Substituent-Driven Electronic Modulation of Azo Bond: Enabling the Design of Ultrafast and Exceptionally Sensitive Fluorescent Probe for Hypoxia

Detecting tumor hypoxia is critical for cancer diagnosis and the evaluation of therapeutic efficacy. However, conventional fluorescent probes for hypoxia imaging often suffer from slow response kinetics and limited sensitivity. To overcome these challenges, we developed a novel strategy enabling rapid and highly sensitive hypoxia detection through substituent effect modulation. This approach involves tuning the electrophilicity of the azo bond (-N═N-) at the azoreductase (AzoR)-responsive site by introducing electron-donating and electron-withdrawing substituents. UV-vis spectral kinetic analyses combined with density functional theory calculations demonstrate that the electron-withdrawing p-nitrophenyl group (-pNO₂) enhances both the electrophilicity and bond dissociation energy (BDE) of the -N═N- moiety, thereby accelerating its enzymatic reduction by AzoR with high sensitivity. In contrast, the electron-donating p-methoxyphenyl group reduces electrophilicity and BDE, resulting in slower reduction kinetics and diminished sensitivity. Based on this insight, a series of fluorescent probes (DCM-Azo-Rs) was designed using a substituent regulation strategy. In this design, fluorescence is quenched in the "off" state due to the presence of the -N═N- bond, and restored upon AzoR-mediated cleavage of the bond. Comparative analysis of eight probe variants revealed that DCM-Azo-pNO₂, featuring the strong electron-withdrawing -pNO₂ group, exhibits superior performance in hypoxia detection, achieving the fastest response time (50 s) and the highest fluorescence enhancement (690-fold). Furthermore, cellular imaging in hypoxic HeLa cells and in vivo studies using mouse models of ischemia-induced hypoxia confirm that the sensitivity and response speed of the DCM-Azo-Rs probes are enhanced in biological systems as the electron-withdrawing strength of the substituent increases.