Combating the Rise of Antimicrobial Resistance: Permeation and Efflux Multiparameter Optimization and a Divergent Total Synthesis of Streptothricin F

Combating the Rise of Antimicrobial Resistance: Permeation and Efflux Multiparameter Optimization and A Divergent Total Synthesis of Streptothricin F

by Matthew G. Dowgiallo

B.S. in Chemistry, Le Moyne College

A dissertation submitted to

The Faculty of the College of Science of Northeastern University in partial fulfillment of the requirements for the degree of Doctor of Philosophy

July 31st, 2020

Dissertation directed by

Roman Manetsch Associate Professor of Chemistry and Chemical Biology & Pharmaceutical Sciences

Dedication

To my mother, Eileen Boron Haley, my stepfather Thomas Haley (June 19, 1942-May 26, 2020), my father Glenn Dowgiallo, my stepmother Chery Johnson Dowgiallo, my sister Meghan Dowgiallo, my nephew Grant Dowgiallo, my grandparents Walter and Toni Dowgiallo and my wonderful bride-to-be, Mallory Munro for your love and support. Thank you for bringing joy to my life and always providing me with a source of optimism.

Acknowledgements

I am extremely appreciative to my mentor and friend, Dr. Roman Manetsch for providing me with the opportunity to pursue my dream to synthesize a complex natural product. The tremendous guidance and trust I received from Roman throughout the years have provided me with the confidence to excel inside and outside of the laboratory setting. Roman has provided me with numerous opportunities to present and learn from prestigious symposiums in the Northeast and across the country, for which I am incredibly grateful. Thank you so much Roman for demonstrating great patience and wisdom in guiding me towards the scientist I have become today.

To my committee members, thank you for your unwavering support and critical supervision. Dr. Michael Pollastri, watching you take initiative and provide consistency as our department has changed over the past years has been inspirational. Your willingness to lead and dedication to research in the face of great adversity are lessons I plan to carry into my professional career. Dr. George O’Doherty, thank you for challenging me to not settle for complacency in my research efforts. I have often enjoyed your insightful criticism across the many meetings and talks at Northeastern and beyond, and you have taught me to be a more meticulous researcher. Dr. James

Kirby, thank you for believing in the long-ignored activity among the streptothricins and trusting our group with the awesome synthetic challenges accompanying the compound class.

To current and former members of the Roman Family, thank you for always listening to my roadblocks throughout synthetic endeavors as well as balancing all the joys of living in the lab together. To Dr. Cynthia Lichorowic and Dr. Iredia Iyamu, thank you for your careful instruction and persistence while I transitioned to my new home in the Manetsch Lab. Thank you to Dr. Fabian

Brockmeyer for demonstrating a relentless work ethic and entertaining me with your celebrations while watching Werder Bremen soccer. To Dr. Abdul Shaik, thank you for sharing invaluable

laboratory techniques and good lessons in relational dynamics. Thank you, Dr. Prakash Parvatkar, for bringing a fun tradition of singing among the group and cultivating a culture of appreciation for everyone. Thank you, Dr. Chungsik Kim, for encouraging me to have confidence in myself and pursue curiosity in my studies as well as setting an example towards building a wonderful, loving family. Alicia Wager, thank you for reminding me of the excitement and challenges that come with being a first-year graduate student. Thank you, Lili Huang for your kind conversations and the amusement that accompanies our mutual appreciation of cute dogs and cats. Ami Asakawa, thank you for taking the time to explain biology questions associated with my project as well as your precious connections to computer savvy friends and family. Brandon Miller, your enthusiasm and determination to achieve were essential to the vitality of the streptothricin project. I am so grateful for your leadership and willingness to take on responsibility both within the scope of the project and across countless lab duties. Thank you to Minte Kassu, your contributions to the streptothricin project towards investigating the synthesis of β-lysine were vital and completion of the total synthesis and would not have been possible without your strong work ethic. To my dear friend David Zhao, I am so appreciative to have studied alongside you throughout this process.

You have been a role model for me in holding yourself responsible for delivering the best quality of work in both your teaching and laboratory results.

Thank you to our collaborators with Beth Israel Deaconess Medical Center / Harvard

Medical School Dr. Kenneth P. Smith and Dr. Lucius Chiaraviglio for your patience while we pursued the synthesis and isolation of streptothricins as well as your rapid response when compounds were ready. A special thank you to Frank Fronczek from LSU for the crystal support throughout the course of synthesizing streptothricin F intermediates. To our collaborators Dr.

Jeffery N. Agar and Dr. Daniel P. Donnelly, thank you for giving me the opportunity to become a

part of the groundbreaking work from your lab. To Sue Abbatiello, thank you for your help in obtaining HRMS data while managing your myriad of projects.

To the NMR staff at Northeastern, I am incredibly appreciative for your mentorship and assistance. Brian D’Amico, thank you for enforcing the NMR scheduling policies and ensuring accessibility was fair to all users. Jason Guo, thank you for transforming the NMR facility into a safe, modern space for the university and especially for assisting with final data collection. To Dr.

Roger Kautz (July 24, 1958-May 27, 2017), thank you so much for your thought-provoking conversation and kindness while teaching me the secrets of Varian. I am so lucky to have studied within the nurturing, stimulating atmosphere you provided to rising graduate students.

To my peers, thank you for your guidance and friendship from the beginning. Dr. Andrew

Spaulding, you were the first face I met when coming to Northeastern for recruitment weekend, and I am so grateful that I got to learn from you during our time in the Aggen group. Thank you for providing me with advice to become the best version of myself both inside and outside of this world. Dr. Westley Tear, thank you for keeping me accountable for staying active, at least in the early days, as well as your camaraderie throughout the course of our research journey

I would like to thank undergraduate researchers Fabiola Caban Bravo, Loren Po, Andrew

Fetigan, Clarissa Santori and Gian-Marco Rossi for helping me explore areas of my projects with fresh, new perspectives and providing me with extra sets of hands.

To my teammates on the ultimate frisbee field both with the Huskies and Nerd Alert, thank you for providing me with a healthy distraction from my work. The peace of mind I have received from chasing plastic has undoubtedly contributed towards my well-being over the last 6 years. To my roommates Dave Reppucci, Allie Hung and Calvin Marie, thank you for a loving, relaxing and entertaining home life in Camberville.

Abstract of Dissertation

The emergence of antimicrobial resistance is a constant threat in the scientific community.

As multidrug resistant organisms approach pan-resistance, Carbapenem-resistant

Enterobacteriaceae (CRE), Acinetobacter baumannii, and Pseudomonas aeruginosa have been categorized as top antimicrobial resistance threats by the Center for Disease Control and

Prevention along with the World Health Organization. Unfortunately, the rate of antimicrobial drug discovery and development has drastically slowed since the 1950s with only three new classes of antimicrobials approved by the Food and Drug Administration in the past two decades. Our research efforts aim to combat the rise of antibiotic resistance through two unique strategies: enhancing the clinical utility of a safe and effective medicine towards extensively resistant organisms as well as tuning the selectivity of a highly potent antimicrobial through the development of a novel total synthesis designed to enable rapid analogue generation.

Chapter 2 of this dissertation explores the use of potentiators to strengthen the activity of meropenem, a commonly administered carbapenem antibiotic, against CRE. We performed a high- throughput screen (HTS) of possible adjunctives to identify a hit series exhibiting synergistic activity alongside meropenem. HTS downselection featured a cheminformatics approach where prioritization of compounds targeted physicochemical properties optimized for Gram-negative bacterial cell penetration and avoidance of extracellular efflux.

Chapter 3 of this dissertation describes a diversity-oriented total synthesis of streptothricin

F (ST-F), an aminoglycoside-like natural product with broad-spectrum antimicrobial activity.

Through a divergent total synthesis, ST-F was synthesized over 35 total steps and 0.0040% overall yield. We hope to derivatize ST-F to tune its selectivity and maintain or even improve its excellent antimicrobial effect, while widening its therapeutic window through an enhanced safety profile.

Table of Contents

Dedication ...... 2 Acknowledgements ...... 3 Abstract of Dissertation ...... 6 Table of Contents ...... 7 List of Figures ...... 10 List of Schemes ...... 11 List of Tables ...... 13 List of Abbreviations ...... 14 Chapter 1: Introduction to Antibiotics Active Against Gram-Negative Bacteria ...... 19 1.1 Gram Negative Bacteria ...... 19 1.2 Carbapenem Antibiotics ...... 22 1.3 Aminoglycoside Antibiotics...... 26 Chapter 2: A Whole-Cell Screen for Adjunctive and Direct Antimicrobials Active Against Carbapenem-Resistant Enterobacteriaceae ...... 29 2.0 Statement of Contribution ...... 30 2.1 Abstract ...... 31 2.2 Introduction ...... 32 2.3 Materials and Methods ...... 33 2.3.1 Primary screening ...... 33 2.3.2 Hit Identification and Confirmation ...... 34 2.3.3 Secondary Analysis using Commercially Available Compounds ...... 35 2.3.4. In-House Synthesis of Confirmed Hits ...... 36 2.3.5. Cheminformatics ...... 36 2.3.6 Spectrum of Activity Testing ...... 38 2.3.7 Construction of Carbapenemase-Expressing E. coli Strains ...... 38 2.4 Results ...... 39 2.4.1 Primary Screening ...... 39 2.4.2 Hit Identification ...... 40 2.4.3 Hit Confirmation ...... 42 2.4.4 Cheminformatics Triage ...... 42 2.4.5 Secondary Analysis Using Commercially Synthesized Compounds ...... 45

2.4.6 Analysis of Re-synthesized Compounds ...... 45 2.4.7 Synergy Testing in a Non-CRE Background ...... 46 2.4.8 Cheminformatic Characterization of High Throughput Screening Libraries ...... 47 2.5 Discussion ...... 48 2.6 Conclusion...... 51 Chapter 3: A Divergent Total Synthesis of Streptothricin F ...... 53 3.1 Introduction to Streptothricins ...... 53 3.1.1 Introduction ...... 53 3.1.2 Total Synthesis of Streptothricin F ...... 55 3.1.3 Streptothricin Biosynthesis ...... 57 3.1.4 Streptothricin Derivatives...... 61 3.1.5 Mechanism of Action by STs ...... 67 3.1.6 Resistance to ST compounds ...... 70 3.2 Total Synthesis Strategy for Streptothricin F ...... 70 3.2.1 Introduction ...... 70 3.2.2 Diamine Enabled Functional Handle ...... 73 3.2.3 Model System Synthesis ...... 75 3.2.4 Synthesis of Carbamoylated ᴅ-Gulosamine ...... 83 3.2.5 Synthesis of Nβ,Nε-Dibenzyloxycarbonyl-ʟ-β-Lysine ...... 89 3.2.6 Synthesis of Thiourea Analogue of Streptolidine Lactam ...... 90 3.2.7 Guanidine Formation Attempts from an Isothiourea Analogue of Streptolidine Lactam ... 98 3.2.8 Guanidine Formation from an Isothiocyanate Analogue of Streptolidine Lactam ...... 101 3.2.9 Deprotection and Isolation of Synthetic Streptothricin F Sulfate ...... 105 3.2.10 Conclusion ...... 106 Chapter 4: Experimental Procedure ...... 112 4.1 General ...... 112 4.2 Chemical Synthesis and Characterization ...... 113 Chapter 5: Supplemental Material for Chapter 2 ...... 176 5.1 List of Compound Libraries Screened ...... 176 5.2 Cheminformatics ...... 176 5.2.1 Permeation and Efflux Multiparameter Optimization (PEMPO) ...... 176 5.2.2 PEMPO Scoring of Known Antibacterial Compounds ...... 177

5.2.3 PEMPO and MPO Scoring for Selected Confirmed Hit Compounds ...... 181 5.3 Supplemental Tables ...... 183 Chapter 6: Supporting Material for Chapter 3 ...... 185 6.1 Purification of NTC and Isolation of STs ...... 185 6.1.1 General ...... 185 6.1.2 Introduction ...... 185 6.1.3 Purification of NTC via Sephadex Size Exclusion Gel ...... 186 6.1.4 Quantification of ST Purity using Elemental Analysis and LC-MS/MS ...... 186 6.2 Chiral Chromatography Supporting Material ...... 188 6.2.1 General ...... 188 6.2.2 Chiral Chromatography for Nitroketone 3.2.78 ...... 189 6.3 Crystallographic Information ...... 190 6.3.1 Crystallographic Information for 3.2.53 ...... 190 6.3.2 Crystallographic Information for 3.2.98 ...... 198 References ...... 202

List of Figures

Figure 1.1 Illustration of the ‘discovery void’ where dates indicate patent or initial discovery ...... 20 Figure 1.2 Comparison between OM of GPB and GNB ...... 21 Figure 1.3 Structures of carbapenems experiencing clinical utility...... 23 Figure 1.4 Structures of β-lactamase and carbapenemase inhibitors ...... 25 Figure 1.5 Structures of relevant AGs ...... 27 Figure 2.1 Overview of high throughput screening hit analysis ...... 40 Figure 2.2 Correlation between in high throughput screen and counterscreen ...... 41 Figure 2.3 Plot of least significant z-scores for duplicate compound testing in the screen and counterscreen ...... 41 Figure 2.4 Representative structures of clusters and selected singletons identified by filtering and PEMPO analysis ...... 44 Figure 2.5 Cheminformatic analyses of screening libraries ...... 48 Figure 3.1 Nourseothricin mixture components and individual moieties ...... 54 Figure 3.2 12-carbamoyl ST-F derivatives and fucothricin ...... 62 Figure 3.3 Streptolidine derivitives of STs ...... 64 Figure 3.4 β-lysine derivatives of STs ...... 65 Figure 3.5 Semi-synthetic, β-lysine analogues of ST-F ...... 66 Figure 3.6 Effect of ST-F on DNA, RNA and protein syntheses in intact cells of E. coli 15 TAU ..... 68 Figure 3.7 Antibiotic docking in the ribosome ...... 69 Figure 3.8 Convergent approach towards total synthesis of ST-F ...... 72 Figure 3.11 Anomeric effect on retention of stereochemistry following alloc deprotection ...... 82 Figure 3.12 Retrosynthetic analysis to construct isothiourea 3.2.76 ...... 92 Figure 3.13 Proposed transition states for enolate-ester 3.2.80 attack of an electrophile ...... 93 Figure 3.14 Retention of stereochemistry at anomeric position upon alloc deprotection ...... 99 Figure 3.15 Proposed mechanism for the desulfurization of a thiourea and guanidine formation ..... 102 Figure 3.16 Proposed mechanism for conversion of diol 3.2.56 to sulfamidate 3.2.57 ...... 107 Figure 3.17 Epimerization of gulosamine 3.2.102 in solution during isothiocyanate coupling ...... 108 Figure 6.1 Calibration curve for DM1 ...... 188 Figure 6.2 Racemic mixture of nitroketone 3.2.78 ...... 189 Figure 6.3 Optically pure nitroketone 3.2.78 ...... 189

List of Schemes

Scheme 3.1 Formation of protected amide 3.1.13 ...... 56 Scheme 3.2 Formation of thiourea 3.1.18 ...... 56 Scheme 3.3 Guanidine cyclization and global deprotection of ST-F 3.1.1 ...... 57 Scheme 3.4 Reported biosynthetic pathway for streptolidine lactam 3.1.5...... 58 Scheme 3.5 Reported biosynthetic pathway for streptothrisamine 3.1.28 ...... 59 Scheme 3.6 Proposed biosynthetic pathway for ʟ-β-lysine 3.1.35 ...... 60 Scheme 3.7 Biosynthetic pathway converging towards ST-F 3.1.1 ...... 61 Scheme 3.8 Semi-synthesis from citromycin 3.1.62 ...... 66 Scheme 3.9 Proposed mechanism for the conversion of diol sugars to 1,2-trans-difunctionalized glycosylamine products ...... 75 Scheme 3.10 Synthesis of azide-opened sulfamidate 3.2.9 ...... 77 Scheme 3.11 Synthesis of streptolidine model 3.2.11 ...... 79 Scheme 3.12 Completion of model system via Route A ...... 80 Scheme 3.13 Partial completion of model system via Route B ...... 81 Scheme 3.14 Synthesis of 3.2.27 using isothiourea 3.2.32 ...... 81 Scheme 3.15 Danishefsky gulal synthesis through Ferrier-type / Mislow Evans rearrangement ...... 83 Scheme 3.16 Conversion of tri-O-acetyl-ᴅ-glucal 3.2.39 to 6-O-benzyl-ᴅ-gulal 3.2.46 ...... 84 Scheme 3.17 Optimization of Crotti route to 6-O-benzyl-ᴅ-gulal 3.2.46...... 85 Scheme 3.18 Reaction sequence and crystal structure towards cyclic sulfamidate 3.2.53 ...... 87 Scheme 3.19 Reaction sequence towards 1,2-trans-difunctionalized gulosamine 3.2.58 ...... 88 Scheme 3.20 Arndt-Eistert homologation of protected ʟ-ornithine derivative 3.2.59 to form protected β-lysine derivative 3.1.11 ...... 89 Scheme 3.21 Synthesis of protected β-lysine derivative 3.1.11 ...... 90 Scheme 3.22 Shiba’s synthesis of streptolidine analogue 3.1.17 over 20 steps ...... 91 Scheme 3.23 Formation of cyclic thiourea 3.2.82 ...... 93 Scheme 3.24 Synthetic attempts towards cyanohydrin 3.2.83 ...... 95 Scheme 3.25 Attempts to form nitroalcohol 3.2.89a via asymmetric Henry reaction ...... 96 Scheme 3.26 Sequence for generating isothiourea analogue of streptolidine lactam 3.2.99 ...... 97 Scheme 3.27 Azide reduction, amidation of protected β-lysine and alloc deprotection ...... 98 Scheme 3.28 Diversion from azido-lactam 3.2.96 to generate isothiocyanate 3.2.107 ...... 101 Scheme 3.29 Coupling of gulosamine 3.2.102 and isothiocyanate 3.2.107 ...... 101

Scheme 3.30 Ishikawa method for guanidine formation from thiocarbamate using DMC ...... 102 Scheme 3.31 Diversion from previous streptolidine protocol to form isothiocyanate 3.2.114 ...... 104 Scheme 3.32 Coupling of streptolidine lactam moiety to gulosamine and guanidine formation ...... 105 Scheme 3.33 Deprotection strategy towards completion of ST-F total synthesis ...... 106 Scheme 3.34 Optimization attempts towards nitroketone 3.2.78 ...... 109 Scheme 3.35 Future work of completion of ST-F total synthesis via Route B...... 110

List of Tables

Table 2.1 Spectrum of activity of commercially synthesized and re-synthesized compounds ...... 45 Table 3.1 Reaction condition optimization for sulfamidate 3.2.19 formation ...... 78 Table 3.2 Optimization of 3-O-position benzylation of 6-O-benzyl-ᴅ-gulal 3.2.46 ...... 86 Table 3.3 Optimization for azide formation from diester 3.2.80 and proposed transition states ...... 94 Table 3.4 Attempts towards guanidine formation from gulosamine 3.2.102 ...... 100 Table 3.5 Reaction optimization attempts towards the formation of guanidine 3.2.109...... 103 Table 5.1 Compound libraries screened ...... 176 Table 5.2 PEMPO scoring of known antibacterial compounds ...... 180 Table 5.3 PEMPO and MPO scoring for selected, confirmed hit compounds...... 182 Table 5.4 Primers used in construction of carbapenemase-expressing E. coli strains ...... 183 Table 5.5 Activity of identified compounds against Escherichia coli DH5α mutant harboring various carbapenemase enzymes ...... 183 Table 5.6 Activity of identified compounds against Escherichia coli tolC mutant harboring various carbapenemase enzymes ...... 184 Table 5.7 Activity of identified compounds against Escherichia coli lptD mutant harboring various carbapenemase enzymes ...... 184 Table 6.1 Raw data for calibration curve determination and DM2 injections ...... 187 Table 6.2 Concentrations and average abundance values for DM1 and DM2 ...... 187

List of Abbreviations

2,4-DMBI 2,4-Dimethoxyisocyanate DMC 2-Chloro-1,3-dimethylimidazolinium chloride Ado● 5’-Deoxyadenosyl radical Ac Acetyl Ac2O Acetic anhydride AcOH Acetic acid AMP Adenosine monophosphate AG Aminoglycoside Ag2O Silver(I) oxide Ala Alanine Alloc Allyloxycarbonyl AME Aminoglycoside modifying enzyme AmpC Ampicillin-hydrolysing β-lactamases Arg Arginine Ba(OH)2 Barium(II) hydroxide BCl3 Boron trichloride BnBr Benzyl bromide BnOH Benzyl alcohol Boc tert-Butyloxycarbonyl Boc2O Di-tert-butyl dicarbonate BocCl tert-Butoxycarbonyl chloride BOM Benzyloxymethyl acetal Bu Butyl Bz Benzoyl BzCl Benzoyl chloride CAMHB Cation-adjusted Mueller–Hinton broth CaSO4 Calcium sulfate Cbz Carboxybenzyl CbzCl Benzyl chloroformate CC50 50% Cytotoxic concentration CDC Center for Disease Control and Prevention CDI 1,1'-Carbonyldiimidazole CFU Colony-forming units CRE Carbapenem-resistant Enterobacteriaceae CrO3 Chromium trioxide CS2 Carbon disulfide DBU 1,8-Diazabicyclo[5.4.0]undec-7-ene DCC N,N′-Dicyclohexylcarbodiimide DCM Dichloromethane

DHP Dehydropeptidase DAP Diaminopimelic acid DIBAL Diisobutylaluminum hydride DIPEA N,N-Diisopropylethylamine DMAP 4-Dimethylaminopyridine DMB 2,4-Dimethoxybenzyl DMF N,N-Dimethylformamide DMP Dess–Martin periodinane DMSO Dimethyl sulfoxide DNA Deoxyribonucleic acid EDCI 1-Ethyl-3-(3-dimethylaminepropyl) carbodiimide E+ Electrophile ESBL Extended-spectrum β-lactamases Et Ethyl Et2NH Diethylamine EtI Ethyl iodide EtOAc Ethyl acetate EtOH Ethanol FIC Fractional inhibitory concentration ratio GES Guiana extended spectrum Gly Glycine GNB Gram-negative bacteria GPB Gram-positive bacteria GulNAc N-Acetyl-ᴅ-gulosamine H2O2 Hydrogen peroxide HBA Hydrogen bond donor HBD Hydrogen bond acceptor HBr Hydrobromic acid HCl Hydrochloric acid Hg(CN)2 Mercury(II) cyanide HgCl2 Mercury(II) chloride HOBT Hydroxybenzotriazole HPLC High performance liquid chromatography HRMS High resolution mass spectrometry IM Inner membrane IMI Imipenem-hydrolyzing β-lactamase Imid Imidazole IMP Imipenem-resistant Pseudomonas aeruginosa ip Intraperitoneal iv Intravenous K2CO3 Potassium carbonate

KCN Potassium cyanide KHMDS Potassium bis(trimethylsilyl)amide KOH Potassium hydroxide KPC Klebsiella pneumoniae carbapenemase KSCN Potassium thiocyanate LA Lewis acid LCMS Liquid chromatography–mass spectrometry LDA Lithium diisopropylamide LiHMDS Lithium bis(trimethylsilyl)amide LLS Longest linear sequence LPS Lipopolysaccharide Lys Lysine M Molar MDR Multidrug resistant Me Methyl MeI Iodomethane MEM 2-Methoxyethoxymethyl MEMCl 2-Methoxyethoxymethyl chloride MeNO2 Nitro methane MeOH Methanol MeONa Sodium methoxide MIC Minimum inhibitory concentration Ms Mesyl MsCl Mesyl chloride MPO Multi-parameter optimization N Normal NaBH4 Sodium borohydride NAc N-acetyl NaClO2 Sodium chlorite NaH Sodium hydride NaHCO3 Sodium bicarbonate NaH2PO4 Monosodium phosphate NaN3 Sodium azide NaOH Sodium hydroxide NBS N-Bromosuccinimide ND Not determined NDM New Delhi metallo-β-lactamase NmcA Not metalloenzyme carbapenemase A NMM N-Methylmorpholine NMO 4-Methylmorpholine N-oxide NMR Nuclear magnetic resonance

NRPS Non-ribosomal peptide synthetase NTC Nourseothricin OD600 Optical density, 600 nm OM Outer membrane ORF Open reading frame o-PhOH ortho-Phenol Orn Ornithine OsO4 Osmium tetroxide PAINS Pan-assay interference compounds PCP2 Penicillin binding protein 2 Pd Palladium PdOAc2 Palladium(II) acetate PEMPO Permeation and efflux multiparameter optimization PG Peptidoglycan Pg Protecting group Ph Phenyl Phe Phenylalanine PhSH Thiophenol PL Phospholipid PLP Pyridoxal phosphate p-NO2PhOH para-Nitro phenol PSA Polar surface area PPh3 Triphenylphosphine p-PhNMe2 para-N,N-Dimethylaniline p-PhOMe para-Methoxy phenyl Pyr Pyridine RNA Ribonucleic acid Sar Sarcosine SAR Structure-activity relationship ST258 Sequence type 258 SFC Serratia fonticola carbapenemase SIM Seoul imipenemase SME Serratia marcescens enzyme SN1 Nucleophilic substitution 1 SN2 Nucleophilic substitution 2 SnCl2Me2 Dichlorodimethylstannane SnCl4 Tin(IV) chloride SnOBu2 Dibutyltin(IV) oxide SOCl2 Thionyl chloride ST Streptothricin ST-D Streptothricin D

ST-E Streptothricin E ST-F Streptothricin F SAT Streptothricin acetyl transferaces TBAF Tetra-n-butylammonium fluoride TBAI Tetra-n-butylammonium iodide TBS tert-Butyldimethyl TBSCl tert-Butyldimethylchlorosilane TBSOTf tert-Butyldimethylsilyl trifluoromethanesulfonate t-Bu tert-Butyl t-BuOH tert-Butyl alcohol t-BuOK Potassium tert-butoxide TCDI Thiocarbonyl diimidazole TEA Triethylamine TFA Trifluoroacetic acid THF Tetrahydrofuran TLC Thin layer chromatography TMS Trimethylsilyl TMSCN Trimethylsilyl cyanide TPPTS Triphenylphosphine-3,3′,3′′-trisulfonic acid trisodium salt Tris-N3 Trisyl azide Ts Tosyl TsCl Tosyl chloride U Uracil UDP Uridine diphosphate Val Valine VIM Verona integron-encoded metallo-β-lactamase WHO World Health Organization XDR Extensively drug-resistant Zn Zinc ZnBr2 Zinc(II) bromide

Chapter 1: Introduction to Antibiotics Active Against Gram-Negative Bacteria

1.1 Gram Negative Bacteria

Resistance to antibiotics is becoming a serious threat in the United States and global community, and new methods of defending against these pathogens must be developed. Each year as a result of bacterial resistance, an excess of $20 billion dollars are spent through healthcare costs and approximately 36,000 people die.1 The United States Center for Disease Control and

Prevention (CDC) has recently classified the growing resistance of bacteria to antibiotics as urgent, the most threatening organisms being multidrug resistant (MDR) Gram-negative (GNB) strains such as Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae.1 As outlined in the 2013 CDC report on antibiotic resistance threats, appropriate prescription of antibiotics is extremely important in controlling resistance. Health professionals are urged to responsibly prescribe antibiotics and appropriately diagnose bacterial infections as opposed to immediately turning to drugs used as the last line of defense. In addition, the CDC has encouraged the development of new antibiotics to keep up with and control growing resistance trends.

Drug discovery for new antibacterials has experienced a serious innovation gap since the

1960s. Other than the oxazolidinones, no new drug class has been discovered for antibiotics in the last 50 years.2 This “discovery void” (Figure 1.1) has been present for a few different reasons; however, the most prominent cause can most likely be tied to a lack of industrial participation. The business opportunity that draws pharmaceutical companies to pursue treatment of a disease area is typically driven by a reliable generation of revenue. Bacterial infects are classically categorized as acute infections whereas pharmaceutical companies are more motivated to pursue chronic disease areas to ensure continued consumption of their product. With the potential for resistance, companies are further discouraged to develop novel antibiotics as there is a risk of short clinical

Figure 1.1 Illustration of the ‘discovery void’ where dates indicate patent or initial discovery.2 utility. Most importantly, industrial researchers in antibacterial discovery have been hesitant to publish findings that do not reflect success as negative results can affect their stakeholders.

However, such information whether successful or not has the potential to narrow the search performed by other groups and come closer to unanswered questions in antibacterial studies.

Bacterial resistance arises from several factors including permeation, efflux, and mutations. GNB differ from Gram-positive bacteria (GPB) with the presence of an additional outer membrane (OM) surrounding an inner membrane (IM). The OM is composed of a lipopolysaccharide layer (LPS) as well as a single layer of phospholipids (PL) (Figure 1.2).3

Figure 1.2 Comparison between OM of GPB and GNB.4

The LPS layer consists of lipid A, core polysaccharides, and O-specific polysaccharide chains.3 Lipid A is responsible for anchoring the LPS layer to the exterior of the GNB while the core and O-specific polysaccharides contain strong hydrophobic groups, important for avoiding phagocytosis from external sources. While both GPB and GNB contain a layer of peptidoglycan

(PG), the OM makes permeation to the bacterial cell very challenging and complicates the delivery of drugs that possess a cytoplasmic target. Most GNB drugs with targets located beneath the OM use porins as the key means to penetration. Porins are specific for importing low molecular weight, hydrophilic molecules necessary for maintaining cell health through the import of nutrients and the export of waste.2 If antibacterial drugs penetrate through porins, efflux pumps present the challenge of the drug remaining inside of the cell. Efflux pumps vary between bacterial species, ranging from single membrane pumps observed in GPB to double-membrane systems observed in some GNB and have the ability to eject drugs from within the bacterial cell.5 Efflux pumps

spanning two membranes have the ability of binding to drugs either in the periplasm or the cytoplasm and ejecting the drug outside of the OM. While efflux pump inhibitors have been considered to combat this method of resistance, complications arise from varying substrate selectivity even within a single bacterial species.5

Finally, bacterial mutations have greatly contributed to resistance. Endogenous mutations spread through horizontal gene transfer where bacterial cells can transmit resistance carrying DNA through plasmids and develop favorable traits of neighboring cells.2 Exogenous mutations are manifested in the imprudent use of antibiotics, resulting in the survival of only the most fit bacterial species. Resulting mutations can include alterations to the target through binding site protection or modification.2 Additional resistance mechanisms include chemical modification of the drug itself, rendering it inactive as observed in the hydrolysis of some β-lactams. The necessity to produce new methods to combat bacteria, most importantly GNB, is crucial as species will continue to evolve and develop new methods of resistance to drugs used for the last line of defense.

Innovations in drug discovery as well as drawing upon past breakthroughs have the potential for discovering novel antibacterial modes of action. Two compound classes that have demonstrated unique mechanisms of actions with prolonged clinical utility are the carbapenems and aminoglycosides (AGs).

1.2 Carbapenem Antibiotics

Carbapenems stem from the β-lactam family of antibiotics and were first identified for their increased stability against β-lactamase resistant enzymes after chemical modification from penicillin.6 Possessing a similar mechanism of action to the β-lactams, carbapenems target penicillin binding protein 2 (PCP2) among other binding domains in the synthesis of peptidoglycan that have resulted in extensive clinical utility.7,8 Carbapenems are less susceptible to modification

by β-lactamases from increased stability; however, resistance has emerged among GNB in the form of similar inactivation by carbapenemases that render carbapenems inactive through hydrolysis of the lactam ring.9 Carbapenems have been successful antimicrobial agents and developed since their discovery to encompass broad-spectrum activity across several scaffolds

(Figure 1.3). The least stable member of the carbapenem family is imipenem, possessing a shorter shelf life compared to other carbapenems as well as requiring co-dosage with cilastatin to prevent hydrolysis by human renal dehydropeptidase (DHP).10 Other carbapenems illustrated in Figure

1.3 have stability to DHP through 1β-methylation (R1) and vary from one another in terms of their activity from broad to narrow scope.11 Meropenem experiences a wide spectrum of activity against

GPB and GNB including good potency towards Enterobacteriaceae as well as activity in

Pseudomonas aeruginosa.12–14 Ertapenem exhibits activity similar to other carbapenems; however, it suffers a loss of activity specifically against Pseudomonas aeruginosa.15 Possessing slightly higher stability than other carbapenems, doripenem exhibits great activity against MDR, GNB.16,17

Figure 1.3 Structures of carbapenems experiencing clinical utility.

Carbapenemases render carbapenem antibiotics inactive similarly to β-lactamases through hydrolysis. There are several classes of carbapenemase enzymes including classes A, B, C and D; however, there is little clinical data surrounding class C carbapenemases.18 Class A carbapenemases can be passed through chromosomes—NmcA (not metalloenzyme carbapenemase A), SME (Serratia marcescens enzyme), IMI-1 (Imipenem-hydrolyzing β- lactamase) and SFC-1 (Serratia fonticola carbapenemase-1), whereas there are others that are spread through horizonal gene transfer—KPC (Klebsiella pneumoniae carbapenemase, KPC-2 through KPC-13), IMI (IMI-1 through IMI-3) and derivatives of GES (Guiana extended spectrum)

(GES-1 through GES-20).19,20 Whereas all other carbapenemase enzymes hydrolyze carbapenems through an active site serine residue, metallo-β-lactamases making up class B carbapenemases rely on interactions with active site zinc ions for hydrolysis.18 Class B carbapenemases include the New

Delhi metallo-β-lactamase 1 (NDM-1), Imipenem-resistant Pseudomonas (IMP)-type carbapenemases, VIM (Verona integron-encoded metallo-β-lactamase), GIM (German imipenemase) and SIM (Seoul imipenemase).18 Class D carbapenemases are similar to class A in terms of their mechanism of action; however, class D carbapenemases are more resistant to typical

β-lactamase and carbapenemase inhibitors. Present in both Acinetobacter baumannii and

Pseudomonas aeruginosa, class D carbapenemases are known for their rapid mutation and wide spectrum of activity associated with the over 102 identified unique genetic sequences.19,21

A widely used method of avoiding hydrolysis of the lactam ring for carbapenems as well as β-lactams is with β-lactam inhibitors. An inhibitor is co-dosed with a β-lactam type antibiotic to inactivate the β-lactamase enzyme within the periplasmic space and allows the antibiotic to reach its active site without modification by the enzyme.22 Using a potentiator helps to extend the clinical life of an established antibiotic, staving off the effects of resistant pathogens.

Figure 1.4 Structures of β-lactamase and carbapenemase inhibitors.

Several types of β-lactamase and carbapenemase inhibitors are illustrated in Figure 1.4. β- lactam-type inhibitors clavulanic acid, sulbactam and tazobactam are used alongside either β- lactam or carbapenem antibiotics against primarily class A carbapenemases.18 Compounds ZINC-

10107204 and ZINC-02318494 potentiated meropenem better than β-lactam-type inhibitors against KPC-2 carbapenemases.23 Diazabicyclooctanes relebactam, avibactam and nacubactam were developed upon observed resistance to previously mentioned ‘suicide’ β-lactamase inhibitors towards KPC enzymes. These inhibitors undergo covalent, reversible interactions with KPC enzymes exhibiting broad spectrum of activity against extended-spectrum β-lactamases (ESBL),

ampicillin-hydrolyzing β-lactamases (AmpC) as well as some class D carbapenemases.24 Boronic acid-based β-lactamase inhibitor, RPX-7009, restores activity of meropenem against KPC containing isolates of K. pneumoniae and Escherichia coli.25 Metallo-β-lactamase inhibitor ME-

1071 and EDTA are able to restore clinical activity of meropenem towards against IMP-1, VIM-2 and NDM-1 produced by P. aeruginosa and Enterobacteriacae; however, EDTA has not been used in the clinic as a result of toxicity.26,27

Discussed in Chapter 2, we describe a method of exploring additional potentiators to restore the activity of meropenem using adjunctives. Just as β-lactamase and carbapenemase inhibitors potentiate carbapenems through avoiding a method of resistance, we performed a high- throughput screen in order to search first for a compound or compound class exhibiting adjunctive activity without identification for the mechanism of action.

1.3 Aminoglycoside Antibiotics

The first aminoglycoside (AG) antibiotic introduced to the clinic in 1944 was streptomycin, isolated from soil Streptomyces griseus (Figure 1.5). When first generation β-lactams started becoming susceptible towards resistance from β-lactamases, AGs were utilized as the last-in-line therapeutic for GNB infections. However, upon introduction of second generation β-lactams, AGs were utilized less in the clinic as they were associated with nephrotoxicity. As resistance became more widespread, clinicians turned to AGs once again for their reliable, bactericidal activity. It was later discovered through dosing studies that as the result of the concentration-dependent nature, higher doses taken less frequently reduce toxicity while maintaining antibacterial efficacy.28

AGs are broad spectrum, bactericidal with activity against Enterobacteriaceae, including

E. coli and K. pneumoniae, Staphylococcus aureus, including methicillin-resistant and

vancomycin-resistant isolates, P. aeruginosa and to a lesser extent Acinetobacter baumannii.29–31

AGs carry out their mechanism of action through inhibition of protein synthesis by binding to the

A-site on the 16S ribosomal RNA of the 30S ribosomal subunit.32 AGs are positively charged at physiological pH are able to penetrate GNB cells through electrostatic interactions of the polycationic AG to the negatively charged components of the lipid A portion of the OM and then displacement of magnesium or calcium ions.33,34 Upon traversing the OM, AGs bind to the 16S ribosomal RNA and induce misreading of amino acid codons, causing a disruption of protein synthesis.35–37 Other known mechanisms of action following initial entrance to the GNB cell include obstruction of amino acid elongation or initiation inhibition.32,34,38

Figure 1.5 Structures of relevant AGs.

GNB have demonstrated resistance to AGs through several methods including enzymatic modification of the AG, target site modification through enzymatic or chromosomal mutation, as well as extracellular efflux. AG modifying enzymes (AMEs) are known to spread through

plasmids and are categorized based on acetylation, phosphorylation, or adenylation of amino / hydroxyl groups at varying moieties on the AG.37 The most prominent target modification occurs through methylation of the 16S ribosomal RNA and sterically blocks AGs from binding and exerting their mechanism of action.39–41

Problems with AGs associated with toxicity or resistance have investigated through isolation of individual compounds within AG mixture or chemical modification of AGs. While the toxicity of AGs had limited their use in the clinic, achieving selectivity is possible within the class.

Among gentamicin congeners (Figure 1.5), C2 retains normal bactericidal activity similar to the others without observed nephrotoxicity within an in vivo rat model, thus separating toxicity from its activity.42 Resistance from AMEs has been avoided through derivatization of AGs as demonstrated from analogues produced by semi-synthesis of 2-deoxystreptamine AGs paromomycin and neomycin.43 Through a series of 4’,6’-O-acetal and 4’-O-ether semi-synthetic derivatives of the 2-deoxystreptamine scaffold, AG compounds demonstrated weakened interactions with eukaryotic mitochondrial or cytosolic ribosomes, yet retained good inhibition for bacterial ribosomes and antibacterial activity.43

Discussed in Chapter 3, we pursue a total synthesis of an AG-like compound mixture, nourseothricin (NTC) which demonstrates excellent activity against MDR, GNB but exhibits nephrotoxicity like the AG class. Just as gentamicin, we observe differences in activity as well as selectivity among the different constituents within the mixture. Through a divergent total synthetic strategy, we hope to derivatize compounds within the NTC mixture to tune its selectivity and maintain its antimicrobial effect, while widening its therapeutic window through an enhanced safety profile.

Chapter 2: A Whole-Cell Screen for Adjunctive and Direct Antimicrobials Active Against

Carbapenem-Resistant Enterobacteriaceae

Reprinted with permission from

Smith, K. P., Dowgiallo, M. G., Chiaraviglio, L., Parvatkar, P., Kim, C., Manetsch, R., & Kirby,

J. E. (2019). A Whole-Cell Screen for Adjunctive and Direct Antimicrobials Active against

Carbapenem-Resistant Enterobacteriaceae. SLAS DISCOVERY: Advancing the Science of Drug

Discovery, 24(8), 842–853. https://doi.org/10.1177/2472555219859592

2.0 Statement of Contribution

Contributions made to this chapter by Matthew G. Dowgiallo are as follows: cheminformatics analysis, characterization and triage, PAINS screening, MPO scoring, PEMPO design and implementation, physicochemical property calculations and comparison, and ICCB library analysis. The manuscript was written by Kenneth P. Smith and Matthew G. Dowgiallo with careful review and contributions made by James E. Kirby, Roman Manetsch, and Lucius Chiaraviglio.

2.1 Abstract

Carbapenem-resistant Enterobacteriaceae (CRE) are an emerging antimicrobial resistance threat for which few if any therapeutic options remain. Identification of new agents that either inhibit CRE or restore activity of existing antimicrobials is highly desirable. Therefore, a high throughput screen of 182,427 commercially available compounds was used to identify small molecules, which either enhanced activity of meropenem against a carbapenem-resistant

Klebsiella pneumoniae ST258 screening strain and/or directly inhibited its growth. The primary screening methodology was a whole cell screen/counterscreen combination assay that tested for reduction of microbial growth in the presence or absence of meropenem, respectively. Screening hits demonstrating eukaryotic cell toxicity based on an orthogonal screening effort or identified as pan-assay interference (PAINS) compounds by computational methods were triaged. Primary screening hits were then clustered and ranked according to favorable physicochemical properties.

Among remaining hits, we found ten compounds that enhanced activity of carbapenems against a subset of CRE. However, direct antimicrobials that passed toxicity and PAINS filters were not identified in this relatively large screening effort. It was previously shown that the same screening strategy was productive for identifying candidates for further development in screening known bioactive libraries inclusive of natural products. Our findings therefore further highlight liabilities of commercially available small molecule screening libraries in the Gram-negative antimicrobial space. In particular, there was especially low yield in identifying compelling activity against a representative, highly multidrug-resistant, carbapenemase-producing Klebsiella pneumoniae.

2.2 Introduction

Enterobacteriaceae are a common cause of bacterial bloodstream, urinary tract, and surgical site infections. Concerningly, these organisms also are commonly associated with resistance to clinically useful first and second-line antimicrobials including penicillins, cephalosporins, fluoroquinolones, and aminoglycosides.44 Carbapenems are the major last line of defense against multidrug-resistant Gram-negative pathogens. Unfortunately, carbapenem- resistant Enterobacteriaceae (CRE) emerged rapidly in the past two decades.45 They have now been detected worldwide46 with particularly high prevalence in Asia47 and Southern Europe.48 CRE are also isolated with increasing frequency in the United States.49 Few, if any treatments remain and those few often have dose-limiting toxicity.50 Recently, truly pandrug-resistant CRE have appeared,51 along with highly pathogenic hypermucoviscous strains that cause metastatic multi- organ infection in otherwise healthy adults,52,53 highlighting the pressing need for new antimicrobials with activity against these pathogens.

Development of new antimicrobials active against multidrug-resistant Gram-negative pathogens has proven difficult due to relative impermeability of the Gram-negative cell membrane54 and ubiquitous expression of efflux pumps.55 However, carbapenems overcome both of these challenges and may retain detectable in vitro56 and in vivo57 activity even in strains expressing enzymes that degrade carbapenems (carbapenemases). Therefore, we hypothesized that this partial activity could be potentiated by small molecules through a variety of mechanisms to restore carbapenem efficacy against otherwise resistant CRE.

We therefore chose to use our previously validated screening/counterscreening approach to evaluate activity of a large collection of small molecules for their ability to either directly inhibit or potentiate activity of a representative carbapenem (meropenem) against a CRE screening

strain.58 To rapidly triage compounds with non-specific activity, we used data from an orthogonal screening effort to eliminate those with eukaryotic cytotoxicity.59 Finally, we identified a series of compounds with optimal physicochemical properties, tested their spectrum of activity against representative CRE strains using commercially available compounds, and confirmed activity upon re-synthesis. Based on our observations, we believe the screening strategy will prove an efficient method for identifying direct and indirect antimicrobials, however, only in libraries optimized for the Gram-negative antimicrobial space.

2.3 Materials and Methods

2.3.1 Primary screening

Our primary screening strain was Klebsiella pneumoniae BIDMC12A, a CRE strain of sequence type 258 (ST258), the most common sequence type of K. pneumoniae CRE strains circulating in the United States, which expresses the KPC-3 carbapenemase, and blaSHV-11, blaSHV-134, and blaTEM-1 -lactamases.60 The screen was performed as a screening/counterscreening experiment as described in our previous work where only known bioactive compounds were examined.58 Briefly, prior to screening, 30 µL of cation-adjusted

Mueller-Hinton broth (CAMHB, BD Diagnostics, Sparks, MD) containing 20 µg mL-1 meropenem

(ArkPharm, Libertyville, IL) (screen) or no antibiotic (counterscreen) was added to clear, untreated polystyrene 384-well plates (Greiner Bio-One, Monroe, NC) using a MultiDrop Combi liquid handler (ThermoFisher Scientific, Waltham, MA). Compounds were added using pin-transfer robot calibrated to deliver 300 nL to each well and screened in duplicate in separate screening plates.

We screened commercially available libraries available at the Institute of Chemistry and

Cell Biology (Harvard Medical School, Boston, MA), listed in Supplemental Material 5.1, which

consist of small molecules without previously characterized activity. Compound concentrations varied by library. For libraries with concentrations expressed in µg mL-1, screening concentrations were 2.5, 10, 25, or 75 µg mL-1. For libraries with concentrations expressed as molarity, screening concentrations were 0.5, 5, 16.5, 44, or 50 µM. Immediately after compound transfer, 30 µL of K. pneumoniae BIDMC12A (1 x 106 colony forming units (CFU) mL-1) in CAMHB was added, bringing the final concentration of cells to approximately 5 x 10 5 CFU mL-1 per CLSI guidelines61 and meropenem (where applicable) to 10 µg mL-1 in a final assay volume of 60 µL.

Plates were incubated for 48 hours at 37 °C in 100% humidity. Bacterial growth was quantified by optical density at 600 nm (OD600) using an EnVision multimode plate reader

(PerkinElmer, Waltham, MA). For each plate, Z’ was calculated based on positive (5 µg mL-1 colistin) and negative controls (CAMHB alone).62 Graphical representations of screening results was created using a custom Python script using the matplotlib library63 with point density calculated using the kernel density function as implemented in the SciPy library.64

2.3.2 Hit Identification and Confirmation

For each well, z-scores were calculated based on average and standard deviation of all experimental wells from the same assay plate. Direct antimicrobial hits were defined as strong (z

< -6), moderate (-3 > z > -6), or weak (-1.5 > z > -3) based on the least significant z-score between replicates. Compounds were defined as potential adjunctives when the z-score for the screen was

>3-fold that of the counterscreen. Based on previous work,58 we selected hits with >50% inhibition in the screen as candidates for follow-up testing.

