Three-Component Glycosylation of Transient Hemiacetals Toward Tunable Aryl-Bisacetal Substrates

Quantitative live cell monitoring of catalytic activity is essential for advancing chemical biology, yet designing substrate probes that combine broad applicability with finely tunable kinetics remains a significant challenge. While glyco-bisacetal-based substrates (BABS) have proven applicable to several enzymes, their alkyl-hemiacetal core can limit turnover rates for certain enzymes. Herein, we report a novel one-pot, three-component glycosylation strategy to synthesize Aryl-BABS through the trapping of transient aryl-hemiacetals. This approach enables rapid diversification of the bisacetal scaffold using various phenols, yielding a library of aryl-bisacetal substrates. Kinetic evaluation of catalytic hydrolysis with a model glycosidase demonstrated that these Aryl-BABS are efficiently processed, with turnover rates up to 2 orders of magnitude faster than analogous alkyl glycosides and approaching those seen for activated <i>p</i>-nitrophenyl glycosides. Simple substitutions to phenol lead to a 20-fold range of kinetic tunability. Crucially, stopped-flow studies combined with kinetic simulations revealed that the breakdown of the enzymatically released aryl-hemiacetal is extremely rapid, at least 100-fold faster than that of alkyl-hemiacetals. This synthetic and kinetic tunability offers a powerful roadmap for developing advanced substrate probes of biocatalysts, eventually enabling quantitative measurement of previously intractable enzymes in living systems.