Novel agents for the in-situ detection of cysteine oxidation states

Cysteine (Cys-SH) is an amino acid, which due to the many stable oxidation states of the sulfur containing thiol moiety, readily undergoes oxidation by reactive oxygen species (ROS) to form sulfenic (Cys-OH), sulfinic (Cys-SO2H) and sulfonic (Cys-SO3H) acids. Thiol modifications of cysteine have been implicated as modulators of cellular processes and represent significant biological modifications that occur during oxidative stress and cell signaling. However, the different oxidation states are difficult to monitor in a physiological setting due to the limited availability of experimental tools. Therefore it is of interest to synthesize a range of chemical probes that selectively recognize specific oxidation states of sulfur and also are cell permeable to allow observation of their formation in real time. The current study is aimed at investigating a synthetic approach for novel fluorescent probe synthesis, for the specific detection of cysteine sulfenic acids. The probe will consist of a dimedone like molecule, 3, 5-diketohexahydrobenzoic which is uniquely reactive with Cys-SOH, a linker and a fluorescent tag which will enable detection by fluorescence spectroscopy. To achieve this, 2-aminoanthracene, a fluorescent molecule has been coupled with chloroacetyl chloride which acts as a bifunctional linker to form N-(anthracen-2-yl)-2-chloroacetamide. This compound has been used to synthesise 2-amino-N-(anthracen-2-yl)acetamide via an amide synthesis. Successful synthesis of these compounds has been confirmed by nuclear magnetic resonance and mass spectrometric analysis. Present work involves the attachment of 3,5-diketohexahydrobenzoic acid synthesise to 2-amino-N-(anthracen-2-yl)acetamide. We will use this for investigation of cysteine oxidation in T cells under oxidative stress. [1-3] Phillips D.C, Dias H.K, Kitas G.D, Griffiths H.R.(2010) Antioxid Redox Signal,12(6):743-85 Grant M.M,Griffiths H.R. (2007) Environmental Pharmacology and Toxicology 23, 335-339 Phillips D.C., Woollard K.J. and Griffiths H.R. (2003) Brit. J. Pharmacol. 138, 501-511.