Polycationic pillar[5]arene derivatives: Interaction with DNA and biological applications

Article abstract of DOI:10.1002/chem.201303029

Dendritic pillar[5]arene derivatives have been efficiently prepared by grafting dendrons with peripheral Boc-protected amine subunits onto a preconstructed pillar[5]arene scaffold. Upon cleavage of the Boc-protected groups, water-soluble pillar[5]arene de

Full text of DOI:10.1002/chem.201303029

DOI: 10.1002/chem.201303029
Polycationic Pillar[5]arene Derivatives: Interaction with DNA and
Biological Applications
Iwona Nierengarten,[a] Marc Nothisen,[b] David Sigwalt,[a, b] Thomas Biellmann,[a]
Michel Holler,[a] Jean-Serge Remy,*[b] and Jean-FranÅois Nierengarten*[a]
Dedicated to Professor Dirk M. Guldi on the occasion of his 50th birthday
Abstract: Dendritic pillar[5]arene de-
rivatives have been efficiently prepared
by grafting dendrons with peripheral
Boc-protected amine subunits onto
these compounds to form stable nano-
particles with plasmid DNA has been
demonstrated by gel electrophoresis,
(DLS) investigations. Transfection effi-
ciencies of the self-assembled 13/
pCMV-Luc and 14/pCMV-Luc poly-
plexes have been evaluated in vitro
with HeLa cells. The transfection effi-
ciencies found for both compounds are
good, and pillar[5]arenes 13 and 14
show very low toxicity if any.
transmission
electron
microscopy
a
preconstructed pillar[5]arene scaf-
(TEM), and dynamic light scattering
fold. Upon cleavage of the Boc-pro-
tected groups, water-soluble pillar[5]ar-
ene derivatives with 20 (13) and 40
(14) peripheral ammonium groups
have been obtained. The capability of
Keywords: click chemistry · den-
drimers · gene technology · pil-
lar[5]arenes · polycations
Introduction
line materials.[5] Pillar[n]arenes have also started to be used
as building blocks for the preparation of new molecules for
bio-oriented applications. For example, artificial transmem-
brane channels obtained from pillar[5]arene monomeric and
dimeric derivatives have been reported by Hou and co-
workers.[6] Single-channel conductance measurements and
isotope effect experiments under acidic conditions showed
selective proton transport through the channels, which were
mediated by water wires formed in the pillar[5]arene back-
bones. The same group has also reported hydrazide-append-
ed pillar[5]arene derivatives.[7] These tubular derivatives
have been inserted into the lipid membranes of vesicles,
leading to the transport of water through the channels pro-
duced by single molecules.[7] Huang and co-workers have
shown that the formation of supramolecular complexes be-
tween percarboxylated pillar[6]arenes and paraquat leads to
a reduction of the toxicity of paraquat.[8] Indeed, the forma-
tion of stable host-guest complexes reduces the interaction
of paraquat with reducing agents in the cell and the genera-
tion of its radical cation is thus more difficult, resulting in
efficient reduction of paraquat toxicity. Pillar[5]arene has
also been used as a multivalent core unit to prepare glyco-
conjugates, and a mannosylated pillar[5]arene derivative has
been assayed as an inhibitor of the adhesion of an uropatho-
genic Esherichia coli strain to red blood cells.[9] Following
this first report on glycopillar[5]arenes, another approach
was reported by Huang and co-workers.[10] They prepared
an amphiphilic pillar[5]arene containing galactose subunits
as the hydrophilic part and alkyl chains as the hydrophobic
part.[10] This compound self assembles in water to produce
nanotubular structures that have been utilized as cell glues
to agglutinate Esherichia coli.
