New non-ionic, highly water-soluble derivatives of C60 designed for biological compatibility

Article abstract of DOI:10.1016/S0040-4039(01)00956-X

The most water-soluble (>240 mg C₆₀ mL^-1) fullerene derivatives to date, the new malonodiserinolamide fullerenes C₆₀[C(COSer)₂]_n (n = 4, 5, 6, Ser = 2-amino-1,3-propanediol), have been synthesized and

Full text of DOI:10.1016/S0040-4039(01)00956-X

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 42 (2001) 5159–5162
New non-ionic, highly water-soluble derivatives of C60 designed
for biological compatibility
Tim Wharton, Vinay U. Kini, Rachel A. Mortis and Lon J. Wilson*
Department of Chemistry and the Center for Nanoscale Science and Technology, MS-60 Rice University, Houston,
TX 77251-1892, USA
Received 8 May 2001; accepted 4 June 2001
Abstract—The most water-soluble (>240 mg C60 mL−1) fullerene derivatives to date, the new malonodiserinolamide fullerenes
C60[C(COSer)2]n (n=4, 5, 6, Ser=2-amino-1,3-propanediol), have been synthesized and characterized. The compounds were
designed to provide maximum water solubility and biological compatibility. The facile synthesis is based on Bingel additions to
C60 and should be applicable to the solubilization of any prospective fullerene-based drug. © 2001 Elsevier Science Ltd. All rights
reserved.
Organically-functionalized derivatives of C60 are cur-
rently being investigated for applications in the fields of
biology and medicine.1 In general, prospective diagnos-
tic/therapeutic agents will be required to have sufficient
biological availability, and thus, adequate water solu-
bility. C60 itself may be coerced to form a colloidal
suspension in water,2 but it is intrinsically insoluble.3
Therefore, finding simple, well-characterized methods
to solubilize fullerene materials in biological environ-
ments is the subject of great current interest.4–8
which are reasonably stable in the absence of oxygen.2b
Similarly, the carbon-based anion Ph5C60 is soluble in
water, with a maximum solubility of ꢀ3.8 mg C60
mL−1.7 For medicinal applications, however, the oxygen
sensitivity and expected biological instability of such
carbon-based anions, coupled with relatively low water
solubility, makes them unattractive for use in vivo.
−
C60-containing host–guest complexes (Method 2) are
also limited in medicinal applicability due to very low
aqueous solubilities (ꢀ0.07 mg C60 mL−1).4b Other
potential problems with this approach include an effec-
tive shielding by the host of a fullerene compound from
biological availability or undesired release and precipi-
tation of C60 in vivo which, in mammals, leads to
accumulation in the liver.10
To date, the methods proposed to water solubilize
fullerenes have taken one of four approaches: (1) chem-
ical/electrochemical reduction of C60 or its derivatives
n−
to a water-soluble carbon-based anion (e.g. C60 or
− 2b,7
Ph5C60
)
(Method 1); (2) complexation of C60 in a
host–guest fashion within hosts such as the
cyclodextrins4a,4b and calixarenes,4c effectively shielding
the hydrophobic fullerene from the polar aqueous
environment9 (Method 2); (3) covalent modification of
C60 with one or more organic functionalities that con-
tain(s) one or more ionic group(s)5 (Method 3), and
finally (4) covalent modification of C60 with one or
more organic functionalities that contain(s) strongly
hydrophilic group(s) that are non-ionic6 (Method 4).
Most literature examples of water-soluble fullerenes
involve functionalization of the fullerene with ionic
organic groups (Method 3).5 The trifluoroacetic acid
salt of an octahedrally-functionalized (Th) derivative of
C60 containing 12 primary amine groups was found to
have a solubility of 89 mg C60 mL−1 in water, the
highest value reported to date.5b A monofunctionalized
dendrimeric C60 derivative with 18 carboxylic acid
groups produced a solubility of 65 mg C60 mL−1 at pH
10 but only 8.7 mg C60 mL−1 at pH 7.5a Although this
approach imparts excellent water solubility, the solubil-
ity is pH dependent with only marginal solubility at
biological pH of 7.4. In addition, the hyperosmolality
caused by the multiple ionic groups (and their counteri-
ons) of these types of fullerene compounds is undesir-
able for medical applications such as magnetic
n−
Using Method 1, aqueous solutions of C60 (n=1, 2)
with concentrations of ꢀ1 mg mL−1 may be obtained,
Keywords: fullerene derivatives; fullerenes; water-soluble fullerenes.