Hits with eukaryotic cell cytotoxicity were identified based on results from a separate orthogonal high throughput screening effort using the same compound libraries.59 Briefly, the cytotoxicity assay consisted of application of compounds to J774A.1 macrophages incubated in

the presence of 125 nM SYTOX Green, a membrane impermeant nucleic acid binding dye. In this assay, cytotoxicity results in increased eukaryotic cell membrane permeability and associated increase in SYTOX Green fluorescence, which is measured relative to controls. The assay was described previously as part of a combined screen for intracellular bacterial growth and eukaryotic cell death.59 Cytotoxic compounds were defined conservatively as those with cytotoxicity z-scores

> 1.5.

Hits were cherry picked for confirmatory testing from library plates using a Tecan EVO75 liquid handler (Tecan, Morrisville, NC). We then used an HP D300 digital dispenser (HP Inc.,

Palo Alto, CA) to add 300 nL of compound to CAMHB or CAMHB containing 10 µg mL-1 meropenem to replicate conditions of the screen and counterscreen. K. pneumoniae BIDMC12A was added to a concentration of 5 x 105 CFU mL-1 with a final assay volume of 60 µL. Plates were incubated at 37 °C in 100% humidity for 48 hours and growth quantitated as described in the primary screen. Adjunctive activity was considered confirmed if it resulted in >25% growth inhibition in the presence of meropenem, but <25% growth inhibition in CAMHB alone, while direct activity was consider confirmed if inhibition were >25% in the absence of meropenem.

2.3.3 Secondary Analysis using Commercially Available Compounds

Select compounds were ordered as powder from ChemDiv (San Diego, CA), ChemBridge

(San Diego, CA), Enamine (Monmouth Jct., NJ), or Asinex (Winston-Salem, NC). Compounds were dissolved in 100% DMSO (Sigma-Aldrich, St. Louis, MO) to a concentration of 5 mg mL-1 and stored at -80 °C. For each compound, we performed two-dimensional synergy assays in combination with meropenem using a previously validated protocol.65,66 Briefly, we used an HP

D300 digital dispenser to prepare combinatorial two-fold orthogonal dilution series of meropenem and compounds of interest. Minimal inhibitory concentrations were defined as the lowest

concentration of antimicrobial resulting in complete growth inhibition (OD600 < 0.08), as previously validated by our laboratory.67

In these experiments, we only tested meropenem potentiators with no detectable MIC value on their own. Therefore, synergy was assessed solely based on the greatest fold reduction of the meropenem MIC in the presence of compound, i.e., the MIC of meropenem in the presence of compound divided by the MIC of meropenem alone, which is expressed as the fractional inhibitory concentration ratio or FIC. FIC values  0.5, consistently observable in biological replicates, were considered to indicate synergy.66,68

2.3.4. In-House Synthesis of Confirmed Hits

Selected compounds were re-synthesized in-house for follow-up activity confirmation experiments.

2.3.5. Cheminformatics

Pan-assay interference compound (PAINS)69 filtering was performed through an available

PAINS filter70 (Eli Lilly, Cambridge MA). Next, using Scaffold Hunter, hit compounds were arranged into clusters based on Tanimoto distance measurements of fingerprints generated for each molecule.71 Physicochemical properties of compounds were predicted with Microsoft Excel

(Microsoft, Redmond WA) using add-ins from ChemDraw (PerkinElmer, Walthan MA) and

ChemAxon (Cambridge MA). Compounds were scored through two multi-parameter optimization

(MPO) tools using these predicted properties.

First, compounds were ranked by a previously reported multi-parameter optimization

(MPO) algorithm for calculating optimal physicochemical properties of drug molecules with good bioavailability.72 Furthermore, an “in-house” MPO algorithm was designed to predict the ability of a compound to penetrate into a bacterial cell and avoid efflux, both characteristics of effective

Gram-negative antimicrobial compounds. For this reason, we refer to our “in-house” MPO algorithm as PEMPO (Permeation and Efflux Multiparameter Optimization).

PEMPO scoring focused on assessing optimal ranges (shown in parentheses) for targeted physicochemical properties of Gram-negative antimicrobials including the isoelectric point (6.1-

8.7), the total polar surface area (100-200 Å2), the number of hydrogen bond donors (2-6), the number of hydrogen bond acceptors (6-11), the partition coefficient cLogP (≤3), and the distribution coefficient cLogD7.4 (≤0.2). Optimal ranges were defined by analysis of average physicochemical properties of 100 known Gram-negative active antimicrobials from a study by

Moser et al.73. Compounds were then scored based on how similar each physicochemical property related to the optimal value (Supplemental Material 5.2.3). Thus, a high scoring compound suggested a high probability for bacterial cell permeation and a low probability for efflux.

Two antibacterial classes were excluded from development of the PEMPO model: macrocycles (such as macrolides or cyclic peptides such as colistin) and aminoglycosides. Both compound classes exhibit a significantly higher molecular weight than most “drug-like” compounds found within screening libraries and as a result would disproportionately influence the scoring of compounds based on extreme characteristics compared with other classes.

Aminoglycosides contain on average 30 HBD/HBA whereas the other 6 classes of antibacterials

(penicillins, cephems, carbapenems, sulfa drugs, fluoroquinolones, and tetracyclines) contain on average 13 HBD/HBA.73 Macrolides display many more lipophilic residues, contributing to a higher average cLogD7.4 of 2.6, whereas an average cLogD7.4 of -2.77 is observed among the other

6 antimicrobial classes.73 Molecular weight was not used to calculate PEMPO scores as molecular weight of known Gram-negative compounds can vary widely based on compound class. The formula for calculating the PEMPO score is described in Supplemental Material 5.2.1.

As physicochemical property calculators vary between platforms, we evaluated PEMPO scores calculated from properties generated by Pipeline Pilot (Accelrys, San Diego, California) and ACD/Labs (Toronto, Ontario, Canada) compared to scores generated from Chemdraw and

ChemAxon. Known Gram-negative antimicrobials had an average PEMPO score of 4.97 and 5.08 out of 6.0, respective to property prediction platform. Results listed by compound can be found in

Supplemental Material 5.2.2. Therefore, we observed an average increased PEMPO score of

+0.11 using the latter compared to the former property generation tools but considered this difference to be negligible.

2.3.6 Spectrum of Activity Testing

Follow-up activity spectrum studies were performed for selected compounds. We tested commercially available or re-synthesized compound in combination with meropenem as described above using thirty de-identified CRE isolates collected at our institution including Escherichia coli

(n = 8), K. pneumoniae (n = 20), Serratia marcescens (n = 1) and Enterobacter cloacae (n = 1).

The genome sequences of all strains are available.60

2.3.7 Construction of Carbapenemase-Expressing E. coli Strains

KPC-2, KPC-3, and NDM-1 carbapenemases were PCR amplified with Q5 DNA polymerase (New England Biolabs, Ipswich, MA, annealing temperature = 60 °C) using primers listed in Supplemental Material 5.3, table 5.4. PCR products were introduced into the pUC19 vector using the NEBuilder HiFi DNA Assembly Kit (New England Biolabs) according to manufacturer’s instructions. Vectors alone or vector containing carbapenemases were transformed into electrocompetent DH5α (New England Biolabs); tolC mutant strain, JW5503-1 (E. coli

Genetic Resources Stock Center, Yale University, New Haven, CT); or lptD mutant strain,

RFM795 (E. coli Genetic Resources Stock Center), and selected with 100 µg mL-1 ampicillin

(ThermoFisher Scientific, Waltham, MA). Cloning fidelity was confirmed by DNA sequencing

(Genewiz, Boston, MA). Carbapenemase expression was confirmed phenotypically by determination of meropenem MICs.67 Select re-synthesized compounds were tested for synergy with meropenem in all constructed strains as outlined in the secondary analysis section.

2.4 Results

2.4.1 Primary Screening

Gram-negative bacteria are intrinsically resistant to a variety of antibiotics owing to the relative impermeability of the cell envelope. Additionally, multidrug-resistant organisms have an extensive system of efflux pumps with broad and unpredictable specificities, which also prevent molecules from reaching the cytoplasm.55 Therefore, we chose to perform a whole cell bacterial growth inhibition screen so that screening hits would have already passed these two significant hurdles. Furthermore, our screening strain was a representative ST258, multidrug-resistant clinical

K. pneumoniae isolate, representative of the most common CRE strains circulating in the United

States, and resistant to a variety of antimicrobial agents including penicillins, cephalosporins, carbapenems, fluoroquinolones, nitrofurantoin, trimethoprim/sulfamethoxazole, tobramycin, and amikacin.58 Thereby, a high bar for activity was set, which is appropriate for identifying efficacy against an emerging multidrug-resistant pathogen target.

Our high throughput screening assay was designed as a screen/counterscreen. Screening wells contained meropenem at a subinhibitory concentration of 10 µg mL-1, and the counterscreen contained no antimicrobial. Therefore, compounds that potentiated meropenem would demonstrate activity in the screen (growth inhibition in the presence of meropenem), but not in the counterscreen (without meropenem). Direct antimicrobials would exhibit inhibitory effects independent of meropenem and therefore demonstrate activity in both the screen and

counterscreen. In total, we screened 182,427 compounds without previously characterized biological activity in duplicate using this two-tiered assay. An overview of the screening effort and secondary analysis is summarized in Figure 2.1. During the screening effort, we found good reproducibility between replicates for both the screen and counterscreen experiments (Figure 2.2).

Average cumulative Z’ was 0.61 for the screen and 0.67 for the counterscreen based on positive and negative screening wells from screening plates.

Figure 2.1 Overview of high throughput screening hit analysis.

2.4.2 Hit Identification

We initially identified 1,531 (0.84% of total compounds screened) total adjunctive and direct antimicrobial screening hits. Of the adjunctive hits, 605 (0.332%), 599 (0.328%) and 43

(0.02%) were weak, medium, and strong, respectively. Of the direct antimicrobial hits 205

(0.11%), 71 (0.04%), and 8 (0.004%) were weak, medium, and strong, respectively. The z-score distribution for the screen/counterscreen is graphically summarized in Figure 2.3.

Figure 2.2 Correlation between in high throughput screen and counterscreen. Assays were performed in duplicate. Screening wells contained 10 g/mL of meropenem, counterscreening wells did not contain antibiotic. Readout was the OD600 of microwells after a 48 h incubation. The values for each pair of duplicate measurements were plotted on X and Y axes for the screen (A) and counterscreen (B), respectively. Higher relative data point density is represented by warmer colors as indicated in legend. Inclusive of control wells, r2 = 0.82 for the screen and 0.92 for the counterscreen, indicating excellent correlation between replicate wells.

Figure 2.3 Plot of least significant z-scores for duplicate compound testing in the screen and counterscreen. Z-criteria hit ranking (strong, medium, weak) are represented in shades of yellow (direct antimicrobials) or blue (adjunctive antimicrobials). Higher relative data point density is represented by warmer colors as indicated in legend.

We previously established that our screening assay yielded a high false positive rate based on z-score criterion alone and that those hits demonstrating <50% inhibition (compared to control wells) were unlikely to confirm in secondary analysis.58 Accordingly, we applied a potency requirement of >50% inhibition for at least one of the duplicate measurements. After applying this filter, 439 (72.6%) weak, 598 (99%) medium, and all strong adjunctive hits and 20 (9.8%) weak,

52 (73.2%) moderate, and all strong direct hits were retained.

Additionally, we filtered out compounds that showed cytotoxicity to eukaryotic cells, a marker for non-specific activity, or a target shared by both prokaryotes and eukaryotes, which as a consequence would not be druggable. Eukaryotic cytotoxicity data were from a previously described screening assay using the same compound libraries59. After applying this filter, 252

(57.4%) weak, 375 (62.7%) moderate, and 31 (72.1%) strong adjunctive hits were retained; 9

(45%) weak, 31 (59.6%) moderate, and no strong direct antimicrobial hits were retained.

2.4.3 Hit Confirmation

We selected 274 filtered adjunctive and direct antimicrobial hits based on primary screening potency for confirmatory testing using cherry picks from commercial library plates in a manner identical to the primary screening assay. Here, we set a less stringent 25% inhibition cutoff in recognition that hits may not recapitulate activity exactly upon secondary analysis. In total, 127

(44.2%) adjunctive hits and no direct antimicrobial hits confirmed on retesting.

2.4.4 Cheminformatics Triage

Cheminformatics filtering was then performed to remove nonspecific, pan-assay interference compounds (PAINS) with features of covalent modifiers (for example electrophiles such as aldehydes, ketones, or boronic acids) or metal binders (for example hydroxamic acids or phosphonates). Even though many antibacterial drugs contain such reactive structural features

(approximately 58% of known antibiotics from our testing set fail the PAINS filter), PAINS are considered to be problematic for hit-to-lead optimization and drug development.69,74 Therefore, of the 127 confirmed, adjunctive hits, 20 compounds were identified as PAINS and excluded from further analysis. The remaining 107 were clustered based on common substructure, which resulted in identification of 15 clusters and 17 singletons. Further prioritization within clusters was performed based on a compound activity profile and scoring of physicochemical properties characteristic of known antibacterials, using a cheminformatic pipeline called PEMPO

(Permeation and Efflux Multiparameter Optimization) described in the materials and methods section. Select singletons were removed after visual inspection because of limited synthetic tractability or the presence of unfavorable functional groups known to possibly pose bioavailability limitations, narrowing our future analysis to 42 compounds representing 15 clusters and 6 singletons. PEMPO and MPO scoring for these compounds is shown in Supplemental Material

5.2.3. Representative structures of top scoring clusters and singletons are shown in Figure 2.4.

Figure 2.4 Representative structures of clusters and selected singletons identified by filtering and PEMPO analysis. For clusters, representative structures shown are the highest PEMPO scoring compounds within each cluster. Compounds highlighted in gray demonstrated a synergistic adjunctive activity against representative CRE strains after repurchase from commercial suppliers.

2.4.5 Secondary Analysis Using Commercially Synthesized Compounds

We ordered these 42 compounds from commercial suppliers and performed synergy assays in combination with meropenem using our primary screening strain. In total, 23.8% (n = 10) had evidence of synergistic activity with meropenem (FIC ≤ 0.5).

All compounds with an FIC ≤ 0.5 were tested for activity spectrum against a panel of CRE strains consisting of E. coli and K. pneumoniae containing either KPC-2 or KPC-3 carbapenemases. All selected compounds had activity against ≥50% of strains tested (Table 2.1). Average FICa % CRE Activityb

Compound Commercialc Re-synthesizedd Commercial Re-synthesized KP40 0.31 0.42 75 23 KP14 0.5 -e 70 - KP17 0.5 - 70 - KP5 0.38 - 60 - KP13 0.5 - 60 - KP11 0.38 0.5 50 27 KP8 0.38 - 50 - KP9 0.38 >1 50 33 KP19 0.5 0.5 50 17 KP56 0.19 0.75 - 17 Table 2.1 Spectrum of activity of commercially synthesized and re-synthesized compounds. aCalculated from quadtruplicate testing of K. pneumoniae BIDMC 12A. bPercent of CRE strains with FIC ≤ 0.5 on combinatorial testing with meropenem. Calculated using at least 10 representative CRE strains for commercial compounds and 30 CRE strains for re-synthesized compounds. cCompounds purchased from commercial suppliers. dCompounds synthesized in our laboratory. eNot determined

2.4.6 Analysis of Re-synthesized Compounds

To this point, we had been using compounds available in limited quantities from the commercial suppliers of our screening libraries. We added an additional layer of confirmation by resynthesizing hit compounds and confirming structural identity and purity by liquid chromatography with diode array detection, mass spectrometry and NMR.

We used potency (based on FIC) and spectrum of activity as primary criteria and predicted physicochemical property data as secondary criteria to select KP40 and KP11 for re-synthesis.

Other compounds chosen for re-synthesis were KP9 and KP19, which displayed excellent PEMPO scores (4.4 and 4.8 respectively), along with KP56, which demonstrated good activity in primary screening. Following synthesis, these compounds were tested again using our screening strain to confirm activity using our standard synergy assay.

Of the re-synthesized compounds, KP11, KP40, and KP19 demonstrated synergy with meropenem against our screening strain (Table 2.1). However, two compounds, KP9 and KP56, did not. We then tested re-synthesized compounds against our 30 strain CRE panel. Re-synthesized compounds showed synergy against 17 to 33% of CRE strains. Interestingly, KP9 and KP56, while not showing synergy against the screening strain, demonstrated synergy against a subset of clinical

CRE strains.

2.4.7 Synergy Testing in a Non-CRE Background

Confirmed adjunctive hits might interfere with carbapenemase activity or alternatively affect the physiology of specific bacterial strains to enhance potency of meropenem by other mechanisms. To distinguish phenotypically between these possibilities, we first constructed isogenic E. coli strains expressing the serine carbapenemases (KPC-2 or KPC-3) or metallo- carbapenemase (NDM-1). The strain background used was DH5α, a laboratory-adapted E. coli K-

12 strain with no intrinsic resistance to -lactams including meropenem. However, we found no synergy of compounds with meropenem, suggesting that effects might be strain specific, and as a result not effective on E. coli K-12.

We therefore considered whether the lack of activity in the E. coli K-12 background might relate to either lack of permeation and/or efflux. To distinguish between these two possibilities, we introduced our KPC-2, KPC-3, and NDM-1 containing plasmids into E. coli strains with defects in the outer membrane permeability barrier (lptD) or efflux activity (tolC). The lptD mutant

expresses a truncated form of LptD, a protein critical in transporting lipopolysaccharide (LPS) to the outer membrane.75 The resulting deficit in LPS in the outer membrane leads to increased permeability. The tolC mutant inactivates a critical shared component of several efflux pumps.76

Interestingly, we did not observe synergy with meropenem in either strain. However, we did observe direct antimicrobial activity of KP40 in the tolC mutant. MICs were independent of carbapenemase production and ranged from 16-128 µM during replicate testing, an unusual degree of biological variability not typical of established antimicrobials with specific mechanisms of action (Supplemental Material 5.3, tables 5.5-5.7).

2.4.8 Cheminformatic Characterization of High Throughput Screening Libraries

The physicochemical properties of screening libraries used in this effort were characterized and compared to the chemical space occupied by 100 known Gram-negative antimicrobials.

Molecular weight, polar surface area, cLogD7.4, and the summation of hydrogen bond donor/acceptors of screening compounds and known antimicrobials were calculated. Plots of physicochemical properties versus molecular weight are shown in Figure 2.5.

From data plots, it is apparent that the screening library consists of compounds with a greater degree of lipophilic substituents. More specifically, partition coefficients (cLogD7.4) for library compounds demonstrate increasing lipophilicity with increasing molecular weight (Figure

2.5A). In contrast, partition coefficients of 100 known Gram-negative active antimicrobials show the opposite trend. Similar but inverted trends for the screening libraries and known antimicrobials were observed in plots of polar surface area (Figure 2.5B) as well as summations of hydrogen bond donors/acceptors (Figure 2.5C).

Figure 2.5 Cheminformatic analyses of screening libraries. (A) Partition coefficient (cLogD7.4) (B) polar surface area (PSA), and (C) the summation of hydrogen bond donors (HBD) and hydrogen bond acceptors (HBA) versus molecular weight for screening library compounds (blue) known Gram-negative antimicrobials (red) and confirmed hit compounds (green). In contrast to Gram-negative antimicrobials, library compounds demonstrated increasing lipophilicity (cLogD7.4) with increasing molecular weight. Opposite trends were observed for polar surface area and summations of hydrogen bond donors/acceptors.

2.5 Discussion

A screen of commercially available small molecule libraries was performed to identify carbapenem potentiators and direct antimicrobial inhibitors of a representative Klebsiella pneumoniae carbapenem-resistant clinical isolate. The goal was to identify hits that could be further improved upon using medicinal chemistry approaches. Furthermore, the whole cell screening approach was agnostic as to potential mechanism of action that could be further delineated at a later time for promising scaffolds.

Hits underwent initial triage based on combined use of cheminformatics approaches and data from an orthogonal screen to eliminate eukaryotic cell toxic compounds. However, based on this initial stringent, but likely appropriate down selection, ultimately only a few hits with potentiating activity and no hits with direct activity remained. Many, but not all, of these adjunctive hits retained activity on re-synthesis. The lack of complete reproducibility on re-synthesis is a well- known finding in commercial library screening efforts and may result from contaminants such as heavy metal catalysts, which may confer antimicrobial activity unrelated to the compound under study. Additionally, we observed reduction in activity spectrum of several of the compounds.

Reasons for this are not immediately obvious, but may relate to borderline adjunctive activity, which did not reach a threshold for phenotypic detection with the resynthesized compound. It may also represent contributions of both compound and contaminants in the original commercial preparations that differed from resynthesized compounds.

We hypothesized that a subset of potentiators would represent hits that either directly or indirectly targeted carbapenemase activity. However, tests in isogenic E. coli strains expressing several types of carbapenemases failed to detect synergy with meropenem suggesting effects were specific to only a subset of clinical strains being tested based on shared regulatory and/or biophysical characteristics, potentially a reflection of the diversity of the CRE strain set.

To address target access, we tested previously well-characterized E. coli K-12 strains with known defects in either permeability barrier (lptD) or in a major class of efflux pumps (tolC).

Neither strain allowed observation of carbapenem potentiation in the E. coli K-12 background, suggesting that differences other than efflux or outer membrane permeability barrier accounted for the observed activity spectrum. Interestingly, one compound, KP40, was noted to have direct, but highly variable, antimicrobial activity against the tolC mutant; this high biological variability suggests non-specific interference with bacterial growth, i.e., hitting multiple targets with total assay variability reflecting the sum of the variability of multiple events.

Our goal was to identify compounds that had already passed the high bar for activity against a multidrug-resistant pathogen. In that way there would be a stringent biological triage with hopes of later improving initial activity using medicinal chemistry approaches. Our clinical screening strain is known to encode multiple antimicrobial resistance elements60 and has a very high baseline carbapenem MIC (50 µg mL-1) which is 16 to 32-fold higher than in a laboratory E. coli strain expressing the same carbapenemase gene (data not shown). Therefore, the carbapenem resistance

phenotype observed in this and other clinical isolates is likely complex and polygenic with contributions from efflux pumps, altered porins, membrane and cell wall characteristics, and/or - lactamases with low-level ability to hydrolyze carbapenems.

Unfortunately, compounds with compelling direct or adjunctive antimicrobial activity were not identified through these efforts. One potential explanation for this is that the biological or cheminformatics triage was too stringent. For example, we did not follow up on compounds that demonstrated statistically significant cytotoxicity for J774A.1 macrophages observed in a separate screening effort. Therefore, it remains possible that some compounds identified as eukaryotic cell toxic may have had some degree of selectivity for bacteria that could have been improved upon during structure-activity relationship studies.

Another possibility is that the commercial screening libraries available did not contain sufficiently diverse compounds with physicochemical properties conducive to Gram-negative antimicrobial activity. For example, a prior screening effort of 500,000 compounds at

GlaxoSmithKline against a efflux competent strain of E. coli yielded no confirmed hits.77 This finding was attributed to lack of chemical diversity. Although the chemical space occupied by the libraries examined was not reported, it is well known that commercial and pharmaceutical libraries historically have been optimized for “drug-like” molecules based on metrics such as Lipinski’s rule of five.78

However, antimicrobials in general and Gram-negative agents in particular rarely satisfy these rules.73 Upon analyses of the physicochemical properties of our screening libraries and representative hits, we observed trends suggesting that compounds with characteristics of Gram- negative antimicrobials were underrepresented. Gram-negative antimicrobials typically possess zwitterionic or polar moieties, which facilitate passage of compounds through water-filled

transmembrane porins and entry into the periplasm. However, our libraries and screening hits had a paucity of such compounds. Instead, they were enriched for compounds with lipophilic substituents (high cLogD), decreased number of hydrogen bond donors and acceptors, and decreased polar surface area, which face an increased enthalpic barrier for entry into the Gram- negative cell. Compounds with these attributes are generally less challenging to synthesize and purify and therefore not unexpectedly are overrepresented in screening libraries.

An alternative screening approach using a screening strain with a lower barrier for activity may have been more productive in identifying lead candidates. For example, a screen of 150,000 small molecules using fully antimicrobial susceptible E. coli and Pseudomonas aeruginosa strains identified several confirmed hits with weak activity.79 However, further development of novel compounds from this screen has not been described to the best of our knowledge. Additional perturbation, such as use of a tolC or lptD mutants may further lower the bar for Gram-negative inhibitor detection80 but later require additional chemistry efforts to address efflux and permeability effects that may or not prove productive.

2.6 Conclusion

Taken together, our results support previous observations that Gram-negative antimicrobial lead candidates may be largely absent from commercially available screening libraries. Certainly, this appeared to be the case for compounds with intrinsic activity against a highly multidrug- and carbapenem-resistant Klebsiella pneumoniae clinical strain. Although we were able to find detectable activity for several compounds, the overall potency was low. Therefore, we provide further data that whole cell screening efforts for Gram-negative antimicrobials should be conducted using libraries with diverse scaffolds and substituents outside the Lipinski space, for example, including compounds with greater hydrophilicity.

Further supporting this view, our prior efforts using the same screening strategy to examine known bioactive libraries with higher diversity inclusive of natural products was highly productive. This led to identification of apramycin and several nucleoside analogues as lead direct antimicrobial candidates for development against highly drug-resistant CRE and for the former against MDR Acinetobacter baumannii, where apramycin is now recognized more generally as a candidate for pre-clinical development.81–83. In this earlier screening effort, we also identified potent meropenem adjunctive activity of triclosan confirming the underlying ability of the screening strategy to detect both direct and adjunctive antimicrobials.58 Therefore, the fundamental ability of the whole cell, high throughput screening assay to detect antimicrobials with activity against CRE, and by extension other MDR Gram-negative pathogens of concern, offers promise as libraries with appropriate physicochemical properties become available.

Future studies will involve a structure-activity relationship (SAR) study of the pyrazole- thiazole core-containing compound, KP40. Direct antimicrobial activity against the tolC mutant and observed synergy with meropenem against K. pneumoniae ST258 implicate interest in exploring the effects of structurally diversifying KP40 through tuning its physicochemical properties and maximizing its PEMPO score. Impressive FIC values observed between the library sample (FIC = 0.31) and the “in-house” synthesized sample (FIC = 0.42) suggest great adjunctive activity which could be improved through SAR modification. In addition, the synthesis of KP40 has some advantages in terms of facile synthesis and easily accessible functional group modifications. Through designing structural diversity in KP40 derivatives, FIC and MAC values would be targeted for reduction, thus improving the synergistic potential of the compounds identified in the original study.

Chapter 3: A Divergent Total Synthesis of Streptothricin F

3.1 Introduction to Streptothricins

3.1.1 Introduction

Strepothricin (ST) compounds have been described in the literature for over 80 years as powerful antibiotics. Since first being discovered in 1942 by Waksman and Woodruff,84 the antimicrobial activity of the compound class has been recognized as being effective towards broad classes of MDR bacteria. Unfortunately, ST compounds exhibit some level of inherent toxicity; therefore, STs have not been considered clinically relevant as potential antibacterial therapies. The actual chemical structure of ST compounds took several decades and research groups to be fully elucidated, and the final configuration was confirmed through total synthesis of streptothricin F

(ST-F, 3.1.1).85 Produced by several Streptomyces sp., ST compounds are known to undergo a complex, convergent biosynthesis that has also produced over 80 various derivatives. Semi- synthesis was performed upon ST compounds during the Golden Age of antibacterial discovery as researchers recognized the importance of activity against GNB but needed to avoid acute toxicity.

While the mechanism of action for ST compounds has still not been accurately described in the literature, it’s well understood that inhibition of prokaryotic translation and induced ribosomal miscoding activity contribute to its bactericidal effect.86 The few reports of resistance among STs has been encouraging, but understanding the mode in which STs are rendered inactive could be important towards designing sustainable and safe therapeutics.

STs are most frequently grown from bacterial fermentation broth and isolated as a mixture of ST compounds, known as nourseothricin (NTC). All STs contain a carbamoylated, ᴅ- gulosamine sugar core 3.1.4 to which the ʟ-β-lysine homopolymer 3.1.6, and the guanidine containing streptolidine lactam 3.1.5 are attached (Figure 3.1). The most abundant ST species

within the NTC mixture are ST-F, streptothricin E (ST-E) 3.1.2 and streptothricin D (ST-D) 3.1.3 with concentrations of 65.5%, 4.9% and 29.6% respectively (these values were determined based on LC-MS data from the Manetsch Lab). Commercial lots of NTC vary in ST distribution but most contain >85% ST-F and ST-D.

Figure 3.1 Nourseothricin mixture components and individual moieties.

Isolated from soil Streptomyces lavenduale, Waksman and Woodruff published their findings of the novel substance, streptothricin, that exhibited selective bactericidal activity against

GNB.84 Notable observations besides from the remarkable activity of the isolate included its great water solubility, stability up to 15 minutes at 100 °C and its similarities to an organic base.84 Since the initial isolation of ST, reports of ST compounds have been identified from various

Streptomyces species.87–91

The structural formula was first proposed by the van Tamelen group in 1961, but was ultimately confirmed through synthetic studies performed by the Shiba group.92 The Shiba group were able to confirm the structure of most ST compounds through first assembling the hydrolyzed

streptolidine lactam,93 devising a method for coupling the streptolidine moiety94 and then completing a total synthesis of ST-F.85

3.1.2 Total Synthesis of Streptothricin F

Synthetic studies by the Shiba group were initiated to contribute towards the structural understanding of STs, starting with ST-F. Recognizing the importance of ST-F’s biological activity, the Shiba group desired to design a total synthesis to better understand the antibacterial and toxicity profile for STs. The group aspired to enable analogue generation from their total synthesis and establish a structure-activity relationship (SAR); however, while the total synthetic was accomplished, no synthetic analogues were produced.

The total synthesis of ST-F performed by the Shiba group is carried out through a convergent route first between gulosamine sugar 3.1.10 and protected β-lysine 3.1.11. The formation of gulosamine sugar 3.1.10 is shown in Scheme 3.1 starting from ᴅ-glucosamine 3.1.7.

Yields and reagents are provided according to the literature. Following an initial Cbz protection, the protected glucosamine was subsequently allylated, isolating only a single epimer, and then selectively benzoylated and mesylated resulting in protected glucosamine 3.1.8. A dilute solution of sodium hydroxide was used to deprotect both benzoyl groups, resulting in the formation of epoxide 3.1.9. The 6-position alcohol was acylated and upon exposure to acetic acid, the neighboring Cbz group opened the epoxide and was subsequently protected to give oxazolidinone

3.1.10 of the desired gulos-conformation. The resulting derivative was hydrolyzed and then underwent amidation with protected β-lysine 3.1.11 and benzyl protected to yield amide 3.1.13.95

Scheme 3.1 Formation of protected amide 3.1.13.

Shown in Scheme 3.2, the 4-position MEM was removed with zinc(II) bromide, and the newly deprotected alcohol reacted with chloroacetyl isocyanate which was reduced with zinc to yield carbamoyl 3.1.14. Removal of the glycosidic allyl group gave anomeric 3.1.15 which was activated with p-NBCl and then converted to isothiocyanate 3.1.16 with KSCN. Electrophilic isothiocyanate 3.1.16 was then treated with amino lactam 3.1.17 which provided thiourea 3.1.18

Scheme 3.2 Formation of thiourea 3.1.18.

and united the final coupling partner. Now, the guanidine ring of streptolidine would be cyclized followed by further global deprotection of protected ST-F.

The endgame for the total synthesis of ST-F is illustrated in Scheme 3.3. The guanidine moiety was cyclized through a three-step procedure. First, thiourea 3.1.18 was activated with ethyl iodide to give an isothiourea. Next, the β-amine of the streptolidine group was deprotected with

TFA to yield the primary amine. Finally, desulfization was completed through nucleophilic attack by the streptolidine β-amine which occurred after the addition of base with TEA. Global deprotection was then carried out via hydrogenation with catalytic Pd black under acidic conditions to yield ST-F 3.1.1. The isolated acetate salt of ST-F 3.1.1 was purified with Sephadex size exclusion chromatography, neutralized with Amberlite resin and then converted to the trihydrochloride salt.

Scheme 3.3 Guanidine cyclization and global deprotection of ST-F 3.1.1.

The overall total synthesis of ST-F was carried out over 46 total steps, a longest linear sequence (LLS) of 25 steps and overall yield of 0.0028%. Like the total synthesis, the biosynthesis of ST-F and other STs occur through a convergent approach via three separate coupling partners.

3.1.3 Streptothricin Biosynthesis

Studies towards the understanding of ST biosynthesis began with an interest in elaborating upon the origins of the unusual amino acid streptolidine lactam 3.1.5. Before the correct structure

of ST-F had been characterized, crystallographic techniques were used to determine the structure and chirality of hydrolyzed streptolidine lactam.96 Initial reports on the formula for streptolidine were confirmed through feeding experiments of ʟ-arginine where the biosynthetic pathway was investigated.97 The Gould group described the single, linear route from ʟ-arginine to streptolidine lactam produced by Streptomyces sp. labeled with either 13C and 15N or with 2H.98–100 The complete sequence was elucidated in 2014 with genomic sequencing of the S. lavendulae ST gene cluster

BCRC 12163, high-resolution crystal structure analysis of intermediates and 13C labeling.101

The biosynthesis for streptolidine lactam begins with dihydroxylation via ʟ-arginine β,γ- dihydroxylase of ʟ-arginine 3.1.19 (Scheme 3.4). β,γ-dihydroxy-ʟ-arginine 3.1.20 undergoes α,β- dehydration and subsequent pyridoxal-phosphate (PLP) dependent cyclase mediated ring closure to afford the non-proteinogenic amino acid intermediate, capreomycidine 3.1.22 through dehydroarginine 3.1.21. Comparison of the 13C labeling patterns in capreomycidine 3.1.22 and streptolidine acid 3.1.23 suggest that the six-membered cyclic guanidine of 3.1.22 is then contracted to a five-membered cyclic guanidine. Nucleophilic addition of the α-amine at the central guanidine carbon then occurs through a hydroxylase-peptidase-non-ribosomal peptide synthetase

(NRPS) mediated reaction cascade to form guanidine 3.1.23.101 Following guanidino-ring contraction, a final condensation step affords streptolidine lactam 3.1.5.

Scheme 3.4 Reported biosynthetic pathway for streptolidine lactam 3.1.5.

ᴅ-glucosamine was determined as a precursor to uridine diphosphate (UDP) N-acetyl-ᴅ- glucosamine through feeding experiments in Streptomyces sp..102 The exact nature of the transformations from ᴅ-glucosamine remain ambiguous. It is known that uridine diphosphate

(UDP) N-acetyl-ᴅ-glucosamine is a common nucleotide sugar found in Streptomyces sp. and is therefore treated as the principle biosynthetic precursor for the gulal sugar core in the ST biosynthesis literature.102,103 It was also concluded that all the gulal sugar stereocenters are not configured until after coupling with the streptolidine lactam, and instead the sugar coupling partner in a glycosyl transfer reaction was actually UPD N-acetyl-ᴅ-galactosamine 3.1.24, derived from

10-position epimerization (Scheme 3.5).103 The first convergent step in the ST biosynthesis is the glycosyl transfer reaction between 3.1.24 and 3.1.5 to give 3.1.25 before epimerization of the 9- position providing gulosamine 3.1.26. Carbamoylation of the 10-position alcohol to 3.1.27 and deacetylation of the 8-position amine afford streptothrisamine 3.1.28.

Scheme 3.5 Reported biosynthetic pathway for streptothrisamine 3.1.28.

The β-lysine moiety of STs is derived directly from α-lysine 3.1.29 through a 2,3- aminomutase reaction catalyzed by the S-adenosyl methionine dependent amino mutase, ʟ-lysine

2,3-amino transferase, shown in Scheme 3.6.98,104–107 This aminomutase reaction is dependent upon the formation of a 5’-deoxyadenosyl radical (Ado●) and a PLP cofactor which stabilizes the amino group participating in the amino transfer.103 Enzyme bound PLP is first transferred to a free lysine to give PLP-bound α-lysine 3.1.30.105–107 Abstraction of a hydrogen from the β-position of

3.1.30 gives radical 3.1.31.105–107 This β-radical then rearranges to the carbonyl stabilized α-radical

3.1.33 through aziridine 3.1.32. The resultant α-radical then abstracts a hydrogen from Ado● to give PLP-bound β-lysine 3.1.34. PLP transfer then liberates free ʟ-β-lysine 3.1.35. The origin

Scheme 3.6 Proposed biosynthetic pathway for ʟ-β-lysine 3.1.35.

of α-lysine in Streptomyces sp. was determined to come from the diaminopimelic acid (DAP) cycle which, is preceded by the tricarboxylic acid cycle in the production of aspartic acid, a necessary precursor of the DAP cycle.98,104

The coupling of streptothrisamine 3.1.28 with the β-lysine moiety 3.1.35 of ST-F proceeds through two non-ribosomal peptide synthetases (NRPS), characterized in Streptomyces rochei

NBRC12908, denoted open reading frame 5 (ORF) and ORF 18 based on coding regions identified in the ST gene cluster (Scheme 3.7).108 ʟ-β-lysine 3.1.35 is first adenylated with adenosine monophosphate (AMP) in the adenylation (A) domain (shown in green) in ORF 5 to AMP-β-lysine

3.1.36. 3.1.36 is then loaded onto the thiolation (T) domain (shown in red) of ORF 18 to produce

3.1.37. Next, 3.1.28 diffuses into ORF 18 and the condensation of 3.1.28 and the 3.1.37 is catalyzed by the adjacent condensation (C) domain (shown in blue) of ORF 18 to give ST-F 3.1.1.108

Scheme 3.7 Biosynthetic pathway converging towards ST-F 3.1.1.

3.1.4 Streptothricin Derivatives

Although there have been over 80 ST compounds identified in the literature, only a handful have been structurally and biologically characterized. NTC is the major component from bacterial isolates; however, Streptomyces sp. produce ST compounds differing from the NTC mixture including regioisomers, stereoisomers and a wide variety of β-lysine chain analogues.

Additionally, a library of semi-synthetic, β-lysine derivatives of ST-F were synthesized and tested for in vivo activity.109–113 The current reports for the activity and toxicity profile of ST compounds

have given some insight towards future SAR, but it’s clear that the lack of systematic SAR studies is a major gap in the ST literature.

Beginning with modifications to the ᴅ-gulosamine sugar core 3.1.4, only 2 variants have been observed in the literature: migration of the 10-carbamoyl group to the 12 position and a deoxy, ᴅ-gulosamine sugar known as fucothricin. The biosynthetic pathway to produce the 12- carbamoyl STs was hypothesized to undergo a similar route to the 10-carbamoyl STs.103 12- carbamoyl ST variants (Figure 3.2) have been isolated and characterized for their biological activity. In terms of their activity, 3.1.38-3.1.40 are orders of magnitude less active against both

GPB and GNB compared the components of the NTC mixture.91,114,115 Stereoisomers 3.1.41-3.1.45

Figure 3.2 12-carbamoyl ST-F derivatives and fucothricin.

differing at the 7 and 2-positions constitute cis-diamino gulosamine or cis-streptolidine. All diastereomers are inactive; however, it’s difficult to attribute the inactivity as a result of their stereochemistry since all are also acetylated at the β-amine. N-acetylation at the β-amine of the β- lysine residue is well known to cause total loss of activity for STs.116 12-carbamoylated, hydrolyzed lactams with varying stereochemistry found among 3.1.46-3.1.48 (also inactive) could be interesting to investigate as inactivity could be a result of carbamoyl migration or acetylation, but not necessarily lactam hydrolysis. Interestingly, fucothricin 3.1.49 maintains some level of activity against GNB (MIC for E. coli 2.5 µg/mL) without observed murine toxicity at 500 mg/kg.117

Streptolidine derivatives include removal of the 4-position alcohol, methylation of the lactam amine and hydrolysis of the lactam ring. Figure 3.3 shows structures of streptolidine analogues first with a lactam ring and then next with a hydrolyzed lactam. ST-F analogue 3.1.51 previously isolated and characterized as the natural product albothricin exhibits potent and broad antibacterial activity, displaying reduced toxicity even at high doses of 200 mg/kg (i.v. administration).118 N-methyl ST-F119 3.1.52 and N-methyl ST-D90 3.1.56 showed strong antimicrobial activity against GPB, GNB and fungi; however, the toxicity profile has never been studied for these derivatives. N,N-Dimethylation at the lactam and β-lysine amide sites were observed in 3.1.53-3.1.55 and experienced antibacterial activity similar to STs. N,N-dimethyl ST-

F120 3.1.53 exhibited a reduced toxicity by two orders of magnitude, but necrotic symptoms were noted with in vivo experiments performed with glycinothricin 3.1.54 and BD-12 3.1.55.121 While only structural data has been recorded for 3.1.57, other hydrolyzed lactam compounds ST-F acid

3.1.58 and ST-D acid 3.1.59 demonstrated a reduction in activity but ST-D acid 3.1.59 in particular exhibited significant increases in selectivity towards GNB in vitro.122,123

Figure 3.3 Streptolidine derivitives of STs.

ST isolates with deviations from NTC with respect to the β-lysine moiety are the most structurally diverse for all the ST functional groups. Deviations from the typical β-lysine found in

NTC were also observed in streptolidine isolates 3.1.54 and 3.1.55. Several of the isolates found in Figure 3.4 were observed to be either inactive (3.1.63-3.1.65) or possessing reduced activity

(3.1.67) while others (3.1.60, 3.1.61 and 3.1.66) have never been biologically characterized.75,108,120,124,125 Only citromycin 3.1.62 was shown to display similar activity to NTC; however, typical necrotic symptoms were observed with in vivo studies.75 More investigation into the SAR of the β-lysine moiety was explored through semi-synthesis from ST-F.

Figure 3.4 β-lysine derivatives of STs.

Semi-synthetic approaches were performed upon both citromycin 3.1.62 and ST-F 3.1.1 from 1972-1974 across a five-part publication by the Taniyama group.109–113 In the first study of citromycin 3.1.62 shown in Scheme 3.8, the parent natural product was treated with methanolic ammonia, 3M HCl or N-acetoxysuccinimide resulting in 3.1.61 and 3.1.68-3.1.69. Activity was void in semi-synthetic compounds 3.1.61 and 3.1.69; however, it was revealed that the toxicity of hydrolyzed lactam 3.1.68 exhibited half the toxicity of parent 3.1.62 although antimicrobial activity for 3.1.68 was not shown.109 ST derivatives 3.1.70-3.1.75 (Figure 3.5) were synthesized from ST-F via condensation with the appropriate aldehyde under basic conditions, and 3.1.74-

3.1.77 were hydrogenated thereafter in the presence of platinum oxide.110 Phenylalkyidene derivatives 3.1.70-3.1.73 presumably hydrolyzed to the parent antibiotic and displayed activity similar to ST-F while 3.1.74-3.1.77 were inactive. Through independent acetylation of both the β- amine or ε-amine of the β-lysine chain of ST-F, a dramatic loss of activity was observed and both amines were deemed to be essential for activity.111,112 Finally, amino-acid semi-synthetic derivatives 3.1.78-3.1.85 (Figure 3.5) were produced from parent ST-F through first reaction with

a Cbz-protected, activated ester of each amino acid. Protected analogues were then hydrogenated and purified before being tested for in vitro and toxicity studies.113 Glycyl and lysyl derivatives

3.1.78 and 3.1.83 had single digit MIC values of 3 ug/mL against E. coli K-12 similar to ST-F and even comparable activity towards the MDR strain of P. aeruginosa (100 ug/mL) while the remaining amino acid analogues showed less notable activity. Toxicity studies showed that both analogues had a reduction in toxicity compared to ST-F; however, prolonged toxicity remained.

Scheme 3.8 Semi-synthesis from citromycin 3.1.62.

Figure 3.5 Semi-synthetic, β-lysine analogues of ST-F.

Past literature regarding ST toxicity use only semi-quantitative measures of activity for impure compound and where very large doses were used presumably exceeding 100mg/kg.126

More recent studies in mice with purified STs showed that toxicity varies with the length of the β-

126,127 lysine chain: ST-F LD50 of 300 mg/kg compared with ST-D LD50 of ~10 mg/kg. For

128 comparison, the murine LD50 for gentamicin is 52 mg/kg i.v.; colistin methanesulfonate 40 mg/kg i.p.;129 and tobramycin 260 mg/kg i.p.130 Although several natural product variants with modifications of the streptolidine ring and replacement of the gulosamine and β-lysine moieties have been described, the lack of systematic SAR comparisons for these variants is a major gap in the streptothricin literature. Understanding the bactericidal mechanism of action by STs and mapping the binding pocket could be the first step towards establishment of a rational SAR strategy.

3.1.5 Mechanism of Action by STs

As ST compounds have a similar spectrum of activity compared to aminoglycosides (AGs), it has been long hypothesized that the two classes carry out their antimicrobial mechanism of action similarly. The ability for ST compounds to inhibit protein synthesis through the miscoding of protein translation has been well characterized; however, its binding site has only been hypothesized and never confirmed. The Kirby group had observed unexpected activity of ST-F to a target altered mutant, otherwise resistant to AG therapeutics [Kirby, unpublished], suggesting the possibility for a novel mechanism of action. This novel mechanism of action was further explored through cryoEM studies indicating that STs bind to the 50S ribosomal subunit in contrast to AGs binding to the 30S subunit [Yu, unpublished]. ST-F isolated for these experiments was prepared by the Manetsch Lab and the protocol is further explained in Supporting Material 6.1.

As GNB inch closer towards pan-resistance, the elucidation of a novel mechanism of action is extremely important to keep pace in the development of antimicrobials.

Following the identification of STs as powerful antimicrobial agents, interest in its mechanism of action arose. It was determined that STs inhibit polyuracil (U) directed poly- phenylalanine synthesis, while ST compound boseimycin, preferentially inhibited protein synthesis in intact bacterial cells.131,132 Additionally, the Haupt research group confirmed that ST-

F specifically altered protein synthesis without affecting DNA or RNA syntheses through poly(U) misreading observed for isoleucine, leucine, serine, and tyrosine (Figure 3.6).86 ST-F was found to be concentration dependent in terms of its miscoding activity as is consistent with AGs and could be attributed to multiple binding sites on the ribosome.133

Figure 3.6 Effect of ST-F on DNA, RNA and protein syntheses in intact cells of E. coli 15 TAU. (○) without antibiotic, (Δ) with 2 x 10-5 M ST-F, (●) with 3 x 10-5 M ST-F.

