The Chemical Basis of Thiol Addition to Nitro-conjugated LinoleicAcid a Protective Cell-signaling Lipid

摘要

Nitroalkene fatty acids are formed in vivo and exert protective and anti-inflammatory effects via reversible Michael addition to thiol-containing proteins in key signaling pathways. Nitro-conjugated linoleic acid (NO2-CLA) is preferentially formed, constitutes the most abundant nitrated fatty acid in humans, and contains two carbons that could potentially react with thiols, modulating signaling actions and levels. In this work, we examined the reactions of NO2-CLA with low molecular weight thiols (glutathione, cysteine, homocysteine, cysteinylglycine, and β-mercaptoethanol) and human serum albumin. Reactions followed reversible biphasic kinetics, consistent with the presence of two electrophilic centers in NO2-CLA located on the β- and δ-carbons with respect to the nitro group. The differential reactivity was confirmed by computational modeling of the electronic structure. The rates (kon and koff) and equilibrium constants for both reactions were determined for different thiols. LC-UV-Visible and LC-MS analyses showed that the fast reaction corresponds to β-adduct formation (the kinetic product), while the slow reaction corresponds to the formation of theδ-adduct (the thermodynamic product). The pH dependence of the rateconstants, the correlation between intrinsic reactivity and thiolpKa, and the absence of deuterium solventkinetic isotope effects suggested stepwise mechanisms with thiolate attack onNO2-CLA as rate-controlling step. Computational modelingsupported the mechanism and revealed additional features of the transitionstates, anionic intermediates, and final neutral products. Importantly, thedetection of cysteine-δ-adducts in human urine provided evidence for thebiological relevance of this reaction. Finally, human serum albumin was found tobind NO2-CLA both non-covalently and to form covalent adducts atCys-34, suggesting potential modes for systemic distribution. These resultsprovide new insights into the chemical basis of NO2-CLA signalingactions.