and further elaborated to 85 (Scheme 2.13). Notice that this particular Bohlmann-Rahtz reaction afforded a pyridine incorporating a single heterocyclic substituent. Even 25 so, this route already appears to be more efficient, convergent, and robust relative to the ones outlined in the previous section. Scheme 2.13. The Moody synthesis of the pyridine core of promothiocin A Bagley’s synthesis of dimethyl sulfomycinamate,87 a product of degradation of the thiopeptide, sulfomycin, demonstrated an improved, “two steps, one pot” sequence that delivered pyridine 88 displaying two heterocyclic substituents. Condensation of ketone 86 with NH4OAc under microwave irradiation generated enamine 87 (not isolated). Addition of ethyl 3-trimethylsilylpropiolate to in situ formed 87 provided 88 in 50% yield (Scheme 2.14). Scheme 2.14. Bagley’s route to dimethyl sulfomycinamate. 26 Ciufolini introduced an additional refinement that permitted the incorporation of all three heterocyclic substituents in one step. Drawing upon Bagley’s finding, a method was developed whereby a “three-component condensation” of ketone 89, ynone 90, and NH4OAc in refluxing acetic acid delivered the complete heterocyclic cluster of thiocillin I, 91 (Scheme 2.15)36,88. Notice that the TBS protecting group in 91 was replaced by an acetate ester in the course of the reaction. This represented a difficulty that, at that time, had to be corrected by releasing the acetyl group and restoring TBS protection. A more appealing solution was developed in the course of the present study and will be described in Chapter 3. Regardless, the Ciufolini approach is now regarded as the definitive Bohlmann-Rahtz avenue to the heterocyclic cluster of D-series thiopeptides. Scheme 2.15. The Ciufolini synthesis of heterocyclic cluster of thiocillin I Ciufolini’s earlier efforts toward micrococcin P134,35,117 relied on a modified Hantzsch pyridine synthesis rather than a Bohlmann-Rahtz reaction, illustrating an alternative route to thiopeptide cores. Thus, the merger of ketone 89 and enone 92 generated 1,5-diketone 93. Subsequent treatment of 93 with NH4OAc and DDQ produced the desired pyridine 94 in excellent yield (Scheme 2.16). Although this method performed well, the Bohlmann-Rahtz sequence is clearly more direct and efficient. 27 Scheme 2.16. Assembly of the MP1 core via a modified Hantzsch pyridine synthesis It will be recalled that the biosynthesis of the pyridine nucleus of thiopeptides involves a noteworthy Diels-Alder-like reaction of an azadiene with a dehydroaminoacid derivative. Independently and almost simultaneously, Moody and Nicolaou elaborated a biomimetic approach that undoubtedly constitutes the most elegant avenue to thiopeptide pyridines yet described. To illustrate, a key step in the Moody synthesis of the core of amythiamycin118 was the reaction of azadiene 95 with enamide 96, leading to pyridine 98 in 33% yield (Scheme 2.17)66. 28 Scheme 2.17. Moody’s biomimetic route to the core of amythiamycin Nicolaou described a conceptually similar approach to the cores of thiostrepton A51,52 and of GE2270 substances.119 Scheme 2.18 depicts the route to the tetrahydropyridine found in thiostrepton (thiopeptide B series), for which the technology is especially valuable. Treatment of thiazolidine 99 with Ag2CO3 and DBU produced transient azadiene 100, which underwent sequential hetero Diels-Alder dimerization – imine hydrolysis to afford the desired 102 in a single step (Scheme 2.18). 29 Scheme 2.18. Nicolaou’s biomimetic approach to the core of thiostrepton 2.4 Objective of the Present Study Collaborative research with a pharmaceutical company has revealed certain clinically interesting properties of micrococcin P2. This work also determined that the macrocyclic core of the natural product is essential for antibacterial activity; that is, little variability is tolerated there. Medicinal chemistry studies will be disclosed in Chapter 3.4. The foregoing observations established a need for a synthetic route to MP2 that would be suitable for structure-activity relationship (SAR) investigations. While the syntheses adumbrated 30 in the previous section were designed to produce a specific end product, the exploration of the SAR of thiopeptide scaffolds required a diversity-oriented avenue to MP2 and congeners. An ideal route would involve transition metal-mediated coupling of macrocyclic substrate 103 or 142 with an appropriate aryl fragment, 104 (Scheme 2.19), despite the fact that similar reactions result in syntheses that fail to meet the guidelines for efficiency, conciseness, economy, and convergence set forth earlier in Chapter 2. Coupling partners 103 or 142 and 104 would have to be matched at the level of substituents X and Y. For instance, X could be a halogen and Y a boryl group, or vice versa. It will be recalled that the coupling reactions outlined earlier employed tin or zinc derivatives of thiazoles. Scheme 2.19. A hypothetical diversity-oriented synthesis of thiopeptides Yet, neither organometallic is contemplated in Scheme 2.19, and this for two important reasons. First, the toxicity of organotin compounds is such that even traces of Sn-containing materials in synthetic products to be assayed for biological activity may produce false results. For that reason, the use of organotin reagents is carefully avoided in medicinal chemistry research. In contrast, organoboron compounds are non-toxic, so much so that Suzuki coupling89 is the method of choice for metal-mediated biaryl synthesis in the pharmaceutical industry.90 Organozinc compounds are 31 also biologically benign, but their pyrophoric nature and moisture-sensitive character complicates their preparation and use, whereas boronic acids are air-stable, insensitive to moisture, readily available, and very easy to handle. A key objective of the present research was thus to establish appropriate conditions for the conduct of Suzuki reactions between 103 and 104. Such a goal, however, came with major unknowns. No examples of Suzuki coupling have seemingly been recorded in the thiopeptide field, raising the possibility that the failure of such reactions may have gone unreported. Also, it was entirely unclear whether macrocycles 103 would be good substrates for Suzuki coupling, given the multitude of metal-coordinating centers (S and N atoms) present in them. An equally important aim was to improve the economy and efficiency of the sequence leading to macrocycles 103 by suppressing as many protection-deprotection, redox, and chromatographic purification steps as possible. This required a thorough re-working of the best avenue yet described to such structures: the Ciufolini route to the thiocillin core. These objectives were successfully attained. The improvements thus devised form the basis of the first total synthesis of MP2 and the exploration of its biological activity, a total synthesis of MP1, and a synthetic effort that produced a very advanced macrocyclic precursor of lactocillin. 32 Chapter 3. Total Synthesis of Micrococcin P2 Micrococcin P2 (3), is the minor component of “micrococcin P” (MP), an antibiotic complex produced by Bacillus pumilus38, and typically consisting of 1:7 mixture of MP2 and MP143, 91. No total synthesis on MP2 has been reported and no biological data for pure MP2 is available because of its rarity and its difficult separation from MP1. We now describe the first total synthesis of MP2 through methodology that builds upon the guiding principles in Chapter 2.4, and that provided the groundwork for a medicinal chemistry venture. We note that medicinal chemistry efforts involving modification of the heterocyclic core of thiopeptides are unprecedented92. 3.1 Retrosynthetic Considerations We set out to devise a route to the target compound through a coupling reaction of macrocycle 103 with segment 106 (Scheme 3.1). Regardless of which permutation of substituents X and Y may ultimately emerge as the appropriate one, group X initially would be, e.g., a bromide. Macrocycle 103, X = Br, would materialize upon the union of carboxylic acid 108 and fragment 107. The published synthesis of the latter is efficient and requires no additional improvement. On the other hand, much refinement of the Bohlmann-Rahtz sequence leading to acid 108, and patterned after Ciufolini’s route to the heterocyclic cluster of thiocillin I, was necessary. A construction of the “northwestern” thiazole in 108 through a White-Siegel condensation would resolve a number of difficulties (vide infra). This approach requires cyanopyridine 109, which in turn would be reached via Bohlmann-Rahtz reaction of the cyanoketone 110 with ynone 111. 33 Scheme 3.1. Retrosynthetic analysis of micrococcin P2 Although these fragments could be prepared by appropriate modifications of the Ciufolini route, such a protocol involves a number of problematic operations.35,36 To illustrate, the synthesis of 34 ketones related to 110 evolves from ester 113 (Scheme 3.2), which is obtained starting with the reaction of L-threonine methyl ester, 112, with hazardous phosgene. The resulting 113 is converted into thioamide 114 by a sequence that employs Lawesson’s reagent. Byproducts arising from the latter are persistent contaminants in 114, which requires purification by flash column chromatography. Ester 115 is then treated with three equivalents of organolithium species 116 (Scheme 3.3). One equivalent of 116 serves to deprotonate the acidic oxazolidinone N-H; a second equivalent adds to the ester, and a third equivalent is required to deprotonate the highly acidic nascent ketone 117. The requirement for 3 equivalents of 116, of which only 1 is incorporated into the final product, is wasteful and had to be circumvented. Scheme 3.2. The Ciufolini synthesis of ester 115 Scheme 3.3. Requirement for excess organolithium species 116 to prepare ketone 117 35 Organolithium reagent 116 is created in situ by deprotonation of 119 with BuLi. In turn, 89 is manufactured from 115 by ester reduction and protection of the resulting alcohol (Scheme 3.4). Ester reduction is necessary to avoid nucleophilic addition to 115 (either by BuLi or by 116) upon C-2 methyl deprotonation. Yet, the TBS-protected alcohol must be oxidized back to a carboxylic group at a later stage of the synthesis by a 3-step sequence: deprotection, oxidation to an aldehyde, Pinnick oxidation to an acid. This five-step redox-protection-deprotection maneuver had to be suppressed. Scheme 3.4. The preparation of ketone 89 Plausible solutions to such problems were envisaged as follows (Scheme 3.5). First, a Garner-type120 protected threonine 120 in lieu of 113 would suppress an acidic N-H bond and remove the need for 1 equivalent of organometallic nucleophile. Second, advancing 120 to thiazole 122 through a White-Siegel reaction, via nitrile 121, would circumvent the need for Lawesson’s reagent. Third, reaction of 122 with two equivalents of economical lithiated acetonitrile, instead of expensive lithiated methylthiazole 116, would produce 110. Notice that the second equivalent is required to deprotonate the highly acidic cyanoketone 134 (Scheme 3.9, p. 39). The latter would 36 then be elaborated to cyanopyridine 109. The “northwestern” thiazole can be annealed onto 109 by the White-Siegel method, thus eliminating the 5-step redox/protection sequence alluded-to earlier. Scheme 3.5. Synthetic logic for pyridine 108 The synthesis of ynones such as 90 also needed improvement. The original avenue to substances of this type started with the oxidation of an appropriate 2-methylthiazole to an aldehyde by the use of toxic selenium dioxide (Scheme 3.6), which is not a permissible reagent in a pharmaceutical environment. Moreover, scale-up of the reaction suffered from diminished yield on account of overoxidation of 124, 125 to carboxylic acids 126, 127 and decarboxylation thereof to 128, 129.88 Luckily, aldehyde 130 is commercially available. Ynone should be readily accessed from 130 by the two-step sequence delineated in Scheme 3.7. Clearly, the problematic selenium dioxide oxidation is no longer necessary. 37 Scheme 3.6. Selenium dioxide oxidation of 2-methylthiazoles en route to ynones Scheme 3.7. Plan for the preparation of ynone 111 38 3.2 Micrococcin P2: Synthesis of the Fragments The known acid 120 was prepared as described in the literature70,78 and converted into amide 132 via a mixed anhydride prepared in situ by treatment with ethyl chloroformate and triethylamine (Scheme 3.8). The action of cyanuric chloride on 132 resulted in dehydration to the corresponding nitrile 121. Scheme 3.8. Preparation of nitrile 121 Reaction of 121 with L-cysteine methyl ester returned the expected thiazoline 133, which was subsequently oxidized by DBU and CBrCl3 to afford the thiazole 122 in 64% yield over 5 steps from N-Boc-L-threonine (Scheme 3.9). Product 122 was estimated to be at least 95% pure by 1H NMR analysis and required no column chromatography. It is worthy of note that the reaction has been repeatedly run on a scale of 1.5 mol (~300 g) without incident and with no erosion of overall yield. Finally, exposure of 122 to the action of two equivalents of lithiated acetonitrile provided the desired cyanoketone 110 in purity up to 95%, determined by 1H NMR analysis. Therefore, 110 was used for a subsequent reaction without chromatography. 39 Scheme 3.9. Preparation of ketone 110 The synthesis of ynone 111 proceeded uneventfully as detailed in Scheme 3.10, setting the stage for the critical Bohmann-Rahtz construction of the pyridine unit. Relevant operations started with the condensation of crude cyanoketone 110 with excess NH4OAc in a refluxing mixture of toluene/AcOH to afford enamine (Scheme 3.11), which underwent Michael addition to ynone 111 upon heating in EtOH solution. Scheme 3.10. Preparation of ynone 111 Initially, the resulting aminodiene 136 was isolated and purified, then heated neat to 165oC to 40 induce E/Z isomerization and cyclodehydration to pyridine 109. It rapidly transpired that addition of acetic acid to the ethanolic solution in which 136 is formed (30% of the volume of EtOH) effectively mediates isomerization and cyclization, leading to cyanopyridine 109 in a single step. This is where the first flash column chromatography in the entire sequence was performed, resulting in a 30% overall yield of 109 from L-threonine over 8 steps. Finally, condensation of cyanopyridine 109 with L-cysteine hydrochloride, and oxidation of the thiazoline 137 by DBU and CBrCl3 delivered clean carboxylic acid 108, which required no purification. Scheme 3.11. Synthesis of pyridine core 108 41 Scheme 3.12. Preparation of fragment 107 by the literature method It is apparent that the present route is considerably more robust, economical, and convenient than the original Ciufolini one. A second crucial fragment of MP2 is the known35,36 compound 107. This material was prepared without difficulty by the literature method (Scheme 3.12). Crude carboxylic acid 108 was now coupled with 107 in the presence of tetramethyl-chloroformamidinium hexafluorophosphate (TCFH) and N-methylimidazole93 (Scheme 3.13). Scheme 3.13. Union of 108 and 107 and macrocyclization sequence to afford 103 42 The use of this coupling reagent resulted in a fast reaction that completed within 15 minutes. A second flash column chromatography was performed at this juncture, affording pure 138 in 56% yield over 3 steps from 109. Two-stage deprotection of the resulting 138 (aqueous LiOH in THF for the saponification of ester functionality; TFA in DCM for the removal of the N-Boc and acetonide moieties) returned seco-intermediate 139, which underwent Yamada marcolactamization94 to 103 in 56% yield over 3 steps. This efficient sequence has delivered decagram quantities of pure 103 in nearly 10% yield over 14 steps. We were now in a position to address the crucial question of whether 103 would be a good substrate for palladium-mediated coupling reaction. Initial investigations focused on Suzuki coupling with commercial arylboronic acids. Such reactions were performed in a mixture of THF / water with K2CO3 and Pd(dtbpf)Cl2 {dtbpf = bis[(tert-Bu)2PPh]ferrocene}. Table 3.1. Suzuki coupling reactions of 103 Entry Ar-B(OR)2 Yield (%) 140a 70 140b 67 140c 65 43 140d 70 140e 64 140f 68 140g 62 140h 61 This catalyst was found to be especially competent in the present case, affording the desired products in 60-70% yield with a wide range of heterocyclic boronic acids. The more economical Pd(PPh3)4 works almost as well. Representative results appear in Table 3.1. As of this writing, this constitutes the first example of Suzuki coupling with a fully formed thiopeptide macrocycle. The foregoing results suggest that an especially efficient synthesis of MP2 would be possible through coupling of 103 with 2-thiazolylboronic acid 141 (Figure 3.1).95,96 However, 2- thiazolylboronic acids are elusive species. For instance, numerous attempts in our group to produce such organometallics, either by C-2 deprotonation-borylation of a thiazole, or by Suzuki-Miyaura borylation of a 2-bromothiazole, consistently met with failure. Figure 3.1 Hypothetical boronic acid 141 44 A reasonable conjecture at this point was to reverse the Suzuki role of the coupling partners. Suzuki-Miyaura borylation of 103 proved to be quite facile and efficient (Scheme 3.14). The ready availability of 142 opened the door to coupling reactions with diverse aryl halides; in particular with 2-bromothiazoles. Scheme 3.14. Successful borylation of 103 Accordingly, compounds 147 and 148 were prepared from commercial 2-bromo-4-carboxylic acid as outlined in Scheme 3.15. Despite the molecular complexity of fragments, 103, 147 and 148, and the presence of many N- and S- atoms therein (all potential ligation sites for Pd species), the coupling reaction worked very well, delivering synthetic MP1 and MP2 in 60% yield in either case (Scheme 3.16). 