Highly selective fluorescent probe for cysteine via a tandem reaction and its bioimaging application in HeLa cells

Cysteine, as a vital endogenous small molecule mercaptan, plays a crucial role in various physiological processes. The high sensitivity and selectivity of fluorescent probes provide a method to monitor cysteine, which is helpful to understand the mechanism of cysteine in physiological processes more comprehensively. However, the detection of cysteine can be interfered by other small molecule biothiols. Therefore, the design of fluorescent probe based on the structural characteristics and reactivity of cysteine has become research focus currently. Given the biological compatibilities, biological targets, the metabolic pathway of 3-hydoxythalidomide, and its unique fluorescent properties, herein, we have designed a chemodosimeter, 2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl acrylate, for the detection of cysteine based on a tandem reaction of thiol-ene click chemistry and aminolysis involving 3-hydroxythalidomide as a parent compound. Experimental data exhibited that the probe showed unique selectivity and sensitivity for cysteine over other amino acid and biothiols. In addition, the fluorescent intensity at 511 nm increased linearly as a function of cysteine concentration in the range of 0-6 × 10^-7 M (regression factor, R² = 0.999), with a limit of detection of 6.1 nM. The sensing mechanism was confirmed through ¹H NMR titration and density functional theory calculations. Additionally, the probe was also successfully utilized for the detection of cysteine in sewage and for bioimaging in HeLa cells.