black solid; yield, 87%. 1H NMR (400 MHz, CDCl3–CD3OD ~
10 : 1 (v/v), TMS, 25 uC): d ~ 10.05–10.30 (m, 4H, meso-H), 8.38
(m, 2H, CHL), 7.72, 7.57, 6.60 (m each, 2H each, ArH), 6.14, 6.37
(m each, 2H each, LCH2), 4.42 (m, 6H, 131, 171-CH2, ArOCH2),
4.29 (s, 1H, NCH(Ar)C60), 3.98 (m, 2H, COOCH2), 3.85, 4.72 (m
each, 1H each, NCH2C60), 2.90–3.30 (m, 12H, 2,7,12,18-CH3),
3.25 (m, 4H, 132,172-CH2), 2.64 (s, 3H, NCH3), 1.12–1.84 (m, 8H,
(CH2)4); IR (KBr): n ~ 2921 (nCH), 1728 (CLO, ester), 1710 (CLO,
carboxylic acid) cm21; MS: 1578.77 (M1). The absorption
maxima of Soret- and Q-bands of 1?Zn inCHCl3–MeOH ~15 : 2
(v/v) appeared at 418 nm and 546 and 584 nm, respectively.
Nanosecond time-resolved absorption spectroscopy
The nanosecond time-resolved absorption spectra were mea-
sured with SHG (532 nm) of a Nd:YAG laser (Spectra-Physics,
Quanta-Ray GCR-130, fwhm 6 ns) as an excitation source.
The transient absorption spectra in the visible and near-IR
region from 600–1600 nm were detected with a Ge-APD
photodiode (Hamamatsu Photonics, B2834).30 All the samples
were contained in a 1 cm quartz cell and were deaerated by Ar
bubbling.
Acknowledgement
Preparation of 1?Fe-Mb
This work was supported, in part, by the Grant-in-Aids from
the Ministry of Education, Culture, Sports, Science and
Technologies, Japan and the Asahi Glass Foundation (both
for N. N.) and Sasagawa Scientific Research Grant from the
Japan Science Society (for H. M.). We thank Professor
S. Shinkai, Professor I. Hamachi and Dr S. Kiyonaka for
the technical advice to perform the axial-ligand exchange
reaction of 1?Fe-Mb.
The apomyoglobin (apo-Mb) was prepared from horse heart
myoglobin by Teale’s acid–butanone method.15 Reconstitution
of 1?Fe and of 1?Zn with apo-Mb was conducted according to a
modified procedure of Yonetani et al.17
A pyridine solution (100 mL) of 1?Fe (0.5 mg, 3.3 6
1027 mol) was added dropwise to an apo-Mb (1.3 mL, 3.3 6
1027 mol) aqueous phosphate buffer solution (10 mM, pH 7.0)
at 4 uC. 20 mL of the buffer solution was added each for one-
drop addition of the pyridine solution to maintain the total
pyridine content at 10 vol% in the buffer. The precipitate
produced was separated by centrifugation (6000 g) at 4 uC and
then the clear supernatant was purified with Sephadex G-25
equilibrated with a 10 mM phosphate buffer at pH 7.5 to
produce 1?Fe-Mb, which was determined spectrophotometri-
cally.
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The preparation of 1?Zn-Mb was performed in the same
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Preparation of 1?Fe-Mb (or 1?Zn-Mb)–lipid modified electrodes
and electrochemical measurements
The preparation of electrodes modified with 1?Fe-Mb (or 1?Zn-
Mb) and didodecyldimethylammonium bromide (DDAB) is as
follows. 10 mL portions of 0.1 M DDAB in CHCl3 were placed
on a basal plane pyrolytic graphite (BPG) electrode (geometric
area, 0.25 cm2) and then allowed to air dry. The lipid-modified
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5 days at 4 uC in the dark. The electrodes in the solutions were
then transferred into a 50 mM phosphate buffer (pH 7.5)
containing 10 mM KBr and 10 mM triethanolamine (TEOA).
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were recorded with an electrochemical analyzer (Bioanalytical
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electrode (SCE) and a Pt plate were used as the reference and
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Measurements of photocurrent and action spectrum
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18 An attempt to reconstitute dicarboxymethano[60]fullerene with
apo-Mb was unsuccessful. This fullerene is expected to be too large
to incorporate in the crevice of apo-Mb.
Time-resolved fluorescence spectroscopy
19 The concentration of 1?Fe-Mb aqueous solution was calculated
with the molecular extinction coefficient of the Soret-band
determined by pyridine-haemochromogen method: see, K. G. Paul,
H. Theorell and A. Akeson, Acta Chem. Scand., 1953, 7, 1284 . The
molecular extinction coefficient of the Soret-band in 1?Fe-Mb
determined was 189 000, which is very close to that of native-Mb
(188 000). Thus, the concentration of 1?Zn-Mb aqueous solution
The time-resolved fluorescence spectra were measured with
second harmonic generation (SHG) (410 nm) of a Ti:sapphire
laser (Spectra-Physics, Tsunami 3950-L2S, full width at half-
maximum (fwhm) 1.5 ns) as an excitation source and detected
with streakscope (Hamamatsu Photonics, C4334-01).32 All
samples were deaerated by Ar bubbling.
2032
J. Mater. Chem., 2002, 12, 2026–2033