COMMUNICATION
Figure 2. Photodegradation of HIV-1 protease and attempted photode-
gradation of other proteins using 2 under long-wavelength UV irradiation
(panels a–e) or visible-light irradiation (panel f). Each protein (1.5 mm)
was incubated with 2 in 50 mm PBS buffer (pH 7.0) containing 10%
DMF at 258C for 2 h under irradiation with a UV lamp (365 nm, 100 W)
or visible light (diffuse sunlight, 75 W) placed 10 cm from the sample,
and the products were analyzed by tricine-SDS-PAGE. The proteins used
are: a, f) HIV-1 protease, b) BSA, c) Lyso, d) HIV reverse transcriptase,
e) HIV-1 protease+BSA. Lanes 1, size marker; lanes 2, protein alone;
lanes 3, protein upon photoirradiation; lanes 4, protein+2 (15 nm) with-
out photoirradiation; lanes 5–8, protein+2 (at concentrations of 15, 5,
1.5, and 0.5 nm, respectively) upon photoirradiation.
Scheme 1. Synthesis of fullerene-sugar hybrid 2. a) PhMe, reflux, 18 h,
57%; b) TFA, CH2Cl2, 08C, 10 min, 89%; c) AgClO4, MS 5 ꢁ, CH2Cl2,
rt, 85 h, 59%, 3:2 dr; d) NaOMe, CH2Cl2/MeOH (3/1), 08C, 1.5 h, 90%;
e) BBr3, CHCl3, rt, 20 h, 45%.
After the chemical synthesis of hybrid 2, photo-induced
degradation assays of four proteins—HIV-1 protease, bovine
serum albumin (BSA), hen egg lysozyme (Lyso), and HIV
reverse transcriptase—were carried out under UV light irra-
diation (365 nm, 100 W), and the progress of the reactions
was monitored by SDS-PAGE (Figure 2). Comparison of
lanes 3 and 4 with lane 2 in Figure 2a showed that neither
photoirradiation of HIV-1 protease in the absence of 2 nor
treatment of HIV-1 protease with 2 without photoirradiation
resulted in a change in the SDS-PAGE profile. By contrast,
no band corresponding to HIV-1 protease could be detected
after exposure to 2 under photoirradiation (lane 5), thus in-
dicating degradation of HIV-1 protease. These results show
that 2 is capable of degrading a target protein, HIV-1 pro-
tease, upon irradiation with long-wavelength UV light. It
was also found that 2 degraded HIV-1 protease in a dose-de-
pendent manner and that a catalytic amount of 2 was suffi-
cient for the degradation. These results were in sharp con-
trast to those obtained using the other proteins (BSA, Lyso,
and HIV reverse transcriptase), which showed no degrada-
tion upon photoirradiation (Figure 2b–d). Furthermore, it
should be noted that when HIV-1 protease and BSA were
both present in the reaction mixture, only HIV-1 protease
was degraded by 2, as shown in Figure 2e. These results
clearly indicate that 2 selectively degraded HIV-1 protease
upon long-wavelength UV irradiation. Moreover, to exam-
ine the possibility of photodegradation of proteins by 2
upon irradiation with visible light, we measured the UV/Vis
spectrum of 2. The results indicated that 2 absorbs not only
in the UV region but also in the visible region (see Fig-
ure S1 in the Supporting Information). These results
prompted us to examine the photodegradation activitity of 2
under visible-light irradiation. As shown in Figure 2 f, similar
results were obtained when visible light (diffuse sunlight, 75
W xenon lamp) was used instead of UV light, thereby clear-
ly indicating that hybrid 2 degraded HIV-1protease upon ir-
radiation not only with long-wavelength UV light but also
with visible light. Moreover, it is noteworthy that the degra-
dation activity of 2 is approximately 100 times higher than
that of 1.[4] The photodegradation of HIV-1 protease using 2
was also confirmed by matrix-assisted laser desorption/ioni-
zation time-of-flight mass spectrometry (MALDI-TOF MS)
analysis (see Figure S2 in the Supporting Information).
To investigate the mechanism behind the photodegrada-
tion of proteins, electron paramagnetic resonance (EPR)
studies were carried out.[9] 5,5-Dimethyl-1-pyrroline-N-oxide
(DMPO) was used as a spin-trapping agent for the detection
of superoxide anions (O2CÀ) or hydroxyl radicals (COH) (Fig-
ure 3a). It was found that photoirradiation of 2 in the pres-
ence of DMPO gave products with EPR spectra characteris-
tic of the DMPO–superoxide anion spin adduct DMPO/
COOH; no peaks corresponding to DMPO/COH were detect-
ed (Figure 3b). It was also confirmed that no peaks corre-
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ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Asian J. 2012, 7, 911 – 914