The most well-known mechanism of action for AGs that inhibit protein synthesis is through binding to the A-site on the 16S ribosomal RNA of the 30S ribosomal subunit.32 Resistance towards AGs can include enzymatic modification of the AG, enzyme or mutation-based target site

modification, and extracellular efflux. In a recent study by the Kirby group, STs were immune to both classes of 16S rRNA methyltransferases that block AG activity [Kirby, unpublished].

Consistent with these observations and in contrast to miscoding AGs that bind to 16S rRNA, cryo-

EM studies found that ST-F binds to a novel site on the 50S ribosomal subunit [Yu, unpublished].

Other AGs are known to bind to the A site of the smaller 30S subunit (Figure 3.7C), forming interactions with 16S ribosomal RNA and causing extensive miscoding.134 However, based on a resolved cryo-EM structure, we now believe that ST-F binds to the large 50S subunit, in its own, discrete pocket. Instead of binding in the ‘AG’ region, STs bind closer to the macrolide binding site on the larger 50S subunit (Figure 3.7B), indicating a novel mechanism of action.

Figure 3.7 Antibiotic docking in the ribosome.134 (A) Three different antibiotic binding sites in the 70S ribosome. (B) The macrolide binding site (pink, azithromycin; yellow, clarithromycin; blue, erythromycin). (C) The aminoglycoside binding site (pink, amikacin; brown, gentamicin; purple, kanamycin). (D) The tetracycline binding site (orange, tetracycline; tan, tigecycline; green, eravacycline). Results for all classes represent known binding modes for the different antibiotics, showing that these binding sites are conserved in the A. baumannii ribosome.

3.1.6 Resistance to ST compounds

From 1981-1988 in Germany, NTC was used as a growth promoter in pigs.116 Less than one year later, plasmid-borne resistance was observed in the gut of humans directly working with the pigs as well as humans living in the surrounding area.116 Since then, streptothricin compounds have been discovered to confer resistance to several streptothricin acetyl transferaces (SATs) that specifically modify the β-lysine, β-amine, resulting in loss of activity.135 Based on genome studies by the Kirby group, MDR/XDR A. baumannii indicate that that ST resistance does not show appreciable overlap with other forms of multidrug-resistance possessing clinical concern (e.g., carbapenemases) [Kirby, unpublished]. Aside from acetylation of the β-lysine, β-amine, the

Takagi group reported hydrolysis of the streptolidine lactam realized through cloning of a gene whose product confers ST resistance as a form of enzymatic modification.123 The product of the sttH gene was found to catalyze the hydrolysis of the amide bond of streptolidine lactam which eliminated activity for ST-F and decreased the activity for ST-D as both were converted to their respective strepotolidine acids. Interestingly, ST-D still maintained some level of activity against prokaryotes, with a major drop in toxicity towards eukaryotes.123 Nevertheless, existence of resistance, some plasmid-borne, suggests that medicinal chemistry efforts should include attempts to block/alter the modification sites on the β-amine of the β-lysine residue and through preservation of the streptolidine lactam.

3.2 Total Synthesis Strategy for Streptothricin F

3.2.1 Introduction

Past experience indicates that it is possible to modify AGs to alter toxicological properties and block inactivation by AG modifying enzymes (AME)42,43 e.g., sisomicin was modified to plazomicin which in clinical trials did not induce ototoxicity, showed zero to minimal renal

toxicity, and is immune to most AME.136–138 Structurally unique AGs such as apramycin exist that presumptively cause neither ototoxic nor renal toxic side effects, likely because of enhanced selectivity for prokaryotic versus mitochrondrial ribosomes43 and are unaffected by almost all

AME.82,139,140 It was also possible to alter the non-selective 50S ribosomal subunit inhibitor, blasticidin, by extending its structure141 to create a pyrrolocytosine series with excellent selectivity for GNB and immunity to existing resistance mechanisms.142 The GNB activity of ST-F appears similar ST-E and ST-D,91 yet is substantially more selective [Kirby, unpublished]. We hypothesize it should be possible, aided by structural and functional data, to derivatize ST-F to further increase its safety margin, block SAT-based inactivation and avoid lactam hydrolysis. Such modifications to the structure of ST-F would require a total synthetic strategy tailored towards medicinal chemistry.

The original ST-F total synthesis is poorly suited for a medicinal chemistry campaign as it is purely linear, lacks practical divergent steps, requires a significant number of steps to set the stereochemistry of the diamines in the ᴅ-gulosamine core, and relies on dated reactions conditions which do not enable rapid analogue generation. Furthermore, the original synthesis of ST-F installs the β-lysine chain early. In contrast, we realized a convenient synthesis enabling structural diversification at the end of the synthesis. We hoped to design and execute a ST synthetic route

(Figure 3.8), by which gulosamine precursor 3.2.1 is first modified by β-lysine and then by streptolidine (Route A) to provide streptolidine analogues or, a route where streptolidine is first added, followed by β-lysine (Route B) to synthesize analogues not otherwise accessible through semi-synthesis or from biosynthetic starting materials. The two protected amines in precursor 3.2.1 would act as functional handles to enable the synthesis of ST-F analogues; however, first a total synthesis would need to be completed from this intermediate.

Figure 3.8 Convergent approach towards total synthesis of ST-F.

We planned to develop and execute an efficient and robust total synthesis for ST-F and determine the relevant properties of ST-F in several in vitro and in vivo systems through analogue generation. These studies would serve as foundation for SAR exploration of the three primary molecular constituents of this molecule to an extent not previously possible. We would then pursue structure-guided derivatization assisted by the solved structure of ST-F associated with the 50S subunit with goals of blocking inactivation by SAT enzymes, preserving the streptolidine lactam and increasing selectivity (increase in vitro CC50, decrease renal toxicity), while at the same time preserving and extending its activity spectrum and potency against target MDR pathogens.

3.2.2 Diamine Enabled Functional Handle

To develop a diaminated functional handle such as 3.2.1, a diamination approach would need to enable discrimination between the anomeric and 8-position amine as well as result in a

7R,8R-trans configuration. The functional handle would need to be incorporated to a gulosamine sugar, involve discretely removed protecting groups, and conserve the stereochemistry at the anomeric position upon deprotection. Most importantly, constructing and maintaining the proper gulosamine sugar conformation would be essential. We decided to begin with the gulosamine sugar as the starting point for development of the synthesis.

Following the initial isolation and structure elucidation for ST-F, it was recognized as the first ᴅ-gulose containing natural product.143 15 years after isolation, two syntheses of ᴅ-gulosamine hydrochloride were performed very close to one another: the first by epimerization from ᴅ- galactosamine and the next from ᴅ-xylose.143,144 N-acetyl-ᴅ-gulosamine was soon after synthesized through a double inversion of a 3,4-dimesylated-N-acetylated-glucosamine derivative via solvolysis assisted by the participating NAc group and through a 3,4-epoxide intermediate.145 2 years after the total synthesis of ST-F, ᴅ-gulosamine hydrochloride was formed from ᴅ- glucosamine over 4 steps.146 After the biosynthetic pathway for ST-F was elucidated, a method for preparation of UDP-ᴅ-GulNAc 3.1.24 was provided in 6 steps.103 Just within the last year, a method for the synthesis of gulosamine thioglycosides was described in 11 steps from glucosamine hydrochloride.147 Unfortunately, each approach contains a hurdle towards the synthesis of an intermediate such as 3.2.1 that either requires deprotection of the anomeric position alcohol

(resulting in a mixture of anomers) or does not enable a systematic installation of the 10-position carbamoyl without extensive regioisomer formation. Moreover, these methods typically lack substrate generality and often result in variable stereoselectivity, especially in complex contexts.148

Therefore, we turned our attention towards glycosylamine synthesis via a sulfamidate intermediate that could provide a more direct approach towards diamination.

The group of K.C. Nicolaou published an extensive review of novel uses of classic and modified Burgess reagents.148 The most appealing aspects of the publication highlighted the formation of cyclic sulfamidates from diol sugars with predictable α:β ratios (Figure 3.9A), followed by opening of the sulfamidate with sodium azide (Figure 3.9B) to yield a 1,2-trans- diamino sugar. Epimerization at the anomeric center could be avoided by gentle deprotection of an alloc protecting group through utilizing excess diethylamine and catalytic amounts of palladium and triphenylphosphine-3,3′,3′′-trisulfonic acid trisodium salt (TPPTS) (Figure 3.9C).

A hypothesized mechanism by the Nicolaou group suggests sulfamidate formation occurs through a two-step procedure shown in Scheme 3.9. Upon addition of 2 equivalents of Burgess reagent 3.2.3 to protected glycol 3.2.2, the sulfamide formation could occur through two different

Figure 3.9 Examples of sulfamidate formation, opening and resulting deprotection.148 (A) Diol sugar are converted to cis-cyclic sulfamidates, dependent upon stereochemistry of the 3-position. (B) Cyclic sulfamidates can be opened resulting in a trans-1,2 diamino bifunctional handle. (C) Gentle deprotection of the alloc protecting group can be carried out and stereochemistry preserved at the anomeric position.

pathways. The 2-position could undergo SN2 attack of the electrophilic, anomeric position through intermediate 3.2.4 or through an oxonium intermediate 3.2.6 by means of α-elimination from 3.2.5.

The 2-position would dictate the preferred stereochemical orientation of the nitrogen attack. Both pathways result in the same, 1,2-trans-difunctionalized glycosylamine product 3.2.8 following nucleophilic attack of the sulfamidate 3.2.7. We envisioned a possibility where the trans-1,2 diamino bifunctional handle found in Figure 3.9B could be utilized as a complex intermediate similar to 3.2.1 where we could carry out a convergent total synthesis or diverge towards various streptolidine or β-lysine analogues. First, we would carry out synthesis of a model system to provide proof of concept that such a design could be applied towards our diversity-oriented, total synthesis for ST-F.

Scheme 3.9 Proposed mechanism for the conversion of diol sugars to 1,2-trans-difunctionalized glycosylamine products.148

3.2.3 Model System Synthesis

Our model system synthesis had two goals to accomplish in order to provide proof of concept towards utilizing the Burgess reagent as a key step towards a ST-F total synthesis. First,

we wanted to be sure that an azide-opened sulfamidate would enable a divergent approach towards two different routes of ST-F analogue synthesis. Next, we wanted to be sure that the resulting anomeric amine would be stable upon deprotection conditions, ensuring a single diastereomer was delivered to the next step.

Our model system would proceed through two different routes shown in Figure 3.10. In

Route A, amidation of an azide-reduced 3.2.9 to β-lysine model 3.2.10 resulting in amide 3.2.12 would occur first followed by installation of the streptolidine model 3.2.11 before global deprotection to yield model system 3.2.14. Alternatively, in Route B, guanidine formation with

3.2.11 would occur first to give intermediate 3.2.13 followed by amidation. Through completion of this divergent approach towards model system 3.2.14, we would have proof of concept that the total synthesis of ST-F could be pursued through a similar method.

Figure 3.10 Divergent strategy towards model system synthesis for proof of concept.

Beginning with the synthesis of 3.2.9, we used deoxy-glucal 3.2.15 as the starting material because the 3S stereochemistry was required to direct the 2-position stereoselectivity for the future dihydroxylation reaction. A protecting group swap was performed from the deoxy-glucal 3.2.15 from acyl to benzyl as incompatibilities with acyl groups had been reported148, and we desired hydrogenation as a global deprotection step. Benzylated deoxy-glucal 3.2.16 was dihydroxylated with OsO4 to give 3.2.17 as a 1:1 mixture of anomers. Alloc-modified Burgess reagent 3.2.18 was prepared over 2 steps starting from chlorosulfonyl isocyanate and reacted with anomeric 3.2.17 to yield cyclic sulfamidate 3.2.19 in moderate yield where only the β-sugar was isolated.148 Finally, cyclic sulfamidate 3.2.19 was opened through warming with sodium azide to yield 1,2-trans-

1 difunctionalized glycosylamine 3.2.9 with a C4 conformation. The stereochemical configurations of 3.2.19 and 3.2.9 were confirmed through analysis of NMR coupling constants between protons located on C-1 and C-2. In 3.2.19, a coupling constant of J1,2 = 4.9 Hz is observed, typical for glycosyl protons with an axial-equatorial relationship.149,150 Upon opening of the cyclic sulfamidate, that relationship is shifted to equatorial-equatorial as sodium azide attacks from the

149,150 bottom face, resulting in a decreased coupling constant of J1,2 = 3.2 Hz.

Scheme 3.10 Synthesis of azide-opened sulfamidate 3.2.9.

We were disappointed with the yield observed in the synthesis of sulfamidate 3.2.19 and performed a series of experiments attempting optimization of the reaction conditions. Shown in

Table 3.1, we varied temperature, time, addition of base, equivalents of Burgess reagent 3.2.18 and solvent system. Based on previous literature151, we suspected that the Burgess reagent could be undergoing thermal decomposition, so we synthesized a ‘thermally-stable’ Burgess reagent

3.2.20 along with DMAP variant 3.2.21 to observe their effect upon sulfamidate yield.

Entry Reagent (eq.) Temperature Solvent Base (eq.) Time Yield 1 3.2.18 (2.5) 80 °C 4:1 THF:DCM - 6 h 31% 2 3.2.18 (2.5) 80 °C 4:1 THF:DCM - 24 h 8% 3 3.2.18 (2.5) 50 °C 4:1 THF:DCM - 24 h 10% 4 3.2.18 (2.5) 80 °C 4:1 THF:DCM TEA (3) 6 h 16.5% 5 3.2.18 (2.5) rt 4:1 THF:DCM - 24 h 7% 6 3.2.18 (4) 80 °C 4:1 THF:DCM - 6 h 35% 7 3.2.18 (8) 80 °C 4:1 THF:DCM - 6 h 10% 8 3.2.18 (4) 80 °C THF - 6 h 47% 9 3.2.20 (4) 80 °C THF - 6 h 42% 10 3.2.21 (4) 80 °C THF - 6 h NR

Table 3.1 Reaction condition optimization for sulfamidate 3.2.19 formation.

Our initial attempts to reproduce the literature yields ranging from 74-91% with exact experimental conditions were met with a yield of 31% shown in entry 1 of Table 3.1. We allowed for longer reaction conditions in entry 2 which ultimately led to decomposition of product. A lowered temperature in entry 3 gave lowered yield even though consumption of starting material was observed. The addition of TEA in entry 4 was used to attempt to accelerate the initial diol

‘attack’ of the Burgess reagent as illustrated in the hypothesized mechanism shown in Scheme

3.10, although an increase in reaction yield was not observed. Increasing the equivalents of

Burgess reagent 3.2.18 from 2.5 to 4 did increase the yield but increasing equivalents to 8

decreased yield. Based on other applications of the Burgess reagent in the literature, we changed solvent systems to just THF, and observed a final increase in reaction yield.152 ‘Thermally stable’

Burgess reagent 3.2.20 performed similarly to 3.2.18 under the most optimized conditions, but

DMAP variant 3.2.21 consumed starting material without producing any product.

We followed a protocol reported for the synthesis of 3.2.11 that would be used as our model for the streptolidine moiety (Scheme 3.11).153 Protected β-lysine model 3.2.10 could be purchased.

Scheme 3.11 Synthesis of streptolidine model 3.2.11.153

Upon collecting all coupling partners, the convergent portion of the model system synthesis was performed. We first attempted to carry out Route A of Figure 3.12 shown in Scheme 3.12 where azide 3.2.9 was first reduced through Staudinger conditions to yield amine 3.2.24 and then amidated with protected β-lysine model 3.2.10 to yield amide 3.2.25.154 Amide 3.2.25 was deprotected using a method previously described to prevent epimerization at the anomeric center.148,155 Although the anomeric proton for amine 3.2.12 was broad and the 2-position signal was crowded by benzyl protons, the 3-position signal gave coupling constant values of J2,3 = 4.2

Hz, suggesting an axial-equitorial relationship between the protons and a conservation in configuration.149,150 Amino sugar 3.2.12 was then coupled with the streptolidine model isothiocyanate 3.2.11 to give thiourea 3.2.26. Cyclization towards guanidine 3.2.27 was carried out over two steps. First, the Boc group was deprotected using TFA, coordination to sulfur was carried out through mercury(II) chloride catalysis, and then elimination with TEA gave guanidine

3.2.27.156 Mercury(II) chloride was selected as the desulfurization agent after attempts with others such mercury(II) oxide and EDCI were unsuccessful.157,158 Global deprotection was attempted

with hydrogenation under acidic conditions; however, incomplete deprotection was observed in all cases.159,160 Instead, global deprotection with boron trichloride resulted in complete deprotection of Bn and Cbz groups.161 The model system product 3.2.14 was purified through reverse-phase chromatography. NMR analysis of the deprotected model system made it clear that stereochemistry of the deprotected alloc group was conserved through to the final product as well

1 149,150 as the C4 conformation made clear by the coupling constant J1,2 = 1.8 Hz.

Scheme 3.12 Completion of model system via Route A.

After the completion of the model system through Route A, we attempted to diverge from azide 3.2.9 towards Route B of Figure 3.12 shown in Scheme 3.13. Alloc deprotection of 3.2.9 gave one single diastereomer for amino sugar 3.2.28. Again, the anomeric signal was broad, and the neighboring proton was crowded with other signals, making it difficult to decipher the coupling constants. Amine 3.2.28 was exposed to streptolidine model isothiocyanate 3.2.11 in DMF, resulting in the formation of thiourea 3.2.29. This thiourea was then converted to guanidine 3.2.31

1 under the same conditions described previously which demonstrated a C4 configuration.

Scheme 3.13 Partial completion of model system via Route B.

Thinking forward to when we would carry out the total synthesis for ST-F, we considered altering the protocol for guanidine formation. Instead of reacting amino sugar 3.2.12 with isothiocyanate 3.2.11 and requiring 2 additional steps to close the cyclic guanidine and form

3.2.27, we could instead react an activated thiourea. Such a sequence would also avoid the use of toxic mercury(II) chloride. Therefore, we developed a route shown in Scheme 3.14 where

Scheme 3.14 Synthesis of 3.2.27 using isothiourea 3.2.32.

isothiourea 3.2.32162 is warmed with amine 3.2.12 to give the same guanidine 3.2.27 from the previous sequence of reactions.

Through our model system studies, we identified both strengths and weaknesses for using the Burgess reagent as the key step for 1,2-trans-difunctionalized glycosylamine formation. We could expect low yield for sulfamidate formation and broad anomeric NMR signals following alloc group deprotection. We recognized that possibly the lack of epimerization could have been a result of the anomeric effect. Based on coupling constants, we know that the NHAlloc group was in the axial position prior to deprotection for both 3.2.9 and 3.2.25. Following the deprotection, the axial position of the heteroatomic, deprotected amine is stabilized by the anomeric effect. Shown in

Figure 3.11, configuration 3.2.33a involves a stabilizing, partial neutralization of dipoles whereas

3.2.33b contributes towards a destabilized, partial intramolecular addition of the two dipoles.

Mostly, the anomeric effect is a caused by hyperconjugation resulting from the anti-periplanar configuration of the amine group. The axial amine in 3.2.33a has its antibonding orbital in plane with the C-O lone pair bonding orbital, allowing for donation into its antibonding σ*-orbital, thus shortening and strengthening the bond. No such delocalizing effect is observed for anomer 3.2.33b, and likely 3.2.33a exists as the major anomer. Going forward with the total synthesis of ST-F, the substituent configuration could be especially critical when considering the importance of maintaining an axial amine that could prevent epimerization from the desired anomer in solution.

Figure 3.11 Anomeric effect on retention of stereochemistry following alloc deprotection.

3.2.4 Synthesis of Carbamoylated ᴅ-Gulosamine

To carry out a total synthesis for ST-F in a method demonstrated by the model system, it would be necessary to perform the key Burgess reagent addition step upon a gulal sugar.

Construction of gulal sugars has not been widely explored in the literature; however, there have been a few recent advances. The first reported synthesis of gulal sugars was carried out by the

Danishefsky group through thio-phenol Ferrier-type/Mislow-Evans [2,3] sigmatropic rearrangements starting from tri-O-acetyl-ᴅ-galactal over 3 steps (Scheme 3.15).163 First, tri-O- acetyl-ᴅ-galactal 3.2.34 is converted to α-thiophenol pseudoglycal 3.2.35 through the Ferrier rearrangement. Oxidation to allylic sulfoxide 3.2.36 then undergoes a Mislow-Evans rearrangement to sulfenate 3.2.37 which is in turn exposed to a thiophilic nucleophile, resulting in gulal 3.2.38.164–166 Danishefsky’s group had used mCPBA and piperidine for the oxidizing agent and thiophilic nucleophile, respectfully; however, DMDO and diethylamine have been used recently for more general applications.167

Scheme 3.15 Danishefsky gulal synthesis through Ferrier-type / Mislow Evans rearrangement.

Another method for gulal sugar formation comes from the Crotti group starting from tri-

O-acetyl-ᴅ-glucal 3.2.39 over 6 steps (Scheme 3.16).168,169 The process begins with deacylation of

3.2.39 to yield ᴅ-glucal 3.2.40. The 6-O-position is selectively protected in 3.2.41 and then silylated with TBS chloride in 3.2.42. The 3-O-TBS protected 3.2.42 was mesylated into 3.2.43 and then deprotected with TBAF to give mesyl alcohol 3.2.44. Upon deprotonation of the allyl alcohol, epoxide 3.2.45 forms which is exposed to tetra-butyl ammonium trimethyl silanolate and then hydrolyzed to give 6-O-benzyl-ᴅ-gulal 3.2.46.

Scheme 3.16 Conversion of tri-O-acetyl-ᴅ-glucal 3.2.39 to 6-O-benzyl-ᴅ-gulal 3.2.46.

Although the method demonstrated by the Danishefsky group is carried out in half the steps of the Crotti method, the cost of starting material vastly differs. Bulk purchase of tri-O-acetyl-ᴅ- galactal 3.2.24 costs $18/g or could be synthesized from β-ᴅ-galactose pentaacetate ($2.5/g) over

2 steps.170 Nonetheless, 4,6-di-O-acetyl-ᴅ-gulal 3.2.28 would require a protecting group swap from acetyl to benzyl and possibly extensive optimization for selectivity between protection of the 4 and 6-position, thus making tri-O-acetyl-ᴅ-glucal 3.2.39 ($0.91/g) our desired starting material.

We set forward with the Crotti route and made some alterations to the method (Scheme

3.17). Initial deacylation was carried out under identical conditions and gave quantitative results.

We deviated from the reported, selective 6-position benzylation and pursued a route performed with the use of the Taylor catalyst 3.2.47.171 The Taylor catalyst 3.2.47 coordinates to the 3 and 4-

O-positions and allowed for reproducible yield more than double of that reported for the same product by the Crotti method. We further optimized the route by reducing the silver(I) oxide equivalents from 2 to 1 while no reductions in yield or selectivity towards benzylated product

3.2.41 were observed. We achieved reported yields through silylation and mesylation. Near quantitative yields were observed for the TBAF deprotected product 3.2.44 and similar yields were observed for epoxide formation and opening towards 6-O-benzyl-ᴅ-gulal 3.2.46. We experienced the best yields when potassium trimethyl silanolate (used in the synthesis of tetra-butyl ammonium trimethyl silanolate) was freshly prepared.172 All reactions could be performed on scales up to 50 g (with the exceptions of the final step as dilute conditions were required) with reproducible yields.

Scheme 3.17 Optimization of Crotti route to 6-O-benzyl-ᴅ-gulal 3.2.46.

Protection and functionalization of the newly produced gulal 3.2.46 was then explored.

Attempts to achieve selective carbamoylation for the 4-O-position via reaction with trichloroacetyl isocyanate provided a mixture of 3 and 4-O-position carbamoylation as well as di-carbamoylation.

We then pursued a method to incorporate selective benzylation of the 3-O-position and carbamoylation of the 4-O-position. The literature for selectivity of the 3 and 4-O-position towards protection of gulal sugars is not thoroughly explored, so initial trials were performed to learn more

about the reactivity of gulal 3.2.46 in Table 3.2.169 Typical benzylation conditions were entirely unselective and resulted in a distribution between mono benzylation products 3.2.48 and 3.2.49 as well as di-O-benzylated product 3.2.50. The Crotti group reported selective 3-O-silylation using

TBSCl, imidazole and DMAP; however, only starting material was observed when using benzyl bromide.169 Gentle benzylation conditions shown in entry 3 also resulted in recovery of starting material. The use of tin catalysts when carrying out selective alkylation of adjacent diols has been reported to proceed smoothly, especially towards the 3-O-position; however, neither examples for gulal nor 3,4-trans sugars have been reported.173,174 Still, we explored the use of tin towards our desired 3-O-benzylation product 3.2.48. In entry 4 with the use of dimethyl tin, we mostly reclaimed starting material, but obtained a small amount of product and the undesired regioisomer.173 Dibutyltin oxide in entry 5 with toluene as the solvent gave better selectivity; however, starting material was not recovered. Neat conditions with dibutyltin oxide, triethylamine and tetra-butyl ammonium iodide gave the best results, providing the highest yield with 2-fold selectivity of 3-O-benzylation relative to benzylation of the 4-O-position.174

Entry Conditions Results 1 BnBr, NaH, TBAI, DMF 48 (20%), 49 (23%), 50 (34%) 2 BnBr, imid, DMAP, DMF 46 (SM recovered) 3 BnBr, Ag2O, DCM 46 (SM recovered) 4 BnBr, SnCl2Me2, Ag2O, MeCN 46 (38%), 48 (7%), 49 (5%) 5 BnBr, SnOBu2, TEA, TBAI, tol, 70 °C 48 (16%), 49 (5%) 6 BnBr, SnOBu2, TEA, TBAI, 70 °C 48 (51%), 49 (25%) Table 3.2 Optimization of 3-O-position benzylation of 6-O-benzyl-ᴅ-gulal 3.2.46.

Following benzylation, we moved forward to carbamoylation through reaction with trichloroacetyl isocyanate and subsequent reduction resulting in carbamoyl 3.2.51 in Scheme

3.18.175 The same dihydroxylation protocol performed in the model system was carried out towards diol 3.2.52; however, instead of a 1:1 mixture of anomers, a 5:1 anomeric mixture was observed.

The direction of anomeric equilibrium between the α and β gulose sugars was difficult to determine as the anomeric protons were crowded by benzylic signals in NMR analysis. The key Burgess reagent addition to yield cyclic sulfamidate 3.2.53 proceeded with poor yield, but a crystal structure was resolved by our collaborators in the Fronczek group which confirmed the stereochemistry of both the gulose sugar and the 1R,2S-cis-fused cyclic sulfamidate. We hypothesized that the poor yield for sulfamidate formation could have been a result of the free, unprotected carbamoyl group that was not present in the model system. It was also at this point in the synthesis of the streptolidine moiety that we realized that global deprotection would require 2 steps: removal of silyl protecting groups and a final hydrogenation. Therefore, an alternate protection and carbamoylation strategy was evaluated for 6-O-benzyl-ᴅ-gulal 3.2.46.

Scheme 3.18 Reaction sequence and crystal structure towards cyclic sulfamidate 3.2.53.

For reasons unexplained in the literature, selective silylation of 6-O-benzyl-ᴅ-gulal 3.2.46 to 3-O-(t-butyldimethylsilyl)-6-O-benzyl-ᴅ-gulal 3.2.54 proceeded smoothly as described by the

Crotti group shown in Scheme 3.19.169 As the same selectivity was observed in 3-O-silylation of glucal 3.2.41, we hypothesized that the electronics as opposed to the sterics of the 3-O-position of

glycal sugars lead to the observed selectivity in silylation; however, no such selectivity was observed in alkylation experiments as investigated in Table 3.1. Carbamoylation was carried out so that a protected carbamoyl group would be delivered to the 4-O-position through reaction with

2,4-dimethoxyisocyanate to yield protected carbamoyl gulal 3.2.55.167 Dihydroxylation to diol

3.2.56 and subsequent Burgess reagent addition to sulfamidate 3.2.57 proceeded with better yield than experienced with a deprotected carbamoyl. An anomeric mixture of 8:1 was observed for diol

3.2.56, and the direction of anomeric equilibrium was presumed to lie towards the α-anomer as revealed by a large coupling constant (J1,2 = 11.3Hz) for the anomeric proton. Opening of cyclic sulfamidate 3.2.57 was performed through nucleophilic attack of the 2-position through warming with sodium azide to yield 1,2-trans-difunctionalized gulosamine 3.2.58. NMR analysis of the

4 anomeric coupling constant suggests a C1 gulosamine conformation, although steric bulk of 3,4

1 and 6-position substituents would predict instead a C4 conformation.

Scheme 3.19 Reaction sequence towards 1,2-trans-difunctionalized gulosamine 3.2.58.

3.2.5 Synthesis of Nβ,Nε-Dibenzyloxycarbonyl-ʟ-β-Lysine

The synthesis of β-amino acids has been extensively explored throughout the literature, and the most straightforward method from α-amino acids is through the Ardnt-Eistert homologation protocol. In the total synthesis of ST-F, protected β-lysine (Nβ,Nε- dibenzyloxycarbonyl-ʟ-β-lysine) 3.1.11 was generated through a Wolff-rearrangement over 4 steps from a protected ʟ-ornithine derivative shown in scheme 3.20.95,176 Protected ʟ-ornithine derivative 3.2.59 was converted to an active ester with ethyl chloroformate and then treated with diazomethane to give diazoketone 3.2.60. Reaction with catalytic silver(I) benzoate enabled the key Wolff-rearrangement step and then trapping of ketene 3.2.60a with methanol to give methyl ester 3.2.62. The final step of basic hydrolysis provided protected β-lysine 3.1.11.

Scheme 3.20 Arndt-Eistert homologation of protected ʟ-ornithine derivative 3.2.59 to form protected β-lysine derivative 3.1.11.

Our group attempted the Arndt-Eistert homologation protocol on small scale and were not able to reproduce the desired protected β-lysine derivative 3.1.11. Instead, a stepwise approach more amenable towards scale-up as opposed to generating large quantities of diazomethane would be desirable. Therefore, we adopted a method of β-amino acid synthesis performed in Scheme

3.21.177

First, protected ʟ-ornithine derivative 3.2.59 was converted to the active amide with CDI and subsequently reduced with sodium borohydride.178 The crude alcohol was mesylated to give mesylate 3.2.62. Refluxing with KCN in MeCN provided nitrile 3.2.63 with great yield.177

Attempts were then made to hydrolyze nitrile 3.2.63 under various conditions. Hydrolysis of nitrile

3.2.63 failed under both acidic and basic conditions as well as with hydrogen peroxide (Scheme

3.21 A-C), resulting in either decomposition of starting material or no reaction.177 Instead, stepwise reduction with DIBAL to give aldehyde 3.2.64 and then Pinnick oxidation resulted in the desired protected β-lysine derivative 3.1.11.

Scheme 3.21 Synthesis of protected β-lysine derivative 3.1.11.

3.2.6 Synthesis of Thiourea Analogue of Streptolidine Lactam

To synthesize amino lactam 3.1.17 in the first total synthesis of ST-F, the Shiba group carried out a 20-step procedure starting from ᴅ-xylose 3.2.65 shown in Scheme 3.22.94 First, construction of xylofuranose 3.2.66 was prepared through a previously reported method.179 Next, the alcohols were tosylated, acetonide deprotected and then benzoylated to yield 3.2.67. After resolution of the anomeric center, benzyl glycoside 3.2.68 was formed. Tosyl leaving groups were next substituted with sodium azide to give diazide 3.2.69. Following benzoyl group removal, the

resulting alcohol was mesylated, mesylate 3.2.70 was exposed to hydrogenation and isolated as dibenzamido derivative 3.2.71. γ-lactone 3.2.72 was formed through benzyl deprotection via hydrogenation and then subsequent oxidation. The mesyl group was substituted resulting in α- azide 3.2.73 which was then converted to lactam 3.2.74 over 4 steps. The alcohol was protected to give 3.2.75 and then a final hydrogenation provided amino lactam 3.1.17 that was ready to couple with the gulose-isothiocyanate.

Scheme 3.22 Shiba’s synthesis of streptolidine analogue 3.1.17 over 20 steps.

We considered this methodology too step wise for preparing our streptolidine moiety and desired a shorter, more convenient route towards a streptolidine-thiourea analogue as was

demonstrated in our model system. Fortunately, there have been several reports for synthesis of both the streptolidine lactam moiety as well as various analogues.93,180–186 We adopted our first generation approach for synthesis of a thiourea analogue of streptolidine lactam to carry out a similar coupling of the ST-F fragments as demonstrated by our model system in Scheme 3.14 via formation of an isothiourea. Retrosynthetically, we envisioned accessing isothiourea 3.2.76 from partially protected aspartic acid 3.2.78 through either a ‘thiourea first’ approach via intermediate

3.2.77 or a ‘lactam first’ approach via lactam 3.2.79 (Figure 3.12). The Maruoka group recently performed a synthesis of a urea-based analogue of streptolidine lactam through a ‘urea first’ approach that inspired our first generation approach shown in Scheme 3.23. 185

Figure 3.12 Retrosynthetic analysis to construct isothiourea 3.2.76.

We began with a microwave reaction of protected aspartic acid 3.2.78 and triethyl orthoacetate to form diester 3.2.80 in near quantitative yield.187 Through optimized conditions found in Table 3.3, the desired azido diester 3.2.81a was formed along with its diastereomer

3.2.81b. Next, the newly formed azide was reduced and Cbz group deprotected through hydrogenation and subsequently cyclized with carbon disulfide to yield cyclic thiourea 3.2.82 which could be separated from its diastereomer through silica gel chromatography.188,189

Scheme 3.23 Formation of cyclic thiourea 3.2.82.

Exploring the literature, we found that the counterion used for ester-enolate formation was especially important for dictating the stereochemistry of the resulting addition product.190,191

Shown in Figure 3.13, a hypothesized transition state suggests that when a lithium-based base is used as seen with 3.2.80a, lithium complexes with the enolate and forms a loose cyclic chelate between the oxygen and protected amine.186,190,192 Such a complex from the Z-enolate would result in addition from the Re face because of the steric bulk of the neighboring ethyl ester moiety. The

Z-enolate was observed through TMS-trapping experiments to undergo addition reactions over just a few minutes while the E-enolate took several days to go to completion.191 Alternatively, use of a potassium base was hypothesized to complex with the enolate through chelation to the enolate oxygen and nearby carbonyl oxygen shown in 3.2.80b and attacks towards the Si face.

Figure 3.13 Proposed transition states for enolate-ester 3.2.80 attack of an electrophile.

Based on the literature, we could attempt direct azidation by use of a potassium base and then in situ addition of an electrophilic source of azide to obtain our desired diasteroselectivity.

Alternatively, we could perform indirect azidation by using a potassium base and adding a leaving group that could be inverted through SN2 attack of an azide nucleophile. Shown in Table 3.3, our first attempt towards direct azidation through ester-enolate formation with KHMDS and trisyl azide used as an electrophilic azide source gave no reaction and only starting material. Increasing the equivalents of base in entry 2 resulted in a diastereomeric mixture, with the ratio shifted towards the undesired (R,S) product 3.2.81b, in contrast with the expected trend.193 Interested to see if the diasteroselectivity of direct azidation would shift with the use of a lithium base, we attempted the same conditions with LiHMDS and observed an even more dramatic shift away from the formation of desired diastereomer 3.2.81a. Indirect azidation attempts beginning with LDA as the base for enolate formation resulted in a 1:1 mixture of diastereomers. Fortunately, use of

LiHMDS significantly shifted the diastereoselectivity towards the formation of our desired stereoisomer 3.2.81a. Slight improvements in yield were also observed by decreasing the equivalents of base from 2.4 to 2.

Entry Base (eq) Azide source Yield 81a (S,S) : 81b (R,S) 1 KHMDS (1) Trisyl azide NR - 2 KHMDS (2.4) Trisyl azide 41% 3 : 7 3 LiHMDS (2.4) Trisyl azide 29% 1 : 9 4 LDA (2.4) NBS, then NaN3 / DMF 37% 1 : 1 5 LiHMDS (2.4) NBS, then NaN3 / DMF 31% 19 : 1 6 LiHMDS (2) NBS, then NaN3 / DMF 42% 19 : 1 Table 3.3 Optimization for azide formation from diester 3.2.80 and proposed transition states.

Continuing in the synthesis of the ‘thiourea first’ approach, we would perform carbon chain elongation through cyanohydrin formation from cyclic thiourea 3.2.82. The Maruoka group reported smooth formation of a mixture of diastereomers from a urea-analogue similar to 3.2.82.185

We attempted the same protocol by first partially and selectively reducing the ethyl ester with

DIBAL to the aldehyde and then adding trimethylsilyl cyanide in situ to obtain cyanohydrin 3.2.83.

Starting material was consumed; however, nothing resembling the desired product was formed.

Next, we tried performing the reaction step wise by obtaining aldehyde 3.2.84, but product did not form. We tried stepwise formation of aldehyde 3.2.84 by selectively reducing ethyl ester 3.2.82 to primary alcohol 3.2.85 with decent yield, but neither oxidation towards aldehyde 3.2.84 nor in situ formation of cyanohydrin 3.2.83 were successful. We hypothesized that the nucleophilicity of the thiourea could be causing undesired side reactions, so we decreased the nucleophilicity of 3.2.82 through formation of Boc-protected thiourea 3.2.86. Reduction with sodium borohydride gave primary alcohol 3.2.87, but oxidation towards aldehyde 3.2.88 was unsuccessful. While we tried multiple methods towards reaching cyanohydrin 3.2.83, we recognized its non-trivial synthetic

Scheme 3.24 Synthetic attempts towards cyanohydrin 3.2.83.

tractability as well as the unaddressed difficulty of diastereoselective control for the resulting cyandohydrin. Therefore, we focused on a ‘lactam first’ approach to build isothiourea 3.2.76.

First attempts towards a ‘lactam first’ approach started with a carbon chain elongation through an asymmetric Henry reaction to access (3S,4R) nitroalcohol 3.2.89a (Scheme 3.25).

Conversion of aspartic acid residue 3.2.78 to the Weinreb amide was followed by DIBAL reduction to give aldehyde 3.2.90.194 The literature suggested that the use of La-Li-(R)-Binol catalyst ((R)-LLB) with similar (S) protected α-amino aldehydes enabled formation of nitroalcohols, favoring the erythro (3S,4R) stereochemistry with >99:1 ee.195–198 Upon application to our system, we observed a 1:1 mixture of the erythro (3S,4R) 3.2.89a and threo (3S,4S) 3.2.89b.

Scheme 3.25 Attempts to form nitroalcohol 3.2.89a via asymmetric Henry reaction.

We next turned to a different approach where activation of carboxylic acid 3.2.78 with CDI to an activate amide was added to a potassium nitromethane salt resulting in nitroketone 3.2.91

(Scheme 3.26). Dilute, ambient conditions were essential for retaining the stereochemistry of the

(S) protected α-amino ketone as warming or higher concentration resulted in racemization.199

Diastereoselective reduction was carried out under Felkin-Ahn conditions, allowing a borohydride attack from the less hindered face and resulted in >9:1 dr of the desired erythro (3S,4R) nitroalcohol 3.2.89a.200–202 A reversal in selectivity was observed through reduction by L-

selectride with >10:1 threo:erythro. Optimization to increase selectivity was performed with a solvent screen of methanol or ethanol to further break up any chelate; however, DCM / MeOH provided the best selectivity. Nitro group reduction and immediate Boc protection was carried out with nickel boride to yield di-carbamate 3.2.92a as a mixture of diastereomers.203 Although the

Boc group was removed in just the next step, carbamate protection enabled higher yield since the free amine is very water soluble and difficult to extract from the aqueous workup. Lactamization was carried out through warming of di-carbamate 3.2.92a in formic acid to give hydroxy lactam

3.2.93 where the diastereomeric mixture could be separated.

Scheme 3.26 Sequence for generating isothiourea analogue of streptolidine lactam 3.2.99.

Silylation of hydroxylactam 3.2.93 with TBS triflate provided protected lactam 3.2.94 with great yield. The lactam was further protected with Boc anhydride resulting in di-carbamate

3.2.95.204 Diastereoselective azidation through formation of a lactam-enolate with KHMDS was exposed to trisyl azide and then quickly quenched with acetic acid to provide azidolactam 3.2.96 isolated as a single diasteromer.205,206 The sterically bulky nature of the trisyl group allowed for delivery of the electrophilic azide from the back face of the enolate. The azidolactam 3.2.96 was

Boc-deprotected with TFA and then hydrogenated resulting in diamino lactam 3.2.97 which was cyclized crude with thiocarbonyl diimidazole to yield thiourea 3.2.98.207 The stereochemical configuration for our synthesized thiourea analogue of streptolidine lactam 3.2.98 was confirmed through a solved crystal structure. Thiourea 3.2.98 was then prepared for coupling to the gulosamine by activation with methyl iodide resulting in isothiourea 3.2.99.

3.2.7 Guanidine Formation Attempts from an Isothiourea Analogue of Streptolidine Lactam

We began merging of the coupling partners starting with Route A; amidation of the protected β-lysine derivative 3.1.11 would be performed first followed by guanidine formation.

1,2-trans-difunctionalized gulosamine 3.2.58 was prepared for coupling to protected β-lysine through Staudinger azide reduction to produce amine 3.2.100.154 EDCI coupling of amine 3.2.100 and carboxylic acid 3.1.11 proceeded smoothly, resulting in the formation of amide 3.2.101. Alloc-

Scheme 3.27 Azide reduction, amidation of protected β-lysine and alloc deprotection.

deprotection was carried out similarly to the model system and, as demonstrated by our model system as well as the Nicolaou literature, stereochemistry was retained at the anomeric position for gulosamine 3.2.102 (Figure 3.14).148

Figure 3.14 Retention of stereochemistry at anomeric position upon alloc deprotection.

Upon deprotection of the alloc group, gulosamine 3.2.102 was ready to be coupled with isothiourea analogue 3.2.99. Our attempts towards guanidine formation are illustrated in Table

3.4. We began with identical conditions to those that performed in our model study by warming the amino sugar with the isothiourea in DMF. However, complete decomposition of guloasmine

3.2.102 was observed in just 2 hours while there was no product formation. We hypothesized that high temperature could have caused decomposition, and we attempted slow warming in trial 2. We gradually warmed the reaction mixture from room temperature up to 60 °C over the course of 2 days; however, we observed only starting material or decomposed gulosamine 3.2.102 with similar results in MeOH or THF as reaction solvents.208 To neutralize any residual hydroiodide salts remaining from the isothiourea synthesis, we added DIPEA but observed no reaction progress. To aid with desulfurization, we added mercury(II) chloride as it has been reported in the literature to assist in amine and isothiourea coupling; however, there was no reaction.156,209–211 Reactivity with various protected isothioureas and thioureas 3.2.98 and 3.2.103-105 gave similar results with no product formation although literature reports suggested an increase of electrophilicity with carbamate protection.212–214 Examples in the literature show glycosyl amines successfully

converted to guanidine sugars coupled to 5-membered, cyclic isothioureas with catalytic acetic acid; however, such a transformation was not observed with our bicyclic isothiorea.215–218

We suspect that the reaction did not progress because of the high-energy transition state required to form the reaction intermediate of a streptolidine carbodiimide. Carbodiimide formation as a result of desulfurization would provide an electrophile sufficiently electron poor enough for attack by nucleophilic gulosamine 3.2.102. However, progression towards a cyclic, 5-member carbodiimide likely requires temperatures higher than 60 °C, in contrast with the ambient conditions necessary for preventing decomposition of gulosamine 3.2.102. Therefore, we shifted our focus towards the more step wise guanidine formation we previously explored in our model system via an isothiocyanate analogue of streptolidine lactam.

Trial Thiourea Solvent Additive Temperature Time Result 1 3.2.99 DMF - 80 °C 2 h decomp 2 3.2.99 DMF - 25, 40, 60 °C 2 d – 2 h NR - decomp 3 3.2.99 MeOH - 25, 40, 60 °C 2 d – 2 h NR - decomp 4 3.2.99 THF - 25, 40, 60 °C 2 d – 2 h NR - decomp 5 3.2.99 DMF DIPEA 25 °C 1 d NR 6 3.2.99 DMF HgCl2, TEA 25 °C 1 d NR 7 3.2.98 DMF HgCl2, TEA 25 °C 1 d NR 8 3.2.103 DMF HgCl2, TEA 25 °C 1 d NR 9 3.2.104 DMF HgCl2, TEA 25 °C 1 d NR 10 3.2.105 DMF HgCl2, TEA 25 °C 1 d NR 11 3.2.105 THF - 25 °C 1 d NR 12 3.2.105 EtOH AcOH 25 °C 1 d NR Table 3.4 Attempts towards guanidine formation from gulosamine 3.2.102.

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3.2.8 Guanidine Formation from an Isothiocyanate Analogue of Streptolidine Lactam

We easily adapted our protocol for the formation for a thiourea analogue of streptolidine lactam to an isothiocyanate. Shown in Scheme 3.28, we could divert from azido lactam 3.2.96 through first Boc deprotection with TFA and then azide reduction to yield α-amino lactam 3.2.106.

The α-amine was next converted to an isothiocyanate upon reaction with thiophosgene under basic conditions.219

Scheme 3.28 Diversion from azido-lactam 3.2.96 to generate isothiocyanate 3.2.107.

Gulosamine 3.2.102 was then reacted with isothiocyanate 3.2.107 in DCM, resulting in the desired β-epimer for thiourea 3.2.108 (Scheme 3.29). The undesired anomer of α-3.2.108 was observed but not isolated or characterized. It’s likely that epimerization of gulosamine 3.2.102 occurred in solution. The next step towards the synthesis of ST-F would involve desulfurization and guanidine cyclization. Through desulfurization of thiourea 3.2.108, the proximal protected amine could add to a more easily formed linear carbodiimide.

Scheme 3.29 Coupling of gulosamine 3.2.102 and isothiocyanate 3.2.107.

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Based on our proposed mechanism for the conversion of a linear thiourea to a cyclic guanidine, we imagine the first step is coordination of the nucleophilic thiourea towards a lewis acid (LA) or covalently bonding to an electrophile (E+) (Figure 3.15). The first and second equivalents of base would deprotonate the thiourea and enable elimination of the sulfur moiety, resulting in the reactive carbodiimide species. With the pKa values of the thiourea and carbamate being approximately the same (~20-22), regeneration of the base should be possible during the final carbamate deprotonation and nucleophilic attack step. Alternatively, if the deprotonation of the carbamate is not completed, a water quench would result in the formation of a urea.