45 Scheme 3.15. Preparation of thiazolyl coupling partners of 147 and 148 46 Scheme 3.16. Total synthesis of MP1 and MP2 MP1 thus produced was spectroscopically identical to the natural product. Spectral data for pure MP2 are unavailable. However, the high-resolution mass spectrum of synthetic MP2 exhibited a strong molecular ion of the correct composition, while its NMR spectra were nearly identical to those of MP1, except for the presence of diagnostic signals for a methyl ketone (1H, 13C) and a ketone carbonyl (13C) (Figure 3.2 and Figure 3.3). Structural proof for synthetic MP2 was achieved by chemoselective reduction of the ketone with NaBH4. The 1H NMR spectrum of the resulting secondary alcohol in DMSO-d6 was superimposable (Figure 3.4) to that of pure MP1 in the same solvent. However, the reduction should have produced a pair of diastereomeric products. Interestingly, the presence of two diastereomeric alcohols in a 1:1 ratio became apparent from the 1H NMR spectrum recorded in CDCl3. One set of resonances coincided with that of pure 47 MP1 in CDCl3, whereas the other one was attributable to epi-MP1, 152. (Figure 3.5). These observations fully support the structural assignment of synthetic MP2. Scheme 3.17. Sodium borohydride reduction of MP2, resulting in 4 and 152 The present avenue is significantly more efficient than any other route to thiopeptide antibiotics. The diversity-oriented synthesis just described paves the way towards a systematic medicinal chemistry venture. The accomplishments described in this dissertation are crucial to an ongoing effort aiming to develop new antibiotic resources for the treatment of a number of infectious diseases. 48 Figure 3.2. 1H NMR of synthetic MP2 Figure 3.3. 13C NMR of synthetic MP2 49 Figure 3.4. 1H NMR spectral comparison of reduced MP2 vs MP1 (DMSO-d6) Figure 3.5. 1H NMR spectrum reduced MP2, containing 1:1 mixture of MP1 and epi-MP1 (152), in CDCl3 (400 MHz) 50 3.3 Biological Studies of Micrococcin P2 Synthetic MP2 thus obtained was tested against a wide range of pathogenic microorganisms. This revealed noteworthy bioactivity and provided numerous opportunities to establish meaningful collaborations with various agencies in S. Korea and Canada to develop new anti-infective resources. It should be noted that thiopeptides that incorporate a 26-membered ring, like MP2, exhibit no cross-resistance97 with clinically used antibacterials. Moreover, their peculiar mode of action renders them effective against bacteria that have become resistant to most other antibiotics. These factors make thiopeptides a potentially valuable source of new antibiotics. The biological assays described below were primarily conducted at A & J Science by Young-Jin Son. Professor Jin-Hwan Kwak, at Handong Global University, and Professor Hyunjoo Pai, at Hanyang University Medical School evaluated MP2 against Clostridioides difficile. The data provided herein constitute the first extensive investigation of the biological activity of MP2. As anticipated for a thiopeptide, MP2 showed significant activity against Gram-positive organisms, but modest or no activity against Gram-negative ones, with the noteworthy exception of the serious human pathogen, Salmonella enterica. Table 3.2 summarizes the observed MIC50 in μg/mL of MP2 against various strains of Staphylococcus, Enterobacter, and Clostridioides organisms, as well as other bacteria. For each species, the potency of MP2 is compared to that of ciprofloxacin (column heading “CIP”), vancomycin (“VAN”) and linezolid (“LIN”). It is apparent that in several cases MP2 is more potent than vancomycin, which is an antibiotic of last resort.121 51 Table 3.2. Activity of MP2 (MIC50 in μg/mL) against various microorganisms Strain Details MP2 CIP VAN LIN Gram-(+) S. aureus ATCC 25923MS 0.125 0.25 0.5 2 ATCC 29213MS 0.125 0.5 0.5 1 ATCC 43300MR 1 1 0.5 4 Clinical isolateVR 1 >32 >32 8 S. epidermidis ATCC 12228 1 0.25 2 4 Clinical isolateMS 0.125 >32 1 1 Clinical isolateMR 0.125 >32 0.5 1 E. faecalis ATCC 19433 0.25 1 1 2 Clinical isolateVR 0.125 >32 >32 2 Clinical isolateLR 0.125 32 1 >32 E. faecium ATCC 19434 0.5 8 0.5 4 Clinical isolateVR 1 32 >32 2 Clinical isolateLR 0.125 16 0.5 32 B. subtilis ATCC 6688 >32 <0.06 0.25 4 C. difficile ATCC 9689 1 1 Gram -(-) E. coli ATCC 25922 >32 <0.06 >32 >32 K. pneumoniae ATCC 10031 >32 <0.06 >32 32 P. aeruginosa ATCC 27853 >32 0.5 >32 >32 A. baumannii ATCC 17904 >32 0.25 >32 >32 S. enterica ATCC 14028 0.5 <0.06 0.25 8 The activity of MP2 and of some analogues thereof against C. difficile was studied in some detail, given the great concern that this microorganism represents in the medical field. C. difficile is a Gram-positive, spore-forming, toxin-producing, anaerobic bacterium that has become a serious health threat.63,122 C. difficile is designated as an urgent threat by the U.S Centers for Disease 52 Control and Prevention (CDC)26. In the U.S alone, more than 220,000 cases of C. difficile infection (CDI) with 12,800 deaths were reported in 2019. Yet, only two U.S FDA approved drugs, vancomycin and fidaxomicin, are available to target C. difficile123. Vancomycin suffers from high rates of relapse (25% to 30%)124, and its broad antibiotic spectrum against Gram-positive bacteria leads to an undesirable reduction in microbiome diversity125. Fidaxomicin has lower recurrence rates because of its better selectivity towards C. difficile, but its high price overshadows its efficacy. The incidence of CDI in South Korea is essentially identical to the U.S, but the lack of national-level support for antimicrobial stewardship undermines public awareness126. The only available CDI treatment options for South Korean patients are vancomycin and metronidazole, both of which exhibit poor clinical prognosis and recurrence rates. Thus, there is a clear and urgent need for new effective antibiotics against C. difficile. Our route to MP2 (Chapter 3.2) secured gram quantities of material, enabling determination of key pharmacological parameters, as well as a thorough investigation of in vitro and in vivo anti CDI activity146. We have determined that MP2 exhibits very low cytotoxicity in various mammalian cell lines (Figure 3.6), and there is no appreciable toxicity even up to 40 μM. Figure 3.6. Cellular toxicity assay of MP2 53 Since C. difficile targets primarily the lumen of the large intestine, a good CDI agent should possess very low bioavailability.99,123 Indeed, vancomycin, fidaxomicin and most of the anti-CDI agents in the current pipeline are poorly bioavailable. Oral administration of MP2 to rats resulted in blood plasma levels below the quantification limit (BQL – lower than 10ng/mL; Table 3.3). The very low oral bioavailability of MP2 is ideal for an oral anti-CDI agent. Table 3.3. Rat pharmacokinetic studies of MP2 Intravenous (2mg/kg) Oral (10mg/kg) T1/2 (h) 0.8 ± 0.2 BQL AUClast (hr * ng/mL) 374.0 ± 97.7 BQL Cl_observed (mL/min/kg) 88.9 ± 23.6 BQL MRT_observed (hr) 0.4 ± 0.1 BQL Vss_observed (L/kg) 2.1 ± 0.1 BQL Micrococcin P2 was then screened against C. difficile isolates collected by Professor Pai at Hanyang Medical School in South Korea from 2008 to 2018. These clinical isolates have been fully characterized by multilocus sequence typing (MLST)127, PCR ribotyping128, and the presence of genes encoding toxin A, toxin B and binary toxin129. The antibiotic activity of MP2 has been tested according to MLST clade and ribotype. This constitutes the first extensive study of the activity of a natural, 26-membered thiopeptide against C. difficile clinical isolates from South Korea. MP2 exhibits superior antibiotic activity relative to vancomycin. (Table 3.4). Furthermore, the high selectivity index of MP2 (antibacterial activity vs. cellular toxicity) is remarkable. 54 Table 3.4. in vitro activity of MP2 against classified 53 C. difficile clinical isolates [minimum inhibitory concentrations (MIC50), μg/ml] Clade Ribotype MLST Toxin VAN 1 Selectivity Index (SI) 1 RT018 (10) ST17 A+B+ 1 0.5 >100 RT001 (4) ST3 A+B+ 2 1 >50 RT002 (4) ST8 A+B+ 1 1 >50 RT012 (3) ST54 A+B+ 2 1 >50 RT014 (3) ST14 A+B+ 1 1 >50 RT015 (3) ST35 A+B+ 1 1 >50 RT293 (2) ST129 A+B+ 1 1 >50 2 RT027 (3) ST1 A+B+CDT+ 1 0.5 >100 3 RT130 (5) ST5 A+B+CDT+ 1 0.5 >100 nontypable (3) ST221 A+B+CDT+ 2 1 >50 4 RT017 (10) ST37 A-B+ 1 0.5 >100 5 RT078 (3) ST11 A+B+CDT+ 1 1 >50 55 Figure 3.7. Twenty-four hour time-kill curves for vancomycin and MP2. Turning next to bacterial killing rates, MP2 was tested against C. difficile PCR ribotype 027130, a hypervirulent strain that is becoming ever more prevalent, and that is of special clinical concern, because of its severe disease presentation and higher recurrence rates. The conduct of 56 time kill assays and in vivo studies (vide infra) has been performed by the laboratory of Professor Kwak at Handong University. The comparative killing kinetics of MP2 and vancomycin against C. difficile ribotype 027 and the killing effect of MP2 and vancomycin are shown in Figure 3.7, together with the reductions in cfu/mL following a 24-hour exposure to the antibiotics. It is apparent that MP2 kills C. difficile ribotype 027 more efficiently than vancomycin, as the significant reduction in bacterial counts (2.5 – 3.3 log10 units) is achieved at doses between 1 and 4 times the MIC value. At 2x and 4x MIC, MP2 exerts a complete bactericidal action against C. difficile. In both cases, the limit of detection (LOD) was reached at 24 hours. On the other hand, vancomycin is bacteriostatic at all the tested concentrations. We note that a regrowth of C. difficile occurred 6 hours after exposure to a dose of MP2 corresponding to 0.5 times the MIC. Based on MP2’s pharmacokinetic properties, bacterial regrowth can be avoided through a suitable oral dosing regimen, and this has been demonstrated in our in vivo C. difficile mouse model. (vide infra). Over the past decade, more clinical cases of CDI patients suffering from inflammatory bowel diseases (IBD) have been documented131. Patients suffering from CDI-IBD comorbidity experience poor clinical outcomes with higher rates of C. difficile recurrence. The development of new therapeutic agents that could target CDI-IBD comorbidity is highly desirable. Based on MP2’s promising in vitro profile against C. difficile, we have adopted the established in vivo mouse model of Zhou, et al132, in which mice are initially treated with dextran sulfate sodium (DSS) in a drinking water for 5 days to cause IBD, and then infected with hypervirulent C. difficile ribotype 027 to induce CDI-IBD comorbidity. The severity of the ensuing infection is apparent from the fact that all untreated mice succumbed within 3 days. Only two U.S FDA approved drugs, vancomycin and fidaxomicin, were used as control. These drugs were administrated once daily at a dose of 30mg/kg via oral gavage. MP2 was been administrated in the same manner. MP2 resulted in a survival rate 57 of 100% (Figure 3.8), which is superior to both vancomycin and fidaxomicin. Figure 3.8. In vivo mouse CDI-IBD efficacy of MP2. Recent clinical studies suggest that the host’s immune response affects pathogen virulence in CDI infection133. Indeed, the severe pathogenesis of CDI is accompanied by an upregulation of certain cytokines134, especially interleukin IL-1β, IL-6, and IL-8. It has been noted that vancomycin upregulates interleukin IL-1β, IL-6135, while fidaxomicin reduces the production of IL-1β136. Recent studies corroborate the notion that downregulation of cytokine production translates into a cytoprotective effect that may be linked to lower rates of CDI recurrence and better clinical outcomes (15.4% vs 25.3%, respectively)137. To our surprise, MP2 significantly diminishes the expression of IL-1β, IL-6 in mouse monocyte-macrophage RAW 264.7 cells. (Figure 3.9). Thus, MP2 possesses some degree of anti-inflammatory and cytoprotecting activity, along with potent antibiotic activity. All these promising results clearly warrant the development of 26-0204060801000 1 2 3 4 5 6 7 8 9 10 11 12 13 14Survival rate(%)DayVehicle MP2 Fidaxomicin Vancomycin 58 membered thiopeptide antibiotics as a new source for C. difficile antibiotics. Figure 3.9. MP2 immune action against cytokines IL-1β and IL-6. Dexamethasone is used as a control. Micrococcin P2 also proved to be quite active against Mycobacterium tuberculosis102, including multi-drug resistant (MTB) and extensively-drug resistant (XTB) strains. (Table 3.5). Table 3.5. Activity of MP2 against M. tuberculosis, including MDR and XDR strains Strain Strain # Resistance Reference Micrococcin P2 MIC50 M. tuberculosis H37Rv SUS INHS, RIFS, MXFS, AMKS 89 nM M. tuberculosis 704 SUS INHS, RIFS, MXFS, AMKS 199 nM M. tuberculosis 1443 SUS INHS, RIFS, MXFS, AMKS 356 nM M. tuberculosis 2577 SUS INHS, RIFS, MXFS, AMKS 354 nM M. tuberculosis 2656 SUS INHS, RIFS, MXFS, AMKS 208 nM M. tuberculosis 2318 MDR INHR, RIFR, MXFS, AMKS 73 nM M. tuberculosis 2400 MDR INHR, RIFR, MXFS, AMKS 169 nM M. tuberculosis 6393 MDR INHR, RIFR, MXFS, AMKS 51 nM M. tuberculosis 5027 MDR INHR, RIFR, MXFS, AMKR 2.3 uM 59 Abbreviations: SUS: susceptible; MDR: Multi-Drug Resistant; XDR: Extensively Drug Resistant; INH: Isoniazid; RIF: Rifampicin; MXF: Moxifloxacin; AMK: Amikacin Furthermore, MP2 exhibits comparable activity against non-tuberculosis Mycobacterium (NTM)64, a pathogen for which no effective treatment is available. NTM is regarded as “tuberculosis of the developed countries” because the incidence of NTM infection is more prevalent in the developed countries. Therefore, NTM constitutes a clear, urgent and unmet medical need. MP2 possesses superior in vitro MIC50 values (Table 3.6) compared to clarithromycin in both M. avium and M. intracellulare. Its remarkable activity against clarithromycin resistant M. avium is noteworthy. These encouraging results suggest that thiopeptide antibiotics can be potentially leveraged for the treatment of various bacterial infections. In vitro assays of M. tuberculosis and NTM have been conducted by Dr. Da-kyum Lee at the Masan National Tuberculosis Hospital in South Korea. Table 3.6 MP2’s in vitro activity against various NTM bacteria. Strain Strain # Micrococcin P2 MIC50 Clarithromycin Mycobacterium avium ATCC 70098 28 nM 76 nM Mycobacterium intracellulare ATCC 13950 33 nM 380 nM Clarithromycin-resistant M. avium clinical isolate 125 nM >50 μM M. tuberculosis 2180 MDR INHR, RIFR, MXFR, AMKS 304 nM M. tuberculosis 4027 MDR INHR, RIFR, MXFR, AMKS 44 nM M. tuberculosis 3836 XDR INHR, RIFR, MXFR, AMKR 375 nM M. tuberculosis 4691 XDR INHR, RIFR, MXFR, AMKR 94 nM M. tuberculosis 30 XDR INHR, RIFR, MXFR, AMKR 114 nM M. tuberculosis 9845 XDR INHR, RIFR, MXFR, AMKR 722 nM M. tuberculosis 8301 XDR INHR, RIFR, MXFR, AMKR 209 nM 60 3.4 Medicinal Chemistry Studies in the Micrococcin P2 Series The robust synthesis illustrated in Chapter 3.2 enabled the conduct of a thorough medicinal chemistry campaign in the micrococcin area. Such an effort revealed that the structural modifications are possible essentially only at the level of the short “eastern” dipeptide side chain. In particular, the bithiazole segment proved to be essential for activity. For example, analogues 140d, 140f, and 140h (Scheme 3.18) were inactive (Table 3.7). Scheme 3.18. MP2 analogue synthesis via Suzuki coupling Table 3.7 Activity profile of simple aromatic substitution of micrococcin series MIC (μg/mL) Cell toxicity (μM) S. aureus E. faecalis E. faecium C. difficile HEK293T SH-SY5Y Van 0.5 1 0.5 1 MP2 0.5 0.5 0.5 1 >50 >50 140d >64 >64 >64 >64 >50 >50 140f >64 >64 >64 >64 >50 >50 61 140h >64 >64 >64 >64 >50 >50 Loss of activity was also observed in analogues that retained the short side chain, but that exhibited a phenyl (compound 150) or a pyridine (151) residue in lieu of the second thiazole ring (Figure 3.10). Figure 3.10. The substitution of second thiazole ring to 150, 151 Conversely, modification of the short side chain produced clinically useful compounds. These constitute intellectual property of A&J Science and are outside the scope of this dissertation. 