Michael addition-elimination-cyclization based turn-on fluorescence (MADELCY TOF) probes for cellular cysteine imaging and estimation of blood serum cysteine and aminoacylase-1

Oxidative stress is a hallmark of a range of diseases including cancer, HIV-AIDS and cardiovascular disorders. Blood plasma cysteine (Cys) is an established biomarker for oxidative stress, rendering the development of sensitive and technically straightforward approaches for its estimation extremely desirable. Herein, we achieve this goal by developing a series of Michael addition-elimination-cyclization based turn-on fluorescence (MADELCY TOF) probes that selectively detect Cys over other amino acids and biothiols such as homocysteine (Hcy) and glutathione (GSH). Cysteine detection by these compounds involves cascaded fluorophore release via the Michael addition of the cysteinyl thiol followed by the elimination of a leaving group and lactamization via the cysteinyl amine. Conveniently, the Cys-reactivity of these probes can be tuned via both inductive and mesomeric effects imparted by their beta ' position-substituents. One of our probes that contains the p-nitrophenyl group at its beta ' position demonstrated rapid kinetics (t(1/2) = 2.9 min) with excellent sensitivity (detection limit = 8.2 nM) towards Cys. This probe also enabled the sensitive (detection limit = 9.5 pM) estimation of the blood plasma levels of the cancer and liver cirrhosis biomarker, the aminoacylase-1 (ACY-1) enzyme. Furthermore, another probe of our library enabled live cell imaging of cellular Cys allowing us to develop an imaging-based assay to monitor hydrogen peroxide-induced oxidative stress in mammalian cells.

Automated summary

Oxidative stress is a characteristic feature of various diseases, and there is a need for sensitive and technically simple methods to measure blood plasma cysteine. In this study, researchers developed a series of fluorescence probes based on Michael addition-elimination-cyclization that can selectively detect cysteine and showed rapid kinetics and excellent sensitivity. One of the probes was able to detect a biomarker for cancer and liver cirrhosis, while another probe enabled live cell imaging to monitor oxidative stress in mammalian cells.