Application of a Stopped-Flow EPR Method for the Detection of Short-Lived Flavonoid Semiquinone Radicals Produced by Oxidation Using ¹⁵N-Labeled Nitrosodisulfonate Radical (Fremy’s Salt)

A stopped-flow-electron paramagnetic resonance (EPR) method was applied for the detection of short-lived radicals of flavonoids bearing a catechol moiety as the B-ring, such as flavonols (quercetin, fisetin, and rutin), flavanones (eriodictyol and taxifolin), flavanols (catechin and epicatechin), and flavone (luteolin). ¹⁵N-labeled sodium salt of nitrosodisulfonate (¹⁵NDS) was employed to obtain the highly resolved EPR hyperfine structure (hfs) of flavonoid-derived semiquinone radicals under stoichiometrically regulated reaction conditions in aqueous media (pH 10). The EPR hfs of these flavonoids radicals, except catechin and epicatechin, were recorded. Based on the g value and the proton hyperfine coupling constants (hfcc), these flavonoid-derived radicals were assigned to be semiquinone radicals of the catechol moiety (B-ring). For example, the observed EPR hyperfine structure (hfs) of the luteolin radical (Lut^−·) was composed of four sets of doublet splitting, which could be ascribed to the three protons of the B-ring (a^2′ = 0.136, a^5′ = 0.102, and a^6′ = 0.272 mT) and a vinyl proton of the C-ring (a³ = 0.099 mT). In addition, the characteristically small doublet splitting resolved for the fisetin anion radical (Fis^−·, 0.028 mT) was assigned to the aromatic proton at the C₅ carbon of the A-ring, indicating that the unpaired electron of the radials was partially delocalized onto the A-ring through the π bonds involved in the vinyl-carbonyl moiety of the C-ring. The hfcc of the methine protons at the C₂ carbon of taxifolin and eriodictyol-derived radicals (Tax^−· and Eri^−·) was, respectively, evaluated to be 0.102 and 0.230 mT. The assignment of the proton hfcc of flavonoid-derived semiquinone radicals will be discussed in relation with the molecular structure of the C-ring.