Synthesis and characterization of water-soluble amino fullerene derivatives

Article abstract of DOI:10.1021/ol990312a

A series of amino-substituted methanofullerene derivatives were prepared by mono-, tris-, and hexa-Bingel-Hirsch reactions using an N-protected malonate derivative. Upon scission of the protecting groups, the e,e,e-tris- and octahedral T_h hexa-methano amino fullerenes were found to be among the most water-soluble fullerene derivatives yet prepared. ³He NMR data on corresponding adducts of ³He@C₆₀ helped verify the assigned structures. UV spectral studies confirmed the influence of aggregation on solubility of these adducts in water.

Full text of DOI:10.1021/ol990312a

ORGANIC
LETTERS
2000
Vol. 2, No. 8
1011-1014
Synthesis and Characterization of
Water-Soluble Amino Fullerene
Derivatives
Christine F. Richardson, David I. Schuster,* and Stephen R. Wilson*
Department of Chemistry, New York UniVersity, New York, New York 10003
steVe.wilson@nyu.edu
Received October 4, 1999 (Revised Manuscript Received March 9, 2000)
ABSTRACT
A series of amino-substituted methanofullerene derivatives were prepared by mono-, tris-, and hexa-Bingel−Hirsch reactions using an N-protected
malonate derivative. Upon scission of the protecting groups, the e,e,e-tris- and octahedral T hexa-methano amino fullerenes were found to
h
3
be among the most water-soluble fullerene derivatives yet prepared. He NMR data on corresponding adducts of 3He@C60 helped verify the
assigned structures. UV spectral studies confirmed the influence of aggregation on solubility of these adducts in water.
Recently, several fullerene derivatives have been shown to
possess significant biological activity.1,2 The extraordinary
chemical and physical properties of C60 and its derivatives
confer activity of these compounds as potential therapeutic
agents against HIV3 and neurodegenerative diseases4 and as
agents for DNA cleavage.5 Most recently, certain C60
derivatives have been shown to be excellent inhibitors of
nitric oxide synthase.6 Potential applications of fullerenes
in medicine is enhanced by the finding that most fullerene
derivatives have unusually low toxicity compared with
typical drugs.7 However, the insolubility of fullerenes in
aqueous media has slowed the realization of these biological
applications. The history of water-soluble fullerene deriva-
tives is limited to only a few compounds, most of which
contain carboxylic acids as the solubilizing group. Herein,
we report the synthesis of a series of amino-substituted
methanofullerenes, two of which exhibit pronounced water
solubility.
Our main objective was to prepare simple fullerene
derivatives containing polar groups to enhance their water
solubility. A series of derivatives containing an increasing
number of malonate appendages with polar groups on the
ends were investigated. The reliable Bingel-Hirsch addition
of malonates to C60 was used since it allowed the introduction
of two polar headgroups for every malonate attached. The
malonate reagent 1 was prepared as in Scheme 1 by treatment
(1) Jensen A. W.; Wilson, S. R.; Schuster, D. I. Biorg. Med. Chem. Lett.
1996, 4, 767-779.
(2) (a) Prato, M.; DelaRosa, M. Chem. Commun. 1999, 663-669; (b)
Wilson, S. R. In The Fullerene Handbook; Kadish, K., Ruoff, R., Eds. In
press.
Scheme 1. Synthesis of Malonate 1
(3) Schinazi, R. F.; Sijbesma, R.; Srdanov. G.; Hill, C. L.; Wudl, F.
Antimicrob. Agents Chemother. 1993, 37, 1707-1710.
(4) Dugan, L. L.; Turetsky, D. M.; Du, C.; Lobner, D.; Wheeler, M.;
Almli, C. R.; Shen, C. K. F.; Luh, T. Y.; Choi, D. W.; Lin, T. S. Proc.
Natl. Acad. Sci. U.S.A. 1997, 94, 9434-9439.
10.1021/ol990312a CCC: $19.00 © 2000 American Chemical Society
Published on Web 03/30/2000

