Rational Design of a Dual-Channel Fluorescent Probe for the Simultaneous Imaging of Hypochlorous Acid and Peroxynitrite in Living Organisms

Hypochlorous acid (HOCl) and peroxynitrite (ONOO^-) are two important highly reactive oxygen/nitrogen species, which commonly coexist in biosystems and play pivotal roles in many physiological and pathological processes. To investigate their function and correlations, it is urgently needed to construct chemical tools that can track the production of HOCl and ONOO^- in biological systems with distinct fluorescence signals. Here, we found that the coumarin fluorescence of coumarin-benzopyrylium (CB) hydrazides (spirocyclic form) is dim, and their fluorescence properties are controlled by their benzopyran moiety via an intramolecular photo-induced electron transfer (PET) process. Based on this mechanism, we report the development of a fluorescent probe CB2-H for the simultaneous detection of HOCl and ONOO^-. ONOO^- can selectively oxidize the hydrazide group of CB2-H to afford the parent dye CB2 (Abs_max/Em_max = 631/669 nm). In the case of HOCl, it undergoes an electrophilic attack on the benzopyran moiety of CB2-H to give a chlorinated product CB2-H-Cl, which inhibits the PET process within the probe and thus affords a turn-on fluorescence response at the coumarin channel (Abs_max/Em_max = 407/468 nm). Due to the marked differences in absorption/emission wavelengths between the HOCl and ONOO^- products, CB2-H enables the concurrent detection of HOCl and ONOO^- at two independent channels without spectral cross-interference. CB2-H has been applied for dual-channel fluorescence imaging of endogenously produced HOCl and ONOO^- in living cells and zebrafish under different stimulants. The present probe provides a useful tool for further exploring the distribution and correlation of HOCl and ONOO^- in more biosystems.