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Document Type

Open Access Dissertation

Degree Name

Doctor of Philosophy (PhD)

Degree Program

Chemistry

Year Degree Awarded

2014

First Advisor

Nathan Schnarr

Second Advisor

Dhandapani Venkataraman

Third Advisor

Michael Knapp

Subject Categories

Biochemistry, Biophysics, and Structural Biology | Chemistry

Abstract

Polyketides are a broad class of natural products that have received attention from the scientific community because they are a rich mine of bioactive structures. The common thread that binds the class together is the method by which they are synthesized, by large enzymatic complexes called polyketide synthases (PKSs) which display assembly line like organization. A great deal of effort has been put into studying PKSs, but their mechanistic steps are still not perfectly understood. In order to further the study of PKSs and their components, we have developed a series of reactive small molecules that covalently modify specific sites of PKS components. We have shown that b-lactones are able to selectively load holo-acyl carrier proteins (ACPs) with polyketide-like functionality while leaving similarly functionalized components such as the ketosynthase domain virtually untouched. We have also shown that b- lactams are able discriminate between apo- and holo-ACPs and have used this difference in reactivity to develop a method of purifying holo-ACPs. Another b-lactam based probe has been shown to directly modify the active portion of holo-ACPs with malonate-like functionality, and may be starting point for introducing novel functionality into difficult to access sites of polyketides.

During the course of these investigations the copper catalyzed azide-alkyne cycloaddition has been used heavily for conjugation reactions involving small molecules and proteins. The presence of copper has been a serious problem, often leading to lost and damaged proteins. Copper-free azide-alkyne conjugation methods exist, but suffer from drawbacks of their own such as slow reaction rates and difficult targetability. In order to maintain the positive aspects of copper free click yet overcome the drawbacks, we have been developing photosensitive probes which upon irradiation rapidly react with azides in a click like reaction. Although this reaction was initially developed with bioconjugation in mind, further experiments have indicated that this may not be the best use. However, experiments are currently being carried out to optimize their performance and explore their usefulness in other chemical conjugation applications.

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