Date of Award

Fall 2022

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Applied Mathematics

First Advisor

Herzon, Seth

Abstract

The human microbiota is now understood to be intimately linked to host physiology. Recent studies indicate that microbiota species which possess a biosynthetic gene cluster – termed clb – contribute to the development and progression of colorectal cancer. Bacteria possessing the clb gene cluster are correlated to colorectal cancer (CRC) in humans, initiate colonic tumor and polyp formation in murine models, and induce DNA interstrand cross-links in cell models. Furthermore, clb+ bacteria induce a unique mutational signature which has been detected in a number of human tumor samples. Early studies by Oswald and co-workers established that the final product of the clb pathway – termed colibactin – was likely driving this carcinogenic phenotype. However, colibactin’s limited stability under laboratory conditions has precluded its isolation and characterization by standard spectroscopic methods. Instead, all knowledge of colibactin’s chemical identity and mechanism of action has derived from indirect means, such as biosynthetic analysis, enzymology, and characterization of shunt metabolites. These diverse lines of inquiry have led to a general understanding of nearly all steps in the clb pathway, provided a deeper understanding of the biological phenotypes induced by clb+ bacteria, and led to characterization of colibactin’s structure and chemical reactivity. This dissertation is a disclosure of our contribution to this global effort through the use of synthetic chemistry.We first discuss our total synthesis of precolibactin 886. These studies explore the unexpected instability of an α-aminomalonate derived C36–C37 1,2-dicarbonyl found in the biosynthetic precursor to precolibactin 886. The observed instability accounts for the low titer of precolibactin 886 and also provides a chemical mechanism for the production of other precolibactin shunt metabolites. We then discuss our structural elucidation and confirmation of colibactin 770 through the use of isotopic labelling studies, mass spectroscopy, and total synthesis. The structure of colibactin 770 accounts for all enzymes in the clb pathway required for genotoxicity and provides a clear chemical rationale for the DNA cross-linking phenotype of the clb gene cluster. The chemical instability of colibactin 770 precluded its direct study in cellular models. As such, we synthesized a stable analog – colibactin 742 – which recapitulates key aspects of the clb genotoxic phenotype. Structural studies of this stable analog revealed the presence of a β-hydroxy lactam, a structural feature not previously detected using mass spectroscopy techniques. Finally, we disclose our preliminary synthetic studies of colibactin 771, the putative biosynthetic precursor to colibactin 770. These studies suggest that the α-aminoketone proposed in the biosynthesis actually exists as an enolamine. This enolamine reacts rapidly with ambient oxygen to provide previously observed 1,2-dicarbonyl motifs. Additionally, this oxidation event appears to produce H2O2 which may further contribute to colibactin’s genotoxic potential in the gut.

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