Development of a Bioorthogonal Click-to-Release Reaction for Hydrogen Polysulfide (H₂S) Detection

In this study, we present an innovative "click-to-release" strategy for the design of highly specific H₂S bioorthogonal probes that undergo a specific click reaction with H₂S and release fluorophores by a following rearrangement. A library of cyclooctyne derivatives was established and successfully demonstrated the availability of the release strategy. Then, a model probe CM-CT was synthesized, which can achieve effective fluorophore release (>80%) in the presence of a H₂S donor. To further validate the application of this class of probes, a new probe QN-RHO-CT based on Rhodamine 110 was developed. This probe showed good water solubility (>160 μM) and fast release kinetics and can achieve selective H₂S detection in living cells. We used this probe to study the process of H₂S-mediated protein S-persulfidation and demonstrated that excess H₂S would directly react with protein persulfides to generate H₂S₂ and reduce the persulfides to thiols. Additionally, we elucidated the click-to-release mechanism in our design through a detailed mechanistic study, confirming the generation of the key intermediate α, β-unsaturated cyclooctanethione. This bioorthogonal click-to-release reaction provides a useful tool for investigating the function of H₂S and paves the way for biological studies on H₂S.