* Corresponding author. Fax: +1-713-348-5155; e-mail: durango@
rice.edu
0040-4039/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(01)00956-X

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T. Wharton et al. / Tetrahedron Letters 42 (2001) 5159–5162
resonance imaging (MRI) and X-ray radiography,
where a contrast-enhancing agent is administered intra-
venously in very high concentration.11
yield with K2CO3 in CH3OH/H2O, followed by removal
of potassium (as confirmed by energy dispersive X-ray
spectroscopy) with 50W-X2 cation exchange resin (H+
form). Anhydrous samples of 3, confirmed by ther-
mogravimetric analysis (TGA), were obtained after dry-
ing over P2O5 at ꢀ100°C under vacuum.
Finally, the covalent modification of C60 with strongly
water-solubilizing, non-ionic groups (Method 4) in the-
ory circumvents the above noted problems of hyperos-
molality, chemical instability, limited water solubility,
and pH dependence of solubility. To date, however,
derivatives of this type have met with only limited
success. Examples are a cyclodextrin–fullerene conju-
gate made by nucleophilic addition to C60 of an amine-
Initial efforts focused on the synthesis of the n=1
adduct, which was isolated in 44% yield as a reddish-
brown solid after column chromatography on silica
(acetone/toluene). The 13C spectrum (Fig. 2) of the
malonodiamide derivative verified a structure com-
pletely analogous to comparable structures containing
malonodiester addends. That is, the malonodiamide
added exclusively across one of the 6–6 double bonds of
C60 forming a cyclopropane ring.17 Deprotection of the
n=1 adduct yielded the expected tetrahydroxy product
which was found to be completely insoluble in water.
modified cyclodextrin,6b
a polyethylene glycol–C60
conjugate,6c a N-(3,6,9-trioxadecyl)-glycine functional-
ized fulleropyrrolidene,6a and the fullerenols.12 In all
these cases, the compounds were either ill-defined or
only slightly soluble in water.
In this work, we report the design, synthesis, and
characterization of new Type 4 water-soluble fullerene
derivatives with exceptionally high water solubility at
biological pH. These new malonodiserinolamide fullere-
nes (Fig. 1) are non-ionic and are based on the biolog-
ically-stable serinolamide structures used to water
solubilize commercially available X-ray contrast agents
such as iohexol13 and iopamidol,14 which are also
designed to be non-ionic and non-toxic. In addition, we
have previously used serinolamides to water-solubilize
MnIII metalloporphyrins for use as MRI contrast
agents.15
Efforts then turned to the multiadduct products. Using
a 10-fold excess of 1 relative to C60, a cherry red
solution of mixed products was obtained. After flash
chromatography on silica (acetone/toluene), the red
solid (69% yield) was analyzed by MALDI-TOF MS
and determined to consist of ꢀ15% tetra-, ꢀ80%
penta-, and ꢀ5% hexa-functionalized product (Fig. 3).
With some difficulty, a sample consisting of only an
isomer mixture of pure n=5 product could be obtained
by multiple passes on silica, as confirmed by HPLC and
MS. However, the bulk solubility and partition coeffi-
cient (KOW) measurements reported below were per-
formed on the deprotected mixture.
