Chemiluminescent Detection of Enzymatically Produced Hydrogen Sulfide: Substrate Hydrogen Bonding Influences Selectivity for H₂S over Biological Thiols

Abstract

Hydrogen sulfide (H₂S) is now recognized as an important biological regulator and signaling agent that is active in many physiological processes and diseases. Understanding the important roles of this emerging signaling molecule has remained challenging, in part due to the limited methods available for detecting endogenous H₂S. Here we report two reaction-based ChemiLuminescent Sulfide Sensors, CLSS-1 and CLSS-2, with strong luminescence responses toward H₂S (128- and 48-fold, respectively) and H₂S detection limits (0.7 ± 0.3, 4.6 ± 2.0 μM, respectively) compatible with biological H₂S levels. CLSS-2 is highly selective for H₂S over other reactive sulfur, nitrogen, and oxygen species (RSONS) including GSH, Cys, Hcy, S₂O₃^2–, NO₂^–, HNO, ONOO^–, and NO. Despite its similar chemical structure, CLSS-1 displays lower selectivity toward amino acid-derived thiols than CLSS-2. The origin of this differential selectivity was investigated using both computational DFT studies and NMR experiments. Our results suggest a model in which amino acid binding to the hydrazide moiety of the luminol-derived probes provides differential access to the reactive azide in CLSS-1 and CLSS-2, thus eroding the selectivity of CLSS-1 for H₂S over Cys and GSH. On the basis of its high selectivity for H₂S, we used CLSS-2 to detect enzymatically produced H₂S from isolated cystathionine γ-lyase (CSE) enzymes (<i>p</i> < 0.001) and also from C6 cells expressing CSE (<i>p</i> < 0.001). CLSS-2 can readily differentiate between H₂S production in active CSE and CSE inhibited with β-cyanoalanine (BCA) in both isolated CSE enzymes (<i>p</i> < 0.005) and in C6 cells (<i>p</i> < 0.005). In addition to providing a highly sensitive and selective reaction-based tool for chemiluminescent H₂S detection and quantification, the insights into substrate–probe interactions controlling the selectivity for H₂S over biologically relevant thiols may guide the design of other selective H₂S detection scaffolds