| Summary: | Activated fluorosugars are covalent inactivators for a number of glycosidases, functioning through accumulation of stable glycosyl-enzyme intermediates. Two approaches were taken in designing new inactivators: more highly fluorinated sugars that could form more stable intermediates, and fluorosugars bearing novel aglycones that could be manipulated to improve selectivity and efficiency.
Six novel difluorosugar fluorides were synthesized and evaluated as covalent inactivators. Four of the compounds were tested as time-dependent inactivators of the β-glucosidase from Agrobacterium sp. (Abg) and, while they were shown to behave as reversible competitive inhibitors, the only time-dependent inactivation was traced to the presence of an extremely small amount (<0.1%) of a contaminating impurity. The other two compounds were evaluated as inactivators of human lysosomal α-iduronidase (Idua) the enzyme deficient in Mucopolysaccharidosis I. No time-dependent inactivation was observed, and only one functioned as a reversible competitive inhibitor for Idua.
Ten novel fluorosugars bearing dialkyl phosphate, phosphonate or phosphinate aglycones were also synthesized and evaluated as covalent inactivators for appropriate model retaining α- and β-glycosidases, but as expected only inactivated retaining β-glycosidases. Some of these compounds were also tested as inactivators of human glucocerebrosidase (GCase), the enzyme that is deficient in Gauchers disease. Activated fluorosugars bearing phosphorus-based aglycones were found to be efficient inactivators of GCase with the best compound, dioctyl (2-deoxy-2-fluoro-β-D-glucopyranosyl)phosphate, being ~4300-fold more efficient as an inactivator of GCase than the previously most efficient inactivator, 2-deoxy-2-fluoro-β-D-glucopyranosyl fluoride.
Two compounds, 2-deoxy-2-fluoro-β-D-glucopyranosyl fluoride and benzyl benzyl-(2-deoxy-2-fluoro-β-D-glucopyranosyl)phosphonate, were evaluated as potential pharmacological chaperones for GCase. Both compounds raised the melting temperature of the enzyme in vitro, and cell-based assays showed an increase of GCase levels in the presence of both compounds, although no increase in enzyme activity was observed.
Finally, a radiosynthetic route was developed starting from 2-deoxy-2-[¹⁸F]-fluoro-D-glucose to produce 2,4-dinitrophenyl 2-deoxy-2-[¹⁸F]-β-D-fluoroglucopyranoside, which behaved as an active-site directed inactivator of both Abg and GCase in vitro. The radiolabelled complex formed with GCase was purified, injected into mice, and imaged using positron emission tomography (PET). This represents a potentially useful technology for non-invasive studies on enzyme replacement therapy (ERT), which is used clinically to treat Gauchers disease.
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