Effects of Pin-up Oxygen on [60]Fullerene for Enhanced Antioxidant Activity

The introduction of pin-up oxygen on C60, such as in the oxidized fullerenes C60O and C60On, induced noticeable increase in the antioxidant activity as compared to pristine C60. The water-soluble inclusion complexes of fullerenes C60O and C60Onreacted with linoleic acid peroxyl radical 1.7 and 2.4 times faster, respectively.


Introduction
Fullerenes and its derivatives are well known as a new class of antioxidants and they have attracted considerable attention in biologic applications due to their high reactivity toward radicals [1], especially reactive oxygen species (ROS) such as superoxide [2], hydroxyl radical [3], peroxyl radicals [4], and nitric oxide [5]. These harmful radicals attack lipids, proteins, DNA, and other biologic tissues and organs. It has been found that watersoluble fullerenes can be used as potential antioxidants and neuroprotective drugs against degenerative diseases related to oxidative stress [6][7][8][9][10][11]. Thus, water-soluble fullerenes, including host-guest inclusion complexes, are promising candidates for practical use as antioxidants.
However, such a radical scavenging ability has not been well investigated systematically for functionalized fullerenes, and the development of more efficient and easily accessible fullerene antioxidant derivatives has become an urgent requirement.
In this article, we first report that the introduction of pinup oxygen on C 60 , such as that in the oxidized fullerene (fullerene epoxide) C 60 O n , induces significant increase in the antioxidant activity as compared to pristine C 60 . The relative radical scavenging rate constant k rrs was kinetically determined using a b-carotene bleaching assay in the presence of water-soluble polyvinylpirrolidone (PVP)entrapped [12] and c-cyclodextrin (CD)-capped [13] C 60 and C 60 O n (n = 1 and 0-4) [14] inclusion complexes (Fig. 1).

Materials and Apparatuses
Fullerene C 60 and oxidized fullerene C 60 O n were purchased from Frontier Carbon Corporation. Polyvinylpirrolidone (PVP K 30) was purchased from Wako Pure Chemical Industries, Ltd. Other reagents and organic solvents as well as pure water were all commercially available and used as received. UV-visible spectra were measured on a JASCO V-550 equipped with a thermal controller. LCMS analysis was performed on a SHIMADZU LCMS-2010EV. Ball mill grinding for the preparation of c-cyclodextrin inclusion complexes was carried out using a FRITSCH pulverisette 6. DFT calculation of molecular orbital energy levels were performed using Spartan '04 software at B3LYP/6-31G* level of theory.

Synthesis of PVP/C 60 and its Oxidized Derivatives
A toluene solution (10 mL) of fullerene C 60 (8 mg) was added to an ethanol solution (5 mL) of PVP (1 g) and stirred for 12 h at room temperature under air. After evaporation of the solvent, drying of the residue under vacuum at room temperature for 18 h gave PVP/C 60 quantitatively (1 g) as a brown solid.
Synthesis of c-CD/C 60 and C 60 O Fullerene C 60 (10 mg) and c-cyclodextrin (70 mg) in an agate vessel (50 mL) together with a mixing ball made of zirconia (0.3 g 9 30) were vigorously mixed by using ball mill at a rate of 650 rpm for 30 min. The milling was repeated by addition of ethanol (5 mL) for 30 min. After drying the ethanol, pure water (5 mL) was added and mixed again for 30 min. The mixture was centrifuged and the obtained solution was filtered through a membrane filter (0.45 and 0.1 lm) to give a clear purple solution. The concentration of solution and the yield were estimated to be 1.40 mM and 31.7%, respectively, by the use of the molar absorption coefficient e = 5.06 9 10 4 M -1 cm -1 determined at k max 329 nm for the cyclohexane solution according to the previously reported method [13b]. The concentration and the yield for C 60 O were 682 lM and 25.1%, respectively (e = 3.25 9 10 4 M -1 cm -1 at k max 322 nm in cyclohexane).

