A ratiometric and near-infrared fluorescent probe for discrimination of Cys/Hcy/GSH in aqueous solution and its intracellular imaging in living cells

Abstract

In this work, a unique ratiometric and NIR fluorescent probe, Hecy-NBD, was designed and synthesized to detect cysteine/homocysteine/glutathione in aqueous solution. Hemicyanine skeleton was chosen as the major fluorophore and nitro benzoxadiazole (NBD) chromophore was selected as the recognition unit to construct a ratiometric fluorescent probe. The fluorescence of probe Hecy-NBD was very weak due to the introduction of quenching group NBD. Upon the addition of 100 μM GSH to 10 μM Hecy-NBD, the fluorescence intensity at 702 nm was very strong with the releasing of compound Hecy-OH. While upon the addition of 100 μM Cys or 100 μM Hcy, probe exhibited distinct fluorescence enhancement at 540 nm and 702 nm, which resulted from the formation of compound NBD-Cys and NBD-Hcy through the 5/6-membered cyclic transition and the fluorescence releasing of compound Hecy-OH. The proposed mechanism based on SNAr reaction and molecular rearrangement, which were confirmed by HRMS spectra. The detection limit of these three biothiols was also calculated (GSH, 50 nM; Cys, 81.0 nM; Hcy, 130 nM). Moreover, probe Hecy-NBD could function well at wide pH conditions from 4 to 10. Besides that, probe Hecy-NBD with low cell toxicity had been successfully applied to dual-channel fluorescence imaging in living cells. Considering the good characteristics, Hecy-NBD had great potential to detect biothiols-associated diseases in the future.