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UBC Theses and Dissertations

Mechanism-based inhibitors of lysosomal glycosidases Wong, Alexander William


The lysosomal storage disorders are genetic diseases arising from a deficiency of a lysosomal protein that is part of a catabolic pathway. Some of these enzymes are exoglycosidases that act in sequence on glycolipids and glycosaminoglycans, i.e. substrates are degraded by a stepwise removal of terminal sugar units. Thus, the deficiency of a single lysosomal enzyme causes the blockage of the entire pathway, leading to the accumulation of that substrate. Because of their clinical significance, interest in the lysosomal glycosidases is widespread. The uronidases are a specific subset of lysosomal glycosidases. According to their sequence-based glycosidase family assignments, they are predicted to hydrolyze their substrates via a double displacement mechanism. In order to perform mechanistic studies upon these glycosidases, two classes of fluoro-sugar inactivators were developed - the 2- deoxy-2-fluoro- and the 5-fluoro-glycopyranosyluronic acids. These compounds function via the trapping of a covalent glycosyl-enzyme intermediate. Five new compounds - 2-deoxy-2-fluoro-P-D-glucopyranosyluronic acid fluoride (2FGlcAF), 2- deoxy-2-fluoro-P-D-mannopyranosyluronic acid fluoride (2FManAF), 2-deoxy-2-fluoroa- L-idopyranosyluronic acid fluoride (2FIdoAF), 5-fluoro-P-D-glucopyranosyluronic acid fluoride (5FGlcAF) and 5-fluoro-a-L-idopyranosyluronic acid fluoride (5FIdoAF) were synthesized and evaluated with the appropriate enzymes. The syntheses of both 2FGlcAF and 2FManAF were accomplished using a TEMPOmediated oxidation of the primary C-6 alcohol group of the parent glycopyranosides while 2FIdoAF, 5FGlcAF and 5FIdoAF were synthesized from the parent glycopyranosyluronic acid fluorides via radical bromination of the C-5 carbon and displacement of the installed bromine with either tributyltin hydride (2FIdoAF) or silver tetrafluoroborate (5FGlcAF and 5FIdoAF). All five inactivators were evaluated using E. coli (^-glucuronidase (EBG) as a model system. Further, labelling and identification of the catalytic nucleophile of EBG as Glu504 within the sequence ₅₀₂ITEYGV₂₀₇ was accomplished by LC/MS analysis of proteolytic digests using 5FGlcAF. Both 2FGlcAF and 2FManAF were tested as inactivators of human P-glucuronidase, with its catalytic nucleophile being identified as Glu540 within the sequence 539SEYGAET545 through the use of 2FGlcAF. Finally, both 5FIdoAF and 2FIdoAF were tested on human a-Liduronidase, with its catalytic nucleophile being identified through the use of 5FIdoAF and 2FIdoAF. Gaucher disease is a lysosomal storage disorder caused by a deficiency in glucocerebrosidase (GCase). Current treatment of the Type I version for this disease involves enzyme replacement therapy, whereby the patient is administered recombinant enzyme intravenously. A non-invasive technique of assessing in vivo localization of the enzyme would be beneficial in patient care. To this end, ¹⁸F-labelled 2,6-dideoxy-2,6- difluoro-p-D-glucopyranosyl fluoride was developed as a potential positron emission tomographic imaging agent. Starting with 3,4-di-0-acetyl-6-0-trifluoromethanesulfonyl- 2-deoxy-2-fluoro-β-D-glucopyranosyl fluoride, the triflate group was displaced with ¹⁸F- fluoride, and the acetyl groups were removed under Zemplen conditions to give the radiolabeled inactivator in 8% radiochemical yield (corrected for decay). Attempts at radiolabelling GCase in vitro have so far failed. Explanations for this result are offered, with suggestions as to what may be done.

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