In a typical synthesis of 3 (Fig. 1), two equivalents of
serinol (2-amino-1,3-propanediol) were condensed with
diethylmalonate at elevated temperature, with the loss
of ethanol, to give the corresponding malonodiamide.16
Esterification of the alcohol groups in pyridine/Ac2O
(76% overall yield for two steps starting from serinol)
was followed by electrophilic bromination (79% yield)
at the central acidic carbon of the malonodiamide with
Br2 in EtOAc/NEt3. The bromomalonodiamide, 1, was
then appended to C60 via a Bingel type reaction17 in the
presence of DBU (1,8-diazabicyclo[5.4.0]undec-7-ene),
in toluene or toluene/acetone to produce the desired
compound 2.18 The stoichiometry was varied depending
on the degree of addition desired. Deprotection of 2
was then effected to yield 3 in essentially quantitative
The water solubility of 3 (n=4–6) was determined using
UV–vis spectrophotometry at pH 7. In a 1 mm quartz
cell containing 400 mL of water, the material was
dissolved in 5.0 mg increments and spectra taken
sequentially. The material continued to dissolve and the
spectrum continued to increase linearly (R2=0.995) in
intensity even after 255 mg of 3 had been added. At this
point, further addition was discontinued because of
solution viscosity. The degree of water solubilization
observed corresponds minimally to a remarkable 240
mg C60 mL−1! Additionally, no visible change in the
solution was noted in the pH 2–12 range. This result
Figure 1. Synthesis of the malonodiserinolamide fullerenes. DBU=1,8-diazabicyclo[5.4.0]undec-7-ene, Ac=acetate.

T. Wharton et al. / Tetrahedron Letters 42 (2001) 5159–5162
5161
Figure 2. 13C NMR of 2 (n=1). 400 MHz, CDCl3, *=solvent impurity.
Figure 3. MALDI-TOF mass spectrum of 2 (n=4, 5, 6) as a product mixture. CS2/S, CHCl3, negative ion mode.
In conclusion, a new synthetic methodology for water
solubilizing fullerene materials has been developed. The
resulting fullerene derivatives are exceedingly soluble in
water without a significant pH dependence. This
straightforward approach for water-solubilizing
fullerene-based materials leads to well-defined, biologi-
cally-compatible products and should be generally
applicable to empty fullerenes, as well as to endohedral
metallofullerenes, M@Cn, with M being any one of
several medically-interesting metal ions.1,20 Water-solu-
bilized metallofullerene materials are presently under
investigation.
suggests no practical solubility limitations to the devel-
opment of fullerene-based diagnostic/therapeutic agents
using malonodiserinolamide addends, even in conjunc-
tion with other active drug components appended to
the same fullerene molecule.
The n-octanol/water partition coefficient of 3 (n=4−6)
was determined at 25°C by the method of Leo.19
Accordingly, a 2.0 mg mL−1 solution of 3 in n-octanol-
saturated water was shaken for 5 min with an equal
volume of n-octanol, followed by centrifugation. The
UV–vis spectrum of the aqueous layer was unchanged
from that of the starting solution, and the organic
phase remained colorless. Thus, for 3, KOW:0 which
predicts that the material will have negligible hydro-
phobic interactions in vivo. This value compares well
with the very small KOW values (<10−3)13 of currently-
used X-ray contrast agents which, in vivo, are restricted
to the extracellular space and are rapidly and com-
pletely cleared from the body. Compound 3, with its
high degree of derivatization (n=4–6), is therefore also
well suited to extracellular applications. However, lesser
degrees of functionalization (n=2–3) may allow a tai-
loring of KOW for intracellular uses, such as drug
delivery, while still providing adequate water solubility.
Acknowledgements
This research was supported by The Robert A. Welch
Foundation (C-0627) and C Sixty, Inc.
References
1. Reviews: (a) Wilson, L. J. Interface 1999, 8, 24–28; (b)
Jensen, A. W.; Wilson, S. R.; Schuster, D. I. Biorg. Med.

5162
T. Wharton et al. / Tetrahedron Letters 42 (2001) 5159–5162
Chem. 1996, 4, 767–779; (c) Da Ros, T.; Prato, M. Chem.
Commun. 1999, 663–669.