Whereas cyclotriveratrylenes (CTV) and calix[n]arenes have
been known for decades, their paracyclophane analogues,
namely pillar[n]arenes, have been only recently discov-
ered.[1,2] These macrocyclic compounds are composed of 1,4-
disubstituted hydroquinone subunits linked by methylene
bridges in their 2,5-positions.[2] In contrast to CTV and cal-
ix[n]arenes, which generally adopt cone-shaped conforma-
tions, pillar[n]arenes are tubular-shaped compounds with
a Dn symmetry. Moreover, both rims of the pillar[n]arene
macrocycle are equivalent and functionalized with n alkoxy
substituents. Pillar[n]arenes are therefore appealing compact
cores for the preparation of unique nanomaterials with
a controlled distribution of functional subunits.[3–5] Examples
include water-soluble polycationic or polyanionic systems
for host-guest chemistry,[3] multichromophoric assemblies
with peculiar photophysical properties,[4] and liquid-crystal-
[a] Dr. I. Nierengarten, Dr. D. Sigwalt, T. Biellmann, Dr. M. Holler,
Dr. J.-F. Nierengarten
Laboratoire de Chimie des Matꢀriaux Molꢀculaires
Universitꢀ de Strasbourg et CNRS (UMR 7509)
Ecole Europꢀenne de Chimie, Polymꢁres et Matꢀriaux (ECPM)
25 rue Becquerel, 67087 Strasbourg Cedex 2 (France)
E-mail: nierengarten@unistra.fr
[b] M. Nothisen, Dr. D. Sigwalt, Dr. J.-S. Remy
Laboratoire V-SAT and laboratory of excellence Medalis
Universitꢀ de Strasbourg et CNRS (UMR 7199)
Facultꢀ de Pharmacie
74 route du Rhin, B.P. 60024, 67401 Illkirch (France)
E-mail: remy@unistra.fr
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201303029.
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Chem. Eur. J. 2013, 19, 17552 – 17558

FULL PAPER
As part of this research on biologically active pillar[n]ar-
ene derivatives, we now report the preparation of polycat-
ionic dendritic pillar[5]arene derivatives and demonstrate
their capacity to interact with DNA. Owing to their efficient
ability to compact DNA, these compounds can be used in
gene transfer experiments,[11,12] thus opening new research
avenues in the field of biological applications with pil-
K2CO3 in acetone at reflux afforded the next generation al-
cohol 5, which was converted into the corresponding benzyl-
ic bromide 6 by treatment with NBS/PPh3. Finally, azide de-
rivatives 7 and 8, which are required for conjugation on the
pillar[5]arene core, were obtained by reaction of the corre-
sponding benzylic bromides with sodium azide (NaN3) in
N,N-dimethylformamide (DMF) at room temperature.
A
As shown in Scheme 2, the complementary pillar[5]arene
building block 10 was prepared from compound 9[17] under
the classical conditions developed by Ogoshi and co-work-
ers.[18] Treatment of 9 and paraformaldehyde with BF3·Et2O
in 1,2-dichloroethane afforded cyclopentamer 10 in 40%
yield.[19] The terminal alkyne function is therefore compati-
ble with these conditions. It should, however, be noted that
only traces of pillar[5]arene 10 were obtained when the re-
action was performed with AlCl3 or FeCl3 as the Lewis acid
catalyst. Under these conditions, mainly polymers were ob-
tained. Grafting of dendrons 7 and 8 onto pillar[5]arene 10
was achieved under the CuAAC conditions we have previ-
ously developed for the functionalization of multi-alkyne
high efficiency and low toxicity remains a major challenge,
and synthetic chemistry is still at the center of this research.
Synthesis: The synthesis of polycationic pillar[5]arene deriv-
atives relies upon the copper-catalyzed alkyne–azide cyclo-
addition (CuAAC) reaction[13] to introduce dendrons with
peripheral benzyloxycarbonyl (Boc)-protected amine subu-
nits on both rims of the macrocyclic core. This methodology
has proven to be a powerful procedure for the functionaliza-
tion of pillar[5]arene derivatives due to its versatility and
the ready availability of starting materials.[4b,5,14] The prepa-
ration of the Boc-protected amine terminated poly(aryl
ether) dendritic branches is depicted in Scheme 1. They
have been obtained by a convergent synthesis by using the
classical methodology developed by Hawker and Frꢀchet.[15]
Compound 1 was prepared as previously described.[16] Lithi-
um aluminum hydride (LAH) mediated reduction of 1 gave
benzylic alcohol 2 in 99% yield. Subsequent bromination
with N-bromosuccinimide (NBS) and triphenylphosphine
(PPh3) gave benzylic bromide 3 in 82% yield. Reaction of 3
with 3,5-dihydroxybenzyl alcohol (4) in the presence of
cores.[20]
A
mixture of 10 (1 equiv),
7
(11 equiv),
CuSO4·5H2O (0.1 equiv), and sodium ascorbate (0.3 equiv)
in CH2Cl2/H2O was stirred for two days. After work-up and
purification, compound 11 was thus obtained in 88% yield.
Similarly, reaction of azide 8 with 10 gave dendronized pil-
lar[5]arene 12 in 81% yield.
