Synthesis of novel fullerene amino acids and their multifullerene peptides

Article abstract of DOI:10.1016/j.tetlet.2004.07.088

[60]-Fullerene functionalized amino acids with 4-6 methylene spacers from α-carbon to the nitrogen atom of fulleropyrolidine and corresponding multifullerene peptides have been synthesized.

Full text of DOI:10.1016/j.tetlet.2004.07.088

Tetrahedron
Letters
Tetrahedron Letters 45 (2004) 7137–7140
Synthesis of novel fullerene amino acids and their
multifullerene peptides
Louis A. Watanabe,a Mohammed P. I. Bhuiyan,b Binoy Jose,b
Tamaki Katob and Norikazu Nishinob,
*
aFaculty of Engineering, Kyushu Institute of Technology, Kitakyushu 808-8550, Japan
bGraduate School of Life Science and Systems Engineering, Kyushu Institute of Technology, Kitakyushu 808-0196, Japan
Received 14 May 2004; revised 7 July 2004; accepted 21 July 2004
Available online 13 August 2004
Abstract—[60]-Fullerene functionalized amino acids with 4–6 methylene spacers from a-carbon to the nitrogen atom of fulleropyro-
lidine and corresponding multifullerene peptides have been synthesized.
Ó 2004 Elsevier Ltd. All rights reserved.
Considerable effort in fullerene chemistry has been
directed to establish this novel form of carbon as a
standard building block in organic synthesis.1,2
Fullerene-based molecules have a wide variety of inter-
esting characteristics including nonlinear optical
properties, superconductivity and biological properties.3
Biological properties of [60]-fullerene derivatives com-
prise of neuroprotective, enzymatic, antiapoptotic, anti-
bacterial, DNA photocleaving, nitric oxide synthase
inhibiting and chemotactic activities.4 Fullerene amino
acids and peptides are interesting targets due to their
importance in biological applications and also in physico-
chemical studies.5 Fullerene peptides were recently
reported as antimicrobial agents6 and also as a molecu-
lar ruler for electron transfer system.7
converted to various functional groups such as amino
group of lysine homologues. By attaching a carboxy-
methyl group to the amino group, they may be reacted
with C60 according to the literature.6 Therefore, we at-
tempted to prepare the novel fullerene amino acid deriv-
atives useful in peptide synthesis (2a–c and 3a–c).
Prato and co-workers reported a C60-functionalized
amino acid (1) derived from glutamic acid and incorpo-
rated it into a peptide.6 This is an important development
to introduce fullerene moiety to bioactive peptides. The
compound 1 has 6-atomed linker from the a-carbon of
glutamic acid to the nitrogen atom of fulleropyrrolidine.
The connecting amide could play important roles in the
solubilities or in the interaction with biomolecules. On
the other hand, we conveniently prepared optically active
a-amino-x-bromoalkanoic acid (Abn) as useful artificial
amino acids.8 The side chain alkyl bromide can be easily
Since these fullerene amino acids were expected to in-
crease the solubility in organic solvents, the synthetic
method for multifullerene peptides were also examined
using 2c or 3c with same spacer length as 1. To our
knowledge, there is no report on the synthesis of a
dipeptide or tripeptide with two or more fullerene
moieties. Here, we report the synthesis of C60-function-
alized amino acids with different methylene spacers
Keywords: Fullerenes; Amino acids; Fullerene peptide.
*
Corresponding author. Tel./fax: +81-93-695-6061; e-mail: nishino@
life.kyutech.ac.jp
0040-4039/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2004.07.088

