Glycoside hydrolase reactivity with cyclohexenyl-based carbasugars: Mechanistic insights, applications, and design of new analogs

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(Thesis) Ph.D.
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Cyclohexenyl carbasugars are natural product-inspired sugar mimics which incorporate a carbon-carbon double bond in their structure, which functions to replace the endocyclic oxygen of natural sugars. This replacement has significant implications for how these sugar mimics are processed by glycoside hydrolases (GHs), a family of biocatalysts important for carbohydrate catabolism in nature. Particularly, most GHs evolved to catalyze the breakdown of carbohydrates by stabilizing a glycosylium ion-like transition state (TS), which involves the delocalization of a positive charge onto the oxygen atom in the ring of the carbohydrate residue. In cyclohexenyl carbasugars, an alkene takes the place of the endocyclic oxygen and this led to slower rates of enzymatic breakdown, which has been exploited in the development of slow substrates and covalent inhibitors for some GHs. In this thesis, we show that cyclohexenyl carbasugars are valuable tools for revealing mechanistic details of how GHs catalyze the breakdown of sugars. Specifically, with the aid of a Brønsted linear free energy relationship (LFER) study, these carbasugars revealed a previously undetermined and non-evident difference in intrinsic nucleophilicities between the enzymatic nucleophiles in two GH97 enzymes: H2O in the inverting -glucosidase and a carboxylate in the retaining -galactosidase. We also show, with the aid of a Bartlett-type LFER study, that the breakdown of these sugar mimics and their analogues proceeds through a TS that is analogous, with similar positive charge development, to that for natural sugars. Finally, we report a progressive quest towards prolonging the lifetime of the covalent intermediate formed by cyclohexenyl carbasugars by various structural modifications on the cyclohexene ring. These new covalent intermediates were made to form faster using chloride and fluoride leaving groups. Our new cyclohexenyl carbasugar analogs show significant improvement as reversible covalent inhibitors which lead to longer lasting, and more rapidly formed, covalent intermediates, so that these compounds target a variety of GHs, and are thus promising lead compounds for new applications, such as in pharmacological chaperone therapy.
275 pages.
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Thesis advisor: Bennet, Andrew
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