Table 1 Selected cyclic voltammetry data for fullerene amino acidsa
presently investigating the cellular uptake, transport through
porcine skin, and inhibitory binding with HIV-1 PR and
carbonic anhydrase of this new class of fullerene amino acids.
Financial support for this work is provided by the Robert
A. Welch Foundation. We thank Dr Jianhua Yang for the
cellular toxicity studies.
1
2
Compound
Formal potential, E /V
Structure
C60
ꢀ0.247, ꢀ0.741, ꢀ1.327
ꢀ0.280, ꢀ0.892b
Boc-aza-C60-Lys (3a)
Boc-aziridino-C60-Lys (3b)
Boc-aza-C60-Phe (1a)
Boc-Baa
5,6-Open
6,6-Closed
5,6-Open
6,6-Closed
ꢀ0.319, ꢀ0.792b
ꢀ0.270, ꢀ0.744, ꢀ1.318
ꢀ0.377, ꢀ0.847, ꢀ1.489
a
Potentials are given in volts versus a reference electrode, Ag|AgCl.
Third reduction not observed.
b
Notes and references
z An amino derivatized, Na-protected amino acid (1 equiv.), imidazole-
sulfonyl-azideꢂHCl,16 triethylamine (2 equiv.) and 4 mol% ZnCl2 were
dissolved in a solution of MeOH : MeCN (3 : 2) and stirred in the
dark under N2 for 12 h.16 The mixture was concentrated then diluted
with ethyl acetate and washed with AcOH (10%, 3ꢃ) and brine (1ꢃ),
dried over MgSO4, and concentrated under vacuum. Flash chromato-
graphy is necessary at the gram scale. C60 (1 equiv.) was vacuum dried
for 45 min and o-dichlorobenzene (ODCB) was added. To this was
added the Na-protected, azido-amino acid (1 equiv.) with stirring.
The temperature was maintained between 80–100 1C for the Phe
derivatives and 60–70 1C for the Lys derivatives. The reaction was
monitored by RP-HPLC (5–95% IPA in 0.1% TFA over 30 min, then
95% IPA for 20 min). Typically, within 24 h the mono addition
product was isolated by column chromatography.
each successive addition to its core. For C60 derivatives, the
first electron transfer is shifted to more negative potentials
while maintaining the characteristic redox properties of
pristine C60.13 In this respect, Baa (6,6-closed) is consistent
with other similar closed ring structures, with its first reduction
occurring at ꢀ0.377 V. In contrast, in the open ring structure
of the aza-C60 derivatives (1a and 3a), only sp2 hybridized
carbons are present on the cage allowing these compounds to
behave more like pristine C60 in their reductive ability under
the specified conditions.14 Table 1 shows the detailed redox
potential data for the aza and aziridino-fullerene derivatives
relative to C60 and the previously reported Baa. For C60 and
the aza-fulleroid derivatives the first reduction occurs between
ꢀ0.248 and ꢀ0.288 V. However, the closed ring structures
with two sp3 C60 carbons do not undergo their first reduction
until more negative potentials, cathodically shifted approxi-
mately 0.04–0.15 V relative to the aza derivatives.
y MALDI/TOF, DCTB matrix, (Mꢀ): Fmoc-aza-C60-Phe (1a) m/z
1120.23 (calcd 1120.14); Boc-aza-C60-Phe (2a) m/z 998.16 (calcd
998.13); Boc-aza-C60-Lys (3a) m/z 964.06 (calcd 964.14); aza-C60-Phe
(4) m/z 898.02 (calcd 898.07); aza-C60-Phe-Gly-OtBu (matrix CHCA)
(5) m/z (M + 1) 1234.11 (calcd 1234.23).
1 J. Yang and A. R. Barron, Chem. Commun., 2004, 2884.
2 J. Yang, L. B. Alemany, J. Driver, J. D. Hartgerink and
A. R. Barron, Chem.–Eur. J., 2007, 13, 2530.
3 T. Da Ros and M. Prato, Chem. Commun., 1999, 663;
N. Tagmatarchis and H. Shinohara, Mini-Rev. Med. Chem.,
2001, 1, 339.
4 J. G. Rouse, J. Yang, A. R. Barron and N. A. Monteiro-Riviere,
Toxicol. in Vitro, 2006, 20, 1313.