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L. A. Watanabe et al. / Tetrahedron Letters 45 (2004) 7137–7140
(Afn, 2-amino-n-fulleropyrrolidinoalkanoic acid, where
n = 6–8) and the synthesis of multifullerene peptides
containing Af8s (for instance, Boc-Af8-Af8-Af8-Ala-
OBzl).
addition of fullerene to azomethine ylide.6 When the
ratio of fullerene is lower, such as, 1:1 or 2:1, there is
a possibility of multiaddition of the amino acid to fulle-
rene resulting in lower yield of the desired product. With
a ratio of 3:1, the possibility of single addition product is
high and therefore we observed a high yield. However,
no further enhancement in yield was observed with in-
crease in fullerene ratio. The excess amount of C60 was
conveniently removed from 8a–c by silica gel column
with toluene/ethyl acetate (95/5, v/v). Compounds 8a–c
were fully characterized by 1H and 13C NMR and
high-resolution FAB MS.11 Compounds 8a–c were
deprotected using trifluoroacetic acid to afford the free
fullerene amino acids (Afn) 9a–c. The amino group of
Afn was then protected to Fmoc-Afn-OH 2a–c. Boc
protection was also carried out to give Boc-Afn-OH
3a–c (Scheme 1).
The synthesis of C60-functionalized amino acids was
started from recently reported Boc-a-amino-x-bromo-
alkanoic acids 4b and 4c (Boc-Abn-OH),8 whose carb-
oxyl group was protected with t-butyl group9 and then
the alkyl bromide of the side chain was transformed to
the amine by the treatment with potassium phthalimide
in the presence of KI and subsequent reaction with hy-
drazine to yield the corresponding amines 5b and 5c.
The 5a was obtained by the protection of the carboxyl
group of Boc-Lys(Z)-OH with t-butyl group9 followed
by hydrogenation. The amino group of 5a–c was then
protected with 4-nitrobenzenesulfonyl (NBS) group
and reacted with benzyl bromoacetate to yield 6a–c.
The NBS protection of the amine allowed 1:1 reaction
with benzyl bromoacetate in quantitative yield. Reac-
tions without protecting with NBS group cause double
or triple incorporation of benzyl bromoacetate to the
amino group of 5a–c resulting in poor yields. Side chain
deprotection using piperidine/n-BuSH10 and subsequent
benzyl ester hydrogenation yielded 7a–c. Amino acids
7a–c were reacted with C60 and paraformaldehyde to
yield fullerene functionalized amino acid 8a–c in about
60% yield. Fullerene addition reaction to 7c was carried
out in six different ratios such as, 1:1, 1.5:1, 2:1, 3:1, 4:1
and 6:1. By careful examination of the ratio of amino
acid 7c and C60, we found that the use of 3equiv of
C60 gives the optimized yield of the desired product
(see Supplementary data for details). This yield is about
20% higher than that reported for the 1,3-dipolar cyclo-
One of the major problems associated with fullerene
compounds is the poor solubility in many common
solvents. Therefore, we checked the solubility of the
N-protected Afn in different solvents. These Afn
derivatives are fairly soluble in dichloromethane,
chloroform, toluene, chlorobenzene and DMF. For
example, 5.5mg of Boc-Af8-OH is soluble in 1mL of
DMF. This solubility allows the N-protected fullerene
amino acids to be used in most of the coupling
conditions. Even under lower solubility conditions, we
observed higher yields for coupling and deprotection
reactions of Afn containing peptides.
As for the peptide synthesis, initially we used solid phase
peptide synthesis with Wang-PEG-resin preloaded with
glycine and Fmoc-Af8-OH was coupled. However, the
Scheme 1. Reagents, conditions and yields: (i) Boc2O, t-BuOH, DMAP, rt, 1h, 80–84%; (ii) potassium phthalimide, KI, DMF, rt, 1h, 80–85%;
(iii) NH2–NH2ÆH2O, EtOH, rt, 1h, 95–100%; (iv) 4-nitrobenzenesulfonyl chloride, Et3N, CHCl3, 0°C, 30min, 90–95%; (v) benzyl bromoacetate,
K2CO3, KI, DMF, 0°C, 24h, 90–95%; (vi) piperidine, DMF, n-BuSH, rt, 6h, 85–90%; (vii) Pd–C, H2, 6h, 95–100%; (viii) C60, (CH2O)n, toluene,
reflux, 1.5h, 60%; (ix) TFA, CH2Cl2, 3h, 100%; (x) Fmoc-OSu, Na2CO3, H2O/dioxane (1/1), 24h, 95–100%; (xi) Boc2O, H2O/dioxane (1/1),
Et3N, 0°C, 24h, 100%.

