Synthesis of a hybrid fullerene-trimethoxyindole-oligonucleotide conjugate

Article abstract of DOI:10.1039/b008744k

The synthesis of novel functionalized fullerene derivatives is reported: a DNA minor groove binder such as trimethoxyindole-2-carboxylate (TMI) and an oligonucleotide chain have been covalently linked to C₆₀ with the aim of duplicating DNA interactions for increasing sequence selectivity.

Full text of DOI:10.1039/b008744k

Synthesis of a hybrid fullerene–trimethoxyindole–oligonucleotide conjugate
Massimo Bergamin,a Tatiana Da Ros,a Giampiero Spalluto,a Alexandre Boutorineb and Maurizio Prato*a
a Dipartimento di Scienze Farmaceutiche, Università di Trieste, Piazzale Europa 1, 34127 Trieste, Italy.
E-mail: prato@univ.trieste.it
b Laboratoire de Biophysique, INSERM U 201 - CNRS UMR 8646, Museum National d’Histoire Naturelle, 43 rue
Cuvier, 75231 Paris Cedex 05, France
Received (in Cambridge, UK) 30th October 2000, Accepted 14th November 2000
First published as an Advance Article on the web 11th December 2000
The synthesis of novel functionalized fullerene derivatives is
reported: a DNA minor groove binder such as trimethoxy-
indole-2-carboxylate (TMI) and an oligonucleotide chain
have been covalently linked to C60 with the aim of
duplicating DNA interactions for increasing sequence se-
lectivity.
linkers such as TMI and oligonucleotide. The TMI unit is
characteristic of a class of natural antibiotics named duocarmy-
cins, possessing antitumor activity in the picomolar range, in
part due to high affinity and selectivity of the TMI group for the
minor groove in AT-rich sequences of DNA.7 The effect of the
oligonucleotide chain could be considered both to induce high
sequence-selectivity and to increase water solubility, one of the
main problems of C60 derivatives for biological application.
The designed compound 1 was prepared following the
general synthetic strategy summarized in Schemes 1–2, accord-
ing to the known procedure for the synthesis of full-
eropyrrolidines, based on the 1,3-dipolar cycloaddition of
azomethine ylides to C60.8,9
Fullerene C60 and its organofunctionalized derivatives have
recently become a topic of interest in bioorganic and medicinal
chemistry. This class of compounds has shown high potential in
a wide variety of biological activities, such as DNA photo-
cleavage, HIV protease inhibition, neuroprotection and apopto-
sis.1 In particular, the excited state properties of C60 may offer
viable routes to novel pharmacological tools.2
One of the problems of photodynamic therapy is the
addressed delivery of a photoactive agent to its target. That is
why conjugates of fullerene with molecules possessing bio-
logical affinity to certain nucleic acids, proteins, cell types,
organelles, etc., might be of particular interest. Additionally,
C60 itself might facilitate the interactions of certain biologically
active molecules with lipophilic membranes of living cells and
consequently improve cellular uptake due to its high hydro-
phobicity.
Recently, some conjugates between C60 and nucleic acid-
specific agents (acridine,3 netropsin4 and complementary
oligonucleotides5,6) have been reported, with the aim of better
understanding the mechanism of action and to increase their
cytotoxic properties and specificity. Coupling of fullerene to an
intercalator or a minor groove binder already leads to higher
affinity and specificity of the derivatives towards target DNA.4
The next step envisages the improvement of the photocleavage
efficiency of the conjugates, possibly by stabilizing their
duplexes and triplexes and increasing their sequence selectiv-
ity.
In order to achieve these goals, we have designed derivative
(1) of fulleropyrrolidine linked to trimethoxyindole (TMI) and
Scheme 1 Reagents: i, dioxane, 0 °C, then rt 18 h; ii, TFA, CH2Cl2, rt, 20
min; iii, NMM, HOBT, EDC, CH2Cl2; iv, Pd/C 10%, H2, MeOH; v, toluene,
reflux, 50 min; vi, TFA, CH2Cl2, rt, 1 h.
an oligonucleotide chain simultaneously. The rational design of
this derivative was based on a synergistic effect of two different
DOI: 10.1039/b008744k
Chem. Commun., 2001, 17–18
This journal is © The Royal Society of Chemistry 2001
17

