Oxidative Transformation of Leucocyanidin by Anthocyanidin Synthase from Vitis vinifera Leads Only to Quercetin

Anthocyanidin synthase from Vitis vinifera ( VvANS) catalyzes the in vitro transformation of the natural isomer of leucocyanidin, 2 R,3 S,4 S- cis-leucocyanidin, into 2 R,4 S-flavan-3,3,4-triol ([M + H]⁺, m/ z 323) and quercetin. The C₃-hydroxylation product 2 R,4 S-flavan-3,3,4-triol is first produced and its C₃,C₄-dehydration product is in tautomeric equilibrium with (+)-dihydroquercetin. The latter undergoes a second VvANS-catalyzed C₃-hydroxylation leading to a 4-keto-2 R-flavan-3,3-gem-diol which upon dehydration gives quercetin. The unnatural isomer of leucocyanidin, 2 R,3 S,4 R- trans-leucocyanidin, is similarly transformed into quercetin upon C₃,C₄-dehydration, but unlike 3,4- cis-leucocyanidin, it also undergoes some C₂,C₃-dehydration followed by an acid-catalyzed hydroxyl group extrusion at C₄ to give traces of cyanidin. Overall, the C₃,C₄- trans isomer of leucocyanidin is transformed into 2 R,4 R-flavan-3,3,4-triol (M + 1, m/ z 323), (+)-DHQ, (-)-epiDHQ, quercetin, and traces of cyanidin. Our data bring the first direct observation of 3,4- cis-leucocyanidin- and 3,4- trans-leucocyanidin-derived 3,3-gem-diols, supporting the idea that the generic function of ANS is to catalyze the C₃-hydroxylation of its substrates. No cyanidin is produced with the natural cis isomer of leucocyanidin, and only traces with the unnatural trans isomer, which suggests that anthocyanidin synthase requires other substrate(s) for the in vivo formation of anthocyanidins.