Design and synthesis of 2’-deoxy-2’-fluorodisaccharides as mechanism-based glycosidase inhibitors that exploit aglycon specificity

Stable, aglycon-specific inactivators of glycosidases have considerable potential as fools in the study of mechanisms of oligosaccharide processing, and possibly as avenues toward new therapeutics. Glycosidases for which the rate-determining step with the natural substrate is the hydrolysis of the glycosyl-enzyme intermediate are shown to be inactivated by the 2’-deoxy-2’-fluoro derivative of this substrate. Thus Agrobacterium faecalis beta-glucosidase is inactivated by 2’-deoxy-2’-fluorocellobiose according to inactivation parameters of k(i) = 0.018 min(-1) and K-i = 20 mM. Inactivation is shown to occur via the accumulation of the same 2-deoxy-2-fluoroglycosyl-enzyme intermediate as that formed using activated 2-deoxy-2-fluoroglycosides by identification of the labeled peptide in proteolytic digests. Thus, interactions between the enzyme and the sugar aglycon provide sufficient transition state stabilization to allow formation and trapping of the glycosyl-enzyme. beta-Glucocerebrosidase, a beta-glucosidase specific for hydrolysis of glucocerebrosides, is not inactivated by 2’-deoxy-2’-fluorocellobiose, thereby demonstrating the aglycon specificity of this class of inactivator.