Cell-trappable fluorescent probes for endogenous hydrogen sulfide signaling and imaging H2O2-dependent H2S production

Hydrogen sulfide (H2S) is a reactive small molecule generated in the body that can be beneficial or toxic owing to its potent redox activity. In living systems, disentangling the pathways responsible for H2S production and their physiological and pathological consequences remains a challenge in part due to a lack of methods for monitoring changes in endogenous H2S fluxes. The development of fluorescent probes with appropriate selectivity and sensitivity for monitoring production of H2S at biologically relevant signaling levels offers opportunities to explore its roles in a variety of systems. Here we report the design, synthesis, and application of a family of azide-based fluorescent H2S indicators, Sulfidefluor-4, Sulfidefluor-5 acetoxymethyl ester, and Sulfidefluor-7 acetoxymethyl ester, which offer the unique capability to image H2S generated at physiological signaling levels. These probes are optimized for cellular imaging and feature enhanced sensitivity and cellular retention compared with our previously reported molecules. In particular, Sulfidefluor-7 acetoxymethyl ester allows for direct, real-time visualization of endogenous H2S produced in live human umbilical vein endothelial cells upon stimulation with vascular endothelial growth factor (VEGF). Moreover, we show that H2S production is dependent on NADPH oxidase-derived hydrogen peroxide (H2O2), which attenuates VEGF receptor 2 phosphorylation and establishes a link for H2S/H2O2 crosstalk.