3.5 A Remarkable Chemical Property of Micrococcin P2 All clinically interesting congeners of MP2 produced to date by A&J scientists are amide derivatives of acid 153 (Figure 3.11), and can be readily accessed by the straightforward 14-step sequence outlined earlier. It should be noted that advances in synthetic organic chemistry render such multistep routes to drug material quite practicable. Two instructive examples are Tetraphase’s eravacycline, 2, and Eisai’s eribulin mesylate, 157, both FDA-approved drugs, and both a 62 testament to the power of contemporary synthetic organic chemistry. Figure 3.11. Acid 153: the precursor of all clinically interesting MP2 analogues Eravacycline (2) is a fully synthetic tetracyclin antibiotic, the synthesis of which relies on technology developed in the laboratories of Prof. A. G. Myers142, and further optimized by process chemists at Tetraphase Pharmaceuticals143. The synthesis of 156 entails the union of enone 155, obtained in turn from benzoic acid by an 11-step sequence and in 10% overall yield, and ester 154. Thus, deprotonation of 154 at the methyl group and reaction of the corresponding anion with 155 results in formation of compound 156 through sequential Michael and Dieckmann reactions (Scheme 3.19). A five-step sequence is then employed to advance 156 to 2, the overall yield of which is comparable to that of micrococcin P2 and congeners. 63 Scheme 3.19. Synthesis of eravacycline (2) Even more impressive is the case of eribulin mesylate144, 157, an exceptionally potent anticancer agent, the synthesis of which relies heavily upon technology developed in the laboratories of Prof. Y. Kishi (Figure 3.12). Interestingly, the chemistry effort leading to 157 also perfected an important transformation called the Nozaki-Hiyama-Kishi reaction (Scheme 3.20),145 which is a key step towards eribulin mesylate. Figure 3.12. Chemical structure of eribulin 64 Scheme 3.20. Nozaki-Hiyama-Kishi reaction applied to the synthesis of eribulin The above notwithstanding, it would be useful to gain access to biosynthetically produced acid 153. The requisite drugs could then be obtained from 153 by semisynthesis, resulting in a more economical route. The history of pharmaceutical science is replete with similar examples, a case in point being the beta-lactam antibiotics. Groundbreaking research on penicillin led to a number of advances, including John C. Sheehan’s discovery of DCC agents104 and Dorothy Hodgkins’ X-ray crystallographic work105. Yet, no synthetic route ever devised proved to be competitive with a fermentation process that yields benzylpenicillin, and thence 6-aminopenicillanic acid (6-APA), at a nearly insignificant cost (few dollars per kg)138. Chemical acylation of the free amino group in 6-APA then resulted in successive generations of beta-lactam antibiotics, many of which remain in use today106. More relevant to the present work is the case of Novartis’ LFF57198,99,100,101, which is a semisynthetic derivative of the thiopeptide, GE2270A, produced, in turn, by fermentation. Consultations with molecular biologists led to the conclusion that it would be difficult to modify a micrococcin-fermenting organism to produce 153. Accordingly, the decision was made to research the selective hydrolysis of the side chain of MP1 and/or MP2 as an avenue to the 65 desired acid. Fully synthetic antibiotics were thus exposed to the action of lithium hydroxide in aqueous THF. Micrococcin P1 proved to be relatively resistant to base hydrolysis, which gave rise to an intractable mixture of unreacted MP1 plus a multitude of products. Signals consistent with the presence of 153 were apparent in an LC-MS spectrum of the crude hydrolysate, but separation of a pure sample of 153 proved to be impossible even by HPLC. In stark contrast, MP2 was rapidly and cleanly converted into the desired 153, which was isolated in pure form and in 74% yield. The remarkable difference in hydrolytic behavior between MP1 and MP2 merits additional comment. It is speculated that base treatment promotes equilibration of MP2 with a hemiamidal, which probably exists as intramolecularly hydrogen-bonded structure 160 (Scheme 3.21). Such an equilibration is impossible in MP1, which displays a secondary alcohol on the side chain, in lieu of a ketone. The hydrogen bond in 160 is likely to activate the C=O group toward nucleophilic addition of hydroxide ion. The resulting tetrahedral intermediate 161 incorporates an N atom (starred) of significantly lower basicity than an ordinary 2-piperazinone, which in turn is considerably less basic than a typical amine.107,108,109 Therefore, the starred N functions as a considerably better leaving group, ultimately departing as 162 and unveiling 153. The latter presumably is rapidly deprotonated under basic conditions, but the corresponding lithium carboxylate is returned to the carboxylic acid state upon acidic workup. 66 Scheme 3.21. Mechanistic hypothesis for the highly chemoselective hydrolysis of MP2 The important result just described induced A & J Science researchers to re-engineer B. subtilis, so as to optimize the fermentation of MP2. Very recently, a genetically modified B. subtilis has been created, that produces only MP2. Briefly, methods of molecular biology revealed that only eight Tcl genes are required to produce MP2. Truncation and reorganization of enzymatic functions in a heterologous host of biologically engineered B. subtilis suppressed the formation of MP1 and resulted in exclusive formation of MP2 (Scheme 3.22). 67 Scheme 3.22. Bioengineering sequences leading to the formation of MP2 using B. subtilis Scientists at A & J Science have improved the fermentation yield of MP2 from an initial level of 0.07 mg / L to the current 5 mg / L, and are relentlessly working to increase it to 25-30 mg / L. This is a break-through discovery that significantly increases the odds of bringing lead compounds to the clinic, and that greatly simplifies the rigorous Chemistry, Manufacturing & Control (CMC)103 issues associated with the advancement of a drug candidate to the clinical stage. 68 Chapter 4. Efforts towards the Total Synthesis of Lactocillin The human microbiome has been implicated in the modulation of human health and metabolism. Accumulating evidence suggests that microbiomes could influence systemic functions including immunity and brain function through the production of secondary metabolites139. In that connection, M. Fischbach and collaborators carried out extensive research on human commensal microorganisms, their secondary metabolites, and their role and impact on human health.37 A noteworthy finding that emerged from their work is that Lactobacillus gasseri JV-V03, which is naturally present in the vagina of healthy women, possesses a crucial biosynthetic gene cluster that encodes for the production of lactocillin, 6: a 26-membered ring thiopeptide antibiotic37 (Figure 4.1). Figure 4.1. The chemical structure of lactocillin 69 4.1 Synthetic Background on Lactocillin The discovery of lactocillin elicited significant attention from the synthetic community. However, a total synthesis of this difficult target remains elusive. The presence of a sensitive thiazoline and a labile thioester contributes to the synthetic difficulty. Furthermore, constitutional and stereochemical uncertainties persist, and in all likelihood, they will have to be settled by total synthesis, just as in the case of MP135. However, it is likely that lactocillin is derived from L-amino acids, since its biosynthesis involves a series of post-translational modifications of a large peptide consisting of naturally occurring L-amino acids.43,44 Siegel and collaborators have reported synthetic study toward lactocillin, but a total synthesis of the natural product has yet to be achieved. Indeed, the most advanced synthetic intermediate that these workers have described to date is compound 163 (Figure 4.2)110. Notice that 163 lacks the macrocyclic ring found in the natural product. Figure 4.2. The Siegel advanced intermediate for lactocillin Siegel’s approach to 163 envisions the union of fragments 164-166 (Scheme 4.1). In particular, 164 and 165 would be merged by Stille coupling, the thioester would be installed 70 through a Mitsunobu reaction111 of thioacid 167 with the serine-derived portion of 163, and that macrocycle formation would be achieved via White-Siegel thiazoline synthesis, hence the nitrile in 166. Scheme 4.1. Siegel’s retrosynthetic logic for lactocillin Siegel’s assembly of fragment 169 aligns with the strategy adopted for the total synthesis of MP1 (Scheme 2.11, p. 23). Thus, 168 was prepared from L-cysteine hydrochloride in 9 steps and 36% overall yield, then united with cyanopyridine 69 through a White-Siegel thiazole synthesis. Through a series of chemical transformations, 169 was afforded in 87% over 3 steps and set the stage for a crucial amide coupling (Scheme 4.2). 71 Scheme 4.2. Construction of the pyridine core of lactocillin by Siegel Micrococcins and lactocillin share the common fragment 170. Hydrolysis of the methyl ester in 170 and coupling with threonine-derived nitrile 171 delivered 172 in 70% yield over 2 steps (Scheme 4.3). However, it rapidly transpired that nitrile 172 was intolerant of the acidic conditions required to deblock the acetonides. Scheme 4.3. Preparation of nitrile 172 To remedy the problem, 173 was coupled with threonine methyl ester, and the ester was subsequently converted into a primary amide, 175 (Scheme 4.4). 72 Scheme 4.4. Synthetic efforts to afford 175 Scheme 4.5. Successful coupling of 169 with 175 and dehydration to yield pyridine 177 73 The merger of carboxylic acid 169 and 175 proceeded smoothly by the action of HATU and iPr2NEt in DMF, affording product 176 in 75% yield. All of the secondary alcohols were then protected as TBS ethers, and the primary amide was dehydrated to nitrile 177 using the Burgess reagent112 (Scheme 4.5). The preparation of Stille coupling partner 186 was rather long and arduous. Ethylene glycol was first elaborated to enantiopure Ellman N-tert-butylsulfinyl imine 181 (Scheme 4.6). Halogen-magnesium exchange of the known dibromothiazole 182 occurred selectively at the C-2 position. Addition of the resulting Grignard reagent to 181 produced the desired 183 as the major component of a 4:1 mixture of diastereomers. Release of the sulfinyl group under acidic conditions, amide formation and stannylation led to the desired 186. 74 Scheme 4.6. Preparation of enantiopure stannylthiazole 186 The Stille coupling of 177 and 186 required much optimization, but in the end it worked well and afforded 163 in 89% yield (Scheme 4.7). Unfortunately, the synthesis stalled at this juncture on account of poor stability of nitrile under the acidic conditions required to deblock the cysteine moiety. That is why Siegel’s effort could not advance beyond 163. 75 Scheme 4.7. Stille coupling of 177 and 186 to afford 163 4.2 Retrosynthetic Analysis of Lactocillin Our own retrosynthetic analysis divides lactocillin into five pieces (Scheme 4.8). Pyridine core 187 can be constructed by the method described earlier for the assembly of the corresponding portion of micrococcins, except that the “eastern” sector of 187 would emanate from serine instead of threonine. The synthesis of 2-bromo thiazole 189 and fragment 173 would also retrace the routes described in Chapter 3. The primary amine and the secondary alcohol in the sensitive thiazoline unit 188 require sturdy protecting groups, such as an allyl carbamate (Alloc) and a t-butyldiphenylsilyl ether, respectively. At this juncture, it was assumed that the stereocenters present in lactocillin are derived from natural L-amino acids. 76 Scheme 4.8. Retrosynthetic analysis of lactocillin 4.3 Efforts towards the Total Synthesis of Lactocillin The construction of pyridine core 187 started from N-Boc-L-serine. Appropriate functional group interconversions produced nitrile 193, which underwent White-Siegel condensation with L-cysteine methyl ester and a subsequent oxidation by CBrCl3 and DBU to afford thiazole, 195. This material reacted with lithiated acetonitrile to afford cyanoketone 196 in 55% yield (Scheme 4.9). Scheme 4.9. Synthesis of serine derived cyanoketone 196 77 In contrast to its L-threonine-derived analogue used in the synthesis of MP2, compound 196 required a column chromatographic purification. The subsequent elaboration of 196 to a cyanopyridine proceeded smoothly and returned the desired 198 in 38% over 2 steps. (Scheme 4.10). A final White-Siegel thiazole construction afforded the complete pyridine core 187 in 78% over 2 steps. Scheme 4.10. Synthesis of the pyridine core of lactocillin Parallel work centered on the preparation of fragment 203 (Scheme 4.11), which would be merged with 187 to form the macrocyclic domain of lactocillin. The synthesis of the sensitive thiazoline 188 started with N- and O-protected L-threonine methyl ester 199. Upon the hydrolysis of methyl ester, coupling of the acid with S-trityl cysteine methyl ester, 200, afforded dipeptide 201 in 75% over 2 steps. Simultaneous release of the trityl group and cyclodehydration to the desired thiazoline 188 occurred in 80% yield upon acid treatment of 201 under Kelly conditions114. 78 Thiazoline 188 and the previously described thiazole 173 (see p. 72) were coupled in a one-pot operation involving release of the Alloc group, followed by EDCI mediated amide formation to afford 202 in 58% yield. Finally, deprotection of 202 by treatment with a mixture of TFA and CH2Cl2 (1 :1) unmasked the amine functionality. The acidic conditions of the deprotection sequence promote formation of some side products that could be separated by column chromatography, affording the desired 203 in 36% yield. Scheme 4.11. Synthesis of sensitive thiazoline fragment 203 The union of carboxylic acid 187 with 203 occurred smoothly in the presence of TCFH and N-methylimidazole to afford 204 in 52% yield (Scheme 4.12). However, the macrocyclization sequence had to be revisited extensively. Release of Boc and acetonide protection from 204 by treatment with TFA or mixtures of TFA / CH2Cl2 simultaneously eliminated TBDPS protected 79 alcohol, resulting in the formation of dehydroaminoacid and the loss of oxygen functionality. Fortunately, deblocking could be achieved by exposure of 204 to 4M HCl solution in dioxane. Still, the reaction had to be conducted carefully, and contact time could not exceed 25 minutes. Under such conditions, the deprotection sequence was satisfactorily replicated on scales of 0.5-1.0 mmol scale to provide amine 205 in 48% yield after purification. Second, hydrolysis of the methyl ester was achieved by the action of Me3SnOH - a well-known reagent to prevent epimerization of the sensitive thiazoline.140 Finally, DPPA mediated Yamada macrolactamization94 afforded the macrocycle 206 in only 15% yield over 2 steps from 204 (Scheme 4.12). Despite the low yield, this constitutes the first successful macrocyclization of the lactocillin core. Due to the time constraint, a full characterization of 206 could not be made (only 1H NMR, and LC/MS provided in the supporting information). At this juncture, the author was driven by the intellectual curiosity to test whether the macrocyclic core containing the fragile thiazoline group would be a good substrate for the late-stage Suzuki coupling. Indeed, macrocyclic core 206 underwent successful Suzuki-Miyaura borylation, enabling coupling with bromothiazole 189, which was obtained through the union of commercial 2-bromothiazole-4-carboxylic acid with L-alanine methyl ester hydrochloride. Despite the low yield, it is certain that the late-stage Suzuki coupling is successfully implemented – substance 208 is the most advanced synthetic precursor of lactocillin described to date (Scheme 4.13). 