Natural and Synthetic Compounds as Tools to Overcome Antibiotic Resistance Open Access

Steele, Andrew (Spring 2018)

Permanent URL: https://etd.library.emory.edu/concern/etds/jq085k021?locale=en
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Abstract

Antibiotic resistance is a pressing challenge that chemists have invested significant resources on. Current antibiotic development has slowed in recent years due to fewer compounds that operate via new mechanisms of action. To this end, we have looked to natural products with unknown, presumably new, mechanisms of action, as well as known scaffolds with newly discovered antibiotic activity to solve this problem. In each of these projects, diverted total synthesis was utilized to access not only the original target compounds, but also analogs in a highly efficient manner. Specifically, promysalin, a Gram-negative selective antibiotic, was synthesized for the first time, and its structure was elucidated. From there, a series of analogs was synthesized to determine preliminary structure-activity data. Utilizing these findings, an analog suitable for affinity-based protein profiling was synthesized and subsequent experiments allowed the discovery of the protein target of promysalin. Further experiments shed light on promysalin’s specificity as well as structural mode of action. CD437 and nTZDpa were known synthetic compounds that received new attention in the laboratory of a collaborator (Mylonakis lab, Brown University) when they were shown to be membrane-disrupting compounds with potent activity against pathogenic Gram-positive bacteria. Through our synthetic studies, we optimized both the potency and toxicity of each compound. Finally, baulamycin A and B were discovered in a high-throughput screen effort to find new inhibitors of siderophore biosynthesis in pathogenic bacteria. However, their activity profile seemed to indicate multiple mechanisms were at play. The originally proposed structure was shown to be incorrect by others working on these molecules. We were the second laboratory to synthesize the corrected structures of baulamycins A and B, and the first to leverage baulamycin analogs in whole-cell assays. These assays allowed us to elucidate the baulamycins’ alternative mechanism of action, which gratifyingly complemented the biological data previously disclosed. These projects attest to the power of total synthesis in new antibiotic discovery and development, and future work could open the door for new therapeutics and tools for further biological discoveries.

Table of Contents

Table of Contents

Chapter 1: Introduction. 1..................................................................................................1

1.1 The Discovery and Rise of Modern Antibiotics 1...............................................................1

1.2 Antibiotic Resistance. 2.................................................................................................2

1.2.1 Introduction. 2..........................................................................................................2

1.2.2 Drug-Inactivating Enzymes. 3.....................................................................................3

1.2.3 Modification of Drug Target 5.....................................................................................5

1.2.4 Prevention of Access to Target 7.................................................................................7

1.3 Chemical Approaches to Antibiotic Resistance. 8...............................................................8

1.3.1 Semisynthesis. 8.......................................................................................................8

1.3.2 Diverted Total Synthesis. 11.......................................................................................11

1.3.3 Conclusions. 13........................................................................................................13

1.4 References 13............................................................................................................13

Chapter 2: Synthesis, Structure Elucidation, and Target Identification of the Gram-negative Selective Antibiotic Promysalin. 16

2.1 Promysalin background. 16...........................................................................................16

2.1.1 Gram-negative and Pseudomonas Clinical Infections. 16.................................................16

2.1.2 Narrow-spectrum Therapeutics. 17..............................................................................17

2.1.3 Promysalin Background. 19........................................................................................19

2.2 Synthesis of Promysalin. 21..........................................................................................21

2.2.1 Retrosynthesis 21....................................................................................................21

2.2.2 Synthesis of Promysalin Acid Fragment 22...................................................................22

2.2.3 Synthesis of alcohol fragment diastereomers. 23...........................................................23

2.2.4 Completion of Promysalin Synthesis. 24.......................................................................24

2.3 Structure Elucidation of Promysalin. 25...........................................................................25

2.3.1 NMR Analysis of Promysalin Diastereomers 25.............................................................25

2.3.2 Biological Testing of Promysalin Diastereomers (–)-2.1a(–)-2.1d. 27..........................27

2.3.3 Observation of Novel Promysalin-Induced Phenotypes. 30...............................................30

2.3.4 Conclusions. 30........................................................................................................30

2.4 Synthesis of Promysalin Analogs. 32..............................................................................32

2.4.1 Promysalin Analog Design. 32.....................................................................................32

2.4.2 Promysalin Proline Analog Synthesis. 34.......................................................................34

2.4.3 Promysalin Salicylate Analog Synthesis. 38...................................................................38

2.4.4 Promysalin Side Chain Analog Synthesis. 43.................................................................43

2.4.5 Synthesis of “Pro-drug” Analogs. 47............................................................................47

2.5 Promysalin Analog Results and Conclusions. 47................................................................47

2.5.1 Promysalin Analog Inhibitory Data. 47.........................................................................47

2.5.2 CAS Assay and Iron-binding. 49..................................................................................49

2.5.3 Conclusions. 51........................................................................................................51