The chemical structures of compounds 11 and 12 were
confirmed by NMR and IR spectroscopic analysis as well as
by mass spectrometry and elemental analysis (see the Sup-
1
porting Information). Their H and 13C NMR spectra, show-
ing one set of signals for all the ten equivalent peripheral
subunits, were fully consistent with their D5-symetrical struc-
tures. The expected molecular
ion peaks were also observed in
the MALDI-TOF mass spectra
of both 11 and 12, despite
a high level of fragmentation
resulting from the cleavage of
terminal Boc-protecting groups
as well as from the cleavage of
ether O–CH2–triazole bonds.
The monodispersity of both 11
and 12 was further supported
by their chromatograms record-
ed with a HPLC instrument
equipped with a PLgel size-ex-
clusion column (Figure 1). The
difference in retention time was
in perfect agreement with the
increasing size of the com-
pounds when going from 11 to
12.
Treatment of 11 and 12 with
a large excess of trifluoroacetic
acid (TFA) gave the corre-
sponding deprotected deriva-
tives as their trifluoroacetate
Scheme 1. Reagents and conditions: i) LAH, THF (99%); ii) NBS, PPh3, THF (3: 82%, 6: 73%); iii) K2CO3,
acetone (97%); iv) NaN3, DMF (7: 93%, 8: 90%).
Chem. Eur. J. 2013, 19, 17552 – 17558
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J.-S. Remy, J.-F. Nierengarten et al.
self-assembled nanostructures
are not well-defined. Com-
pounds 13 and 14 are indeed
bolaamphiphilic
compounds
and aggregation results most
probably from hydrophobic in-
teractions between the central
cores of the molecules.[21] At
lower concentration (<1.5 nm),
compound 14 no longer aggre-
gates, whereas compound 13
still forms nanoparticles with an
average diameter of ca. 70 nm.
This difference in behavior be-
tween 13 and 14 results from
differences in the size of their
polar subunits. When the gener-
ation number of the peripheral
dendrons is increased, intermo-
lecular contacts become more
difficult for steric reasons and
less favorable due to increased
electrostatic repulsion.
Interaction with DNA: The
ability of compounds 13 and 14
to bind plasmid DNA (pCMV-
Luc) was first assessed through
the use of gel electrophoresis.
The polyplexes[22] were pre-
pared from pCMV-Luc and 13
or 14 with increasing N/P
Scheme 2. Reagents and conditions: i) BF3·Et2O, (CH2O)n, ClCH2CH2Cl (40%); ii) CuSO4·5H2O, sodium as-
corbate, CH2Cl2/H2O (11: 88%, 12: 81%); iii) TFA (13: quantitative, 14: quantitative).
(vector amine per DNA phosphate) ratios in 5% aqueous
glucose solutions. Ethidium bromide fluorescence revealed
full DNA condensation at and above N/P=2 for both pil-
lar[5]arenes (Figure 2). Effectively, the condensed plasmids
remain in the well as soon as N/Pꢀ2. Furthermore, starting
from N/P=3 and above, the polyplexes are not visible in the
gel because the staining agent (ethidium bromide) can no
longer bind the highly condensed DNA. Polyplexes were
also prepared from pCMV-Luc and 13 or 14 at different N/P
ratios in aqueous 150 mm NaCl solutions. Agarose gel elec-
trophoresis (gel shift) of these polyplexes also revealed full
DNA condensation from N/P=2 for both 13 and 14 (see
the Supporting Information).
Figure 1. HPLC traces on a size exclusion column (PLgel, CH2Cl2, UV
Polyplexes obtained from pCMV-Luc and 13 or 14 in
either isotonic 150 mm NaCl or iso-osmotic 5% glucose sol-
utions in water were further characterized by transmission
electron microscopy (TEM). As typical examples, TEM
images observed for polyplexes prepared at N/P=5 in 5%
glucose aqueous solutions are shown in Figure 2. Discrete
spherical nanostructures were observed in both cases and
the size distribution was quite narrow. These results were
also confirmed by DLS measurements made on these poly-
plexes (see the Supporting Information). Interestingly, the
polyplexes obtained from both 13 and 14 were smaller in
size irrespective of the N/P ratios when compared with the
detector at l=254 nm) obtained for compounds 11 and 12.
