Total synthesis and biological investigations of natural product inspired antimicrobials Open Access

Wilt, Ingrid (Spring 2022)

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Natural products have a rich history of antimicrobial use. Chemodiversity and complexity of these structures often culminate in unique biological properties. Divergent and convergent synthetic approaches allow rapid diversification of natural product core scaffolds to evaluate the influence of structure on biological activity. The first chapter provides insight on antimicrobial development inspired by natural products, isolation of novel bioactive natural products, and synthetic approaches to generate compound libraries to evaluate key chemical moieties involved in microbial inhibition.

           The second chapter describes the synthesis of phenolic bisabolanes isolated from deep sea sediment that display potent activity towards microbial pathogens. Despite structural similarities, these natural products are reported to have narrow spectrum bioactivity suggesting minor changes in structure can result in unique mechanisms of action. Initially, the chapter focuses on the synthesis of peniciaculin A, a natural product with reported species specific activity against the plant fungal pathogen Alternaria brassicae. Investigations of the unique reported bioactivity leveraging synthetic derivatives of the natural product suggest peniciaculin A may be inhibiting fungal growth as a ubiquinone mimic. Identification of a key transformation in the synthesis of peniciaculin A allowed for rapid generation of 1-hydroxyboivinianin A, a lactone derivative with reported species specific activity against aquatic bacteria Vibrio harveyi.

           The third chapter explores the development of a common intermediate strategy for the synthesis of antifungal natural products, the purpurides. A key tricyclic intermediate was accessed through a tethered intramolecular Diels-Alder cycloaddition. The desired trans bicyclic system was synthesized via heterogeneous hydrogen atom transfer. Notably, heterogeneous catalysis provided solely cis isomer, invalidating previous reports of obtaining the trans isomer of analogous systems using this approach.

The fourth chapter investigates natural product metabolites as narrow spectrum antimicrobial agents. Epoxy isonitrile containing natural products demonstrate specific and potent antibacterial activity against gram-positive pathogens, in particular Staphylococcus aureus. This scaffold, however, is extremely labile under acidic and basic conditions, undergoing a Payne rearrangement to produce a stable epoxy ketone metabolite and releasing hydrogen cyanide. Toxicity associated with the release of hydrogen cyanide renders these molecules unusable as antibiotics. When compared to the activity of sodium cyanide in vivo, epoxy isonitriles, including amycomicin, aerocyandin, and YM-47515, were shown to be more potent. Thus, the epoxy isonitriles were thought to act as prodrugs, undergoing the Payne rearrangement to produce active epoxy ketone metabolites. These metabolites would not only reduce the toxicity of the parent compounds by eliminating the release of hydrogen cyanide, but could be accessed via a more facile synthetic route. We synthesized and performed biological assays with epoxy ketone containing metabolites and identified that the epoxy isonitrile moiety is pertinent for biological activity. Serendipitously, we discovered an α,β-unsaturated epoxy ketone analogue that exhibited moderate activity against S. aureus.

The fifth chapter includes discussion of a subpopulation of bacteria, persisters. Diminished response to antibacterials is often exacerbated by the presence of persister or metabolically dormant populations of bacteria. Membrane perturbing small molecules have the potential to eliminate persister populations, but use has been limited due to the low selectivity of these compounds for bacterial over mammalian membranes. Bithionol, a previously approved anthelmintic drug, is shown to inhibit methicillin-resistant Staphylococcus aureus (MRSA) persister cells via disruption of the membrane lipid bilayer at nontoxic levels to mammalian cells. Investigation of the structure activity relationship of bithionol and its tendency to interact with bacterial membranes suggests bioactivity correlates with the ability to increase membrane fluidity. This work demonstrates that membrane perturbing small molecules can be selective antibacterial agents and further investigations of this mechanism of action is warranted. 