Figure 3.15 Proposed mechanism for the desulfurization of a thiourea and guanidine formation.

Our trials for the formation of guanidine 3.2.109 are described in Table 3.5. We began with the use of 2-chloro-1,3-dimethylimidazolinium chloride (DMC) as an electrophile for guanidine formation. Shown in Scheme 3.30 is formation of a guanidine moiety from a thiourea

Scheme 3.30 Ishikawa method for guanidine formation from thiocarbamate using DMC.

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ethyl carbamate with the use of DMC as a desulfurization agent. This method was reported through a series of publications by the Ishikawa group where they demonstrated 19 examples of the reaction succeeding with 1.2 equivalents of DMC and 3 equivalents of base.220–225 Using MeCN freshly dried over molecular sieves, we attempted the same protocol as described by the Ishikawa group in entry 1. We observed complete consumption of starting material over 12 h but did not observe any product formation via LCMS analysis. Instead, we observed the formation of urea

3.2.110, suggesting that the carbodiimide reactive intermediate did form, although final deprotonation towards guanidine 3.2.109 was incomplete. We recognized that our system was significantly more complex than the reported thiourea ethyl carbamate derivatives described by the Ishikawa group, so we attempted to increase the equivalents of TEA from 3 to 8; however, we still only observed the formation of urea. Reaction in THF with DMC and TEA gave the same result. We then attempted to increase the strength of the base from TEA to DBU or LiHMDS and

Entry LA / E+ Base (eq) Solvent Temperature Time Result (yield) 1 DMC TEA (3) MeCN 85 °C 12 h 3.2.110 2 DMC TEA (8) MeCN 85 °C 12 h 3.2.110 3 DMC TEA (3) THF 70 °C 12 h 3.2.110 4 DMC DBU (3) MeCN 85 °C 12 h 3.2.110 5 DMC LiHMDS (3) MeCN 0 °C - rt 2 h 3.2.109 (trace) 6 DMC LiHMDS (2.5) MeCN 0 °C - rt 12 h 3.2.110 7 DMC LiHMDS (3.5) MeCN 0 °C - rt 2 h - 8 EDCI LiHMDS (3) THF -78 °C - rt 2 h 3.2.109 (trace) 9 HgCl2 LiHMDS (3) DMF 0 °C - rt 2 h 3.2.109 (trace) Table 3.5 Reaction optimization attempts towards the formation of guanidine 3.2.109.

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we were excited to observe the formation of guanidine product with the use of LiHMDS by LCMS.

However, only trace product was observed while the remainder of the mass balance was comprised of unidentified impurities. Varying equivalents of LiHMDS resulted in either urea 3.2.110 or unidentified impurities. Other desulfurization agents, EDCI and mercury(II) chloride, gave similar results. LiHMDS was so strong a base that any excess was leading to undesired side reactions; however, weaker bases resulted with incomplete formation of product. We decided that the amine would have to be deprotected to allow for a weaker base to allow reaction progression, but selective

Cbz deprotection would not be straightforward since other Cbz groups are present. Therefore, we diverted the synthesis of streptolidine isothiocyanate once again to allow for a more selective deprotection.

Diversion from the previous isothiocyanate synthesis started from protected lactam 3.2.94 which was hydrogenated resulting in near quantitative yield of β-amino lactam 3.2.111. With slow, portion-wise addition of Boc anhydride and DMAP as a base, both the primary amine and lactam nitrogen were carbamate protected. Similar azidation conditions were employed to provide azido lactam 3.2.113 which was subsequently converted to isothiocyanate 3.2.114 under Staudinger conditions where carbon disulfide was employed as a cosolvent.226

Scheme 3.31 Diversion from previous streptolidine protocol to form isothiocyanate 3.2.114.

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Coupling of isothiocyanate 3.2.114 proceeded smoothly under similar conditions used for the formation of the previous thiourea to yield thiourea 3.2.115. Boc groups as well as the 2,4- dimethoxybenzyl carbamoyl protecting group were removed with TFA in DCM and then the crude reaction mixture was cyclized to guanidine 3.2.116 though mercury(II) chloride mediated desulfurization with TEA as the base. Great yield was observed, and the guanidine product could be purified through silica gel chromatography. Final deprotection and purification would be required to complete the total synthesis of ST-F.

Scheme 3.32 Coupling of streptolidine lactam moiety to gulosamine and guanidine formation.

3.2.9 Deprotection and Isolation of Synthetic Streptothricin F Sulfate

Two consecutive deprotections from guanidine 3.2.116 would complete the total synthetic sequence. First, TBAF would be used to deprotect secondary alcohols on the streptolidine and gulosamine moieties (Scheme 3.33). With freshly distilled THF, a solution of TBAF enabled silyl removal and provided partially deprotected ST-F 3.2.117 in good yield as the penultimate step.

Under conditions similar to the Shiba total synthesis, Bn and Cbz groups were removed in a single step through hydrogenation in acidic solvent to provide the acetate salt of ST-F 3.1.1.85 We desired

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to convert the acetate salt of ST-F 3.1.1 to the sulfate salt for consistency when the activity of the synthetic ST-F is compared to naturally isolated ST-F sulfate. Through a sulfate salt swap protocol in a patent focused on aminoglycoside derivative synthesis, we converted ST-F acetate to ST-F sulfate by precipitation following sulfuric acid treatment.227 The empirical formula for ST-F sulfate

228 was previously determined by elemental analysis to be C19H43N8O17S1.5.

Scheme 3.33 Deprotection strategy towards completion of ST-F total synthesis.

3.2.10 Conclusion

The first total synthesis of ST-F completed by the Shiba group in 1982 consisted of 46 total steps (LLS 25 steps) and an overall yield of 0.0028%. We have completed the second total synthesis for ST-F across 35 steps (LLS 19 steps) with an overall yield of 0.0040%. While we are pleased with the convergent nature of the coupling partners, we hope to improve upon the efficiency of the process, particularly focusing on the optimization for the LLS and streptolidine synthesis. Notable areas of improvement include addition of the Burgess reagent towards cyclic sulfamidate 3.2.57 formation, epimerization of gulosamine 3.2.102 towards generating thiourea

3.2.115 and scalability for nitroketone 3.2.91.

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When considering improvement of the Burgess reagent addition step, it’s important to contemplate the reaction mechanism along with the configuration of diol 3.2.56. As mentioned previously, diol 3.2.56 is comprised of an 8:1 mixture of anomers with the equilibrium favoring the α-anomer suggested by analysis of coupling constant values (J = 11.3Hz) which also suggests

1 4 a C4 gulose configuration. The resulting sulfamidate 3.2.57 possesses a β- C1 conformation where

1 4 a C4- C1 transition is likely required progressing towards product formation (Figure 3.16).

Assuming the addition of Burgess reagent 3.2.18 occurs first followed by the ring flip, the mechanism can occur either through an SN2 route or SN1 reaction via an oxonium intermediate.

1 4 Through either path, a C4- C1 transition is energetically disfavored, resulting in 1,3-diaxial strain between most substituents. A ring flip would proceed slowly, potentially resulting in decomposition of either or both of starting material and thermally unstable Burgess reagent 3.2.18.

Using smaller, less bulky protecting groups could result in a lower energy transition state and enable a faster ring flip, limiting the amount of decomposition occurring with starting material or reagent. Using a less polar solvent could even stabilize a ring flip through increasing the anomeric effect, therein promoting the heteroatomic, anomeric position to an axial orientation.

Figure 3.16 Proposed mechanism for conversion of diol 3.2.56 to sulfamidate 3.2.57.

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Alternatively, weakening the anomeric effect using a polar solvent could be beneficial towards preventing epimerization during isothiocyanate coupling to gulosasmine 3.2.102 (Figure

3.17). While anomerization was relatively low (>10:1 β:α), the overall yield for this step could be improved through a reduction in the anomeric effect. A more polar solvent such as MeCN or

MeOH would weaken the anomeric effect, leading to less epimerization towards the α-anomer.

Figure 3.17 Epimerization of gulosamine 3.2.102 in solution during isothiocyanate coupling.

Scalability for the formation of nitroketone 3.2.91 poses a problem as a result of the dilute, ambient conditions required to prevent racemization. Such conditions have provided poor reproducibility for the activated amide formation unless dry conditions on large scale are carefully performed. Reaction scales for 5 grams of starting material require >500 mL of dry THF to be freshly distilled with the best yields observed for when the Na/benzophenone THF still is purple in color. Blue color for the Na/benzophenone THF still or drying via 4Å molecular sieves resulted in poor yield or reduced ee. Occasionally, formation of the active amide reaction intermediate can stall and even degenerate to starting material after long reaction times. Heating was used to promote reaction progress from the carboxylic acid to the activate amide intermediate; however, racemization was always observed with reaction heating. The sensitivity of the reaction arises from the acidic proton at the lone stereocenter. The pKa for Ha is ~20 while the pKa for Hb is ~7.7

(Scheme 3.34).229 Even with sub-stoichiometric equivalents of t-BuOK (pKa ~17) and excess nitromethane (pKa ~10), racemization was observed with any warming above rt or increases in

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concentration. Routes utilizing a weaker base (DBU), changes in reaction solvent (DMSO) or attempts to divert from CDI as the activating agent provided poor reaction yield, racemization or a combination of the two.202,230–233

Scheme 3.34 Optimization attempts towards nitroketone 3.2.78.

Future directions for the synthesis involve completion of the divergent route. We envision this total synthesis to be divergent from 1,2-trans difunctionalized gulosamine 3.2.58 towards building derivatives through both Route A and Route B described in Figure 3.8. Such a diverse route allows rapid analogue generation of either streptolidine or β-lysine derivatives. While we have demonstrated a completed total synthesis via Route A, we will still like to perform a completed reaction sequence to ST-F through Route B (Scheme 3.35). Possible challenges posed by this route include epimerization at the anomeric center upon alloc deprotection, amidation of

β-lysine in the presence of a deprotected carbamoyl and difficulties with purification as a result of the highly polar guanidine moiety.

Upon alloc deprotection of gulosamine 3.2.58, special attention should be paid to the conformation of the resulting amine 3.2.118. Assuming the stereochemistry of the anomeric

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position is conserved as previously experienced, the sugar conformation could be helpful in

4 determining the solvent for the next reaction with isothiocyanate 3.2.114. If a C1 conformation

(3.2.118a) is conserved from gulosamine 3.2.58, polar solvents such as MeOH or MeCN could be used to weaken the anomeric effect and minimize epimerization to the undesired α-anomer.

Scheme 3.35 Future work of completion of ST-F total synthesis via Route B.

Alternatively, if a ring flip is observed, less polar solvents such as toluene or ether could further

1 emphasize the anomeric effect and stabilize the β-anomer for a C4 conformation (3.2.118b)

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For the transformation of thiourea 3.2.119 to guanidine 3.2.120, function groups appear to be compatible and allow for similar conditions performed in Route A through Boc deprotection and mercury-mediated guanidine cyclization. Searching through previous literature, there appears to be only one example of an amidation reaction in the presence of a deprotected carbamoyl with the use of HATU as the coupling reagent.234 We recognize the lack of literature precedent for the amidation; however, we expect side reactions to be minimal since the nucleophilicity of the guloasmine is stronger than the deprotected carbamoyl.

Lastly, purification of intermediate 3.2.120 and its azide-reduced product could be challenging with silica gel chromatography as a result of the highly polar functionalities.

Separation of the azide-reduced product from triphenylphosphine oxide will be challenging as well, but the use with alumina as a stationary phase in the model system with particularly polar functional groups proved to be successful in practice.

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Chapter 4: Experimental Procedure

4.1 General

Unless otherwise noted all reactions were performed in flame-dried round bottom flasks under argon atmosphere. THF was distilled from benzophenone and sodium metal before use.

Other dry solvents were purchased from VWR. All other reagents were purchased from Aldrich

Chemical Co. or Fisher Scientific and used without further purification. Analytical thin layer chromatography was performed on 0.25 mm silica gel 60 F254 precoated plates from EMD

Millipore. Plates were visualized with ultraviolet light (254 nm) or by treatment with iodine, cerium ammonium molybdate, potassium permanganate or ninhydrin stain followed by heating.

Sorbtec silica gel 60Å (particle size 40-63 μm) mesh was used for all silica gel column chromatography. Sephadex LH-20 size exclusion gel was purchased from GE Healthcare. Reverse phase, preparatory chromatography was performed using a Phenomenex Luna 5µm C18(2) 100Å

LC column with dimensions of 250 x 30 mm NMR spectra were recorded at ambient temperature on a 400 MHz or 500 MHz Varian NMR spectrometer in the solvent indicated. All 1H NMR experiments are reported in δ units, parts per million (ppm) downfield of TMS, and were measured relative to the signals of chloroform (7.26 ppm), methanol (3.31 ppm), acetone (2.05 ppm) and dimethylsulfoxide (2.50 ppm) with 1H decoupled observation. 13C NMR spectra were recorded with 1H decoupled observation at ambient temperature on a Varian NMR spectrometer operating at 125 MHz or 100 MHz in the solvent indicated with the signal of the residual solvent as internal standard. Data for 1H NMR are reported as follows: chemicals shift (δ ppm), multiplicity (s = singlet, bs = broad singlet, d = doublet, dd = doublet of doublets, ddd = doublet of doublet of doublets, dt = doublet of triplets, ddt = doublet of doublet of triplets, dq = doublet of quartets, t = triplet, td = triplets of doublets, q = quartet, qd = quartet of doublets, qt = quartet of triplets, p =

112

pentet, m = multiplet), integration and coupling constant (Hz) whereas 13C NMR analyses were reported in terms of chemical shift. NMR data was analyzed by using MestReNova Software version 12.0.1. Low-resolution mass spectra were performed on an Agilent 6120 LC/MSD with electrospray ionization. High-resolution mass spectra were performed on an LTQ Orbitrap XL via loop injection with an RSLC nano pump. Microwave heating was performed on a single-mode

Anton Paar Monowave 300 and all microwave-irradiated reactions were conducted in heavy- walled glass vials sealed with Teflon septa. Polarimeter analysis was performed on a Jasco P-2000

Polarimeter using Spectra Manager 2.13.00 software.

4.2 Chemical Synthesis and Characterization

Tert-butyl ((1R,2R)-2-aminocyclohexyl)carbamate (3.2.23)

To a stirring solution of (1R,2R)-cyclohexane-1,2-diamine 3.2.22 (4.24 g, 37.13 mmol) in anhydrous DCM (10 mL) was added a solution of di-tert-butyl dicarbonate (2.67 g, 12.23 mmol) in anhydrous DCM (30 mL) at 0 °C under a stream of argon. After 15 mins, the reaction mixture was warmed to rt and stirred for 12 h until consumption of starting material was observed through

TLC analysis. The reaction mixture was diluted in DCM (25 mL) and water (50 mL), was extracted with DCM (25 mL) and the organic layer was concentrated to a yellow oil. The residue was diluted with ether (25 mL) and water (25 mL) and the aqueous was acidified to a pH of 5 with 4M HCl.

The aqueous layer was removed and washed with ether (2 x 25 mL). The aqueous layer was then basified to a pH of 10 with 2N NaOH. EtOAc (3 x 25 mL) was used to extract from the aqueous layer, the combined organics were dried over sodium sulfate, filtered and concentrated to a residue yielding 1.72 g (8.03 mmol, 66%) of 3.2.23 that was used directly in the next step with no further

1 purification. H NMR (400 MHz, (CD3)2SO) δ 6.66 (d, J = 8.4 Hz, 1H), 2.91 – 2.84 (m, 1H), 2.31

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(td, J = 10.3, 4.0 Hz, 1H), 1.76 – 1.73 (m, 1H), 1.62 – 1.55 (m, 2H), 1.53 – 1.49 (m, 1H), 1.38 (s,

9H), 1.12 (d, J = 10.8 Hz, 2H), 1.03 (d, J = 12.0 Hz, 2H). The 1HNMR data of 3.2.23 are in

153 13 accordance with those reported previously. C NMR (100 MHz, (CD3)2SO) δ 155.6, 77.3, 57.0,

+ 53.8, 34.4, 32.1, 28.3, 24.9, 24.7. LCMS-ESI (m / z): [M + H] calcd for C11H22N2O2, 215.17 Da; found 215.1 Da.

Tert-butyl ((1R,2R)-2-isothiocyanatocyclohexyl)carbamate (3.2.11)

To a stirring solution of N,N′-dicyclohexylcarbodiimide (0.439 g, 2.13 mmol) and carbon disulfide

(0.918 mL, 15.19 mmol) in freshly distilled THF (10 mL) was added 3.2.23 (0.456 g, 2.13 mmol) in freshly distilled THF (14 mL) at 0 °C under a stream of argon over 30 mins. After the addition was complete, the reaction mixture was warmed to rt and stirred for 12 h until consumption of starting material was observed through TLC analysis. The reaction mixture was concentrated, diluted in ether (50 mL), filtered and the filtrate was concentrated to a residue. The crude product was purified by silica gel chromatography with 5:1 hexanes:EtOAc to yield 0.399 g (1.56 mmol,

1 73%) of 3.2.11 as a white solid. Rf (2:1 hexanes/EtOAc) = 0.52; H NMR (400 MHz, CDCl3) δ

4.55 (bs, 1H), 3.66 – 3.54 (m, 1H), 3.54 – 3.41 (m, 1H), 2.11 (d, J = 13.3 Hz, 1H), 2.06 – 1.97 (m,

1H), 1.75 – 1.64 (m, 2H), 1.63 – 1.57 (m, 1H), 1.47 (s, 9H), 1.39 – 1.33 (m, 1H), 1.30 – 1.22 (m,

2H). The 1HNMR data of 3.2.11 are in accordance with those reported previously.153 13C NMR

(100 MHz, CDCl3) δ 155.2, 132.1, 80.1, 60.5, 53.9, 32.4, 31.6, 28.6, 24.1, 23.6. LCMS-ESI (m /

+ z): [M + Na] calcd for C12H20N2O2SNa, 279.11 Da; found 279.3 Da.

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(2S,3S,4S)-3,4-bis(benzyloxy)-2-methyl-3,4-dihydro-2H-pyran (3.2.16)

To a stirring solution of 3,4-di-O-acetyl-6-deoxy-L-glucal 3.2.15 (4.19 g, 22.91 mmol) in MeOH

(130 mL) was added potassium carbonate (0.126 g, 0.916 mmol) at rt and the reaction mixture was stirred for 12 h until consumption of starting material was observed through TLC analysis. The reaction mixture was concentrated to an amber paste and then diluted in DMF (80 mL). To the reaction mixture was added tetra-n-butylammonium iodide (0.846 g, 2.29 mmol) and was cooled to 0 °C before added sodium hydride (1.54 g, 64.14 mmol) portion wise over 30 mins. Benzyl bromide (7.62 mL, 64.14 mmol) was added dropwise over 30 mins, warmed to rt and then stirred for 12 until consumption of starting material was observed through TLC analysis. The reaction mixture was poured into a saturated solution of ammonium chloride (100 mL) and extracted with ether (4 x 50 mL). The combined organic extracts were washed with water (2 x 50 mL) and brine

(2 x 50 mL), dried over magnesium sulfate, filtered and concentrated to a residue. The crude product was purified by silica gel chromatography with 24:1 hexanes:EtOAc to yield 5.48 g (17.66

1 mmol, 77%, 2 steps) of 3.2.16 as a yellow oil. Rf (1:1 hexanes/EtOAc) = 0.78; H NMR (400 MHz,

CDCl3) δ 7.41 – 7.30 (m, 10H), 6.38 (dd, J = 6.1, 1.4 Hz, 1H), 4.91 (d, J = 11.1 Hz, 1H), 4.88 (dd,

J = 6.2, 2.4 Hz, 1H), 4.72 (d, J = 11.3 Hz, 1H), 4.68 (d, J = 11.6 Hz, 1H), 4.59 (d, J = 11.8 Hz,

1H), 4.23 (ddd, J = 6.4, 2.5, 1.4 Hz, 1H), 3.97 (dq, J = 8.6, 6.4 Hz, 1H), 3.50 (dd, J = 8.9, 6.5 Hz,

1H), 1.40 (d, J = 6.5 Hz, 3H). The 1HNMR data of 3.2.16 are in accordance with those reported

235 13 previously. C NMR (100 MHz, CDCl3) δ 144.9, 138.5, 138.4, 128.6, 128.1, 127.9, 127.8,

+ 100.3, 79.7, 76.6, 74.2, 74.1, 70.7, 17.6. LCMS-ESI (m / z): [M + Na] calcd for C20H22O3Na,

333.15 Da; found 333.0 Da.

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(3S,4S,5S,6S)-4,5-Bis(benzyloxy)-6-methyltetrahydro-2H-pyran-2,3-diol (3.2.17)

To a stirring solution of 3.2.16 (5.48 g, 17.66 mmol) in THF (56 mL), t-BuOH (24 mL) and water

(8 mL) was added N-methylmorpholine N-oxide (6.2 g, 52.97 mmol) and osmium tetraoxide (4% in H2O, 5.61 mL) and continued to stir for 12 h. Consumption of starting material was determined through TLC analysis, and H2O (100 mL) was used to dilute the reaction mixture. Sodium sulfite

(11.1 g, 88.28 mmol) was added to the reaction mixture and stirred for an additional 2 h. EtOAc

(3 x 100 mL) was used to extract the aqueous layer and the combined organic layers were washed with water (2 x 100 mL), dried over sodium sulfate, filtered and concentrated to a residue. The crude product was purified by silica gel chromatography with 2:1 hexanes:EtOAc to yield 5.19 g

1 (15.06 mmol, 85%) of 3.2.17 as a white solid: mp 81-83 °C. Rf (1:1 EtOAc/hexanes) = 0.30; H

NMR (400 MHz, CDCl3, 1:1 mixture of anomers) δ 7.41 – 7.25 (m, 16H), 5.19 (d, J = 3.7 Hz,

1H), 4.93 – 4.81 (m, 6H), 4.67 (d, J = 4.7 Hz, 1H), 4.64 (d, J = 4.6 Hz, 1H), 4.53 (d, J = 7.6 Hz,

1H), 4.02 (dq, J = 9.4, 6.3 Hz, 1H), 3.78 (dd, J = 9.0 Hz, 1H), 3.66 (dd, J = 9.3, 3.8 Hz, 1H), 3.58

– 3.40 (m, 3H), 3.22 (dd, J = 9.1 Hz, 1H), 3.16 (dd, J = 9.1 Hz, 1H), 1.32 (d, J = 6.2 Hz, 3H), 1.28

13 (d, J = 6.3 Hz, 3H); C NMR (100 MHz, CDCl3) δ 138.63, 138.58, 138.19, 138.12, 128.64, 128.62,

128.56, 128.08, 128.05, 127.99, 127.95, 127.93, 96.5, 92.3, 84.3, 83.5, 83.3, 82.3, 75.9, 75.5, 75.4,

+ 75.4, 75.3, 73.1, 71.8, 67.4, 18.0, 17.9. LCMS-ESI (m / z): [M + Na] calcd for C20H24NaO5, 367.15

Da; found 367.0 Da.

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Allyl (3aR,5S,6S,7R,7aS)-6,7-bis(benzyloxy)-5-methyltetrahydropyrano[2,3-d][1,2,3] oxathiazole-3(3aH)-carboxylate 2,2-dioxide (3.2.19)

To a stirring solution of 3.2.17 (5.18 g, 15.03 mmol) in freshly distilled THF (150 mL) was added alloc-modified burgess reagent 3.2.18 (15.89 g, 60.11 mmol) and immediately placed in an oil bath pre-warmed to 80 °C. The reaction mixture was refluxed for 6 h and consumption of starting material was determined through TLC analysis. Upon cooling to rt, the reaction mixture was diluted with DCM (100 mL) and was added to saturated ammonium chloride (100 mL). The aqueous layer was extracted with DCM (3 x 100 mL) and the combined organic extracts were washed with water (300 mL). The organic layer was dried over sodium sulfate, filtered and concentrated to a residue. The crude product was purified by silica gel chromatography with 8:1 hexanes:EtOAc to yield 3.44 g (7.03 mmol, 47%) of 3.2.19 as an off-white wax. Rf (1:1

1 EtOAc/hexanes) = 0.92; H NMR (400 MHz, CDCl3) δ 7.43 – 7.22 (m, 10H), 6.02 – 5.91 (m, 1H),

5.89 (d, J = 5.2 Hz, 1H), 5.45 (dq, J = 17.1, 1.5 Hz, 1H), 5.32 (dq, J = 10.9, 1.5 Hz, 1H), 4.90 (ddd,

J = 4.9, 3.5, 1.1 Hz, 1H), 4.85 (ddt, J = 13.2, 5.6, 1.4 Hz, 1H), 4.78 (ddt, J = 13.2, 5.5, 1.5 Hz, 1H),

4.57 (d, J = 2.6 Hz, 2H), 4.50 (d, J = 11.9 Hz, 1H), 4.41 (d, J = 11.9 Hz, 1H), 4.03 (dd, J = 3.5,

2.1 Hz, 1H), 3.95 (dq, J = 8.9, 6.1 Hz, 1H), 3.37 (ddd, J = 8.9, 2.1, 1.1 Hz, 1H), 1.29 (d, J = 6.1

13 Hz, 3H); C NMR (100 MHz, CDCl3) δ 149.4, 137.4, 136.7, 130.5, 128.9, 128.7, 128.6, 128.20,

128.18, 128.1, 119.7, 81.6, 80.8, 75.4, 74.6, 72.7, 72.5, 68.6, 68.0, 19.4. LCMS-ESI (m / z): [M +

+ Na] calcd for C24H27NNaO8S, 512.14 Da; found 512.0 Da.

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Allyl ((2R,3R,4S,5S,6S)-3-azido-4,5-bis(benzyloxy)-6-methyltetrahydro-2H-pyran-2- yl)carbamate (3.2.9)

To a stirring solution of 3.2.19 (3.44 g, 7.03 mmol) in anhydrous DMF (70 mL) was added sodium azide (2.28 g, 35.14 mmol) at rt under a stream of argon. The reaction mixture was warmed to 60

°C and stirred for 5 h. LCMS analysis was performed to determine reaction progress and upon completion, the reaction mixture was cooled to rt and quenched with 10% sulfuric acid (70 mL).

The reaction mixture was stirred for an additional 30 mins and then poured into brine (50 mL).

The brine layer was extracted with ether (3 x 100 mL) and the combined organic extracts were washed with water (2 x 100 mL). The organic layer was dried over magnesium sulfate, filtered and concentrated to a residue. The crude product was purified by silica gel chromatography with

10:1 toluene:EtOAc to yield 1.97 g (4.34 mmol, 62%) of 3.2.9 as a white solid: mp 77-82 °C. Rf

1 (7:1 toluene/EtOAc) = 0.43; H NMR (500 MHz, CDCl3) δ 7.37 – 7.27 (m, 10H), 5.90 (ddt, J =

17.2, 10.4, 5.6 Hz, 1H), 5.44 – 5.41 (m, 1H), 5.31 (dq, J = 17.2, 1.5 Hz, 1H), 5.23 (dd, J = 10.6,

1.6 Hz, 1H), 4.77 (d, J = 11.2 Hz, 1H), 4.73 (d, J = 11.5 Hz, 1H), 4.66 (d, J = 11.7 Hz, 1H), 4.62

– 4.59 (m, 3H), 3.88 – 3.83 (m, 1H), 3.83 – 3.79 (m, 1H), 3.75 (p, J = 6.6 Hz, 1H), 3.48 (dd, J =

13 7.3 Hz, 1H), 1.34 (d, J = 6.4 Hz, 3H); C NMR (100 MHz, CDCl3) δ 155.0, 137.9, 137.4, 132.2,

128.7, 128.65, 128.61, 128.3, 128.1, 118.4, 78.8, 78.7, 77.2, 74.7, 72.9, 70.1, 66.4, 60.6, 17.7.

+ LCMS-ESI (m / z): [M + Na] calcd for C24H28N4NaO5, 475.20 Da; found 475.1 Da.

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Allyl ((2R,3R,4S,5S,6S)-3-amino-4,5-bis(benzyloxy)-6-methyltetrahydro-2H-pyran-2- yl)carbamate (3.2.24)

To a stirring solution of 3.2.9 (0.086 g, 0.190 mmol) in THF (2.2 mL) and water (0.127 mL) was added triphenylphosphine (0.070 g, 0.266 mmol) at rt. The reaction mixture was warmed to reflux and stirred for 12 h. LCMS analysis was performed to determine reaction progress and upon completion, the reaction mixture was concentrated to a residue. The crude product was purified by silica gel chromatography with 3% MeOH/DCM to yield 0.069 g (0.162 mmol, 85%) of 3.2.24 as

1 a clear film. Rf (3% MeOH/DCM) = 0.31; H NMR (400 MHz, CDCl3) δ 7.40 – 7.25 (m, 10H),

5.90 (ddt, J = 16.5, 10.9, 5.6 Hz, 1H), 5.60 (bs, 1H), 5.34 – 5.28 (m, 1H), 5.27 – 5.23 (m, 1H),

5.21 (d, J = 10.5 Hz, 1H), 4.71 (d, J = 11.4 Hz, 1H), 4.60 – 4.56 (m, 4H), 3.93 – 3.85 (m, 1H),

3.75 (dd, J = 6.2, 3.6 Hz, 1H), 3.43 (dd, J = 6.1 Hz, 1H), 3.11 (dd, J = 4.3 Hz, 1H), 1.60 (bs, 2H),

13 1.36 (d, J = 6.6 Hz, 3H). C NMR (100 MHz, CDCl3) δ 155.7, 138.0, 137.8, 132.5, 128.6, 128.5,

128.1, 128.01, 127.96, 127.90, 118.2, 79.2, 77.6, 73.8, 72.3, 70.4, 66.1, 51. 6, 29. 8, 17.5. LCMS-

+ ESI (m / z): [M + H] calcd for C24H31N2O5, 427.22 Da; found 427.2 Da.

Dibenzyl ((S)-6-(((2R,3R,4S,5S,6S)-2-(((allyloxy)carbonyl)amino)-4,5-bis(benzyloxy)-6- methyltetrahydro-2H-pyran-3-yl)amino)-6-oxohexane-1,5-diyl)dicarbamate (3.2.25)

To a stirring solution of 3.2.24 (0.428 g, 1.00 mmol) in anhydrous DCM (8.2 mL) was added

N2,N6-bis((benzyloxy)carbonyl)-L-lysine 3.2.10 (0.416 g, 1.00 mmol), a solution of 4- dimethylaminopyridine (0.007 g, 0.05 mmol) in anhydrous DCM (0.5 mL) and EDCI·HCl (0.192

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g, 1.00 mmol) at rt under a stream of argon. The reaction mixture was stirred for 6 h and reaction completion was determined through TLC analysis. The reaction mixture was diluted with DCM

(20 mL) and then poured into water (50mL). The water layer was extracted with DCM (3 x 25 mL) and the combined organic extracts were washed with water (100 mL). The organic layer was dried over sodium sulfate, filtered and concentrated to a residue. The crude product was purified by silica gel chromatography with 2:1 hexanes:EtOAc to yield 0.602 g (0.732 mmol, 73%) of 3.2.25

1 as a colorless oil. Rf (1:1 hexanes/EtOAc) = 0.52; H NMR (400 MHz, (CD3)2SO) δ 8.38 (bs, 1H),

8.11 (d, J = 8.8 Hz, 1H), 7.38 – 7.24 (m, 20H), 7.18 (t, J = 5.7 Hz, 1H), 5.94 (ddt, J = 16.1, 10.6,

5.4 Hz, 1H), 5.32 (dd, J = 17.3, 1.9 Hz, 1H), 5.20 (d, J = 10.4 Hz, 1H), 5.07 – 5.04 (m, 1H), 5.02

(d, J = 2.5 Hz, 2H), 4.99 (s, 2H), 4.75 (d, J = 11.3 Hz, 1H), 4.57 – 4.49 (m, 4H), 4.43 – 4.39 (m,

2H), 4.19 (dd, J = 8.8, 5.0 Hz, 1H), 4.14 (dd, J = 8.8, 4.5 Hz, 1H), 3.59 (dt, J = 12.3, 6.2 Hz, 1H),

3.46 (dd, J = 8.7 Hz, 1H), 2.92 – 2.86 (m, 2H), 1.60 – 1.40 (m, 3H), 1.32 – 1.27 (m, 3H), 1.20 (d,

13 J = 6.1 Hz, 3H); C NMR (100 MHz, (CD3)2SO) δ 172.6, 156.0, 138.6, 138.5, 137.28, 137.0,

133.3, 128.3, 128.2, 128.1, 127.8, 127.7, 127.4, 127.32, 127.27, 117.4, 79.1, 76.7, 73.6, 70.2, 67.4,

67.3, 65.4, 65.1, 64.7, 54.45, 48.8, 40.1, 32.0, 29.1, 22.8, 17.9. LCMS-ESI (m / z): [M + Na]+ calcd for C46H54N4NaO10, 845.37 Da; found 845.4 Da.

Dibenzyl ((S)-6-(((2R,3R,4S,5S,6S)-2-amino-4,5-bis(benzyloxy)-6-methyltetrahydro-2H- pyran-3-yl)amino)-6-oxohexane-1,5-diyl)dicarbamate (3.2.12)

To a stirring solution of 3.2.25 (1.04 g, 1.26 mmol) diluted in THF (8.8 mL) and water (3.5 mL) was added diethylamine (5.23 mL, 50.55 mmol) at rt. The reaction mixture was stirred for 10 mins, and then triphenylphosphine-3,3′,3′′-trisulfonic acid trisodium salt (0.143 g, 0.253 mmol) was

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added at rt and stirred for 1 hr. Consumption of starting material was determined through TLC analysis, the reaction mixture was diluted with EtOAc (25 mL) and poured into water (50 mL).

The water layer was extracted with EtOAc (3 x 25 mL) and the combined organic extracts were washed with brine (100 mL). The organic layer was dried over sodium sulfate, filtered and concentrated to a residue. The crude product was purified by silica gel chromatography with 3%

MeOH/DCM to yield 0.814 g (1.10 mmol, 87%) of 3.2.12 as a colorless gum. Rf (5%

1 MeOH/DCM) = 0.31; H NMR (400 MHz, CDCl3) δ 7.38 – 7.22 (m, 20H), 6.24 (d, J = 10.1 Hz,

1H), 5.56 (d, J = 7.5 Hz, 1H), 5.09 – 4.98 (m, 4H), 4.92 (d, J = 10.9 Hz, 1H), 4.82 – 4.76 (m, 2H),

4.72 (dd, J = 6.2 Hz, 1H), 4.57 (d, J = 10.9 Hz, 1H), 4.48 (d, J = 11.0 Hz, 1H), 4.28 – 4.23 (m,

1H), 4.12 (q, J = 7.1 Hz, 1H), 3.66 (dd, J = 9.2, 4.3 Hz, 1H), 3.36 (dt, J = 12.2, 6.2 Hz, 1H), 3.10

– 3.01 (m, 2H), 2.97 – 2.90 (m, 1H), 2.11 (bs, 2H), 1.88 – 1.76 (m, 2H), 1.68 – 1.54 (m, 2H), 1.38

13 – 1.30 (m, 4H), 1.28 (d, J = 6.0 Hz, 3H); C NMR (100 MHz, CDCl3) δ 172.9, 156.6, 156.5,

138.4, 138.0, 136.7, 136.3, 128.6, 128.6, 128.5, 128.4, 128.3, 128.2, 128.1, 127.9, 127.8, 82.7,

81.3, 80.2, 75.3, 72.8, 71.1, 67.1, 66.7, 55.4, 50.8, 40.4, 32.3, 29.5, 22.4, 18.5. LCMS-ESI (m / z):

+ [M + H] calcd for C42H51N4O8, 739.37 Da; found 739.4 Da.

Dibenzyl ((S)-6-(((2R,3R,4S,5S,6S)-4,5-bis(benzyloxy)-2-(3-((1R,2R)-2-((tert- butoxycarbonyl)amino)cyclohexyl)thioureido)-6-methyltetrahydro-2H-pyran-3-yl)amino)-

6-oxohexane-1,5-diyl)dicarbamate (3.2.26)

121

To a stirring solution of 3.2.12 (0.794 g, 1.07 mmol) in anhydrous DMF (10.7 mL) was added

3.2.11 (0.550 g, 2.15 mmol) at rt under a stream of argon. The reaction mixture was warmed to 65

°C and stirred for 12 h until consumption of starting material was observed through LCMS analysis. The reaction mixture was diluted with EtOAc (25 mL) and poured into brine (25 mL).

The aqueous layer was extracted with EtOAc (3 x 25 mL) and the combined organic extracts were washed with water (3 x 50 mL) and brine (50 mL). The organic layer was dried over sodium sulfate, filtered and concentrated to a residue. The crude product was purified by silica gel chromatography with 2:1 hexanes:EtOAc to yield 0.791 g (0.795 mmol, 74%) of 3.2.26 as a

1 colorless gum. Rf (1:1 hexanes:EtOAc) = 0.53; H NMR (400 MHz, CDCl3) δ 7.49 (d, J = 8.8 Hz,

1H), 7.37 – 7.24 (m, 20H), 6.76 (d, J = 8.9 Hz, 1H), 6.43 (d, J = 10.1 Hz, 1H), 5.69 (d, J = 7.6 Hz,

1H), 5.66 (d, J = 5.5 Hz, 1H), 5.36 (d, J = 8.0 Hz, 1H), 5.22 (d, J = 12.3 Hz, 1H), 5.12 – 4.97 (m,

4H), 4.91 (d, J = 11.0 Hz, 1H), 4.81 – 4.72 (m, 3H), 4.60 (d, J = 11.0 Hz, 1H), 4.50 (d, J = 10.8

Hz, 1H), 4.36 – 4.26 (m, 1H), 3.88 – 3.83 (m, 1H), 3.77 (dd, J = 9.5, 4.3 Hz, 1H), 3.60 – 3.53 (m,

1H), 3.24 – 3.18 (m, 1H), 3.13 (dd, J = 9.2 Hz, 1H), 3.07 – 2.99 (m, 1H), 2.97 – 2.89 (m, 1H), 2.10

– 2.05 (m, 1H), 2.00 – 1.95 (m, 1H), 1.80 – 1.75 (m, 1H), 1.66 – 1.58 (m, 3H), 1.42 (s, 9H), 1.36

– 1.35 (m, 1H), 1.33 (d, J = 6.0 Hz, 3H), 1.29 – 1.21 (m, 2H), 1.21 – 1.11 (m, 2H), 1.00 – 0.92 (m,

13 1H); C NMR (100 MHz, CDCl3) δ 183.6, 172.6, 157.1, 156.8, 156.2, 138.4, 137.8, 136.5, 135.7,

128.7, 128.6, 128.4, 128.4, 128.2, 128.0, 127.9, 127.8, 81.1, 80.4, 80.1, 79.3, 75.1, 73.1, 71.1, 67.5,

66.8, 57.5, 56.3, 55.7, 50.0, 40.1, 32.8, 32.6, 30.9, 29.5, 28.6, 24.9, 24.7, 22.3, 18.3. LCMS-ESI

+ (m / z): [M + H] calcd for C54H71N6O10S, 995.50 Da; found 995.5 Da.

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Dibenzyl ((S)-6-(((2R,3R,4S,5S,6S)-4,5-bis(benzyloxy)-6-methyl-2-(((3aR,7aR)-octahydro-

2H-benzo[d]imidazol-2-ylidene)amino)tetrahydro-2H-pyran-3-yl)amino)-6-oxohexane-1,5- diyl)dicarbamate (3.2.27)

To a stirring solution of 3.2.26 (0.362 g, 0.364 mmol) in anhydrous DCM (3.6 mL) was added

TFA (1.8 mL) at 0 °C under a stream of argon. The reaction mixture was stirred for 2 h at 0 °C, and consumption of starting material was observed through TLC analysis. While maintaining a temperature of at 0 °C, the reaction mixture was quenched through the addition of a saturated solution of sodium bicarbonate (10 mL). The reaction mixture was warmed to rt, diluted with DCM

(10 mL) and extracted with DCM (3 x 10 mL). The organic extracts were washed with water (50 mL), dried over sodium sulfate, filtered and concentrated to a residue. The crude product (0.313 g) was used directly in the next step. To a stirring solution of crude amine (0.313 g) in anhydrous

DMF (7.7 mL) was added triethylamine (0.143 mL, 1.05 mmol) and mercury(II) chloride (0.284 g, 1.05 mmol) successively at 0 °C under a stream of argon. The reaction mixture was stirred for

15 mins, warmed to rt and stirred for 3 h when consumption of starting material was observed through TLC analysis. The reaction mixture was diluted with DCM (10 mL), filtered through celite and then poured into brine (25 mL). The brine layer was extracted with DCM (3 x 10 mL) and the combined organic extracts were washed with water (3 x 50 mL). The organic layer was dried over sodium sulfate, filtered and concentrated to a residue. The crude product was purified by alumina chromatography with 0.5% MeOH/DCM to yield 0.232 g (0.270 mmol, 77% over 2 steps) of

123

1 3.2.27 as a white film. Rf (12% MeOH/DCM) = 0.05; H NMR (400 MHz, CDCl3) δ 7.42 – 7.15

(m, 20H), 6.26 (bs, 1H), 5.59 (bs, 1H), 5.10 – 4.99 (m, 4H), 4.99 – 4.95 (m, 1H), 4.90 (d, J = 11.1

Hz, 1H), 4.72 (d, J = 11.0 Hz, 1H), 4.57 (d, J = 10.6 Hz, 2H), 4.49 (d, J = 10.8 Hz, 1H), 4.22 (dd,

J = 9.2, 4.4 Hz, 1H), 4.15 (d, J = 7.2 Hz, 1H), 3.86 (dq, J = 11.8, 6.2 Hz, 1H), 3.13 (dd, J = 9.2

Hz, 1H), 3.03 – 2.89 (m, 4H), 1.98 (dd, J = 12.0 Hz, 2H), 1.82 – 1.76 (m, 3H), 1.63 – 1.55 (m,

13 1H), 1.40 – 1.27 (m, 8H), 1.22 (d, J = 6.2 Hz, 3H); C NMR (100 MHz, CDCl3) δ 171.9, 161.7,

156.6, 156.2, 138.8, 138.5, 136.8, 136.4, 128.6, 128.6, 128.4, 128.3, 128.22, 128.20, 128.1, 127.9,

127.6, 127.6, 85.8, 81.0, 78.0, 75.1, 71.2, 67.0, 66.8, 66.6, 62.3, 60.7, 55.1, 51.8, 40.5, 32.9, 29.8,

+ 29.8, 29.7, 29.5, 24.1, 22.4, 18.8. LCMS-ESI (m / z): [M + H] calcd for C49H61N6O8, 861.46 Da; found 861.5 Da.

(S)-2,6-diamino-N-((2R,3R,4S,5R,6S)-4,5-dihydroxy-6-methyl-2-(((3aR,7aR)-octahydro-2H- benzo[d]imidazol-2-ylidene)amino)tetrahydro-2H-pyran-3-yl)hexanamide (3.2.14)

To a stirring solution of 3.2.27 (0.111 g, 0.129 mmol) in anhydrous DCM (1.3 mL) was added a solution of boron trichloride (1M in DCM, 4.56 mmol) dropwise over 30 mins at -78 °C under a stream of argon. The reaction mixture was stirred at -78 °C for 1 h, warmed to 0 °C over 1 h, stirred at 0 °C and then warmed to rt. The reaction mixture was stirred at rt for 12 h and the consumption of starting material was observed through TLC analysis. The reaction mixture was cooled to 0 °C, quenched with MeOH (3 mL) warmed to rt and concentrated to a residue. The crude product was purified by reverse phase chromatography 100%-2% water/MeCN over 32 mins and pure fractions

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were freeze dried to yield 0.018 g (0.042 mmol, 33%) of 3.2.14 as a white foam. 1H NMR (500

MHz, CD3OD) δ 5.01 (d, J = 1.8 Hz, 1H), 4.58 (dd, J = 4.7, 1.8 Hz, 1H), 4.15 – 4.09 (m, 1H), 3.72

(dd, J = 9.7, 4.7 Hz, 1H), 3.46 (dq, J = 9.4, 6.1 Hz, 1H), 3.31 – 3.28 (m, 2H), 3.26 (dd, J = 9.6 Hz,

1H), 2.95 (dd, J = 7.6 Hz, 2H), 2.16 (d, J = 11.5 Hz, 2H), 1.91 – 1.86 (m, 4H), 1.76 – 1.63 (m,

3H), 1.59 – 1.52 (m, 3H), 1.44 – 1.38 (m, 2H), 1.35 (d, J = 6.1 Hz, 3H); 13C NMR (100 MHz,

CD3OD) δ 172.1, 162.5, 81.0, 75.7, 73.3, 64.4, 54.6, 54.1, 40.4, 31.9, 30.7, 29.7, 28.2, 24.8, 22.7,

+ 17.8. LCMS-ESI (m / z): [M + H] calcd for C19H37N6O4, 413.29 Da; found 413.3 Da.

(2R,3R,4S,5S,6S)-3-azido-4,5-bis(benzyloxy)-6-methyltetrahydro-2H-pyran-2-amine

(3.2.28)

To a stirring solution of 3.2.9 (0.294 g, 0.650 mmol) diluted in MeCN (8 mL) and water (8 mL) was added diethylamine (2.69 mL, 25.99 mmol) at rt. The reaction mixture was stirred for 10 mins, and then triphenylphosphine-3,3′,3′′-trisulfonic acid trisodium salt (0.074 g, 0.130 mmol) was added at rt and stirred for 1 hr. Consumption of starting material was determined through TLC analysis, the reaction mixture was diluted with EtOAc (25 mL) and poured into water (50 mL).

1 (0.631 mmol, 97%) of 3.2.28 as a light brown oil. H NMR (400 MHz, CDCl3) δ 7.42 – 7.27 (m,

10H), 4.93 (d, J = 10.8 Hz, 1H), 4.78 (d, J = 11.6 Hz, 1H), 4.72 (d, J = 11.6 Hz, 1H), 4.65 (d, J =

10.8 Hz, 1H), 4.17 – 4.12 (m, 1H), 3.98 (d, J = 3.6 Hz, 1H), 3.72 (dd, J = 9.1, 3.6 Hz, 1H), 3.44

(dd, J = 9.2 Hz, 1H), 3.31 (dq, J = 9.2, 6.1 Hz, 1H), 2.14 (bs, 2H), 1.29 (d, J = 6.1 Hz, 3H); 13C

125

NMR (100 MHz, CDCl3) δ 138.3, 137.6, 128.7, 128.60, 128.55, 128.20, 128.18, 128.1, 128.01,

127.95, 82.9, 81.8, 80.0, 75.7, 72.7, 72.6, 63.7, 18.2. LCMS-ESI (m / z): [M + H]+ calcd for

C20H25N4O3, 369.19 Da; found 369.2 Da.