1
salts in quantitative yields. The H and 13C NMR spectra of
13 and 14 clearly show the disappearance of the Boc-pro-
tecting groups. The broadening observed in the 1H NMR
spectra recorded in D2O is ascribed to aggregation (see the
Supporting Information). This view was supported by dy-
namic light scattering (DLS) measurements. At a concentra-
tion higher than 3 nm, large aggregates were evidenced for
both compounds. The distribution in size was rather large
(from ca. 100 to 1000 nm in diameter), suggesting that the
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Polycationic Pillar[5]arene Derivatives
FULL PAPER
Transfection experiments: The transfection efficiency of
the self-assembled 13/pCMV-Luc and 14/pCMV-Luc poly-
plexes prepared either in 5% glucose or 150 mm NaCl aque-
ous solutions was evaluated in vitro on HeLa cells. A com-
mercially available gene delivery system (Jet-PEI, Polyplus-
Transfection, Illkirch France) and naked pCMV-Luc were
used as positive and negative controls, respectively, for com-
parison purposes. The transfection capabilities were higher
for polyplexes prepared under iso-osmotic conditions than
those formulated under isotonic conditions (see the Sup-
porting Information). This is consistent with the TEM and
DLS results; the polyplexes prepared in glucose have the
appropriate size for an optimum cellular uptake and should
be of interest for in vivo experiments.[23]
For polyplexes prepared under iso-osmotic conditions, 13
and 14 had practically the same level of luciferase expres-
sion at their optimal N/P ratio, that is, between 109 and
1010 RLU per mg of protein (Figure 3). Although being less
Figure 2. Top: Electrophoresis mobility shift assays performed with poly-
plexes prepared in 5% glucose solutions in water with plasmid DNA
(pCMV-Luc) (0.4 mg) and A) 13 or B) 14 at increasing N/P (indicated
above the gel); the control is plasmid pCMV-Luc without vector. Bot-
tom: TEM images of C) 13/pCMVLuc (scale bar: 2 mm) and D) 14/
pCMVLuc polyplexes (scale bar: 1 mm) at N/P 5 in water with 5% glu-
cose.
aggregates observed for 13 and 14 alone under the same
conditions. It appears that the electrostatic interactions of
the negatively charged pCMV-Luc plasmid with the aggre-
gates of 13 or 14 is capable of disrupting/remodeling, at
least in part, the hydrophobic interactions of the bolaamphi-
philic pillar[5]arene derivatives, thus showing that they must
be rather weak. However, the addition of electrostatic and
van der Waals interactions leads to strong complexation of
plasmid DNA. Nevertheless, the bolaamphiphilic character
of 13 and 14 plays an important role in the transfection ex-
periments (see below). ZÞta (&) potential measurements
showed positively charged polyplexes from N/P=2 and
above, in agreement with the transfection experiments.
The polyplexes prepared from both 13 and 14 in 150 mm
aqueous NaCl are larger in size than those obtained in 5%
glucose aqueous solutions. At N/P ratios higher than 5, the
polyplexes are clumped and highly irregular in shape, which
are actually made of aggregated spheres (see the Supporting
Information). A similar behavior has been also reported for
polyplexes prepared from DNA and polyethylenimine
(PEI),[23] and results from ionic strengths effects (i.e., a de-
crease in the electrostatic repulsions between particles). The
results of DLS measurements on the particles formulated in
NaCl were consistent with the TEM observations for both
13 and 14. It should be noted that the & potentials could not
be determined under the saline conditions.
Figure 3. In vitro gene delivery experiments on HeLa cells of pCMVLuc
with pillar[5]arenes 13 and 14 at various N/P (polyplexes prepared in 5%
glucose solutions). Luciferase expression (bars) and percentage of total
cellular proteins (squares) are given for negative control (untreated
HeLa cells), positive control (PEI), 13, and 14. Means and standard devi-
ation of separate triplicates are given.
efficient than the ꢃgolden standardꢄ Jet-PEI, both 13 and 14
are by far less toxic, as attested by much higher levels of
total cellular proteins. Importantly, in contrast to classical
dendritic vectors, for which high efficiency requires general-
ly high generation numbers,[24] the first-generation dendri-
mer (13) already has optimum gene delivery capabilities.
This is certainly associated with the bolaamphiphilic charac-
ter of compound 13 and its ability to aggregate, thus increas-
ing the stability of the polyplexes. Indeed, globular systems
with an isotropic repartition of charges requires sufficient
numbers of peripheral amino groups to ensure DNA com-
paction into stable and positively charged polyplexes.[25,26]
For example, an analogous hexasubstituted fullerene deriva-
Chem. Eur. J. 2013, 19, 17552 – 17558
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17555

J.-S. Remy, J.-F. Nierengarten et al.
(75 MHz, CDCl3): d=160.0, 156.0, 143.9, 105.5, 100.6, 79.7, 67.3, 65.0,
tive substituted with 24 peripheral ammonium groups has
shown low gene transfer capabilities whereas the corre-
sponding next generation compound with 48 peripheral am-
monium groups is highly efficient.[26]
À1
À
À
40.1, 28.5 ppm; IR (neat): n˜ =3347 (O H), 3360 (N H), 1689 cm (C=
O); elemental analysis calcd (%) for C21H34N2O7 (426.50): C 59.14, H
8.03, N 6.57; found: C 58.56, H 8.04, N 6.31.
