Antioxidative activity of alcohol-soluble fullerene derivative

Abstract

To utilize the radical scavenging property of fullerene (C₆₀), hydrophilic poly(ethylene glycol) (PEG) was chemically attached by Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC). The resulting C₆₀-PEG derivatives were soluble in methanol when the molecular weight of PEG was 5,000. The antioxidative activity was evaluated for the alcohol-soluble C₆₀ derivative. The C₆₀-PEG has sufficient antioxidative activity with the β-carotene-attributed chromaticity of 76% for the C₆₀-PEG derivative (9.96 mM). Further chemical modification will produce water-soluble fullerenes with higher antioxidative activities.

Keywords:

• Antioxidant
• click chemistry
• cycloaddition
• fullerene
• poly(ethylene glycol)

1. Introduction

The radical scavenging property of fullerene (C₆₀) has attracted much attention with a view to commercialize the product by blending it into cosmetics.^[1] However, the hydrophobic feature of C₆₀ makes it difficult to homogeneously distribute in an aqueous solution, which also hinders application studies of forming biopolymer complexes. In order to enhance the hydrophilicity and water solubility, chemical modification has been often adopted. For example, water-soluble fullerenes with numerous hydroxyl groups on their surface have been developed and given the trademark name “radical sponge,” and they have been proven to show excellent radical scavenging properties.^[2] Therefore, cosmetics containing hydroxylated fullerenes are already available on the market, and they promote anti-aging and whitening effects on the skin. It is generally said that water-soluble fullerenes have approximately 170 times the antioxidant effect of vitamin C. However, in the chemical structure of the “radical sponge,” a part of the C₆₀ conjugated system disappeared due to the introduction of hydroxyl groups, and it is thought that the properties of fullerenes, represented by their antioxidant properties, accordingly decreased. To solve this problem, a more controlled chemical modification of fullerenes can be adopted.^[3] For example, it is known that the water solubility issue can be solved by substituting a giant water-soluble (high) molecular weight chain into a fullerene. However, a new problem arises: as the molecular weight of the substituent increases, the reactivity of the polymer terminal functional group generally decreases and the fullerene unit density in the resulting water-soluble fullerene derivative also decreases. In this study, we decided to control the solubility balance and selected the Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) click chemistry reaction to introduce a single poly(ethylene glycol) (PEG) chain, a water-soluble polymer, to C₆₀. The applicability of CuAAC to C₆₀ was previously demonstrated.^[4] The resulting PEGylated C₆₀ has the good solubility in methanol, depending on the length of the PEG chain. Finally, the antioxidant activity of the obtained PEG-fullerene derivative was evaluated. Since the antioxidants in natural products include not only water-soluble substances but also many lipophilic components, it is significant to evaluate the antioxidant activity of various synthetic materials.

2. Experimental section

2.1. General procedures

All chemicals were purchased from Kanto Chemical Co., TCI, and Aldrich and used without further purification. Nuclear magnetic resonance (NMR) spectra were recorded using a JEOL model AL300 (300 MHz) at room temperature. Deuterated chloroform was used as the solvent. Chemical shifts of NMR were reported in ppm (parts per million) relative to the residual solvent peak at 7.26 ppm for ¹H NMR spectroscopy. Coupling constants (J) were given in Hz. The resonance multiplicity was described as s (singlet), br s (broad singlet), and m (multiplet). Fourier transform infrared (FT-IR) spectra were recorded by a JASCO FT/IR-4200 spectrometer in the range from 4000 to 600 cm⁻¹. MALDI − TOF mass spectra were measured on a Shimadzu/Kratos AXIMACFR mass spectrometer equipped with a nitrogen laser (λ = 337 nm) and pulsed ion extraction, which was operated in a linear-positive ion mode at an accelerating potential of 20 kV. Tetrahydrofuran (THF) solutions containing 1 g L⁻¹ of a sample, 10 g L⁻¹ of dithranol, and 1 g L⁻¹ of sodium trifluoroacetate were mixed to a ratio of 1:1:1; and then 1 μL aliquot of this mixture was deposited onto a sample target plate. UV–vis absorption spectra were recorded on a JASCO V-670 spectrophotometer.

