A dual-emission fluorescent probe for discriminating cysteine from homocysteine and glutathione in living cells and zebrafish models.

Cellular biothiols function crucially and differently in physiological and pathological processes. However, it is still challenging to detect and discriminate thiols within a single one molecule, especially for cysteine (Cys) and homocysteine (Hcy). In this study, a simple two-emission turn-on fluorescent biothiol probe (ICN-NBD) was rationally designed and synthesized through a facile ether bond linking 7-nitro-1,2,3-benzoxadiazole (NBD) and phenanthroimidazole containing a cyano tail. The probe in the presence of Cys elicited two fluorescence responses at 470 nm and 550 nm under excitation at 365 nm and 480 nm, respectively, because of the concomitant generation of both the fluorophore and NBD-N-Cys. In contrast, addition of Hcy and glutathione (GSH) could result in only a blue fluorescence enhancement at 470 nm. which was reasonably attributed to rearrangement from NBD-S-Hcy/GSH to NBD-N-Hcy/GSH as a result of geometrical constraints or solvent effects. Therefore, the fluorescent probe with the NBD scaffold could detect biothiols and simultaneously discriminate Cys from Hcy/GSH in both blue and green channels. The probe has been successfully applied for visualizing biothiols in living cells and zebrafish.