Presentation Solvent Effect on the Radical-Scavenging Mechanism of Ascorbic Acid and Its Derivatives

Nakanishi, Ikuo  ,  Ohkubo, Kei  ,  Inami, Keiko  ,  Nomura, Shogo  ,  Fukuhara, Kiyoshi  ,  Matsumoto, Kenichiro  ,  Ozawa, Toshihiko  ,  Fukuzumi, Shunichi  ,  Mochizuki, Masataka

Ascorbic acid (AscH2) is a representative water-soluble antioxidant and shows diverse biological activities, such as antitumor and radioprotective activities. However, the mechanism of the radical-scavenging reaction of AscH2 has yet to be fully clarified. Recently, we have reported that a water-insoluble 2,2-diphenyl-1-picrylhydrazyl radical (DPPH•) frequently used as a reactivity model of reactive oxygen species was successfully solubilized in water using β-cyclodextrin (β-CD) (Chem. Commun. 2015, 51, DOI: 10.1039/c5cc02236c). This enables us to investigate the radical-scavenging mechanism of AscH2 and its derivatives both in aqueous and non-aqueous media using DPPH•.AscH2 efficiently scavenged DPPH• both in MeOH and aqueous buffer solutions. The decay of the absorbance at 516 nm due to DPPH• monitored by a stopped-flow technique in MeOH obeyed pseudo-first-order kinetics, when the concentration of AscH2 ([AscH2]) was maintained at more than a 10-fold excess of DPPH• concentration. The pseudo-first-order rate constants (kobs) increased with increasing [AscH2] to reach a constant value. On the other hand, the kobs values determined in a phosphate buffer (0.1 M, pH 7.4) from the decay of the absorbance at 527 nm due to β-CD-solubilized DPPH• (DPPH•/β-CD) linearly increased with increasing [AscH2]. Because the pKa value of AscH2 is reported to be 4.1, AscH2 undergoes deprotonation and exists in its anionic form, AscH–, in the phosphate buffer solution (0.1 M, pH 7.4). In such a case, AscH– scavenged DPPH• to produce Asc•– and DPPH-H via a hydrogen-atom transfer. On the other hand, little deprotonation of AscH2 occurs in MeOH. Thus, the saturation behavior of the kobs values in MeOH may reflect a pre-equilibrium between AscH2 and AscH–. The effects of aprotic solvents as well as pH in aqueous systems using AscH2 and its derivatives were also investigated to clarify the detailed radical-scavenging mechanism of AscH2.

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