2.6 Synthesis of Promysalin Photoprobe and Affinity-based Protein Profiling. 51.........................51

2.6.1 Affinity-based Protein Profiling in Natural Product Target Identification. 51........................51

2.6.2 Promysalin Photoaffinity Probe Design and Synthesis. 54................................................54

2.6.3 Biological Evaluation of Promysalin Photoprobe and Proteomic Experiments. 55.................55

2.6.4 Succinate Dehydrogenase and its Role in Pseudomonas Metabolism.. 57............................57

2.7 References 60..................................................................................................................60

Chapter 3: Diverted Total Synthesis of CD437 and nTZDpa Analogs – Effective Membrane Disrupting Agents Against Gram-positive Pathogens.........................................................64 64

3.1 Introduction and Background. 64...................................................................................64

3.1.1 Staphylococcus aureus – Resistance and Clinical Relevance. 64.......................................64

3.1.2 Persister Cells 65.....................................................................................................65

3.2 The Discovery of New Antibiotic Activity of CD437 and nTZDpa. 67.....................................67

3.2.1 C. elegans-MRSA High-throughput Screen. 67...............................................................67

3.2.2 Biological Activity of CD437 and nTZDpa. 68.................................................................68

3.2.3 CD437 Simulations and Proposed Mechanism of Action. 70.............................................70

3.3 Diverted total synthesis of CD437 and analogs. 71...........................................................71

3.3.1 First-generation CD437 analogs from adapalene. 71......................................................71

3.3.2 Retrosynthetic Analysis and CD437 Analog Design. 73...................................................73

3.3.3 Synthesis of Boronic Acid Building Blocks. 73................................................................73

3.3.4 Synthesis of Unsubstituted Naphthalene CD437 Analogs. 74...........................................74

3.3.5 Unsuccessful attempts at accessing oxidized naphthalene analogs of CD437. 75................75

3.3.6 Synthesis of oxidized naphthalene analogs of CD437. 78................................................78

3.3.7 SAR data of CD437 analogs. 80...................................................................................80

3.3.8 Conclusions. 82.................................................................................................................82

3.4 Synthetic and Biological Studies of nTZDpa. 82................................................................82

3.4.1 Synthetic Route Design and Prior Art 82......................................................................82

3.4.2 Optimized Synthesis of nTZDpa and Analogs. 83...........................................................83

3.4.2 SAR data of nTZDpa analogs. 90.................................................................................90

3.5 Conclusions. 92.................................................................................................................92

3.6 References 94............................................................................................................94

Chapter 4: Diverted Total Synthesis of Baulamycin A, B, and Analogs Provides Evidence to a Newly Identified Mechanism of Action. 96.............................................................................96

4.1 Introduction and background. 96...................................................................................96

4.1.1 Bacterial Iron Acquisition. 96......................................................................................96

4.1.2 Siderophores of Gram-positive and Gram-negative Bacteria. 98.......................................98

4.1.3 Biosynthesis of Citrate-containing Siderophores 99.......................................................99

4.1.4 Baulamycin A & B Isolation and Structural Ambiguity. 101..............................................101

4.1.5 Hypothesized Model of Binding and Absolute Configuration. 103......................................103

4.2 Other Syntheses of Baulamycin A.. 107............................................................................107

4.2.1 Total Synthesis of Originally Reported Structure of Baulamycin A by Goswami et al. 107....107

4.2.2 Synthesis of the Proposed Baulamycin Carbon Framework by Chandrasekhar, et al. 111....111

4.2.3 Total Synthesis and Stereochemical Assignment of the Baulamycins by Aggarwal and Co-workers  113....................................................................................................................113

4.2.4 Synthesis and in vitro SAR of Baulamycin Structures by Sim and Co-workers. 118.............118

4.3 Total Synthesis of Baulamycin A and B.. 122.....................................................................122

4.3.1 Retrosynthesis 122...................................................................................................122

4.3.2 Synthesis of Originally Proposed Left Half Fragment 123................................................123

4.3.3 Synthesis of Corrected Structure. 124..........................................................................124

4.3.4 Completion of the Total Synthesis of Baulamycin A, Baulamycin B, and Analogs. 127..........127

4.4 Biological evaluation of Baulamycin A, B, and Analogs. 131................................................131

4.4.1 S. aureus Inhibition. 131............................................................................................131

4.4.2 Hemolysis and SYTOX Uptake Assay Results. 133..........................................................133

4.5 Conclusions. 134...............................................................................................................134

4.6 References 136...........................................................................................................136

5.1 Experimental Details. 140.............................................................................................140

5.1.1 Chemistry: Instrumentation and General Notes. 140......................................................140

5.1.2 Chemistry: Experimental Procedures and Characterization Data. 141...............................141

5.1.3 Biology: Bacterial Strains and Culture Conditions. 319....................................................319

5.1.4 Biology: Assay Procedures. 319..................................................................................319

5.1.5 SYTOX Uptake Assay Data. 322...................................................................................322

Appendix: NMR Spectra. 328..............................................................................................328

 

 

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