of 5 g (2 equiv) of tert-butyl N-(3-hydroxypropyl)carbamate
in 250 mL of dry methylene chloride with 1.97 g (1 equiv)
of distilled malonyl chloride and 2.21 g (2 equiv) of pyridine.
Following purification by column chromatography on silica
gel using 1:1 hexane/ethyl acetate as the eluant, 3.20 g
(54.7%) of malonate 1 was obtained (Rf ) 0.41, silica gel,
1:1 EtOAc:hexane): 1H NMR (200 MHz, CDCl3) δ 4.85
(s, 1H), 4.19 (t, 4H), 3.37 (s, 2H), 3.17 (q, 4H), 1.82 (m,
4H), 1.41 (s, 18H); CI-MS calcd for C19H34N2O8 [M + NH3]
419, found 419; 13C NMR (200 MHz, CDCl3 δ 166.9, 156.3,
79.7, 63.6, 42.0, 37.9, 29.5, 28.5 ppm).
Preparation of the C60 monoadduct 2a proceeded smoothly
according to the general procedure of Hirsch and co-
workers.8 To that end, 500 mg (1 equiv) of C60 and 331 mg
(1.5 equiv) of CBr4 were dissolved in 300 mL of toluene by
sonication, followed by the addition of 418 mg (1.5 equiv)
of malonate 1 and 0.3 mL (3 equiv) of DBU. The reaction
was complete in 30 min. After conventional workup, the
crude reaction mixture was chromatographed on a silica gel
column using toluene as eluent to remove unreacted C60,
followed by 10:1 toluene/ethyl acetate to yield, after evapo-
ration and drying, 334.8 mg (51.8%) of the desired mono-
adduct 2a (Rf ) 0.09, silica gel, 100:5 toluene:ethyl
acetate): 1H NMR (200 MHz CDCl3) δ 4.90 (bs), 4.57 (t),
3.32 (q), 2.06 (m), 1.46 (s); FAB-MS calcd for C79H32N2O8
1137.129, found 1137.4). The synthesis of 2a was repeated
Figure 1. 3He-NMR Spectrum of e,e,e-tris methanofullerene 3a.
3
3
Signal A is He@C60 (0 ppm) and B is He@3a (-11.966 ppm).
gel column using 2:1 toluene/ethyl acetate and an orange-
yellow band was isolated. This band was then rechromato-
graphed on a preparative TLC plate using 2:1 toluene/ethyl
acetate to yield 4a as a yellow solid (Rf ) 0.42, silica gel,
2:1 ethyl acetate:toluene); 13C NMR (200 MHz, CDCl3) δ
164.13, 156.44, 146.18, 141.51, 69.00, 65.28, 37.91, 29.23,
29.04; FAB-MS calcd for C174H192O48N12 3216, found
3217.9). In the 13C NMR spectrum, only three distinct signals
appear at δ 69, 141, and 145 ppm, corresponding, respec-
tively, to the three types of sp3 and sp2 carbon atoms
remaining on the fullerene core. This spectrum compares well
with the literature report of the analogous Th-symmetrical
hexa adduct of diethyl malonate to C60 reported by Hirsch
(see Figure 2).11 Further evidence supporting the octahedral
3
3
using He@C60 to give the corresponding He@2a, which
3
showed a sharp singlet at δ -8.045 ppm in its He NMR
spectrum, which is in the expected range for a monomethano
adduct across a 6,6 bond of C60.9
The number of malonate substituents connected to the
fullerene core was then increased to three by using 3.3 equiv
of CBr4, 3.3 equiv of malonate 1, and 6.6 equiv of DBU.
After workup, the crude residue was chromatographed on
silica gel using 10:1 toluene/ethyl acetate followed by 2:1
toluene:ethyl acetate to give the e,e,e isomer of the tris
malonate adduct 3a as a red-orange solid in 10.4% yield (Rf
) 0.25, silica gel, 2:1 toluene:ethyl acetate): FAB-MS calcd
for C117H96O48N12 1968, found 1969). The UV-vis absorp-
tion spectrum of 3a was identical with that of the e,e,e Hirsch
tris-ethyl ester.10 The synthesis of 3a was repeated with
3He@C60 to give the corresponding 3He@3a which showed
a sharp singlet in its 3He NMR spectrum at δ -11.966 ppm,
which is in the expected position for an e,e,e-tris adduct to
C60 (see Figure 1).9d
Next, we turned our attention to the synthesis of the
octahedral (Th) hexa-malonate derivative, 4b, a derivative
with 12 amino groups, which was expected to show even
greater hydrophilicity. This symmetrical addition pattern is
well-known from the work of Hirsch.11 Thus, 10 equiv of
CBr4, 10 equiv of malonate 1, and 20 equiv of DBU were
subjected to the same reaction conditions as above. Following
workup, the crude reaction mixture was separated on a silica
Figure 2. Comparison of 13C NMR spectrum of hexa-adducts.
symmetry of 4a is its UV spectrum which is in excellent
agreement with that reported for the Hirsch hexa-adduct.10
The 3He NMR spectrum of 3He@4a showed a sharp singlet
at δ -12.1 ppm, which is consistent with that reported for
a Th-symmetrical hexa-adduct.9d
(5) Ikeda, A.; Hatano, H.; Kawaguchi, M.; Suenaga, H.; Shinkai, S. Chem.
Commun. 1999, 1403-1404.
(6) Wolf, D. J.; Mialkowski, M.; Papoiu, A.; Richardson, C. F.; Schuster,
D. I.; Wilson, S. R., submitted for publication.
(7) Schuster, D. I.; Wilson, S. R.; Schinazi, R. F. Biorg. Med. Chem.
Lett. 1996, 1253-1256.
The carbamate protecting groups on 2a, 3a, and 4a were
removed using trifluoroacetic acid (see Scheme 2). Following
1012
Org. Lett., Vol. 2, No. 8, 2000