Table of Contents

1.    Chapter 1. Introduction to natural product inspired antimicrobials 1

1.1.Traditional medicine1

1.2. Synthetic approaches to modern antimicrobials 1

1.3. Development of antimicrobial resistance5

1.4. Approaches to combating antimicrobial resistance and development of novel therapies8

1.4.1.    Combination therapy and regulated use8

1.4.2.    Analogue design8

1.4.3.    Novel natural products10

1.4.4.    Wuest lab’s approach to combating antimicrobial resistance13 Natural product inspired analogues14 Repurposing16

1.5. Conclusion17

1.6. Chapter 1 references18

2.    Chapter 2. Application of a key transformation for the synthesis of phenolic bisabolanes and biological investigations of their derivatives25

2.1. Introduction25

2.1.1.    Antifungal ubiquinone mimics25

2.1.2.    Antimicrobial phenolic bisabolanes29

2.1.3.    Previous synthetic approaches to phenolic bisabolanes31 Aggarwal’s 1,2-migration 32’s asymmetric Grignard 33 Diastereoselective nucleophilic addition to chiral imidazolidinone 34 Yajima’s synthesis of the peniciaculins34

2.2. Results and discussion35

2.2.1.    1,2-migration35

2.2.2.    Asymmetric Grignard39

2.2.3.    Chiral imidazolidinone43

2.2.4.    Analogue design and synthesis54 Ethoxy-acrylate analogues55 Ketone analogues56 Diorcinol analogues58

2.2.5.    Application of key transformation for the synthesis of other phenolic bisabolanes59

2.2.6.    Antimicrobial bioactivity66

2.3. Conclusion68

2.4. Chapter 2 references69

3.    Chapter 3. Leveraging core scaffold for the synthesis of amino acid-sesquiterpene conjugates and antimicrobial investigations of their derivatives73

3.1. Introduction73

3.1.1.    Development of antifungals for clinic use73

3.1.2.    Antimicrobial purpurides: a common intermediate in sight77

3.1.3.    Previous approaches to 6-6 trans bicycles79

3.2. Results and discussion82

3.2.1.    Construction of common intermediate via intramolecular Diels-Alder cycloaddition82

3.2.2.    Building trans fused bicycle via HAT 83

3.2.3.    Esterification88

3.2.4.    Analogue design92

3.2.5.    Preliminary biological investigations94

3.3. Conclusion95

3.4. Chapter 3 references96

4.    Chapter 4. Synthesis and biological investigations of epoxy-isonitrile metabolites100

4.1. Introduction100

4.1.1.    Narrow spectrum antibiotics100

4.1.2.    Epoxy isonitrile containing natural products101

4.2. Results and discussion104

4.2.1.    Synthesis of metabolites104

4.2.2.    Antimicrobial activity106

4.3. Conclusion109

4.4. Chapter 4 references 110

5.    Chapter 5. Membrane perturbing small molecules as novel antimicrobials112

5.1. Introduction112

5.1.1.    Persister cells112

5.1.2.    Membrane perturbation as a mechanism of action115

5.1.3.    Repurposing bithionol as a novel antimicrobial116

5.2. Results and discussion119

5.2.1.    Synthesis of bithionol analogues119

5.2.2.    Antimicrobial activity122

5.2.3.    Mechanism of action126

5.3. Conclusion128

5.4. Chapter 5 references 128

6.    Chapter 6. Experimental details132

6.1. Supplementary figures, schemes, and tables132

6.1.1.    Chapter 2132

6.1.2.    Chapter 3133

6.1.3.    Chapter 4133

6.2. Biology: general notes136

6.3. Biology: Procedures and supplemental information137

6.3.1.    Chapter 4 IC50 assay curves138

6.4. Chemistry: General notes 139

6.5. Chemistry: Synthesis procedures and characterization 140

6.5.1.    Chapter 2140

6.5.2.    Chapter 3194

6.5.3.    Chapter 4205

6.5.4.    Chapter 5208

6.6. Crystal analysis213

6.7. SI references 233

7.    Appendix235

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