Synthesis and characterization of highly water-soluble dendrofulleropyrrolidine bisadducts with DNA binding activity

Article abstract of DOI:10.1021/ol301946r

The synthesis, characterization and DNA binding studies of a series of polycationic fullerene adducts are reported. These cationic species, exhibiting reasonably high water solubility and a heterogeneous distribution of positive charges, can efficiently c

Full text of DOI:10.1021/ol301946r

ORGANIC
LETTERS
XXXX
Vol. XX, No. XX
000–000
Synthesis and Characterization of Highly
Water-Soluble Dendrofulleropyrrolidine
Bisadducts with DNA Binding Activity
†
Alejandro Montellano Lopez, Francesco Scarel, Noelia Rubio Carrero,
†
‡
ꢀ
Ester Vazquez, Aurelio Mateo-Alonso,§, ,^,# Tatiana Da Ros,† and Maurizio Prato*,†
‡
ꢀ
Center of Excellence for Nanostructured Materials (CENMAT), Department of Chemical
and Pharmaceutical Sciences, and INSTM, unit of Trieste, University of Trieste, Piazzale
´
Europa 1, 34127 Trieste, Italy, Departamento de Quımica Organica, Facultad de Ciencias
Quımicas-IRICA, Universidad de Castilla-La Mancha, 13071 Ciudad Real, Spain, Freiburg
ꢀ
´
Institute for Advanced Studies (FRIAS), School of Soft Matter Research, Albert-Ludwigs-
€
Universitat, Freiburg Albertstrasse 19, D-79105 Freiburg im Breisgau, Germany, Institut fur
Organische Chemie und Biochemie (IOCB), Albert-Ludwigs-Universitat, Freiburg,
€
Albertstrasse 21, D-79104, Freiburg im Breisgau, Germany, POLYMAT, University of the
Basque Country UPV/EHU, Avenida de Tolosa 72, E-20018 Donostia-San Sebastian,
Spain, and Ikerbasque, Basque Foundation for Science, E-48011 Bilbao, Spain
prato@units.it
Received July 14, 2012
ABSTRACT
The synthesis, characterization and DNA binding studies of a series of polycationic fullerene adducts are reported. These cationic species, exhibiting
reasonably high water solubility and a heterogeneous distribution of positive charges, can efficiently complex plasmid DNA. Electrophoresis studies
show different DNA binding efficiencies for different adducts, some of which can be considered excellent candidates for DNA binding therapies.
Nonviral approaches for modulating gene expression
and for treating genetic diseases rely on the efficient intra-
cellular release of foreign genetic material. This is usually
achieved by using polycationic species, such as dendrimers,1
polymers,2 nanoparticles,3 and nanotubes,4 which can
effectively bind oligonucleotides by ionic interactions with
the phosphate anions. Cationic fullerene derivatives have
shown to be particularly effective gene delivery vectors
† University of Trieste.
‡ Universidad de Castilla-La Mancha.
Freiburg Institute for Advanced Studies, Albert-Ludwigs-Universitat.
(2) (a) Lim, D. W.; Yeom, Y. I.; Park, T. G. Bioconjugate Chem. 2000,
11, 688. (b) Vinogradov, S. V.; Bronich, T. K.; Kabanoz, A. V. Biocon-
jugate Chem. 1998, 9, 805.
(3) (a) He, X. X.; Wang, K.; Tan, W.; Liu, B.; Lin, X.; He, C.; Li, D.;
Huang, S.; Li, J. J. Am. Chem. Soc. 2003, 125, 7168.
(4) (a) Herrero, M. A.; Toma, F. M.; Al-Jamal, K. T.; Kostarelos, K.;
Bianco, A.; Da Ros, T.; Bano, F.; Casalis, L.; Scoles, G.; Prato, M.
J. Am. Chem. Soc. 2009, 131, 9843. (b) Singh, R.; Pantarotto, D.;
McCarthy, D.; Chaloin, O.; Hoebeke, J.; Partidos, C. D.; Briand,
J. P.; Prato, M.; Bianco, A.; Kostarelos, K. J. Am. Chem. Soc. 2005,
127, 4388. (c) Pantarotto, D.; Singh, R.; McCarthy, D.; Erhardt, M.;
Briand, J. P.; Prato, M.; Kostarelos, K.; Bianco, A. Angew. Chem., Int.