80 Scheme 4.12. Successful formation of the lactocillin macrocycle 81 Scheme 4.13. Late-stage Suzuki coupling to install the tail fragment The final remaining task in the synthesis of thiocillin would be the thioesterification of serine-derived primary alcohol in 208. This objective would be achieved via a Mitsunobu reaction111 of 208 with thioacid 190 to install the indole group containing 209. Sequential deprotection of TBDPS group and indole N-Boc group, such as TASF and 4M HCl in dioxane, will render lactocillin-methyl ester, 210, which can be spectroscopically compared with the work by Fischbach.37 (Scheme 4.14). 82 Scheme 4.14. Anticipated completion of lactocillin methyl ester 83 5 Conclusion This dissertation describes the first total synthesis of micrococcin P2, a synthesis of micrococcin P1, and synthetic efforts towards lactocillin that led to the most advanced synthetic precursor of the natural products yet described. The new technology for MP2 synthesis established a practical approach to medicinal chemistry work and SAR studies in the thiopeptide field. These synthetic efforts provide an avenue to the exploitation of unexplored 26-membered thiopeptides. In that connection, fully synthetic analogues of MP2 with more biologically and pharmacologically desirable properties than the natural product itself have been identified. A noteworthy chemical property of MP2 opens the door to semisynthetic routes to clinical candidates based on thiopeptide motifs. The newly established availability of multi-gram quantities of MP2 has enabled us to conduct in-depth studies of its biological activity against bacterial pathogens that are identified as serious or urgent threats by the US Centers for Disease Control and Prevention. Firstly, MP2 exerts potent antibacterial activity against various extensively classified C. difficile clinical isolates. C. difficile primarily targets the large intestines, and it is best treated with antibiotics that exhibit very low oral bioavailability. The drugs thus remain in the intestinal lumen, greatly reducing the likelihood of systemic toxicity. Indeed, MP2 possesses very low oral bioavailability, which greatly limits systemic absorption. Therefore, the chances of the drug eliciting systemic toxicity become negligible. While MP2 does not penetrate mammalian cells (determined by in vitro PAMPA permeability studies; data not shown), it easily penetrates C. difficile bacterial cells, subsequently inhibiting protein synthesis and ultimately killing the organism. This effect was clearly observed in our extensive in vitro studies. To illustrate, the time-kill assay studies have shown that MP2 kills 84 C. difficile ribotype 027 more efficiently than vancomycin. Moreover, these encouraging results translated well into in vivo mouse models. Given the significant increase in the incidence of CDI in patients suffering from inflammatory bowel disease (IBD), a new clinical agent that could target both indications are highly desirable. We have utilized the established CDI-IBD comorbidity mouse model, and have demonstrated that MP2 has better in vivo efficacy than fidaxomicin and vancomycin. Secondly, the incidence of patients suffering from Mycobacterium avium complex (MAC) pulmonary diseases is rising globally, which constitutes urgent medical needs to develop new, and effective antibiotics. MP2 exerts potent antibacterial activity against MAC species. Lastly, the activity of MP2 against Mycobacterium tuberculosis, including multi-drug and extensively-drug resistant strains, is noteworthy. However, the low oral bioavailability of MP2 constitutes a liability in the present case, because the drug will not be systemically available to reach the target organs: the lungs. On the other hand, direct delivery of the drug to the lungs, for instance by inhalation of a nebulized suspension of a lipid nanoparticle (LNP) formulation, could circumvent bioavailability issues. A relevant example is the recently approved Arikayce (liposomal amikacin), which is administered by nebulization for NTM-MAC indications, thus overcoming poor systemic absorption. Moreover, LNPs tend to accumulate in phagocytic cells and macrophages, which are key hosts of NTM and TB bacteria. Encapsulation of antibiotics in LNPs thus offers enhanced opportunity for pharmacological intervention. The observation that MP2 undergoes clean base hydrolysis to MP acid has induced molecular biologists at A & J Science to engineer B. subtilis to produce only MP2. Antibiotic obtained by fermentation can then be converted into MP acid, which is the precursor of virtually 85 all of the clinically interesting analogues of the natural product. Such compounds thus become accessible by semi-synthetic routes that greatly simplify the rigorous Chemistry, Manufacturing & Control (CMC)103 issues associated with the advancement of a drug candidate to the clinical stage. Antimicrobial Resistance (AMR) constitutes a pressing medical problem. The world needs new antibiotics. 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General Experimental Procedures Unless otherwise noted, all reactions were carried out under Ar in flamed-dried glassware using anhydrous solvents. Anhydrous solvents were prepared by distillation over the indicated drying agents prior to use and were transferred under Ar: THF, Et2O (Mg/anthracene), toluene (Na/K), CH2Cl2, MeOH (Mg); DMF and Et3N were dried by an adsorption solvent purification system based on molecular sieves. Thin layer chromatography (TLC): Macherey-Nagel precoated plates (POLYGRAM®SIL/UV254). Flash chromatography: Merck silica gel 60 (40−63 µm) with technical grade solvents. NMR: Spectra were recorded on Bruker AV VIII 400 or 600 spectrometers in the solvents indicated. The solvent signals were used as references, and the chemical shifts were converted to the TMS scale (CDCl3: δC = 77.0 ppm; residual CHCl3 in CDCl3: δH = 7.26 ppm; CD3OD: δC = 49.0 ppm; residual CHD2OD in CD3OD: δH = 3.31 ppm; CD2Cl2: δC = 54.0 ppm; residual CHDCl2 in CD2Cl2: δH = 5.32 ppm). FT-IR spectra were obtained on Thermo Scientific Nicolet 6700 and reported in frequency of the absorption (cm−1) High resolution mass spectra (HRMS) were recorded on an AB SCIEX Q-TOF 5600 mass spectrometer. Optical rotation ([𝛼]𝐷20 and [𝛼]𝐷25): Krüss P8000-T, 10 cm/1 mL cell. Unless otherwise noted, all commercially available compounds (Acros, Aldrich, Alfa Aesar, TCI) were used as received. Melting points were determined on A. KRÜSS OPTRONIC M3000. MPLC purifications are performed by CombiFlash RF+ UV. For some of the final compounds, further purification was performed by preparative HPLC on Kinetex 5μm biphenyl, 100 Å (GX-281 HPLC system, Gilson, Middletown, WI, USA; column tube 250 mm × 21.2 mm i.d.), with ACN/H2O as eluent. The purity of target compounds was determined to be >95% by analytical HPLC using dual different wavelength detector. 106 B. Synthesis and characterization of various intermediates To a solution of N-Boc-L-threonine (100 mmol, 22.0 g) in THF (200 mL) was added 2,2-dimethoxypropane (1000 mmol, 123 mL, 10 equiv) and pyridinium p-toluenesulfonate (30 mmol, 7.50 g, 0.3 equiv) were added. The mixture was brought up to reflux and stirred for 18 hours. The mixture was cooled to room temperature, and concentrated in vacuo. The residue was partitioned between water (250 mL) and ethyl acetate (250 mL). The aqueous layer was further extracted with ethyl acetate (200 mL x 2). The combined organic layer was dried over Na2SO4, and concentrated in vacuo to afford the desired 120 as a white solid. [Rf – 0.30 (20% ethyl acetate : 80% hexane)]. Yield : 25.4 g (98%) 1H NMR (600 MHz, CDCl3, ppm): δ 4.26 (d, J = 40.4 Hz, 1H), 4.05 – 3.86 (m, 1H), 1.68 – 1.52 (m, 6H), 1.51 – 1.37 (m, 12H) (note: An exchangeable proton is not present in the 1H NMR); 13C NMR (151 MHz, CDCl3, ppm): δ 176.0, 174.1, 152.8, 151.1, 95.5, 95.0, 81.7, 81.0, 74.0, 73.5, 66.1, 65.8, 28.4, 27.9, 26.7, 25.1, 24.1, 19.1. Due to restricted rotations, a greater number of 13C peaks is observed. To a solution of 120 (26.2 g, 101.4 mmol) and triethylamine (111.5 mmol, 15.5 mL, 1.1 equiv) in THF (200 mL) at 0 oC was carefully added ethyl chloroformate (111.5 mmol, 10.6 mL, 1.1 equiv) and the mixture was stirred for 1.5 hours. Then, aqueous ammonia (18M, 152 mmol, 8.40 mL, 1.5 107 equiv) was added and the mixture was allowed to warm to room temperature and stirred for 22 hours. The mixture was concentrated in vacuo, and the resulting aqueous suspension was extracted with ethyl acetate (150 mL x 3). The combined organic layer was washed with water (100 mL), brine (100 mL), dried over Na2SO4, and concentrated in vacuo to afford the desired 132 as a yellow solid. Yield : 22.8 g (87%), 1H NMR (600 MHz, CDCl3, ppm): δ 5.47 (s, 1H), 4.40 (qd, J = 6.5, 2.3 Hz, 1H), 3.78 (d, J = 17.9 Hz, 1H), 1.62 (s, 3H), 1.59 (s, 3H), 1.46 (s, 9H), 1.41 (d, J = 6.1 Hz, 3H); 13C NMR (151 MHz, CDCl3, ppm): δ 207.2, 174.0, 156.7, 95.1, 81.3, 80.7, 66.6, 61.3, 57.7, 34.6, 31.4, 31.1, 28.4, 25.7, 19.1, 18.3, 14.7. Due to restricted rotations, a greater number of 13C peaks is observed. To a solution of 132 in DMF (88 mL) was carefully added cyanuric chloride (44.1 mmol, 8.13 g, 0.5 equiv) in portions. The mixture was stirred at room temperature for 30 minutes. Ice water (50 mL) was added and whereupon the solid was formed. The solids were filtered, and further washed with water (100 mL) and dried in vacuo to afford the desired 121 as a white solid. [Rf – 0.70 (25% ethyl acetate : 75% hexane)]. Yield : 19.5 g (92%) 1H NMR (600 MHz, CDCl3, ppm): δ 4.47 – 4.40 (m, 1H), 4.02 (d, J = 7.4 Hz, 1H), 1.66 – 1.55 (m, 6H), 1.52 (s, 9H), 1.44 (d, J = 6.1 Hz, 3H); 13C NMR (151 MHz, CDCl3, ppm): δ 150.7, 117.4, 96.0, 95.4, 82.4, 74.3, 74.0, 53.2, 28.4, 27.9, 26.6, 25.6, 24.6, 18.5. Spectral data are similar with the reported data.1 1 Christy, M. P.; Johnson, T.; McNerlin, C. D.; Woodard, J.; Nelson, A. T.; Lim, B.; Hamilton. T. L.; Freiberg, K. M.; Siegel, D. Org. Lett, 2020, 22, 6, 2365-2370. 108 To a solution of 121 in the mixture of isopropyl alcohol : 0.1 M pH 6 sodium phosphate buffer (3 : 2 v/v 240 mL : 160 mL) was added L-cysteine methyl ester hydrochloride (122 mmol, 20.9 g, 1.5 equiv). The mixture was degassed with N2 for a period of 10 minutes, and the mixture was stirred at 50 oC for 15 hours. The mixture was concentrated in vacuo to remove excess isopropyl alcohol, and the aqueous layer was extracted with ethyl acetate (150 mL x 2). The organic layer was washed with 1M HCl (60 mL), water (60 mL), dried over Na2SO4 and concentrated in vacuo to afford the thiazoline 133 as a brown solid. The crude thiazoline 133 (77.6 mmol, 27.8 g) was dissolved in CH2Cl2 (260 mL) at 0 oC and was treated with BrCCl3 (116.4 mmol, 11.5 mL, 1.5 equiv). DBU (116.4 mmol, 17.4 mL, 1.5 equiv) was carefully added dropwise. The mixture was then brought up to reflux and stirred for 4 hours. The mixture was quenched by the addition of 1M HCl (100 mL) and aqueous layer was extracted with CH2Cl2 (100 mL x 2). The combined organic layer was washed with water (100 mL), brine (100 mL), dried over Na2SO4, and concentrated in vacuo. The isolated viscous crude material was kept in water (200 mL) for 30 minutes whereupon the crude was solidified. Then, the crude was washed through a suction filtration method by water (100 mL x 3), followed by hexane (100 mL x 3). The desired 122 was isolated as a dark brown solid. [Rf – 0.60 (20% ethyl acetate : 80% hexane)]. Yield : 23.7 g (82% over 2 steps), 1H NMR (600 MHz, CDCl3, ppm, measured at 50 oC): δ 8.13 (s, 1H), 4.78 (d, J = 7.6 Hz, 1H), 4.23 – 4.16 (m, 1H), 3.93 (s, 3H), 1.68 (d, J = 5.0 Hz, 6H), 1.41 (d, J = 6.1 Hz, 3H), 1.24 (brs, 9H) ; 13C NMR (151 MHz, CDCl3, ppm, measured at 50 oC): δ 173.5, 161.8, 151.7, 146.8, 127.5, 95.4, 81.0, 77.9, 66.2, 109 52.4, 28.3, 27.0, 26.3, 18.2. ; Melting point: 121.4-122.6; IR(neat): 3099, 2980, 2934, 1699, 1490 (cm-1); HRMS: Calcd for C16H25N2O5S + [M+H]+ 357.1479, found 357.1480; [𝛼]𝐷20: -48.8 (c=0.25, CHCl3). A solution of a commercial 4-bromo-2-formyl thiazole (104.0 mmol, 20.0 g) in THF (200 mL) was added dropwise to a commercial 0.5M solution of ethynylmagnesium bromide in THF (1.2 equiv, 125 mmol, 250 mL) at room temperature. The mixture was stirred for 30 min, and then quenched with aqueous saturated NH4Cl solution (250 mL). The organic layer was separated and the aqueous phase was extracted with ethyl acetate (2 x 150 mL). The combined extracts were dried over Na2SO4 and concentrated in vacuo. MPLC (gradient 10 → 30% ethyl acetate : 90 → 70% hexane) of the residue afforded the desired 131 as a white solid. (87 mmol, 19.0 g, Yield: 84%). ([Rf – 0.30 (30% ethyl acetate : 70% hexane)]. The resulting 131 (87 mmol, 19.0 g) was dissolved in CH2Cl2 (400 mL). Manganese dioxide (11.3 equiv, 987 mmol, 85.0 g) was added at room temperature and the mixture was stirred for 12 hours. The mixture was filtered through Celite and the bed was further washed with ethyl acetate (300 mL) and CH2Cl2 (200 mL). The filtrate was concentrated in vacuo to afford the desired 111 as a brown solid. [Rf – 0.42 (30% ethyl acetate : 70% hexane)]. Crude is clean enough for a subsequent reaction. However, a clean sample is obtained by MPLC (30 → 50% ethyl acetate : 70 → 50% hexane) as a brown solid. [Rf – 0.42 (30% ethyl acetate : 70% hexane)]. Yield: 16.0 g (71% over 2 steps); 1H NMR (600 MHz, CDCl3, ppm): δ 7.66 (s, 1H), 3.64 (s, 1H); 13C NMR (151 MHz, CDCl3, ppm): δ 168.1, 165.8, 128.7, 125.9, 84.5, 110 78.9; Melting point: 148.2-149.3 ℃; IR(neat): 3192, 3084, 2641, 2092, 1633 cm-1; HRMS: Calcd for C6H3BrNOS + [M+H]+ 215.9113, found 215.9107. A commercial solution of n-BuLi (2.40 M in hexanes, 54.6 mL, 131 mmol, 2.1 equiv) was added over a period of 10 minutes to a cold (–78 °C) solution of acetonitrile (135 mmol, 2.15 equiv 7.04 mL) in THF (120 mL), the mixture was stirred at -78oC for 20 minutes, then a solution of 122 (62.6 mmol, 22.3 g) in THF (120 mL) was slowly added over a period of 10 minutes. The mixture was slowly brought up to room temperature over 1 hour, then it was carefully quenched with 1M HCl (250 mL). The mixture was diluted with ethyl acetate (300 mL), transferred to a separatory funnel and further acidified with 1M HCl to pH 3. Aqueous layer was further extracted with ethyl acetate (200 mL). The organic layer was collected, dried over Na2SO4 and concentrated in vacuo. Crude 110 is clean enough for a subsequent reaction. However, authentic clean sample is obtained by MPLC (30 → 50% ethyl acetate : 70 → 50% hexane) as a brown solid. [Rf – 0.36 (30% ethyl acetate : 70% hexane)]. 1H NMR (600 MHz, CDCl3, ppm, measured at 50 ℃) δ 8.24 (s, 1H), 4.68 (brs, 1H), 4.21 – 4.10 (m, 3H), 1.67 (s, 3H), 1.65 (s, 3H), 1.59 – 0.97 (m, 12H); 13C NMR (151 MHz, CDCl3,ppm, measured at 50 ℃) δ 206.5, 182.2, 173.2, 151.6, 127.3, 113.8, 95.4, 81.2, 68.6, 65.9, 30.4, 28.3, 26.1, 18.1; Melting point: 98.1-100.6 ℃; IR(neat): 3133, 2977, 2934, 2260, 1697, 1491 (cm-1); HRMS: Calcd for C17H24N3O4S + [M+H]+ 366.1482, found 366.1482; [𝛼]𝐷20: -32.3 (c=0.25, CHCl3). 111 To a solution of 110 (61.0 mmol, 22.3 g) in the mixture of toluene / acetic acid (5 :1 v/v 300 mL : 60 mL) was added ammonium acetate (610 mmol, 47.0 g, 10 equiv). The mixture was brought up to reflux and stirred for 1 hour. The mixture was cooled down, diluted with ethyl acetate (600 mL) and carefully washed with sat. aq. NaHCO3 (350 mL). The organic layer was collected, dried over Na2SO4 and concentrated in vacuo to afford the desired 135 as a yellow oil (9:1 mixture of geometric isomers). Crude material is used for a subsequent reaction without further purification. However, an authentic clean sample is obtained by MPLC (30 → 50% ethyl acetate : 70 → 50% hexane) as a brown foam. [Rf – 0.36 (30% ethyl acetate : 70% hexane)]. 1H NMR (600 MHz, CDCl3, measured at 50 ℃) δ 7.54 (s, 1H), 5.46 (s, 2H), 4.68 (s, 1H), 4.53 (s, 1H), 4.23 – 4.16 (m, 1H), 1.69 (s, 3H), 1.67 (s, 3H), 1.40 (d, J = 6.1 Hz, 3H), 1.35 – 1.14 (m, 9H). 