Tandem activated caged galactoside prodrugs: advancing beyond single galactosidase dependence.

β-Galactoside prodrugs, activated by β-galactosidase (β-gal) highly expressed in some cancer cells, have been explored as anticancer agents for three decades. However, the distribution of β-gal lacks sufficient specificity to ensure precise drug release at cancer sites. By utilizing the highly stringent substrate specificity of β-gal, we chose the naturally occurring hydroxyl group of galactose as a prodrug modification site and developed a new class of tandem activated caged galactoside (TACG) prodrugs that require an additional trigger for more controlled on-demand drug release. We demonstrated that attaching various masking groups to the 6-hydroxyl group of galactose renders the galactosides resistant to β-gal hydrolysis. Focusing on the photosensitive mask 4,5-dimethoxy-2-nitrobenzyl (DMNB), we synthesized 6-DMNB modified galactosides of combretastatin A4 and 8-hydroxyquinoline, showcasing their UV/β-gal-dependent anticancer activities. We further established synthetic routes for 2-, 3-, and 4-DMNB modified TACGs. Comparative intracellular studies highlighted the 2-DMNB modified TACG as the most effective positional isomer, offering superior light-dependent selectivity. This insight led to the discovery of the 2-DMNB modified galactoside of combretastatin A4 as a potent UV-dependent microtubule assembly inhibitor. Our work provides a straightforward, effective, and universally applicable strategy for constructing dual-stimulus responsive galactoside prodrugs, extendable to various glycoside prodrugs, advancing carbohydrate-based drug discovery.