Ed. 2004, 43, 5242.
§
€
Institut fur Organische Chemie und Biochemie, Albert-Ludwigs-
€
Universitat.
^ POLYMAT, University of the Basque Country.
# Ikerbasque.
(1) (a) Lim, Y.-B.; Kim, S.-M.; Lee, Y.; Lee, W.-K.; Yang, T.-G.;
Lee, M.-J.; Suh, H.; Park, J.-S. J. Am. Chem. Soc. 2001, 123, 2460. (b)
Kabanov, A. V. Adv. Drug Delivery 1998, 30, 49. (c) Toncheva, V.;
Wolfert, M. A.; Dash, P. R.; Oupicky, D.; Ulbrich, K. L.; Seymour, W.;
Schact, E. H. Biochim. Biophys. Acta, Gen. Subj. 1998, 1380, 354. (d)
Tang, M. X.; Redemann, C. T.; Szoka, F. C. J. Bioconjugate Chem. 1996,
7, 703.
r
10.1021/ol301946r
XXXX American Chemical Society

both in vitro5 and in vivo.6 Recent studies5a have demon-
strated that for efficient transfection the size of such
fullerene/oligonucleotide complexes must be kept to a
submicrometer scale in order to be internalized through
endocytosis. The various complexes obtained so far pres-
ent sizes that span over several orders of magnitude (from
several nm to μm), making it difficult to understand the
molecular factors that control the transfection mechanism
and thus to rationalize the design of more efficient vectors.
Moreover, the vectors must be capable of releasing the
delivered oligonucleotide in the cytoplasm or in the nu-
cleus. Therefore, it is crucial to design cationic fullerenes
that form submicrometer complexes with a good oligonu-
cleotide complexation/decomplexation balance, in order
to ensure efficient transfection.7 C60 bisadducts are parti-
cularly interesting for this purpose, since the eight different
regioisomers can potentially display different oligonucleo-
tide affinities and thus provide a versatile platform for
studying, tuning, and improving delivery vectors for spe-
cific oligonucleotides.
Herein, we reporta synthetic strategy that hasallowed us
to prepare a library of regioisomerically pure bisadducts
that can be further decorated with cationic dendrons.
Remarkably such bisadducts exhibit a high solubility in
water and DNA binding activity. In fact, electrophoresis
studies demonstrate that cationic regioisomerically pure
bisadducts exhibit different affinities to DNA and thus that
they can be used to control and modulate ionic interactions
with oligonucleotides.
To pursue our objectives, we have designed and synthe-
sized a series of fulleropyrrolidine monoadduct and bisad-
ducts equipped with a first generation of polyamidoamine
dendrons (PAMAM) that upon protonation provide
highly water-soluble polycationic fullerenes. PAMAM
dendrons have been selected because of their relatively
easy synthesis and also because their dendrimeric analogs
are known to interact with DNA.8 To ensure that the
separation of the bisadducts is possible, we have designed
a convergent synthetic strategy, in which the different
fullerene adducts with a terminal carboxylic group are
first synthesized and isolated. Then the individual isolated
fullerene adducts are coupled to a first generation
PAMAM dendron with a terminal free amine through
the formation of an amide.
Scheme 1. Synthesis of Fulleropyrrolidines 4 and 5
times (2À3 h) result in the formation of tris and higher
adducts and their presence renders the recovery of pure
isomers difficult and time-consuming. In this way, it was
possible to prepare and isolate mono- 2 and bis-fullero-
pyrrolidines 3aÀe (trans-1, trans-2, trans-3, trans-4, and
equatorial). Cis isomers were isolated as a mixture and
obtained in a low quantity as revealed by HPLC. As a
result, five different bisadducts containing tert-butyl ester
groups were obtained (Scheme 1). Mono- and bis-full-
eropyrrolidines (trans-1, trans-2, trans-3, trans-4, and
equatorial) have been characterized by 1H and 13C NMR
and UVÀvis absorption spectroscopies, which are in per-
fect agreement with previous work10 (Supporting Informa-
tion, Figure S1). The deprotection of the tert-butyl group
was easilyachieved inquantitativeyields using a mixture of
CHCl3 and trifluoroacetic acid (TFA), obtaining mono-
adduct 4 and bisadducts 5aÀe containing free carboxylic
acid residues.
