Intestinal Microbial Metabolic Activation Converts Soy Isoflavones into Potent Direct Antioxidants: First Total Synthesis and Mechanistic Characterization of 5-Hydroxydehydroequol

Soy isoflavones exhibit diverse health-promoting effects, including antioxidant and anti-inflammatory activities. However, these biological effects have largely been attributed to the modulation of cellular signaling pathways, whereas the influence of intestinal microbial metabolism on their direct radical-scavenging properties remains poorly understood. In this study, synthetic routes were established for the major intestinal bacterial metabolites daidzein and genistein, equol (Eq), dehydroequol (DEq), 5-hydroxyequol (5-OH-Eq), and, for the first time, 5-hydroxydehydroequol (5-OH-DEq), and their direct radical-scavenging activities, reaction mechanisms, radical-scavenging capacities, and DNA protective effects were systematically evaluated. Introduction of a C-ring double bond markedly enhanced direct radical-scavenging activity, whereas the parent isoflavones and their reduced metabolites exhibited little or no activity. Among the compounds examined, 5-OH-DEq exhibited the highest antioxidant activity, scavenging radicals predominantly through hydrogen atom transfer, with a second-order rate constant of 1.44 × 103 M−1 s−1, exceeding that of Trolox and a radical-scavenging capacity of approximately three radical equivalents per molecule. Moreover, 5-OH-DEq protected plasmid DNA against radiation-induced oxidative damage. These findings demonstrate that intestinal microbial metabolism can function as a form of metabolic activation, converting dietary isoflavones into potent direct antioxidants. 5-OH-DEq was identified as a previously unexplored antioxidant metabolite and a promising lead scaffold for further development of next-generation phenolic antioxidants.