2. (a) Scrivens, W. A.; Tour, J. M. J. Am. Chem. Soc. 1994,
116, 4517–4518; (b) Wei, X.; Migfei, W.; Qi, L.; Xu, Z. J.
Chem. Soc., Perkin Trans. 2 1997, 1389–1393.
3. (a) Sivaraman, N.; Dhamodaran, R.; Kaliappan, I.; Srini-
vasan, T. G.; Rao, P. R. V.; Mathews, K. C. J. Org.
Chem. 1992, 57, 6077–6079; (b) Ruoff, R. S.; Tse, D. S.;
Malhotra, R.; Larents, D. C. J. Phys. Chem. 1993, 97,
3379–3383; (c) Scrivens, W. A.; Tour, J. M. J. Chem.
Soc., Chem. Commun. 1993, 15, 1207–1209.
4. (a) Andersson, T.; Nilsson, K.; Sundahl, M.; Westman,
G.; Wennerstrom, O. J. Chem. Soc., Chem. Commun.
1992, 604–606; (b) Priyadarsini, K. I.; Mohan, H.; Tyagi,
A. K.; Mittal, J. P. J. Phys. Chem. 1994, 98, 4756–4759;
(c) Ikeda, A.; Hatano, T.; Kawaguchi, M.; Suenaga, H.;
Shinkai, S. Chem. Commun. 1999, 1403–1404.
5. (a) Brettreich, M.; Hirsch, A. Tetrahedron Lett. 1998, 39,
2731–2734; (b) Richardson, C. F.; Schuster, D. I.;
Wilson, S. R. Org. Lett. 2000, 2, 1011–1014; (c) Oka-
mura, H.; Miyazono, K.; Minoda, M.; Komatsu, K.;
Fukuda, T.; Miyamoto, T. J. Polym. Sci. A: Pol. Chem.
2000, 38, 3578–3585; (d) Chi, Y.; Bhonsle, J. B.; Canteen-
wala, T.; Huang, J.-P.; Shiea, J.; Chen, B.-J.; Chiang, L.
Y. Chem. Lett. 1998, 465–466; (e) Sijbesma, R.; Srdanov,
G.; Wudl, F.; Castoro, J. A.; Wilkins, C.; Friedman, S.
H.; DeCamp, D. L.; Kenyon, G. L. J. Am. Chem. Soc.
1993, 115, 6510–6512; (f) Cerar, J.; Cerkovnik, J.; Sker-
janc, J. J. Phys. Chem. B 1998, 102, 7377–7381; (g)
Schinazi, R. F.; Sijbesma, R.; Srdanov, G.; Hill, C. L.;
Wudl, F. Antimicrob. Agents Chemother. 1993, 37, 1707–
1710; (h) Takenaka, S.; Yamashita, K.; Takagi, M.;
Hatta, T.; Tsuge, O. Chem. Lett. 1999, 321–322.
6. (a) Da Ros, T.; Prato, M. J. Org. Chem. 1996, 61,
9070–9072; (b) Samal, S.; Geckeler, K. E. Chem. Com-
mun. 2000, 1101–1102; (c) Tabata, Y.; Ikada, Y. Pure
Appl. Chem. 1999, 71, 2047–2053.
11. Sovak, M.; Hoey, G. B.; Smith, K. R. In Handbook of
Experimental Pharmacology; Sovak, M., Ed.; Springer-
Verlag: Berlin, 1984; Vol. 73, pp. 1–125.
12. Li, J.; Takeuchi, A.; Ozawa, M.; Li, X.; Saigo, K.;
Kitawaza, K. J. Chem. Soc., Chem. Commun. 1993, 1784.
13. Haavaldsen, J.; Nordal, V.; Kelly, M. Acta Pharm. Suec.
1983, 20, 219–232. German Offen. 2,727,196. 1977.
14. Felder, E.; Pitre, D. E. German Offen. 2,547,789. Jan. 24,
1976. Chem. Abstr. 8s:94103r, 1976.
15. Bradshaw, J. E.; Gillogly, K. A.; Wilson, L. J.; Kumar,
K.; Wan, X.; Tweedle, M. F.; Hernandez, G.; Bryant, R.
