Oxidative conjugation of chlorogenic acid with glutathione. Structural characterization of addition products and a new nitrite-Promoted pathway

Chlorogenic acid (1), a cancer chemopreventive agent widely found in fruits, tea and coffee, undergoes efficient conjugation with glutathione (GSH), in the presence of horseradish peroxidase/H(2)O(2) or tyrosinase at pH 7.4, to yield three main adducts that have been isolated and identified as 2-S-glutathionylchlorogenic acid (3), 2,5-di-S-glutathionylchlorogenic acid (4) and 2,5,6-tri-S-glutathionylchlorogenic acid (5) by extensive NMR analysis. The same pattern of products could be obtained by reaction of 1 with GSH in the presence of nitrite ions in acetate buffer at pH 4. Mechanistic experiments suggested that oxidative conjugation reactions proceed by sequential nucleophilic attack of GSH on ortho-quinone intermediates. Overall, these results provide the first complete spectral characterization of the adducts generated by biomimetic oxidation of 1 in the presence of GSH, and disclose a new possible nitrite-mediated conjugation pathway of 1 with GSH at acidic pH of physiological relevance.