Compound 3:
A solution of 2 (3.52 g, 8.25 mmol), PPh3 (2.81 g,
10.73 mmol), and N-bromosuccinimide (1.91 g, 10.73 mmol) in THF
(50 mL) was stirred at RT. After 15 min, H2O was added and the result-
ing aqueous layer was extracted with CH2Cl2 (3ꢅ). The combined organic
layers were dried (MgSO4), filtered, and evaporated. Column chromatog-
raphy (SiO2; cyclohexane/EtOAc 8:2) gave 3 (3.31 g, 82%) as a colorless
solid. 1H NMR (300 MHz, CDCl3): d=6.54 (d, J=2 Hz, 2H), 6.38 (t, J=
2 Hz, 1H), 4.94 (br. s, 2H), 4.39 (s, 2H), 4.00 (t, J=5 Hz, 4H), 3.52 (m,
Conclusion
Dendritic pillar[5]arene derivatives 11 and 12 have been ef-
ficiently prepared by grafting dendrons with peripheral Boc-
protected amine subunits onto a preconstructed pillar[5]ar-
ene scaffold. Upon cleavage of the Boc-protected groups,
water-soluble pillar[5]arene derivatives 13 and 14 with 20
and 40 peripheral ammonium groups, respectively, have
been obtained. The capacity of these compounds to form
stable nanoparticles with plasmid DNA has been demon-
strated by gel electrophoresis, TEM, and DLS investigations.
Finally, the capability of both 13 and 14 to efficiently con-
dense plasmid DNA into stable and positively charged poly-
plexes has been exploited in gene delivery experiments. The
transfection efficiencies found for both 13 and 14 are good,
albeit slightly lower than that obtained for PEI. What is
more, these pillar[5]arene derivatives exhibit good efficien-
cy, while maintaining low toxicity. Both 13 and 14 are by far
less toxic than PEI. Owing to the possibility of preparing ef-
ficiently rotaxanes from pillar[5]arene derivatives,[14b,27] addi-
tional functional groups (e.g., specific sugars for cell target-
ing or fluorescent moieties for monitoring cell uptake)
should be easily incorporated onto the molecular thread of
rotaxane derivatives incorporating 13 or 14 as their macro-
cyclic component. Therefore, the results reported herein
pave the way towards the development of a new generation
of vectors capable of carrying out several tasks. Work in this
direction is underway in our laboratories.
4H), 1.45 ppmACTHNUTRGNEUNG
(s, 18H); 13C NMR (75 MHz, CDCl3): d=159.8, 155.5,
139.9, 107.8, 101.4, 79.8, 67.3, 40.0, 33.3, 28.4 ppm; IR (neat): n˜ =3366
(N H), 1677 cmÀ1 (C=O); elemental analysis calcd (%) for
À
C21H33BrN2O6 (489.40): C 51.54, H 6.80, N 5.72; found: C 51.27, H 6.59,
N 5.57.
Compound 5: A mixture of 3 (3.24 g, 6.62 mmol), 4 (0.40 g, 2.88 mmol),
and K2CO3 (1.59 g, 11.54 mmol) in acetone (55 mL) was heated to reflux.
After 2 days, the mixture was filtered and evaporated. Column chroma-
tography (SiO2; CH2Cl2/EtOAc 7:3) gave 5 (2.67 g, 97%) as a colorless
1
solid. H NMR (300 MHz, CDCl3): d=6.61 (d, J=2 Hz, 2H), 6.56 (d, J=
2 Hz, 4H), 6.50 (t, J=2 Hz, 1H), 6.39 (t, J=2 Hz, 2H), 5.00 (br. s, 4H),
4.97 (s, 4H), 4.64 (d, J=4 Hz, 2H), 4.00 (t, J=5 Hz, 8H), 3.51 (m, 8H),
À1
À
À
1.45 ppm (s, 36H); IR (neat): n˜ =3346 (O H/N H), 1686 cm (C=O);
elemental analysis calcd (%) for C49H72N4O15 (957.12): C 61.49, H 7.58, N
5.85; found: C 61.60, H 7.66, N 5.70.
