Ratiometric Measurement of Hydrogen Sulfide and Cysteine/Homocysteine Ratios Using a Dual-Fluorophore Fragmentation Strategy

[Image: see text] Hydrogen sulfide (H(2)S) is an integral signaling molecule in biology with complex generation, translocation, and metabolism processes that are intertwined with cellular thiols. Differentiating the complex interplay between H(2)S and biological thiols, however, remains challenging due to the difficulty of monitoring H(2)S and thiol levels simultaneously in complex redox environments. As a step toward unraveling the complexities of H(2)S and thiols in sulfur redox homeostasis, we present a dual-fluorophore fragmentation strategy that allows for the ratiometric determination of relative H(2)S and cysteine (Cys) or homocysteine (Hcy) concentrations, two important metabolites in H(2)S biosynthesis. The key design principle is based on a nitrobenzofurazan-coumarin (NBD-Coum) construct, which fragments into spectroscopically differentiable products upon nucleophilic aromatic substitution with either H(2)S or Cys/Hcy. Measurement of the ratio of fluorescence intensities from coumarin and the NBD-Cys or NBD-Hcy adducts generates a sigmoidal response with a dynamic range of 3 orders of magnitude. The developed scaffold displays a rapid response (<1 min) and is selective for sulfhydryl-containing nucleophiles over other reactive sulfur, oxygen, and nitrogen species, including alcohol- and amine-functionalized amino acids, polyatomic anionic sulfur species, NO, and HNO. Additionally, NBD-Coum is demonstrated to differentiate and report on different oxidative stress stimuli in simulated sulfur pools containing H(2)S, Cys, and cystine.