13C NMR (151 MHz, CDCl3, measured at 50 ℃) δ 172.1, 153.5, 149.1, 147.1, 127.3, 122.0, 119.3, 117.3, 95.4, 81.1, 66.1, 61.9, 28.4, 26.0, 18.2; IR(neat): 3469, 3351, 3232, 3103, 2979, 2934, 2189, 1692, 1617, 1590 (cm-1); HRMS: Calcd for C17H25N4O3S+ [M+H]+ 365.1642, found 365.1647; [𝛼]𝐷20: -55.7 (c=0.25, CHCl3). 112 To a solution of 135 (57.5 mmol, 21.0 g) in ethanol (300 mL) was added 111 (106.3 mmol, 22.9 g, 1.85 equiv) and the mixture was stirred at 60 oC for 1.5 hours. At this time, starting material is consumed and Michael adduct is clearly seen by TLC and LC/MS system. Then, acetic acid (30 % volume, 90 mL) was added and the mixture was brought up to reflux and stirred 16 hours to mediate the cyclization and aromatization. The excess ethanol and acetic acid was removed in vacuo. The crude was redissolved in CH2Cl2 (450 mL), and carefully quenched with sat. aq. NaHCO3 (200 mL). The organic layer was collected and dried over Na2SO4, and concentrated in vacuo. MPLC (5 → 15% ethyl acetate : 95 → 85% hexane) of the residue afforded the desired 109 as a yellow solid. [Rf – 0.40 (20% ethyl acetate : 80% hexane)]. Yield : 16.5 g (47% over 3 steps); 1H NMR (600 MHz, CDCl3,ppm, measured at 50 ℃) δ 8.24 (s, 1H), 8.22 – 8.14 (m, 2H), 7.45 (s, 1H), 4.83 (d, J = 7.7 Hz, 1H), 4.39 (s, 1H), 1.72 (s, 6H), 1.54 – 1.49 (m, 3H), 1.47 – 1.21 (m, 9H); 13C NMR (151 MHz, CDCl3, ppm, measured at 50 ℃)2 δ 167.8, 153.3, 152.0, 151.7, 144.3, 144.0, 127.4, 121.8, 121.6, 118.0, 117.0, 107.6, 95.5, 80.9, 66.1, 28.5, 26.1, 18.2; Melting point: 185.3-187.7 ℃; IR(neat): 3124, 3062, 2979, 2933, 2221, 1681, 1582, 1558, 1490 (cm-1); HRMS: Calcd for C23H25BrN5O3S2+ [M+H]+ 562.0577, found 562.0558; [𝛼]𝐷20: -45.2 (c=0.25, CHCl3) 2 Due to atropisomerism, a 13C NMR spectrum was recorded at 50 °C. A set of carbon resonances was tentatively assigned. 113 To a solution of 109 (24.0 mmol, 13.5 g) in the mixture of ethanol / pH 7 sodium phosphate buffer / water (6 : 2 : 1 v/v 210 mL : 70 mL : 35 mL) was added L-cysteine (168 mmol, 20.3 g, 7 equiv), sodium bicarbonate (96.0 mmol, 8.07 g, 4 equiv) and the mixture was stirred at 100 oC overnight. Upon completion, excess solvent was removed in vacuo and mixture was diluted with ethyl acetate (500 mL). Organic layer was washed with 1M HCl (150 mL x 3) to get rid of excess cysteine, and dried over Na2SO4 to afford the crude desired thiazoline 137 as a brown gum. The crude 137 is pushed for a subsequent oxidation reaction. The crude thiazoline 137 (23.5 mmol, 15.6 g) was dissolved in CH2Cl2 (260 mL) and treated with BrCCl3 (94 mmol, 9.2 mL, 4.0 equiv). DBU (117.5 mmol, 17.4 mL, 5.0 equiv) was carefully added dropwise. The mixture was then brought up to reflux and stirred for 3 hours. The mixture was quenched by the addition of 1M HCl (200 mL) and aqueous layer was extracted with CH2Cl2 (200 mL). The combined organic layer was dried over Na2SO4, and concentrated in vacuo. The resulting crude thiazole 108 was used for a subsequent amide coupling. The crude thiazole 108 (23.5 mmol, 15.6 g) in acetonitrile (240 mL) was added 107 (23.5 mmol, 11.3 g, 1.0 equiv) and N-methylimidazole (70.5 mmol, 5.61 mL, 3 equiv). Then, TCFH (30.6 mmol, 8.58 g, 1.3 equiv) was added and the mixture was stirred at room temperature for 30 minutes. Water (50 mL) was added to quench the reaction, and aqueous layer was extracted with CH2Cl2 (150 mL x 2). The organic layer was collected, dried over Na2SO4 and concentrated in vacuo. The crude was subjected to MPLC (1.5% → 2.3% MeOH : 98.5% → 97.7% CH2Cl2) of 114 the residue afforded the desired 138 as a yellow solid. [Rf – 0.22 (4% MeOH : 96% CH2Cl2)]. Yield : 15.2 g (66% over 3 steps); 1H NMR (600 MHz, CDCl3, ppm) δ 8.60 (s, 1H), 8.48 (d, J = 9.6 Hz, 1H), 8.32 – 8.08 (m, 4H), 8.07 (s, 1H), 7.96 (s, 1H), 7.94 – 7.90 (m, 1H), 7.40 (s, 1H), 6.45 (q, J = 7.0 Hz, 1H), 5.37 (dd, J = 9.6, 7.0 Hz, 1H), 4.85 (s, 1H), 4.63 (qd, J = 6.3, 2.6 Hz, 1H), 4.52 (s, 1H), 4.11 – 4.03 (m, 1H), 3.87 (s, 3H), 2.45 – 2.37 (m, 1H), 2.17 (brs, 1H), 1.87 (d, J = 7.1 Hz, 3H), 1.68 – 1.56 (m, 6H), 1.48 – 1.42 (m, 3H), 1.27 – 1.19 (m, 12H), 1.04 (d, J = 6.8 Hz, 3H), 0.95 (d, J = 6.7 Hz, 3H); 13C NMR (100 MHz, CDCl3, ppm) δ 171.0, 168.9, 168.6, 166.2, 165.7, 161.6, 161.2, 160.5, 160.4, 152.5, 151.0, 150.2, 149.4, 148.9, 146.7, 140.0, 128.9, 128.6, 127.2, 126.9, 126.5, 125.8, 123.2, 120.6, 118.5, 95.2, 80.7, 67.4, 65.7, 58.1, 58.0, 52.4, 38.6, 34.1, 28.2, 26.5, 25.7,19.3, 18.7, 18.4, 17.8, 14.7; Melting point: 145.0-148.5 ℃; IR(neat): 3381, 3116, 2973, 2932, 1656, 1579, 1531, 1477, 1429 (cm-1); HRMS: Calcd for C46H52BrN10O9S5+ [M+H]+ 1127.1700, found 1127.1725; [𝛼]𝐷20: +22.4(c=0.25, CHCl3) 138 (21.2 g, 18.8 mmol) was dissolved in the mixture of THF (90 mL) / H2O (90 mL) and LiOH · H2O (56.3 mmol, 2.36 g, 3.0 equiv) was added and stirred at room temperature for 1.5 hours before it was acidified with 1M HCl (150 mL). The compound was then extracted with ethyl acetate (3 x 150 mL), dried over Na2SO4, and concentrated in vacuo to afford the crude carboxylic acid. This was then dissolved in the mixture of CH2Cl2 (75 mL) / TFA (75 mL) and stirred at room temperature for 30 minutes before it was concentrated in vacuo to give the 139 trifluoroacetate salt. 115 This crude amine 139 was then dissolved in DMF (135 mL), DPPA (28.2 mmol, 6.87 mL, 1.5 equiv) and sodium bicarbonate (225 mmol, 18.9 g, 12 equiv) were added and stirred overnight at room temperature before being diluted with 350 mL super solvent (4:1 CHCl3 : isopropyl alcohol). The organic layer was washed with water four times (150 mL x 4), dried over Na2SO4, and concentrated in vacuo. The crude was subjected to MPLC (2% → 5% MeOH : 98% → 95% CH2Cl2) to afford the desired 103 as a yellow solid. [Rf – 0.25 (5% MeOH : 95% CH2Cl2)]. Yield: 10.1 g (56% over 3 steps);1H NMR (400 MHz, CDCl3, ppm): δ 8.74 (s, 1H), 8.38 (d, J = 9.6 Hz, 1H), 8.20 – 8.17 (m, 2H), 8.15 – 8.06 (m, 3H), 8.00 – 7.86 (m, 3H), 7.39 (s, 1H), 6.37 (q, J = 7.0 Hz, 1H), 5.23 – 5.18 (m, 2H), 4.91 (d, J = 7.8 Hz, 1H), 4.71 – 4.55 (m, 2H), 4.39 – 4.28 (m, 1H), 2.93 (s, 1H), 2.46 (td, J = 13.4, 6.6 Hz, 1H), 1.78 (d, J = 7.0 Hz, 3H), 1.55 (d, J = 5.9 Hz, 3H), 1.17 (d, J = 6.4 Hz, 3H), 1.12 (d, J = 6.7 Hz, 3H), 0.93 (d, J = 6.6 Hz, 3H); (13C NMR (151 MHz, CDCl3, ppm) δ 170.6, 170.0, 168.8, 168.5, 166.5, 166.3, 161.4, 161.0, 160.5, 153.0, 150.6, 150.4, 150.2, 149.5, 149.0, 141.2, 129.0, 128.4, 127.9, 126.8, 125.1, 124.0, 123.9, 122.7, 120.9, 118.3, 69.0, 67.8, 58.5, 55.8, 54.8, 34.0, 20.1, 19.9, 19.2, 18.9, 14.6; Melting point: 237.7-239.9 ℃; IR(neat): 3388, 3114, 2969, 2931, 1654, 1579, 1530, 1478, 1429 (cm-1); HRMS: Calcd for C38H36BrN10O6S5+ [M+H]+ 955.0601, found 955.0628; [𝛼]𝐷20: +31.0 (c=0.25, CHCl3) N-Boc-L-threonine (9.20 mmol, 2.00 g) and 143 (11.0 mmol, 3.46 g, 1.2 equiv) was dissolved in CH2Cl2 (40 mL). EDCI (11.0 mmol, 2.10 g, 1.2 equiv), HOBt (11.0 mmol, 1.48 g, 1.2 equiv) and diisopropylethylamine (18.4 mmol, 3.20 mL, 2 equiv) was added and the mixture was stirred at 116 room temperature for 2 hours. The mixture was quenched by the addition of water (10 mL), and further extracted with CH2Cl2 (40 mL). The crude mixture was purified by MPLC (20% → 40% ethyl acetate : 80% → 60% hexane) to afford the desired 144 as a white solid. [Rf – 0.50 (50% hexane : 50% ethyl acetate)]. Yield: 2.80 g (60%). 1H NMR (600 MHz, CDCl3, ppm): δ 7.69 (m, 4H), 7.49 – 7.44 (m, 2H), 7.43 – 7.39 (m, 4H), 6.89 (brs, 1H), 5.47 (d, J = 7.9 Hz, 1H), 4.34 (qd, J = 6.4, 2.0 Hz, 1H), 4.05 – 3.97 (m, 2H), 3.34 – 3.30 (m, 1H), 3.24 (dt, J = 13.5, 4.5 Hz, 1H), 2.87 (brs, 1H), 1.48 (s, 9H), 1.18 (d, J = 6.4 Hz, 3H), 1.10 (s, 9H), 1.06 (d, J = 6.2 Hz, 3H); 13C NMR (151 MHz, CDCl3, ppm): δ 172.0, 156.7, 135.9, 135.9, 134.1, 133.8, 130.0, 129.9, 127.9, 127.8, 80.4, 68.2, 66.8, 58.1, 46.4, 28.4, 27.1, 20.9, 19.3, 18.5. IR (film): 3337, 3070, 2968, 2931, 2893, 2859, 1814, 1658, 1488 (cm-1); HRMS: Calcd for C28H43N2O5Si+ [M+H]+ 515.2936, found 515.2923. [𝜶]𝑫𝟐𝟎 = –3.7 (c = 1.0, CHCl3) 2-bromothiazole-4-carboxylic acid (8 mmol, 1.66 g) was added to the solution containing the Boc-deprotected 144 trifluoroacetic acid salt which was synthesized by exposing 144 in CH2Cl2 / TFA and used immediately> (8.8 mmol, 3.64 g, 1.1 equiv) and diisopropylethylamine (24 mmol, 4.20 mL, 3 equiv) in CH2Cl2 (20 mL). EDCI (8.8 mmol, 1.68 g, 1.1 equiv) and HOBt (8.8 mmol, 1.19 g, 1.1 equiv) were added, and the mixture was stirred at room temperature overnight. The mixture was quenched by the addition of water (10 mL), and extracted with CH2Cl2 (30 mL). The organic layer was then dried with Na2SO4 and concentrated in vacuo. The crude was subjected to MPLC (20% → 50% ethyl acetate: 80% → 50% hexane) to afford the desired 145 as a yellow foam. [Rf 117 – 0.25 (45% ethyl acetate : 55% hexane)]. Yield: 3.00 g (63%) 1H NMR (600 MHz, CDCl3, ppm) δ 8.08 (s, 1H), 8.01 (d, J = 8.4 Hz, 1H), 7.69 – 7.65 (m, 4H), 7.48 – 7.44 (m, 2H), 7.42 – 7.37 (m, 4H), 6.92 – 6.90 (m, 1H), 4.44 (qd, J = 6.5, 1.9 Hz, 1H), 4.39 (dd, J = 8.4, 1.9 Hz, 1H), 4.02 – 3.97 (m, 1H), 3.55 (brs, 1H), 3.29 (app.t, J = 5.3 Hz, 2H), 1.22 (d, J = 6.5 Hz, 3H), 1.05 (s, 9H), 1.03 (d, J = 6.5 Hz, 3H); 13C NMR (151 MHz, CDCl3, ppm): δ 171.3, 160.8, 149.2, 136.4, 136.0, 135.9, 134.1, 133.9, 130.0, 129.9, 127.9, 127.8, 127.7, 68.3, 66.5, 56.8, 46.6, 27.1, 21.1, 19.3, 18.6; IR (Cast Film): 3375, 3071, 2931, 2857, 1734, 1652, 1533 (cm-1); HRMS: Calcd for C27H35BrN3O4SSi+ [M+H]+ 604.1295, found 604.1281; [𝜶]𝑫𝟐𝟎 = -45.0 (c=0.1, CHCl3). To a solution of 145 (5.00 mmol, 1.50 g) in CH2Cl2 (40 mL) was added methanesulfonyl chloride (14.9 mmol, 1.15 mL, 3 equiv) and Et3N (14.9 mmol, 2.10 mL, 3 equiv). The mixture was stirred at room temperature for 1 hour. Then, DBU (74.8 mmol, 11.2 mL, 15 equiv) was added and the mixture was further stirred at room temperature for 2 hours. The reaction was quenched by the addition of sat. aq. NH4Cl (40 mL), and extracted with CH2Cl2 (100 mL). The organic layer was dried over Na2SO4. The crude mixture was purified by MPLC (30% → 50% ethyl acetate: 70 → 50% hexane) to afford the desired 146 as a white foam. [Rf – 0.38 (50% ethyl acetate : 50% hexane)] Yield: 2.20 g (75%) 1H NMR (600 MHz, CDCl3, ppm): δ 8.32 (s, 1H), 8.12 (s, 1H), 7.66 – 7.64 (m, 4H), 7.48 – 7.41 (m, 2H), 7.40 – 7.37 (m, 4H), 6.53 (q, J = 7.1 Hz, 1H), 6.39 (app.t, J = 5.3 Hz, 1H), 4.05 – 4.01 (m, 1H), 3.41 (ddd, J = 13.6, 5.4, 3.6 Hz, 1H), 3.33 (dt, J = 13.6, 5.9 Hz, 1H), 1.79 (dd, J = 7.1, 0.7 Hz, 3H), 1.10 (d, J = 6.2 Hz, 3H), 0.99 (s, 9H); 13C NMR (151 MHz, CDCl3, ppm): δ 164.3, 158.1, 149.3, 136.4, 135.9, 135.8, 133.9, 133.9, 130.2, 130.0, 129.9, 129.1, 128.1, 118 128.0, 127.8, 68.8, 46.8, 27.0, 21.2, 19.3, 14.1; IR (Cast Film): 3351, 3070, 2929, 2855, 1666, 1639, 1522 cm-1; HRMS: Calcd for C27H33BrN3O3SSi+ [M+H]+ 586.1190, found 586.1198. [𝜶]𝑫𝟐𝟎 = +18.3 (c=0.25, CHCl3). To a solution of 146 (3.74 mmol, 2.20 g) in THF (30 mL) was added a 1 M solution of TBAF in THF (6.74 mmol, 6.74 mL, 1.8 equiv) and the mixture was stirred for 30 minutes before being diluted with ethyl acetate (80 mL) and sat. aq. NH4Cl (40 mL). The organic layer was extracted, dried over Na2SO4 and concentrated in vacuo. The crude mixture was purified by MPLC (2% → 5% MeOH : 98% → 95% CH2Cl2) to afford the desired 147 as a colorless oil. [Rf – 0.23 (5% MeOH : 95% CH2Cl2)]. Yield: 800 mg (61%) 1H NMR (600 MHz, CD3OD, ppm)3: δ 8.30 (d, J = 0.9 Hz, 1H), 6.71 (q, J = 7.1 Hz, 1H), 3.94 – 3.85 (m, 1H), 3.30 (dd, J = 13.5, 4.7 Hz, 1H), 3.20 (dd, J = 13.4, 7.1 Hz, 1H), 1.79 (d, J = 7.1 Hz, 3H), 1.16 (d, J = 6.3 Hz, 3H) Exchangeable protons (3H in total) are invisible in this case due to the deuterated methanol used.; 13C NMR (151 MHz, CD3OD, ppm): δ 167.2, 161.0, 150.4, 137.8, 132.2, 131.0, 130.2, 67.4, 48.1, 20.8, 13.7; IR (Cast Film): 3312, 2970, 2929, 1657, 1628, 1523 cm-1; HRMS: Calcd for C11H15BrN3O3S+ [M+H]+ 348.0012, found 348.0008; [𝛼]𝐷20 = -12.2 (c=0.5, CH3OH). 3 Exchangeable protons (3H in total) are invisible in this case due to the deuterated methanol used. 119 To a solution of 147 (1.12 mmol, 386 mg) in CH2Cl2 (10 mL) was added Dess-Martin Periodinane (1.66 mmol, 706 mg, 1.5 equiv). The mixture was stirred at room temperature for 2 hours. The mixture was diluted with CH2Cl2 (20 mL) before being washed with sat. aq. NaHCO3 (10 mL). The organic layer was dried over Na2SO4 and concentrated in vacuo. The crude mixture was purified by MPLC (2% → 5% MeOH : 98% → 95% CH2Cl2) to afford the desired 148 as a white gum. [Rf – 0.33 (5% MeOH : 95% CH2Cl2)]. Yield: 356 mg (89%); 1H NMR (600 MHz, CD3OD, ppm)4: δ 8.30 (s, 1H), 6.77 (q, J = 7.1 Hz, 1H), 4.07 (s, 2H), 2.18 (s, 3H), 1.81 (d, J = 7.1 Hz, 3H) Exchangeable protons (2H in total) are invisible in this case due to the deuterated methanol used.; 13C NMR (151 MHz, CD3OD, ppm): δ 206.5, 167.2, 161.0, 150.4, 137.8, 133.2, 130.2, 127.9, 50.6, 27.0, 13.8; IR (Cast Film): 3401, 3347, 3083, 2937, 2908, 1712, 1679, 1642, 1530 (cm-1); HRMS: Calcd for C11H13BrN3O3S+ [M+H]+ 345.9856, found 345.9849. To a solution of 103 (1.30 mmol, 1.24 g) in dioxane (10 mL) was added B2Pin2 (1.69 mmol, 428 mg, 1.3 equiv), potassium acetate (1.95 mmol, 191 mg, 1.5 equiv), XPhos (0.195 mmol, 93.0 mg, 4 Exchangeable protons (3H in total) are invisible in this case due to the deuterated methanol used. 120 0.15 equiv) and purged with N2 for few minutes. Then, Pd2(dba)3 (0.130 mmol, 119 mg, 0.10 equiv) was added and the mixture was stirred at 90 oC for 1.5 hours. The mixture was cooled to room temperature, diluted with CH2Cl2 (30 mL) and washed with H2O (10 mL). The organic layer was collected and filtered through Celite. Then, the filtrate was dried over Na2SO4 and concentrated in vacuo. The resulting crude boronic acid is immediately treated with (1.30 mmol) 148 (1.43 mmol, 494 mg, 1.1 equiv), K2CO3 (3.90 mmol, 539 mg, 3 equiv) in THF (10 mL) / H2O (2.5 mL) and purged with N2 for few minutes. Then, Pd(dtbpf)Cl2 (0.13 mmol, 84.7 mg, 0.1 equiv) was added and the mixture was brought up to 60 oC and stirred for 12 hours. It was cooled to room temperature and was filtered through Celite, and the bed was further washed with CH2Cl2 (50 mL). The organic layer was washed with 1M HCl (1 mL), dried over Na2SO4, and concentrated in vacuo. The crude was subjected to MPLC (2% → 5% MeOH : 98% → 95% CH2Cl2) to afford MP2 (3) as a white solid. [Rf – 0.34 (5% MeOH : 95% CH2Cl2)]. Yield: 890 mg (60% over 2 steps) 1H NMR (600 MHz, CD3OD, ppm): δ 8.40 – 8.31 (m, 4H), 8.27 (s, 1H), 8.22 (s, 1H), 8.13 (s, 1H), 7.96 (s, 1H), 6.82 (q, J = 7.1 Hz, 1H), 6.60 (q, J = 6.8 Hz, 1H), 5.24 (d, J = 2.9 Hz, 1H), 5.22 (d, J = 8.8 Hz, 1H), 4.78 (d, J = 3.1 Hz, 1H), 4.62 – 4.55 (m, 1H), 4.30 (qd, J = 6.3, 2.8 Hz, 1H), 4.10 (d, J = 13.0 Hz, 1H), 2.63 (dq, J = 13.4, 6.7 Hz, 1H), 2.19 (s, 3H), 2.20 – 2.14 (m, 1H), 1.92 (s, 1H) 1.86 (app. d, J = 7.0 Hz, 6H), 1.55 (d, J = 6.4 Hz, 3H), 1.18 (d, J = 6.3 Hz, 3H), 1.13 (d, J = 6.7 Hz, 3H), 0.99 (d, J = 6.7 Hz, 3H). Exchangeable protons (3H in total) are invisible in this case due to the deuterated methanol used. 13C NMR (151 MHz, CD3OD, ppm): δ 206.6, 172.4, 171.6, 171.0, 170.1, 168.0, 167.3, 166.2, 164.0, 163.2, 162.5, 162.5, 162.3, 155.2, 152.9, 151.8, 151.2, 151.2, 151.1, 150.4, 149.7, 141.3, 133.2, 131.1, 130.8, 130.6, 130.2, 126.5, 126.4, 125.8, 125.4, 122.3, 121.8, 120.0, 69.6, 69.2, 59.3, 57.4, 57.4, 50.6, 34.4, 27.1, 20.8, 20.7, 20.2, 19.3, 14.3, 13.9; Melting point: above 360 ℃, IR (film): 2923, 2851, 1590, 1464, 1352, 1134 cm-1 HRMS: Calcd 121 for C48H48N13O9S6+ [M+H]+ 1142.2017, found 1142.2045. [𝜶]𝑫𝟐𝟎 = +120.0 (c = 0.1, CD3OD). To a solution of 103 (1.30 mmol, 1.24 g) in dioxane (10 mL) was added B2Pin2 (1.69 mmol, 428 mg, 1.3 equiv), potassium acetate (1.95 mmol, 191 mg, 1.5 equiv), XPhos (0.195 mmol, 93.0 mg, 0.15 equiv) and purged with N2 for few minutes. Then, Pd2(dba)3 (0.130 mmol, 119 mg, 0.10 equiv) was added and the mixture was stirred at 90 oC for 1.5 hours. The mixture was cooled to room temperature, diluted with CH2Cl2 (30 mL) and washed with H2O (10 mL). The organic layer was collected and filtered through Celite. Then, the filtrate was dried over Na2SO4 and concentrated in vacuo. The resulting crude boronic acid is immediately treated with (1.30 mmol) 147 (1.43 mmol, 496 mg, 1.1 equiv), K2CO3 (3.90 mmol, 539 mg, 3 equiv) in THF (10 mL) / H2O (2.5 mL) and purged with N2 for few minutes. Then, Pd(dtbpf)Cl2 (0.13 mmol, 84.7 mg, 0.1 equiv) was added and the mixture was brought up to 60 oC and stirred for 12 hours. It was cooled to room temperature and was filtered through Celite, and the bed was further washed with CH2Cl2 (50 mL). The organic layer was washed with 1M HCl (1 mL), dried over Na2SO4, and concentrated in vacuo. The crude was subjected to MPLC (2% → 5% MeOH : 98% → 95% CH2Cl2) to afford MP1 (4) as a white solid. [Rf – 0.30 (5% MeOH : 95% CH2Cl2)]. Yield: 892 mg (60% over 2 steps). This is synthesized to double confirm the feasibility of Suzuki coupling method to make MP1. 