Simultaneous Quantitation of Persulfides, Biothiols, and Hydrogen Sulfide through Sulfur Exchange Reaction with Trityl Spin Probes

Reactive sulfur species (RSS) including persulfides (RSSHs), biothiols, and hydrogen sulfide (H₂S) are key regulators in various physiological processes. To better understand the symbiotic relationship and interconversion of these RSS, it is highly desirable but challenging to develop analytical techniques that are capable of detecting and quantifying them. Herein, we report the rational design and synthesis of novel trityl-radical-based electron paramagnetic resonance (EPR) probes dubbed CT02-TNB and OX-TNB. CT02-TNB underwent fast sulfur exchange reactions with two reactive RSSHs (PS1 and PS2) which were released from their corresponding donors PSD1 and PSD2 to afford the specific conjugates. The resulting conjugates exhibit characteristic EPR spectra, thus enabling discriminative detection and quantitation of the two RSSHs. Moreover, CT02-TNB showed good response toward other RSS including glutathione (GSH), cysteine (Cys), H₂S, and sulfite as well. Importantly, based on the updated EPR spectral simulation program, simultaneous quantitation of multiple RSS (e.g., PS1/GSH/Cys or PS1/GSH/H₂S) by CT02-TNB was also achieved. Finally, the levels of released PS1 from PSD1 and endogenous GSH in isolated mouse livers were measured by the hydrophilic OX-TNB. This work represents the first study achieving discriminative and quantitative detection of different persulfides and other RSS by a spectroscopic method.