Synthesis of C60 derivatives for photoaffinity labeling

Article abstract of DOI:10.1016/j.tetlet.2003.10.212

In order to study the interaction of fullerenes with biological molecules, a novel photoaffinity labeling agent derived from C₆₀ was designed and synthesized. As photosensitive functional groups, azide group, and aziridine group are utilized. A convenient synthetic route via fulleropyrrolidine 2 was employed to obtain compounds labeling agents 5 and 9.

Full text of DOI:10.1016/j.tetlet.2003.10.212

Tetrahedron
Letters
Tetrahedron Letters 45 (2004) 527–529
Synthesis of C derivatives for photoaffinity labeling
60
a,b
Eiji Okada, Yuka Komazawa, Masaaki Kurihara, Hideshi Inoue, Naoki Miyata,
a,b
a
b
a
a
Haruhiro Okuda, Toshie Tsuchiya and Yoko Yamakoshi
a
a,
*
a
National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya, Tokyo 158-8501, Japan
b
School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, 143201 Horinouchi, Hachioji, Tokyo 192-03, Japan
Received 9 October 2003; accepted 31 October 2003
Abstract—In order to study the interaction of fullerenes with biological molecules, a novel photoaffinity labeling agent derived from
60 was designed and synthesized. As photosensitive functional groups, azide group, and aziridine group are utilized. A convenient
C
synthetic route via fulleropyrrolidine 2 was employed to obtain compounds labeling agents 5 and 9.
Ó 2003 Published by Elsevier Ltd.
The biological activities of fullerenes have attracted
considerable attention due to their potential medicinal
phobic amino acid residues, of HIV-1 protease. In our
own work, we calculated that C60 binds to GST at a cleft
between two subunits of the enzyme, although the
specific residues, which make up the active site are
1–3
applications. Their novel and unexploited properties
stem from their bulky hydrophobic shape and their
4
–7
8–11
20
photosensitivity
ing
tron system.
and radical-generating /-quench-
unclear.
12;13
activities enabled by highly conjugated p-elec-
In order to clarify the more detailed binding site of C ,
60
two solutions are possible as follows. One is to isolate
pure enzyme–fullerene complex and determine the
structure by NMR or crystallographic methods.
Another potential method for identifying the active site
area is photoaffinity labeling, which is particularly useful
for identifying the active site in solution under physio-
logical conditions.
As a most remarkable activity, direct inhibition
of enzymes by C60 has been reported. The first example,
HIV-1 protease inhibition by a water soluble fullerene
14–16
derivative, was reported in 1993
et al. Independently, Toniollo et al. has reported C -
by Wudl, Wilkins,
60
peptide conjugates and identified activity of these com-
pounds against HIV-1 protease and chemotatic activity
17
against human monocytes.
developed new procedures for solubilizing C60 in water
Separately, we have
We now report the design and synthesis of the first C60-
18
derived photoaffinity labeling reagents. Our synthetic
route to photoaffinity reagents 5 and 9 provide a con-
cise, flexible route to fullerenes functionalized with
photoreactive pendant groups such as phenylazide and
phenyldiazirine, which generate aryl nitrene and aryl
and assayed unfunctionalized C60 for direct enzymatic
inhibtion. These studies led to the discovery that aque-
ous solutions of C inhibit glutathione-S-transferase
60
19
(
GST).
21
carbene, respectively (Eqs. 1 and 2).
ꢀ
The ability of C , which is large (7 A id) hydrophobic
60
molecules, to bind to biological compounds, was ini-
tially surprising and several groups have attempted to
identify and calculate the binding sites. Based on a
computer simulated docking study, Wudl, Wilkins, et al.
speculated that the C60 core was enclosed in the cylin-
drical active site, which consists primarily of hydro-
hν
RH
RH
H
N3
N
N
R
ð1Þ
ð2Þ
CF3
CF3
CF3
R
h
ν
N
N
In order to develop an efficient and flexible synthetic
method, which would allow the late-stage introduction
of a variety of photoaffinity labels, we chose to utilize
*
Corresponding author. Tel.: +81-3-3700-1141; fax: +81-3-3707-6950;
e-mail addresses: yamakosh@nihs.go.jp, yamakoshi@chem.ucsb.edu
dimethylfulleropyrrolidine (2). This
C60 derivative
0
040-4039/$ - see front matter Ó 2003 Published by Elsevier Ltd.
doi:10.1016/j.tetlet.2003.10.212

