The Regioselectivity of Glutathione Adduct Formation with Flavonoid Quinone/Quinone Methides Is pH-Dependent

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Abstract
In the present study, the formation of glutathionyl adducts from a series of 3‘,4‘-dihydroxy flavonoid o-quinone/p-quinone methides was investigated with special emphasis on the regioselectivity of the glutathione addition as a function of pH. The flavonoid o-quinones were generated using horseradish peroxidase, and upon purification by HPLC, the glutathionyl adducts were identified by LC/MS as well as 1H and 13C NMR. The major pH effect observed for the glutathione conjugation of taxifolin and luteolin quinone is on the rate of taxifolin and luteolin conversion and, as a result, on the ratio of mono- to diglutathione adduct formation. With fisetin, 3,3‘,4‘-trihydroxyflavone, and quercetin, decreasing the pH results in a pathway in which glutathionyl adduct formation occurs in the C ring of the flavonoid, being initiated by hydration of the quinone and H2O adduct formation also in the C ring of the flavonoid. With increasing pH, for fisetin and 3,3‘,4‘-trihydroxyflavone glutathione adduct formation of the quinone occurs in the B ring at C2‘ as the preferential site. For quercetin, the adduct formation of its quinone/quinone methide shifts from the C ring at pH 3.5, to the A ring at pH 7.0, to the B ring at pH 9.5, indicating a significant influence of the pH and deprotonation state on the chemical electrophilic behavior of quercetin quinone/quinone methide. Together the results of the present study elucidate the mechanism of the pH-dependent electrophilic behavior of B ring catechol flavonoids, which appears more straightforward than previously foreseen.