Tert-butyl ((1R,2R)-2-(3-((2R,3R,4S,5S,6S)-3-azido-4,5-bis(benzyloxy)-6-methyltetrahydro-

2H-pyran-2-yl)thioureido)cyclohexyl)carbamate (3.2.29)

To a stirring solution of 3.2.28 (0.224 g, 0.608 mmol) in anhydrous DMF (6.5 mL) was added

3.2.11 (0.311 g, 1.22 mmol) at rt under a stream of argon. The reaction mixture was warmed to 65

°C and stirred for 12 h until consumption of starting material was observed through LCMS analysis. The reaction mixture was diluted with EtOAc (25 mL) and poured into brine (25 mL).

The aqueous layer was extracted with EtOAc (3 x 25 mL) and the combined organic extracts were washed with water (3 x 50 mL) and brine (50 mL). The organic layer was dried over sodium sulfate, filtered and concentrated to a residue. The crude product was purified by silica gel chromatography with 4:1 hexanes:EtOAc to yield 0.221 g (0.356 mmol, 58%) of 3.2.29 as an off

1 white solid: mp 169-171 °C. Rf (2:1 hexanes:EtOAc) = 0.56; H NMR (400 MHz, CDCl3) δ 7.39

– 7.27 (m, 10H), 7.00 (d, J = 6.9 Hz, 1H), 6.12 (bs, 1H), 5.74 (d, J = 9.0 Hz, 1H), 4.90 (d, J = 10.9

Hz, 1H), 4.76 – 4.71 (m, 2H), 4.69 (d, J = 7.8 Hz, 1H), 4.65 (d, J = 10.9 Hz, 1H), 3.98 – 3.93 (m,

1H), 3.86 – 3.79 (m, 1H), 3.48 – 3.41 (m, 3H), 2.40 (d, J = 12.5 Hz, 1H), 1.97 (d, J = 10.3 Hz,

1H), 1.81 – 1.68 (m, 3H), 1.44 (s, 9H), 1.30 (d, J = 5.4 Hz, 3H), 1.28 – 1.22 (m, 2H), 1.17 – 1.09

13 (m, 1H); C NMR (100 MHz, CDCl3) δ 177.4, 157.5, 138.2, 137.5, 128.7, 128.6, 128.2, 128.1,

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128.1, 128.0, 82.7, 80.3, 79.8, 75.7, 73.5, 72.9, 62.6, 53.5, 32.3, 28.4, 25.2, 24.3, 18.1. LCMS-ESI

+ (m / z): [M + H] calcd for C32H45N6O5S, 625.32 Da; found 625.3 Da.

(3aR,7aR)-N-((2R,3R,4S,5S,6S)-3-azido-4,5-bis(benzyloxy)-6-methyltetrahydro-2H-pyran-

2-yl)octahydro-2H-benzo[d]imidazol-2-imine (3.2.31)

To a stirring solution of 3.2.29 (0.526 g, 0.842 mmol) in anhydrous DCM (9.5 mL) was added

TFA (2.5 mL) at 0 °C under a stream of argon. The reaction mixture was stirred for 2 h at 0 °C, and consumption of starting material was observed through TLC analysis. While maintaining a temperature of at 0 °C, the reaction mixture was quenched through the addition of a saturated solution of sodium bicarbonate (25 mL). The reaction mixture was warmed to rt, diluted with DCM

(25 mL) and extracted with DCM (3 x 25 mL). The organic extracts were washed with water (100 mL), dried over sodium sulfate, filtered and concentrated to a residue. The crude product (0.427 g) was used directly in the next step. To a stirring solution of crude amine (0.427 g) in anhydrous

DMF (18 mL) was added triethylamine (0.333 mL, 2.44 mmol) and mercury(II) chloride (0.662 g, 2.44 mmol) successively at 0 °C under a stream of argon. The reaction mixture was stirred for

15 mins, warmed to rt and stirred for 3 h when consumption of starting material was observed through TLC analysis. The reaction mixture was diluted with DCM (25 mL), filtered through celite and then poured into brine (50 mL). The brine layer was extracted with DCM (3 x 25 mL) and the combined organic extracts were washed with water (3 x 100 mL). The organic layer was dried over sodium sulfate, filtered and concentrated to a residue. The crude product was purified by

127

silica gel chromatography with 10% MeOH/DCM to yield 0.298 g (0.607 mmol, 73% over 2 steps)

1 of 3.2.31 as colorless oil. Rf (8% MeOH/DCM) = 0.22; H NMR (400 MHz, CDCl3) δ 7.40 – 7.25

(m, 10H), 4.92 (d, J = 10.8 Hz, 1H), 4.82 – 4.79 (m, 1H), 4.75 (d, J = 11.7 Hz, 1H), 4.69 (d, J =

11.7 Hz, 1H), 4.63 (d, J = 10.8 Hz, 1H), 3.98 (d, J = 3.6 Hz, 1H), 3.71 (dd, J = 8.6, 3.6 Hz, 1H),

3.46 – 3.41 (m, 2H), 3.01 – 2.98 (m, 2H), 1.99 (d, J = 11.1 Hz, 2H), 1.78 (d, J = 8.4 Hz, 2H), 1.47

13 – 1.33 (m, 4H), 1.31 (d, J = 5.6 Hz, 3H); C NMR (100 MHz, CDCl3) δ 160.6, 138.4, 137.8,

128.7, 128.5, 128.2, 128.09, 128.06, 128.0, 85.0, 82.0, 80.0, 75.7, 73.5, 72.2, 64.2, 62.9, 29.8, 24.3,

+ 18.4. LCMS-ESI (m / z): [M + H] calcd for C27H35N6O3, 491.28 Da; found 491.3 Da.

(3aR,7aR)-2-(methylthio)-3a,4,5,6,7,7a-hexahydro-1H-benzo[d]imidazole (3.2.27)

To a stirring solution of (3aR,7aR)-octahydro-2H-benzo[d]imidazole-2-thione (0.037 g, 0.237 mmol) in anhydrous MeOH (2.5 mL) was added iodomethane (0.073 mL, 1.18 mmol) at rt under a stream of argon. The reaction mixture was warmed to 85 °C and stirred for 2 h. Upon consumption of starting material observed through TLC analysis, the reaction mixture was cooled to rt and concentrated to a residue yielding 0.069 g (0.231 mmol, 97%) of 3.2.27 that was used

1 directly in the step without further purification. Rf (10% MeOH/DCM) = 0.29; H NMR (400 MHz,

CDCl3) δ 9.38 (bs, 2H), 3.54 – 3.50 (m, 2H), 2.88 (s, 3H), 2.51 (d, J = 11.8 Hz, 2H), 1.88 (d, J =

13 9.5 Hz, 2H), 1.60 – 1.50 (m, 2H), 1.40 – 1.30 (m, 2H); C NMR (100 MHz, CDCl3) δ 175.2, 65.8,

28.9, 23.8, 16.3. The 1HNMR and 13CNMR data of 3.2.27 are in accordance with those reported previously.236

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4-(tert-butyl) 1-ethyl ((benzyloxy)carbonyl)-L-aspartate (3.2.80)

N-Cbz-L-aspartic acid 4-tert-butyl ester 3.2.78 (10.02 g, 29.35 mmol) was dissolved in triethyl orthoacetate (13.87 mL, 73.73 mmol) and heated to 200 °C in a microwave reactor for 2 mins. The reaction was extracted with EtOAc (3 x 25 mL), washed with brine (50 mL) and dried over Na2-

SO4. The solvent is removed to yield 9.95 g (28.32 mmol, 96%) of pure diester 3.2.80 as an amber

24 -1 oil. Rf (3:1 hexanes/EtOAc) = 0.44; [α]ᴅ = +16.02 (c = 0.4, DCM); IR (thin film, cm ): 2979,

1 1724, 1505, 1367, 1337, 1212, 1151, 1043, 846, 753, 698; H NMR (500 MHz, CDCl3) δ 7.37 –

7.31 (m, 5H), 5.74 (d, J = 8.7 Hz, 1H), 5.13 (s, 2H), 4.57 (dt, J = 9.0, 4.6 Hz, 1H), 4.26 – 4.17 (m,

2H), 2.93 (dd, J = 16.8, 4.7 Hz, 1H), 2.74 (dd, J = 16.9, 4.6 Hz, 1H), 1.42 (s, 9H), 1.26 (t, J = 7.1

13 Hz, 3H); C NMR (100 MHz, CDCl3) δ 170.9, 167.0, 156.0, 136.3, 128.5, 128.2, 128.1, 81.7,

+ 67.0, 61.7, 50.6, 37.8, 28.0, 14.1. HRMS-ESI (m / z): [M + Na] calcd for C18H25NO6Na, 374.1580

Da; found 374.1575 Da.

1-(tert-butyl) 4-ethyl (2S,3S)-2-azido-3-(((benzyloxy)carbonyl)amino)succinate (3.2.81a)

A stirring solution of X (2.01 g, 5.73 mmol) in freshly distilled THF (14.5 mL) was cooled to -78

°C under argon. To the solution was added a solution of lithium bis(trimethylsilyl)amide (1.3 M in THF, 11.46 mmol) dropwise over 10 mins and then stirred for 50 mins. The solution was warmed slightly to -50 °C and then N-bromosuccinimide (3.06 g, 17.19 mmol) was added swiftly in one portion. The reaction mixture was stirred at -50 °C for 50 mins and then warmed to room temperature. A saturated solution of ammonium chloride (40 mL) was added and then the organic

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layer was extracted with EtOAc (3 x 25 mL). The combined organic extracts were washed with water (50 mL), dried over magnesium sulfate and concentrated to an amber residue. Upon drying, the amber residue was dissolved in anhydrous DMF (25 mL), sodium azide (0.555 g, 8.54 mmol) was added and the reaction mixture stirred for 12 h at room temperature. EtOAc (50 mL) was added and the organic layer was washed with water (3 x 50 mL). The organic layer was washed with brine (50 mL) and dried over Na2SO4. The crude product was purified by silica gel chromatography with 10:1 hexanes/EtOAc to yield 0.946 g (2.41 mmol, 42% over 2 steps) of azide

24 3.2.81a as a clear oil. Rf (3:1 hexanes/EtOAc) = 0.58; [α]ᴅ = -22.66 (c = 0.1, DCM); IR (thin film, cm-1): 3311, 2921, 2851, 2115, 1734, 1507, 1369, 1214, 1151, 1057, 697; 1H NMR (500 MHz,

CDCl3) δ 7.37 – 7.31 (m, 5H), 5.38 (d, J = 9.7 Hz, 1H), 5.14 (d, J = 12.2 Hz, 1H), 5.09 (d, J = 12.3

Hz, 1H), 4.95 (dd, J = 9.9, 2.2 Hz, 1H), 4.60 (d, J = 2.3 Hz, 1H), 4.25 (q, J = 7.0 Hz, 2H), 1.45 (s,

13 9H), 1.30 (t, J = 7.1 Hz, 3H) ; C NMR (100 MHz, CDCl3) δ 169.0, 166.3, 156.0, 136.2, 128.6,

128.3, 128.2, 84.5, 67.4, 63.5, 62.5, 55.4, 27.92 14.2. HRMS-ESI (m / z): [M + Na]+ calcd for

C18H24N4O6Na, 415.1594 Da; found 415.1593 Da.

4-(Tert-butyl) 5-ethyl (4S,5S)-2-thioxoimidazolidine-4,5-dicarboxylate (3.2.82)

To a stirring solution of 3.2.81a (2.40 g, 6.13 mmol) diluted in methanol (50 mL) was added

Pearlman’s catalyst (Pd 20% on carbon, nominally 50% water, 0.516 g). The reaction vessel was evacuated and purged with argon gas (7x), evacuated and purged with hydrogen gas (7x) and then stirred overnight under a hydrogen atmosphere (balloon). The reaction mixture was filtered over a celite bed and concentrated to a residue which was used in the next step without further purification. The resulting yellow residue was dissolved in ethanol (30 mL) and transferred to a

130

sealable tube. Carbon disulfide (0.937 g, 12.31 mmol) was added, the reaction vessel was sealed, and the reaction mixture was heated to reflux for 12 h. Upon consumption of starting material observed by TLC, the reaction was cooled to room temperature and then concentrated to a residue.

The crude product was purified by silica gel chromatography eluting with 10:1 – 2:1 hexanes/EtOAc to yield 0.851 g (3.10 mmol, 50% over 2 steps) of the thiourea 3.2.82 as a yellow

25 -1 oil. Rf (1:1 hexanes/EtOAc) = 0.76; [α]ᴅ = +35.72 (c = 0.1, DCM); IR (thin film, cm ): 2924,

1 1738, 1522, 1370, 1263, 1153, 732, 702; H NMR (500 MHz, CDCl3): δ 6.48 (s, 1H), 6.47 (s, 1H),

4.78 (d, J = 4.8 Hz, 1H), 4.72 (d, J = 4.8 Hz, 1H), 4.28 (m, 2H), 1.50 (s, 9H), 1.32 (t, J = 7.1 Hz,

13 3H) ; C NMR (100 MHz, CDCl3) δ 182.8, 168.7, 167.4, 84.4, 62.8, 61.4, 60.6, 28.0, 14.2. HRMS-

+ ESI (m / z): [M + H] calcd for C11H19N2O4, 275.1066 Da; found 275.1063 Da.

Tert-butyl (4S,5S)-5-(hydroxymethyl)-2-thioxoimidazolidine-4-carboxylate (3.2.85)

To a stirring solution of 3.2.82 (0.801 g, 2.92 mmol) in anhydrous MeOH (29 mL) was added dropwise a solution of sodium borohydride (0.331 g, 8.76 mmol) in anhydrous MeOH (29 mL) over 10 mins at 0 °C under a stream of argon. The reaction mixture was stirred for 1 h at 0 °C and an additional portion of sodium borohydride was added (0.110 g, 2.92 mmol). The reaction mixture was stirred for 2 h at 0 °C and consumption of starting material was observed by LCMS analysis.

The reaction mixture was diluted with DCM (50 mL) and poured into a saturated solution of ammonium chloride (100 mL). DCM (3 x 25 mL) was used to extract the aqueous layer and the combined organic extracts were washed with water (100 mL). The organic layer was dried over sodium sulfate, filtered, concentrated to a residue and purified by silica gel chromatography with

1:1 hexanes:EtOAc to yield 0.391 g (1.68 mmol, 58%) of 3.2.85 as a colorless gum. Rf (3:7

131

1 hexanes/EtOAc) = 0.32; H NMR (500 MHz, (CD3)2CO) δ 7.41 (bs, 1H), 7.30 (bs, 1H), 4.33 (d, J

= 5.8 Hz, 1H), 4.23 (t, J = 6.0 Hz, 1H), 4.08 (q, J = 4.9 Hz, 1H), 3.73 – 3.68 (m, 1H), 3.66 – 3.60

13 (m, 1H), 1.47 (s, 9H); C NMR (100 MHz, (CD3)2CO) δ 184.9, 170.4, 82.6, 63.7, 63.1, 61.2, 28.1.

+ LCMS-ESI (m / z): [M + H] calcd for C9H17N2O3S, 233.1 Da; found 233.1 Da.

Tert-butyl (S)-3-(((benzyloxy)carbonyl)amino)-5-nitro-4-oxopentanoate (3.2.91)

To a stirring solution of carbonyl diimidazole (2.60 g, 16.04 mmol) in freshly distilled THF (165 mL) was added a solution of N-Cbz-L-aspartic acid 4-tert-butyl ester 3.2.78 (4.94 g, 15.28 mmol) in freshly distilled THF (55 mL) at 22 °C under argon and stirred for 5 h. A solution of the nitro methane, potassium salt was prepared by diluting potassium tert-butoxide (1.89 g, 16.81 mmol) in freshly distilled THF (220 mL) which was placed in an ice bath at 0 °C. A solution of nitromethane

(8.18 mL, 152.78 mmol) in THF (88 mL) was added to the potassium tert-butoxide solution at 0

°C and stirred for 30 mins at 0 °C, and then allowed to warm to room temperature over 30 mins.

The vessel containing the activated mixed anhydride was added to this solution via cannula and then stirred for 12 h. The reaction mixture was quenched with a 1 M HCl solution (400 mL), diluted with EtOAc (100 mL), and then extracted with EtOAc (3 x 100 mL). The combined organic layers were washed with brine (200 mL) and dried over Na2SO4. The solvent is removed to yield 4.6 g

(12.6 mmol, 93%) of pure nitro ketone 3.2.91 as an amber solid: mp 88-91 °C. Rf (5%

21 -1 MeOH/DCM) = 0.36; [α]ᴅ = -43.12 (c = 0.5, DCM); IR (thin film, cm ): 3366, 2979, 2934, 1717,

1 1562, 1510, 1455, 1369, 1316, 1243, 1155, 1053, 998, 844, 752, 698; H NMR (500 MHz, CDCl3)

δ 7.42 – 7.33 (m, 5H), 5.85 (d, J = 8.6 Hz, 1H), 5.60 (d, J = 15.3 Hz, 1H), 5.49 (d, J = 15.4 Hz,

1H), 5.16 (s, 2H), 4.61 (dt, J = 8.4, 4.1 Hz, 1H), 3.06 (dd, J = 17.5, 4.2 Hz, 1H), 2.72 (dd, J = 17.5,

132

13 4.7 Hz, 1H), 1.43 (s, 9H); C NMR (100 MHz, CDCl3): δ 195.9, 170.4, 156.2, 135.7, 128. 8,

128.7, 128.4, 82.9, 81.9, 77.5, 77.2, 76.8, 68.0, 55.7, 36.8, 28.0. HRMS-ESI (m / z): [M + Na]+ calcd for C17H22N2O7Na, 389.1325 Da; found 389.1325 Da.

Tert-butyl (3S,4R)-3-(((benzyloxy)carbonyl)amino)-4-hydroxy-5-nitropentanoate (3.2.89a) and Tert-butyl (3S,4S)-3-(((benzyloxy)carbonyl)amino)-4-hydroxy-5-nitropentanoate

(3.2.89b)

To a stirring solution of 3.2.91 (10.7 g, 29.23 mmol) in a solvent mixture of anhydrous DCM (35 mL) and anhydrous MeOH (25 mL) at 0 °C was added sodium borohydride (1.11 g, 29.23 mmol) portion wise over 1 h under argon. The reaction mixture was allowed to stir for 30 mins following the final addition of sodium borohydride. While stirring at 0 °C, the reaction mixture was quenched by the addition of 10% KHSO4 solution, diluted with DCM (100 mL) and then extracted with

DCM (3 x 50 mL). The combined organic layers were washed with water (100 mL), dried over sodium sulfate, and concentrated. The crude product was purified by silica gel chromatography eluting with 5:1 – 3:1 hexanes/EtOAc to yield 9.3 g (25.25 mmol, 86%) of the nitro alcohol 3.2.89a and 3.2.89b as a diastereomeric mixture of a clear oil (dr: >6:1 a:b); Rf (2:1 hexanes/EtOAc) =

23 -1 0.38; [α]ᴅ = +1.88 (c = 1.03, DCM); IR (thin film, cm ): 3339, 1697, 1554, 1368, 1253, 1154,

1 1039, 697; H NMR (500 MHz, CD3OD) δ 7.38 – 7.27 (m, 5H), 5.12 (d, J = 12.3 Hz, 1H), 5.04

(d, J = 12.5 Hz, 1H), 4.62 (dd, J = 12.7, 2.6 Hz, 1H), 4.41 – 4.36 (m, 1H), 4.22 – 4.16 (m, 1H),

3.95 (td, J = 9.6, 3.9 Hz, 1H), 2.74 (dd, J = 15.4, 4.1 Hz, 1H), 2.38 (dd, J = 15.4, 9.9 Hz, 1H), 1.40

13 (s, 9H); C NMR (100 MHz, CD3OD) δ 172.1, 158.2, 138.1, 129.5, 129.0, 128.9, 82.1, 80.2, 72.2,

133

+ 67.7, 52.7, 38.3, 28.3. HRMS-ESI (m / z): [M + Na] calcd for C17H24N2O7Na, 391.1481 Da; found

391.1478 Da.

Tert-butyl (3S,4R)-3-(((benzyloxy)carbonyl)amino)-5-((tert-butoxycarbonyl)amino)-4- hydroxypentanoate (3.2.92a) and tert-butyl (3S,4S)-3-(((benzyloxy)carbonyl)amino)-5-

((tert-butoxycarbonyl)amino)-4-hydroxypentanoate (3.2.92b)

To a stirring solution of 3.2.89a and 3.2.89b (10.85 g, 29.46 mmol) and nickel(II) chloride hexahydrate (7.00 g, 29.46 mmol) in MeOH (150 mL) and THF (150 mL) was added sodium borohydride (5.57 g, 147.29 mmol) portion wise over 10 mins at 0 °C under a stream of argon.

The reaction mixture was stirred for 30 mins and reaction progress was determined by TLC analysis. Upon consumption of the starting material, di-tert-butyl dicarbonate (19.29 g, 88.37 mmol) was added at 0 °C, stirred at 0 °C for 15 mins, warmed to rt and then stirred for 12 h. A

10% solution of sodium bicarbonate (200 mL) was added and the reaction mixture was filtered over celite, concentrated to half its volume and added to water (100 mL). The aqueous layer was extracted with EtOAc (3 x 100 mL) and the combined organic extracts were washed with brine

(200 mL). The organic layer was dried over sodium sulfate, filtered and concentrated to a residue.

The crude product was purified by silica gel chromatography with 5:1 – 3:1 hexanes/EtOAc to yield 11.0 g (25.08 mmol, 85%) of a diastereomeric mixture of 3.2.92a and 3.2.92b as a clear oil.

25 -1 Rf (1:1 hexanes/EtOAc) = 0.50; [α]ᴅ = +25.53 (c = 1.3, DCM); IR (thin film, cm ): 3339, 2977,

1 1691, 1511, 1455, 1392, 1366, 1248, 1157, 1040, 845, 738, 697; H NMR (500 MHz, CDCl3) δ

7.37 – 7.33 (m, 5H), 5.68 (d, J = 9.1 Hz, 1H), 5.46 (bs, 1H), 5.09 (s, 2H), 3.95 – 3.88 (m, 1H),

3.65 – 3.53 (m, 2H), 3.02 (dt, J = 14.9, 4.7 Hz, 1H), 2.67 (dd, J = 16.4, 5.8 Hz, 1H), 2.61 – 2.54

134

13 (m, 1H), 1.44 (s, 9H), 1.42 (s, 9H). C NMR (126 MHz, CDCl3) δ 171.6, 157.8, 156.5, 136.3,

128.5, 128.1, 128.0, 81.2, 79.8, 73.0, 66.8, 50.2, 43.2, 36.2, 28.4, 28.0. HRMS-ESI (m / z): [M +

+ Na] calcd for C22H34N2O7Na, 461.2264 Da; found 461.2260 Da.

Benzyl ((4S,5R)-5-hydroxy-2-oxopiperidin-4-yl)carbamate (3.2.93)

A stirring solution of 3.2.92a and 3.2.92b (5.33 g, 12.15 mmol) in formic acid (118 mL, 3.11 mol) was heated to 60 °C for 8 h under argon. The solvent was removed under reduced pressure, and the residue was concentrated in heptane (3 x 100 mL) to remove residual formic acid. The residue was diluted in MeOH (100 mL) and sodium carbonate (12.88 g, 121.54 mmol) was added and the reaction mixture was stirred for 1 h. The reaction mixture was diluted in DCM (100 mL), filtered through a celite pad and concentrated to a residue. The crude product was purified by silica gel chromatography with 10% MeOH/EtOAc to yield 1.30 g (4.92 mmol, 40%) of 3.2.93 as a white

28 solid: mp 166-171 °C. Rf (12% MeOH/EtOAc) = 0.28; [α]ᴅ = -22.17 (c = 0.1, MeOH); IR (thin film, cm-1): 3307, 2922, 1699, 1649, 1540, 1495, 1455, 1333, 1257, 1042, 738, 697; 1H NMR (500

MHz, CD3OD) δ 7.40 – 7.27 (m, 5H), 5.09 (s, 2H), 4.09 (q, J = 2.7 Hz, 1H), 4.02 – 3.94 (m, 1H),

3.44 (dd, J = 13.4, 3.0 Hz, 1H), 3.28 (dd, J = 13.4, 2.8 Hz, 1H), 2.52 (dd, J = 17.4, 11.1 Hz, 1H),

13 2.45 (dd, J = 17.4, 6.6 Hz, 1H); C NMR (126 MHz, (CD3)2SO) δ 169.1, 155.6, 137.1, 128.4,

+ 127.9, 127.9, 65.4, 63.4, 48.9, 46.1, 33.0. HRMS-ESI (m / z): [M + Na] calcd for C13H16N2O4Na,

287.1007 Da; found 287.1005 Da.

135

Benzyl ((4S,5R)-5-((tert-butyldimethylsilyl)oxy)-2-oxopiperidin-4-yl)carbamate (3.2.94)

To a stirring solution of crude 3.2.93 (1.34 g, 5.05 mmol) in anhydrous DMF (21.9 mL) was 2,6- lutidine (2.37 mL, 20.21 mmol) and tert-butyldimethylsilyl trifluoromethanesulfonate (2.37 mL,

10.10 mmol) at 0 °C under a stream of argon. After 20 mins, the reaction mixture was warmed to rt and stirred for 12 h. Upon consumption of starting material determined by TLC, the reaction was quenched with the addition of brine (50 mL). Ether (50 mL) was added to the mixture and used to extract the organic layer (3 x 50 mL), and then the organic extracts were washed with water

(2 x 50 mL) and brine (50 mL). The combined organic layers were dried over sodium sulfate, filtered and concentrated to a residue. The crude product was purified by silica gel chromatography with 2:1 – 10:1 EtOAc/hexanes to yield 1.46 g (3.86 mmol, 76%) of protected lactam 3.2.94 as a

22 white solid: mp 57-59 °C. Rf (10:1 EtOAc/hexanes) = 0.17; [α]ᴅ = -26.40 (c = 1.0, DCM); IR (thin

-1 1 film, cm ): 2927, 1705, 1664, 1494, 1336, 1258, 1088, 1047, 835; H NMR (500 MHz, CDCl3) δ

7.39 – 7.30 (m, 5H), 6.06 (bs, 1H), 5.10 (s, 2H), 4.83 (d, J = 7.9 Hz, 1H), 4.19 – 4.15 (m, 1H),

4.08 – 3.99 (m, 1H), 3.44 (d, J = 12.5 Hz, 1H), 3.23 (dt, J = 13.0, 3.0 Hz, 1H), 2.53 (dd, J = 17.0,

6.3 Hz, 1H), 2.46 (dd, J = 17.0, 11.5 Hz, 1H), 0.87 (s, 9H), 0.05 (d, J = 7.5 Hz, 6H); 13C NMR

(100 MHz, CDCl3) δ 170.5, 155.6, 136.3, 128.7, 128.4, 128.3, 67.1, 65.7, 49.2, 47.2, 33.2, 25.8,

+ 18.1, -4.7, -4.8. HRMS-ESI (m / z): [M + Na] calcd for C19H30N2O4SiNa, 401.1872 Da; found

401.1868 Da.

136

Tert-butyl (4S,5R)-4-(((benzyloxy)carbonyl)amino)-5-((tert-butyldimethylsilyl)oxy)-2- oxopiperidine-1-carboxylate (3.2.95)

To a stirring solution of 3.2.94 (0.730 g, 1.93 mmol) in anhydrous MeCN (25 mL) was added 4- dimethylaminopyridine (0.012 g, 0.096 mmol) and di-tert-butyl dicarbonate (0.210 g, 0.964 mmol) at 0 °C under a stream of argon. The reaction mixture was warmed to room temperature and stirred for 3 h. An additional portion of di-tert-butyl dicarbonate (0.210 g, 0.964 mmol) was added at 0

°C. Following 30 mins, consumption of starting material was observed by TLC, and the reaction was quenched with a saturated solution of ammonium chloride (50 mL). The organic layer was diluted with EtOAc and extracted (3 x 30 mL), and then the organic extracts were washed with brine. The combined organic layers were dried over sodium sulfate, filtered and concentrated to a residue. The crude product was purified by silica gel chromatography with 5:1 hexanes/EtOAc to yield 0.742 g (1.55 mmol, 87%) of Boc protected lactam 3.2.95 as a white solid: mp 95-97°C. Rf

22 -1 (1:1 hexanes/EtOAc) = 0.73; [α]ᴅ = -15.90 (c = 1.0, DCM); IR (thin film, cm ): 2929, 1772, 1712,

1 1527, 1368, 1251, 1139, 1065, 979, 835, 776, 697; H NMR (500 MHz, CDCl3) δ 7.38 – 7.31 (m,

5H), 5.11 (s, 2H), 4.82 (d, J = 8.6 Hz, 1H), 4.23 – 4.18 (m, 1H), 4.09 – 4.02 (m, 1H), 3.93 (dd, J

= 13.9, 3.4 Hz, 1H), 3.47 (d, J = 13.8 Hz, 1H), 2.72 (dd, J = 16.9, 6.5 Hz, 1H), 2.60 (dd, J = 16.9,

13 11.3 Hz, 1H), 1.51 (s, 9H), 0.87 (s, 9H), 0.07 (s, 3H), 0.06 (s, 3H); C NMR (100 MHz, CDCl3) δ

168.5, 155.6, 152.5, 136.3, 128.7, 128.4, 128.3, 83.5, 67.2, 66.2, 50.4, 49.2, 36.6, 28.1, 25.8, 18.1,

+ -4.7, -4.8. HRMS-ESI (m / z): [M + Na] calcd for C24H38N2O6SiNa, 501.2397 Da; found

501.23867 Da.

137

Tert-butyl (3S,4S,5R)-3-azido-4-(((benzyloxy)carbonyl)amino)-5-((tert- butyldimethylsilyl)oxy)-2-oxopiperidine-1-carboxylate (3.2.96)

To a flame dried, 25 mL round bottom flask was added freshly distilled THF (7.5 mL) under argon and then cooled to -78 °C. Potassium bis(trimethylsilyl)amide (1 M in THF, 3.47 mmol) was added to the round bottom flask and stirred. A separate solution was prepared by diluting 3.2.95 (0.503 g, 1.05 mmol) in freshly distilled THF (2.65 mL) and then cooled to -78 °C in a pear-shaped flask under argon. The solution of 3.2.95 was then added dropwise via cannula to the potassium bis(trimethylsilyl)amide solution and continued stirring for 40 mins. A separate solution was prepared by diluting 2,4,6-triisopropylbenzenesulfonyl azide (0.650 g, 2.10 mmol) in freshly distilled THF (4.0 mL) and then cooled to -78 °C in a pear-shaped flask under argon. The newly prepared solution was then added via cannula to the reaction mixture and allowed to stir for 2 mins.

The reaction was then quenched with acetic acid (0.276 mL, 4.83 mmol) at - 78 °C, the cold bath was removed, and the reaction mixture was warmed to room temperature over 3 h. A saturated solution of sodium bicarbonate (20 mL) was added and then EtOAc was used to extract the organic layer (3 x 25 mL). The organic extracts were washed with brine, dried over sodium sulfate, filtered and concentrated to a residue. The crude product was purified by silica gel chromatography with

12:1 hexanes/EtOAc to yield 0.237 g (0.456 mmol, 43%) of azido lactam 3.2.96 as a white solid:

23 - mp 117-119 °C. Rf (2:1 hexanes/EtOAc) = 0.79; [α]ᴅ = -54.90 (c = 1.0, DCM); IR (thin film, cm

1 1 ): 2930, 2113, 1774, 1723, 1525, 1369, 1257, 1140, 1067, 838, 779; H NMR (500 MHz, CDCl3)

δ 7.39 – 7.31 (m, 5H), 5.17 – 5.10 (m, 2H), 4.98 (d, J = 7.8 Hz, 1H), 4.31 – 4.25 (m, 1H), 4.15 (d,

138

J = 11.1 Hz, 1H), 3.91 (dd, J = 14.0, 3.0 Hz, 1H), 3.86 (d, J = 10.0 Hz, 1H), 3.49 (d, J = 13.7 Hz,

13 1H), 1.52 (s, 9H), 0.87 (s, 9H), 0.07 (s, 6H); C NMR (100 MHz, CDCl3) δ 167.4, 155.7, 152.2,

136.2, 128.7, 128.5, 128.3, 84.3, 67.4, 66.6, 62.6, 54.5, 49.9, 28.0, 25.7, 18.1, -4.8, -4.9. HRMS-

+ ESI (m / z): [M + Na] calcd for C24H37N5O6SiNa, 542.2411 Da; found 542.2409 Da.

(3aS,7R,7aS)-7-((tert-butyldimethylsilyl)oxy)-2-thioxooctahydro-4H-imidazo[4,5-c]pyridin-

4-one (3.2.98)

To a stirring solution of 3.2.96 (1.10 g, 2.12 mmol) in anhydrous DCM (10 mL) was added trifluoroacetic acid (5 mL) at 0 °C under a stream of argon. The reaction mixture was stirred for 2 h at 0 °C, and consumption of starting material was observed through TLC analysis. While maintaining a temperature of at 0 °C, the reaction mixture was quenched through the addition of a saturated solution of sodium bicarbonate (20 mL). The reaction mixture was warmed to rt, diluted with EtOAc (20 mL) and extracted with EtOAc (3 x 10 mL). The organic extracts were washed with brine (50 mL), dried over sodium sulfate, filtered and concentrated to a residue. The crude lactam product was used directly in the next step. To a stirring solution of crude lactam (0.887 g) diluted in methanol (21 mL) was added Pearlman’s catalyst (Pd 20% on carbon, nominally 50% water, 0.267 g). The reaction vessel was evacuated and purged with argon gas (7x), evacuated and purged with hydrogen gas (7x) and then stirred for 12 h under a hydrogen atmosphere (balloon).

The reaction mixture was filtered over a celite bed and concentrated to a residue. The crude product

3.2.97 was used directly in the next step. To a stirring solution of 3.2.97 (0.539 g) diluted in dry

MeCN (46 mL) was added imidazole (0.110M in MeCN, 0.010 g, 0.145 mmol) followed by thiocarbonyl diimidazole (0.481 g, 2.70 mmol). The reaction vessel was fitted with a Vigreux

139

column capped with a drying tube containing Drierite®. Upon warming to 50 °C, the suspension went entirely into solution and the reaction mixture was stirred for 3.5 h when consumption of starting material was observed through TLC analysis. The reaction mixture was concentrated to a residue and the crude product was purified by silica gel chromatography with 3:1 EtOAc/hexanes to yield 0.443 g (1.47 mmol, 70% over 3 steps) of thiourea lactam 3.2.98 as a white solid: mp 237-

28 -1 241 °C. Rf (3:1 EtOAc/hexanes) = 0.20; [α]ᴅ = -120.41 (c = 0.1, MeOH); IR (thin film, cm ):

3200, 2928, 2856, 1684, 1502, 1470, 1330, 1296, 1253, 1181, 1140, 1007, 895, 836, 780, 731,

1 571, 548, 443; H NMR (500 MHz, (CD3)2SO) δ 8.56 (s, 1H), 8.44 (s, 1H), 7.34 (s, 1H), 4.39 (bs,

1H), 4.17 (d, J = 14.7 Hz, 1H), 3.68 (d, J = 14.9 Hz, 1H), 3.53 (ddd, J = 13.6, 3.5, 2.2 Hz, 1H),

13 2.99 (d, J = 13.5 Hz, 1H), 0.86 (s, 9H), 0.10 (s, 3H), 0.10 (s, 3H). C NMR (100 MHz, (CD3)2SO)

δ 187.5, 167.7, 63.2, 61.8, 55.2, 50.0, 25.7, 17.8, -4. 6, -4.9. HRMS-ESI (m / z): [M + H]+ calcd for C12H24N3O2SSi, 302.1359 Da; found 302.1358 Da.

(3aS,7R,7aS)-7-((tert-butyldimethylsilyl)oxy)-2-(methylthio)-1,3a,5,6,7,7a-hexahydro-4H- imidazo[4,5-c]pyridin-4-one (3.2.99)

To a stirring solution of 3.2.98 (0.103 g, 0.342 mmol) in freshly distilled THF (6 mL) and anhydrous MeCN (6 mL) was added iodomethane (0.213 mL, 3.42 mmol) at rt and then the reaction vessel was sealed with a glass stopper. The reaction mixture was warmed to 45 °C and stirring for 1 h until consumption of starting material was observed through TLC analysis. The reaction mixture was cooled to rt, concentrated to a residue and the crude product was purified by silica gel chromatography with 3% MeOH/DCM to yield 0.078 g (0.247 mmol, 72%) of

140

28 isothiourea lactam 3.2.99 as a colorless oil. Rf (5% MeOH/DCM) = 0.20; [α]ᴅ = -41.17 (c = 0.2,

MeOH); IR (thin film, cm-1): 3242, 2927, 2854, 1677, 1550, 1462, 1255, 1142, 1084, 1004, 898,

1 836, 779; H NMR (500 MHz, (CD3)2SO) δ 7.12 (s, 1H), 7.05 (bs, 1H), 4.58 – 4.55 (m, 1H), 4.04

(d, J = 15.5 Hz, 1H), 3.57 – 3.51 (m, 1H), 3.49 (dd, J = 15.5, 2.2 Hz, 1H), 2.98 (d, J = 13.4 Hz,

13 1H), 2.37 (s, 3H), 0.85 (s, 9H), 0.11 (s, 3H), 0.08 (s, 3H); C NMR (100 MHz, (CD3)2SO) δ 170.0,

166.3, 65.0, 50.4, 25.7, 18.0, 12.8, -4.4, -4.8. HRMS-ESI (m / z): [M + H]+ calcd for

C13H26N3O2SSi, 316.1515 Da; found 316.1511 Da.

Benzyl ((3S,4S,5R)-3-amino-5-((tert-butyldimethylsilyl)oxy)-2-oxopiperidin-4-yl)carbamate

(3.2.106)

To a stirring solution of 3.2.96 (0.205 g, 0.394 mmol) in anhydrous DCM (2 mL) was added trifluoroacetic acid (1 mL) at 0 °C under a stream of argon. The reaction mixture was stirred for 2 h at 0 °C, and consumption of starting material was observed through TLC analysis. While maintaining a temperature of at 0 °C, the reaction mixture was quenched through the addition of a saturated solution of sodium bicarbonate (5 mL). The reaction mixture was warmed to rt, diluted with EtOAc (5 mL) and extracted with EtOAc (3 x 10 mL). The organic extracts were washed with brine (50 mL), dried over sodium sulfate, filtered and concentrated to a residue. The crude lactam product was used directly in the next step. To a stirring solution of crude lactam (0.155 g) diluted in THF (4 mL) and water (0.785 mL) was added triphenylphosphine (0.128 g, 0.490 mmol) at rt and the reaction mixture was stirred for 12 h. Consumption of starting material was observed through TLC analysis, the reaction mixture was concentrated to a residue and the crude product

141

was purified by silica gel chromatography with 1:1 hexanes:EtOAc, then 5-10% MeOH/DCM to yield 0.132 g (0.335 mmol, 85% over 2 steps) of 3.2.106 as a white solid: mp 56-61 °C. Rf (5%

25 -1 MeOH/DCM) = 0.16; [α]ᴅ = -93.38 (c = 0.2, DCM); IR (thin film, cm ): 2928, 2856, 1666, 1490,

1 1263, 1119, 1050, 1000, 940, 836, 777, 733, 700; H NMR (500 MHz, CDCl3) δ 7.38 – 7.30 (m,

5H), 5.90 (bs, 1H), 5.32 (bs, 1H), 5.14 (d, J = 12.1 Hz, 1H), 5.08 (d, J = 12.3 Hz, 1H), 4.37 – 4.34

(m, 1H), 3.76 (m, 1H), 3.53 (d, J = 9.1 Hz, 1H), 3.50 (d, J = 12.7 Hz, 1H), 3.20 (dt, J = 12.7, 2.8

13 Hz, 1H), 2.04 (bs, 2H), 0.86 (s, 9H), 0.04 (s, 3H), 0.03 (s, 3H); C NMR (126 MHz, CDCl3) δ

173.2, 156.3, 136.4, 128.6, 128.31, 128.30, 67.0, 66.4, 55.8, 51.4, 47.2, 25.8, 18.0, -4.8, -4.9.

+ HRMS-ESI (m / z): [M + H] calcd for C19H32N3O4Si, 394.2162 Da; found 394.2154 Da.

Benzyl ((3S,4S,5R)-5-((tert-butyldimethylsilyl)oxy)-3-isothiocyanato-2-oxopiperidin-4- yl)carbamate (3.2.107)

To a stirring solution of 3.2.106 (0.103 g, 0.262 mmol) in DCM (5 mL) was added a saturated solution of sodium bicarbonate (5 mL) at 0 °C. The reaction mixture was stirred for 10 mins at 0

°C before the stirring was stopped, and thiophosgene (0.027 mL, 0.293 mmol) was added to the lower, organic layer via pipette. The reaction mixture was warmed to rt and then stirring for an hour before consumption of starting material was observed through TLC analysis. The reaction mixture was diluted with DCM (10 mL) and poured into a saturated solution of sodium bicarbonate

(10 mL). The aqueous layer was extracted with DCM (3 x 10 mL), washed with water (50 mL) dried over sodium sulfate, filtered and concentrated to a residue. The crude product was purified by silica gel chromatography with 2:1 hexanes:EtOAc to yield 0.102 g (0.234 mmol, 89%) of

142

26 3.2.107 as a white film. Rf (1:1 EtOAc/hexanes) = 0.46; [α]ᴅ = -114.85 (c = 0.7, DCM); IR (thin film, cm-1): 3270, 2928, 2856, 2057, 1684, 1486, 1260, 1219, 1111, 1047, 1002, 943, 837, 809,

1 778, 697, 534; H NMR (500 MHz, CDCl3) δ 7.41 – 7.30 (m, 5H), 6.27 (bs, 1H), 5.18 (d, J = 12.4

Hz, 1H), 5.14 (d, J = 12.1 Hz, 1H), 5.07 (d, J = 8.7 Hz, 1H), 4.54 (d, J = 11.4 Hz, 1H), 4.28 – 4.25

(m, 1H), 4.17 (td, J = 8.8, 4.2 Hz, 1H), 3.51 (dd, J = 13.2, 2.7 Hz, 1H), 3.22 (dt, J = 13.2, 2.9 Hz,

13 1H), 0.87 (s, 9H), 0.06 (s, 3H), 0.06 (s, 3H); C NMR (126 MHz, CDCl3) δ 165.9, 155.6, 140.6,

136.0, 128.7, 128.4, 128.4, 67.4, 66.8, 58.2, 54.7, 46.9, 25.7, 18.0, -4.8, -4.9. HRMS-ESI (m / z):

+ [M + Na] calcd for C20H29N3O4SSiNa, 458.1546 Da; found 458.1541 Da.

(4S,5R)-4-amino-5-((tert-butyldimethylsilyl)oxy)piperidin-2-one (3.2.111)

To a stirring solution of 3.2.94 (1.37 g, 3.63 mmol) in anhydrous MeOH (1.77 mL) was added palladium on carbon (Pd 10% on carbon, 0.386 g). The reaction vessel was evacuated and purged with argon gas (7x), evacuated and purged with hydrogen gas (7x) and then stirred for 12 h under a hydrogen atmosphere (balloon). The reaction mixture was filtered over a celite bed, concentrated to 0.887 g (3.63 mmol, 99%) of 3.2.111 as a white solid and used directly in the next step without

25 further purification. mp 125-127 °C. Rf (10:1 EtOAc/hexanes) = 0.20; [α]ᴅ = -24.47 (c = 0.3,

MeOH); IR (thin film, cm-1): 3269, 2928, 2855, 1641, 1492, 1343, 1250, 1099, 1046, 991, 937,

1 832, 773, 705, 489; H NMR (500 MHz, CD3OD) δ 4.08 (q, J = 3.1 Hz, 1H), 3.36 (dd, J = 13.2,

3.1 Hz, 1H), 3.28 (dd, J = 13.2, 3.8 Hz, 1H), 3.15 (ddd, J = 9.7, 5.9, 2.1 Hz, 1H), 2.49 (dd, J =

17.5, 5.8 Hz, 1H), 2.29 (dd, J = 17.5, 9.7 Hz, 1H), 0.93 (s, 9H), 0.16 (s, 3H), 0.15 (s, 3H); 13C

143

NMR (126 MHz, CDCl3) δ 171.7, 68.2, 49.4, 45.9, 37.0, 25.8, 18.1, -4.5, -4.7. HRMS-ESI (m / z):

+ [M + H] calcd for C11H25N2O2Si, 245.1685 Da; found 245.1681 Da.

Tert-butyl (4S,5R)-4-((tert-butoxycarbonyl)amino)-5-((tert-butyldimethylsilyl)oxy)-2- oxopiperidine-1-carboxylate (3.2.112)

To a stirring solution of 3.2.111 (0.725 g, 2.97 mmol) in anhydrous DMF (14.8 mL) was added 4- dimethylaminopyridine (0.036 g, 0.297 mmol) at 0 °C under a stream of argon. The reaction mixture was stirred for 10 mins before di-tert-butyl dicarbonate (1.29 g, 5.93 mmol) was added.