1H NMR (600 MHz, DMSO-d6, ppm): δ 9.53 (br s, 1H), 9.52 (br s, 1H), 8.60 (s, 1H), 8.47 (d, J = 8.1 Hz, 2H), 8.45 (s, 1H), 8.38 (s, 1H) , 8.35 (d, J = 8.1 Hz, 1H), 8.30 (s, 1H), 8.24 (s, 1H), 8.21 (s, 1H), 8.12 (s, 1H), 122 7.92 (t, J = 5.6 Hz, 1H), 7.87 (d, J = 7.5 Hz, 1H), 6.53 – 6.46 (m, 2H), 5.16 – 5.12 (m,1 H), 5.11 – 5.08 (m, 1H), 4.70 (dd, J = 7,2 Hz, 3.0 Hz 1H), 4.41 – 4.34 (m, 2H), 4.03 – 3.96 (m, 2H), 3.74 – 3.69 (m, 2H), 3.12 – 2.95 (m, 2H), 1.75 (d, J = 6.9 Hz, 3H), 1.71 (d, J = 7.0 Hz, 3H), 1.39 (d, J = 5.9 Hz, 3H), 1.04 (d, J = 6.2 Hz, 3H), 1.03 (d, J = 6.2 Hz, 6H), 0.98 (d, J = 6.5 Hz, 3H), 0.87 (d, J = 6.6 Hz, 3H). Exchangeable protons were not detected in this case. Sodium Borohydride Reduction of Micrococcin P2 To a solution of micrococcin P2 (10 mg, 0.009 mmol) in the mixture of CH2Cl2 / MeOH (1:1 v/v 100 μL:100 μL) was added NaBH4 (4 eq, 0.036 mmol, 1.5 mg) at -78 oC. The mixture was stirred for 10 minutes and brought up to room temperature. The reaction was quenched by the addition of water, and the aqueous layer was extracted with CH2Cl2. The combined organic layer was collected, dried over Na2SO4, and concentrated in vacuo to afford the desired product. Yield: 10 mg (50%). The comparison of 1H NMR is provided. 123 1H-NMR (400 MHz, CDCl3) – epimer is seen in 1H NMR in CDCl3 1H-NMR (400 MHz, DMSO-d6) - epimer is not seen in 1H NMR in DMSO-d6 124 General Suzuki Coupling Protocol and Characterization of Compounds 103 (0.031 mmol, 30 mg), a corresponding aryl boronic acid (1.1 equiv) and K2CO3 (0.093 mmol, 12.8 mg, 3.0 equiv) were dissolved in the mixture of THF (0.25mL) / water (0.05 mL) and purged with N2 for few minutes. Then, Pd(dtbpf)Cl2 (0.0031 mmol, 2 mg, 10 mol %) was added and the mixture was brought up to 60 oC and stirred overnight. The mixture was filtered through Celite, and the bed was further washed with ethyl acetate (5 mL). The organic layer was washed with 1M HCl (1 mL), dried over Na2SO4, and concentrated in vacuo. The crude was subjected to MPLC (2% → 5% MeOH : 98% → 95% CH2Cl2) to afford the desired product (140a – 140h). 1H NMR (600 MHz, CDCl3, ppm): δ 8.46 – 8.43 (m, 1H), 8.40 – 8.34 (m, 2H), 8.26 (s, 1H), 8.17 (s, 1H), 8.15 – 8.12 (m, 3H), 8.11 (s, 1H), 8.08 (m, 3H), 7.98 (m, 2H), 7.79 (s, 1H), 6.40 (q, J = 6.9 Hz, 1H), 5.31 – 5.22 (m, 2H), 4.85 (d, J = 7.5 Hz, 1H), 4.65 – 4.61 (m, 1H), 4.49 – 4.45 (m, 1H), 3.95 (s, 3H), 3.81 – 3.49 (brs, 2H), 2.49 – 2.41 (m, 1H), 1.82 (d, J = 6.9 Hz, 3H), 1.54 (d, J = 6.3 Hz, 3H), 1.20 (d, J = 5.9 Hz, 3H), 1.13 (d, J = 6.7 Hz, 3H), 0.96 (d, J = 6.7 Hz, 3H); 13C NMR (151 MHz, CDCl3, ppm): δ 170.0, 168.8, 168.0, 167.0, 166.7, 166.4, 161.4, 161.1, 160.6, 156.1, 151.7, 150.2, 149.4, 148.9, 140.9, 138.4, 130.4, 130.4, 129.9, 129.0, 128.6, 127.6, 126.4, 125.3, 124.2, 124.0, 122.5, 118.7, 118.5, 69.0, 67.8, 58.5, 55.8, 54.9, 52.4, 33.9, 29.8, 22.8, 20.0, 19.9, 19.2, 19.0, 14.6; Melting point: 233.9-237.8℃; IR(neat): 3386, 3120, 2928, 1667, 1611, 1482, 1437, 1375, 1282 (cm-1); HRMS: Calcd for C45H43N10O8S5+ [M+H]+ 1011.1863, found 1011.1867; 125 [𝜶]𝑫𝟐𝟎: -3.3 (c=1, CHCl3). 1H NMR (600 MHz, DMSO-d6, ppm)5: δ 9.66 (d, J = 2.4 Hz, 1H), 9.53 (s, 1H), 9.09 (s, 2H), 8.71 (s, 1H), 8.48 (s, 2H), 8.37 (s, 1H), 8.30 (s, 1H), 8.25 – 8.20 (m, 2H), 8.17 – 8.08 (m, 4H), 7.87 (d, J = 8.0 Hz, 1H), 7.83 (ddd, J = 8.4, 6.7, 1.5 Hz, 1H), 7.70 (t, J = 7.5 Hz, 2H), 6.47 (q, J = 6.8 Hz, 1H), 5.20 – 5.05 (m, 2H), 4.69 (dd, J = 8.0, 3.6 Hz, 1H), 4.40 – 4.36 (m, 1H), 4.06 – 3.99 (m, 1H), 1.76 (d, J = 6.9 Hz, 3H), 1.38 (d, J = 6.4 Hz, 3H), 1.04 (d, J = 6.2 Hz, 3H), 0.98 (d, J = 6.8 Hz, 3H), 0.86 (d, J = 6.7 Hz, 3H); 13C NMR (151 MHz, DMSO-d6, ppm): δ 171.6, 170.0, 169.8, 168.5, 167.7, 166.3, 164.2, 160.5, 159.8, 153.0, 152.6, 151.0, 150.3, 149.6, 149.1, 148.5, 148.2, 146.5, 140.6, 133.0, 130.2, 129.6, 128.7, 128.4, 128.2, 127.7, 127.5, 127.0, 126.0, 125.3, 124.8, 124.4, 121.4, 120.0, 118.7, 67.5, 67.1, 59.8, 57.6, 56.1, 32.3, 20.6, 19.9, 19.6, 18.5, 13.8. ; Melting point: 246.0-247.7 ℃; IR(neat): 3385, 3120, 2972, 1665, 1485, 1376, 1205 (cm-1); HRMS: Calcd for C46H42N11O6S5+ [M+H]+ 1004.1918, found 1004.1904; [𝜶]𝑫𝟐𝟎: +46.4 (c=0.25, DMSO) 5 Two exchangeable protons are invisible in this case 126 1H NMR (600 MHz, CDCl3, ppm): δ 8.46 (s, 1H), 8.37 (d, J = 8.9 Hz, 1H), 8.33 (d, J = 8.2, 1H), 8.23 (s, 1H), 8.18 (s, 1H), 8.16 – 8.11 (m, 1H), 8.09 (s, 1H), 7.99 – 7.81 (m, 3H), 7.54 (dd, J = 3.6, 1.2 Hz, 1H), 7.52 (s, 1H), 7.33 (dd, J = 5.0, 1.1 Hz, 1H), 7.11 (dd, J = 5.0, 3.6 Hz, 1H), 6.39 (q, J = 7.0 Hz, 1H), 5.29 – 5.22 (m, 2H), 4.84 (dd, J = 8.0, 2.1 Hz, 1H), 4.63 (m, 1H), 4.47 – 4.43 (m, 1H), 3.63 – 2.88 (brs, 2H), 2.45 (m, 1H), 1.81 (d, J = 7.0 Hz, 3H), 1.54 (d, J = 6.5 Hz, 3H), 1.19 (d, J = 6.3 Hz, 3H), 1.13 (d, J = 6.7 Hz, 3H), 0.96 (d, J = 6.7 Hz, 3H); 13C NMR (151 MHz, CDCl3, ppm): δ 170.8, 170.0, 168.8, 167.6, 166.7, 166.3, 161.4, 161.0, 160.6, 153.1, 151.8, 151.6, 150.3, 149.8, 149.5, 149.0, 140.9, 138.2, 129.1, 128.3, 128.0, 127.4, 125.8, 125.0, 124.6, 124.0, 123.8, 122.5, 118.6, 115.3, 69.0, 67.8, 58.5, 55.8, 54.8, 33.9, 20.1, 19.9, 19.2, 18.9, 14.6; Melting point: 278.7-282.0℃; IR(neat): 3383, 2971, 1667, 1536, 1477 (cm-1); HRMS: Calcd for C41H39N10O6S6+ [M+H]+ 959.1373, found 959.1374; [𝜶]𝑫𝟐𝟎: +15.3 (c=0.5, CHCl3). 1H NMR (600 MHz, CDCl3, ppm): δ 8.40 (d, J = 8.1 Hz, 1H), 8.39 – 8.34 (m, 2H), 8.26 – 8.22 (m, 1H), 8.21 (s, 1H), 8.18 – 8.10 (m, 1H), 8.09 (s, 1H), 7.96 – 7.95 (m, 2H), 7.94 (m, 1H), 7.92 – 127 7.89 (m, 2H), 7.54 (s, 1H), 7.06 – 6.97 (m, 2H), 6.40 (q, J = 7.0 Hz, 1H), 5.31 – 5.23 (m, 2H), 4.81 (dd, J = 8.1, 2.0 Hz, 1H), 4.68 – 4.59 (m, 1H), 4.46 – 4.40 (m, 1H), 3.87 (s, 3H), 2.44 (m, 1H), 1.82 (d, J = 7.0 Hz, 3H), 1.53 (d, J = 6.5 Hz, 3H), 1.20 (d, J = 6.4 Hz, 3H), 1.13 (d, J = 6.7 Hz, 3H), 0.97 (d, J = 6.6 Hz, 3H); 13C NMR (151 MHz, CDCl3, ppm): δ 170.6, 170.0, 168.8, 167.5, 166.9, 166.4, 161.4, 161.1, 160.5, 160.1, 157.1, 153.1, 151.9, 150.4, 149.6, 149.0, 141.0, 129.8, 129.0, 128.3, 127.9, 127.3, 125.0, 124.0, 123.8, 122.6, 118.6, 115.0, 114.4, 114.0, 69.1, 67.8, 58.5, 55.8, 55.5, 54.7, 34.0, 20.2, 19.9, 19.2, 18.9, 14.6; Melting point: 222.8-226.7 ℃; IR(neat): 3386, 2968, 1667, 1612, 1534, 1467, 1418, 1304, 1249 (cm-1); HRMS: Calcd for C44H43N10O7S5+ [M+H]+ 983.1914, found 983.1916; [𝜶]𝑫𝟐𝟎: +1.6 (c=0.5, CHCl3). 1H NMR (600 MHz, CDCl3, ppm): δ 8.86 (t, J = 2.0 Hz, 1H), 8.50 (s, 1H), 8.37 (d, J = 8.0 Hz, 1H), 8.35 (d, J = 9.8 Hz, 1H), 8.31 (dt, J = 7.8, 1.3 Hz, 1H), 8.25 – 8.19 (m, 2H), 8.15 (s, 1H), 8.10 (s, 1H), 8.01 (d, J = 8.1 Hz, 1H), 7.97 (s, 1H), 7.94 – 7.90 (m, 2H), 7.83 (s, 1H), 7.63 (t, J = 8.0 Hz, 1H), 6.41 (q, J = 6.9 Hz, 1H), 5.33 (d, J = 9.0 Hz, 1H), 5.25 (t, J = 9.2 Hz, 1H), 4.87 (d, J = 8.0, 1H), 4.66 – 4.62 (m, 1H), 4.52 (d, J = 6.3 Hz, 1H), 3.61 (br s, 2H), 2.45 (h, J = 6.8 Hz, 1H), 1.82 (d, J = 7.0 Hz, 3H), 1.56 (d, J = 6.5 Hz, 3H), 1.20 (d, J = 6.3 Hz, 3H), 1.13 (d, J = 6.7 Hz, 3H), 0.96 (d, J = 6.7 Hz, 3H); 13C NMR (151 MHz, CDCl3, ppm): δ 171.4, 170.0, 168.8, 168.3, 166.4, 161.4, 161.2, 161.0, 160.5, 154.6, 152.7, 151.5, 150.3, 149.5, 149.1, 148.9, 140.8, 136.0, 132.1, 130.0, 130.0, 129.0, 128.6, 127.9, 125.2, 124.2, 123.8, 123.1, 122.4, 121.4, 118.8, 118.5, 128 69.0, 67.7, 58.4, 55.8, 55.0, 33.9, 20.0, 19.9, 19.3, 19.1, 14.6 ; Melting point: 234.7-238.9℃; IR(neat): 3394, 3119, 2970, 1663, 1534, 1483, 1419, 1349, 1246 (cm-1); HRMS: Calcd for C43H40N11O8S5+ [M+H]+ 998.1659, found 998.1653; [𝜶]𝑫𝟐𝟎: +7.43 (c=1, CHCl3). 1H NMR (600 MHz, CDCl3, ppm): δ 8.39 (d, 1H), 8.35 (s, 1H), 8.32 (d, 1H), 8.25 (s, 1H), 8.22 (s, 1H), 8.15 – 8.07 (m, 4H), 8.00 – 7.88 (m, 3H), 7.82 (s, 1H), 7.79 – 7.74 (m, 2H), 6.40 (qd, J = 6.8, 2.3 Hz, 1H), 5.28 – 5.19 (m, 2H), 4.83 (dd, J = 8.1, 2.1 Hz, 1H), 4.64 (td, J = 6.8, 2.2 Hz, 1H), 4.39 – 4.35 (m, 1H), 4.15 (d, J = 7.3 Hz, 1H), 3.08 (d, J = 3.1 Hz, 1H), 2.49 – 2.41 (m, 1H), 1.82 (d, J = 7.0 Hz, 3H), 1.54 (d, J = 6.5 Hz, 3H), 1.19 (d, J = 6.3 Hz, 3H), 1.13 (d, 3H), 0.96 (d, J = 6.5 Hz, 3H); 13C NMR (151 MHz, CDCl3, ppm): δ 170.3, 170.0, 168.8, 168.5, 166.7, 166.3, 161.4, 161.0, 160.5, 155.1, 153.3, 151.5, 150.5, 150.3, 149.6, 149.0, 141.2, 138.4, 132.9, 129.1, 128.3, 127.6, 127.0, 124.9, 123.9, 123.8, 122.7, 119.1, 119.0, 118.3, 111.8, 69.1, 67.8, 58.5, 55.8, 54.7, 33.9, 29.8, 20.5, 20.2, 19.9, 19.2, 18.8, 14.6 ; Melting point: 246.3-245.7 ℃; IR(neat): 3402, 2969, 2923, 2227, 1666, 1537, 1484, 1420, 1208 cm-1; HRMS: Calcd for C44H40N11O6S5+ [M+H]+ 978.1761, found 978.1776; [𝜶]𝑫𝟐𝟎: -1.443 (c=0.46, CHCl3). 129 1H NMR (400 MHz, CDCl3, ppm): δ 9.17 (s, 1H), 8.56 (d, J = 3.5 Hz, 1H), 8.45 (s, 1H), 8.34 (t, J = 9.9 Hz, 1H), 8.25 – 8.11 (m, 4H), 8.09 – 7.94 (m, 3H), 7.91 – 7.82 (m, 2H), 7.67 (s, 1H), 7.27 (dd, J = 7.8, 4.9 Hz, 1H), 6.31 (q, J = 6.9 Hz, 1H), 5.29 – 5.07 (m, 2H), 4.79 (d, J = 8.3 Hz, 1H), 4.62 – 4.49 (m, 1H), 4.34 – 4.18 (m, 1H), 3.07 (s, 1H), 2.39 (dd, J = 14.8, 7.0 Hz, 1H), 1.75 (d, J = 7.0 Hz, 3H), 1.48 (d, J = 6.5 Hz, 3H), 1.12 (d, J = 6.4 Hz, 3H), 1.06 (d, J = 6.7 Hz, 3H), 0.89 (d, J = 6.6 Hz, 3H); 13C NMR (151 MHz, CDCl3, ppm): δ 170.3, 170.0, 168.9, 166.8, 166.3, 161.3, 161.1, 160.5, 153.2, 151.5, 150.5, 150.2, 149.6, 149.0, 147.9, 143.2, 142.9, 141.2, 134.6, 130.6, 129.1, 128.3, 127.5, 125.0, 124.1, 123.9, 123.8, 122.7, 118.3, 118.1, 69.2, 67.8, 58.6, 55.8, 54.7, 34.0, 20.5, 20.3, 19.9, 19.2, 18.8, 14.6; Melting point: 230.7-233.9 ℃; IR(neat): 2972, 2901, 1664, 1536, 1478, 1408, 1249 cm-1; HRMS: Calcd for C42H40N11O6S5+ [M+H]+ 954.1761, found 954.1783; [𝜶]𝑫𝟐𝟎: +49.3 (c=0.5, CH3OH). 130 1H NMR (400 MHz CDCl3, ppm)6: δ 9.13 (s, 2H), 8.57 (s, 1H), 8.43 (d, J = 9.6 Hz, 1H), 8.34 – 8.30 (m, 2H), 8.26 (s, 1H), 8.22 (s, 1H), 8.16 – 8.09 (m, 3H), 7.99 (d, J = 7.0 Hz, 1H), 7.69 (s, 1H), 6.41 (q, J = 6.9 Hz, 1H), 5.31 – 5.21 (m, 2H), 4.89 (dd, J = 8.0, 1.6 Hz, 1H), 4.72 – 4.65 (m, 1H), 4.42 (dd, J = 15.5, 6.6 Hz, 1H), 4.10 (s, 3H), 2.48 (td, J = 13.3, 6.5 Hz, 1H), 2.12 – 1.96 (m, 1H), 1.89 – 1.77 (m, 3H), 1.57 (d, J = 6.5 Hz, 3H), 1.21 (d, J = 6.4 Hz, 3H), 1.15 (d, J = 6.7 Hz, 3H), 0.97 (d, J = 6.6 Hz, 3H); 13C NMR (100 MHz, CDCl3, ppm): δ 170.3, 169.9, 168.7, 168.6, 166.6, 166.2, 165.3, 161.2, 160.9, 160.4, 157.1, 153.2, 151.3, 151.1, 150.3, 150.3, 149.5, 148.9, 141.0, 129.0, 128.1, 127.4, 124.8, 123.9, 123.7, 122.5, 118.2, 116.4, 68.9, 67.6, 63.7, 58.3, 55.7, 55.2, 54.7, 33.8, 19.8, 19.8, 19.2, 18.7, 14.5 ; Melting point: 238.8-242.5 ℃; IR(neat): 3368, 3119, 1667, 1603, 1539, 1482, 1404, 1330, 1219 cm-1; HRMS: Calcd for C42H41N12O7S5+ [M+H]+ 985.1819, found 985.1834; [𝜶]𝑫𝟐𝟎: +76.4 (c=0.25, DMSO). 1H NMR (400 MHz, CDCl3) δ 8.51 (s, 1H), 8.42 (d, J = 9.7 Hz, 1H), 8.36 (d, J = 8.1 Hz, 1H), 8.26 (s, 1H), 8.22 (s, 1H), 8.19 – 8.08 (m, 6H), 8.03 – 7.93 (m, 3H), 7.84 (s, 1H), 7.08 (t, J = 5.0 Hz, 1H), 6.41 (q, J = 7.0 Hz, 1H), 5.26 (t, J = 9.3 Hz, 2H), 4.87 (dd, J = 8.0, 1.8 Hz, 1H), 4.66 (d, J = 5.5 Hz, 1H), 4.49 – 4.32 (m, 2H), 4.22 (d, J = 4.5 Hz, 1H), 3.45 (d, J = 6.9 Hz, 1H), 3.07 (s, 1H), 2.47 (dt, J = 13.3, 6.6 Hz, 1H), 2.27 (s, 3H), 1.84 (d, J = 7.0 Hz, 3H), 1.60 – 1.49 (m, 6H), 6 Two exchangeable protons are invisible in this case 131 1.21 (d, J = 6.4 Hz, 3H), 1.15 (d, J = 6.7 Hz, 3H), 0.98 (d, J = 6.6 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ 202.3, 177.7, 170.2, 169.8, 168.6, 168.1, 166.8, 166.6, 166.2, 161.2, 160.9, 160.4, 155.7, 153.3, 151.5, 150.3, 150.2, 149.5, 148.9, 141.0, 138.8, 131.6, 130.0, 129.0, 128.1, 127.3, 126.5, 124.8, 123.8, 123.7, 122.5, 118.6, 68.8, 67.6, 58.3, 55.7, 54.7, 53.4, 49.3, 42.1, 40.0, 33.7, 29.7, 27.3, 19.8, 19.7, 19.1, 18.7, 14.5, 13.4 ; HRMS (FAB+) Calcd for C51H50N12O9S5+ [M+H]+ 1135.2500, found 1135.2510 ; HPLC: flow rate 1.0 mL / min, tR (5% -> 100 % acetonitrile / 95% -> 0% water (containing 0.1% TFA )) = 6.9 min, purity 99%. 1H NMR (400 MHz, MeOD) δ 9.24 (s, 1H), 9.04 (s, 1H), 8.83 (s, 1H), 8.56 (d, J = 9.6 Hz, 1H), 8.47 (d, J = 10.0 Hz, 1H), 8.41 – 8.19 (m, 4H), 8.17 – 8.02 (m, 2H), 7.85 (s, 1H), 6.91 – 6.76 (m, 1H), 6.68 – 6.51 (m, 1H), 5.26 – 5,17 (m, 2H), 4.77 (s, 1H), 4.57 (s, 1H), 4.24 – 4.09 (m,3H), 2.64 – 2.58 (m, 1H), 2.23 (s, 3H), 1.92 (d, J = 6.8 Hz, 3H), 1.86 (d, J = 7.1 Hz, 3H), 1.56 (d, J = 6.1 Hz, 3H), 1.18 – 1.14 (m, 6H), 0.98 (d, J = 6.5 Hz, 3H).; HRMS (FAB+) Calcd for C50H49N13O9S5+ [M+H]+ 1136.2453, found 1136.2458 ; HPLC: flow rate 1.0 mL / min, tR (5% -> 100 % acetonitrile / 95% -> 0% water (containing 0.1% TFA )) = 6.0 min, purity 99%. 132 To a solution of micrococcin P2 (23 mg, 0.020 mmol) in the mixture of THF / H2O (1 : 1 v/v 100 μL : 100 μL) was added LiOH · H2O (8 eq, 0.160 mmol, 6.7 mg), and the mixture was stirred at 50 oC for 16 hours. Upon completion, the mixture was quenched by the addition of 1M HCl (300 μL). The mixture was transferred to a separatory funnel, and organic compounds were extracted with 1 mL x 2 super solvent (4:1 CHCl3 : isopropyl alcohol). 153 is obtained as a white solid and purification is not necessary. Yield: 14.8 mg (74%). 1H NMR (400 MHz, DMSO-d6, ppm) δ 9.55 (s, 1H), 8.59 – 8.51 (s, 2H), 8.50 – 8.35 (m, 3H), 8.35 – 8.27 (m, 2H), 8.28 – 8.17 (m, 2H), 8.13 (s, 1H), 7.88 (d, J = 7.6 Hz, 1H), 6.47 (q, J = 6.5 Hz, 1H), 5.20 – 4.96 (m, 2H), 4.73 – 4.63 (m, 1H), 4.42 – 4.33 (m, 1H), 4.04 – 3.96 (m, 1H), 1.76 (d, J = 6.3 Hz, 3H), 1.38 (d, J = 6.0 Hz, 3H), 1.00 (dd, J = 25.3, 6.1 Hz, 6H), 0.86 (d, J = 6.4 Hz, 3H) ; 13C NMR (100 MHz, DMSO-d6, ppm): δ 170.4, 170.3, 169.0, 168.6, 166.8, 164.5, 162.5, 162.3, 160.9, 160.2, 152.9, 151.5, 150.2, 150.0, 149.7, 149.5, 148.7, 148.7, 141.1, 130.0, 129.8, 129.1, 129.1, 126.5, 125.2, 124.8, 122.1, 122.0, 119.1, 68.0, 67.5, 58.1, 56.5, 55.9, 32.7, 21.1, 20.3, 20.1, 18.9, 14.2 ; Melting point: 258.9-261.6 ℃; IR(neat): 3384, 3121, 2931, 1663, 1539, 1485, 1377 cm-1; HRMS: Calcd for C42H41N12O7S5+ [M+H]+ 1004.1224, found 1004.1216 ; [𝜶]𝑫𝟐𝟎: +42.0 (c=0.25, MeOH). 133 To a solution of N-Boc-L-serine (100 mmol, 20.6 g) in THF (200 mL) was added 2,2-dimethoxypropane (1000 mmol, 123 mL, 10 equiv) and pyridinium p-toluenesulfonate (30 mmol, 7.50 g, 0.3 equiv) were added. The mixture was brought up to reflux and stirred for 18 hours. The mixture was cooled to room temperature, and concentrated in vacuo. The residue was partitioned between water (250 mL) and ethyl acetate (250 mL). The aqueous layer was further extracted with ethyl acetate (200 mL x 2). The combined organic layer was dried over Na2SO4, and concentrated in vacuo to afford the carboxylic acid 191 as a white solid. To a solution of the carboxylic acid 191 and triethylamine (110 mmol, 15.3 mL, 1.1 equiv) in THF (200mL) at 0 oC was carefully added ethyl chloroformate (111 mmol, 10.5 mL, 1.1 equiv) and the mixture was stirred for 1.5 hours. Then, aqueous ammonia (18M, 150 mmol, 8.33 mL, 1.5 equiv) was added and the mixture was allowed to warm to room temperature and stirred for 22 hours. The mixture was concentrated in vacuo, and the resulting aqueous suspension was extracted with ethyl acetate (150 mL x 3). The combined organic layer was washed with water (100 mL), brine (100 mL), dried over Na2SO4, and concentrated in vacuo to afford the desired 192 as a white solid. Yield : 20.0 g (98%) 1H NMR (400 MHz, CDCl3, ppm): δ 7.03 - 5.77 (m, 2H), 4.42 – 4.11 (m, 2H), 4.10 - 3.96 (m, 1H), 1.62 (s, 3H), 1.50 (s, 3H), 1.46 (s, 9H). 13C NMR (100 MHz, CDCl3, ppm): δ 176.7, 98.6, 83.20, 68.6, 67.2, 61.9, 29.1, 28.5. Due to restricted rotations, a greater number of 13C peaks is observed. 