Compound 6:
A solution of 5 (2.68 g, 2.80 mmol), PPh3 (0.95 g,
3.63 mmol), and N-bromosuccinimide (0.65 g, 3.64 mmol) in THF
(25 mL) was stirred at RT. After 20 min, H2O was added and the result-
ing aqueous layer was extracted with CH2Cl2 (3ꢅ). The combined organ-
ic layers were dried (MgSO4), filtered, and evaporated. Column chroma-
tography (SiO2; cyclohexane/EtOAc 7:3) gave 6 (2.08 g, 73%) as a color-
1
less solid. H NMR (300 MHz, CDCl3): d=6.63 (d, J=2 Hz, 2H), 6.56 (d,
J=2 Hz, 4H), 6.52 (t, J=2 Hz, 1H), 6.40 (t, J=2 Hz, 2H), 4.98 (br. s,
4H), 4.96 (s, 4H), 4.41 (s, 2H), 4.01 (t, J=5 Hz, 8H), 3.52 (m, 8H),
À1
À
1.45 ppm (s, 36H); IR (neat): n˜ =3347 (N H), 1691 cm (C=O); elemen-
tal analysis calcd (%) for C49H71BrN4O14·0.6 CH2Cl2: C 55.69, H 6.81, N
5.24; found: C 55.76, H 6.86, N 5.25.
Compound 7: A solution of 3 (0.92 g, 1.88 mmol) and NaN3 (0.247 g,
3.8 mmol) in DMF (20 mL) was stirred at RT. After 12 h, H2O was
added and the resulting aqueous layer was extracted with Et2O (3ꢅ). The
combined organic layers were washed with H2O, dried (MgSO4), filtered,
and evaporated. Column chromatography (SiO2; cyclohexane/EtOAc
7:3) gave 7 (0.79 g, 93%) as a colorless solid. 1H NMR (300 MHz,
CDCl3): d=6.46 (d, J=2 Hz, 2H), 6.41 (t, J=2 Hz, 1H), 4.96 (br. s, 2H),
4.26 (s, 2H), 4.01 (t, J=5 Hz, 4H), 3.52 (m, 4H), 1.45 ppm (s, 18H);
13C NMR (75 MHz, CDCl3): d=160.2, 156.0, 138.0, 107.0, 101.3, 79.7,
Experimental Section
General: All reagents were used as purchased from commercial sources
without further purification. Compounds 1[16] and 9[17] were prepared ac-
cording to previously reported procedures. Evaporation and concentra-
tion were performed at water aspirator pressure and drying in vacuo at
10À2 Torr. Column chromatography: silica gel 60 (230–400 mesh, 0.040–
0.063 mm) was purchased from E. Merck. Thin-layer chromatography
(TLC) was performed on glass sheets coated with silica gel 60 F254 pur-
chased from E. Merck, visualization by UV light. NMR spectra were re-
corded with a Bruker AC 300 or AC 400, with solvent peaks as reference.
IR spectra were recorded with a Spectrum Two PerkinElmer FTIR spec-
trometer. Elemental analyses were performed by the analytical service of
the Faculty of Chemistry (University of Strasbourg). MALDI-TOF mass
spectra were recorded by the analytical service of the School of Chemis-
try (Strasbourg, France).
À
67.5, 54.9, 40.2, 28.5 ppm; IR (neat): n˜ =3345 (N H), 2098 (N3),
1680 cmÀ1 (C=O); elemental analysis calcd (%) for C21H33N5O6 (451.52):
C 55.86, H 7.37, N 15.51; found: C 55.50, H 7.09, N 15.11.
Compound 8: This compound was prepared as described for 7 starting
from 6 (600 mg, 0.59 mmol) and NaN3 (76 mg, 1.17 mmol) in DMF
(10 mL). After work-up, column chromatography (SiO2; cyclohexane/
EtOAc 7:3) gave
8 (521 mg, 90%) as a
colorless solid. 1H NMR
(300 MHz, CDCl3): d=6.55–6.57 (m,7H), 6.40 (t, J=2 Hz, 2H), 4.99
(br. s, 4H), 4.97 (s, 4H), 4.27 (s, 2H), 4.01 (t, J=5 Hz, 8H), 3.52 (m, 8H),
1.45 ppm (s, 36H); 13C NMR (75 MHz, CDCl3): d=160.2, 160.1, 156.0,
139.3, 137.8, 107.4, 106.1, 101.9, 101.0, 79.6, 70.0, 67.4, 54.9, 40.2,
À1
À
28.5 ppm; IR (neat): n˜ =3347 (N H), 2100 (N3), 1694 cm (C=O); ele-
Compound 2: LAH (1m in THF, 8.2 mL, 8.23 mmol) was added dropwise
to a solution of 1 (3.74 g, 8.23 mmol) in anhydrous THF (100 mL) at 08C
under argon. After 3 h, MeOH (1 mL) was slowly added, followed by
H2O (several drops). The resulting mixture was filtered through Celite
and evaporated. Column chromatography (SiO2; CH2Cl2/EtOAc 1:1)
gave 2 (3.48 g, 99%) as a colorless solid. 1H NMR (300 MHz, CDCl3):
d=6.53 (d, J=2 Hz, 2H), 6.37 (t, J=2 Hz, 1H), 4.95 (br. s, 2H), 4.63 (s,
2H), 4.01 (t, J=5 Hz, 4H), 3.52 (m, 4H), 1.45 ppm (s, 18H); 13C NMR
mental analysis calcd (%) for C49H71N7O14 (982.13): C 59.92, H 7.29, N
9.98; found: C 59.93, H 7.34, N 9.68.