Given that the aza-C60-Phe and aza-C60-Lys are easier to
prepare on a large scale, and therefore of much greater utility
for subsequent studies as compared to Baa, it is important to
ensure that the change in linkage unit does not alter the cellular
toxicity significantly. The toxic response of the deprotected
aza-C60-Phe (4) was determined by MTT assay on three
different neuroblastoma cell lines (JF, SK-N-AS, and LAN-1)
at concentrations from 0.004–0.4 mg mLꢀ1. No appreciable
toxicity was observed for aza-C60-Phe at any of the tested
concentrations after a 24 hour incubation period.
5 J. G. Rouse, J. Yang, J. P. Ryman-Rasmussen, A. R. Barron and
N. A. Monteiro-Riviere, Nano Lett., 2007, 7, 155.
6 S. Durdagi, C. T. Supuran, T. A. Strom, N. Doostdar,
M. K. Kumar, A. R. Barron, T. Mavromoustakos and
M. G. Papadopoulos, J. Chem. Inf. Model., 2009, 49, 1139;
A. Innocenti, S. Durdagi, N. Doostdar, T. A. Strom,
A. R. Barron and C. T. Supuran, Bioorg. Med. Chem., 2010, 18,
2822.
To demonstrate the feasibility of using aza-C60-Phe for SPPS
the coupling reaction of compound 2a to a tert-butyl ester of
glycine (NH2-Gly-OtBu) was carried out in 4 : 1 DCM/DMF
in the presence of HBTU and NEt3 in a sonication bath for 2 h
(5, Scheme 1). The reaction products were characterized by
MALDI MS.y After 2 hours, only starting materials and
products were detected. Given the rapid oxidation of 3a in
air, only Lys derivative 3b will be useful for SPPS.
7 M. Prato, C. Li and F. Wudl, J. Am. Chem. Soc., 1993, 115, 1148.
8 A. Hirsch and M. Brettreich, Fullerenes: Chemistry and Reactions,
Wiley-VCH Verlag GmbH & Co., Weinheim, Germany, 2005,
p. 134.
9 D. Pantarotto, A. Bianco, F. Pellarini, A. Tossi, A. Giangaspero,
I. Zelezetsky, J. Briand and M. Prato, J. Am. Chem. Soc., 2002,
124, 12543.
10 E. F. Scriven, Azides and Nitrenes: Reactivity and Utility,
Academic Press, Inc, Orlando, FL, 1984, p. 2.
11 J. C. Hummelen, M. Prato and F. Wudl, J. Am. Chem. Soc., 1995,
117, 7003; S. Xiao, Y. Li, H. Fang, H. Li, H. Liu, Z. Shi, L. Jiang
and D. Zhu, Org. Lett., 2002, 4, 3063; D. M. Guldi,
In summary, we report a general route to fullerene deriva-
tized amino acids through the dipolar addition of azido amino
acids, both alkyl and aryl. The ease of this procedure, including
purification, is evident over previously reported methods.1,2
The newly reported C60 amino acids differ in their linkage
to C60, resulting in mostly a 5,6-open, aza-fulleroid structure
(1a, 2a, 3a) rather than a 6,6-closed cyclohexyl ring on C60. We
have demonstrated the coupling ability of 2a indicating its
ability for incorporation into a peptide sequence, and the
unprotected phenylalanine derivative has shown inhibitory
binding with HIV-1 PR at nM concentrations.15 We are
H. Hungerbuhler, I. Carmichael, K.-D. Asmus and M. Maggini,
¨
J. Phys. Chem. A, 2000, 104, 8601.
12 C. S. Foote, Top. Curr. Chem., 1994, 169, 347.
13 C. Boudon, J. Gisselbrecht, M. Gross, A. Herrmann,
M. Ruttimann, J. Crassous, F. Cardullo, L. Echegoyen and
¨
F. Diederich, J. Am. Chem. Soc., 1998, 120, 7860.
14 C. Boudon, J. Cisselbrecht and M. Cross, Helv. Chim. Acta, 1995,
78, 1334.
15 S. Durdagi, C. T. Supuran, N. Doostdar, T. A. Strom,
A. R. Barron, T. Mavromoustakos and M. G. Papadopoulos,
2010, submitted.
16 E. D. Goddard-Borger and R. V. Stick, Org. Lett., 2007, 9, 3797.
ꢁc
This journal is The Royal Society of Chemistry 2010
4766 | Chem. Commun., 2010, 46, 4764–4766