L. A. Watanabe et al. / Tetrahedron Letters 45 (2004) 7137–7140
7139
tendency of the fullerene moiety to embed onto the resin
made it difficult to recover the product in reasonable
yield. A similar problem of solid phase peptide synthesis
with fullerene amino acids was reported by Prato and
co-workers.6 Due to this drawback with solid phase syn-
thesis of fullerene peptides, we proposed to use solution
phase synthesis.
The Fmoc-Af8-OH was coupled with H-Gly-OBzl using
HATU in DMF to yield Fmoc-Af8-Gly-OBzl (10) in al-
most quantitative yield. The product was characterized
by high-resolution FAB MS. As we know that the com-
mon Fmoc deprotection reagents such as piperidine and
morpholine result in a 1,4-addition to fullerene in the
presence of oxygen, leading to the generation of bis-
and tetra-adducts,12 the deprotection of Fmoc group
was carried out using 5% DBU in DMF. However, for-
mation of bis-adduct of fullerene with DBU was ob-
served during the deprotection reaction. Therefore, we
changed the strategy to the Boc method using Boc-
Af8-OH.
Figure 1. Conformational outline of 13.
effect. One of the possible conformations was obtained
as shown in Figure
1
(/1 = À75.1°, w1 = 68.0°;
/2 = À172°, w2 = À83.6°; /3 = À68.5°, w3 = 179°; /4 =
À29.8°, w4 = À177°). The main chain conformation is
not similar to a-helix or b-strand. The fullerene moieties
of the peptide may disrupt the conformation of 13 in the
energy-minimized structure.
In summary, we described the side chain modification of
a-amino-x-bromoalkanoic acid to lysine homologues
and to the fullerene amino acids. Further, we success-
fully synthesized multifullerene peptides using Boc pro-
tected fullerene amino acids by solution phase method.
The attraction of this synthetic approach is the general-
ity of the method for the preparation of fullerene amino
acids with different spacer lengths between the a-carbon
and the nitrogen atom of the fulleropyrolidine. These
novel amino acids have potential as building blocks
for the synthesis of peptides with interesting biological
and physicochemical properties.
Boc-Af8-OH was coupled with H-Ala-OBzl using
HBTU/HOBt in DMF to yield Boc-Af8-Ala-OBzl (11)
quantitatively. The dipeptide 11 was precipitated from
the reaction solvent as the coupling reaction proceeded.
The product was simply filtered and washed with DMF
and small volume of CH2Cl2. Since the product was
pure enough for further step, the analytical amount
was passed through a silica gel column with toluene/
ethyl acetate (95/5, v/v). The Boc group of 11 was suc-
cessfully removed by treatment with trifluoroacetic acid.
The dipeptide benzyl ester was again coupled with
Boc-Af8-OH to give Boc-Af8-Af8-Ala-OBzl (12) as
described above. The tripeptide 12 was also precipitated
from the reaction solvent in 97% yield as filtration. Rep-
etition of the deprotection and coupling steps gave Boc-
Af8-Af8-Af8-Ala-OBzl (13) as a precipitate from DMF
in 95% yield. A part of the product was applied to silica
gel column with toluene/ethyl acetate (95/5, v/v). The
protected peptides were characterized by MALDI
TOF MS and 1H NMR using deuterized o-dichlorobenz-
ene, data being given in Ref. 11 and Supplementary
data. The tetrapeptide 13 resisted the deprotection with
trifluoroacetic acid resulting in failure in further elonga-
tion. However, we will find out appropriate conditions
in the future study.
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.tetlet.
1
2004.07.088. This contains H and 13C NMR spectra
of selected compounds and optimization data for
fullerene to amino acid coupling reaction.
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(m, 2H), 1.58–1.49 (m, 4H), 1.47 (s, 9H), 1.42 (s, 9H); 13C
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146.1, 145.7, 145.6, 144.9, 143.5, 142.9, 142.7, 142.5, 142.3,
140.5, 136.6, 81.8, 79.8, 71.2, 68.4, 55.2, 33.3, 29.7, 29.2,
28.5, 28.2, 27.9, 25.7; high-resolution FAB MS (M+1)+
1077.2719, calcd for C79H37N2O4 1077.2753; 9a: high-
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C68H17N2O2 893.1290; 9b: high-resolution FAB MS
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high-resolution FAB MS (M+1)+ 921.1592, calcd for
C70H21N2O2 921.1603; 3a: high-resolution FAB MS
(M+1)+ 993.1804, calcd for C68H17N2O2 993.1814; 3b:
high-resolution FAB MS (M+1)+ 1007.1962, calcd for
C74H27N2O4 1007.1971; 3c: high-resolution FAB MS
(M+1)+ 1021.2093, calcd for C75H29N2O4 1021.2127; 12:
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(m, 2H), 4.71–4.68 (m, 2H), 4.29–4.24 (m, 9H), 2.98–2.95
(m, 4H), 1.98–1.45 (m, 32H); MALDI TOF MS (M+1)+
2084.92, calcd for C155H57N5O6 2085.14; 13: 1H NMR
(500MHz, C6D4Cl2): d 5.21–5.11 (m, 3H), 4.84–4.76 (m,
2H), 4.56–4.45 (m, 2H), 4.30–4.23 (m, 12H), 3.02–2.93 (m,
6H), 1.98–1.45 (m, 42H); MALDI TOF MS (M+1)+
2989.81, calcd for C225H76N7O7 2989.05.
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11. All the new compounds were characterized by NMR and
mass spectral data. Selected spectroscopic data: 8a: 1H
NMR (500MHz, CD2Cl2): d 5.05 (d, 1H), 4.41 (s, 4H),
4.22 (m, 1H), 3.09 (m, 2H), 1.95 (m, 2H), 1.82 (m, 2H),
1.69 (m, 2H), 1.49 (s, 9H), 1.45 (s, 9H); 13C NMR
(CD2Cl2): d 172.3, 155.8, 147.6, 146.6, 146.4, 146.1, 145.8,
145.7, 145.0, 143.5, 143.0, 142.7, 142.5, 142.3, 142.2, 140.5,
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CD2Cl2): d 5.06 (d, 1H), 4.4 (s, 4H), 4.18 (m, 1H), 3.07
(m, 2H), 1.97–1.85 (m, 4H), 1.76–1.61 (m, 2H), 1.58–1.48
(m, 2H), 1.45 (s, 9H), 1.41 (s, 9H); 13C NMR (CD2Cl2): d
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