An O-GlcNAcase-specific inhibitor and substrate engineered by the extension of the N-acetyl moiety

A novel analogue of PUGNAc, a potent O-GlcNAcase inhibitor, was synthesized and analyzed as an inhibitor of O-GlcNAcase, hexosaminidase A, and hexosaminidase B. While PUGNAc does not demonstrate selective inhibition of these related enzymes, the extension of the acetyl moiety to the longer butyl chain provided a compound with depressed inhibition of O-GlcNAcase and no observed inhibition of either hexosaminidase A or hexosaminidase B. Further, we applied this knowledge of substrate recognition at the N-acetyl group to our recently reported fluorogenic substrate for monitoring O-GlcNAcase activity. Gratifyingly, this altered small molecule was demonstrated to be a potent substrate for O-GlcNAcase while possessing no activity at hexosaminidase A. This reagent provides, for the first time, a means for monitoring O-GlcNAcase activity independent of the related enzymes hexosaminidase A and hexosaminidase B.