FRET-based mitochondria-targetable dual-excitation ratiometric fluorescent probe for monitoring hydrogen sulfide in living cells.

Hydrogen sulfide (H2S) is connected with various physiological and pathological functions. However, understanding the important functions of H2S remains challenging, in part because of the lack of tools for detecting endogenous H2S. Herein, compounds Ratio-H2S 1/2 are the first FRET-based mitochondrial-targetable dual-excitation ratiometric fluorescent probes for H2S on the basis of H2S-promoted thiolysis of dinitrophenyl ether. With the enhancement of H2S concentration, the excitation peak at λ≈402 nm of the phenolate form of the hydroxycoumarin unit drastically increases, whereas the excitation band centered at λ≈570 nm from rhodamine stays constant and can serve as a reference signal. Thus, the ratios of fluorescence intensities at λ=402 and 570 nm (I402/I570) exhibit a drastic change from 0.048 in the absence of H2S to 0.36 in the presence of 180 μM H2S; this is a 7.5-fold variation in the excitation ratios. The favorable properties of the probe include the donor and acceptor excitation bands, which exhibit large excitation separations (up to 168 nm separation) and comparable excitation intensities, high sensitivity and selectivity, and function well at physiological pH. In addition, it is demonstrated that the probe can localize in the mitochondria and determine H2S in living cells. It is expected that this strategy will lead to the development of a wide range of mitochondria-targetable dual-excitation ratiometric probes for other analytes with outstanding spectral features, including large separations between the excitation wavelengths and comparable excitation intensities.