Investigation of the Relationship Between H₂O₂ and HClO in Living Cells by a Bifunctional, Dual-ratiometric Responsive Fluorescent Probe

Abstract

As two important reactive oxygen species, hydrogen peroxide (H₂O₂) and hypochlorous acid (HClO) play vital roles in many physiological and pathological processes. However, the relationship between these two species is seldom investigated, in part, because of the lack of robust molecular tools that can simultaneously visualize HClO and H₂O₂ in biosystems. In this work, we present a design strategy to construct a single fluorescent probe that can detect H₂O₂ and HClO by simultaneously monitoring two distinct detection channels. In the design, one phenothiazine-based coumarin serves as a chromophore and sensor for HClO, while a second coumarin precursor containing a boronate ester acts as a sensor for H₂O₂. After a head-to-head screening of three candidates differing in their coumarin precursor moieties, probe <b>CSU1</b> was found to have the optimal characteristics. As shown experimentally, it is able to detect them selectively and sensitively to generate distinct fluorescence signals and patterns in living cells. Furthermore, the endogenous generation of HClO from H₂O₂ and Cl^- catalyzed by myeloperoxidase enzyme in living cells can be clearly monitored by the probe. These studies demonstrate the potential of the probe as a powerful tool to investigate the interplay of HClO and H₂O₂ in oxidative stress.