Similarly, hexa-adducts 4a and 4b were also compared.
The UV spectra were compared in methanol and water (see
Figure 4), and a broadening of the bands in water was
Scheme 2. Deprotection of tBoc-Protected Amines
deprotection, the water solubility of 2b, 3b, and 4b was
determined spectroscopically by UV analysis. Extinction
coefficients for each adduct were determined by analysis of
standard solutions. The corresponding extinction coefficient
was then used to calculate the amount in mg/mL of each
derivative that dissolved in water. To that end, saturated
solutions of each adduct in water were prepared and allowed
to stir for 36 h. Each solution was then centrifuged and a
portion was analyzed by UV spectroscopy. Thus, 0.64 mg/
mL of monoadduct 2b, 152 mg/mL of tris adduct 3b, and
418 mg/mL of hexa adduct 4b were found to be soluble in
water at room temperature.12 Previously, Hirsch and Brett-
reich reported a polycarboxylated fullerene dendrimer with
a water solubility of 254 mg/mL using a similar method.13
It has been known for some time that fullerene derivatives
aggregate and this property might enhance their solubility.
In 1995, Guldi and co-workers reported that evidence of
clustering could be obtained from UV spectra, specifically,
broadening of the bands is characteristic of aggregation.14
To investigate whether aggregation was influencing the
solubility of our compounds, UV studies were initiated.
The UV spectra of 2b, 3b, and 4b were examined over a
range of concentrations from 8 to 20 µM at 257 nm.
Pronounced red shifts were observed as the concentration
decreased for 2b, 3b, and 4b. For a given absorbance,
hypochromism on the order of 10-50% is observed in
aggregates.15 The UV spectrum of 3a in methanol was
compared to that of 3b in water (see Figure 3). In water, the
spectrum is characterized by broader bands compared to that
in methanol.
Figure 4. Comparison of UV spectra of hexa-adducts 4a in MeOH
and 4b in water.
observed. Similar band broadening was observed when the
organic solvent was THF. A table of values for λmax and the
corresponding extinction coefficients in both organic solvent
(THF) and water for compounds 2-4a,b are presented below
(see Table 1).
Table 1. λmax and Values for ꢀ for Compounds 2a, 3a, and 4a
in THF and 2b, 3b, and 4b in Water
λmax
ꢀ
2a
2b
3a
3b
4a
248
289
368
212
276
342
239
263
287
212
248
265
213
245
278
319
336
243
265
290
316
335
118052
44789
19842
36750
37600
32850
37062
39125
46200
76000
67250
46900
75000
33950
32000
17550
15700
79500
49400
53600
48250
39600
4b
In summary, a series of mono, tris, and hexa adducts to
C60 of malonates with N-protected amino headgroups have
been synthesized and characterized. Following removal of
the protecting groups, the tris- and hexa-amino adducts were
Figure 3. Comparison of UV spectra of tris-adducts 3a in MeOH
and 3b in water.
(8) Camps, X.; Hirsch, A. J. Chem. Soc., Perkin Trans. 1 1997, 1595-
1596.
Org. Lett., Vol. 2, No. 8, 2000
1013

