Nucleophilic Ring-Opening of N-Benzoylaziridine Combined with Spirolactamization of Rhodol Enables Ratiometric Fluorescence Imaging of Hydropersulfides in Arabidopsis thaliana

Hydropersulfides (RSSH) are an important class of reactive sulfur species (RSS) involved in a variety of physiological processes in biological systems. However, selective detection of RSSH is challenging since the persulfide group (-SSH) exhibits reactivity akin to biologically abundant biothiols. To address this issue, we designed CRBA as a ratiometric fluorescent probe for RSSH by integrating the nucleophilic ring-opening of an N-benzoylaziridine and spirolactamization of a rhodol. The ratiometric sensing of RSSH is realized by modulating Förster resonance energy transfer (FRET) in the coumarin-rhodol dyad. Nucleophilic attack of the N-benzoylaziridine moiety in CRBA by RSSH affords the corresponding secondary amide ring-opened product, which subsequently undergoes a spontaneous intramolecular spirolactamization to disrupt the π-conjugation structure of the rhodol. The tandem reaction decreases the intramolecular FRET efficiency within the probe and causes a clear dual-emission signal change. Probe CRBA possesses outstanding selectivity toward RSSH over biothiols and H₂S, and is capable of tracking RSSH levels in Arabidopsis thaliana roots. Moreover, we observed the upregulation of RSSH levels in heavy metal (HM)-induced stress of A. thaliana with probe CRBA, and revealed the correlation between S-persulfidation and H₂S levels in living plants.