134 To a solution of 192 (50 mmol, 11.2 g) in DMF (50 mL) was carefully added cyanuric chloride (25 mmol, 4.60 g, 0.5 equiv) in portions. The mixture was stirred at room temperature for 30 minutes. Ice water (50 mL) was added and whereupon the solid was formed. The solids were filtered, and further washed with water (100 mL) and dried in vacuo to afford the desired 193 as a white solid. Yield : 10.5 g (94%) 1H NMR (400 MHz, CDCl3, ppm): δ 4.80 – 4.43 (m, 1H), 4.28 – 4.21 (m, 1H), 4.17 – 3.94 (m, 1H), 1.65 (dd, J = 11.7, 9.1 Hz, 3H), 1.60 – 1.34 (m, 12H); 13C NMR (100 MHz, CDCl3, ppm): δ 151.7, 144.2, 122.1, 121.6, 117.7, 117.0, 107.2, 95.3, 81.0, 69.1, 59.5, 28.3, 26.6, 23.0. Due to restricted rotations, a greater number of 13C peaks is observed. To a solution of 193 (50.0 mmol, 11.3 g) in the mixture of isopropyl alcohol : 0.1 M pH 6 sodium phosphate buffer (3 : 2 v/v 150 mL : 100 mL) was added L-cysteine methyl ester hydrochloride (75 mmol, 12.9 g, 1.5 equiv). The mixture was degassed with N2 for a period of 10 minutes, and the mixture was stirred at 50 oC for 15 hours. The mixture was concentrated in vacuo to remove excess isopropyl alcohol, and the aqueous layer was extracted with ethyl acetate (100 mL x 2). The organic layer was washed with 1M HCl (50 mL), water (50 mL), dried over Na2SO4 and concentrated in vacuo to afford the thiazoline 194 as a brown solid. The crude thiazoline 194 (46.0 135 mmol, 15.8 g) was dissolved in CH2Cl2 (150 mL) at 0 oC and was treated with BrCCl3 (69.0 mmol, 6.77 mL, 1.5 equiv). DBU (69.0 mmol, 10.3 mL, 1.5 equiv) was carefully added dropwise. The mixture was then brought up to reflux and stirred for 4 hours. The mixture was quenched by the addition of 1M HCl (80 mL) and aqueous layer was extracted with CH2Cl2 (80 mL x 2). The combined organic layer was washed with water (80 mL), brine (80 mL), dried over Na2SO4, and concentrated in vacuo. MPLC (gradient 10 → 30% ethyl acetate : 90 → 70% hexane) of the residue afforded the desired 195 as a yellow solid. [Rf – 0.60 (20% ethyl acetate : 80% hexane)]. Yield : 13.8 g (80% over 2 steps), 1H NMR (400 MHz, CDCl3, ppm): δ 8.14 (s, 1H), 5.58 – 5.09 (m, 1H), 4.36 – 4.07 (m, 2H), 3.96 (s, 3H), 1.78 (d, J = 26.4 Hz, 3H), 1.56 (d, J = 26.0 Hz, 6H), 1.36 – 1.01 (m, 6H); 13C NMR (100 MHz, CDCl3, ppm): δ 175.5, 174.6, 161.8, 152.4, 151.5, 146.8, 127.3, 95.3, 94.8, 81.5, 81.0, 69.3, 69.0, 60.4, 59.5, 52.4, 28.2, 27.4, 26.6, 24.0, 22.8. Due to restricted rotations, a greater number of 13C peaks is observed; Melting point: 112.4 - 114.4 oC; IR(neat): 1734 cm-1; HRMS: Calcd for C15H22N2O5S+H+ [M+H]+ 343.1322, found 343.1331; [𝛼]𝐷25: -42.8 (c= 1.0, CDCl3). A commercial solution of n-BuLi (2.10 M in hexanes, 20.0 mL, 42.0 mmol, 2.1 equiv) was added over a period of 10 minutes to a cold (–78 °C) solution of acetonitrile (43.0 mmol, 2.15 equiv 2.23 mL) in THF (30 mL), the mixture was stirred at -78oC for 20 minutes, then a solution of 195 (20 mmol, 6.85 g) in THF (30 mL) was slowly added over a period of 10 minutes. The mixture was slowly brought up to room temperature over 1 hour, then it was carefully quenched with 1M HCl 136 (50 mL). The mixture was diluted with ethyl acetate (100 mL), transferred to a separatory funnel and further acidified with 1M HCl to pH 3. Aqueous layer was further extracted with ethyl acetate (100 mL). The organic layer was collected, dried over Na2SO4 and concentrated in vacuo. MPLC (gradient 10 → 30% ethyl acetate : 90 → 70% hexane) of the residue afforded the desired 196 as a faint yellow oil. Yield: 4.21g (60%) [Rf – 0.40 (30% ethyl acetate : 70% hexane)]. 1H NMR (400 MHz, CDCl3, ppm) δ 8.24 (d, J = 11.4 Hz, 1H), 5.34 – 5.09 (m, 1H), 4.30 (dt, J = 14.0, 7.0 Hz, 1H), 4.27 – 4.03 (m, 3H), 1.72 (d, J = 18.9 Hz, 3H), 1.63 – 1.42 (m, 7H), 1.33 (s, 5H); 13C NMR (100 MHz, CDCl3, ppm) δ 182.3, 175.1, 174.3, 152.4, 151.6, 151.3, 127.6, 127.5, 114.0, 95.4, 94.9, 81.7, 81.1, 68.9, 68.4, 59.2, 30.7, 30.5, 28.3, 27.2, 26.5, 24.1, 22.9. Due to restricted rotations, a greater number of 13C peaks is observed; IR(neat): 2980, 2257, 1693 (cm-1); HRMS: Calcd for C16H21N3O4S+H+ [M+H]+ 352.1326, found 352.1329; [𝛼]𝐷25: -53.0 (c=1.0, CDCl3). To a solution of 196 (10.0 mmol, 3.51 g) in the mixture of toluene / acetic acid (5 :1 v/v 40 mL : 8 mL) was added ammonium acetate (100 mmol, 7.71 g, 10 equiv). The mixture was brought up to reflux and stirred for 1 hour. The mixture was cooled down, diluted with ethyl acetate (50 mL) and carefully washed with sat. aq. NaHCO3 (30 mL). The organic layer was collected, dried over Na2SO4 and concentrated in vacuo to afford the desired enamine 197 as a yellow oil. Then, the resulting enamine 197 is used for a subsequent reaction without further purification. To a solution of enamine 197 in ethanol (100 mL) was added 111 (18.5 mmol, 3.99 g, 1.85 equiv) and the mixture 137 was stirred at 60 oC for 1.5 hours. At this time, starting material is consumed and Michael adduct is clearly seen by TLC and LC/MS system. Then, acetic acid (30 % volume, 30 mL) was added and the mixture was brought up to reflux and stirred 16 hours to mediate the cyclization and aromatization. The excess ethanol and acetic acid was removed in vacuo. The crude was redissolved in CH2Cl2 (100 mL), and carefully quenched with sat. aq. NaHCO3 (50 mL). The organic layer was collected and dried over Na2SO4, and concentrated in vacuo. MPLC (5 → 15% ethyl acetate : 95 → 85% hexane) of the residue afforded the desired 198 as a yellow solid. [Rf – 0.45 (20% ethyl acetate : 80% hexane)]. Yield : 2.08 g (38% over 3 steps); 1H NMR (400 MHz, CDCl3,ppm,) δ 8.28 (s, 1H), 8.20 (s, 2H), 7.49 (s, 1H), 5.48 – 5.26 (m, 1H), 4.61 – 4.28 (m, 2H), 1.86 – 1.64 (m, 3H), 1.61 – 1.02 (m, 6H), 1.00 – 0.89 (m, 6H) 13C NMR (100 MHz, CDCl3, ppm) δ 171.7, 171.3, 171.1, 151.4, 151.2, 150.4, 117.9, 95.4, 95.0, 94.8, 94.4, 82.2, 82.1, 80.9, 80.3, 66.3, 66.2, 66.0, 60.4, 59.3, 59.2, 52.4, 52.3, 47.5, 47.4, 28.4, 28.3, 28.3, 28.1, 27.3, 26.6, 26.0, 25.6, 25.2, 24.9, 24.8, 24.4, 24.1, 21.0, 14.2. Due to restricted rotations, a greater number of 13C peaks is observed; Melting point: 110.4 - 112.7 ℃; IR(neat): 2978, 2380, 2224, 1696 (cm-1); HRMS: Calcd for C22H22BrN5O3S2+H+ [M+H]+ 548.0420, found 548.0423; [𝛼]𝐷25: -65.4 (c=1.0, CDCl3) To a solution of 198 (5.00 mmol, 2.74 g) in the mixture of ethanol / pH 7 sodium phosphate buffer / water (6 : 2 : 1 v/v 24 mL : 8 mL : 4 mL) was added L-cysteine (35.0 mmol, 4.24 g, 7 138 equiv), sodium bicarbonate (20.0 mmol, 1.68 g, 4 equiv) and the mixture was stirred at 100 oC overnight. Upon completion, excess solvent was removed in vacuo and mixture was diluted with ethyl acetate (50 mL). Organic layer was washed with 1M HCl (20 mL x 3) to get rid of excess cysteine, and dried over Na2SO4 to afford the crude desired thiazoline as a brown gum. The crude thiazoline (4.36 mmol, 2.84 g) was dissolved in CH2Cl2 (40 mL) and treated with BrCCl3 (17.4 mmol, 1.71 mL, 4.0 equiv). DBU (21.8 mmol, 3.25 mL, 5.0 equiv) was carefully added dropwise. The mixture was then brought up to reflux and stirred for 3 hours. The mixture was quenched by the addition of 1M HCl (30 mL) and aqueous layer was extracted with CH2Cl2 (60 mL). The combined organic layer was dried over Na2SO4, and concentrated in vacuo. The resulting crude 187 was clean enough for a subsequent reaction and was isolated as a dark brown solid. Yield : 2.54 g (78% over 2 steps); 1H NMR (400 MHz, CDCl3,ppm,) δ 9.37 (s, 1H), 8.58 – 8.09 (m, 3H), 7.90 (s, 1H), 7.42 (s, 1H), 5.24 (dd, J = 46.4, 5.2 Hz, 1H), 4.24 – 4.19 (m, 1H), 4.02 (dd, J = 18.4, 9.0 Hz, 1H), 1.75 (d, J = 20.1 Hz, 3H), 1.65 – 1. 49 (m, 7H), 1.46 – 1.16 (m, 5H); 13C NMR (100 MHz, CDCl3, ppm) δ 172.6, 171.6, 166.5, 163.9, 150.7, 150.3, 149.6, 148.6, 144.3, 138.4, 128.7, 128.5, 126.5, 124.7, 119.9, 119.6, 118.9, 116.8, 93.5, 93.0, 79.9, 79.3, 67.1, 66.6, 57.1, 26.4, 25.3, 24.6, 22.1, 21.0. Due to restricted rotations, a greater number of 13C peaks is observed; Melting point: 161.2 - 163.9 ℃; IR(neat): 3100, 2977, 1690 (cm-1); HRMS: Calcd for C25H24BrN5O5S3+H+ [M+H]+ 650.0196, found 650.0203; [𝛼]𝐷25: -16.9 (c=1.0, CDCl3) 139 To a solution of L-Threonine methyl ester hydrochloride (124 mmol, 21.0g) in CH2Cl2 (500 mL) / DMF (10 mL) was added TBDPS-Cl (149 mmol, 38.2 mL, 1.2 equiv) and imidazole (372 mmol, 25.2 g, 3 equiv) in small portions over 5 minutes. The mixture was stirred at room temperature overnight. The mixture was quenched by the addition of water (200 mL), and further extracted with CH2Cl2 (200 mL x 2). The crude mixture was purified by MPLC (30% → 60% ethyl acetate : 70% → 40% hexane) to afford the desired product as a colorless oil. Then, TBDPS protected alcohol (74.4 mmol, 27.6 g) in THF (300 mL) at 0 ℃ was added iPr2NEt (223 mmol, 38.9 mL, 3 equiv) and allyl chloroformate (89.2 mmol, 9.48 mL, 1.2 equiv) was added dropwise over a period of 10 minutes. The mixture was slowly brought up to room temperature for 2 hours. The mixture was quenched by the addition of sat. aq. NaHCO3 (200 mL), and was extracted with ethyl acetate (200 mL x 2). The crude mixture was purified by MPLC (5% → 20% ethyl acetate : 95% → 80% hexane) to afford the desired 199 as a colorless oil. [Rf – 0.43 (20% hexane : 80% ethyl acetate)]. Yield: 28.2 g (50% over 2 steps). 1H NMR (400 MHz, CDCl3, ppm): δ 7.83 – 7.54 (m, 4H), 7.54 – 7.32 (m, 6H), 6.00 (ddd, J = 22.8, 10.8, 5.6 Hz, 1H), 5.57 (d, J = 9.7 Hz, 1H), 5.39 (dd, J = 17.2, 1.4 Hz, 1H), 5.29 (dd, J = 10.4, 1.2 Hz, 1H), 4.65 (d, J = 5.6 Hz, 2H), 4.48 (qd, J = 6.3, 1.7 Hz, 1H), 4.27 (dd, J = 9.8, 1.8 Hz, 1H), 3.65 (s, 3H), 1.08 (d, J = 6.2 Hz, 3H), 1.03 (s, 9H); 13C NMR (100 MHz, CDCl3, ppm): δ 173.1, 158.5, 137.7, 135.6, 134.7, 134.6, 131.8, 131.7, 129.6, 129.4, 119.8, 72.0, 67.9, 61.7, 54.1, 28.7, 22.5, 21.1; IR (film): 2954, 2103, 1726 (cm-1); HRMS: Calcd for C25H33NO5Si+H+ [M+H]+ 456.2201, found 456.2210. [𝜶]𝑫𝟐𝟓 = –6.9 (c = 1.0, CDCl3) 140 199 (9.11 g, 20.0 mmol) was dissolved in the mixture of THF (50 mL) / H2O (50 mL) and LiOH · H2O (60.0 mmol, 2.52 g, 3.0 equiv) was added and stirred at room temperature for 1.5 hours before it was acidified with 1M HCl (100 mL). The compound was then extracted with ethyl acetate (3 x 150 mL), dried over Na2SO4, and concentrated in vacuo to afford the crude carboxylic acid. Then, to a solution of the crude carboxylic acid (19.1 mmol, 8.43 g) in CH2Cl2 (150 mL) was added amine 200 (22.9 mmol, 8.64 g, 1.2 equiv), iPr2NEt (57.3 mmol, 10.0 mL, 3 equiv), EDCI (22.9 mmol, 4.38 g, 1.2 equiv) and HOBt (22.9 mmol, 3.09 g, 1.2 equiv). The mixture was stirred overnight at room temperature before being diluted with CH2Cl2 (200 mL). The organic layer was washed with water (150 mL), dried over Na2SO4, and concentrated in vacuo. The crude mixture was purified by MPLC (5% → 20% ethyl acetate : 95% → 80% hexane) to afford 201 as white solid. [Rf – 0.46 (25% hexane : 75% ethyl acetate)]. Yield: 12.4 g (78% over 2 steps). 1H NMR (400 MHz, CDCl3, ppm): δ 7.98 – 7.54 (m, 4H), 7.46 – 7.27 (m, 12H), 7.26 – 7.20 (m, 9H), 6.07 – 5.81 (m, 1H), 5.72 (d, J = 6.0 Hz, 1H), 5.32 (d, J = 17.2 Hz, 1H), 5.22 (d, J = 10.3 Hz, 1H), 4.69 – 4.47 (m, 2H), 4.43 (broad peak, 1H), 4.33 (broad peak, 1H), 3.72 (s, 3H), 2.74 – 2.61 (m, 1H), 2.58 – 2.48 (m, 1H), 1.10 (s, 9H), 0.98 (d, J = 6.3 Hz, 3H); 13C NMR (100 MHz, CDCl3, ppm): δ 172.3, 171.1, 157.6, 146.1, 137.8, 137.7, 134.5, 131.9, 131.7, 131.4, 129.9, 129.7, 129.6, 128.7, 119.7, 71.8, 68.8, 67.7, 60.8, 54.4, 53.4, 35.6, 29.0, 21.1, 19.2; Melting point: 65.1 - 67.9 ℃; IR (film): 2931, 2123, 1720, 1677 (cm-1); HRMS: Calcd for C47H52N2O6SSiNa+ [M+Na]+ 823.3213, found 823.3210 [𝜶]𝑫𝟐𝟎 = –6.9 (c = 1.0, CDCl3) 141 To a solution of triphenylphosphine oxide (30 mmol, 8.38 g, 3 equiv) in CH2Cl2 (100 mL) at 0 ℃ was carefully added Tf2O (15 mmol, 2.52 mL, 1.5 equiv) dropwise and stirred at this temperature for 10 minutes. Then, the solution of 201 (10 mmol, 8.00 g) in CH2Cl2 (100 mL) was added dropwise at 0 ℃ and brought up to room temperature, and stirred for 1 hour. The mixture was quenched by the slow addition of sat. aq. NaHCO3 (300 mL) and the aqueous layer was extracted with CH2Cl2 (200 mL). The combined organic layer was dried over Na2SO4, and concentrated in vacuo. The crude mixture was purified by MPLC (15% → 30% ethyl acetate : 85% → 70% hexane) to afford 188 as a colorless gum. [Rf – 0.38 (35% hexane : 65% ethyl acetate)]. Yield: 4.32 g (80%). 1H NMR (400 MHz, CDCl3, ppm): δ 7.92 – 7.56 (m, 4H), 7.56 – 7.29 (m, 6H), 6.00 (ddd, J = 22.6, 10.8, 5.6 Hz, 1H), 5.81 (d, J = 9.1 Hz, 1H), 5.39 (dd, J = 17.2, 1.1 Hz, 1H), 5.28 (d, J = 10.5 Hz, 1H), 4.96 – 4.76 (m, 1H), 4.72 – 4.62 (m, 2H), 4.58 (d, J = 7.1 Hz, 2H), 3.81 (s, 3H), 3.72 – 3.52 (m, 1H), 3.48 – 3.39 (m, 1H), 1.18 – 0.87 (m, 12H); 13C NMR (100 MHz, CDCl3, ppm): δ 176.3, 171.1, 156.1, 136.0, 135.9, 133.7, 133.1, 132.7, 129.8, 127.9, 127.6, 127.6, 117.9, 78.1, 71.2, 66.0, 59.5, 52.7, 35.0, 27.0, 20.1, 19.3.; IR (film): 2857, 2118, 1725, 1617 (cm-1); HRMS: Calcd for C28H36N2O5SSiH+ [M+H]+ 541.2187, found 541.2194; [𝜶]𝑫𝟐𝟓 = +12.9 (c = 1.0, CDCl3) 142 To a solution of 173 in (4.42 mmol, 1.88 g) in CH2Cl2 (30 mL) at 0 ℃ was added 188 (5.30 mmol, 2.86g, 1.2 equiv), EDCI (5.50 mmol, 1.05g, 1.25 equiv) and HOBt (5.50 mmol, 747 mg, 1.25 equiv). The mixture was stirred at 0 ℃ for 1 hour, and brought up to room temperature for 1 hour. Then, Pd(PPh3)4 (0.053 mmol, 620 mg, 0.12 equiv) and DABCO (22.1 mmol, 2.48 g, 5.0 equiv) was added to the mixture and the mixture was stirred at room temperature for 2 hours. The mixture was quenched by sat. aq. NaHCO3 (30 mL) and the aqueous layer was extracted with CH2Cl2 (50 mL). The combined organic layer was dried over Na2SO4, and concentrated in vacuo. The crude mixture was purified by MPLC (30% → 70% ethyl acetate : 70% → 30% hexane) to afford 202 as a light greenish solid. [Rf – 0.57 (35% hexane : 65% ethyl acetate)]. Yield: 2.22 g (58%). 1H NMR (400 MHz, CDCl3, ppm): ) δ 8.44 (s, 1H), 8.07 (s, 1H), 7.77 – 7.57 (m, 4H), 7.52 – 7.34 (m, 6H), 6.84 (broad peak, 1H), 4.97 (d, J = 9.1 Hz, 1H), 4.88 (t, J = 9.2 Hz, 1H), 4.79 – 4.67 (m, 1H), 4.47 (s, 1H), 4.03 (d, J = 6.8 Hz, 1H), 3.81 (s, 3H), 3.66 – 3.48 (m, 2H), 3.43 (t, J = 10.5 Hz, 1H), 1.84 (d, J = 7.2 Hz, 3H), 1.68 (s, 6H), 1.50 (s, 9H), 1.49 (s, 3H), 1.13 (s, 9H), 1.05 (d, J = 6.2 Hz, 3H) ; 13C NMR (100 MHz, CDCl3, ppm): δ 171.1, 168.6, 167.0, 161.0, 150.0, 136.0, 136.0, 135.2, 134.8, 133.7, 133.1, 129.8, 129.8, 129.6, 127.7, 127.6, 127.3, 123.3, 95.2, 81.5, 81.3, 78.3, 57.5, 52.7, 35.1, 28.4, 27.0, 26.6, 26.0, 20.5, 19.4, 19.0, 14.2.; Melting point: 112.4 - 115.3 ℃; IR (film): 2931, 2123, 1741, 1685 (cm-1); HRMS: Calcd for C43H57N5O8S2Si+H+ [M+H]+ 864.3491, found 864.3402 [𝜶]𝑫𝟐𝟓 = -39.5 (c = 1.0, CDCl3) 143 To a solution of 202 (1.0 mmol, 865 mg) in CH2Cl2 (5 mL) was added TFA (5 mL) and the mixture was stirred at room temperature for 30 minutes. Excess solvent was removed in vacuo. The crude mixture was purified by MPLC (0.5% → 2.5% MeOH : 99.5% → 97.5% CH2Cl2) to afford 203 as a white solid. Yield: 260 mg (36%) [Rf – 0.30 (95% CH2Cl2 : MeOH)]. 1H NMR (400 MHz, CDCl3, ppm): δ 9.22 (s, 1H), 8.44 (d, J = 9.1 Hz, 1H), 8.03 (s, 1H), 7.77 – 7.68 (m, 4H), 7.50 – 7.34 (m, 6H), 6.65 (q, J = 7.1 Hz, 1H), 5.07 (dt, J = 9.1, 2.1 Hz, 1H), 5.02 – 4.85 (m, 1H), 4.64 (qd, J = 6.2, 2.2 Hz, 1H), 4.37 (qd, J = 6.4, 3.1 Hz, 1H), 3.82 (s, 3H), 3.61 – 3.54 (m, 1H), 3.53 – 3.40 (m, 2H), 1.84 (d, J = 7.2 Hz, 3H), 1.26 (d, J = 6.4 Hz, 3H), 1.12 (s, 9H), 1.05 (d, J = 6.3 Hz, 3H); 13C NMR (100 MHz, CDCl3, ppm): δ 175.8, 172.7, 171.2, 166.9, 160.8, 149.6, 136.0, 135.9, 133.9, 133.2, 129.8, 129.7, 128.3, 127.6, 127.6, 126.4, 123.1, 71.2, 68.2, 59.8, 57.4, 52.7, 35.3, 27.0, 26.9, 19.8, 19.4, 19.1, 14.3; IR (film): 2932, 1742, 1675 (cm-1); HRMS: Calcd for C35H46N5O8S2Si+H+ [M+H]+ 724.2653, found 724.2663 [𝜶]𝑫𝟐𝟓 = +4.4 (c = 0.5, CDCl3) 144 To a solution of 187 (0.510 mmol, 333 mg) in acetonitrile (3 mL) was added 203 (0.510 mmol, 366 mg) and N-methylimidazole (1.53 mmol, 0.122 mL, 3 equiv). Then, TCFH (0.663 mmol, 186 mg, 1.3 equiv) was added and the mixture was stirred at room temperature for 30 minutes. Water (2 mL) was added to quench the reaction, and aqueous layer was extracted with CH2Cl2 (10 mL x 2). The organic layer was collected, dried over Na2SO4 and concentrated in vacuo. The crude was subjected to MPLC (1.5% → 2.3% MeOH : 98.5% → 97.7% CH2Cl2) of the residue afforded the desired 204 as a yellow solid. Yield : 356 mg (52%) [Rf – 0.30 (20% hexane : 80% ethyl acetate)]. 