Photocaging the C6-carboxylate of β-glucuronide prodrugs enables spatiotemporally controlled release of anticancer agents via a dual activation strategy

Traditional β-glucuronide prodrugs are limited by high hydrophilicity, which hinders cellular uptake, and by insufficient extracellular enzyme levels, which restrict effective drug release. To address these bottlenecks, we developed a dual-stimuli-responsive strategy by photocaging the C6-carboxylate of the glucuronic acid moiety with 2-hydroxymethyl-3-N,N-dimethylamino-naphthalene (DANE). This modification enhances lipophilicity for efficient passive cellular entry while preventing premature enzymatic hydrolysis. Subsequent intracellular UV irradiation regenerates the hydrophilic carboxyl group, effectively trapping the intermediate inside the cell for specific cleavage by lysosomal β-glucuronidase (β-GUS). We validated this design using 8-hydroxyquinoline (HQ) and gemcitabine (Gem). The resulting prodrugs exhibited excellent hydrolytic stability in the dark. Upon sequential UV/β-GUS activation, they exhibited potent cytotoxicity with high light-dependent selectivity indices, successfully triggering apoptosis, cell cycle arrest, and suppression of colony formation and migration. This work establishes C6-carboxylate caging as a robust strategy for the spatiotemporally controlled activation of β-glucuronide prodrugs.