Bioresponsive pseudoGlucosinolates (psGSLs) Release Isothiocyanates (ITCs) in the Presence of Nitroreductases

Glucosinolates (GSLs) are plant secondary metabolites that release bioactive isothiocyanates (ITCs) upon myrosinase-mediated activation. While ITCs display diverse antimicrobial and chemoprotective activities, their application is limited by dependence on myrosinase and intrinsic hydrolytic instability. Here, we introduce pseudoglucosinolates (psGSLs), a synthetic platform that mimics the natural GSL activation mechanism but replaces the thioglucosidic trigger with an enzyme-responsive para-aminobenzylthiol motif. Using nitroreductase (NfsB) as a noncanonical activating enzyme, we synthesized and characterized a series of nitro-masked psGSLs, including azide-, alkyne-, and fluorophore-functionalized derivatives. Enzymatic reduction induces a self-immolative 1,6-elimination and subsequent thio-Lossen rearrangement, releasing ITCs under physiological conditions. The liberated ITCs covalently modify peptides and proteins, showing predominant lysine reactivity in chemoproteomic analyses of the Staphylococcus aureus proteome, including functional sites of essential proteins. Fluorescent probes enabled visualization of enzyme-dependent protein labeling and demonstrated nitroreductase-triggered ITC release in vivo in Caenorhabditis elegans. Together, psGSLs establish a modular, bioresponsive prodrug and chemical biology platform for enzyme-controlled ITC delivery, expanding the scope of ITC-based covalent modification beyond natural myrosinase-dependent systems.