Development of large Stokes shift, near-infrared fluorescence probe for rapid and bioorthogonal imaging of nitroxyl (HNO) in living cells.

Nitroxyl (HNO), as an electron reduced and protonated form of nitric oxide, is emerging as a potential diagnostic and therapeutic biomarker. It is still of great interest to develop probes of desirable properties to study its biological functions. Here we develop a near infrared fluorescence probe for detecting and visualizing exogenous and endogenous HNO in living cells. The probe is designed by coupling a HNO-responsive moiety, diphenylphosphinobenzoyl group, with a near infrared fluorophore with large of Stokes shift via an ester linker. The probe was initially nonfluorescent. HNO-catalyzed oxidation reaction generates an aza-ylide, which intramolecularly attacks the carbonyl carbon, liberating the initial fluorophore with activated fluorescence signals. The probe is proportional to the concentrations of HNO in the range of 2.0-80 μM with a limit of detection of 0.05 μM. Furthermore, the probe also exhibits high selectivity and fast response (reaching plateau within 600 s) towards HNO in vitro. Moreover, imaging studies reveal that the probe is capable of detecting exogenous HNO with dose-dependent fluorescence signals. Its ability to image endogenous HNO without or with induction is also demonstrated in living cells. This turn-on fluorescence probe provides a useful tool for studying HNO in living cells.