Quinone methide based organophosphate hydrolases inhibitors: Trans proximity labelers versus Cis labeling activity-based probes.

Quinone methide (QM) chemistry is widely applied including in enzyme inhibitors. Typically, enzyme-mediated bond-breaking releases a phenol product that rearranges into an electrophilic QM that in turn covalently modifies protein side-chains. However, the factors that govern the reactivity of QM-based inhibitors, and their mode of inhibition, have not been systematically explored. Foremost, enzyme inactivation may occur in cis , whereby one of the QM molecules inactivates the very same enzyme molecule that released it, or by trans , if the released QMs diffuse away and inactivate other enzyme molecules. We examined QM-based inhibitors for enzymes exhibiting phosphoester hydrolase activity. We tested different phenolic substituents and benzylic leaving groups, thereby modulating the rates of enzymatic hydrolysis, phenolate-to-QM rearrangement, and the electrophilicity of the resulting QM. By developing assays that distinguish between cis versus trans inhibition, we have identified certain combinations of leaving groups and phenyl substituents that lead to inhibition in the cis mode, while other combinations led to trans inhibition. Our results suggest that cis acting QM-based substrates may be used as activity-based probes for identifying various phospho- and phosphono-ester hydrolases, and potentially other hydrolases.