A multifunctional near-infrared fluorescence probe with multi-reactive sites for real-time tracking Cys to SO₂ and GSH to SO₂ metabolism in vivo

Reactive sulfur species (RSS), including sulfur dioxide (SO2), glutathione (GSH), homocysteine (Hcy) and cysteine (Cys), and their associated metabolic processes, are essential for biological systems to maintain normal life activities. However, due to the similarity of their chemical properties and the diversity of the metabolic pathways, using a single fluorescence probe to differentiate RSS and monitor the Cys-SO2 and GSH-SO2 metabolic pathways in real time is rather difficult. Herein, we developed a multifunctional near-infrared (NIR) fluorescence probe NIR-COU with multiple response sites, in which aldehyde and ether groups served as the specific recognition sites for GSH and Cys/Hcy, and active C--C as the specific response site for SO2. Moreover, C--N+ with another new reactive site formed after the reaction of GSH with the probe, together with C--C was applied as the specific reactive site for SO2 for real-time monitoring of Cys to SO2 and GSH to SO2 metabolic processes. The structures of the NIR-COU and intermediates were characterized by NMR and HRMS. Based on the FRET mechanism, the NIR-COU exhibited different fluorescence signals under different excitations (385, 460 and 580 nm), not only for GSH (enhanced green and NIR emissions), Cys/Hcy, (enhanced blue and NIR emissions) and SO2 (enhanced green emissions only), but also ratiometric fluorescence responses to the metabolic pathways of GSH to SO2 and Cys to SO2. Moreover, NIR-COU could also be utilized for imaging GSH, Cys/Hcy, SO2 and visual tracking metabolic pathways from Cys to SO2 and GSH to SO2 in living cells as well as zebrafish.