Rational design of a dual-reactive probe for imaging the biogenesis of both HS and GSH from l-Cys rather than d-Cys in live cells.

Biothiols and their interconversion are involved in cellular redox homeostasis as well as many physiological processes. Here, a dual-reactive dual-quenching fluorescent probe was rationally developed based on thiolysis reactions of 7-nitrobenzoxadiazole (NBD) tertiary amine and 7-cyanobenzoxadiazole (CBD) arylether for imaging of the biothiol interconversion. We demonstrate that the NBD-CBD probe exhibits very weak background fluorescence due to the dual-quenching effects, and can be dual-activated by HS and GSH with an over 500-fold fluorescence increase at 525 nm. In addition, the probe shows high sensitivity, excellent selectivity, and good biocompatibility, all of which promote the simultaneous detection of both HS and GSH in live cells. Importantly, probe 1 was successfully employed to reveal the biogenesis of both HS and GSH from l-Cys rather than from d-Cys, and therefore, d-Cys would be solely converted into HS, which may help understand the more HS generation from d-Cys than from l-Cys in live cells.