Year of Publication

2011

Degree Name

Doctor of Philosophy (PhD)

Document Type

Dissertation

College

Pharmacy

Department

Pharmaceutical Sciences

First Advisor

Doctor Jürgen Rohr

Abstract

Natural products occupy a central role as the majority of currently used antibiotic and anticancer agents. Among these are type-II polyketide synthase (PKS)-derived molecules, or polyketides, which are produced by many representatives of the genus Streptomyces. Some type-II polyketides, such as the tetracyclines and the anthracycline doxorubicin, are currently employed as therapeutics. However, several polyketide molecules exhibit promising biological activity, but due to toxic side effects or solubility concerns, remain undeveloped as drugs.

Gilvocarcin V (GV) (topoisomerase II inhibitor) has a novel mechanism of action: [2+2] cycloaddition to thymine residues by the 8-vinyl side chain and cross-linking of histone H. Mithramycin blocks transcription of proto-oncogenes c-myc and c-src by forming an Mg2+-coordinated homodimer in the GC-rich minor groove of DNA. The purpose of this research was to investigate the biosynthesis of several type II polyketide compounds (e.g. chrysomycin, elloramycin, and mithramycin) with the goal of improving the bioactivities of these drugs through combinatorial biosynthesis. Alteration of the glycosylation pattern of these molecules is one promising way to improve or alter the bioactivities of these molecules. To this end, an understanding of the glycosyltransferases and post-polyketide tailoring enzymatic steps involved in these biosynthetic pathways must be established. Four specific aims were established to meet these goals.

In specific aim 1, the biosynthetic locus of chrysomycin A was successfully cloned and elucidated, which afforded novel biosynthetic tools. Chrysomycin monooxygenases were found to catalyze identical roles to their gilvocarcin counterparts. Cloning of deoxysugar constructs (plasmids) which could direct biosynthesis of ketosugars, NDP-D-virenose, and NDP-D-fucofuranose in foreign pathways was undertaken in specific aim 2. Finally, these “sugar” plasmids were introduced into producer organisms of elloramycin and mithramycin pathways in specific aims 3 and 4 to interrogate the endogenous glycosyltransferases in order to alter their glycosylation patterns. These experiments resulted in the successful generation of a newly glycosylated tetracenomycin, as well as premithramycin, and mithramycin analogues. In specific aim 4, a new mithramycin analogue with an altered sugar pattern rationally designed and improved structural features was generated and structurally elucidated.

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