The reaction mixture was warmed to rt and stirred for 3 h, cooled again to 0 °C and was added di- tert-butyl dicarbonate (0.647 g, 2.97 mmol). The reaction mixture was warmed to rt, stirred for 3 h and then consumption of starting material was observed through TLC analysis. The reaction was quenched with the addition of brine (50 mL). EtOAc (50 mL) was added to the mixture and used to extract the organic layer (3 x 30 mL), and then the organic extracts were washed with water (2 x 50 mL) and brine (50 mL). The combined organic layers were dried over sodium sulfate, filtered and concentrated to a residue. The crude product was purified by silica gel chromatography with

5:1 hexanes:EtOAc to yield 1.00 g (2.25 mmol, 76%) of 3.2.112 as an off-white solid: mp 107-

27 -1 110 °C. Rf (2:1 hexanes:EtOAc) = 0.50; [α]ᴅ = -22.66 (c = 0.5, DCM); IR (thin film, cm ): 2930,

1774, 1714, 1501, 1391, 1367, 1298, 1253, 1158, 1095, 1070, 974, 837, 778; 1H NMR (500 MHz,

CDCl3) δ 4.57 (d, J = 8.6 Hz, 1H), 4.22 – 4.19 (m, 1H), 3.99 – 3.94 (m, 1H), 3.92 (dd, J = 13.8,

3.3 Hz, 1H), 3.45 (dd, J = 13.8, 2.1 Hz, 1H), 2.68 (dd, J = 16.9, 6.4 Hz, 1H), 2.57 (dd, J = 17.0,

11.5 Hz, 1H), 1.51 (s, 9H), 1.44 (s, 9H), 0.88 (s, 9H), 0.10 (s, 3H), 0.09 (s, 3H); 13C NMR (126

144

MHz, CDCl3) δ 168.8, 155.0, 152.5, 83.4, 80.1, 66.1, 50.5, 48.6, 36.4, 28.4, 28.1, 25.8, 18.1, -4.76,

+ -4.83. HRMS-ESI (m / z): [M + Na] calcd for C21H40N2O6SiNa, 467.2553 Da; found 467.2545

Da.

Tert-butyl (3S,4S,5R)-3-azido-4-((tert-butoxycarbonyl)amino)-5-((tert-butyldimethylsilyl) oxy)-2-oxopiperidine-1-carboxylate (3.2.113)

To a flame dried, round bottom flask was added freshly distilled THF (14 mL) under argon and then cooled to -78 °C. Potassium bis(trimethylsilyl)amide (1 M in THF, 6.75 mmol) was added to the round bottom flask and stirred. A separate solution was prepared by diluting 3.2.112 (1.00 g,

2.25 mmol) in freshly distilled THF (5.6 mL) and then cooled to -78 °C in a pear-shaped flask under argon. The solution of 3.2.112 was then added dropwise via cannula to the potassium bis(trimethylsilyl)amide solution and continued stirring for 40 mins. A separate solution was prepared by diluting 2,4,6-triisopropylbenzenesulfonyl azide (1.39 g, 4.5 mmol) in freshly distilled

THF (8.7 mL) and then cooled to -78 °C in a pear-shaped flask under argon. The newly prepared solution was then added via cannula to the reaction mixture and allowed to stir for 2 mins. The reaction was then quenched with acetic acid (0.592 mL, 10.35 mmol) at - 78 °C, the cold bath was removed, and the reaction mixture was warmed to room temperature over 3 h. A saturated solution of sodium bicarbonate (40 mL) was added and then EtOAc was used to extract the organic layer

(3 x 50 mL). The organic extracts were washed with brine, dried over sodium sulfate, filtered and concentrated to a residue. The crude product was purified by silica gel chromatography with 9:1 hexanes/EtOAc to yield 0.522 g (1.07 mmol, 48%) of azido lactam 3.2.113 as a white solid: mp

145

27 -1 135-139 °C. Rf (4:1 hexanes/EtOAc) = 0.42; [α]ᴅ = -68.95 (c = 0.2, DCM); IR (thin film, cm ):

2930, 2857, 2112, 1776, 1723, 1506, 1472, 1392, 1368, 1286, 1256, 1157, 1067, 976, 837, 780;

1 H NMR (500 MHz, CDCl3) δ 4.75 (d, J = 7.9 Hz, 1H), 4.32 – 4.28 (m, 1H), 4.13 (d, J = 11.2 Hz,

1H), 3.90 (dd, J = 13.8, 3.3 Hz, 1H), 3.77 (dd, J = 9.4 Hz, 1H), 3.47 (dd, J = 13.9, 2.0 Hz, 1H),

13 1.52 (s, 9H), 1.46 (s, 9H), 0.89 (s, 9H), 0.11 (s, 3H), 0.09 (s, 3H); C NMR (126 MHz, CDCl3) δ

167.6, 155.1, 152.2, 84.2, 80.5, 66.4, 62.4, 54.1, 49.9, 28.4, 28.0, 25.7, 18.1, -4.8, -4.9. HRMS-

+ ESI (m / z): [M + Na] calcd for C21H40N2O6SiNa, 508.2567 Da; found 508.2565 Da.

Tert-butyl (3S,4S,5R)-4-((tert-butoxycarbonyl)amino)-5-((tert-butyldimethylsilyl)oxy)-3- isothiocyanato-2-oxopiperidine-1-carboxylate (3.2.114)

To a stirring solution of 3.2.113 (0.279 g, 0.574 mmol) in freshly distilled THF (1.15 mL) was added carbon disulfide (0.288 mL, 4.6 mmol) and triphenylphosphine (0.151 g, 0.574 mmol) at rt.

The reaction vessel was sealed with a glass stopper and stirred at rt for 12 h when consumption of starting material was observed through TLC analysis. The reaction mixture was concentrated to a residue and purified by silica gel chromatography with 5% acetone, 5% DCM in hexanes to yield

0.195 g (0.389 mmol, 68%) of 3.2.114 as a colorless gum. Rf (18:1:1 hexanes/acetone/DCM) =

28 -1 0.11; [α]ᴅ = -37.47 (c = 0.4, DCM); IR (thin film, cm ): 2930, 2857, 2056, 1776, 1719, 1500,

1 1391, 1368, 1290, 1256, 1152, 1124, 1057, 969, 838, 779, 491; H NMR (500 MHz, CDCl3) δ

4.79 (d, J = 8.7 Hz, 1H), 4.60 (d, J = 11.5 Hz, 1H), 4.29 – 4.26 (m, 1H), 4.08 (dd, J = 9.5 Hz, 1H),

3.87 (dd, J = 13.9, 3.2 Hz, 1H), 3.50 (dd, J = 14.0, 2.3 Hz, 1H), 1.51 (s, 9H), 1.47 (s, 9H), 0.88 (s,

13 9H), 0.12 (s, 3H), 0.09 (s, 3H); C NMR (126 MHz, CDCl3) δ 164.8, 154.8, 152.3, 141.1, 84.4,

146

80.9, 66.6, 60.7, 54.7, 49.9, 28.5, 28.0, 25.7, 18.1, -4.8, -4.9. HRMS-ESI (m / z): [M + Na]+ calcd for C22H39N3O6SSiNa, 524.2227 Da; found 524.2225 Da.

(S)-2,5-Bis(((benzyloxy)carbonyl)amino)pentyl methanesulfonate (3.2.62)

To a stirring solution of (S)-2,5-bis(((benzyloxy)carbonyl)amino)pentanoic acid 3.2.59 (0.200 g,

0.50 mmol) in freshly distilled THF (1.7 mL) was added carbonyl diimidazole (0.081 g, 0.50 mmol) and the solution was stirred at room temperature for 30 mins under a stream of argon. The reaction mixture was cooled to 0 °C then an aqueous solution of NaBH4 (1.5 M, 0.50 mmol) was added dropwise over 10 mins. The solution was brought to room temperature and stirred for 1 hour. The reaction mixture was then neutralized with 4 M HCl (10 mL) and extracted with EtOAc

(3 x 10 mL). The combined organic extracts were washed with brine (50 mL), dried over sodium sulfate, and concentrated under reduced pressure to yield the crude alcohol. To a solution of the crude alcohol (0.193 g) in anhydrous DCM (2.5 mL) was added triethylamine (0.108 mL, 0.75 mmol). The mixture was cooled to 0 °C then methanesulfonyl chloride (0.120 mL, 1.55 mmol) was added dropwise. The solution was then brought to rt and stirred for 12 h under a stream of argon. The reaction mixture was quenched with the addition of water (10 mL) and extracted with

DCM (3 x 25 mL). The organic layer was dried over sodium sulfate, filtered and concentrated to a residue. The crude product was purified by silica gel chromatography with 1:1 hexanes/EtOAc to yield 0.183 g (0.394 mmol, 79% over 2 steps) of 3.2.62 as a white solid: mp 105-106 °C. Rf (2:1

29 -1 EtOAc/hexanes) = 0.46; [α]ᴅ = -16.96 (c = 0.3, DCM); IR (thin film, cm ): 3320, 2933, 1697,

1 1527, 1454, 1352, 1248, 1173, 1025, 960, 820, 740, 698, 528; H NMR (500 MHz, CDCl3) δ 7.37

– 7.30 (m, 10H), 5.11 – 5.07 (m, 4H), 4.90 (bs, 1H), 4.24 (dd, J = 10.6, 3.8 Hz, 1H), 4.17 (dd, J =

10.5, 4.1 Hz, 1H), 3.95 – 3.88 (m, 1H), 3.23 – 3.16 (m, 2H), 2.95 (s, 3H), 1.63 – 1.51 (m, 4H); 13C

147

NMR (126 MHz, CDCl3) δ 156.7, 156.2, 136.5, 136.3, 128.5, 128.5, 128.2, 128.10, 128.05, 70.9,

+ 66.9, 66.6, 50.0, 40.4, 37.1, 28.0, 26.2. HRMS-ESI (m / z): [M + Na] calcd for C22H28N2O7SNa,

487.1515 Da; found 487.1509 Da.

Dibenzyl (5-cyanopentane-1,4-diyl)(S)-dicarbamate (3.2.63)

To a stirring solution of 3.2.62 (3.27 g, 7.03 mmol) in MeCN (88 mL) was carefully added 18- crown-6-ether (2.23 g, 8.4 mmol) and (KCN (1.37 g, 21.1 mmol) at rt. The reaction was warmed to 90 °C and stirred for 1 h then quenched with a saturated solution of sodium bicarbonate (100 mL) and extracted with EtOAc (4 x 50 mL). The organic layer was washed with brine (100 mL), dried over sodium sulfate, filtered and concentrated to a residue. The crude product was purified by silica gel chromatography with 5:1 hexanes:EtOAc to yield 2.65 g (6.70 mmol, 95%) of 3.2.63

29 as a white solid: mp 78-79 °C. Rf (2:1 hexanes/EtOAc) = 0.74; [α]ᴅ = -30.51 (c = 0.6, DCM); IR

(thin film, cm-1): 3321, 2949, 1693, 1528, 1454, 1252, 1136, 1026, 739, 697; 1H NMR (500 MHz,

CDCl3) δ 7.38 – 7.30 (m, 10H), 5.12 – 5.08 (m, 4H), 4.86 (t, J = 6.2 Hz, 1H), 3.94 – 3.86 (m, 1H),

3.26 – 3.17 (m, 2H), 2.72 (dd, J = 16.9, 5.5 Hz, 1H), 2.53 (dd, J = 16.8, 4.3 Hz, 1H), 1.64 – 1.51

13 (m, 4H); C NMR (100 MHz, CDCl3) δ 156.7, 155.9, 136.5, 136.1, 128.6, 128.6, 128.3, 128.2,

128.1, 117.4, 67.0, 66.7, 47.7, 40.3, 30.4, 26.5, 23.9. HRMS-ESI (m / z): [M + Na]+ calcd for

C22H25N3O4Na, 418.1743 Da; found 418.1741 Da.

Dibenzyl (6-oxohexane-1,4-diyl)(S)-dicarbamate (3.2.64)

To a stirring solution of 3.2.63 (1.00g, 2.53 mmol) in anhydrous DCM (23 mL) was added diisobutylaluminum hydride (1M in toluene, 7.59 mmol) dropwise at -78 °C under a stream of argon over 15 mins. The reaction mixture was stirred at -78 °C for 3 h until consumption of starting

148

material was observed through TLC analysis. The reaction was quenched by dropwise addition of cold MeOH (25 mL) before an aqueous solution of saturated potassium sodium tartrate (50 mL) was added. The mixture was left to stir for 12 h then was extracted with DCM (3 x 25 mL). The organic layer was dried over sodium sulfate, filtered and concentrated to a residue. The crude product was purified by silica gel chromatography with 3% MeOH/DCM to yield 0.466 g (1.17

29 mmol, 46%) of 3.2.64 as a colorless film. Rf (5% MeOH/DCM) = 0.32; [α]ᴅ = -14.16 (c = 0.7,

DCM); IR (thin film, cm-1): 3325, 2930, 1690, 1525, 1454, 1247, 1073, 738, 697; 1H NMR (500

MHz, CDCl3) δ 9.73 (s, 1H), 7.36 – 7.31 (m, 10H), 5.08 (s, 2H), 5.07 (s, 2H), 4.87 (bs, 1H), 4.10

– 4.04 (m, 1H), 3.22 – 3.17 (m, 2H), 2.68 – 2.63 (m, 2H), 1.60 – 1.52 (m, 4H); 13C NMR (126

MHz, CDCl3) δ 201.0, 156.6, 156.0, 136.6, 136.4, 128.57, 128.55, 128.2, 128.14, 128.11, 128.09,

+ 66.8, 66.6, 48.8, 46.7, 40.5, 31.9, 26.7. HRMS-ESI (m / z): [M + Na] calcd for C22H26N2O5Na,

421.1739 Da; found 421.1736 Da.

(S)-3,6-bis(((benzyloxy)carbonyl)amino)hexanoic acid (3.1.11)

To a stirring solution of 3.2.64 (0.710 g, 1.78 mmol) in t-BuOH (60 mL) was added 2-methyl-2- butene (3.78 mL, 35.64 mmol) followed by solutions of NaOCl (1M in water, 15.2 mmol) and

NaH2PO4 (1M in water, 15.2 mmol). The reaction mixture was stirred for 1 h at rt then quenched a saturated solution of sodium sulfite (100 mL) and 1 M HCl until the solution was acidic (pH <

4). The aqueous layer was extracted with EtOAc (3 x 50 mL), washed with brine, then concentrated to a residue. The crude material was suspended in DCM and the resulting precipitate was filtered off to afford 0.718 g (1.73 mmol, 97%) of 3.2.11 as a white solid: mp 146-149 °C (lit. mp 155

95 25 15 95 -1 °C ). [α]ᴅ = +1.78 (c = 0.2, DMF) (lit [α]ᴅ = +1.0 (c = 1.0, DMF) ); IR (thin film, cm ): 3316,

1 2924, 1699, 1534, 1454, 1257, 1026, 738, 697; H NMR (500 MHz, (CD3)2SO) δ 7.38 – 7.28 (m,

149

10H), 7.23 (t, J = 5.7 Hz, 1H), 7.19 (d, J = 8.7 Hz, 1H), 5.00 (s, 4H), 3.85 – 3.71 (m, 1H), 2.96 (q,

J = 5.9 Hz, 2H), 2.34 (qd, J = 15.4, 6.9 Hz, 2H), 1.46 – 1.33 (m, 4H); 13C NMR (126 MHz,

(CD3)2SO) δ 172.5, 156.1, 155.7, 137.34, 137.29, 128.4, 127.81, 127.78, 127. 7, 65.2, 65.1, 47.9,

+ 40.3, 31.8, 31.4, 26.2. HRMS-ESI (m / z): [M + Na] calcd for C22H26N2O6Na, 437.1689 Da; found

437.1683 Da.

Alloc N-(triethylammoniumsulfonyl)carbamate (3.2.18)

To a stirring solution of chlorosulfonylisocyanate (1.4 mL, 16.07 mmol) in anhydrous DCM (4 mL) was added a solution of allyl alcohol (1.16 mL, 16.88 mmol) in anhydrous DCM (4 mL) over

30 mins at 0 °C under a stream of argon. Upon complete addition, the reaction mixture was immediately concentrated to a residue and then diluted in anhydrous benzene (32 mL). This newly prepared solution was added dropwise to a solution of triethylamine (5.03 mL, 36.07 mmol) in anhydrous benzene (20 mL) over 10 mins at rt under a stream of argon. The reaction mixture was stirred for 1 h at rt and then cooled to 4 °C for 20 mins before being filtered. The filtrate was concentrated to a residue to yield 3.98 g (14.3 mmol, 89% over 2 steps) of 3.2.18 as a clear oil that

1 solidified upon standing. H NMR (500 MHz, CDCl3) δ 5.85 (ddt, J = 16.3, 10.8, 5.6 Hz, 1H),

5.23 (d, J = 17.3 Hz, 1H), 5.11 (d, J = 10.5 Hz, 1H), 4.47 (d, J = 5.8 Hz, 2H), 3.37 (q, J = 7.3 Hz,

13 6H), 1.31 (t, J = 7.3 Hz, 9H); C NMR (100 MHz, CDCl3) δ 157.4, 132.6, 118.0, 66.9, 50.6, 9.5.

The 1HNMR and 13CNMR data of 3.2.18 are in accordance with those reported previously.148

Alloc N-(N-methyl-piperidiniumsulfonyl)carbamate (3.2.20)

150

3.2.20 was prepared in a similar manner to 3.2.18 yielding 2.35 g (8.95 mmol, 75%) of 3.2.20 as

1 a clear oil that solidified upon standing. H NMR (500 MHz, CDCl3) δ 5.94 (ddt, J = 17.2, 10.4,

5.7 Hz, 1H), 5.34 (dq, J = 17.2, 1.6 Hz, 1H), 5.21 (dq, J = 10.5, 1.3 Hz, 1H), 4.57 (dt, J = 5.7, 1.4

Hz, 2H), 3.61 (td, J = 13.2, 3.5 Hz, 2H), 3.43 (dq, J = 13.3, 2.2 Hz, 2H), 3.12 (s, 3H), 1.97 – 1.88

(m, 3H), 1.86 – 1.76 (m, 2H), 1.48 (qt, J = 12.9, 4.0 Hz, 1H). The 1HNMR data of MISC6 are in accordance with those reported previously.151

((Allyloxy)carbonyl)((4-(dimethylamino)pyridin-1-ium-1-yl)sulfonyl)amide (3.2.21)

3.2.21 was prepared in a similar manner to 3.2.18 yielding 0.459 g (1.61 mmol, 23%) of 3.2.21 as

1 a clear oil that solidified upon standing. H NMR (400 MHz, CDCl3) δ 8.66 (d, J = 7.2 Hz, 2H),

6.68 (d, J = 7.2 Hz, 2H), 5.88 (ddt, J = 16.4, 10.8, 5.7 Hz, 1H), 5.27 (d, J = 17.5 Hz, 1H), 5.14 (d,

J = 10.5 Hz, 1H), 4.45 (d, J = 5.5 Hz, 2H), 3.28 (s, 6H).

D-Glucal (3.2.40)

Sodium methoxide (0.10 g, 1.80 mmol) was added to a solution of 3.2.39 (8.0 g, 29.4 mmol) in

MeOH (80 mL) and the reaction mixture was stirred at rt for 5 h. The solvent was concentrated to a residue to yield 4.20 g (4.20 g, 98%) of D-glucal 3.2.40 as an amber oil and was carried on to

1 the next step without further purification. Rf (9:1 DCM/MeOH) = 0.27; H NMR (500 MHz,

CD3OD) δ 6.35 (dd, J = 6.1, 1.8 Hz, 1H), 4.68 (dd, J = 6.1, 2.2 Hz, 1H), 4.11 (dt, J = 7.1, 2.0 Hz,

1H), 3.88 (dd, J = 12.0, 2.5 Hz, 1H), 3.79 (dd, J = 12.0, 5.4 Hz, 1H), 3.72 (ddd, J = 9.8, 5.5, 2.5

13 Hz, 1H), 3.56 (dd, J = 9.7, 7.1 Hz, 1H); C NMR (100 MHz, CD3OD) δ 144.9, 104.5, 80.3, 70.9,

151

70.5, 62.2. The 1HNMR and 13CNMR data of GA0 are in accordance with those reported

168 previously.

6-O-(benzyl)-D-glucal (3.2.41)

Silver oxide (32.06 g, 138.33 mmol) and 2-aminoethyl diphenylborinate (3.11 g, 13.93 mmol) were added successively to a stirred solution of 3.2.40 (20.22 g, 138.33 mmol) diluted in acetonitrile (680 mL). Benzyl bromide (32.86 mL, 276.66 mmol) was added dropwise via addition funnel, and the reaction mixture was stirred at rt for 12 h. Following reaction completion, the reaction mixture was filtered on a celite pad and washed with EtOAc, and then concentrated to remove most of the acetonitrile. EtOAc (300 mL) was added and then brine (300 mL) was used to wash the organic layer. The organic layer was dried over sodium sulfate, filtered and concentrated to a residue. The crude product was purified by silica gel chromatography with 1:1 hexanes:EtOAc to yield 22.97 g (97.22 mmol, 70%) of 3.2.41 as an off-white solid: mp 37-38 °C. Rf (1:1

1 EtOAc/hexanes) = 0.20; H NMR (500 MHz, CDCl3) δ 7.38 – 7.30 (m, 5H), 6.35 (dd, J = 6.1, 1.8

Hz, 1H), 4.74 (dd, J = 6.1, 2.3 Hz, 1H), 4.65 (d, J = 12.0 Hz, 1H), 4.57 (d, J = 12.0 Hz, 1H), 4.25

13 (m, 1H), 3.92 (dt, J = 9.3, 4.1 Hz, 1H), 3.85 – 3.78 (m, 3H). C NMR (100 MHz, CDCl3) δ 144.5,

137.7, 128.7, 128.1, 128.0, 102.8, 76.6, 73.9, 71.6, 69.8, 69.5. The 1HNMR and 13CNMR data of

GA1 are in accordance with those reported previously.168

3-O-(t-Butyldimethylsilyl)-6-O-(benzyl)-D-glucal (3.2.42)

152

A solution of 3.2.41 (11.25 g, 47.62 mmol) in anhydrous DMF (112 mL) was cooled to 0 °C with an ice bath under a stream of argon. Imidazole (6.48 g, 95.23 mmol) and 4-dimethylaminopyridine

(290 mg, 2.38 mmol) were added successively and allowed to stir for 5 mins. Next, tert- butyldimethylsilyl chloride (7.37 g, 47.62 mmol) was added and the reaction was stirred for 15 minutes before removing the ice bath. The reaction mixture was stirred for 12 h and reaction completion was determined through TLC analysis. The reaction mixture was diluted with diethyl ether (200 mL) and then quenched with brine (200 mL). The brine layer was extracted with diethyl ether (3 x 100 mL) and the combined organic extracts were washed with water (2 x 300 mL) and brine (300 mL). The organic layer was dried over sodium sulfate, filtered and concentrated to a residue. The crude product was purified by silica gel chromatography with 12:1 hexanes:EtOAc

1 to yield 13.37 g (38.14 mmol, 80%) of 3.2.42 as a clear oil. Rf (9:1 hexanes/EtOAc) = 0.31; H

NMR (500 MHz, CDCl3) δ 7.38 – 7.27 (m, 5H), 6.30 (dd, J = 6.1, 1.6 Hz, 1H), 4.64 (dd, J = 6.1,

2.5 Hz 1H), 4.63 (d, J = 12.2 Hz, 1H), 4.58 (d, J = 12.2 Hz, 1H), 4.22 (dt, J = 6.5, 2.0 Hz, 1H),

3.99 (ddd, J = 8.8, 5.4, 3.3 Hz, 1H), 3.84 – 3.76 (m, 3H), 0.90 (s, 9H), 0.11 (s, 6H); 13C NMR (100

MHz, CDCl3) δ 143.3, 137.8, 128.4, 127.8, 127.7, 103.5, 77.1, 73.6, 70.4, 69.6, 69.0, 25.8, 18.1, -

4.48, -4.53. The 1HNMR and 13CNMR data of 3.2.42 are in accordance with those reported previously.168

3-O-(t-Butyldimethylsilyl)-4-O-mesyl-6-O-(benzyl)-D-glucal (3.2.43)

A solution of 3.2.42 (20.37 g, 58.11 mmol) in anhydrous pyridine (118 mL) was cooled to 0 °C with an ice bath under a stream of argon. Mesyl chloride (9.00 mL, 116.22 mmol) was added dropwise to the reaction mixture, and a color change from light yellow to dark amber following

153

the remaining addition. The reaction was stirred for 15 mins before removing the ice bath, and the stirring was continued for 12 h before reaction completion was determined through TLC analysis.

The reaction mixture was diluted with diethyl ether (300 mL) and then quenched with water (200 mL). The water layer was extracted with diethyl ether (3 x 100 mL) and the combined organic extracts were washed with water (2 x 300 mL) and 10% cupric sulfate pentahydrate (5 x 100 mL).

The organic layer was dried over sodium sulfate, filtered and concentrated to a residue. The crude product was purified by silica gel chromatography with 15:1 hexanes:EtOAc to yield 21.01 g

1 (49.02 mmol, 84%) of 3.2.43 as a clear oil. Rf (9:1 hexanes/EtOAc) = 0.15; H NMR (500 MHz,

CDCl3) δ 7.36 – 7.27 (m, 5H), 6.39 (dd, J = 6.2, 1.1 Hz, 1H), 4.79 – 4.75 (m, 2H), 4.60 (d, J =

11.9 Hz, 1H), 4.56 (d, J = 11.9 Hz, 1H), 4.40 – 4.33 (m, 1H), 4.31 – 4.26 (m, 1H), 3.82 (dd, J =

10.9, 7.1 Hz, 1H), 3.71 (dd, J = 10.9, 3.5 Hz, 1H), 3.06 (s, 3H), 0.87 (s, 9H), 0.10 (s, 6H); 13C

NMR (100 MHz, CDCl3) δ 143.5, 137.7, 128.4, 127.9, 127.76, 101.5, 77.2, 75.3, 73.5, 67.8, 64.7,

38.8, 25.7, 17.9, -4.55, -4.63. The 1HNMR and 13CNMR data of GA3 are in accordance with those reported previously.168

6-O-(Benzyl)-4-O-Mesyl D-glucal (3.2.44)

A solution of 3.2.43 (7.20 g, 16.79 mmol) in THF (80 mL) was cooled to 0 °C with an ice bath under a stream of argon. Tetra-n-butylammonium fluoride (1M in THF, 16.79 mmol) was added dropwise and the solution was stirred at 0 °C for 20 mins before reaction completion was determined through TLC analysis. The reaction mixture was poured into water (100 mL) and extracted with diethyl ether (3 x 80 mL). The organic layer was dried over sodium sulfate, filtered and concentrated to a residue. The crude product was purified by silica gel chromatography with

154

2:1 hexanes:EtOAc to yield 5.27 g (16.76 mmol, 99%) of 3.2.44 as a clear oil which was used in

1 the next step immediately. Rf (1:1 hexanes/EtOAc) = 0.29; H NMR (500 MHz, CDCl3) δ 7.39 –

7.26 (m, 5H), 6.42 (dd, J = 6.0, 1.6 Hz, 1H), 4.88 – 4.78 (m, 2H), 4.60 (s, 2H), 4.47 (dt, J = 6.6,

2.3 Hz, 1H), 4.11 – 4.07 (dt, J = 9.3, 3.7 Hz, 1H), 3.84 – 3.78 (m, 2H), 3.11 (s, 3H); 13C NMR

(100 MHz, CDCl3) δ 144.7, 137.5, 128.6, 128.1, 128.0, 102.2, 79.3, 75.2, 74.0, 68.3, 67.5, 38.7.

The 1HNMR and 13CNMR data of GA4 are in accordance with those reported previously.168

6-O-Benzyl-D-gulal (3.2.46)

A solution of 3.2.44 (1.99 g, 6.33 mmol) in freshly distilled THF (40 mL) was added potassium tert-butoxide (781 mg, 6.96 mmol) and stirred for 30 mins at rt. Meanwhile, to a solution of tetra- n-butylammonium bromide (8.20 g, 25.45 mmol) in freshly distilled THF (100 mL) was added potassium trimethylsilanolate172 (3.26 g, 25.45 mmol) and stirred for 10 mins at rt. The reagent solution was filtered, concentrated to half its volume (50 mL) and added dropwise to the solution of 6-O-(benzyl)-4-O-mesyl D-glucal and potassium tert-butoxide over 30 mins at rt. The reaction mixture was stirred for 5 h before reaction completion was determined through TLC analysis.

Diethyl ether (100 mL) was added and the reaction mixture was poured into brine. The layers were separated, and the aqueous layer was extracted with diethyl ether (4 x 50 mL). The organic layer was dried over sodium sulfate, filtered and concentrated to a residue. The crude product was purified by silica gel chromatography with 1:1 hexanes:EtOAc to yield 1.02 g (4.32 mmol, 68%)

1 of 3.2.46 as an amber solid: mp 50-52 °C. Rf (3:7 hexanes/EtOAc) = 0.29; H NMR (500 MHz,

CDCl3) δ 7.37 – 7.29 (m, 5H), 6.62 (d, J = 6.1 Hz, 1H), 4.99 (m, 1H), 4.66 (d, J = 11.9 Hz, 1H),

13 4.60 (d, J = 12.0 Hz, 1H), 3.96 – 3.85 (m, 5H); C NMR (125 MHz, CDCl3) δ 147.0, 137.3, 128.7,

155

128.1, 127.9, 100.6, 74.0, 71.7, 71.1, 70.1, 63.9. The 1HNMR and 13CNMR data of GA5 are in accordance with those reported previously.169

3,6-di-O-Benzyl-D-gulal (3.2.48) & 4,6-di-O-Benzyl-D-gulal (3.2.49)

To a flame dried, round bottom flask was added 3.2.46 (0.406 g, 1.72 mmol), dibutyltin oxide

(0.043 g, 0.172 mmol), tetra-n-butylammonium bromide (0.159 g, 0.515 mmol), N, N- diisopropylethylamine (0.600 mL, 3.44 mmol) and benzyl bromide (0.408 mL, 3.44 mmol) successively. The round bottom was sealed with a glass stopper and submerged in an oil bath that was warmed to 80 °C for 5.5 h. The neat reaction mixture was cooled to rt and was added N, N- diisopropylethylamine (0.450 mL, 2.58 mmol) and benzyl bromide (0.306 mL, 2.58 mmol) successively. The round bottom was sealed with a glass stopper and submerged in an oil bath that was warmed to 80 °C for 12 h. The neat reaction mixture was cooled to rt and concentrated to a residue. The crude product was purified by silica gel chromatography with 4:1 hexanes:EtOAc to yield 0.285 g (0.873 mmol, 51%) of 3.2.48 as a white solid, 0.143 g (0.438 mmol, 25%) of the regioisomer 3.2.49 as a clear oil.

26 3.2.48: mp 56-59 °C; Rf (1:1 hexanes/EtOAc) = 0.67; [α]ᴅ = +165.31 (c = 0.5, DCM); IR (thin film, cm-1): 3444.70, 3030.09, 2871.19, 1642.03, 1496.37, 1454.02, 1245.27, 1097.03, 1064.72,

1 1027.91, 738.97, 698.14; H NMR (500 MHz, CDCl3) δ 7.38 – 7.28 (m, 10H), 6.64 (d, J = 6.2 Hz,

1H), 4.99 (ddd, J = 6.1, 5.2, 1.7 Hz, 1H), 4.66 (d, J = 12.1, 1H), 4.65 (d, J = 11.8, 1H), 4.61 (d, J

= 12.0, 1H), 4.58 (d, J = 11.8, 1H), 4.04 – 4.01 (m, 2H), 3.91 (dd, J = 10.7, 3.4 Hz, 1H), 3.85 (dd,

13 J = 10.7, 4.4 Hz, 1H), 3.70 (dd, J = 5.3, 2.3 Hz, 1H); C NMR (100 MHz, CDCl3) δ 147.2, 138.4,

156

137.4, 128.7, 128.6, 128.1, 128.0, 127.88, 127.86, 98.8, 74.1, 72.3, 71.4, 70.6, 70.1, 68.2. HRMS-

+ ESI (m / z): [M + Na] calcd for C20H22O4Na, 349.1415 Da; found 349.1414 Da.

26 -1 3.2.49: Rf (1:1 hexanes/EtOAc) = 0.54; [α]ᴅ = +71.05 (c = 0.8, DCM); IR (thin film, cm ):

3411.39, 3030.08, 2869.35, 1642.86, 1496.30, 1453.85, 1369.85, 1243.30, 1095.58, 1043.70,

1 1026.91, 905.02, 737.42, 697.54, 596.86, 480.66; H NMR (500 MHz, CDCl3) δ 7.35 – 7.28 (m,

10H), 6.58 (d, J = 6.1 Hz, 1H), 4.97 (ddd, J = 6.1, 5.1, 1.7 Hz, 1H), 4.65 (d, J = 12.0 Hz, 1H), 4.57

(d, J = 12.0 Hz, 1H), 4.50 (d, J = 12.0 Hz, 1H), 4.47 (d, J = 12.0 Hz, 1H), 4.10 (ddd, J = 7.1, 5.4,

1.8 Hz, 1H), 4.03 (dd, J = 5.2, 2.7 Hz, 1H), 3.78 (dd, J = 9.9, 7.0 Hz, 1H), 3.60 (m, 1H), 3.59 (dd,

13 J = 9.9, 5.3 Hz, 1H); C NMR (100 MHz, CDCl3) δ 146.9, 137.9, 137.8, 128.6, 128.5, 128.2,

128.1, 128.0, 127.9, 100.7, 74.9, 73.6, 72.49, 72.48, 69.1, 60.9. HRMS-ESI (m / z): [M + Na]+ calcd for C20H22O4Na, 349.1415 Da; found 349.1414 Da.

3,6-di-O-Benzyl-4-carbamoyl-D-gulal (3.2.51)

A solution of 3.2.48 (0.650 g, 1.99 mmol) in anhydrous dichloromethane (15 mL) was cooled to 0

°C with an ice bath under a stream of argon. Trichloroacetyl isocyanate (0.380 mL, 3.19 mmol) was added dropwise, and the reaction was stirred for 15 mins before removing the ice bath. The stirring was continued for 1 h at rt before consumption of starting material was determined through

TLC analysis. Methanol (10 mL) was added to quench the reaction, and the solvent was evaporated. The resulting residue was dissolved in methanol and was added powdered zinc (1.95 g, 29.87 mmol). The stirring was continued for 1 h before consumption of the intermediate was determined through TLC analysis. The suspension was filtered through a celite pad and washed

157

with DCM and concentrated. The crude product was purified by silica gel chromatography with

4:1 hexanes:EtOAc to yield 0.650 g (1.76 mmol, 88%) of 3.2.51 as a white solid: mp 76-79 °C. Rf

27 -1 (2:1 hexanes/EtOAc) = 0.30; [α]ᴅ = +78.75 (c = 0.3, DCM); IR (thin film, cm ): 3356, 2923,

1 1729, 1644, 1601, 1496, 1454, 1380, 1334, 1245, 1048, 738, 698; H NMR (500 MHz, CDCl3) δ

7.41 – 7.26 (m, 10H), 6.60 (d, J = 6.2 Hz, 1H), 5.06 – 5.02 (m, 1H), 4.98 – 4.89 (m, 1H), 4.76 (bs,

2H), 4.72 (d, J = 12.0 Hz, 1H), 4.68 (d, J = 12.1 Hz, 1H), 4.63 (d, J = 12.1 Hz, 1H), 4.55 (d, J =

12.0 Hz, 1H), 4.30 – 4.24 (m, 1H), 3.76 – 3.74 (m, 1H), 3.70 (dd, J = 10.1, 7.0 Hz, 1H), 3.65 (dd,

13 J = 10.1, 5.4 Hz, 1H); C NMR (100 MHz, CDCl3) δ 155.9, 146.8, 138.3, 137.8, 128.6, 128.5,

127.95, 127.94, 127.9, 127.8, 99.1, 73.6, 71.8, 70.3, 68.9, 68.1, 68.0. HRMS-ESI (m / z): [M +

+ Na] calcd for C21H23NO5Na, 392.1474 Da; found 392.1472 Da.

(2R,3S,4S,5S)-4-(Benzyloxy)-2-((benzyloxy)methyl)-5,6-dihydroxytetrahydro-2H-pyran-3- yl carbamate (3.2.52)

To a stirring solution of 3.2.51 (0.767 g, 2.08 mmol) in THF (5.7 mL), t-BuOH (3.8 mL) and H2O

(0.945 mL) was added osmium tetraoxide (4% in H2O, 1.32 mL) and continued to stir for 12 h.

Consumption of starting material was determined through TLC analysis, and H2O (20 mL) was used to dilute the reaction mixture. Sodium sulfite (1.31 g, 10.38 mmol) was added to the reaction mixture and stirred for an additional 2 h. DCM (3 x 25 mL) was used to extract the aqueous layer and the combined organic layers were washed with water (100 mL), dried over sodium sulfate, filtered and concentrated to a residue. The crude product was purified by silica gel chromatography with 3% MeOH/DCM to yield 0.427 g (1.06 mmol, 51%) of 3.2.52 as a white gum. Rf (8%

158

27 -1 MeOH/DCM) = 0.47; [α]ᴅ = -5.66 (c = 0.2, DCM); IR (thin film, cm ): 3354, 2919, 1717, 1604,

1 1454, 1387, 1094, 1029, 738, 698; H NMR (500 MHz, CDCl3, 5:1 mixture of anomers, reporting for major) δ 7.38 – 7.26 (m, 10H), 4.99 (d, J = 10.9 Hz, 1H), 4.86 – 4.82 (m, 1H), 4.68 (d, J = 11.8

Hz, 1H), 4.58 (d, J = 11.8 Hz, 1H), 4.53 (d, J = 11.7 Hz, 1H), 4.45 (d, J = 11.9 Hz, 1H), 4.22 (dd,

J = 5.9 Hz, 1H), 3.88 (m, 1H), 3.70 – 3.64 (m, 1H), 3.62 – 3.54 (m, 2H); 13C NMR (100 MHz,

CDCl3) δ 156.2, 137.6, 137.5, 128.7, 128.6, 128.5, 128.4, 128.10, 128.05, 128.00, 127.96, 127.90,

127.7, 93.4, 75.5, 73.5, 72.4, 72.4, 69.2, 68.3, 67.5. HRMS-ESI (m / z): [M + Na]+ calcd for

C21H25NO7Na, 426.1528 Da; found 426.1525 Da.

Allyl-(3aR,5R,6S,7R,7aS)-7-(benzyloxy)-5-((benzyloxy)methyl)-6-(carbamoyloxy) tetrahydropyrano[2,3-d][1,2,3]oxathiazole-3(3aH)-carboxylate 2,2-dioxide (3.2.53)

To a stirring solution of 3.2.52 (0.140 g, 0.347 mmol) in freshly distilled THF (7.5 mL) was added alloc-modified burgess reagent 3.2.18 (0.367 g, 1.39 mmol) and immediately placed in an oil bath pre-warmed to 80 °C. The reaction mixture was refluxed for 6 h and consumption of starting material was determined through TLC analysis. Upon cooling to rt, the reaction mixture was diluted with DCM (25 mL) and was added to saturated ammonium chloride (25 mL). The aqueous layer was extracted with DCM (3 x 15 mL) and the combined organic extracts were washed with water (50 mL). The organic layer was dried over sodium sulfate, filtered and concentrated to a residue. The crude product was purified by silica gel chromatography with 3:1 hexanes:EtOAc to yield 0.030 g (0.054 mmol, 15%) of 3.2.53 as a white solid: mp 178-179 °C. Rf (1:1

28 -1 hexanes/EtOAc) = 0.65; [α]ᴅ = -18.6 (c = 0.3, DCM); IR (thin film, cm ): 2924, 1732, 1454,

159

1 1384, 1302, 1193, 1064, 991, 833, 745, 699, 575; H NMR (500 MHz, (CD3)2CO) δ 7.48 – 7.24

(m, 9H), 5.97 (ddt, J = 17.3, 10.6, 5.2 Hz, 1H), 5.91 (d, J = 2.4 Hz, 1H), 5.45 (dd, J = 17.2, 1.7

Hz, 1H), 5.26 (d, J = 10.6 Hz, 1H), 4.95 (m, 1H), 4.92 – 4.79 (m, 5H), 4.58 (d, J = 12.1 Hz, 1H),

4.53 (d, J = 12.1 Hz, 1H), 4.29 (m, 1H), 4.27 (dt, J = 6.3, 1.5 Hz, 1H), 3.68 (dd, J = 10.0, 6.1 Hz,

13 1H), 3.62 (dd, J = 10.0, 6.6 Hz, 1H); C NMR (126 MHz, (CD3)2CO) δ 156.8, 149.5, 139.4, 138.3,

132.1, 129.2, 129.1, 128.83, 128.80, 128.4, 128.3, 118.7, 82.1, 75.9, 73.7, 73.5, 71.9, 71.8, 69.0,

+ 68.6, 65.9; HRMS-ESI (m / z): [M + Na] calcd for C25H28N2O10SNa, 571.1362 Da; found

571.1357 Da.

3-O-(t-Butyldimethylsilyl)-6-O-benzyl-D-gulal (3.2.54)

A solution of 3.2.46 (0.562 g, 2.38 mmol) in anhydrous DMF (6.3 mL) was cooled to 0 °C with an ice bath under a stream of argon. Imidazole (0.323 g, 4.76 mmol) and tert-butyldimethylsilyl chloride (0.430 g, 2.85 mmol) were added successively and the reaction was stirred for 15 minutes before removing the ice bath. The reaction mixture was stirred for 12 h and reaction completion was determined through TLC analysis. The reaction mixture was diluted with diethyl ether (20 mL) and then quenched with brine (200mL). The brine layer was extracted with diethyl ether (3 x

10 mL) and the combined organic extracts were washed with water (2 x 30 mL) and brine (30 mL).

The organic layer was dried over sodium sulfate, filtered and concentrated to a residue. The crude product was purified by silica gel chromatography with 4:1 hexanes:EtOAc to yield 0.584 g (1.67

1 mmol, 70%) of 3.2.54 as a clear oil. Rf (1:1 hexanes/EtOAc) = 0.63; H NMR (500 MHz, CDCl3)

δ 7.40 – 7.28 (m, 5H), 6.55 (d, J = 6.1 Hz, 1H), 4.89 – 4.83 (m, 1H), 4.66 (d, J = 12.0 Hz, 1H),

4.59 (d, J = 12.0 Hz, 1H), 4.03 – 3.98 (m, 1H), 3.93 – 3.87 (m, 2H), 3.85 (dd, J = 10.7, 4.6 Hz,

160

13 1H), 3.80 – 3.76 (m, 1H), 0.87 (s, 9H), 0.09 (s, 6H); C NMR (126 MHz, CDCl3) δ 145.8, 137.5,

128.7, 128.1, 127.9, 101.8, 74.1, 71.64, 71.62, 71.0, 64.6, 26.0, 18.2, -4.2, -4.51. The 1HNMR and

13CNMR data of 3.2.54 are in accordance with those reported previously.169

(2R,3S,4S)-2-((Benzyloxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-3,4-dihydro-2H-pyran-3- yl (2,4-dimethoxybenzyl)carbamate (3.2.55)

To a stirring solution of 3.2.54 (3.93 g, 11.21 mmol) in anhydrous DCM (44 mL) was added a solution of freshly prepared 2,4-dimethoxybenzyl isocyanate237 (4.33 g, 22.43 mmol) in anhydrous

DCM (44 mL) and stirred for 1 h at rt under a stream of argon. LCMS analysis was performed to determine reaction progress and upon completion, the reaction mixture was diluted with DCM (50 mL) and quenched with a 10% solution of sodium bicarbonate (100 mL). The aqueous layer was extracted with DCM (3 x 30 mL) and the combined organic extracts were washed with water (200 mL). The organic layer was dried over sodium sulfate, filtered and concentrated to a residue. The crude product was purified by silica gel chromatography with 13:1 hexanes:EtOAc to yield 5.83 g

28 (10.73 mmol, 96%) of 3.2.55 as a clear oil. Rf (5:1 hexanes/EtOAc) = 0.39; [α]ᴅ = +66.11 (c =

0.8, DCM); IR (thin film, cm-1): 2929, 2856, 1723, 1643, 1614, 1590, 1507, 1462, 1289, 1244,

1 1208, 1156, 1130, 1040, 937, 863, 836, 778, 739, 698; H NMR (500 MHz, CDCl3) δ 7.35 – 7.26

(m, 5H), 7.16 (d, J = 8.2 Hz, 1H), 6.51 (d, J = 6.1 Hz, 1H), 6.46 – 6.38 (m, 2H), 5.17 (m, 1H), 4.83

(m, 1H), 4.78 – 4.73 (m, 1H), 4.59 (d, J = 12.1 Hz, 1H), 4.52 (d, J = 12.2 Hz, 1H), 4.29 – 4.23 (m,

3H), 3.93 (dd, J = 5.4, 2.5 Hz, 1H), 3.80 (s, 3H), 3.79 (s, 3H), 3.67 (dd, J = 10.3, 7.6 Hz, 1H), 3.62

13 (dd, J = 10.5, 4.6 Hz, 1H), 0.89 (s, 9H), 0.13 (s, 3H), 0.10 (s, 3H); C NMR (126 MHz, CDCl3) δ

161

160.6, 158.5, 155.3, 145.4, 137.9, 130.2, 128.4, 127.74, 127.65, 118.9, 103.8, 101.7, 98.5, 73.4,

71.3, 70.4, 69.4, 62.0, 55.4, 55.3, 40.7, 25.9, 18.0, -4.4, -4.5. HRMS-ESI (m / z): [M + Na]+ calcd for C29H41NO7SiNa, 566.2550 Da; found 566.2545 Da.