found to be highly water soluble. In addition to conventional
spectroscopic methods, He NMR was used to confirm the
structures of these new compounds. Through UV studies,
aggregation was found to affect the solubility of the mono,
tris, and hexa adducts.
3
(9) (a) Schuster, D. I.; Cao, J.; Kaprinidis, N.; Wu, Y.; Jensen, A. W.;
Lu, Q.; Wang, H.; Wilson, S. R. J. Am. Chem. Soc. 1996, 118, 5639-
5647. (b) Cross, R. J.; Jime´nez-Va´zquez, H. A.; Lu, Q.; Saunders: M.;
Schuster, D. I.; Wilson, S. R.; Zhao, H. J. Am. Chem. Soc. 1996, 118,
11454-11459. (c) Jensen, A. W.; Khong, A.; Saunders: M.; Wilson, S.
R.; Schuster, D. I. J. Am Chem. Soc. 1997, 119, 7303-7307. (d) Ru¨ttimann,
M.; Haldimann, LR. F.; Isaacs, L.; Diederich, F.; Khong, A.; Jime´nez-
Va´zquez, H.; Cross, R. J.; Saunders: M. Chem. Eur. J. 1997, 3, 1071-
1076.
(10) Hirsch, A.; Lamparth, I.; Grosser, T.; Karfunkel, H. R. J. Am. Chem.
Soc. 1994, 116, 9385-9386.
(11) Lamparth, I.; Herzog, A.; Hirsch, A. Tetrahedron 1996, 52, 5065-
5075.
Acknowledgment. We thank Dr. Rene´ Bensasson (Mu-
se´um National d’Histoire Naturelle, Paris) for his helpful
comments regarding aggregation. C.F.R. thanks Ms. Ute
Schurrath (Novartis) for help with the solubility experiments.
3
We thank Anthony Khong (Yale University) for the He
NMR data. We are grateful for support of this work by the
National Science Foundation, CHE 9712735, and by the
donors of the Petroleum Research Fund, administered by the
American Chemical Society.
(12) Values reported for maximum solubility are apparent and do not
take into consideration aggregation which is obviously playing a role.
(13) Brettreich, M.; Hirsch, A. Tetrahedron Lett. 1998, 2731-2734.
(14) Guldi, D. M.; Hungerbuhler, H,; Asmus, K. D. J. Phys. Chem. 1995,
99, 13487-13493.
OL990312A
(15) Cantor, C. R.; Schimmel, P. R. Biophysical Chemistry Part II; WH
Freeman and Company: New York, 1980; pp 399-406.
1014
Org. Lett., Vol. 2, No. 8, 2000