Compound 10: BF3·Et2O (2.82 g, 19.87 mmol) was added to a stirred so-
lution of
9
(3.70 g, 19.87 mmol) and paraformaldehyde (1.79 g,
59.61 mmol) in 1,2-dichloroethane (200 mL). The reaction mixture was
concentrated after being heated at 308C for 3 h. Column chromatography
(SiO2; cyclohexane/CH2Cl2, 1:1) gave 10 (1.59 g, 40%) as a colorless
17556
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Chem. Eur. J. 2013, 19, 17552 – 17558

Polycationic Pillar[5]arene Derivatives
FULL PAPER
solid. The analytical data of 10 were in complete agreement with litera-
nanoZS apparatus with the following specifications: sampling time=
120 s; refractive index of medium (water with 5% glucose)=1.340; re-
fractive index of particles=1.43; medium viscosity=1.0140 cP; tempera-
ture=258C. Data were analyzed by using the multimodal number distri-
bution software included with the instrument. & potentials were measured
with the same apparatus and with the following specifications: 20 meas-
urements per sample; dielectric constant=80; temperature=258C; beam
mode F(Ka)=1.5 (Smoluchowski model). Polyplexes (1 mL volume)
were prepared as described for delivery experiment.
ture data.[4b,19]
Compound 11:
A mixture of 10 (200 mg, 0.20 mmol), 7 (1.0 g,
2.22 mmol), CuSO4·5H2O (5 mg, 0.02 mmol), and sodium ascorbate
(12 mg, 0.06 mmol) in CH2Cl2/H2O (1:1, 16 mL) was vigorously stirred at
RT under Ar. After two days, H2O was added and the aqueous layer was
extracted with CH2Cl2 (3ꢅ) and the combined organic layers were dried
(MgSO4), filtered, and concentrated. Column chromatography (SiO2;
CH2Cl2 containing 3% methanol) followed by gel permeation chroma-
tography (Biobeads SX-1, CH2Cl2) gave 11 (973 mg, 88%) as a colorless
glassy product. 1H NMR (CD2Cl2, 300 MHz): d=7.88 (s, 10H), 6.96 (s,
10H), 6.36 (br. s, 20H), 6.35 (d, J=2 Hz, 10H), 5.46 (br. s, 20H), 5.36 (m,
20H), 4.80 (AB, J=12 Hz, 20H), 3.90 (t, J=5 Hz, 40H), 3.73 (s, 10H),
3.42 (m, 40H), 1.44 ppm (s, 180H); 13C NMR (CDCl3, 75 MHz): d=
160.1, 156.0, 149.7, 144.6, 137.3, 128.7, 123.6, 115.8, 106.9, 100.9, 79.4,
Electron microscopy analysis: Images were taken with a TEM Delong
LVEM5 Instrument (Cordouan Technologies, Pessac, France). Polyplexes
were transferred onto 300 mesh ultrathin carbon film copper grids (EMS,
Washington, USA) by placing the grid on top of 10 mL drop for 1 min.
Grid with adherent particles was wicked from one side and air dried
before imaging.
À
67.3, 62.5, 53.9, 53.2, 39.9, 28.4 ppm; IR (neat): n˜ =3348 (N H),
Cell culture: HeLa cells (ATCC, Eurobio, Courtabœuf, France) were
grown in Eagleꢄs MEM supplemented with 10% FBS, l-glutamine
(2 mm), penicillin (100 units per mL), and streptomycin (100 mgmLÀ1).
Cells were maintained at 378C in a 5% CO2 humidified atmosphere and
all experiments were performed in triplicate. The day before experiment,
cells were seeded in 24-multiwell plates at 50.103 cells/well in fresh com-
plete medium (1 mL).