The antioxidative property of the C₆₀ derivative was evaluated according to a literature procedure.^[7,8] First, a methanol solution of the C₆₀ derivative was prepared at the concentration of 0.0023 M (solution A). Next, a chloroform solution of β-carotene (1 g L⁻¹) (solution B), a chloroform solution of linoleic acid (0.1 g mL⁻¹) (solution C), and a chloroform solution of Tween 40 (0.2 g mL⁻¹) (solution D) were prepared. The solution B (0.25 mL), solution C (0.1 mL), and solution D (0.5 mL) were placed into a sample bottle, and chloroform was evaporated by spaying nitrogen gas. Pure water (50 mL) and 0.2 M phosphate buffer (4.45 mL) were added, and the mixture was stirred at room temperature for 30 min in the dark (solution E). To a UV cuvette, the solution E (2.85 mL) and a fixed amount of solution A were added. The absorbance of β-carotene at 470 nm was tracked over time with stirring at 50 °C. The relative intensity of 470 nm absorption was calculated according to the following Equation (1)(1) $$100–\{(A_{\text{test at}0\text{min}}\hspace{0.17em}–\hspace{0.17em}{\text{A}}_{\text{test at t\hspace{0.17em}min}})/(A_{\text{control at}0\text{\hspace{0.17em}min}}\hspace{0.17em}–{{\text{\hspace{0.17em}A}}_{\text{control at 50}}}_{\text{\hspace{0.17em}min}}\left)\right\}\times 100$$(1) : (1) $$100–\{(A_{\text{test at}0\text{min}}\hspace{0.17em}–\hspace{0.17em}{\text{A}}_{\text{test at t\hspace{0.17em}min}})/(A_{\text{control at}0\text{\hspace{0.17em}min}}\hspace{0.17em}–{{\text{\hspace{0.17em}A}}_{\text{control at 50}}}_{\text{\hspace{0.17em}min}}\left)\right\}\times 100$$(1)

2.2. Synthetic procedures

PEGylated C₆₀ was prepared by CuAAC of the C₆₀ derivative 1⁵ and PEG-N₃. To a 50 mL round bottle flask, 1 (24 mg, 0.022 mmol) and THF (7.2 mL) were added and the solution was stirred at 0 °C. After a THF solution of (nC₄H₉)₄NF (1 M, 0.055 mL) was added, the solution was further stirred at 1.5 h at 0 °C. The solution was gradually heating to room temperature, and brine (200 mL) was added. Organic layers were extracted with CHCl₃ (400 mL), washed with brine (200 mL × 2), and dried over Na₂SO₄. After filtration, the filtrate was concentrated to approximately 15 mL. To this solution, α-methoxy-ω-azide-poly(ethylene glycol) (M_n 5,000, 111 mg, 0.0222 mmol)) and Cu(PPh₃)₃Br (2.06 mg, 0.00221 mmol) were added and the mixture was stirred under N₂ at 60 °C for 22 h. After cooling to room temperature, chloroform, and water were added. The organic layer was washed with water, dried over Na₂SO₄, and filtered. Evaporation and column chromatography (Bio-Beads S-X2, chloroform) afforded the target compound (89 mg, 68%). The resulting PEGylated C₆₀ was soluble in methanol but limited solubility in water at 20–37 °C.

¹H NMR (CDCl₃, 300 MHz): δ = 7.68 ppm (br s, 1H), 4.29 (br s, 4H), 3.44–3.80 (m, 4(n + 1)H), 3.37 (s, 3H), 1.8–1.6 (m, 4H). IR (neat): 3243.7, 2884.0, 2130.0, 1717.3, 1591.0, 1483.0, 1466.6, 1451.2, 1358.6, 1342.2, 1280.5, 1241.0, 1144.6, 1105.0, 1060.7, 1006.7, 962.3, 879.4, 841.8, 717.4, 700.0, 664.4 cm⁻¹.

3. Results and discussion

C₆₀ derivative 1 was synthesized using the Bingel reaction.^[5] After deprotection of the trimethylsilyl groups of 1, one equivalent of methoxy-terminated polyethylene glycol (PEG) azide was added in the presence of a copper catalyst for cycloaddition reaction (the CuAAC click reaction) (Scheme 1). The unreacted starting material and bis-PEG adduct were removed by column chromatography on Bio-Beads SX-2, and the target product of mono-PEGylated fullerenes was isolated. The solubility of the fullerene derivatives varied greatly depending on the molecular weight of the PEG chain: C₆₀-PEG2000 was soluble in organic solvents, such as chloroform and toluene, but insoluble in alcohols. On the other hand, C₆₀-PEG5000 had a good solubility in methanol and chloroform but a limited solubility in water. Accordingly, the mono-PEGylated fullerenes can be further reacted with biomolecules in alcohol or aqueous solutions, although such application studies are out of the scope of this study. In this study, we describe the C₆₀-PEG5000 in detail.