b-Caroten Bleaching Method
Chloroform solutions of 11 lL of b-carotene (1.0 mg/mL), 4.4 lL of linoleic acid (0.1 g/mL) and 22 lL of Tween 40 (0.2 g/mL) were mixed in a quartz cell equipped with a screw-on cap, and then the solvent was removed in vacuo. An aliquot of the emulsion was immediately diluted with 2.4 mL of phosphate buffer solution (0.018 M, pH 7.0), and 0.1 mL of antioxidant (7.5-75 nmol, equivalent to C 60 ) in deionized water was added to the diluted mixture. The solution was mixed well and heated at 50°C under air in a quartz cell on a UV spectrometer in order to monitor the decrease in the absorbance of b-carotene at 460 nm.

Results and Discussion
The water-soluble fullerene inclusion complexes were synthesized by modified literature method [12]. The formation of c-CD/C 60 O has been confirmed only by a mass spectrum [15]. Thus, we confirmed its formation (obtained as a brownish water solution including an excess of free c-CD) and determined the concentration of solution using a UV-vis spectrometer by comparison of the peak absorbance around 360 nm in water to that of pristine C 60 O in cyclohexane (Fig. 2a). On the other hand, PVP/C 60 O and C 60 O n have not been reported so far and this is the first report (Fig. 2b).
The b-carotene bleaching assay is one of the common methods used in the field of food chemistry for evaluating antioxidant activity. The method is based on the discoloration of the yellowish color of a b-carotene solution due to the breaking of p-conjugation by the addition of lipid peroxyl radical (LOO • ) generated from the autoxidation of linoleic acid under air atmosphere [16][17][18]. The assay was performed according to an optimally modified procedure (Fig. 3) [19]. Figure 4 shows the dependency of the pseudo-first-order rate constants, k obs , for the discoloration of b-carotene on the antioxidant concentration of PVP and CD complexes of C 60 and oxidized C 60 O. Here, the rate (R f ) of discoloration of b-carotene by the LOO • radical is given by Eq. 1 [18], where k c and k f denote the second-order rate constants for the radical scavenging of b-carotene and fullerene antioxidant, respectively.
It was found that the b-carotene bleaching was significantly suppressed by the increasing amount of antioxidants, although C 60 O was more effective than C 60 in all tested ranges of concentration. It was also noted that the entrapped PVP and CD exerted no appreciable effect on the antioxidant activity of guest fullerenes. To the best of our knowledge, this is the first result of the higher antioxidant activity of C 60 O in comparison with pristine C 60 , despite the decreasing of p-conjugation. The concentration-dependent antioxidant activities %AOA [19] (= 100 9 {k obs of controlk obs of fullerene}/k obs of control) of PVP/C 60 and C 60 O were 50% and 68% in 10 lM for antioxidant, and 73% and 81% in 30 lM, respectively.
Here, it is more convenient to define the absolute antioxidant activity of fullerenes toward the LOO • radical by considering the relative radical scavenging rate constants k rrs (= k f /k c ) of fullerenes versus b-carotene, as given in Eq. 2 [18], where R 0 is the bleaching rate in the absence of antioxidants ([fullerene] = 0 in Eq. 1).
As shown in In order to clarify the reason for the significant effect of the pin-up oxygen on the antioxidant activity of C 60 , we calculated the energy level of LUMO and HOMO for the C 60 , C 60 O, and C 60 O 2 as well as the energy level of SOMO for the LOO • and L • radical (Fig. 6). It was found that the pin-up oxygen lowers the LUMO level relative to those of pristine C 60 . According to the Klopman and Salem equation [20] as well as the frontier molecular orbital (FMO) theory, the energy (DE) gained in the orbital interactions is inversely proportional to the energy difference |LUMO-SOMO|.

Conclusion
In conclusion, we have found a meaningful key in developing new applicable antioxidants using fullerenes by means of a simple and conventional technique that can enhance their antioxidant activity by simply introducing pin-up oxygen on the fullerene cage.