At this stage, dendron 12 was synthesized with a free
amine, as anchoring points suitable for coupling to the
fullerene building blocks 4and 5aÀe, andtwotert-butyloxy-
carbonyl(Boc)-protectedamines. Five steps were needed to
afford the desired compound 12 as outlined in Scheme 2.
Ethylenediamine was protected with a carbobenzyloxy
group (CBz) bytreatment withN-(benzyloxycarbonyloxy)-
succinimide. Thus, a large excess of the ethylenediamine
was used in order to avoid bisprotection, obtaining mono-
protected ethylendiamine 8. Then, a double aza-Michael
reaction was carried out on 9 in the presence of an excess of
methyl acrylate, resulting in bis-methyl ester derivative 10.
Attempts to react compound 9 with N-Boc-ethylenedia-
mine to afford directly 12 result in very low yields despite
the use of various conditions. To overcome this, an excess
of ethylenediamine was added to a solution of 9 and stirred
for 7 days, yielding 10. Then, the terminal amines were
protected by treatment with di-tert-butyl dicarbonate ob-
taining a mixture of unreacted, mono-, and bis-protected
A 1,3-dipolar cycloaddition was performed on C60 using
amino acid9 1 and p-formaldehyde. The mixture was
allowed to react for only 45 min, since longer reaction
(5) (a) Isobe, H.; Nakanishi, W.; Tonita, N.; Jinno, S.; Okayama, H.;
Nakamura, E. Chem.-Asian J. 2006, 1, 167. (b) Klumpp, C.; Lacerda, L.;
Chaloin, O.; Da Ros, T.; Kostarelos, K.; Prato, M.; Bianco, A. Chem.
Commun. 2007, 3762. (c) Sigwalt, D.; Holler, M.; Iehl, J.; Nierengarten,
J.-F.; Nothisen, M.; Morin, E.; Remy, J.-S. Chem. Commun. 2011, 47,
4640.
(6) Maeda-Mamiya, R.; Noiri, E.; Isobe, H.; Nakanishi, W.; Okamoto,
K.; Doi, K.; Sugaya, T.; Izumi, T.; Homma, T.; Nakamura, E. Proc. Natl.
Acad. Sci. U. S. A. 2010, 12, 5339.
(7) Montellano, A.; Da Ros, T.; Bianco, A.; Prato, M. Nanoscale
2011, 3, 4035.
(8) Fu, H.-L.; Cheng, S.-X.; Zheng, X.-Z.; Zhuo, R.-X. J. Gene Med.
2008, 101, 1334.
(9) Milic, D.; Prato, M Eur. J. Org. Chem. 2010, 476.
(10) Kordatos, K.; Bosi, S.; Da Ros, T.; Zambon, A.; Lucchini, V.;
Prato, M. J. Org. Chem. 2001, 66, 2802.
B
Org. Lett., Vol. XX, No. XX, XXXX

Scheme 2. Synthesis of First-Generation PAMAM Dendron 12
Scheme 3. Coupling and Deprotection Reactions
Figure 1. Water solutions (pH = 7.0, 10À3 M) and molecular
structure of 15, 16a, and 16b.
The same procedure was adopted in the coupling with
the bisadducts 5aÀe, but in this case a much lower yield
was obtained (20%), most probably due to the limited
solubility of the bisadducts in pyridine. The change of the
solvent to a CHCl3/pyridine mixture allowed to obtain
homogeneous solutions of the fullerene derivatives and
yields as much as 70% for bisadducts 5d and 5e. Because of
the high yields obtained for 14a and 14b, only these were
deprotectedfor performing the DNA bindingexperiments.
Bisadducts 16a and 16b were obtained quantitatively from
14a and 14b after 48and 72h of reaction respectively, in the
presence of TFA.
