An hMAO-B-activatable mitochondrial binding fluorescent probe in live-cell via enzyme-anchored and charge-driven dual...

Enzyme-responsive (ER) probes trageting human monoamine oxidase B (hMAO-B) represent pivotal tools for investigating a variety of diseases, including neurodegenerative pathologies, liver diseases, heart failure, metabolic disorders and cancers. Herein, we report THT-MTP-a thiochromone-based fluorescent probe exhibiting highly sensitivity and specificity for hMAO-B that undergoes hMAO-B-catalyzed oxidative conversion of its MTP moiety to MPy⁺, along with >100-fold fluorescence enhancement. Its activation mechanism was validated through safinamide-inhibition assays in both enzyme and cellular models. The hMAO-B-activatable imaging capability demonstrates intensity proportionality to enzyme levels across cell lines with differential MAO-B expression (HepG2 > SH-SY5Y ≫ NIH-3T3). THT-MTP exhibited excellent mitochondrial accumulation via enzyme-anchored and charge-driven dual targeting. This work establishes a design paradigm for hMAO-B-selectvie probes combining high-precision subcellular targeting, activatable imaging capability and wash-free operation, offering a robust molecular tool for investigating hMAO-B-associated diseases.