Development of chemical tools for studying human O-GlcNAcase activity

In recent years, the post-translational modification of nuclear and cytoplasmic proteins with O-linked N-acetylglucosamine (O-GlcNAc) has emerged as playing diverse roles in health and disease. Interestingly, this modification is regulated by only two enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). A method to study the effects of elevated levels of O-GlcNAc is to selectively target OGA. Herein, we describe the structure-activity relationships of a family of 2'-aminothiazoline-based inhibitors, one of which shows to be among the most potent inhibitors of human OGA (hOGA) known to date. We present the selectivity ratios of these compounds for hOGA over the structurally-related lysosomal β-hexosaminidases, define them as transition state analogues and rationalize their potencies by using linear free energy analyses. We also identify two fluorescence quenched substrates for hOGA bearing thioamide quenchers having different fluorogenic leaving groups, which reveal design features for substrates to monitor hOGA activity in live cells.