| Summary: | Two new classes of specific, mechanism-based glycosidase inactivators were
developed: 2,2-dihalo glycosyl chlorides and 5-fluoro glycosyl fluorides. Both classes
were effective against x-glucosidases, which had been hitherto resistant to similar
inactivation strategies. Incubation of yeast -glucosidase with 2-chloro-2-deoxy-2-fluoroa-
D-glucopyranosyl chloride or 2-deoxy-2,2-difluoro-α-D-arabinohexopyranosyl chloride
resulted in time-dependent inactivation of the enzyme, presumably by formation of
extremely stabilized 2,2-dihalo glycosyl-enzyme intermediates that are essentially incapable
of turnover. Similar inhibition of Agrobacterium faecalis β-glucosidase and yeast α
glucosidase was seen with the corresponding 5-fluoro glycosyl fluorides. 5-Fluoro-β- and
5-fluoro-α-D-glucosyl fluorides form catalytically competent intermediates with the
appropriate glucosidases that are capable of turnover, but at rates reduced 10⁵ - and 10³ -
fold, respectively, with respect to the β- and α-D-glucosyl fluoride parent substrates. The
corresponding 5-fluoro-L-idosyl fluorides, the C5 epimers of the glucosyl compounds,
show even greater reductions in turnover rates, the kcat values of the 5-fluoro-α- and 5-
fluoro-β-L-idosyl fluorides with the appropriate enzymes being reduced a
further 1.5- and
3000-fold. The spontaneous hydrolysis rates of these 5-fluoro glycosyl fluorides, and
those of the corresponding 2-deoxy-2-fluoro compounds, were determined to probe the
effects of the various fluorine substitutions on the transition states for hydrolysis.
A novel mass spectrometric technique for the identification and sequencing of
labelled active site peptides without the need for radiolabels has been developed. Briefly,
the technique involves enzyme inactivation, proteolytic digestion of the enzyme, and
identification of the modified peptide using electrospray tandem mass spectrometry by
exploiting the lability of the inhibitor-peptide bond which is selectively cleaved by collisioninduced
fragmentation. The key catalytic nucleophiles in the clinically important human
lysosomal β-glucosidase (deficient in Gaucher disease), human acid β-galactosidase (deficient in GM1 gangliosidosis), and yeast α-glucosidase were identified, as Glu-340,
Glu-268, and Asp-214, respectively.
β-Glucosidase and β-mannosidase inhibitors, labelled with the positron-emitting
isotope ¹⁸F, were synthesized for use in a novel approach to the in vivo imaging of
glycosidase activity using positron emission tomography (PET). This may be useful in the
diagnosis and treatment of abnormal glycosidase activity associated with disease (e.g.
Gaucher disease, the inherited deficiency of lysosomal β-g1ucosidase). Initially,
Agrobacterium faecalis β-glucosidase was labelled in vitro with an ¹⁸F-labelled
mechanism-based enzyme inactivator 2-deoxy-2-[¹⁸F]fluoro--β-D-mannosyl[¹⁸F]fluoride,
the first such labelling of a glycosidase with this positron-emitting isotope, and reactivation
of the labelled enzyme was observed by monitoring release of radioactivity. Non-labelled
versions of these inhibitors were administered to rats. Rapid inactivation of the appropriate
enzymes was observed in each tissue assayed, and the clearance of the inhibitors was
demonstrated by their slow release from the enzymes both in vitro and in vivo. Uptake and
clearance of the inhibitors was probed using ¹⁹F NMR and ¹⁸F-radiolabelled compounds,
revealing little hydrolysis but non-specific uptake of the inhibitor. Preliminary imaging
results were obtained in rats using PET, demonstrating the potential of this approach for
imaging glycosidase activity in vivo.
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