(2R,3S,4S,5S)-2-((Benzyloxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-5,6- dihydroxytetrahydro-2H-pyran-3-yl (2,4-dimethoxybenzyl)carbamate (3.2.56)

To a stirring solution of 3.2.55 (13.0 g, 23.91 mmol) in THF (70 mL), t-BuOH (40 mL) and H2O

(10 mL) was added N-methylmorpholine N-oxide (8.4 g, 71.73 mmol) and osmium tetraoxide (4% in H2O, 15.2 mL) and continued to stir for 12 h. Consumption of starting material was determined through TLC analysis, and H2O (200 mL) was used to dilute the reaction mixture. Sodium sulfite

(15.1 g, 119.54 mmol) was added to the reaction mixture and stirred for an additional 2 h. DCM

(3 x 100 mL) was used to extract the aqueous layer and the combined organic layers were washed with water (150 mL), dried over sodium sulfate, filtered and concentrated to a residue. The crude product was purified by silica gel chromatography with 4:1 hexanes:EtOAc to yield 12.12 g (21.01

28 mmol, 88%) of 3.2.56 as a clear, colorless oil. Rf (1:1 hexanes/EtOAc) = 0.46; [α]ᴅ = +0.22 (c =

0.4, DCM); IR (thin film, cm-1): 3367, 2929, 2857, 1725, 1614, 1590, 1508, 1463, 1253, 1208,

1 1156, 1095, 1035, 937, 838, 780, 736, 698; H NMR (500 MHz, CDCl3, 8:1 mixture of anomers, reporting for major) δ 7.31 – 7.27 (m, 5H), 7.14 (d, J = 8.2 Hz, 1H), 6.44 (d, J = 2.3 Hz, 1H), 6.39

(dd, J = 8.3, 2.4 Hz, 1H), 5.32 (m, 1H), 4.99 (d, J = 11.2 Hz, 1H), 4.66 – 4.62 (m, 1H), 4.52 (d, J

= 11.8 Hz, 1H), 4.41 (d, J = 11.9 Hz, 1H), 4.27 (dd, J = 6.1, 2.9 Hz, 2H), 4.22 (m, 1H), 4.16 – 4.13

(m, 1H), 3.97 (d, J = 11.7 Hz, 1H), 3.80 (s, 3H), 3.78 (s, 3H), 3.59 (dd, J = 10.0, 6.6 Hz, 1H), 3.53

162

(dd, J = 10.0, 5.9 Hz, 1H), 3.42 (d, J = 8.9 Hz, 1H), 2.64 (d, J = 10.5 Hz, 1H), 0.88 (s, 9H), 0.16

13 (s, 3H), 0.12 (s, 3H); C NMR (125 MHz, CDCl3) δ 160.5, 158.4, 154.9, 137.8, 129.9, 128.4,

127.8, 127.7, 118.8, 103.9, 98.6, 93.1, 73.5, 72.0, 70.8, 70.0, 69.2, 69.1, 55.36, 55.35, 40.5, 25.7,

+ 17.8, -4.9, -5.2. HRMS-ESI (m / z): [M + Na] calcd for C29H43NO9SiNa, 600.2605 Da; found

600.2602 Da.

Allyl (3aR,5R,6S,7R,7aS)-5-((benzyloxy)methyl)-7-((tert-butyldimethylsilyl)oxy)-6-(((2,4- dimethoxybenzyl)carbamoyl)oxy)tetrahydropyrano[2,3-d][1,2,3]oxathiazole-3(3aH)- carboxylate 2,2-dioxide (3.2.57)

To a stirring solution of 3.2.56 (2.39 g, 4.14 mmol) in freshly distilled THF (42 mL) was added alloc-modified burgess reagent 3.2.18 (2.73 g, 10.34 mmol) and immediately placed in an oil bath pre-warmed to 80 °C. The reaction mixture was refluxed for 2 h and consumption of starting material was determined through TLC analysis. Upon cooling to rt, the reaction mixture was diluted with DCM (50 mL) and was added to saturated ammonium chloride (100 mL). The aqueous layer was extracted with DCM (3 x 50 mL) and the combined organic extracts were washed with water (100 mL). The organic layer was dried over sodium sulfate, filtered and concentrated to a residue. The crude product was purified by silica gel chromatography with 15:1 hexanes:EtOAc

28 to yield 0.771 g (1.07 mmol, 26%) of 3.2.57 as a clear oil. Rf (3:1 hexanes/EtOAc) = 0.36; [α]ᴅ =

-14.99 (c = 0.3, DCM); IR (thin film, cm-1): 2928, 2856, 1754, 1722, 1614, 1590, 1508, 1455,

1388, 1321, 1288, 1258, 1207, 1102, 1036, 1000, 937, 837, 809, 784, 699, 571; 1H NMR (500

MHz, CDCl3) δ 7.33 – 7.26 (m, 5H), 7.15 (d, J = 8.2 Hz, 1H), 6.45 (d, J = 2.3 Hz, 1H), 6.43 – 6.36

163

(m, 2H), 5.93 (ddt, J = 16.3, 10.8, 5.4 Hz, 1H), 5.71 – 5.68 (m, 1H), 5.44 (d, J = 16.7 Hz, 1H),

5.35 (m, 1H), 5.30 (d, J = 10.3 Hz, 1H), 4.83 (dd, J = 13.4, 5.4 Hz, 1H), 4.78 (dd, J = 13.4, 5.4 Hz,

1H), 4.61 – 4.58 (m, 1H), 4.54 (d, J = 12.0 Hz, 1H), 4.43 (d, J = 12.1 Hz, 1H), 4.40 – 4.38 (m,

2H), 4.28 (d, J = 6.0 Hz, 2H), 4.22 (m, 1H), 3.81 (s, 3H), 3.79 (s, 3H), 3.62 – 3.55 (m, 2H), 0.90

13 (s, 9H), 0.21 (s, 3H), 0.17 (s, 3H); C NMR (126 MHz, CDCl3) δ 160.7, 158.7, 155.3, 148.8,

138.0, 130.4, 130.1, 128.5, 127.8, 127.7, 119.7, 118.7, 103.8, 98.6, 80.3, 77.4, 73.5, 70.2, 68.6,

67.3, 64.6, 55.5, 55.4, 40.9, 29.8, 25.7, 17.9, -4.8, -5.2. HRMS-ESI (m / z): [M + Na]+ calcd for

C33H46N2O12SSiNa, 745.2438 Da; found 745.2439 Da.

(2R,3S,4S,5R,6R)-6-(((Allyloxy)carbonyl)amino)-5-azido-2-((benzyloxy)methyl)-4-((tert- butyldimethylsilyl)oxy)tetrahydro-2H-pyran-3-yl (2,4-dimethoxybenzyl)carbamate (3.2.58)

To a stirring solution of 3.2.57 (1.77 g, 2.44 mmol) in anhydrous DMF (24.4 mL) was added sodium azide (0.794 g, 12.22 mmol) at rt under a stream of argon. The reaction mixture was warmed to 60 °C and stirred for 3 h. LCMS analysis was performed to determine reaction progress and upon completion, the reaction mixture was cooled to rt and quenched with 10% sulfuric acid

(24 mL). The reaction mixture was stirred for an additional 30 mins and then poured into brine (50 mL). The brine layer was extracted with EtOAc (3 x 50 mL) and the combined organic extracts were washed with water (2 x 100 mL) and brine (100 mL). The organic layer was dried over sodium sulfate, filtered and concentrated to a residue. The crude product was purified by silica gel chromatography with 0.5% MeOH/DCM to yield 0.757 g (1.10 mmol, 45%) of 3.2.58 as a clear

29 -1 oil. Rf (0.5% MeOH/DCM) = 0.20; [α]ᴅ = -9.19 (c = 0.5, DCM); IR (thin film, cm ): 3334, 2930,

164

2857, 2106, 1728, 1614, 1589, 1508, 1463, 1255, 1209, 1131, 1099, 1037, 939, 835, 780, 737,

1 698; H NMR (500 MHz, (CD3)2CO) δ 7.36 (d, J = 9.9 Hz, 1H), 7.32 – 7.25 (m, 5H), 7.15 (d, J =

8.3 Hz, 1H), 6.54 (d, J = 2.3 Hz, 1H), 6.53 – 6.49 (m, 1H), 6.42 (dd, J = 8.3, 2.3 Hz, 1H), 5.95

(ddt, J = 16.2, 10.5, 5.3 Hz, 1H), 5.35 – 5.29 (m, 2H), 5.18 (d, J = 10.5 Hz, 1H), 4.77 (d, J = 2.2

Hz, 1H), 4.58 (d, J = 5.4 Hz, 2H), 4.53 (d, J = 12.2 Hz, 1H), 4.48 (d, J = 12.1 Hz, 1H), 4.33 (m,

1H), 4.29 (m, 1H), 4.25 (d, J = 6.1 Hz, 2H), 3.83 (s, 3H), 3.77 (s, 3H), 3.62 (d, J = 8.9 Hz, 1H),

13 3.58 – 3.51 (m, 2H), 0.98 (s, 9H), 0.25 (s, 3H), 0.23 (s, 3H); C NMR (100 MHz, (CD3)2CO) δ

161.3, 159.1, 156.4, 156.3, 139.4, 134.0, 129.9, 129.0, 128.13, 128.10, 120.03, 117.5, 104.9, 99.0,

78.6, 73.5, 72.5, 71.3, 70.7, 68.7, 65.9, 60.3, 55.7, 55.5, 40.4, 26.1, 18.5, -4.7, -5.0. HRMS-ESI (m

+ / z): [M + Na] calcd for C33H47N5O9SiNa, 708.3041 Da; found 708.3040 Da.

Allyl ((2R,3R,4S,5S,6R)-3-amino-6-((benzyloxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-5-

(((2,4-dimethoxybenzyl)carbamoyl)oxy)tetrahydro-2H-pyran-2-yl)carbamate (3.2.100)

To a stirring solution of 3.2.58 (0.864 g, 1.26 mmol) in THF (11.3 mL) and water (1.1 mL) was added triphenylphosphine (0.991 g, 3.78 mmol) at rt. The reaction mixture was warmed to 50 °C and stirred for 12 h. LCMS analysis was performed to determine reaction progress and upon completion, the reaction mixture was cooled to rt and diluted with EtOAc (30 mL). The reaction mixture was added to water (100 mL) and extracted with EtOAc (3 x 30 mL). The organic layer was washed with brine (100 mL), dried over sodium sulfate, filtered and concentrated to a residue.

The crude product was purified by silica gel chromatography with 1:1 hexanes:EtOAc, 0.1% TEA to yield 0.812 g (1.23 mmol, 98%) of 3.2.100 as a clear oil. Rf (1:1 hexanes/EtOAc, 0.1% TEA) =

165

29 -1 0.34; [α]ᴅ = -42.10 (c =0.5, DCM); IR (thin film, cm ): 3310, 2928, 1726, 1615, 1536, 1507,

1 1463, 1364, 1233, 1208, 1135, 1074, 1036, 935, 836, 779, 739, 698; H NMR (500 MHz, CDCl3)

δ 7.31 – 7.24 (m, 5H), 7.10 (d, J = 8.0 Hz, 1H), 6.43 – 6.38 (m, 2H), 6.27 (s, 1H), 5.87 (ddt, J =

16.3, 10.5, 5.5 Hz, 1H), 5.29 (d, J = 17.3 Hz, 1H), 5.19 (dd, J = 10.5, 1.6 Hz, 1H), 5.03 (m, 1H),

4.84 – 4.80 (m, 1H), 4.61 – 4.53 (m, 2H), 4.51 (d, J = 11.8 Hz, 1H), 4.41 (d, J = 12.0 Hz, 1H),

4.34 (dd, J = 15.0, 6.0 Hz, 1H), 4.30 – 4.24 (m, 2H), 4.06 – 4.01 (m, 1H), 3.78 (s, 3H), 3.77 (s,

3H), 3.61 – 3.51 (m, 2H), 2.96 (d, J = 8.7 Hz, 1H), 0.90 (s, 9H), 0.18 (s, 3H), 0.00 (s, 3H); 13C

NMR (126 MHz, CDCl3) δ 160.2, 158.0, 156.8, 156.0, 138.0, 132.5, 128.8, 128.4, 128.0, 127.7,

119.4, 117.7, 103.9, 98.7, 81.8, 73.6, 72.09, 70.8, 70.1, 68.4, 65.8, 55.4, 55.3, 50.7, 39.5, 25.8,

+ 18.0, -4.6, -5.3. HRMS-ESI (m / z): [M + H] calcd for C33H50N3O9Si, 660.3316 Da; found

660.3317 Da.

Dibenzyl ((S)-6-(((2R,3R,4S,5S,6R)-2-(((allyloxy)carbonyl)amino)-6-((benzyloxy)methyl)-4-

((tert-butyldimethylsilyl)oxy)-5-(((2,4-dimethoxybenzyl)carbamoyl)oxy)tetrahydro-2H- pyran-3-yl)amino)-6-oxohexane-1,4-diyl)dicarbamate (3.2.101)

To a stirring solution of 3.2.100 (0.220 g, 0.333 mmol) in anhydrous DCM (2 mL) and anhydrous

DMF (1 mL) was added (S)-3,6-bis(((benzyloxy)carbonyl)amino)hexanoic acid 3.1.11 (0.152 g,

0.367 mmol), 4-dimethylaminopyridine (0.049 g, 0.40 mmol) and EDCI·HCl (0.128 g, 0.667 mmol) at rt under a stream of argon. The reaction mixture was stirred for 12 h and reaction completion was determined through TLC analysis. The reaction mixture was diluted with EtOAc

(20 mL) and then poured into brine (50mL). The brine layer was extracted with EtOAc (3 x 25

166

mL) and the combined organic extracts were washed with water (2 x 30 mL) and brine (30 mL).

The organic layer was dried over sodium sulfate, filtered and concentrated to a residue. The crude product was purified by silica gel chromatography with 1:1 hexanes:EtOAc to yield 0.330 g (0.312

29 mmol, 94%) of 3.2.101 as a clear oil. Rf (1:1 hexanes/EtOAc) = 0.24; [α]ᴅ = -26.76 (c = 0.6,

DCM); IR (thin film, cm-1): 3324, 2930, 1719, 1614, 1509, 1455, 1253, 1209, 1101, 1040, 834,

1 779, 738, 697; H NMR (500 MHz, CDCl3) δ 7.36 – 7.27 (m, 12H), 7.26 – 7.23 (m, 3H), 7.14 (d,

J = 8.2 Hz, 1H), 6.45 (d, J = 2.3 Hz, 1H), 6.38 (dd, J = 8.2, 2.4 Hz, 1H), 5.96 (d, J = 8.5 Hz, 1H),

5.91 – 5.87 (m, 1H), 5.87 – 5.79 (m, 1H), 5.40 (d, J = 8.6 Hz, 1H), 5.33 (m, 1H), 5.22 (d, J = 17.1

Hz, 1H), 5.15 – 5.11 (m, 2H), 5.08 (s, 2H), 5.04 – 4.98 (m, 2H), 4.96 – 4.89 (m, 1H), 4.76 (d, J =

3.6 Hz, 1H), 4.56 – 4.44 (m, 3H), 4.40 (d, J = 12.2 Hz, 1H), 4.27 (dd, J = 6.0, 3.9 Hz, 2H), 4.21

(m, 1H), 4.14 – 4.09 (m, 1H), 4.04 (m, 1H), 3.96 – 3.89 (m, 1H), 3.81 (s, 3H), 3.78 (s, 3H), 3.54

(dd, J = 9.7, 6.0 Hz, 1H), 3.51 – 3.44 (m, 1H), 3.20 – 3.15 (m, 2H), 2.44 (d, J = 12.0 Hz, 1H), 2.36

(d, J = 11.5 Hz, 1H), 1.57 – 1.49 (m, 4H), 0.94 (s, 9H), 0.23 (s, 3H), 0.16 (s, 3H); 13C NMR (101

MHz, CDCl3) δ 171.5, 160.8, 158.7, 156.5, 156.0, 155.9, 155.2, 138.1, 136.7, 136.6, 132.6, 130.3,

128.6, 128.4, 128.2, 128.1, 127.7, 127.6, 118.8, 117.9, 103.9, 98.7, 79.9, 73.3, 71.4, 69.8, 69.7,

67.7, 66.8, 66.6, 65.9, 55.5, 55.4, 48.8, 48.3, 41.0, 40.8, 40.6, 31.4, 26.7, 25.9, 18.1, -4.4, -4.9.

+ HRMS-ESI (m / z): [M + Na] calcd for C55H73N5O14SiNa, 1078.4821 Da; found 1078.4835 Da.

Dibenzyl ((S)-6-(((2R,3R,4S,5S,6R)-2-amino-6-((benzyloxy)methyl)-4-((tert- butyldimethylsilyl)oxy)-5-(((2,4-dimethoxybenzyl)carbamoyl)oxy)tetrahydro-2H-pyran-3- yl)amino)-6-oxohexane-1,4-diyl)dicarbamate (3.2.102)

167

To a stirring solution of 3.2.101 (0.410 g, 0.388 mmol) diluted in MeCN (3.5 mL) and water (3.5 mL) was added diethylamine (1.61 mL, 15.53 mmol) at rt. The reaction mixture was stirred for 10 mins, and then triphenylphosphine-3,3′,3′′-trisulfonic acid trisodium salt (0.044 g, 0.078 mmol) was added at rt and stirred for 1 hr. Consumption of starting material was determined through TLC analysis, the reaction mixture was diluted with EtOAc (20 mL) and poured into water (50 mL).

29 -1 iPrOH/DCM) = 0.15; [α]ᴅ = -25.07 (c = 0.6, DCM); IR (thin film, cm ): 3304, 2928, 1700, 1508,

1 1454, 1253, 1209, 1114, 1028, 833, 778, 737, 697; H NMR (500 MHz, CD3OD) δ 7.36 – 7.23

(m, 15H), 7.12 (d, J = 8.3 Hz, 1H), 6.49 (d, J = 2.3 Hz, 1H), 6.40 (dd, J = 8.3, 2.4 Hz, 1H), 5.05

(s, 4H), 4.73 (d, J = 2.5 Hz, 1H), 4.49 (d, J = 11.8 Hz, 1H), 4.44 (d, J = 11.9 Hz, 1H), 4.33 (d, J =

9.7 Hz, 1H), 4.20 (s, 2H), 4.16 (m, 1H), 4.08 (m, 1H), 3.99 – 3.95 (m, 1H), 3.87 (d, J = 9.7 Hz,

1H), 3.79 (s, 3H), 3.74 (s, 3H), 3.57 – 3.49 (m, 2H), 3.14 – 3.08 (m, 2H), 2.43 (dd, J = 14.2, 6.1

Hz, 1H), 2.37 (dd, J = 14.3, 6.8 Hz, 1H), 1.62 – 1.54 (m, 2H), 1.52 – 1.44 (m, 2H), 0.94 (s, 9H),

13 0.16 (s, 3H), 0.11 (s, 3H); C NMR (101 MHz, CDCl3) δ 170.6, 160.8, 158.7, 156.5, 156.2, 155.2,

138.0, 136.8, 136.7, 130.3, 128.57, 128.55, 128.4, 128.12, 128.08, 128.04, 128.00, 127.7, 118.8,

103.9, 98.6, 82.9, 73.5, 71.6, 70.7, 70.0, 69.2, 66.6, 55.5, 55.4, 50.7, 48.6, 41.3, 41.0, 40.7, 31.4,

+ 29.8, 26.8, 25.8, 18.0, -4.4, -5.0. HRMS-ESI (m / z): [M + H] calcd for C51H70N5O12Si, 972.4790

Da; found 972.4805 Da.

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Dibenzyl ((S)-6-(((2R,3R,4S,5S,6R)-2-(3-((3S,4S,5R)-4-(((benzyloxy)carbonyl)amino)-5-

((tert-butyldimethylsilyl)oxy)-2-oxopiperidin-3-yl)thioureido)-6-((benzyloxy)methyl)-4-

((tert-butyldimethylsilyl)oxy)-5-(((2,4-dimethoxybenzyl)carbamoyl)oxy)tetrahydro-2H- pyran-3-yl)amino)-6-oxohexane-1,4-diyl)dicarbamate (3.2.108)

To a stirring solution of benzyl ((3S,4S,5R)-5-((tert-butyldimethylsilyl)oxy)-3-isothiocyanato-2- oxopiperidin-4-yl)carbamate 3.2.107 (0.057 g, 0.131 mmol) in anhydrous DCM (0.250 mL) was added a solution of 3.2.102 (0.128 g, 0.131 mmol) in anhydrous DCM (1 mL) at 0 °C under a stream of argon. The reaction mixture was stirred for 15 mins, warmed to rt, fitted with a glass stopper and stirred for 12 h. The reaction mixture was concentrated to a residue and the crude product was purified by silica gel chromatography with 1% MeOH/CHCl3 to yield 0.087 g (0.062

29 mmol, 47%, 77% brsm) of 3.2.108 as a colorless gum. Rf (3% MeOH/CHCl3) = 0.17; [α]ᴅ = -

16.64 (c = 0.3, DCM); IR (thin film, cm-1): 3316, 2927, 2855, 1719, 1508, 1454, 1344, 1258, 1208,

1 1098, 1027, 833, 803, 778, 735, 696; H NMR (500 MHz, CDCl3) δ 7.36 – 7.28 (m, 12H), 7.25 –

7.22 (m, 8H), 7.13 (d, J = 8.3 Hz, 1H), 6.41 (s, 1H), 6.37 (d, J = 8.3 Hz, 1H), 5.85 (bs, 1H), 5.17

– 5.08 (m, 3H), 5.05 (d, J = 11.0 Hz, 1H), 5.02 – 4.95 (m, 1H), 4.79 (d, J = 12.2 Hz, 1H), 4.77 –

4.75 (m, 1H), 4.55 (dd, J = 21.9, 12.1 Hz, 1H), 4.37 – 4.25 (m, 4H), 4.25 – 4.14 (m, 3H), 4.12 –

4.07 (m, 1H), 3.90 – 3.83 (m, 1H), 3.76 (s, 3H), 3.75 (s, 3H), 3.56 – 3.49 (m, 2H), 3.20 – 3.14 (m,

2H), 2.66 – 2.44 (m, 1H), 2.33 – 2.12 (m, 1H), 2.06 – 1.97 (m, 2H), 1.67 – 1.48 (m, 4H), 0.95 (s,

169

9H), 0.87 (s, 9H), 0.21 (s, 3H), 0.14 (s, 3H), -0.04 (d, J = 16.4 Hz, 6H); 13C NMR (101 MHz,

CDCl3) δ 187.4, 172.1, 170.3, 160.7, 158.6, 156.7, 156.6, 156.2, 155.4, 138.36, 136.9, 136.8,

136.6, 130.3, 128.8, 128.6, 128.5, 128.44, 128.39, 128.3, 128.2, 128.1, 127.9, 127.8, 127.7, 127.5,

119.1, 103.9, 98.7, 81.8, 73.2, 72.3, 70.3, 69.8, 68.1, 66.8, 66.3, 56.6, 56.4, 55.5, 55.4, 52.8, 50.2,

49.4, 47.2, 46.7, 42.0, 40.7, 40.4, 31.8, 31.3, 29.8, 25.9, 25.8, 18.1, -4.5, -4.8, -4.9, -5.0. HRMS-

+ ESI (m / z): [M + Na] calcd for C71H98N8O16SSi2Na, 1429.6258 Da; found 1429.6262 Da.

Tert-butyl (3S,4S,5R)-3-(3-((2R,3R,4S,5S,6R)-6-((benzyloxy)methyl)-3-((S)-3,6- bis(((benzyloxy)carbonyl)amino)hexanamido)-4-((tert-butyldimethylsilyl)oxy)-5-(((2,4- dimethoxybenzyl)carbamoyl)oxy)tetrahydro-2H-pyran-2-yl)thioureido)-4-((tert- butoxycarbonyl)amino)-5-((tert-butyldimethylsilyl)oxy)-2-oxopiperidine-1-carboxylate

(3.2.115)

To a stirring solution of 3.2.114 (0.104 g, 0.209 mmol) in anhydrous DCM (0.420 mL) was added a solution of 3.2.102 (0.190 g, 0.195 mmol) in anhydrous DCM (1.5 mL) at 0 °C under a stream of argon. The reaction mixture was stirred for 15 mins, warmed to rt, fitted with a glass stopper and stirred for 12 h. The reaction mixture was concentrated to a residue and the crude product was purified by silica gel chromatography with 1% MeOH/CHCl3 to yield 0.197 g (0.062 mmol, 68%,

29 76% brsm) of 3.2.115 as an off-white solid: mp 60-63 °C. Rf (3% MeOH/CHCl3) = 0.21; [α]ᴅ = -

17.93 (c = 0.2, DCM); IR (thin film, cm-1): 3333, 2927, 2855, 1717, 1534, 1456, 1368, 1256, 1132,

170

1 1038, 835, 779, 738, 698; H NMR (500 MHz, CDCl3) δ 7.76 – 7.71 (m, 1H), 7.57 – 7.54 (m, 1H),

7.43 – 7.40 (m, 1H), 7.36 – 7.28 (m, 16H), 7.23 (d, J = 6.7 Hz, 1H), 7.16 (d, J = 8.1 Hz, 1H), 6.45

(s, 1H), 6.40 (d, J = 9.5 Hz, 1H), 6.11 – 6.00 (m, 1H), 5.17 – 5.11 (m, 2H), 5.10 – 5.04 (m, 2H),

4.81 – 4.77 (m, 1H), 4.67 (d, J = 12.1 Hz, 1H), 4.46 – 4.36 (m, 2H), 4.33 – 4.25 (m, 5H), 4.13 –

4.09 (m, 3H), 3.99 – 3.89 (m, 1H), 3.81 (s, 3H), 3.79 (s, 3H), 3.77 – 3.76 (m, 1H), 3.60 – 3.57 (m,

1H), 3.51 – 3.41 (m, 1H), 3.26 – 3.11 (m, 2H), 2.60 – 2.47 (m, 1H), 2.34 – 2.24 (m, 1H), 1.74 –

1.72 (m, 2H), 1.64 – 1.62 (m, 2H), 1.34 (s, 9H), 1.27 (s, 9H), 0.96 (s, 9H), 0.92 (s, 9H), 0.23 (s,

13 3H), 0.16 (s, 3H), 0.14 (s, 3H), 0.08 (s, 3H); C NMR (101 MHz, CDCl3) δ 187.2, 171.7, 167.8,

160.7, 158.7, 156.8, 156.7, 155.6, 155.3, 151.9, 138.4, 136.9, 136.8, 132.5, 131.0, 130.3, 129.0,

128.6, 128.5, 128.3, 128.2, 128.1, 127.9, 127.7, 127.5, 119.0, 103.9, 98.7, 83.5, 79.7, 77.4, 73.4,

71. 9, 69.9, 68.2, 66.8, 66.6, 66.0, 55.5, 55.4, 50.5, 49.6, 40.8, 32.0, 29.8, 29.5, 28.5, 28.0, 27.8,

26.5, 25.9, 25.8, 22.8, 19.3, 18.11, 18.07, 14.2, -4.5, -4.8, -4.9. HRMS-ESI (m / z): [M + Na]+ calcd for C73H108N8O18SSi2Na, 1495.6939 Da; found 1495.6983 Da.

Dibenzyl ((S)-6-(((2R,3R,4S,5S,6R)-6-((benzyloxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-

2-(((3aS,7R,7aS,Z)-7-((tert-butyldimethylsilyl)oxy)-4-oxooctahydro-2H-imidazo[4,5- c]pyridin-2-ylidene)amino)-5-(carbamoyloxy)tetrahydro-2H-pyran-3-yl)amino)-6- oxohexane-1,4-diyl)dicarbamate (3.2.116)

To a stirring solution of 3.2.115 (0.074 g, 0.050 mmol) in anhydrous DCM (5 mL) was added TFA

(2.5 mL) at 0 °C under a stream of argon. The reaction mixture was stirred for 4 h at 0 °C, and

171

consumption of starting material was observed through TLC analysis. While maintaining a temperature of at 0 °C, the reaction mixture was quenched through the addition of a saturated solution of sodium bicarbonate (20 mL). The reaction mixture was warmed to rt, diluted with

EtOAc (20 mL) and extracted with EtOAc (3 x 20 mL). The organic extracts were washed with brine (100 mL), dried over sodium sulfate, filtered and concentrated to a residue. The crude amine

(0.064 g) was used directly in the next step. To a stirring solution of crude amine (0.064 g) in anhydrous DMF (1.2 mL) was added triethylamine (0.024 mL, 0.171 mmol) and mercury(II) chloride (0.046 g, 0.171 mmol) successively at 0 °C under a stream of argon. The reaction mixture was stirred for 15 mins, warmed to rt and stirred for 3 h when consumption of starting material was observed through TLC analysis. The reaction mixture was diluted with EtOAc (10 mL), filtered through celite and then poured into brine (25 mL). The brine layer was extracted with

EtOAc (3 x 10 mL) and the combined organic extracts were washed with water (3 x 50 mL) and brine (50 mL). The organic layer was dried over sodium sulfate, filtered and concentrated to a residue. The crude product was purified by silica gel chromatography with 6% MeOH/DCM to yield 0.045 g (0.041 mmol, 73%, 2 steps) of 3.2.116 as a white film. Rf (12% MeOH/DCM) =

29 -1 0.49; [α]ᴅ = -4.56 (c = 0.8, MeOH); IR (thin film, cm ): 3295, 2925, 2854, 2030, 1983, 1965,

1 1700, 1538, 1462, 1255, 1074, 836, 780, 697, 472; H NMR (500 MHz, CD3OD) δ 7.37 – 7.29 (m,

15H), 5.13 (d, J = 12.5 Hz, 1H), 5.07 (d, J = 8.9 Hz, 3H), 5.01 (d, J = 9.0 Hz, 1H), 4.73 – 4.68 (m,

1H), 4.68 – 4.61 (m, 1H), 4.58 – 4.54 (m, 3H), 4.42 – 4.39 (m, 1H), 4.19 (m, 1H), 4.11 (d, J = 9.2

Hz, 1H), 3.99 (m, 1H), 3.91 (d, J = 14.5 Hz, 1H), 3.71 – 3.64 (m, 2H), 3.61 (dd, J = 13.9, 4.7 Hz,

1H), 3.23 (d, J = 13.9 Hz, 1H), 3.15 – 3.09 (m, 2H), 2.41 (dd, J = 13.4, 4.3 Hz, 1H), 2.18 (dd, J =

13.4, 9.7 Hz, 1H), 1.60 – 1.54 (m, 2H), 1.52 – 1.45 (m, 2H), 0.97 (s, 9H), 0.92 (s, 9H), 0.18 (s,

13 3H), 0.15 (s, 3H), 0.14 (s, 3H), 0.11 (s, 3H); C NMR (101 MHz, CDCl3) δ 172.0, 167.8, 163.2,

172

157.0, 156.9, 155.9, 136.9, 136.8, 136.6, 128.6, 128.5, 128.4, 128.2, 128.1, 128.0, 127.9, 127.6,

127.5, 127.3, 78.8, 73.9, 72.1, 70.2, 69.4, 68.7, 66.5, 66.3, 62.9, 61.4, 54.5, 50.9, 50.8, 50.7, 41.9,

40.4, 31.8, 26.2, 25.8, 25.7, 18.1, 17.9, -4.5, -4.7, -5.0, -5.2. HRMS-ESI (m / z): [M + H]+ calcd for C54H81N8O12Si2, 1089.5512 Da; found 1089.5528 Da.

Dibenzyl ((S)-6-(((2R,3R,4S,5R,6R)-6-((benzyloxy)methyl)-5-(carbamoyloxy)-4-hydroxy-2-

(((3aS,7R,7aS,Z)-7-hydroxy-4-oxooctahydro-2H-imidazo[4,5-c]pyridin-2-ylidene) amino) tetrahydro-2H-pyran-3-yl)amino)-6-oxohexane-1,4-diyl)dicarbamate (3.2.117)

To a stirring solution of 3.2.116 (0.051 g, 0.047 mmol) in freshly distilled THF (3.5 mL) was added tetra-n-butylammonium fluoride (1M in THF, 0.500 mL) dropwise and the solution was stirred at

0 °C for 20 mins before reaction completion was determined through TLC analysis. The reaction mixture was poured into brine (10 mL) and extracted with EtOAc (3 x 10 mL). The organic layer was dried over sodium sulfate, filtered and concentrated to a residue. The crude product was purified by silica gel chromatography with 12% MeOH/DCM to yield 0.032 g (0.037 mmol, 79%)

29 of 3.2.117 as a white film. Rf (12% MeOH/DCM) = 0.33; [α]ᴅ = -6.20 (c = 0.3, MeOH); IR (thin

-1 1 film, cm ): 2922, 2852, 1659, 1463; H NMR (500 MHz, CD3OD) δ 7.36 – 7.32 (m, 12H), 7.30 –

7.26 (m, 3H), 5.12 (d, J = 12.5 Hz, 1H), 5.07 – 5.02 (m, 4H), 4.78 (d, J = 2.1 Hz, 1H), 4.57 (d, J

= 14.4 Hz, 1H), 4.54 (s, 2H), 4.51 – 4.46 (m, 1H), 4.42 (m, 1H), 4.16 (dd, J = 9.7, 2.9 Hz, 1H),

4.00 (m, 1H), 3.97 – 3.93 (m, 1H), 3.86 (dd, J = 14.6, 2.5 Hz, 1H), 3.66 (d, J = 6.2 Hz, 2H), 3.64

– 3.59 (m, 1H), 3.30 – 3.26 (m, 1H), 3.10 (t, J = 5.9 Hz, 2H), 2.47 – 2.40 (m, 1H), 2.30 (dd, J =

173

+ 13.8, 8.8 Hz, 1H), 1.57 – 1.47 (m, 4H). HRMS-ESI (m / z): [M + H] calcd for C42H53N8O12,

861.3783 Da; found 861.3785 Da.

Streptothricin F Sulfate (3.1.1)

To a stirring solution of 3.2.117 (0.020 g, 0.023 mmol) in AcOH (0.4 mL), water (0.4 mL) and

MeOH (0.2 mL) was added palladium (10% on carbon, 5 mg). The reaction vessel was evacuated and purged with argon gas (7x), evacuated and purged with hydrogen gas (7x) and then stirred overnight under a hydrogen atmosphere (balloon). The reaction was filtered through a 0.45 µm

PVDF filter, the filter was washed with water (2 x 1 mL) and concentrated to a residue. The crude product was diluted in water (0.5 mL) and purified by Sephadex (LH-20) size exclusion gel with a mobile phase of 90% water and 10% methanol at a rate of 0.47 mL / min. Fractions testing positive for the ninhydrin stain were analyzed by LCMS. Pure fractions were collected and freeze dried overnight, isolating the acetate salt. To a stirring solution of ST-F acetate in water (0.5 mL) was added 1 M H2SO4 dropwise until pH = 2. The resulting solution was added dropwise to vigorously stirring MeOH (20 mL) and Et2O (10 mL). The slurry was stirred for 20 mins and then solids were collected by centrifugation, washed with 1:1 MeOH:Et2O (10 mL) and Et2O (10 mL).

The resulting precipitate was collected by centrifugation to yield 5.71 mg of ST-F sulfate 3.1.1

21 24 (0.00823 mmol, 49%) as a white solid: mp >210 °C. [α]ᴅ = -43.45 (c = 0.1, H2O) ([α]ᴅ = -41.06

20 228 (c = 0.2, H2O) ST-F sulfate isolated from NTC by Manetsch Lab, [α]ᴅ = -53 (c = 1.0, H2O) ,

174

238 1 [α]ᴅ = -34.8 (H2O) ); H NMR (500 MHz, D2O) δ 5.09 (d, J = 9.8 Hz, 1H), 4.75 (d, J = 3.4 Hz,

1H), 4.73 – 4.69 (m, 1H), 4.61 (d, J = 14.7 Hz, 1H), 4.32 (t, J = 5.9 Hz, 1H), 4.24 (dd, J = 9.8, 3.0

Hz, 1H), 4.15 (t, J = 3.5 Hz, 1H), 4.06 (d, J = 13.8 Hz, 1H), 3.79 (dd, J = 14.6, 5.7 Hz, 1H), 3.71

(d, J = 5.7 Hz, 2H), 3.69 – 3.66 (m, 1H), 3.38 (d, J = 14.6 Hz, 1H), 3.05 – 3.01 (m, 2H), 2.79 (dd,

J = 16.7, 4.4 Hz, 1H), 2.67 (dd, J = 16.7, 8.3 Hz, 1H), 1.82 – 1.74 (m, 4H). HRMS-ESI (m / z): [M

+ 1 + H] calcd for C19H35N8O8, 503.2577 Da; found 503.2574 Da. The HNMR data of 3.1.1 are in accordance with those reported previously.91

175

Chapter 5: Supplemental Material for Chapter 2

5.1 List of Compound Libraries Screened

Library Name Supplier ChemDiv Targeted Diversity Library ChemDiv Enamine 2 Enamine Enamine2a Enamine ActiMolTimTec1 Biomol-TimTec Asinex 1 Asinex Bionet (Ryan Scientific) 2 Bionet ChemBridge3 ChemBridge ChemDiv1 (Combilab and International) ChemDiv ChemDiv3 ChemDiv ChemDiv4 ChemDiv ChemDiv Targeted Diversity Library ChemDiv Enamine 2 Enamine IFLab1 IFLab Life Chemicals 1 Life Chemicals Maybridge4 Maybridge Maybridge5 Maybridge Table 5.1 Compound libraries screened.

5.2 Cheminformatics

5.2.1 Permeation and Efflux Multiparameter Optimization (PEMPO)

Multiparameter optimization (MPO) scoring was first reported by Wager et al. to rank order compounds that would be effective as CNS drugs to penetrate the blood brain barrier.72 The authors also suggested that compounds with a higher MPO scores demonstrate better in vitro

ADME and safety characteristics.

We developed a variation of MPO scoring known as PEMPO (Permeation and Efflux

Multiparameter Optimization), focused on identifying compounds with physicochemical properties ideal for permeating the lipopolysaccharide layer (LPS) of a Gram-negative bacterial cell, as well as avoiding extracellular efflux.

176

PEMPO scores were determined using the physicochemical properties below with optimal values shown in parentheses and suboptimal in brackets, outside the parentheses. A property within the optimal range received a score of 1, whereas suboptimal values were scored as a linear function from the undesired value to the optimal. Undesirable values, defined as values outside of suboptimal, received a score of 0. Physicochemical properties and desirable ranges are as follows: isoelectric point [4.0-6.0 (6.1-8.7) 8.8-10], total polar surface area [60-99

(100-200 Å2) 201-240], number of hydrogen bond donors [0-1 (2-6) 7-9], number of hydrogen bond acceptors [0-5 (6-11) 12-19], partition coefficient clogP [5-3.1 (≤3)], and distribution coefficient clogD7.4 [3-0.3 (≤0.2)].

Isoelectric point was chosen because compounds with zwitterionic character at physiological pH (7.4) exhibit charge that can be desirable for entry into porin proteins on the

LPS as well as an uncharged state to aid with absorption in the gut, essential for bioavailability.

Compounds that can exist in both a charged, and uncharged state are observed with a pKa close to 7.4. The remainder of the physicochemical properties were chosen to reflect the polarity of each compound, awarding higher scores to more hydrophilic molecules.

5.2.2 PEMPO Scoring of Known Antibacterial Compounds

Compound name PEMPO Scorea PEMPO Scoreb Chloramphenicol 4.60 4.71 Loracarbef 5.47 5.64 Ertapenem 5.00 5.00 Imipenem 6.00 6.00 Meropenem 6.00 6.00 R-115685 5.57 4.54 Cefetamet 5.00 4.98 Ceftibuten 5.00 4.59 Cefaclor 5.44 5.61 Cefadroxil 5.71 5.71 Cefamandole 5.00 4.97 Cefazolin 5.00 4.00

177

Cefdinir 5.00 4.71 Cefditoren 5.07 5.00 Cefixime 5.00 3.92 Cefmetazole 5.00 4.25 Cefoperazone 4.58 3.25 Cefotaxime 5.00 4.20 Cefotetan 4.58 3.63 Cefoxitin 5.00 4.96 Cefpodoxime 5.00 4.75 Cefprozil 5.72 5.72 Ceftizoxime 5.00 4.98 Ceftriaxone 4.66 3.50 Cefuroxime 5.00 4.88 Cephalexin 5.54 5.71 Cephalothin 4.83 5.00 Cephradine 5.63 5.80 T-91825 6.00 4.75 Cefepime 5.00 4.98 Cefpirome 6.00 5.88 Ceftazidime 5.00 3.75 Ceftobiprole 6.00 4.63 Iclaprim 4.25 4.36 Trimethoprim 4.68 4.86 Nalidixic Acid 4.19 4.20 ABT-492 4.79 5.00 Ciprofloxacin 5.32 5.32 Clinafloxacin 5.67 5.67 Danofloxacin 4.60 4.60 Difloxacin 4.09 4.24 DX-619 5.90 5.90 Enoxacin 5.63 5.64 Fleroxacin 4.46 4.46 Garenoxacin 4.96 5.47 Gatifloxacin 5.55 5.55 Gemifloxacin 6.00 6.00 Grepafloxacin 5.32 5.32 Levofloxacin 4.77 4.77 Lomefloxacin 5.32 5.32 Moxifloxacin 5.55 5.55 Nadifloxacin 4.40 4.53 Norfloxacin 5.32 5.32

178

Pefloxacin 4.60 4.60 Rufloxacin 4.57 5.20 Sitafloxacin 5.67 5.67 Sparfloxacin 5.97 5.97 Temafloxacin 5.01 5.21 Trovafloxacin 5.98 5.99 Azithromycin 4.75 4.01 Aztreonam 4.97 3.80 Fosfomycin 3.92 4.16 Faropenem 4.51 4.68 Doripenem 6.00 6.00 Amoxicillin 5.66 5.66 Ampicillin 5.50 5.66 Carbenicillin 5.00 5.00 Mezlocillin 5.00 4.57 Ticarcillin 5.00 5.00 Azlocillin 5.00 5.00 Piperacillin 5.00 4.88 Sulfabenzamide 4.24 4.77 Sulfacetamide 4.40 4.77 Sulfachlorpyridazine 4.90 5.15 Sulfadiazine 4.99 5.24 Sulfadimethoxine 5.00 5.16 Sulfaguanidine 5.00 5.00 Sulfamerazine 4.99 5.24 Sulfameter 5.24 5.24 Sulfamethazine 4.99 5.10 Sulfamethizole 4.92 5.16 Sulfamethoxazole 4.54 5.03 Sulfamethoxypyridazine 5.21 5.21 Sulfamonomethoxine 5.36 5.42 Sulfanitran 4.41 4.08 Sulfaphenazole 4.30 4.86 Sulfapyridine 4.35 4.75 Sulfaquinoxaline 4.69 5.22 Sulfathioazole 4.40 5.06 Sulfisoxazole 4.51 5.00 Demeclocycline 5.00 4.67 Doxycycline 5.07 4.73 Meclocycline 5.00 4.67 Methacycline 5.03 4.69

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Minocycline 5.69 5.69 Oxytetracycline 4.63 4.29 PTK-0796 6.00 5.67 Tetracycline 5.03 4.70 Tigecycline 5.52 4.94 Chlortetracycline 5.00 4.67

Average 5.08 4.97 Table 5.2 PEMPO scoring of known antibacterial compounds. Scoring was performed across two property calculation platforms to reveal any discrepancies between scoring when properties were calculated between different engines. aPhysicochemical properties calculated by ChemAxon, ChemDraw bPhysicochemical properties calculated by Pipeline Pilot, ACD/Labs.

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5.2.3 PEMPO and MPO Scoring for Selected Confirmed Hit Compounds

SMILES Compound ID Cluster MPO Score PEMPO Score n1(CC(=O)Nc2onc(c3cccc(Cl)c3)c2)ncc(COc(cccc4)c45)c15 KP10 1 4.489 2.794 n(CC(=O)NCC1COc(c2O1)cccc2)(c(c3ccc(cc3)C)cc4C(OCC)=O)c4C KP11 1 3.544 2.774 N(C(C)C(=O)Nc(cc1)ccc1OCC)(N=Nc2c3c4c(s2)CCCC4)C3=O KP16 2 3.492 2.547 c1(C(c(cccn2)c2)N([H])c3nc(C)cc(C)n3)c(C)c(sc1NC(c4ccccc4)=O)C KP26 2 2.946 2.911 c1(cc(sc1NC(c2ccccc2)=O)C)C(c3ccccn3)N([H])c4ccccn4 KP27 2 3.228 1.870 c1(C(c2cccc(OC)c2)N([H])c3nc(C)cc(C)n3)c4c(sc1NC(c5ccccc5)=O)CCCC4 KP31 2 2.524 2.860 c12c(NC(=O)NC1=O)[nH]c(c3ccc(c4c3)cccc4)c2C5c6c(NC5=O)cccc6 KP44 2 2.950 3.037 N1(CCc2onc(c(ccc(c34)OCO3)c4)n2)C(=O)Nc(c5C1=O)cc(c6c5)OCO6 KP52 3 3.501 3.626 S(=O)(=O)(N(CC1)CCC1C(=O)N2CCc(c3C2)cccc3)c4c(C)noc4\C=C/c(cc5)ccc5OC KP7 3 4.302 2.583 n1(nc(c(C([H])([H])c2ccccc2)c1O)C)c(nc(c3C(=O)N4CCc(c5C4)cccc5)C)s3 KP37 5 3.618 1.607 n1(nc(c(C([H])([H])c2ccccc2)c1O)C)c(nc(c3C(=O)Nc4cccc(Cl)c4)C)s3 KP40 5 2.967 2.198 S(=O)(=O)(c1c(C)cc(c(C)c1)OCC)N2CCc(cc(c3n4)ccc(OC)c3)c24 KP34 7 3.870 1.274 S(=O)(=O)(c(ccc1c2OC(N1CC(=O)NCc(cc3)ccc3OC)=O)c2)N4CCC(CC4)C KP9 7 4.454 4.424 c1(C(c2ccccc2)NC(COc(cc3)ccc3C)=O)nnc(c4ccccc4)o1 KP6 9 4.021 2.480 o1c(c(ccc2c3c(C)c([nH]2)C)c3)nnc1SC(C)C(=O)Nc(ccc(c45)OCCO4)c5 KP18 10 3.237 4.262 c1(nnc(SCC(=O)NC(C)c2[nH]c(c3n2)cccc3)o1)c4cc(c5n4C)cccc5 KP19 10 4.208 4.750 c1(Cc2ccccc2)nc(c3n1CC(O)COc(cc4)ccc4F)cccc3 KP29 11 3.850 1.431 c1(C2)n(CCN2Cc3c(OC)ccc(c3)NC(=O)c4ccc(cc4)OC)c5c(cccc5)n1 KP33 11 3.853 2.151 n1(CC2CC)c(c(O2)ccc3Cl)c3cc1C(=O)N4CCC(CC4)C(OCC)=O KP50 11 4.879 1.347 n1(CC2CC)c(c(O2)ccc3Cl)c3cc1C(NCCCOC(C)C)=O KP51 11 4.933 2.834 n1(Cc(cc2)ccc2OC)c3c(nc1CCC(=O)Nc(ccc(c4C)C)c4)cccn3 KP8 11 3.574 2.644 c(nc(C)cc1NCc(cccn2)c2)(c3c4ccc(cc4)F)n1nc3C(F)(F)F KP32 12 4.058 1.700 c(N(CCOc1ccccc1)S(=O)(=O)c2ccc(cc2)C)(nn(c34)c(C)cc(C)n3)n4 KP42 12 3.915 2.199 N1(Cc(cc2)ccc2C(NCCc(cc3)ccc3C)=O)c4c(cccc4)N(C(=O)C1=O)CC=C KP13 14 3.393 2.528 N(CC(=O)Nc1cccc(C)c1C)(c2c3cccc2)C(=O)C=C3C(=O)NCc4ccccc4 KP43 15 3.435 3.025

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N1(C)C(=O)CSc(ccc(c2)NC(=O)Nc3cc(C)ccc3C)c12 KP14 16 4.735 2.215 c12c(ncnc1N(C)C)n(cc2c3ccccc3)c4ccc(cc4)F KP28 17 3.752 0.554 n1(c2ccccc2Cl)cc(c3c1ncnc3NC4CC4)c5ccccc5 KP30 17 3.759 1.000 n1c(SCc2ccccc2)nc(cc1N(CC3)CCC3C(N)=O)c4ccc(cc4)C KP45 18 3.350 1.500 c1(scc(c2ccccc2)n1)NC(=O)Nc(ccc(c3Cl)F)c3 KP21 20 3.500 1.333 n1c(c(cc2)ccc2NC(=O)c3ccccc3C)onc1c4ccccc4 KP22 21 3.750 2.201 n1c(c(cccc2NC(=O)C3CC3)c2)onc1c4ccccc4 KP23 21 4.127 2.569 n1c(c2ccc(cc2)Cl)onc1N([H])C(COc(cc3)ccc3OC)=O KP35 22 4.739 2.700 C1(NC(=O)CC2c3cc(F)cc(F)c3)=C2C(NC(SCc(cc4)ccc4F)=N1)=O KP17 23 3.845 2.330 N1=C(SC([H])([H])c2ccccc2)C3=C(N(CCc4ccccc4)C1=O)CCC3 KP36 23 3.668 0.369 N1(N(C(C)C)C=N2)C2=NC(CSc(cc3)ccc3NC(=O)Nc4cccc(Cl)c4)=CC1=O KP39 24 3.315 3.980 c1(nnc(NC(CSc(cc2)ccc2Cl)=O)s1)S(=O)(=O)N3CCC(CC3)C KP49 25 4.788 3.249 n1(nc(s2)COc3ccccc3)c2nnc1CSc4ccccc4 KP47 26 4.923 1.361 n12c(nnc1c3ccccc3Cl)sc(c4ccc(c5n4)cccc5)n2 KP48 27 3.988 0.667 [nH](c(ccc(c1)CNC(=O)Nc(cc2)ccc2Cl)c1c3C)c3C KP15 28 3.629 2.017 C(=O)(c1cccc(c1)Nc2cnc(c3n2)cccc3)Nc(cc4)ccc4CC KP5 28 3.455 2.840 c1(NC(=O)Nc(cc2)ccc2OC)sc(c3n1)cc(c(C)c3)C KP20 29 3.594 1.673 Table 5.3 PEMPO and MPO scoring for selected, confirmed hit compounds. Physicochemical properties were calculated by ChemAxon, ChemDraw.