528
E. Okada et al. / Tetrahedron Letters 45 (2004) 527–529
H
N
CH3CHO
Me
Me
Me
OH
H2N
O
1
,2-Cl2C6H4
1
20 °C
0 h
0.0 %
2
2
cis-, trans-2
N3
N3
Me
Me
N
cis-, trans-2
pyridine
SOCl2
N3
COOH
N3
COCl
N
CH2Cl2 / DMF
rt
benzene
O
O
40 - 50 °C
3
4
Me
1
h
Me
2
h
cis-5
8 %
trans-5
57 %
3
Cl
Cl
N
N
N
N
ClOC
Cl
CF3
CF3
O
O
Cl
Me
N
cis-, trans-2
Me
N
F3C
N
Et3N
O
DMAP
COOH
F3C
toluene
rt
2 d
toluene
rt
1.5 h
N
Cl
Cl
O
O
N
Me
6
N
Me
8
cis-9
trans-9
44 %
2
0 %
Scheme 1. Synthesis of C60 derivatives with phenylazide (5) and phenylaziridine (9) group.
is readily prepared by the method of Prato and
23;24
biological molecules with high sensitivity. For example,
an acidic isozyme of GST is specified as cancer
22
co-workers and Wilson and co-workers.
This route
29;30
provides a convenient approach to C60 derivatives with a
secondary amine as an ideal site for the incorporation of
further functionalization.
expressing marker in liver cancers.
selectively tag such diagnostic enzymes with C , which
The ability to
60
has unique and useful chemical and photophysical
properties, may offer a novel and rapid detection
method for identifying trace amounts of enzyme present
in a biological sample. These and other applications of
the reported photoaffinity labeling reagents currently in
progress.
The synthesis of phenylazide derivative of fullerene was
achieved as shown in Scheme 1. Dimethyl fulleropyr-
rolidine 2 (cis- and trans-mixture) was prepared by 1,3-
25
dipolar cycloaddition and then acylated with acid
chloride 4 to give cis- and trans-C -phenylazide deriv-
60
atives 5, which can be easily separated by silica gel
column chromatography.
In conclusion, we have described a concise and flexible
route to fullerene-derived photoaffinity labels with
potential utility in enzyme tagging and the elucidation
26
To synthesize the C60-phenyldiazirine derivative 9, we
first attempted the reaction of fulleropyrrolidine 2 with
an acid chloride, but this reaction did not give useful
amounts of the desired product. Despite attempts to
activate the acyl moiety by a succinimide group using
of the binding sites of protein to C60.
Acknowledgements
4
-(3-trifluoromethylazirino)benzoicsuccinimide, product
formation was not observed. In sharp contrast, how-
ever, the use of Yamaguchi reagent 8 to couple 6 and 2
gave good yields of cis- and trans-C60-phenyldiazirine
We are grateful to thank Dr. Nobuo Ikota in National
Institute of Radiological Sciences for his fruitful dis-
cussions on the synthetic methodologies. We also thank
Prof. Dr. Jeffrey W. Bode at University of California
Santa Barbara for his advice. This research was sup-
ported in part by Grant-in-Aid for Research on Ad-
vanced Medical Technology from Ministry of Labor,
Health and Welfare (TT, YY), Grant-in-Aid for
Encouragement of Young Scientists from Ministry of
Education, Science, Sports and Culture, Japan [Nos.
08772153 (YY), 09772037 (YY) and 13771418 (YY)],
Grant-in-Aid for young researchers from Ministry of
Health and Welfare (YY) and Grant-in-Aid for young
researchers from Human Science Foundation (YY).
27
derivatives 9. These stereoisomers are readily sepa-
rated by silica gel chromatography. Compounds 5 and 9
28
were characterized by spectroscopic methods. The cis-
and trans-stereochemistry of each compounds were
23;24
determined according to the reported studies.
In addition to the potential utility of fullerene-derived
photoaffinity labels for elucidating the active site of C60
binding to enzymes such as GST and HIV-1 protease,
the ability to selectively tag a protein or enzyme with
fullerene may offer a new approach to the detection of

E. Okada et al. / Tetrahedron Letters 45 (2004) 527–529
25. To a solution of C60 (36 mg, 0.05 mmol) and
529
References and Notes
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,
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1
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2
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, (9.7 mg, 0.042 mmol) with Et N (10 lL) in toluene
2 mL), 2,4,6-trichlorobenzoyl chloride (10 lL) was added
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(
3
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1
1
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1
1
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2
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,
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7
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710.
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.21(d, J ¼ 6:0, 6H), 5.74 (q, J ¼ 6:6, 2H), 7.22 (d,
3
1
3
J ¼ 8:4, 2H), 7.99 (d, J ¼ 8:4, 2H); C NMR (CDCl
,
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1
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ꢀ
1
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ꢀ1
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3
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1
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ꢀ
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ꢀ
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