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UBC Theses and Dissertations

Fluoro-glycosyl acridinones as sensitive active site titrating agents for glycosidases Duo, Tianmeng


9H-(1,3-dichloro-9,9-dimethylacridin-2-one) (DDAO) is a fluorescent and chromophoric molecule with a relatively low pKa (5.3) and moderate water solubility. DDAO was evaluated as a leaving group for mechanism-based inhibitors and a suitable reporter molecule for active site titrations of glycosidases. The Koenigs-Knorr coupling of DDAO to a protected α-glucosyl bromide resulted in two connectivities of DDAO to the glycone – DDAOY β-D-glucopyranoside (DDAOY-Glc) with chlorines close to the glycone and DDAOR β-D-glucopyranoside (DDAOR-Glc) with the chlorines distal. Kinetic evaluation of DDAOY-Glc and DDAOR-Glc revealed that both compounds were excellent substrates for a model β-glucosidase from an Agrobacterium sp. (Abg), reacting at or near diffusion-controlled rates. Several novel fluorogenic DDAO 2-deoxy-2-fluoro-β-D-glycopyranosides were synthesized and kinetic parameters for inactivation of a variety of retaining glycosidases were determined. DDAOY 2-deoxy-2-fluoro-β-D-glucopyranoside (DDAOY-2FGlc) was found to be the most effective inactivator of Abg reported to date (ki/Ki = 10⁶ mM-¹ min-¹), also reacting at, or near to, diffusion controlled rates and approximately 10⁶-fold faster than the DDAOR analogue (DDAOR-2FGlc). Differences in reactivity are partially due to the different inherent reactivities of DDAOY and DDAOR aglycones since the rate constant for spontaneous hydrolysis of DDAOY-Glc was found to be 700 times greater than that for DDAOR-Glc. DDAOY 2-deoxy-2-fluoro-β-D-galactopyranosides, D-xylopyranosides and cellobiosides were also shown to be effective inactivators of a variety of cognate glycosidases, including Escherichia coli β-galactosidase (LacZ), Trichoderma reesei endo-glucanase I (EG I), Cellulomonas fimi endo-xylanase (Cex), and Bacillus halodurans β-xylosidase (β-Xyl). Finally, DDAOY-2FGlc was shown to be a valuable tool for determining the concentration of Abg by active site titration. Through fluorescence detection, enzyme concentrations down to 3 nM could be reliably measured. DDAOY fluorosugar inactivators and DDAOR glycosides may serve as excellent substrates and probes in metagenomic and directed evolution screens.

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