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5.3 Supplemental Tables

Primer Name Sequence (5’-3’)

NDM-1R GTTGGCGGGTGTCGGGGCTGGCTTAATCAGCGCAGCTTGTCGGCC

NDM-1F GATAACAATTTCACACAGGAAACAGCTATGGAATTGCCCAATATTATGCAC

KPC-2and3F GATAACAATTTCACACAGGAAACAGCTATGTCACTGTATCGCCGTCTAGT

KPC-2and3R GTTGGCGGGTGTCGGGGCTGGCTTAACTTACTGCCCGTTGACGCCC pUC18R TTAAGCCAGCCCCGACACCCGCC pUC18F AGCTGTTTCCTGTGTGAAATTGTTATCCGCTCAC

Table 5.4 Primers used in construction of carbapenemase-expressing E. coli strains.

Carbapenemase Measurement KP9 KP11 KP19 KP40 KP56 Meropenem

None MIC (µg/mL) >128 >128 >128 >128 >128 0.13

FIC >1 >1 >1 >1 >1 N/A

KPC2 MIC >128 >128 >128 >128 >128 2

FIC >1 >1 >1 >1 >1 N/A

KPC3 MIC >128 >128 >128 >128 >128 2

FIC >1 >1 >1 >1 >1 N/A

NDM1 MIC >128 >128 >128 >128 >128 16

FIC >1 >1 >1 >1 >1 N/A

Table 5.5 Activity of identified compounds against Escherichia coli DH5α mutant harboring various carbapenemase enzymes.

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Carbapenemase Measurement KP9 KP11 KP19 KP40 KP56 Meropenem

None MIC (µg/mL) >128 >128 >128 16-32 >128 0.063

FIC >1 >1 >1 >1 >1 N/A

KPC2 MIC >128 >128 >128 32-128 >128 2

FIC >1 >1 >1 0.25 >1 N/A

KPC3 MIC >128 >128 >128 64-128 >128 4

FIC >1 >1 >1 0.25 >1 N/A

NDM1 MIC >128 >128 >128 32-128 >128 >16

FIC >1 >1 >1 >1 >1 N/A

Table 5.6 Activity of identified compounds against Escherichia coli tolC mutant harboring various carbapenemase enzymes.

Carbapenemase Measurement KP9 KP11 KP19 KP40 KP56 Meropenem

None MIC (µg/mL) >128 >128 >128 >128 >128 0.03

FIC >1 >1 >1 >1 >1 N/A

KPC2 MIC >128 >128 >128 >128 >128 1

FIC >1 >1 >1 >1 >1 N/A

KPC3 MIC >128 >128 >128 >128 >128 0.5

FIC >1 >1 >1 >1 >1 N/A

NDM1 MIC >128 >128 >128 >128 >128 8

FIC >1 >1 >1 >1 >1 N/A

Table 5.7 Activity of identified compounds against Escherichia coli lptD mutant harboring various carbapenemase enzymes.

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Chapter 6: Supporting Material for Chapter 3

6.1 Purification of NTC and Isolation of STs

6.1.1 General

NTC was purchased from Gold Bio. Analysis was performed using an Agilent 6460 Triple

Quad LC/MS with Infinity 1290 LC with an electrospray ionization source. Data was obtained using MassHunter Data Acquisition version B.07.00 and analyzed using MassHunter Qualitative

Analysis version B.06.00. A Phenomenex Luna 5µm HILIC 200Å LC column was used with dimensions of 250 x 4.6 mm. A mobile phase was used consisting of Solvent A: 50% MeCN 50%

Water 5mM ammonium formate buffer pH 1.6, Solvent B: 90% MeCN 10% Water 5mM ammonium formate buffer pH 1.6. The gradient begins with a 50% Solvent A and ramps up to

100% Solvent A over 10 mins. The gradient then returns to 50% Solvent A at 11 mins and is held for 1 min. The flow rate for the method is 0.6 mL/min and the injection volume is 10 uL. The dynamic MRM method is as follows: precursor ion: 503.5; product ion: 171 and 138; retention time: 7.53 min; delta retention time: 2.24; fragmentor energy: 135; collision energy: 28 and 48 (for product ions 171 and 138 respectively); cell accelerator voltage: 4. The pH for the mobile phase was measured using a Fisher Scientific pH/Ion 510. Elemental analysis was performed by Intertek.

6.1.2 Introduction

ST products have been isolated through different methods of chromatography and varying stationary phases. Resolution of the ST components within NTS was accomplished by circular paper chromatography by the Horowtiz group.239 Stationary phases implemented in the literature include Amberlite IRA-400240, cellulose241, carboxy methyl cellulose242 (CMC), activated carbon and dextran gel.228,243 Each method has defects associated with the stationary phase, decomposition being the most prominent in all. Our group was interested in isolating ST compounds from the

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NTS mixture, and we decided upon purification via dextran gel as it has been the most widely explored method in the literature.243

6.1.3 Purification of NTC via Sephadex Size Exclusion Gel

Purification to separate streptothricin compounds of the nourseothricin mixture was performed according to a previously reported method.228 A glass column (150 cm x 2.4 cm) was packed with Sephadex size exclusion gel (LH-20) using a mobile phase of 10% methanol / water and the flow rate was adjusted to 0.6 mL / min. For each separation, approximately 300 mg of

NTC sulfate was diluted in 0.6 mL of water before loading to the column. A mobile phase of 10% methanol / water was used and fraction sizes of 3 mL were collected. Pure streptothricin D began eluting after approximately 120 mL of mobile phase, followed by mixed fractions of ST-D, ST-E and ST-F and then pure streptothricin F. Purity of samples were characterized with LCMS. Pure sample were combined, frozen using dry ice / acetone, and finally lyophilized.

6.1.4 Quantification of ST Purity using Elemental Analysis and LC-MS/MS

The literature suggests, and our elemental analysis confirms, that the ST-F sulfate salt

228 contains an empirical formula of C19H43N8O17S1.5. While our analysis does not summate to

100% (Figure 6.1), the remaining impurities (5.26%) is an unknown impurity that is not C, H, N,

O, or S and consider our initial sample have a purity of 94.74% (sample ID: DM1).

Figure 6.1 Elemental analysis performed on sample DM1.

We based the purity of future batches upon the purity of DM1 and quantified future batches through LC-MS/MS analysis (Tables 6.1-6.2). A calibration curve was established with 5 known

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concentrations of DM1 (0.5, 2.5, 5.0, 25 and 50 ug/mL) performed in triplicate (Figure 6.1). A sample of a known concentration for DM2 (25 ug/mL) was injected for MRM and its average abundance value (178070.5) was used to determine its calibrated concentration (26.28406361 ug/mL). The ratio of the calibrated concentration to the known concentration

(26.28406361/25=1.051362544) was then multiplied by the DM1 purity to obtain DM2 purity.

Name File Area m/z (prod.) Precursor Algorithm streptothricin F1 DM1_0.5ugmL1.d 1616 171 503.1.5 MRM streptothricin F1 DM1_0.5ugmL2.d 1226 171 503.1.5 MRM streptothricin F1 DM1_0.5ugmL3.1.d 982 171 503.1.5 MRM streptothricin F1 DM1_2.5ugmL1.d 7505 171 503.1.5 MRM streptothricin F1 DM1_2.5ugmL2.d 6426 171 503.1.5 MRM streptothricin F1 DM1_2.5ugmL3.1.d 5960 171 503.1.5 MRM streptothricin F1 DM1_5ugmL1.d 20812 171 503.1.5 MRM streptothricin F1 DM1_5ugmL2.d 19822 171 503.1.5 MRM streptothricin F1 DM1_5ugmL3.1.d 18509 171 503.1.5 MRM streptothricin F1 DM1_25ugmL1.d 196495 171 503.1.5 MRM streptothricin F1 DM1_25ugmL2.d 193483 171 503.1.5 MRM streptothricin F1 DM1_25ugmL3.1.d 187500 171 503.1.5 MRM streptothricin F1 DM1_50ugmL1.d 326274 171 503.1.5 MRM streptothricin F1 DM1_50ugmL2.d 320872 171 503.1.5 MRM streptothricin F1 DM1_50ugmL3.1.d 313164 171 503.1.5 MRM streptothricin F1 DM2_1.d 180609 171 503.1.5 MRM streptothricin F1 DM2_2.d 175532 171 503.1.5 MRM Table 6.1 Raw data for calibration curve determination and DM2 injections.

DM1 conc (ug/mL) Avg. Abundance 0 0 0.5 1421 2.5 7505 5 20812 25 187500 50 326274 DM2 conc (ug/mL) Avg. Abundance Slope DM1 Purity DM2 Purity 25 178070.5 6774.8 94.74 99.6067732 Table 6.2 Concentrations and average abundance values for DM1 and DM2.

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DM1 Concentration vs Abundance 400000 350000 300000 250000 200000 150000

Abundance 100000 y = 6774.8x - 3133.4 50000 R² = 0.9926 0 0 10 20 30 40 50 60 -50000 Concentration (ug/mL)

Figure 6.1 Calibration curve for DM1.

6.2 Chiral Chromatography Supporting Material

6.2.1 General

Chiral chromatography was performed using an Agilent 1200 series HPLC with OpenLab software version C.01.07. A Phenomenex Lux Cellulose-4 5µm chiral LC column was used with dimensions of 250 x 4.60 mm. A mobile phase was used consisting of Solvent A: Water 0.1%

TFA, Solvent B: MeOH 0.1% TFA. The gradient begins with 70% Solvent B and ramps up to 82%

Solvent B over 18 mins. The gradient then ramps up to 100% Solvent B over 1 min and holds for

1 min before returning to 70% Solvent B over 1 min. The flow rate for the method is 1 mL/min and the injection volume is 5 uL.

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6.2.2 Chiral Chromatography for Nitroketone 3.2.78

Figure 6.2 Racemic mixture of nitroketone 3.2.78.

Figure 6.3 Optically pure nitroketone 3.2.78.

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6.3 Crystallographic Information

6.3.1 Crystallographic Information for 3.2.53

Data collection: Bruker APEX3; cell refinement: Bruker SAINT; data reduction: Bruker SAINT; program(s) used to solve structure: SHELXT 2014/5 (Sheldrick, 2014); program(s) used to refine structure: SHELXL2014/7 (Sheldrick, 2014).

Crystal data C25H28N2O10S Mr = 548.55 Monoclinic, P21 a = 13.469 (5) Å b = 6.806 (3) Å c = 13.751 (5) Å β = 96.63 (2)° V = 1252.1 (8) Å3 Z = 2

Data collection Bruker Kappa APEX-II DUO diffractometer Radiation source: fine-focus sealed tube TRIUMPH curved graphite monochromator φ and w scans Absorption correction: multi-scan SADABS (Sheldrick, 2004) Tmin = 0.841, Tmax = 0.996

Refinement Refinement on F2 Least-squares matrix: full R[F2 > 2σ(F2)] = 0.053 wR(F2) = 0.118 S = 1.07 4264 reflections 404 parameters 278 restraints Hydrogen site location: mixed

Special details F(000) = 576 -3 Dx = 1.455 Mg m Mo Ka radiation, λ = 0.71073 Å Cell parameters from 2227 reflections ϴ = 3.0-23.5° µ = 0.19 mm-1 T = 90 K Needle, colourless 0.66 x 0.05 x 0.02 mm

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8418 measured reflections 4264 independent reflections 3328 reflections with I > 2σ(I) Rint = 0.054 ϴmax = 25.4°, ϴmin = 1.5° h = -16---16 k = -7---8 l = -16---16

H atoms treated by a mixture of independent and constrained refinement 2 2 2 2 2 w = 1/[σ (Fo ) + (0.0382P) + 0.2547P] where P = (Fo + 2Fc )/3 (Δ/σ)max < 0.001 -3 Δpmax = 0.30 e Å -3 Δpmin = -0.49 e Å Absolute structure: Flack x determined using 1128 quotients [(I+)-(I-)]/[(I+)+(I-)] (Parsons, Flack and Wagner, Acta Cryst. B69 (2013) 249-259). Absolute structure parameter: -0.03 (10)

Geometry. All e.s.d.'s (except the e.s.d. in the dihedral angle between two l.s. planes) are estimated using the full covariance matrix. The cell e.s.d.'s are taken into account individually in the estimation of e.s.d.'s in distances, angles and torsion angles; correlations between e.s.d.'s in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell e.s.d.'s is used for estimating e.s.d.'s involving l.s. planes.

2 Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å ) x y z Uiso*/Ueq Occ. (<1) S1 0.67635 (14) 0.9925 (2) 0.43672 (10) 0.0442 (5) O1 0.7793 (3) 0.6285 (6) 0.3230 (2) 0.0264 (9) O2 0.6170 (3) 0.9064 (6) 0.3417 (2) 0.0296 (9) O3 0.7388 (4) 1.1449 (7) 0.4096 (3) 0.0588 (14) O4 0.6126 (4) 1.0292 (7) 0.5095 (3) 0.0607 (15) O5 0.6784 (2) 0.7844 (5) 0.1487 (2) 0.0161 (7) O6 0.5352 (2) 0.7731 (6) 0.0446 (2) 0.0230 (9) O7 0.5363 (2) 0.4175 (6) 0.2704 (2) 0.0276 (9) O8 0.8161 (2) 0.3424 (5) 0.1100 (2) 0.0186 (8) N1 0.7402 (4) 0.7841 (8) 0.4637 (3) 0.0408 (13) N2 0.6271 (3) 1.0529 (7) 0.0655 (3) 0.0179 (10) H21N 0.584 (4) 1.121 (9) 0.026 (4) 0.022* H22N 0.673 (4) 1.127 (9) 0.094 (4) 0.022* C1 0.7122 (4) 0.6285 (9) 0.3944 (4) 0.0324 (14) H1 0.7111 0.4981 0.4277 0.039* C2 0.6081 (5) 0.6905 (9) 0.3513 (4) 0.0312 (14) H2 0.559 0.6588 0.3983 0.037*

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C3 0.5711 (4) 0.6094 (8) 0.2509 (4) 0.0257 (13) H3 0.5149 0.6916 0.2191 0.031* C4 0.6542 (4) 0.5920 (8) 0.1834 (3) 0.0202 (12) H4 0.6313 0.5053 0.1265 0.024* C5 0.7463 (4) 0.5055 (8) 0.2404 (3) 0.0207 (11) H5 0.7285 0.3737 0.2655 0.025* C6 0.8375 (3) 0.4820 (8) 0.1869 (3) 0.0205 (11) H6A 0.8553 0.61 0.1594 0.025* H6B 0.8949 0.4364 0.2329 0.025* C7 0.9070 (3) 0.2919 (9) 0.0692 (3) 0.0236 (12) H7A 0.9571 0.2413 0.1215 0.028* H7B 0.9349 0.4103 0.0406 0.028* C8 0.8846 (3) 0.1391 (8) -0.0080 (3) 0.0183 (11) C9 0.8956 (3) 0.1775 (9) -0.1052 (3) 0.0216 (12) H9 0.9193 0.3023 -0.1233 0.026* C10 0.8723 (3) 0.0354 (9) -0.1759 (4) 0.0269 (14) H10 0.8799 0.0631 -0.2424 0.032* C11 0.8377 (3) -0.1484 (9) -0.1503 (4) 0.0292 (14) H11 0.8202 -0.2448 -0.1993 0.035* C12 0.8290 (3) -0.1895 (9) -0.0533 (4) 0.0273 (13) H12 0.806 -0.3146 -0.0351 0.033* C13 0.8541 (3) -0.0465 (8) 0.0176 (4) 0.0238 (13) H13 0.8502 -0.0764 0.0845 0.029* C14 0.6072 (3) 0.8668 (8) 0.0823 (3) 0.0156 (11) C15 0.4544 (4) 0.3589 (9) 0.2043 (4) 0.0371 (15) H15A 0.476 0.3426 0.1384 0.045* H15B 0.4024 0.4624 0.2003 0.045* C16 0.4110 (4) 0.1691 (9) 0.2358 (3) 0.0224 (12) C17 0.4040 (4) 0.1281 (11) 0.3341 (4) 0.0400 (17) H17 0.4265 0.2217 0.383 0.048* C18 0.3644 (5) -0.0492 (11) 0.3605 (4) 0.0485 (19) H18 0.3625 -0.079 0.4278 0.058* C19 0.3274 (4) -0.1838 (11) 0.2896 (4) 0.0408 (17) H19 0.2982 -0.3034 0.3079 0.049* C20 0.3335 (3) -0.1421 (9) 0.1928 (4) 0.0262 (13) H20 0.3081 -0.2334 0.1438 0.031* C21 0.3762 (3) 0.0319 (8) 0.1657 (3) 0.0209 (12) H21 0.3815 0.0572 0.0986 0.025* C22A 0.7867 (12) 0.741 (2) 0.5469 (12) 0.025 (5) 0.332 (10) O9A 0.7967 (12) 0.865 (2) 0.6074 (9) 0.040 (4) 0.332 (10) O10A 0.8266 (11) 0.5631 (19) 0.5451 (9) 0.024 (3) 0.332 (10)

192

C23A 0.8829 (11) 0.493 (3) 0.6346 (10) 0.036 (5) 0.332 (10) H23A 0.8834 0.3479 0.6346 0.044* 0.332 (10) H23B 0.8497 0.5376 0.6914 0.044* 0.332 (10) C24A 0.9859 (19) 0.566 (5) 0.645 (2) 0.066 (9) 0.332 (10) H24A 0.9941 0.702 0.6333 0.079* 0.332 (10) C25A 1.0622 (18) 0.473 (5) 0.667 (2) 0.073 (8) 0.332 (10) H25A 1.059 0.3358 0.6794 0.087* 0.332 (10) H25B 1.1252 0.537 0.6712 0.087* 0.332 (10) C22B 0.8274 (8) 0.7970 (17) 0.5376 (7) 0.039 (3) 0.668 (10) O9B 0.8462 (7) 0.9394 (14) 0.5874 (7) 0.065 (3) 0.668 (10) O10B 0.8763 (6) 0.6270 (14) 0.5436 (5) 0.044 (2) 0.668 (10) C23B 0.9679 (10) 0.628 (2) 0.6136 (10) 0.067 (4) 0.668 (10) H23C 0.9535 0.6822 0.6772 0.081* 0.668 (10) H23D 1.0198 0.7099 0.5882 0.081* 0.668 (10) C24B 1.0043 (12) 0.418 (3) 0.6266 (8) 0.062 (4) 0.668 (10) H24B 1.0681 0.3996 0.6627 0.075* 0.668 (10) C25B 0.9613 (9) 0.273 (2) 0.5964 (10) 0.093 (6) 0.668 (10) H25C 0.8972 0.2835 0.5598 0.111* 0.668 (10) H25D 0.9911 0.148 0.6088 0.111* 0.668 (10)

2 Atomic displacement parameters (Å ) U11 U22 U33 U12 U13 U23 S1 0.0896 (13) 0.0174 (8) 0.0243 (7) 0.0135 (9) 0.0007 (7) -0.0018 (6) O1 0.042 (2) 0.017 (2) 0.0192 (17) 0.0052 (18) 0.0003 (15) 0.0000 (15) O2 0.051 (2) 0.017 (2) 0.0223 (18) 0.0065 (19) 0.0090 (16) 0.0018 (15) O3 0.102 (4) 0.015 (3) 0.055 (3) -0.006 (3) -0.013 (3) 0.000 (2) O4 0.126 (4) 0.033 (3) 0.024 (2) 0.036 (3) 0.016 (2) -0.0009 (19) O5 0.0156 (16) 0.014 (2) 0.0178 (16) -0.0001 (16) -0.0004 (12) 0.0022 (15) O6 0.0162 (18) 0.029 (2) 0.0231 (17) -0.0044 (19) -0.0008 (14) -0.0089 (17) O7 0.0233 (18) 0.027 (3) 0.0339 (19) -0.0045 (18) 0.0100 (15) 0.0077 (17) O8 0.0129 (16) 0.020 (2) 0.0231 (16) -0.0001 (15) 0.0049 (13) -0.0042 (14) N1 0.073 (3) 0.023 (3) 0.025 (2) 0.019 (3) 0.000 (2) -0.003 (2) N2 0.013 (2) 0.017 (3) 0.023 (2) 0.000 (2) -0.0034 (17) 0.0002 (19) C1 0.057 (4) 0.023 (3) 0.018 (2) 0.008 (3) 0.007 (2) 0.003 (2) C2 0.058 (4) 0.014 (3) 0.025 (3) -0.007 (3) 0.020 (3) -0.001 (2) C3 0.030 (3) 0.017 (3) 0.033 (3) -0.002 (3) 0.016 (2) 0.001 (2) C4 0.027 (3) 0.013 (3) 0.022 (2) -0.006 (2) 0.008 (2) 0.001 (2) C5 0.032 (3) 0.013 (3) 0.017 (2) -0.002 (3) 0.003 (2) 0.001 (2) C6 0.022 (3) 0.015 (3) 0.023 (2) -0.002 (2) -0.0005 (19) 0.001 (2) C7 0.011 (2) 0.023 (3) 0.038 (3) -0.001 (2) 0.007 (2) -0.004 (2) C8 0.0094 (19) 0.022 (2) 0.0241 (19) 0.0003 (18) 0.0044 (16) -0.0020 (18)

193

C9 0.011 (2) 0.021 (3) 0.034 (3) 0.006 (2) 0.005 (2) 0.005 (2) C10 0.015 (3) 0.038 (4) 0.028 (3) 0.003 (3) 0.003 (2) -0.004 (2) C11 0.011 (2) 0.040 (4) 0.037 (3) 0.003 (3) -0.001 (2) -0.015 (3) C12 0.016 (2) 0.024 (4) 0.043 (3) -0.003 (3) 0.005 (2) -0.004 (3) C13 0.015 (2) 0.027 (4) 0.030 (3) 0.001 (3) 0.005 (2) 0.002 (2) C14 0.014 (2) 0.021 (3) 0.013 (2) 0.002 (2) 0.0056 (18) -0.002 (2) C15 0.056 (4) 0.024 (4) 0.029 (3) -0.003 (3) -0.009 (3) -0.001 (2) C16 0.016 (3) 0.027 (3) 0.023 (2) -0.005 (2) -0.003 (2) 0.000 (2) C17 0.040 (3) 0.050 (4) 0.031 (3) -0.021 (3) 0.008 (2) -0.014 (3) C18 0.060 (4) 0.058 (5) 0.031 (3) -0.036 (4) 0.021 (3) -0.005 (3) C19 0.045 (3) 0.045 (4) 0.035 (3) -0.031 (3) 0.015 (2) -0.005 (3) C20 0.016 (3) 0.035 (4) 0.028 (3) -0.004 (3) 0.007 (2) -0.006 (2) C21 0.017 (2) 0.026 (4) 0.020 (2) 0.001 (2) 0.0018 (19) 0.001 (2) C22A 0.038 (9) 0.023 (9) 0.017 (7) -0.006 (8) 0.014 (7) 0.003 (6) O9A 0.047 (9) 0.041 (10) 0.033 (7) -0.002 (8) 0.009 (6) -0.001 (7) O10A 0.026 (7) 0.016 (7) 0.030 (6) 0.006 (6) 0.008 (5) 0.009 (5) C23A 0.040 (9) 0.038 (9) 0.033 (7) 0.005 (7) 0.009 (6) 0.009 (7) C24A 0.064 (13) 0.057 (13) 0.074 (13) -0.014 (10) 0.000 (9) 0.021 (10) C25A 0.060 (14) 0.082 (17) 0.084 (15) 0.008 (14) 0.037 (12) 0.013 (14) C22B 0.038 (6) 0.042 (7) 0.036 (5) -0.004 (6) -0.005 (4) -0.013 (5) O9B 0.065 (6) 0.062 (7) 0.063 (5) -0.011 (5) -0.014 (4) -0.046 (5) O10B 0.035 (4) 0.060 (6) 0.034 (3) 0.009 (4) -0.015 (3) -0.016 (4) C23B 0.050 (6) 0.092 (9) 0.053 (7) 0.016 (6) -0.020 (5) -0.038 (7) C24B 0.066 (7) 0.088 (9) 0.032 (5) 0.020 (7) -0.002 (5) 0.013 (6) C25B 0.050 (7) 0.103 (12) 0.122 (11) -0.002 (8) -0.007 (7) 0.090 (10)

Geometric parameters (Å, °) S1-O3 1.412 (5) C10-H10 0.95 S1-O4 1.414 (4) C11-C12 1.381 (7) S1-O2 1.565 (4) C11-H11 0.95 S1-N1 1.677 (5) C12-C13 1.391 (7) O1-C1 1.409 (6) C12-H12 0.95 O1-C5 1.440 (6) C13-H13 0.95 O2-C2 1.481 (7) C15-C16 1.502 (8) O5-C14 1.366 (5) C15-H15A 0.99 O5-C4 1.444 (6) C15-H15B 0.99 O6-C14 1.225 (6) C16-C21 1.384 (7) O7-C15 1.403 (6) C16-C17 1.395 (7) O7-C3 1.424 (7) C17-C18 1.385 (9) O8-C6 1.426 (6) C17-H17 0.95 O8-C7 1.445 (5) C18-C19 1.388 (8)

194

N1-C22A 1.274 (18) C18-H18 0.95 N1-C1 1.445 (7) C19-C20 1.373 (7) N1-C22B 1.465 (11) C19-H19 0.95 N2-C14 1.320 (7) C20-C21 1.386 (7) N2-H21N 0.88 (5) C20-H20 0.95 N2-H22N 0.85 (5) C21-H21 0.95 C1-C2 1.517 (8) C22A-O9A 1.178 (18) C1-H1 1 C22A-O10A 1.328 (17) C2-C3 1.517 (8) O10A-C23A 1.450 (17) C2-H2 1 C23A-C24A 1.46 (2) C3-C4 1.539 (6) C23A-H23A 0.99 C3-H3 1 C23A-H23B 0.99 C4-C5 1.509 (7) C24A-C25A 1.21 (3) C4-H4 1 C24A-H24A 0.95 C5-C6 1.511 (6) C25A-H25A 0.95 C5-H5 1 C25A-H25B 0.95 C6-H6A 0.99 C22B-O9B 1.197 (12) C6-H6B 0.99 C22B-O10B 1.329 (12) C7-C8 1.493 (7) O10B-C23B 1.474 (13) C7-H7A 0.99 C23B-C24B 1.513 (19) C7-H7B 0.99 C23B-H23C 0.99 C8-C9 1.386 (6) C23B-H23D 0.99 C8-C13 1.386 (8) C24B-C25B 1.19 (2) C9-C10 1.381 (8) C24B-H24B 0.95 C9-H9 0.95 C25B-H25C 0.95 C10-C11 1.395 (8) C25B-H25D 0.95 O3-S1-O4 119.1 (3) C11-C10-H10 119.8 O3-S1-O2 108.5 (2) C12-C11-C10 119.6 (5) O4-S1-O2 111.3 (3) C12-C11-H11 120.2 O3-S1-N1 111.9 (3) C10-C11-H11 120.2 O4-S1-N1 109.3 (3) C11-C12-C13 119.6 (6) O2-S1-N1 93.9 (2) C11-C12-H12 120.2 C1-O1-C5 112.6 (4) C13-C12-H12 120.2 C2-O2-S1 109.6 (3) C8-C13-C12 120.9 (5) C14-O5-C4 115.2 (4) C8-C13-H13 119.5 C15-O7-C3 113.0 (4) C12-C13-H13 119.5 C6-O8-C7 109.8 (3) O6-C14-N2 126.3 (5) C22A-N1-C1 119.3 (9) O6-C14-O5 122.0 (5) C1-N1-C22B 129.0 (6) N2-C14-O5 111.7 (4) C22A-N1-S1 125.3 (8) O7-C15-C16 111.2 (4) C1-N1-S1 112.7 (4) O7-C15-H15A 109.4

195

C22B-N1-S1 116.9 (6) C16-C15-H15A 109.4 C14-N2-H21N 119 (4) O7-C15-H15B 109.4 C14-N2-H22N 130 (4) C16-C15-H15B 109.4 H21N-N2-H22N 111 (5) H15A-C15-H15B 108 O1-C1-N1 108.7 (5) C21-C16-C17 119.0 (5) O1-C1-C2 111.5 (4) C21-C16-C15 119.5 (4) N1-C1-C2 102.3 (5) C17-C16-C15 121.5 (5) O1-C1-H1 111.4 C18-C17-C16 120.0 (6) N1-C1-H1 111.4 C18-C17-H17 120 C2-C1-H1 111.4 C16-C17-H17 120 O2-C2-C3 107.5 (4) C17-C18-C19 120.6 (5) O2-C2-C1 103.3 (5) C17-C18-H18 119.7 C3-C2-C1 116.5 (5) C19-C18-H18 119.7 O2-C2-H2 109.7 C20-C19-C18 119.2 (6) C3-C2-H2 109.7 C20-C19-H19 120.4 C1-C2-H2 109.7 C18-C19-H19 120.4 O7-C3-C2 104.1 (4) C19-C20-C21 120.7 (5) O7-C3-C4 108.8 (4) C19-C20-H20 119.7 C2-C3-C4 113.0 (4) C21-C20-H20 119.7 O7-C3-H3 110.3 C16-C21-C20 120.5 (4) C2-C3-H3 110.3 C16-C21-H21 119.8 C4-C3-H3 110.3 C20-C21-H21 119.8 O5-C4-C5 108.8 (4) O9A-C22A-N1 118.4 (15) O5-C4-C3 109.7 (4) O9A-C22A-O10A 130.6 (17) C5-C4-C3 109.2 (4) N1-C22A-O10A 110.5 (13) O5-C4-H4 109.7 C22A-O10A-C23A 117.2 (15) C5-C4-H4 109.7 O10A-C23A-C24A 111.7 (16) C3-C4-H4 109.7 O10A-C23A-H23A 109.3 O1-C5-C4 110.1 (4) C24A-C23A-H23A 109.3 O1-C5-C6 104.6 (4) O10A-C23A-H23B 109.3 C4-C5-C6 117.0 (4) C24A-C23A-H23B 109.3 O1-C5-H5 108.3 H23A-C23A-H23B 107.9 C4-C5-H5 108.3 C25A-C24A-C23A 128 (3) C6-C5-H5 108.3 C25A-C24A-H24A 116 O8-C6-C5 109.0 (4) C23A-C24A-H24A 116 O8-C6-H6A 109.9 C24A-C25A-H25A 120 C5-C6-H6A 109.9 C24A-C25A-H25B 120 O8-C6-H6B 109.9 H25A-C25A-H25B 120 C5-C6-H6B 109.9 O9B-C22B-O10B 126.8 (10) H6A-C6-H6B 108.3 O9B-C22B-N1 123.1 (10) O8-C7-C8 109.4 (4) O10B-C22B-N1 110.0 (8)

196

O8-C7-H7A 109.8 C22B-O10B-C23B 114.2 (9) C8-C7-H7A 109.8 O10B-C23B-C24B 108.1 (10) O8-C7-H7B 109.8 O10B-C23B-H23C 110.1 C8-C7-H7B 109.8 C24B-C23B-H23C 110.1 H7A-C7-H7B 108.2 O10B-C23B-H23D 110.1 C9-C8-C13 119.0 (5) C24B-C23B-H23D 110.1 C9-C8-C7 121.3 (5) H23C-C23B-H23D 108.4 C13-C8-C7 119.7 (4) C25B-C24B-C23B 126.8 (13) C10-C9-C8 120.4 (5) C25B-C24B-H24B 116.6 C10-C9-H9 119.8 C23B-C24B-H24B 116.6 C8-C9-H9 119.8 C24B-C25B-H25C 120 C9-C10-C11 120.4 (5) C24B-C25B-H25D 120 C9-C10-H10 119.8 H25C-C25B-H25D 120 O3-S1-O2-C2 141.8 (4) C7-O8-C6-C5 -169.9 (4) O4-S1-O2-C2 -85.3 (4) O1-C5-C6-O8 173.0 (4) N1-S1-O2-C2 27.2 (4) C4-C5-C6-O8 -65.0 (6) O3-S1-N1-C22A 85.7 (9) C6-O8-C7-C8 177.5 (4) O4-S1-N1-C22A -48.5 (10) O8-C7-C8-C9 115.4 (5) O2-S1-N1-C22A -162.7 (9) O8-C7-C8-C13 -65.8 (6) O3-S1-N1-C1 -113.2 (4) C13-C8-C9-C10 2.7 (7) O4-S1-N1-C1 112.6 (4) C7-C8-C9-C10 -178.5 (4) O2-S1-N1-C1 -1.6 (4) C8-C9-C10-C11 -0.1 (7) O3-S1-N1-C22B 54.2 (6) C9-C10-C11-C12 -1.5 (8) O4-S1-N1-C22B -79.9 (6) C10-C11-C12-C13 0.5 (7) O2-S1-N1-C22B 165.9 (5) C9-C8-C13-C12 -3.7 (7) C5-O1-C1-N1 -168.4 (4) C7-C8-C13-C12 177.4 (4) C5-O1-C1-C2 -56.5 (6) C11-C12-C13-C8 2.1 (7) C22A-N1-C1-O1 -102.6 (9) C4-O5-C14-O6 -10.1 (6) C22B-N1-C1-O1 -70.6 (8) C4-O5-C14-N2 170.2 (4) S1-N1-C1-O1 95.0 (5) C3-O7-C15-C16 -171.9 (4) C22A-N1-C1-C2 139.4 (9) O7-C15-C16-C21 -142.3 (5) C22B-N1-C1-C2 171.5 (7) O7-C15-C16-C17 38.3 (7) S1-N1-C1-C2 -23.0 (5) C21-C16-C17-C18 1.2 (9) S1-O2-C2-C3 -167.8 (3) C15-C16-C17-C18 -179.5 (6) S1-O2-C2-C1 -44.1 (4) C16-C17-C18-C19 -2.8 (10) O1-C1-C2-O2 -76.5 (5) C17-C18-C19-C20 2.1 (10) N1-C1-C2-O2 39.4 (5) C18-C19-C20-C21 0.1 (9) O1-C1-C2-C3 41.1 (7) C17-C16-C21-C20 1.1 (8) N1-C1-C2-C3 157.0 (5) C15-C16-C21-C20 -178.3 (5) C15-O7-C3-C2 147.0 (4) C19-C20-C21-C16 -1.8 (8) C15-O7-C3-C4 -92.3 (5) C1-N1-C22A-O9A -169.5 (11)

197

O2-C2-C3-O7 -163.2 (4) S1-N1-C22A-O9A -9.5 (17) C1-C2-C3-O7 81.6 (6) C1-N1-C22A-O10A 17.8 (14) O2-C2-C3-C4 79.0 (6) S1-N1-C22A-O10A 177.8 (8) C1-C2-C3-C4 -36.2 (7) O9A-C22A-O10A-C23A 8 (2) C14-O5-C4-C5 169.4 (3) N1-C22A-O10A-C23A 179.5 (13) C14-O5-C4-C3 -71.2 (5) C22A-O10A-C23A-C24A -83 (2) O7-C3-C4-O5 169.9 (4) O10A-C23A-C24A-C25A -133 (3) C2-C3-C4-O5 -75.0 (5) C1-N1-C22B-O9B 175.3 (9) O7-C3-C4-C5 -70.9 (5) S1-N1-C22B-O9B 10.2 (12) C2-C3-C4-C5 44.1 (6) C1-N1-C22B-O10B -8.3 (11) C1-O1-C5-C4 67.9 (5) S1-N1-C22B-O10B -173.3 (6) C1-O1-C5-C6 -165.6 (4) O9B-C22B-O10B-C23B -6.0 (17) O5-C4-C5-O1 60.4 (5) N1-C22B-O10B-C23B 177.7 (9) C3-C4-C5-O1 -59.3 (5) C22B-O10B-C23B-C24B 169.5 (10) O5-C4-C5-C6 -58.7 (6) O10B-C23B-C24B-C25B -9 (2) C3-C4-C5-C6 -178.4 (4)

Hydrogen-bond geometry (Å, °) D-H...A D-H H...A D...A D-H...A N2-H21N...O6i 0.88 (5) 2.06 (6) 2.924 (5) 168 (4) N2-H22N...O8ii 0.85 (5) 2.41 (6) 3.222 (6) 158 (4) Symmetry codes: (i) -x+1, y+1/2, -z; (ii) x, y+1, z.

6.3.2 Crystallographic Information for 3.2.98

Crystal data C12H23N3O2SSi Mr = 301.48 Orthorhombic, P212121 a = 7.2579 (4) Å b = 11.8753 (8) Å c = 18.1943 (13) Å 3 V = 1568.16 (18) Å Z = 4

Data collection Bruker Kappa APEX-II DUO diffractometer Radiation source: IµS microfocus QUAZAR multilayer optics monochromator φ and ω scans Absorption correction: multi-scan SADABS (Sheldrick, 2004) Tmin = 0.675, Tmax = 0.926

198

Refinement Refinement on F2 Least-squares matrix: full R[F2 > 2σ(F2)] = 0.049 wR(F2) = 0.125 S = 1.05 2213 reflections 186 parameters 3 restraints Hydrogen site location: mixed

Special details F(000) = 648 -3 Dx = 1.277 Mg m Cu Kα radiation, λ = 1.54184 Å Cell parameters from 1907 reflections θ = 4.5–60.7° µ = 2.59 mm-1 T = 90 K Needle, colourless 0.21 × 0.06 × 0.03 mm 4953 measured reflections 2213 independent reflections 1954 reflections with I > 2σ(I) Rint = 0.047 θmax = 61.8°, θmin = 4.5° h = −6→8 k = −12→13 l = −20→19

H atoms treated by a mixture of independent and constrained refinement w = 1/[σ2(F2) + (0.0767P)2] where P = (F2 + 2F2)/3 (Δ/σ)max < 0.001 -3 Δρmax = 0.50 e Å -3 Δρmin = −0.22 e Å Absolute structure: Flack x determined using 686 quotients [(I+)-(I-)]/[(I+)+(I-)] (Parsons, Flack and Wagner, Acta Cryst. B69 (2013) 249-259). Absolute structure parameter: 0.04 (2)

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Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) x y z Uiso*/Ueq S1 0.75726 (19) 0.61261 (10) 0.45110 (7) 0.0436 (4) Si1 0.2926 (2) 0.61597 (12) 0.74430 (7) 0.0402 (4) O1 0.2504 (5) 0.2560 (3) 0.48738 (18) 0.0373 (8) O2 0.2721 (5) 0.5433 (3) 0.66719 (17) 0.0396 (8) N1 0.4280 (6) 0.6174 (4) 0.5208 (2) 0.0395 (10) H1N 0.417 (9) 0.686 (2) 0.510 (3) 0.047* N2 0.5210 (6) 0.4471 (3) 0.4894 (2) 0.0376 (10) H2N 0.597 (7) 0.397 (4) 0.486 (3) 0.045* N3 0.0661 (6) 0.3617 (3) 0.5600 (2) 0.0368 (10) H3N −0.019 (6) 0.317 (4) 0.553 (3) 0.044* C1 0.5630 (8) 0.5589 (4) 0.4879 (3) 0.0385 (13) C2 0.3658 (7) 0.4308 (4) 0.5390 (3) 0.0344 (11) H2 0.415273 0.420501 0.589878 0.041* C3 0.2260 (8) 0.3416 (4) 0.5255 (3) 0.0360 (12) C4 0.0125 (7) 0.4640 (4) 0.6009 (3) 0.0370 (12) H4A −0.020447 0.441783 0.651649 0.044* H4B −0.099626 0.49561 0.577744 0.044* C5 0.1578 (7) 0.5568 (4) 0.6049 (3) 0.0389 (12) H5 0.096842 0.632305 0.606166 0.047* C6 0.2694 (8) 0.5447 (4) 0.5355 (3) 0.0367 (12) H6 0.184818 0.546037 0.492159 0.044* C7 0.2171 (10) 0.5270 (5) 0.8227 (3) 0.0526 (15) H7A 0.100406 0.490018 0.810278 0.079* H7B 0.200017 0.574218 0.866291 0.079* H7C 0.310986 0.469723 0.832744 0.079* C8 0.1512 (8) 0.7471 (4) 0.7405 (3) 0.0477 (14) H8A 0.180911 0.789008 0.695642 0.072* H8B 0.178355 0.79393 0.783536 0.072* H8C 0.020138 0.727308 0.740382 0.072* C9 0.5460 (8) 0.6507 (4) 0.7524 (3) 0.0444 (13) C10 0.5811 (11) 0.7066 (8) 0.8268 (4) 0.083 (2) H10A 0.711846 0.726132 0.831055 0.124* H10B 0.54745 0.654413 0.866337 0.124* H10C 0.506428 0.775107 0.830795 0.124* C11 0.5996 (10) 0.7301 (5) 0.6897 (4) 0.067 (2) H11A 0.522723 0.797888 0.691757 0.100* H11B 0.5808 0.692017 0.642514 0.100* H11C 0.729465 0.751257 0.694711 0.100* C12 0.6595 (8) 0.5429 (5) 0.7471 (4) 0.0538 (15)

200

H12A 0.63723 0.506807 0.699509 0.081* H12B 0.62366 0.491466 0.786727 0.081* H12C 0.790693 0.561216 0.751737 0.081*

2 Atomic displacement parameters (Å ) U11 U22 U33 U12 U13 U23 S1 0.0353 (8) 0.0405 (7) 0.0551 (7) −0.0048 (7) 0.0043 (7) 0.0020 (6) Si1 0.0323 (8) 0.0394 (7) 0.0491 (7) 0.0013 (7) −0.0008 (7) −0.0057 (6) O1 0.027 (2) 0.0332 (16) 0.0518 (17) 0.0011 (15) 0.0004 (17) −0.0074 (14) O2 0.032 (2) 0.0388 (17) 0.0480 (17) 0.0038 (17) −0.0035 (17) −0.0045 (14) N1 0.033 (3) 0.032 (2) 0.054 (2) 0.001 (2) 0.001 (2) 0.003 (2) N2 0.030 (3) 0.036 (2) 0.048 (2) −0.001 (2) 0.002 (2) −0.001 (2) N3 0.027 (3) 0.037 (2) 0.046 (2) −0.0026 (19) 0.0000 (19) −0.0040 (19) C1 0.034 (3) 0.041 (3) 0.040 (3) −0.005 (2) −0.002 (2) 0.002 (2) C2 0.029 (3) 0.034 (2) 0.041 (2) 0.001 (2) −0.003 (2) −0.001 (2) C3 0.028 (3) 0.037 (3) 0.043 (2) 0.002 (2) −0.002 (2) 0.005 (2) C4 0.028 (3) 0.041 (3) 0.042 (3) 0.002 (2) −0.001 (2) −0.005 (2) C5 0.033 (3) 0.036 (3) 0.048 (3) 0.001 (2) −0.005 (2) −0.001 (2) C6 0.032 (3) 0.032 (2) 0.046 (2) 0.001 (2) 0.000 (2) 0.0002 (19) C7 0.049 (4) 0.056 (3) 0.053 (3) 0.003 (3) 0.004 (3) −0.001 (3) C8 0.033 (3) 0.042 (3) 0.068 (3) 0.003 (2) 0.000 (3) −0.010 (3) C9 0.036 (3) 0.041 (3) 0.056 (3) 0.003 (2) −0.003 (3) −0.006 (2) C10 0.043 (4) 0.113 (6) 0.093 (5) 0.000 (4) −0.013 (4) −0.054 (5) C11 0.038 (4) 0.058 (4) 0.105 (5) −0.012 (3) −0.014 (4) 0.018 (4) C12 0.032 (3) 0.051 (3) 0.079 (4) 0.004 (3) −0.002 (3) −0.002 (3)

Hydrogen-bond geometry (Å, °) D—H···A D—H H···A D···A D—H···A N1—H1N···S1i 0.84 (3) 2.75 (4) 3.475 (5) 146 (5) N2—H2N···O1ii 0.82 (3) 2.18 (3) 2.962 (6) 159 (5) N3—H3N···O1iii 0.83 (3) 2.02 (3) 2.819 (6) 162 (5) Symmetry codes: (i) -x+1, y+1/2, -z; (ii) x, y+1, z.

201

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