Monoadduct 15 and bisadducts 16a and 16b exhibit a high
solubility in water and their characteristic brown, greenish
and redish color as depicted in Figure 1. In addition, the
aggregation profiles determined by dynamic light scatter-
ing (DLS) measurements confirm that no aggregation is
observedupto10À3 M (see the SupportingInformation for
dendron, which waseasily separated bychromatographyto
afford 11. The CBz group was easily removed by hydro-
genation using Pd/C as catalyst giving 12 in quantitative
yields. To optimize the coupling between the fullerene
derivatives and the dendron moieties, several conditions
were tested using as modelmonoadduct4. The activation of
its carboxylic residue with 10 equiv of EDC and DMAP in
pyridine was the most convenient procedure to obtain 13,
leading to the remarkable yield of 70% (Scheme 3). Finally,
TFA was added to a solution of 13in chloroform to achieve
full deprotection of the Boc-group in a few hours, as
revealed by TLC and MS spectra, obtaining 15.
Org. Lett., Vol. XX, No. XX, XXXX
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details). The solubility of monoadducts and bisadducts
varies drastically as expected from the different number of
solubilizing dendrons. Monoadduct 15 presents a max-
imum solubility of 1.02 mg/mL, while isomer 16a (trans-4)
presents a solubility 10 times higher (10.02 mg/mL). Inter-
estingly, the equatorial isomer 16b is much more soluble
than its trans-4 analogue with a remarkable solubility
value of 23.90 mg/mL. The solutions of 15, 16a, and 16b
were perfectly stable after several weeks, and neither
visual precipitation nor changes on their absorption
characteristics was observed.
Different concentrations of fullerene adducts 15, 16a,
and 16b were incubated with increasing amounts of plas-
mid DNA (pDNA), and gel electrophoresis was used to
estimatethe presenceofunboundpDNA, bothsupercoiled
pDNA (form I) and free nicked circular pDNA (form II).
Studies on monoadduct 15 showed mostly free pDNA
and some binding to form II at high concentrations (316.8 μM)
corresponding to an amine/phosphate ratio (N/P) of 15
(Figure 2, left panel). Remarkably, in the case of bisad-
ducts 16a and 16b, a concentration 21 times lower
(14.9 μM, N/P ratio = 1) was enough to bind completely
both forms of pDNA. A shorter range concentration
experiment was carried out in order to study the differ-
ence between isomers 16a and 16b (Figure 2, right panel),
which shows clear differences in their DNA binding
activity. The equatorial isomer 16b exhibits a remarkable
higher performance with respect to trans-4 isomer 16a. In
fact, in the case of 16b only traces of unbound form II
pDNA were observed at concentrations as low as 9.9 μM.
Instead, in the case of 16a both traces of unbound forms I
and II can be appreciated.
Figure 2. Images of electrophoretic gels of pDNA with mono-
adduct 15 (left panel) and bisadducts 16a (right panel, top) and
16b (right panel, bottom).
demonstrated by gel electrophoresis. In addition, remark-
able differences between bisadduct isomers have been found
concerning solubility, aggregation profile, and DNA com-
plexation ability, opening the door to a new family of
tunable fulleropyrrolidine-based gene transfection vectors.
The study of the activity of all different bisadducts with
higher dendron generations is currently underway, and the
results will be reported in due course.
Acknowledgment. Financial support from MIUR
(PRIN No. 20085M27SS and FIRB No. RBAP11ETKA),
Associazione Italiana Ricerca sul Cancro (AIRC), Special
Program Molecular Clinical Oncology, 5x1000 (No.
12214), and Vigoni Program (Deutscher Akademischer
Austausch Dienst), the Freiburg Institute for Advanced
Studies (Junior Research Fellowship), the Verband der
Chemischen Industrie (SK 185/13), POLYMAT, and
Ikerbasque.
In conclusion, we have synthesized, isolated, and fully
characterized a fulleropyrrolidine monoadduct and five
bisadducts with terminal carboxylic acids, three of them
(monoadduct and bisadducts trans-4 and equatorial)
further decorated with a first generation PAMAM den-
dron to obtain highly water-soluble cationic derivatives.
The terminal positive charges contained in their struc-
ture lead to a very efficient pDNA complexation as
Supporting Information Available. Synthetic proce-
1
dures of all compounds and H NMR, 13C NMR, and
HPLC spectra of key compounds. DLS characterization
graphics of 15, 16a, and 16b. This material is available free
of charge via the Internet at http://pubs.acs.org.
The authors declare no competing financial interest.
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Org. Lett., Vol. XX, No. XX, XXXX