Vitamin K analogue as a new fluorescence probe for quantitative antioxidant assay

A synthesized vitamin K model compound, NQ-6 (2-hexyloxy-1,4-naphthoquinone) showed weak fluorescence around 440 nm in ethanol. Addition of antioxidants such as vitamin E to a NQ-6 solution suppressed the NQ-6 emission quantitatively. A kinetic study on the quenching of the NQ-6 emission by hydrogen-donor type antioxidants (three tocopherol analogues, catechin, and 2,6-di-tert-butyl-4-methylphenol) in ethanol was performed. The quenching rate constant obtained from the Stern–Volmer plots for the steady-state fluorescence intensity was consistent with the second-order rate constant of each antioxidant for the free-radical scavenging. The NQ-6 emission is thought to be the delayed fluorescence caused by the thermal population to the excited singlet state from the triplet state. Thus, the fluorescence quenching occurred through a hydrogen atom transfer reaction from an antioxidant to NQ-6 in the excited triplet state (3NQ-6*). The second-order rate constant for the reaction between 3NQ-6* and α-tocopherol was estimated to be 1.2 × 1010 M−1 s−1 from the quenching parameter and the 3NQ-6* lifetime in ethanol (1.2 μs) measured with the transient absorption. From the high reactivity of 3NQ-6* to the antioxidants and the amphiphilic property of NQ-6 as a vitamin K model, NQ-6 is applicable to the quantitative antioxidant assay as a new fluorescence probe.