1H NMR (400 MHz, CDCl3, ppm): δ 8.86 – 8.64 (m, 1H), 8.51 – 8.39 (m, 2H), 8.37 – 8.25 (m, 1H), 8.23 – 8.21 (m, 1H), 8.05 (s, 1H), 8.02 – 7.94 (m, 1H), 7.73 – 7.69 (m, 4H), 7.46 – 7.35 (m, 7H), 6.81 – 6.44 (m, 1H ), 5.17 – 5.06 (m, 2H), 5.04 – 4.95 (m, 1H), 4.88 – 4.71 (m, 1H), 4.68 – 4.50 (m, 2H), 4.21 – 4.11 (m, 1H), 4.06 – 3.92 (m, 1H), 3.85 (d, J = 5.4 Hz, 3H), 3.69 – 3.57 (m, 1H), 3.57 – 3.43 (m, 1H), 1.92 – 1.80 (m, 4H), 1.78 – 1.68 (m, 2H), 1.59 – 1.43 (m, 5H), 1.41 – 1.39 (m, 4H), 1.35 - 1.23 (m, 6H), 1.21 (s, 9H), 1.02 (d, J = 6.2 Hz, 3H); 13C NMR (100 MHz, CDCl3, ppm): δ175.8, 175.5, 171.2, 171.0, 169.0, 168.8, 165.1, 160.5, 150.3, 149.3, 149.2, 141.3, 140.0, 139.9, 136.0, 135.9, 133.9, 133.1, 131.5, 129.8, 129.7, 128.9, 128.4, 128.3, 128.0, 127.7, 127.6, 126.6, 125.8, 123.1, 121.1, 120.6, 118.7, 95.3, 92.4, 81.0, 71.1, 69.1, 66.7, 66.4, 60.4, 59.2, 57.8, 57.3, 53.1, 52.9, 35.2, 28.4, 26.9, 26.8, 26.6, 22.9, 21.1, 19.6, 19.4, 19.4, 18.6, 14.7, 14.5, 145 14.2.; HRMS: Calcd for C60H67BrN10O10S5Si+H+ [M+H]+ 1355.2671, found 1355.2670 [𝜶]𝑫𝟐𝟓 = +31.0 (c = 1.0, CDCl3) 204 (0.265 mmol, 356 mg) was directly treated with 4M HCl in dioxane (4 mmol, 1 mL, 15 equiv). The mixture was stirred at room temperature for 30 minutes. Excess solvent was removed in vacuo. It was then redissolved in CH2Cl2 (10 mL) and washed with sat. aq. NaHCO3 (5 mL). The combined organic layer was dried over Na2SO4 and concentrated in vacuo. The crude material was subjected to MPLC (2% → 5% MeOH : 98% → 95% CH2Cl2) to afford 205 as a white solid. Yield : 154 mg (48%) [Rf – 0.30 (3% MeOH : 97% CH2Cl2)]. To a solution of 205 in (0.127 mmol, 154 mg) in DCE (1.5 mL) was added Me3SnOH (0.343 mmol, 61.7 mg, 2.7 equiv) and the mixture was stirred at 80oC for 1.5 hrs. Excess DCE is removed in vacuo, and redissolved in ethyl acetate (10 mL). The organic layer was washed with 1M HCl (3 mL x 5), dried over Na2SO4 and concentrated in vacuo. The crude carboxylic acid obtained was dissolved in DMF (4 mL) and was treated with NaHCO3 (1.27 mmol, 107 mg, 10 equiv) and DPPA (0.228 mmol, 0.049 mL, 1.8 equiv). The mixture was stirred at room temperature for 15 hours. Upon completion, the mixture was diluted with ethyl acetate (10 mL) and washed with water (4 mL x 5). The crude material was subjected to MPLC (2% → 5% MeOH : 98% → 95% CH2Cl2) to 146 afford 206 as a white solid [Rf – 0.35 (4% MeOH : 97% CH2Cl2)]. Yield : 22.0 mg (7% over 3 steps). 1H NMR (400 MHz, CDCl3) δ 8.77 (s, 1H), 8.30 (s, 1H), 8.23 (d, J = 8.1 Hz, 1H), 8.05 – 7.88 (m, 4H), 7.76 (d, J = 6.8 Hz, 2H), 7.70 (d, J = 6.9 Hz, 2H), 7.56 (dd, J = 19.6, 6.7 Hz, 1H), 7.48 – 7.31 (m, 8H), 6.48 (q, J = 6.9 Hz, 1H), 5.16 (broad peak, 1H), 5.10 (d, J = 7.9 Hz, 1H), 5.05 – 4.90 (m, 1H), 4.76 (d, J = 6.8 Hz, 1H), 4.58 – 4.43 (m, 2H), 3.96 (broad peak, 1H), 3.89 – 3.73 (m, 1H), 3.64 (dq, J = 16.0, 11.2 Hz, 3H), 3.43 (s, 1H), 1.80 (d, J = 7.0 Hz, 3H), 1.34 – 1.21 (m, 3H), 1.15 (d, J = 6.1 Hz, 3H), 1.08 (s, 9H). LCMS: [M+H]+ 1185.3 To a solution of 206 (18.6 μmol, 22.0 mg) in dioxane (250 μL) was added B2Pin2 (24.2 μmol, 6.1 mg, 1.3 equiv), potassium acetate (27.9 μmol, 2.7 mg, 1.5 equiv), Xphos (2.8 μmol, 1.3 mg, 0.15 equiv) and purged with N2 for few minutes. Then, Pd2(dba)3 (1.90 μmol, 1.8 mg, 0.11 equiv) was added and the mixture was stirred at 90 ℃ for 1.5 hours. The mixture was cooled to room temperature, diluted with CH2Cl2 (1 mL) and washed with H2O (0.5 mL). The organic layer was dried over Na2SO4 and concentrated in vacuo. The resulting crude boronic acid is immediately treated with compound 189 (18.6 μmol, 5.4 mg), K2CO3 (55.8 μmol, 7.7 mg, 3 equiv) in THF 147 (200 μL) / H2O (50 μL) and purged with N2 for few minutes. Then, Pd(dtbpf)Cl2 (1.90 μmol, 1.2 mg, 0.11 equiv) was added and the mixture was brought up to 60 ℃ and stirred for 12 hours. It was cooled to room temperature and diluted with CH2Cl2 (2 mL). The organic layer was washed with 1M HCl (0.5 mL), dried over Na2SO4, and concentrated in vacuo. The crude was subjected to MPLC (2% → 5% MeOH : 98% → 95% CH2Cl2) to afford the desired 208 as a white solid. [Rf – 0.35 (3% MeOH : 97% CH2Cl2)]. Yield: 4.9 mg (20%); 1H NMR (400 MHz, CDCl3) δ 8.65 (s, 1H), 8.39 – 8.33 (m, 2H), 8.32 – 8.25 (m, 1H), 8.25 – 8.16 (m, 2H), 8.16 – 8.06 (m, 1H), 8.04 – 7.94 (m, 2H), 7.91 (d, J = 7.9 Hz, 1H), 7.81 – 7.74 (m, 2H), 7.71 (d, J = 6.7 Hz, 2H), 7.52 – 7.33 (m, 8H), 6.52 (q, J = 6.9 Hz, 1H), 5.18 – 5.08 (m, 2H), 5.02 – 4.96 (m, 1H), 4.86 (dt, J = 14.8, 7.3 Hz, 1H), 4.77 – 4.69 (m, 1H), 4.54 (d, J = 23.3 Hz, 1H), 3.84 (s, 3H), 3.80 – 3.73 (m, 2H), 3.73 – 3.67 (m, 1H), 3.67 – 3.62 (m, 2H), 3.62 – 3.56 (m, 2H), 1.83 (d, J = 7.0 Hz, 3H), 1.31 (d, J = 6.5 Hz, 3H), 1.26 (t, J = 8.3 Hz, 3H), 1.16 (d, J = 6.2 Hz, 3H), 1.09 (s, 9H). LCMS: [M+H]+ 1317.8 148 C. 1H and 13C NMR Spectra, HPLC traces and LC/MS 1H-NMR (600 MHz, CDCl3) of 120 13C-NMR (151 MHz, CDCl3) of 120 149 1H-NMR (600 MHz, CDCl3) of 132 13C-NMR (151 MHz, CDCl3) of 132 150 1H-NMR (600 MHz, CDCl3) of 121 13C-NMR (151 MHz, CDCl3) of 121 151 1H-NMR of 122 (600 MHz, CDCl3, Measured at 50 oC) 13C-NMR of 122 (151 MHz, CDCl3, Measured at 50 oC) 152 1H-NMR (600 MHz, CDCl3) of 111 13C-NMR (151 MHz, CDCl3) of 111 153 1H-NMR (600 MHz, CDCl3, Measured at 50 oC) of 110 13C-NMR (151 MHz, CDCl3, Measured at 50 oC) of 110 154 1H-NMR (600 MHz, CDCl3, Measured at 50 oC) of 135 13C-NMR (151 MHz, CDCl3, Measured at 50 oC) of 135 155 1H-NMR (600 MHz, CDCl3, Measured at 50 oC) of 109 13C-NMR (151 MHz, CDCl3, Measured at 50 oC) of 109 156 1H-NMR (600 MHz, CDCl3) of 138 13C-NMR (100 MHz, CDCl3) of 138 157 1H-NMR (400 MHz, CDCl3) of 103 13C-NMR (151 MHz, CDCl3) of 103 158 1H-NMR (600 MHz, CDCl3) of 144 13C-NMR (151 MHz, CDCl3) of 144 159 1H-NMR (600 MHz, CDCl3) of 145 13C-NMR (151 MHz, CDCl3) of 145 160 1H-NMR (600 MHz, CDCl3) of 146 13C-NMR (151 MHz, CDCl3) of 146 161 1H-NMR (600 MHz, CD3OD) of 147 13C-NMR (151 MHz, CD3OD) of 147 162 1H-NMR (600 MHz, CD3OD) of 148 13C-NMR (151 MHz, CD3OD) of 148 163 1H-NMR (600 MHz, CD3OD) of Micrococcin P2 (3) 13C-NMR (151 MHz, CD3OD) of Micrococcin P2 (3) 164 1H-NMR (600 MHz, DMSO-d6) of Micrococcin P1 (4) 165 1H-NMR (600 MHz, CDCl3) of 140a 13C-NMR (151 MHz, CDCl3) of 140a 166 1H-NMR (600 MHz, DMSO-d6) of 140b 13C-NMR (151 MHz, DMSO-d6) of 140b 167 1H-NMR (600 MHz, CDCl3) of 140c 13C-NMR (151 MHz, CDCl3) of 140c 168 1H-NMR (600 MHz, CDCl3) of 140d 13C-NMR (151 MHz, CDCl3) of 140d 169 1H-NMR (600 MHz, CDCl3) of 140e 13C-NMR (151 MHz, CDCl3) of 140e 170 1H-NMR (600 MHz, CDCl3) of 140f 13C-NMR (151 MHz, CDCl3) of 140f 171 1H-NMR (400 MHz, CDCl3) of 140g 13C-NMR (151 MHz, CDCl3) of 140g 172 1H-NMR (400 MHz, CDCl3) of 140h 13C-NMR (100 MHz, CDCl3) of 140h 173 1H-NMR (400 MHz, CDCl3) of 150 13C-NMR (100 MHz, CDCl3) of 150 174 HPLC Traces of 150 175 1H-NMR (400 MHz, MeOD) of 151 HPLC Traces of 151 176 1H-NMR (400 MHz, DMSO-d6) of 153 13C-NMR (400 MHz, DMSO-d6) of 153 177 1H-NMR (400 MHz, CDCl3) of 192 13C-NMR (100 MHz, CDCl3) of 192 178 1H-NMR (400 MHz, CDCl3) of 193 13C-NMR (100 MHz, CDCl3) of 193 179 1H-NMR (400 MHz, CDCl3) of 195 13C-NMR (100 MHz, CDCl3) of 195 180 1H-NMR (400 MHz, CDCl3) of 196 13C-NMR (100 MHz, CDCl3) of 196 181 1H-NMR (400 MHz, CDCl3) of 198 13C-NMR (100 MHz, CDCl3) of 198 182 1H-NMR (400 MHz, CDCl3) of 187 13C-NMR (100 MHz, CDCl3) of 187 183 1H-NMR (400 MHz, CDCl3) of 199 13C-NMR (100 MHz, CDCl3) of 199 184 1H-NMR (400 MHz, CDCl3) of 201 13C-NMR (100 MHz, CDCl3) of 201 185 1H-NMR (400 MHz, CDCl3) of 188 13C-NMR (100 MHz, CDCl3) of 188 186 1H-NMR (400 MHz, CDCl3) of 202 13C-NMR (100 MHz, CDCl3) of 202 187 1H-NMR (400 MHz, CDCl3) of 203 13C-NMR (100 MHz, CDCl3) of 203 188 1H-NMR (400 MHz, CDCl3) of 204 13C-NMR (100 MHz, CDCl3) of 204 189 1H-NMR (400 MHz, CDCl3) of 206 LC-MS Trace and Mass Spectrum of 206 190 1H-NMR (400 MHz, CDCl3) of 208 LC-MS Trace and Mass Spectrum of 208 191 D. Experimental Protocol for Biological Evaluations Isolation of C. difficile and detection of toxin genes by multiplex PCR After alcohol shock treatment, stool specimens were cultivated on C. difficile moxalactam–norfloxacin–taurocholate agar (CDMN-TA agar; Oxoid Ltd., Cambridge, UK) supplemented with 7% horse blood. Colonies of C. difficile were identified by Rapid ID 32A (BioMerieux SA, Marcy I’Etoile, France). To identify toxin genes, multiplex PCR was performed using template DNA, as described previously.31 The positive controls were ATCC 43598 (PCR RT017), ATCC 9689 (PCR RT027), VPI 10643 (ATCC 43255, PCR RT087) and ATCC 700057, representing A–B+CDT–, A+B+CDT+, A+B+CDT–, and A–B–CDT– RTs, respectively. PCR ribotyping of C. difficile strains The PCR ribotyping was performed using genomic DNA, as described elsewhere. After electrophoresis of the amplified products, the clustering of banding patterns was checked visually. Each unique pattern was assigned its own RT code and was compared with the PCR RTs of the reference strains RT027 [ATCC 9689], RT017 [ATCC 43598] and standard strains from the European Centre for Disease Prevention and Control–Brazier collection. MLST (Multilocus Sequence Typing) Based on the sequences of seven conserved house-keeping gens adk, atpA, dxr, glyA, recA, sodA, and tpi. Isolates were assigned to STs and clades in the C. difficile MLST database (https://pubmlst.org/organisms/clostridioides-difficile). Clonal relationships among STs, based on allelic polymorphisms, were determined using the Molecular Evolutionary Genetics Analysis 192 software, version 7 (MEGA7). Cellular Toxicity Cytotoxicity was tested against the human cell lines SH-SH5Y, HEK293T using CCK8 cell viability assay. Each cell line was grown in Dulbecco’s Modified Eagles Medium (DMEM; Gibco) supplemented with 1 % penicillin/streptomycin and 10 % fetal bovine serum (Gibco) at humidified incubator containing 5 % CO2 at 37℃. For test, cells were plated at a density of 5000 cells/well in 100 µL of cell culture medium in a 96-well microplate. Micrococcin P2(MP2) were treated in triplicate with 1.25 μM to 40 μM to the cells for 48 h at 37℃. Finally, 10 μL of the CCK-8 reagent (Dojindo) was added into each well, and OD (Optical Density) at 450 nm was measured using a microplate reader (Biotek) after incubation for 2 h at 37℃. The cell viability was normalized to DMSO control. Rat Pharmacokinetic Studies Rat pharmacokinetic studies was conducted at DGMIF (Daegu-Gyeongbuk Medical Innovation Foundation). SD-rats (7 – 8-week-old; five males and females per group) were administrated either 10mg/kg orally or 2mg/kg via IV of 1 which was formulated with 2.5% DMSO, 47.5% Labrasol®, 30% PEG-400, and 20% deionized water. Blood samples were collected via jugular vein at pre-dose and post-dose at 5, 15, and 30 min, 1, 2, 4, 6, and 8 hours. Plasma was prepared and stored frozen at -80oC until bioanalysis. To each plasma sample of 20 μL was added 180 μL 193 of acetonitrile with the internal standard and was subsequently vortexed at 4℃ for 5 minutes followed by centrifugation at 15,000 rpm for 5 minutes. The resulting supernatant 1 was measured using liquid chromatography-tandem mass spectrometry (LC-MS)/MS (Triple Quad 5500 by Applied Biosystems, USA) assay with a lower limit of quantification (LLOQ) of 10ng/mL. Minimum Inhibitory Concentrations Minimal inhibitory concentrations (MICs) of the antimicrobial agents were determined by performing a two-fold agar dilution method as described by the Clinical and Laboratory Standards Institute (CLSI). Vancomycin was diluted in distilled water and MP2 was diluted in DMSO. Doubling dilutions of vancomycin and MP2 (0.06 ~ 8μg/mL) were made in Brucella agar supplemented with 5% laked sheep blood, 1μg/mL hemin and 5μg/mL vitamin K1. Test organisms were grown on Brain Heart Infusion broth (BHI, Difco™, USA) plates containing 5% defibrinated sheep blood and sub-cultured into Brain Heart Infusion broth (BHI, Drifco™, USA) with 0.5% yeast extract and incubated for 24 hours at 37℃ in an anaerobic chamber. The cultured bacteria were diluted using the same fresh medium to achieve a bacterial cell density of approximately 107 colony-forming units (CFU)/mL. All test organisms were seeded in a supplemented Brucella agar at a density of 105 CFU / spot. The plates were incubated at 35℃ for 48 hours and were examined for bacterial growth. 194 Time-Kill Assays The time-kill studies were performed by the method of the CLSI. Test organisms were incubated in BHI broth with 0.5% yeast extract for 18 hours at 37℃ in an anaerobic chamber were diluted with fresh broth to approximately 105 CFU/mL. Vancomycin and MP2 were added to the cultures of 1/2, 1, 2, and 4x the MIC. Samples were removed at 0, 3, 6, and 24-hour post-inoculation, and serial 10-fold dilutions were performed. The number of viable cells were determined on BHI agar containing 5% defibrinated sheep blood after 24 hours of incubation. The plates were incubated at 37℃ in an anaerobic chamber. Antimicrobials were considered bactericidal at the lowest concentration that reduced the original inoculum by 3 log 10 CFU / mL at each of the time periods and bacteriostatic if the inoculum was reduced by 0 to 3 log 10 CFU / mL. In vivo bacteria inoculation C. difficile ribotype 027 was grown on blood agar plate and incubated for two days at 37℃ in an anaerobic chamber and sub-cultured on 70:30 sporulation medium to facilitate the sporulation of C. difficile ribotype 027. Briefly, the sporulation medium consists of 70% SMC (Bacto peptone 90g, protease peptone 5g, NH4SO4 1g, tris base 1.5g, agar 15g per liter) and 30% BHIS (BHI agar with 0.5% yeast extract) agar.37 For inoculation, bacterial colonies were suspended in 0.9% saline solution and further diluted with saline solutions to adjust OD (Optical Density) 1.0 at 1/10 of original saline solutions. Groups of five male C57BL/6J mice were injected orally with a single 0.5 mL dose of the bacterial suspension. 195 Mouse IBD-C. difficile comorbidity infection model All animal experiments were conducted in accordance with the ethical guidelines of the Ethics Review Committee for Animal Experimentation at Handong Global University (Republic of Korea, protocol #HGU-20211214-20). C57BL/6J male (4 weeks old male weighing 20 to 22g, Daehan Bio Link Co., Ltd., Republic of Korea) 5 mouse per group was used in C. difficile infection model. Mice were maintained in animal chambers kept at 23℃ ± 2℃ with 55% ± 20% relative humidity. 2% dextran sodium sulfate (DSS, MP biomedical, USA) was inoculated by drinking water for 5 days to cause colitis in mice. A single dose of clindamycin (20mg/kg) was injected intraperitonially a day before C. difficile ribotype 027 challenge. 30mg/kg of each drug (vancomycin, fidaxomicin, micrococcin P2) were dissolved in a specified vehicle and 0.25 mL was inoculated after 6 hours of infection through oral gavage, and in every 24 hours for 5 days. Anti-Inflammation Assay Mouse monocyte-macrophage RAW 264.7 cells were maintained in RPMI-1640 medium supplemented with 10 % heat-inactivated FBS at 37℃ in a humidified incubator with 5 % CO2. To investigate the effect of Micrococcin P2 on cytokine levels from LPS-treated cells, RAW 264.7 cells (2 × 105 cells/mL) seeded into 24-well plates were treated with 5, 10, 20, 40 μg/mL of micrococcin P2 with 1 mg/L LPS. After 24h, the concentrations of IL-6 and IL-1β in the supernatants of RAW 264.7 cell cultures were determined using an ELISA kit, according to the manufacturer’s instructions (R&D system, BD). 196 Bacterial Inhibition activity against Mycobacterium tuberculosis M. tuberculosis stain H37Rv was obtained from ATCC (ATCC 27294). Clinical M. tuberculosis isolated strains was obtained from TB Specimen Biobank., Masan National Tuberculosis Hospital. All the isolates were identified as M. tuberculosis using Acid-Fast Bacilli (AFB) stain and MTB immunochromatographic detection kit (SD BIOSENSOR). The drug susceptibility of M. tuberculosis strains was determined using the resazurin microtiter assay (REMA) under aerobic conditions. The resazurin solution was prepared in 0.02 % (wt/vol) solution in sterile distilled water using resazurin sodium salt powder (Sigma-Aldrich) and filter sterilized. Bacteria from exponential-phase cultures were harvested and adjusted to an OD600 of 0.0001. in wells of a 96-well microtiter plate. Two-fold serial dilutions of compounds were prepared from 10 uM to 10 nM Micrococcin P2. Plates were then incubated at 37°C. After 7 to 12 days of incubation, 40 μL of 0.025% resazurin was added to the wells. Fluorescence was measured using a SpectraMax® M3 Multi-Mode Microplate Reader (Molecular Devices, Sunnyvale, CA). Concentrations required to inhibit bacterial growth by 50% (IC50) were determined by fitting the curves with a sigmoidal dose-response using GraphPad Prism software (version 6.05).
Thesis/Dissertation
eng
Chemistry
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Ciufolini, Marco A.
Chemical and biological studies on thiopeptide antibiotics : total synthesis of micrococcin P2 and synthetic efforts towards lactocillin
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