Synthesis and Glycosidase Inhibition Studies of Novel Exoglycals Targeting GH3 Family Enzymes: Insights from Comparative Analysis with Macrolide Antibiotics

Glycosidases are key enzymes involved in carbohydrate metabolism. Members of the GH3 family have emerged as therapeutic targets due to their roles in natural product biosynthesis and disease. Transition-state analogues represent a powerful strategy for glycosidase inhibition, and exoglycals (C-glycosylidenes) have gained interest as conformational mimics of glycosidic bond hydrolysis. Herein, we report the synthesis of novel exoglycals bearing diverse substituents, including terminal alkynes and thymidyl residues, via a modified Julia-olefination strategy from sugar-derived lactones and substituted sulfones. Selected alkyne-containing exoglycals were further functionalized through cycloaddition with azidothymidine. Inhibition studies against EryBI, a GH3 glycosyl hydrolase, were performed. Kinetic analysis revealed diverse inhibitory mechanisms among the exoglycals, displaying competitive inhibition, with K_i values spanning micromolar to millimolar affinities. To contextualize the inhibitory potential of exoglycals, we evaluated three clinically relevant macrolide antibiotics-erythromycin, clarithromycin, and azithromycin. Intriguingly, these macrolides exhibited competitive or uncompetitive inhibition, contrasting with the consistent competitive behavior of exoglycals. This comparative analysis highlights the scaffold-dependent selectivity of GH3 inhibition. Our results demonstrate exoglycals as tunable scaffolds for glycosidase inhibition of GH3 glycosidases and provide mechanistic distinctions between carbohydrate mimics and macrolide antibiotics. These insights could guide the development of next-generation glycosidase inhibitors with improved specificity.