Construction of a mitochondria-targeted probe to monitor cysteine levels in cancer cells and zebrafish.

Cysteine (Cys) plays an indispensable role as an antioxidant in the maintenance of bioredox homeostasis. We have constructed an efficient fluorescent probe Mito-Cys based on the binding of indole and naphthol. The acrylic ester group serves as a recognition switch for specific detection of Cys, which undergoes Michael addition and intramolecular cyclization reactions, thereby ensuring the chemical kinetics priority of Cys compared to other biothiols. The probe has good water solubility, large Stokes shift (137 nm), with a detection limit of 21.81 nM. In addition, cell imaging experiments have shown that the probe has excellent mitochondrial targeting ability (R = 0.902). The probe can distinguish between Cys, homocysteine (Hcy) and glutathione (GSH), and can detect Cys specifically and quickly (100 s) to ensure accurate quantitative analysis of Cys changes in cells. More importantly, the probe confirms that ferroptosis inducing factors trigger thiol starvation in mitochondria, which helps to gain a deeper understanding of the physiological and pathological functions related to Cys and ferroptosis.