G. Inorg. Chim. Acta 1998, 275–276, 106–116. US Patent
No. 5-262-532.
16. Backes, J. V.; West, R. W.; Whiteley, M. A. J. Chem.
Soc. 1921, 119, 359–379.
17. Bingel, C. Chem. Ber. 1993, 126, 1957–1959.
18. All new compounds gave satisfactory spectral data. For 2
(n=1): reddish brown powder; TLC Rf 0.26 in 9:1 tolu-
ene:MeOH; 1H NMR (400 MHz, CDCl3) l (ppm) 2.10 (s,
12H, CH3), 4.34–4.41 (m, 8H, CH2), 4.68–4.72 (m, 2H,
CH), 7.37 (br d, J=6.4 Hz, 2H, NH); 13C NMR (400
MHz, CDCl3) l (ppm) 170.95 (ester CꢀO), 162.90 (amide
CꢀO), 145.57, 145.44, 145.49, 145.40, 145.06, 144.99,
144.80, 144.01, 143.41, 143.32, 143.24, 142.40, 142.29,
141.30, 138.21 (C60 sp2 C), 73.45 (C60 sp3 C), 62.72
(CH2), 58.28 (bridgehead), 49.30 (CH), 20.96; FT-IR
(KBr) w (cm−1) 1745 (s, ester CꢀO), 1685 (m, amide CꢀO),
1228 (s, asym. -OCOCH3), 526 (m); HPLC (Cosmosil 5
PBB column), retention time 3.43 min in toluene:MeOH
(40:1), detection at 290 nm; UV (40:1 toluene:MeOH)
umax 285 nm; MALDI-TOF MS calcd for C77H24O10N2
(M+) 1136, found 1136. Anal. (C77H24O10N2 acetone
(C3H6O)) calcd: C, 80.4; H, 2.53; N, 2.34, found: C,
79.79; H, 2.41; N, 2.45.
19. The n-octanol/water partition coefficient (KOW) of com-
pounds has proven to be useful for the determination of
biological structure–activity relationships. It is defined as
7. Sawamura, M.; Nagahama, N.; Toganoh, M.; Hackler,
U. E.; Isobe, H.; Nakamura, E.; Zhou, S.-Q.; Chu, B.
Chem. Lett. 2000, 1098–1099.
8. Cagle, D. W.; Kennel, S. J.; Mirzadeh, S.; Alford, J. M.;
Wilson, L. J. Proc. Natl. Acad. Sci. USA 1999, 96,
5182–5187.
9. This category also includes solutions/dispersions of C60
formed by the use of surfactants. See: (a) Guldi, D. M. J.
Phys. Chem. A 1997, 101, 3895–3900; (b) Eastoe, J.;
Crooks, E. R.; Beeby, A.; Heenan, R. K. Chem. Phys.
Lett. 1995, 245, 571–577.
K
OW=cocw−1, where co and cw are the equilibrium con-
centrations of an analyte in n-octanol and water, respec-
tively. The partition coefficient has been used to predict
the rate of biological uptake,18b the degree of biomagnifi-
cation,18b and the degree of binding of solutes to serum
albumin and other blood proteins.18a (a) Leo, A.; Han-
sch, C.; Elkins, D. Chem. Rev. 1971, 71, 525–554; (b)
Chiou, C. T.; Freed, V. H.; Schmedding, D. W.; Kohnert,
R. L. Environ. Sci. Technol. 1977, 11, 475–478.
20. Wilson, L. J.; Cagle, D. W.; Thrash, T. P.; Mirzadeh, S.;
Alford, J. M.; Ehrhardt, G. J. Coord. Chem. Rev. 1999,
190–192, 199–207.
10. Bullard-Dillard, R.; Creek, K. E.; Scrivens, W. A.; Tour,
J. M. Bioorg. Chem. 1996, 24, 376–385.
.