In summary, we have described the synthesis of the bis-
functionalized fulleropyrrolidine 1 containing two different
linkers capable of modulating and inducing high sequence
selectivity. Results on duplex and triplex helicex DNA
formation and the synthesis of an enlarged series of compounds
for structure–activity relationship studies will be reported in due
course.
This work was supported by MURST (cofin. ex 40%, prot. n.
mm03198284), by Regione Friuli-Venezia Giulia, fondo Anno
1998, and by the European Community (TMR program
BIOFULLERENES, contract No. ERB FMRX-CT98-0192).
Scheme 2 Reagents: i, PPh3, DMSO, DMAP, Py2S2; ii, DMSO; iii, PPh3,
DMSO, Py2S2; iv, Et3N, DMSO, 11.
The appropriate O-benzylaminoacid 4 was prepared by
alkylation of the mono protected (Boc) ethylenediamine 2 with
benzyl a-bromoacetate 3 (Scheme 1).
Notes and references
† Analytical and spectroscopic data of derivative 10: C86H40N4O6 (MW
1225.30), yield: 19%. 1H-NMR: d 9.27 (br s, 1H), 7.30 (br s, 1H), 6.93 (s,
1H), 6.75 (s, 1H), 4.98 (d, J = 10.3 Hz, 1H), 4.52 (m, 1H), 4.37 (t, J = 5.9
Hz, 1H), 4.30 (d, J = 10.5 Hz 1H), 4.08–3.95 (m, 4H), 4.05 (s, 3H), 3.89
(s, 3H), 3.85 (s, 3H), 3.25 (m, 1H), 3.11 (m, 2H), 2.45 (m, 2H), 1.89 (m, 2H),
1.49 (m, 2H), 1.43 (s, 9H). 13C-NMR: d 166.9, 156.4, 154.4, 153.2, 150.0,
147.1, 146.3, 146.2, 146.1, 146.0, 145.9, 145.5, 145.2, 145.1, 144.6, 144.4,
144.3, 143.0, 142.6, 142.2, 142.1, 142.0, 141.9, 141.7, 140.2, 139.7, 136.7,
135.8, 135.6, 135.4, 130.6, 123.3, 97.1, 71.2, 67.4, 65.4, 61.1, 56.2, 44.1,
41.1, 31.0, 29.9, 28.5. ES-MS (THF–MeOH 1+1): m/z 1225 (MH+). UV–
Deprotection of the amino functionality by TFA in 8 led to
amine 5, which was coupled to trimethoxyindole-2-carboxylic
acid 610 to afford amino ester 7. The latter was deprotected at
the carboxylic function by catalytic hydrogenation and the
resulting acid 8 was allowed to react with C60 and N-Boc-
6-aminohexanal 9 to yield the desired multifunctional full-
eropyrrolidine 10.† The latter compound was in turn depro-
tected to obtain the desired salt 11 by treatment with TFA
(Scheme 1).
VIS (cyclohexane) lmax: nm 254, 312, 431, 703. IR (KBr, DRIFT) cm21
3330, 2935, 2850, 1690, 1645, 1245, 1169, 526.
:
The designed product 1 was synthesized following a general
synthetic strategy for the preparation of oligonucleotide con-
jugates previously reported11–13 and summarized in Scheme 2,
but the new procedure contained several modifications. Origi-
nally the reaction was initiated by activation of the oligonucleo-
tide terminal phosphate by means of Mukaiyama reagents,
triphenylphosphine–dipyridyl-2,2A-disulfide in the presence of
DMAP.14 However, it appeared that interaction of the activated
phosphate with the amino group of the fullerene derivative did
not lead to satisfactory yields of conjugation. Thus, it was
necessary to use 6-aminocaproic acid as a spacer between the
two moieties.
1 T. Da Ros and M. Prato, Chem. Commun., 1999, 663.
2 D. Guldi and M. Prato, Acc. Chem. Res., 2000, 33, 695.
3 Y. N. Yamakoshi, T. Yagami, S. Sueyoshi and N. Miyata, J. Org.
Chem., 1996, 61, 7236.
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Bull. Chem. Soc. Jpn., 1996, 69, 2143.
5 A. Boutorine, H. Tokuyama, M. Takasugi, H. Isobe, E. Nakamura and
C. Hélène, Angew. Chem., Int. Ed. Engl., 1994, 33, 2462.
6 Y.-Z. An, C.-H. B. Chen, J. L. Anderson, D. S. Sigman, C. S. Foote and
Y. Rubin, Tetrahedron, 1996, 52, 5179.
7 D. L. Boger and D. S. Johnson, Angew. Chem., Int. Ed. Engl., 1996, 35,
1438.
The phosphorylated oligonucleotide (16-mer) 12 was acti-
vated at its terminal phosphate, precipitated by lithium
perchlorate in acetone according to the method described by
Knorre et al.11 and attached to the e-amino group of 6-aminoca-
proic acid 13 in water in the presence of triethylamine to afford
the carboxylic acid derivative of oligonucleotide 14 in quantita-
tive yield. Coupling of 14 with fullerene derivative 11 was
performed in a similar way, by activation of the carboxylic
group with Mukaiyama reagents in organic media to obtain the
desired conjugate 1. Purification of 1 was performed by
electrophoresis in 1% agarose–0.1% triton X-100 gel using tris-
acetate buffer, followed by excision of the colored band and
electroelution, or digestion of agarose with b-agarase.5,6
8 M. Maggini, G. Scorrano and M. Prato, J. Am. Chem. Soc., 1993, 115,
9798.
9 M. Prato and M. Maggini, Acc. Chem. Res., 1998, 31, 519.
10 D. Boger, T. Ishizaki, H. Zarrinmayeh, P. Kitos and O. Suntornwat,
J. Org. Chem., 1990, 55, 4499.
11 D. G. Knorre, P. V. Alekseyev, Y. V. Gerassimova, V. N. Silnikov,
G. A. Maksakova and T. S. Godovikova, Nucleosides, Nucleotides,
1998, 17, 397.
12 T. S. Godovikova, V. F. Zarytova, T. V. Maltseva and L. M.
Khalimskaya, Bioorg. Khim., 1989, 15, 1246.
13 A. S. Boutorine, T. Le Doan, J. P. Battioni, D. Mansuy, D. Dupré and C.
Hélène, Bioconj. Chem., 1990, 1, 350.
14 T. Mukaiyama, R. Matsueda and M. Suzuki, Tetrahedron Lett., 1970,
22, 1901.
18
Chem. Commun., 2001, 17–18