The synthesis of a reconstituted C60-modified protein

Article abstract of DOI:10.1246/cl.2000.46

We designed and synthesized reconstituted C₆₀-modified myoglobin (C₆₀-Mb) and found that fundamental characteristics, except autoxidation rate constants, of C₆₀-Mb and native Mb were almost identical.

Full text of DOI:10.1246/cl.2000.46

46
Chemistry Letters 2000
The Synthesis of a Reconstituted C60-Modified Protein
Hiroto Murakami, Yuko Okusa, Shigeki Kiyonaka,† Itaru Hamachi,† Seiji Shinkai,† and Naotoshi Nakashima*
Department of Applied Chemistry, Faculty of Engineering, Nagasaki University, Bunkyo, Nagasaki 852-8521
†Department of Chemistry and Biochemistry, Graduate School of Engineering,
Kyushu University, Hakozaki, Fukuoka 812-8581
(Received September 24, 1999; CL-990816)
maceutical and medical applications.
We designed and synthesized reconstituted C60-modified
A protoporphyrin IX-C60 conjugate, 2, and a hemin-C60
conjugate, 1, were synthesized according to Scheme 1. The
reaction of C60 with N-methylglycine and 4-((6-hydroxy-
hexyl)oxy)benzaldehyde in chlorobenzene produced N-methyl-
2-((6-hydroxyhexyl)oxyphenyl)-3,4-fulleropyrrolidine, which
was condensed with protoporphyrin IX (acid chloride form) in
chloroform to give compound 2. Iron insertion to 29 produced
compound 110.
Apo-Mb from met-Mb (horse heart, Sigma) was prepared
according to the literature.11,12 The purity of apo-Mb was 99%
as determined by spectrophotometry.11-12 Preparation of C60-
Mb was conducted as follows. A pyridine solution (100 µL) of
1 (0.5 mg, 3.3 × 10-7 mol) was added drop-wise to an apo-Mb
(1.3 mL, 3.3 × 10-7 mol) aqueous phosphate buffer (10 mmol
dm-3, pH 7.0) at 4 °C. Twenty µL of the buffer solution was
added each for one-drop addition of the pyridine solution. In
this procedure, the total pyridine content in the buffer was var-
ied between 5-15 vol% and 10 vol% resulted in highest yield of
C60-Mb. The precipitate was separated by centrifugation (6000
g), followed by through column-chromatography purification
(Sephadex G-25, eluent: 50 mmol dm-3 phosphate buffer, pH
7.5) to produce C60-Mb.
The molecular extinction coefficient the Soret-band of
C60-Mb (met form) was determined to be 189000,13 which is
very close to that of native Mb (188000)14. Figure 1 shows
UV-vis absorption spectra of C60-Mb alone and the presence of
potassium fluoride or sodium azide. The absorption maxima of
the Soret- and Q-bands of C60-Mb (met-form) appeared at 408
nm and 504 and 630 nm, respectively, which are very close to
those of native Mb(met-form, Soret-band: 408 nm, Q-bands:
502 and 630 nm, respectively)14. The Soret- and Q-bands of
C60-Mb in the presence of potassium fluoride were 407 and
602 nm, which are close to those of corresponding native Mb
(Soret-band: 406 nm, Q-bands: 604 nm) 14 in the presence of
potassium fluoride. The Soret- and Q-bands of C60-Mb in the
presence of sodium azide appeared at 419 nm and 538 and 571
myoglobin (C60-Mb) and found that fundamental characteris-
tics, except autoxidation rate constants, of C60-Mb and native
Mb were almost identical.
We report here the first synthesis of a reconstituted C60-
modified protein. Fullerenes possess wide ranging biological
reactivity including antiviral activity againt HIV1,2, selective
DNA cleavage2, and promotion of chondrogenesis3. Chen and
coworkers4 recently described the preparation of antibodies
specific for fullerenes and discussed protein-fullerene interac-
tions. Fullerenes are also strong photosensitizing reagents and
the photophysics, photochemistry and photobiochemistry of
fullerenes are of exciting areas.5 Our objective is to introduce a
fullerene moiety into proteins to create novel proteins with spe-
cific function. The construction of such systems using proteins
may open new fullerene fields in chemistry and biochemistry.
There are two reports related to this area.6,7 Methano[C60]
fullerene dicarboxylic acid was covalently attached, by Son
and coworkers6, to the amino-residues of native and denatured
bovine serum albumin and the modified proteins were purified
and characterized. Hill and coworkers7 recently described the
preparation of a fullerene-modified redox protein, azurin
mutant S118C, prepared by site-directed mutagenesis methods
followed by chemical modification and reported the electro-
chemical behavior of the protein.
Our strategy for preparing a fullerene-modified protein is
to use the heme protein reconstitution method8. By substitu-
tion of the heme moiety of a protein with hemin derivatives,
reconstituted artificial proteins can be prepared. We describe
here the preparation of a reconstituted C60-modified myoglobin
(C60-Mb), in which the prosthetic group consists of a hemin-
fullerene conjugate. This modified protein is of interest as the
functional component of artificial photosynthetic systems. The
construction of biodevices based on fullerene-protein conjugat-
ed novel materials has possible biological, biochemical, phar-
Copyright © 2000 The Chemical Society of Japan

