A fluorescent probe for antioxidants

The n,pi* singlet-excited azoalkane 2,3-diazabicyclo[2.2.2]-oct-2-ene (DBO) is efficiently quenched by the natural antioxidants alpha-tocopherol (k(q)= 5.3 x 10(9) M-1 s(-1)), uric acid (3.4 x 10(9)), ascorbic acid (2.05 x 10(9)), and glutathione (0.82 x 10(9)). This trend in reactivity is the same as that observed for alternative probes for antioxidants, e.g., triplet-excited benzophenone and the trichloromethylperoxyl radical. Since the quenching of singlet-excited DBO dan be readily quantified by means of its long-lived (325 ns in aerated water) and strong fluorescence (lambda(max) ca. 425 nm), this azoalkane serves as the first fluorescent probe for the reactivity of antioxidants. The effects of pH and deuterium substitution and kinetic solvent effects were also examined. The data suggest the involvement of hydrogen atom transfer in the fluorescence quenching of DBO by antioxidants. The reaction efficiency for radical formation in the quenching;of singlet-excited DBO by cl-tocopherol was found to be one order of magnitude lower (only ca. 10%) than for triplet-excited benzophenone. This contrast is attributed to the variation in spin multiplicity, since reactions of singlet-excited states encounter additional deactivation channels. The potential of the fluorescent probe DBO in providing direct information on antioxidant activity in biological systems is evaluated.