Regulating Sensing Patterns in Fluorescent Probes for Discriminative Detection of Biothiols

Discerning and quantifying the critical biothiols cysteine (Cys), homocysteine (Hcy), and glutathione (GSH) are vital for understanding their synergistic roles in biological systems. In this study, we synthesized a series of phenylethynylcoumarin fluorescent probes with varied structures to investigate the mechanisms underlying biothiol detection. We found that different substituents (−OCH3, −H, −CN) at the para-position of the phenylacetylene, combined with an aldehyde group at the 3-position of the coumarin, significantly affected the probes' reactivity and produced distinct response patterns toward biothiols. These insights enable the strategic development of fluorescent probes tailored to provide the personalized and discriminative detection of these biothiols. Additionally, probes CPOMe and CPCN were specifically evaluated for their efficacy in physiological environments, demonstrating their ability to accurately distinguish between Cys, Hcy, and GSH in living cells through unique fluorescent signals.