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In this study, we present an innovative “click-to-release” strategy for the design of highly specific H2Snbioorthogonal probes that undergo a specific click reaction with H2Snand 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-CTwas synthesized, which can achieve effective fluorophore release (>80%) in the presence of a H2Sndonor. To further validate the application of this class of probes, a new probe QN-RHO-CTbased on Rhodamine 110 was developed. This probe showed good water solubility (>160 μM) and fast release kinetics and can achieve selective H2Sndetection 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 H2S2and 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 H2Snand paves the way for biological studies on H2Sn.

The knowledge of ligand–protein interactions is essential for understanding fundamental biological processes and for the rational design of drugs that target such processes. Carbene footprinting efficiently labels proteinaceous residues and has been used with mass spectrometry (MS) to map ligand–protein interactions. Nevertheless, previous footprinting studies are typically performed at the residue level, and therefore, the resolution may not be high enough to couple with conventional crystallography techniques. Herein we developed a subresidue footprinting strategy based on the discovery that carbene labeling produces subresidue peptide isomers and the intensity changes of these isomers in response to ligand binding can be exploited to delineate ligand–protein topography at the subresidue level. The established workflow combines carbene footprinting, extended liquid chromatographic separation, and ion mobility (IM)–MS for efficient separation and identification of subresidue isomers. Analysis of representative subresidue isomers located within the binding cleft of lysozyme and those produced from an amyloid-β segment have both uncovered structural information heretofore unavailable by residue-level footprinting. Lastly, a “real-world” application shows that the reactivity changes of subresidue isomers at Phe399 can identify the interactive nuances between estrogen-related receptor α, a potential drug target for cancer and metabolic diseases, with its three ligands. These findings have significant implications for drug design. Taken together, we envision the subresidue-level resolution enabled by IM–MS-coupled carbene footprinting can bridge the gap between structural MS and the more-established biophysical tools and ultimately facilitate diverse applications for fundamental research and pharmaceutical development.