Development of a Bioorthogonal Click-to-Release Reaction for Hydrogen Polysulfide (H₂S_<i>n</i>) Detection

In this study, we present an innovative "click-to-release" strategy for the design of highly specific H₂S_<i>n</i> bioorthogonal probes that undergo a specific click reaction with H₂S_<i>n</i> 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 <b>CM-CT</b> was synthesized, which can achieve effective fluorophore release (>80%) in the presence of a H₂S_<i>n</i> donor. To further validate the application of this class of probes, a new probe <b>QN-RHO-CT</b> based on Rhodamine 110 was developed. This probe showed good water solubility (>160 μM) and fast release kinetics and can achieve selective H₂S_<i>n</i> 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_<i>n</i> and paves the way for biological studies on H₂S_<i>n</i>.