Scheme 1. Synthesis of PEGylated fullerenes by CuAAC.

The structure of the obtained C₆₀-PEG5000 derivative was confirmed by ¹H NMR, IR, and MALDI-TOF MS spectra. The ¹H NMR revealed new peaks for the PEG chain and triazole ring, although most peaks other than PEG were very broadened and the terminal alkyne peak was not detected (Figure 1). IR showed H–C≡C and C≡C stretching vibrations of the terminal alkyne at 3243.7 and 2130.0 cm⁻¹, respectively (Figure 2). MALDI-TOF MS revealed that PEG5000 has a molecular weight distribution with a peak top molecular weight (Mass/Charge) of approximately 5000 (Figure 3a), whereas the peak top molecular weight of the C₆₀-PEG5000 derivative was slightly shifted toward the higher molecular weight side (Figure 3b). In addition, the entire peak became broadened in the positive mode (Figure 3b). This is characteristic of the MALDI-TOF MS spectra of C₆₀ derivatives with n-type semiconductor properties. All these spectroscopic data indicated that the target mono-PEGylated product was indeed obtained.

Figure 1. ¹H NMR spectrum of C₆₀-PEG5000 in CDCl₃.

Figure 2. IR spectrum of C₆₀-PEG5000.

Figure 3. MALDI-TOF MS spectra of (a) PEG5000 and (b) C₆₀-PEG5000 (matrix: dithranol).

The antioxidant activity of the C₆₀-PEG5000 derivative was evaluated. It is known that when peroxide radicals generated by the oxidation of linoleic acid attack β-carotene, the absorption intensity of β-carotene at 470 nm decreases.^[6] The addition of an antioxidant to the mixture of linoleic acid and β-carotene prevents the attack to β-carotene. Here the change in the absorption intensity at 470 nm was assumed to be directly related to the antioxidant activity. A rapid decrease in the 470 nm absorption intensity started about 10 min after the addition of linoleic acid to the β-carotene solution (see the plots of the control sample in Figure 4). However, the decrease was milder when 4.79 mM of the C₆₀-PEG5000 derivative was present (see the plots of 4.79 mM in Figure 4). Furthermore, when the concentration of the C₆₀-PEG5000 derivative was increased to 9.96 mM, the decrease in absorbance intensity was further mitigated, suggesting that the PEGylated fullerene derivative has a clear antioxidant effect. The β-carotene-attributed chromaticity at the time of 30 min was 55% for C₆₀-PEG5000 (4.79 m) and 76% for C₆₀-PEG5000 (9.96 m). Although experiments at higher concentrations have not been conducted due to the limited solubility of the developed fullerene derivatives in methanol and water, it is thought that the antioxidant activity of the C₆₀-PEG5000 derivative is similar to those of the previously reported sugar-appended fullerenes.^[7]

Figure 4. Time-dependent 470 nm absorbance change of a β-carotene aqueous solution.

4. Conclusions

In conclusion, we have introduced hydrophilic PEG substituents without compromising the π-electron system of fullerenes. Although azide groups are known to react with fullerenes, the CuAAC reaction proceeded much faster and the target compound was selectively obtained. It was revealed that the solubilities of the fullerene derivatives greatly changed by the molecular weight of the attached PEG chain. The C₆₀-PEG5000 derivative was soluble in methanol. It was thought that a longer PEG chain was necessary to make it completely water-soluble. However, the antioxidant activity of the C₆₀-PEG5000 derivative could be successfully evaluated. The addition of small amounts (4.79–9.96 mM) of the C₆₀-PEG5000 derivative explicitly inhibited the attack of peroxide radicals to β-carotene, indicating the potent antioxidant properties. In addition, the C₆₀-PEG derivatives synthesized in this study have one more unreacted terminal alkyne. Since the solubility in alcohols is ensured by substituting PEG with a molecular weight of more than 5000, we believe that these fullerene derivatives can be covalently linked to azidated biomolecules by using the second CuAAC reaction. This will create novel bio-nanostructures, such as biomaterials decorated by PEGylated C₆₀ derivatives.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This work was partly supported by the Kose Cosmetology Research Foundation.