Cell-trappable fluorescent probes for endogenous hydrogen sulfide signaling and imaging H ₂ O ₂ -dependent H ₂ S production

Hydrogen sulfide (H 2 S) 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 H 2 S production and their physiological and pathological consequences remains a challenge in part due to a lack of methods for monitoring changes in endogenous H 2 S fluxes. The development of fluorescent probes with appropriate selectivity and sensitivity for monitoring production of H 2 S 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 H 2 S indicators, Sulfidefluor-4, Sulfidefluor-5 acetoxymethyl ester, and Sulfidefluor-7 acetoxymethyl ester, which offer the unique capability to image H 2 S 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 H 2 S produced in live human umbilical vein endothelial cells upon stimulation with vascular endothelial growth factor (VEGF). Moreover, we show that H 2 S production is dependent on NADPH oxidase–derived hydrogen peroxide (H 2 O 2 ), which attenuates VEGF receptor 2 phosphorylation and establishes a link for H 2 S/H 2 O 2 crosstalk.