Rational design of a dual-reactive probe for imaging the biogenesis of both H2S and GSH from l-Cys rather than d-Cys in live cells

Abstract

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 H₂S 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 H₂S and GSH in live cells. Importantly, probe 1 was successfully employed to reveal the biogenesis of both H₂S and GSH from l-Cys rather than from d-Cys, and therefore, d-Cys would be solely converted into H₂S, which may help understand the more H₂S generation from d-Cys than from l-Cys in live cells.

Scheme 1. The metabolism and biotransformation pathways of different biothiols.

Scheme 2. (a) Schematic illustration of the design for a dual-quenching probe 1 based on the combination of NBD amine and CBD arylether, which can only be activated by both H₂S and GSH. FRET, fluorescence resonance energy transfer; ICT, intramolecular charge transfer. (b) Synthetic routine and conditions for probe 1.

Fig. 1. Time-dependent fluorescence responses of 1 (1 μM) toward H₂S (100 μM) and/or GSH (2 mM) in PBS buffer. (a) 1 was treated with H₂S and GSH, or only with H₂S (b) or GSH (c). (d) Time-dependent emissions at 525 nm for 1 treated with H₂S and GSH (black), H₂S (green), GSH (red) or probe alone (blue). Excitation, 469 nm.

Fig. 2. (a) Fluorescence spectra and (b) emission at 525 nm of 1 (1 μM) toward different concentrations of H₂S in the presence of GSH (2 mM). (c) Fluorescence spectra and (d) emission at 525 nm of 1 (1 μM) toward different concentrations of GSH in the presence of H₂S (10 μM). Excitation, 469 nm.

Fig. 3. (a) Emissions at 525 nm of 1 (1 μM) toward various biologically relevant species (100 μM) in the presence of GSH (5 mM). (b) Emissions at 525 nm of 1 (1 μM) toward 5 mM GSH and other various biologically relevant species (100 μM) in the presence of H₂S (100 μM). Excitation, 469 nm.

Fig. 4. Confocal microscopy images of HeLa cells with using probe 1 in the presence of endogenous GSH and exogenous H₂S. (A–C) Cells were only incubated with 10 μM probe 1 for 60 min. (D and E) HeLa cells were pre-incubated with 100 μM Na₂S for 60 min, and then washed and incubated with 10 μM probe 1 for another 60 min. After the incubation, the cells were treated with DAPI (2 μg mL⁻¹) for 10 min. (A and D) The blue fluorescence of DAPI (450–500 nm) with 405 nm excitation. (B and E) The green fluorescence of probe 1 (500–550 nm) with 488 nm excitation. (C and F) The merged images of blue and green fluorescence. Scale bar, 50 μm.

Fig. 5. Confocal microscopy images of HeLa cells after co-incubation with probe 1 and d-Cys or l-Cys. (a) After pre-treated with NEM (1 mM) for 30 min, (A and B) cells co-stimulated with 10 μM probe 1 and 500 μM d-Cys for 60 min; (C and D) cells co-stimulated with 10 μM probe 1 and 500 μM l-Cys for 60 min; (E and F) cells pre-incubated with AOAA (200 μM) for 30 min, then co-stimulated with 10 μM probe 1 and 500 μM l-Cys for 60 min. Then, the cells were incubated with DAPI (2 μg ml⁻¹) for 10 min. The merged images of the blue fluorescence of DAPI and green fluorescence of probe 1 are also indicated. Scale bar, 50 μm. (b) Relative green fluorescence of images from (a), N = 3 fields of cells, error bars are means ± S.D. (c) Schematic representation of the chiral Cys metabolism in live cells.