Hydrogen sulfide (H₂S), a gaseous species produced by both bacteria and higher eukaryotic organisms, including mammalian vertebrates, has attracted attention in recent years for its contributions to human health and disease. H₂S has been proposed as a cytoprotectant and gasotransmitter in many tissue types, including mediating vascular tone in blood vessels as well as neuromodulation in the brain. The molecular mechanisms dictating how H₂S affects cellular signaling and other physiological events remain insufficiently understood. Furthermore, the involvement of H₂S in metal-binding interactions and formation of related RSS such as sulfane sulfur may contribute to other distinct signaling pathways. Owing to its widespread biological roles and unique chemical properties, H₂S is an appealing target for chemical biology approaches to elucidate its production, trafficking, and downstream function. In this context, reaction-based fluorescent probes offer a versatile set of screening tools to visualize H₂S pools in living systems. Three main strategies used in molecular probe development for H₂S detection include azide and nitro group reduction, nucleophilic attack, and CuS precipitation. Each of these approaches exploits the strong nucleophilicity and reducing potency of H₂S to achieve selectivity over other biothiols. In addition, a variety of methods have been developed for the detection of other reactive sulfur species (RSS), including sulfite and bisulfite, as well as sulfane sulfur species and related modifications such as S-nitrosothiols. Access to this growing chemical toolbox of new molecular probes for H₂S and related RSS sets the stage for applying these developing technologies to probe reactive sulfur biology in living systems.

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.