The regioselectivity of glutathione adduct formation with flavonoid quinone/quinone methides is pH-dependent

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 H-1 and C-13 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.