Author ORCID Identifier

https://orcid.org/0000-0003-1434-2960

Date Available

5-26-2024

Year of Publication

2023

Degree Name

Doctor of Philosophy (PhD)

Document Type

Doctoral Dissertation

College

Pharmacy

Department/School/Program

Pharmaceutical Sciences

First Advisor

Jon S. Thorson

Abstract

Methyltransferases (MTs) are ubiquitous enzymes commonly involved in biosynthesis and regulation. The fundamental goal of this thesis was to explore the permissive nature of methyltransferases and leverage this unique biocatalytic feature to develop a new platform for chemoselective intramolecular cyclization reactions. To realize this ambitious goal, this thesis project set out to probe the substrate specificity of multiple natural product tailoring enzymes (MTs and glycosyltransferases), identify model substrates common to multiple such tailoring enzymes and subsequently establish multi-enzyme tandem reactions that would set the stage for a subsequent chemoselective intramolecular cyclization reaction. Chapter One highlights current state of the art on enzyme-mediated bioorthogonal technologies in which the key considerations for selecting enzyme catalysts and chemoselective reactions most amenable to bioorthogonal platforms were assessed and reviewed (published in Curr. Opin. Biotechnol., 2021). Chapter Two (manuscript submitted) describes a comprehensive study on the substrate scope of the carminomycin 4-O-MT DnrK, a final enzymatic step in the biosynthesis of daunomycin. This study revealed DnrK as a notably permissive MT and highlighted a functionally and structurally diverse array of validated new DnrK substrates. Bioactivity assays of a select set of the new molecules produced also expanded existing anthraquinone SAR. The third chapter (manuscript submitted) is a follow up on the intriguing discovery of DnrK’s ability to catalyze S- or N-alkylation of heteroatom-containing aromatics. This work opens the door to the intriguing possibility of substrate nucleophilicity/reactivity, rather than or in addition to ligand-binding, as potential driver of MT selectivity and also implicates DnrK as a potentially versatile tool for synthetic biology. The final chapter briefly summarizes the range of other tailoring enzymes evaluated and molecules identified that serve as facile complementary substrates for two or more orthogonal tailoring enzymes as a starting point for realizing the vision of bioorthogonal intramolecular cyclization reactions.

Digital Object Identifier (DOI)

https://doi.org/10.13023/etd.2023.261

Funding Information

This study was supported by National Institutes of Health Grants (no.: R01 GM115261, R37 AI52218, P20 130456)

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