Real‐Time Detection of Reduced Nitroreductase with a Reversible Fluorescent Probe

Nitroreductase (NTR), a class of flavin-dependent redox enzymes, is a key biomarker for hypoxic tumors. Numerous fluorescent NTR probes have been developed to study hypoxia and associated tumors; however, they are reaction-based and provide only static information on the accumulated enzyme activity at a given time. Reversible binding probes are needed to monitor the enzyme level in real time. Here, the first reversible binding probe is presented that selectively detects the active, reduced form of NTR (red-NTR) with a fluorescence turn-on response. This probe, a benzocoumarin dye functionalized with a (nitrobenzyl)pyridinium moiety, is stabilized through hydrogen bonding between its nitro group and the reduced cofactor flavin mononucleotide (FMNH₂). This interaction suppresses both the enzymatic reduction and fluorescence quenching by photoinduced electron transfer. The probe selectively distinguishes red-NTR from its oxidized form (ox-NTR), allowing the observation of active enzyme levels in hypoxic cells, mouse tumor tissues, and cells undergoing premature senescence. The probe offers a unique and valuable tool for studying dynamic biological processes involving NTR under redox homeostasis.