Chemistry Letters 2000
47
is currently underway in our laboratory to define fully the
structural characteristics and physicochemical and biological
properties and function of C60-Mb. We are also preparing zinc-
substituted C60-Mb as well as fullerene-modified proteins using
another proteins instead of Mb.
This work was supported, in part, by the Grant-in-Aids
from the Ministry of Education, Science, Sports, and Culture,
Japan (for N. N.). We thank Prof. Fred Hawkridge for helpful
discussion and Mr. Shinya Tsukiji for technical assistance.
References and Notes
1
S. H. Friedman, D. L. DeCamp, R. P. Sijbesma, G. Srdanov,
F. Wudl, and G. L. Kenyon, J. Am. Chem. Soc., 115, 6506
(1993).
2
E. Nakamura, H. Tokuyama, S. Yamago. T. Shiraki, and Y.
Sugiura, Bull. Chem. Soc. Jpn., 69, 2143 (1996) and refer-
ences therein.
3
4
5
T. Tsuchiya, Y. N. Yamakoshi, and N. Miyata, Biochem.
Biophys. Res. Commun., 206, 885 ( 1995).
B.-X. Chen, S. R. Wilson, M. Das, D. J. Coughlin, and B. F.
Erlanger, Proc. Natl. Acad. Sci. U.S.A, 95, 10809 (1998).
For a review, see: N. S. Sariciftci, A. J. Heeger, in
“Handbook of Organic Conductive Molecules and
Polymers,” ed by H. S. Nalwa, John Wiley & Sons,
Chichester (1997), pp. 414-486.
6
Y.-P. Sun, G. E. Lawson, N. Wang, B. Liu, D. K. Moton,
and R. Dabestani, Proc. Electrochem. Soc., 97-14, 645
(1997).
7
8
A. Kurz, C. M. Halliwell, J. J. Davis, H. A. O. Hill, and G.
W. Canters, J. Chem. Soc., Chem. Commun. 1998, 433.
a) J. R. Winkler and H. B.Gray, Chem. Rev., 92, 369 (1992).
b) I. Hamachi, S. Tanaka, and S. Shinkai,. J. Am. Chem.
Soc., 115, 10458 (1993). c) I. Hamachi, Y. Matsugi, K.
Wakigawa, and S. Shinkai, Inorg. Chem., 37, 1592 (1998).
IR (KBr): 1728 (C=O, ester), and 1710 (C=O, carboxylic
9
1
acid) cm-1. H NMR (400 MHz, CDCl3, TMS): δ 1.12-1.84
nm, respectively, which are also close to those of correspon-
ding native Mb (Soret-band: 418 nm, Q-bands: 540 and 572
nm) 14. These results indicate that the heme moieties in both
proteins are located in similar microenvironments.
(m, 8H, (CH2) 4), 2.55 (s, 3H, NCH3), 3.25 (t, 4H, 132, 172,
CH2), 3.58 – 3.74 (m, 12H, 2,7,12,18,CH3), 3.85, 4.72 (d, J
= 9.3 Hz, 1H each , NCH2C60), 3.98 (t, 2H, COOCH2), 4.35
(s, 1H, NCH(Ar)C60), 4.40 (m, 6H, 131, 171, CH2, ArOCH2),
6.19, 6.36 (d each, J = 11.0 Hz, J = 16.9 Hz, 2H each,
=CH2), 6.54, 7.55, 7.72 (d, J = 7.33 Hz, m, m, 2H, 1H, 1H,
ArH), 8.26 (m, 2H, CH=), 10.05 - 10.20 (m, 4H, meso-H).
Anal. Calcd for C109H55N5O5 + 8H2O: C, 78.93; H, 4.31; N,
4.22 %. Found: C, 78.57; H, 4.42; N, 4.62 %. The absorp-
tion maxima of Soret- and Q-bands of 2 in CHCl3/CH3OH =
10/1 (v/v) appeared at 407.0 nm and 506.0, 541.5, 575.5 and
688.5 nm, respectively.
Mb is known to act as an oxygen carrier in mammalian
cells. The oxygen binding properties of C60-Mb and native Mb
were compared. The addition of sodium dithionate to the
buffer solution of C60-Mb (met-form) produced C60-Mb(FeII)15
, and then dioxygen was introduced. Reduced C60-Mb was
found to form the oxygen complex (Figure 2), whose Soret-
and Q-bands appeared at 415 nm and 543 and 578 nm, respec-
tively, which are very close to those of the oxygen complex of
native Mb (Soret-band: 418 nm, Q-bands: 541 and 578 nm) 14
.
10 The absorption maxima of Soret- and Q-bands of 1 in
CHCl3/CH3OH = 10/1 (v/v) appeared at 402.5 nm and 489.0
and 589 nm, respectively.
11 F. W. Teale, Biochim. Biophys. Acta, 35, 543 (1959).
12 T. Yonetani, J. Biol. Chem., 242, 5008 (1967).
13 The hemin moiety of the protein was converted to pyridine
haemochromogen: see, K. G. Paul, H. Theorell, and A.
Akeson, Acta Chem. Scand., 7, 1284 (1953).
14 T. Asakura and T. Yonetani, J. Biol. Chem., 262, 6725
(1969).
15 Soret- and Q-bands appeared at 431 and 557 nm, respective-
ly, which are close to those of corresponding native Mb(FeII)
(Soret-band: 435 nm, Q-band: 556 nm).
The stability of the oxygen complex of C60-Mb was monitored
by the time course in absorbance of Q-band at 578 nm. The
oxy-form of C60-Mb(FeII) gradually autoxidized to the met-
form with the first-order rate constant of 0.2 h-1, which is ca. 6-
times faster than that of native Mb measured under the same
experimental condition. Introduction of the bulky fullerene
moiety to the protein probably contributes to this faster autoxi-
dation.
In conclusion, we have succeeded in the synthesis of
reconstituted C60-modified myoglobin and found that funda-
mental characteristics, except the autoxidation rate constants,
of C60-Mb and native Mb were almost identical. Intense effort