Fluorinated Sugars as Probes of Glycosidase Mechanisms

Fluorinated sugars have proved to be valuable mechanistic probes of glycosidases in several ways. This is illustrated for Agrobacterium β-glucosidase, a retaining glycosidase which hydrolyses glycosidic bonds with net retention of anomeric configuration via a double displacement mechanism involving a glycosyl-enzyme intermediate. Both ground state and transition state interactions with the individual hydroxyl groups have been probed by use of a series of deoxy- and deoxyfluoroglycosides revealing the importance of interactions at the 2-position. Two new approaches to the trapping of the glycosyl-enzyme intermediate are now described. Incorporation of two fluorines at C-2 slows both the glycosylation and deglycosylation steps enormously, and allows the incorporation of an exceptionally good leaving group at the anomeric centre without undue lability. Thus the 2,4,6-trinitrophenyl α-D-2-deoxy-2,2-difluoro arabino and malto glycosides are time-dependent inactivators of yeast α-glucosidase and human pancreatic α-amylase, respectively. Alternatively, installation of a single fluorine at C-5 along with a fluoride leaving group at the anomeric centre provides a novel class of mechanism-based inactivators of both α- and β-glycosidases, and has allowed identification of the active site nucleophile in yeast α-glucosidase.