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UBC Theses and Dissertations

Replacement of oxocarbenium ions by allylic cations in both enzymatic and spontaneous reactions of glycosides Danby, Phillip M.


Oxocarbenium ions play a pivotal role in the chemistry and biochemistry of glycosides. In this thesis I prepared carbocyclic glycoside analogs that can generate allylic cations in the place of oxocarbenium ions. In these unsaturated cyclitols the C5-O5 bond of a pyranoside is replaced by a C-C double bond. Phenolic leaving groups were introduced at the pseudo anomeric carbon to form allyl ether linkages analogous to glycosidic linkages. Spontaneous heterolysis of these aryl carbasugars was shown, by measurement of secondary kinetic isotope effects, to proceed via an allylic cation. Not only was this mechanism of cleavage similar to that seen for glycosides but the rate constants for solvolysis of aryl glycosides and unsaturated aryl carbasugars were found to be nearly identical. To explore if these close similarities in structure and inherent reactivities would allow glycoside hydrolases to act on non-glycosidic linkages I screened a library of over 150 glycoside hydrolases with a fluorogenic allyl-carbasugar substrate. This screen identified several beta-retaining glycosidases capable of unsaturated cyclitol ether hydrolysis. Detailed analysis of a model enzyme demonstrated that the same mechanism was operative for cleavage of unsaturated cyclitol ethers as glycosidic linkages. Once again, kinetic isotope effects implicated an allylic cation at the rate-limiting transition state for the enzymatic reaction. The observed kinetic parameters for aryl carbasugar cleavage by glycosidases served as a reminder that these enzymes have evolved to act upon glycosides, and not carbocyclic imitators. Turnover constants were 100 – 1000-fold lower for aryl carbasugar hydrolysis compared to hydrolysis of aryl glycosides. Directed evolution has begun to bridge this gap in activity and yield glycosidases better capable of cleaving aryl unsaturated cyclitol linkages. The mechanistic insights gained by these studies have proven useful in preparing novel glycosidase inactivators. Specifically, unsaturated pseudosugars, with the vinyl hydrogen replaced by electron withdrawing halogens, react with retaining glycosidases to accumulate the covalent species and accomplish enzyme inactivation. Taken together these studies highlight the parallels between allylic cations and oxocarbenium ions. Not only do allylic carbasugars have similar inherent reactivities to glycosides but glycosidases are capable of allylic cation stabilization at their transition states.

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