Promysalin and CD437: Compounds Investigating New Antibacterial Mechanisms translation missing: zh.hyrax.visibility.files_restricted.text

Keohane, Colleen (Spring 2019)

Permanent URL: https://etd.library.emory.edu/concern/etds/fx719n450?locale=zh
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Abstract

The growing crisis surrounding antibiotic resistance continues to provide scientists with exciting and impactful research opportunities. The work reported herein will encompass two different approaches at investigating this monumental problem. Promysalin investigates the problem from a narrow-spectrum approach, wherein selectively killing one bacterial species instead of the entire community holds promise for dampening the rate of resistance. Alternatively, CD437 acts via a new mechanism wherein we can hopefully reestablish bioactivity by incorporating new approaches. Promysalin is a natural product isolated from the rhizosphere, a microbiome housing a wealth of microbial diversity, produced by a common plant beneficial bacteria, Pseudomonas putida. Our interest lies in the highly specific activity of the natural product wherein it solely has inhibitory activity against the Gram-negative pathogen Pseudomonas aeruginosa. We have spent the past five years working to understand the mechanism of inhibition and in doing so have completed the first total synthesis of the natural product, allowing for the determination of the natural stereochemistry; carried out a structure-activity relationship investigation which lead to the synthesis of a probe compound and used the probe to putatively determine the target. During this time, a second compound was investigated for antibacterial activity, CD437. CD437 was identified in a high-throughput screen by the Mylonakis Lab (Brown University) to have activity against Methicillin Resistant Staphylococcus aureus (MRSA) persister cells. This activity is attributed to a new mechanism of membrane permeabilization and a library of analogs was synthesized to support this discovery. The entirety of the work highlights the ability to use synthetic organic chemistry as a means to investigate biological problems.

Table of Contents

Table of Contents

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

1.1      Antibiotics................................................................................................................. 1

1.1.1    Mechanism of inhibition........................................................................................... 2

1.1.2    Mechanism of resistance........................................................................................... 7

1.2      Rhizosphere microbiome..........................................................................................11

1.2.1    Diverse microbiome..................................................................................................11

1.2.2    Types of metabolites found in the rhizosphere......................................................... 12

1.3      References................................................................................................................. 16

Chapter 2 Promysalin............................................................................................................. 19

2.1      Introduction............................................................................................................... 19

2.1.1    Narrow-spectrum antibiotics and Gram-negative pathogens.................................... 19

2.1.2    Promysalin isolation................................................................................................. 22

2.2      Synthesis of promysalin diastereomers..................................................................... 24

2.2.1    Synthetic strategy...................................................................................................... 24

2.2.2    Side chain synthesis.................................................................................................. 24

2.2.3    Acid synthesis........................................................................................................... 25

2.2.4    End Game................................................................................................................. 26

2.3      Structure elucidation................................................................................................. 28

2.3.1    NMR analysis........................................................................................................... 28

2.3.2    Biological confirmation............................................................................................ 29

2.3.3    Investigation of swarming phenotype....................................................................... 30

2.4      Discovery of new phenotypes................................................................................... 31

2.4.1    Fluorescent phenotype.............................................................................................. 31

2.4.2    CAS Assay................................................................................................................ 32

2.4.3    Conclusions............................................................................................................... 32

2.5      SAR investigation..................................................................................................... 33

2.5.1    Design of SAR study................................................................................................ 33

2.5.2    Proline analogs.......................................................................................................... 36

2.5.3    Salicylate analogs..................................................................................................... 40

2.5.4    Side chain analogs.................................................................................................... 46

2.5.5    Biologically relevant analogs................................................................................... 50

2.5.6    Biological Results..................................................................................................... 51

2.5.7    Conclusions............................................................................................................... 53

2.6      Investigation of iron relevance to activity................................................................ 53

2.7      Affinity-based protein profiling................................................................................ 55

2.7.1    Intro........................................................................................................................... 55

2.7.2    Synthesis of probe..................................................................................................... 58

2.7.3    Outline of experiments.............................................................................................. 61

2.7.4    Results....................................................................................................................... 62

2.8      Investigation of primary metabolism as target......................................................... 63

2.8.1    In vitro inhibition of SdhC........................................................................................ 63

2.8.2    Docking studies......................................................................................................… 64

2.8.3    Resistance selection.................................................................................................. 67

2.8.4    Feeding experiments................................................................................................. 69

2.8.5    Conclusions............................................................................................................... 71

2.9      Side chain analogs.................................................................................................... 73

2.9.1    Synthesis of side chain analogs................................................................................ 74

2.9.2    Biological results...................................................................................................... 78

2.9.3    Future directions....................................................................................................... 80

2.10     Transcriptomics .........................................................................................................80

2.11     References................................................................................................................. 90

Chapter 3 CD437................................................................................................................... 95

3.1      Introduction............................................................................................................... 95

3.1.1    Staphylococcus aureus.............................................................................................. 95

3.1.2    Persister cells............................................................................................................ 96

3.2      CD437....................................................................................................................... 98

3.2.1    C. Elegans high throughput screen identifies CD437............................................... 98

3.2.2    Proposed mechanism................................................................................................ 99

3.3      Synthesis of CD437 Analogs.................................................................................... 100

3.3.1    First series of analogs............................................................................................... 100

3.3.2    Rationale for second series of analogs...................................................................... 103

3.3.3    Boronic acid fragments............................................................................................. 104

3.3.4    Cyclization – first strategy........................................................................................ 106

3.3.5    Cyclization – second strategy................................................................................... 107

3.3.6    Final cyclization and assembly................................................................................. 109

3.4      Biological data.......................................................................................................... 112

3.5      Conclusions............................................................................................................... 115

3.6      References................................................................................................................. 115

Chapter 4 Conclusion............................................................................................................. 117

Chapter 5 Experimental Details............................................................................................. 120

5.1      Biology: General notes............................................................................................. 120

5.2      Biology: Procedures and supplemental information................................................. 121

5.3      Chemistry: General notes.......................................................................................... 152

5.4      Chemistry: Procedures and characterization............................................................. 153

Appendix: NMR Spectra........................................................................................................ 305

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