1692 cmÀ1 (C=O); MALDI-TOF-MS: m/z calcd for C275H381N50O70
:
5507.27 [M+H]+; found: 5507; elemental analysis calcd (%) for
C275H380N50O70·2CH2Cl2 (5676.13): C 58.61, H 6.82, N 12.34; found: C
58.91, H 6.55, N 12.34.
Compound 12:
A mixture of 10 (100 mg, 0.10 mmol), 8 (1.09 g,
1.11 mmol), CuSO4·5H2O (2.5 mg, 0.01 mmol), and sodium ascorbate
(6 mg, 0.03 mmol) in CH2Cl2/H2O (1:1, 6 mL) was vigorously stirred at
RT under Ar. After 6 days, H2O was added and the aqueous layer was
extracted with CH2Cl2 (3ꢅ) and the combined organic layers were dried
(MgSO4), filtered, and concentrated. Column chromatography (SiO2;
CH2Cl2 containing 2% methanol) followed by gel permeation chroma-
tography (Biobeads SX-1, CH2Cl2) gave 12 (930 mg, 81%) as a colorless
glassy product. 1H NMR (CD2Cl2, 300 MHz): d=7.85 (s, 10H), 6.93 (s,
10H), 6.40 (s, 60H), 6.30 (s, 30H), 5.39 (br. s, 40H), 5.33 (s, 20H), 4.70
(m, 60H), 3.86 (s, 80H), 3.66 (s, 10H), 3.38 (d, J=3 Hz, 80H), 1.38 ppm
(s, 360H); 13C NMR (CD2Cl2, 75 MHz): d=160.5, 160.3, 156.3, 149.8,
145.0, 139.5, 137.9, 129.1, 124.0, 115.3, 107.4, 106.4, 102.2, 100.9, 79.5,
Polyplexe formation for pCMVLuc delivery: The procedure is for a 24-
multiwell plate (ref. 3524, Corning, NY, USA) experiment. Typically, an
aqueous solution of 13 or 14 (volume depending on the desired N/P
ratio) was diluted to 50 mL in water containing 5% glucose or 150 mm
NaCl. The solution was vortexed and left for 10 min. Separately, an aque-
ous solution of pCMVLuc (corresponding to 2 mg pCMVLuc) was diluted
to 50 mL in water containing 5% glucose or 150 mm NaCl. The solution
was then vortexed and left for 10 min, after which time the 13 or 14 solu-
tion was added to the pCMVLuc solution, and vigorously mixed (15 s).
Finally, the polyplexes were incubated for 30 min at RT and added to
each well by dilution with the cell medium without serum (1 mL). After
4 h, each well was completed with serum (0.1 mL). The gene expression
profiles were analyzed 24 h after addition of polyplexes.
À
70.1, 67.6, 62.5, 54.3, 54.1, 40.4, 28.6 ppm; IR (neat): n˜ =3348 (N H),
1692 cmÀ1 (C=O); MALDI-TOF-MS: m/z calcd for C555H761N70O150
:
10812.43 [M+H]+; found: 10812; elemental analysis calcd (%) for
C555H760N70O150 A4CH2Cl2 (11152.10): C 60.20, H 6.94, N 8.79; found: C
Quantification of luciferase gene expression: Luciferase gene expression
was determined 24 h after delivery with a commercial kit, using the man-
ufacturerꢄs protocol (Luciferase Assay System, Promega, Charbonniꢁres,
France). The luminescence was measured from 2 mL of lysate during 1 s
with a luminometer (Centro LB960 XS; Berthold, Thoiry, France). Luci-
ferase activity is expressed as the mean of light units integrated over 10 s
(RLU) and normalized per mg of cell protein by using the BCA assay
(Pierce, Brebiꢁres, France). The errors bars represent standard deviation
derived from triplicate experiments.
·CHTUNGTRENNUNG
60.28, H 6.59, N 8.75.
Compound 13: Compound 11 (297 mg, 0.055 mmol) was dissolved in
TFA (5 mL). After 5 h, the mixture was evaporated and dried under
vacuum to afford 13 as its trifluoroacetate salt (317 mg, quantitative) as
a colorless solid. 1H NMR (300 MHz, D2O): d=7.71 (s, 10H), 6.39 (s,
30H), 6.29 (s, 10H), 5.31 (s, 20H), 4.17 (d, J=12 Hz, 10H), 3.98 (m,
50H), 3.48 (s, 10H), 3.17 ppm (s, 40H); 13C NMR (75 MHz, D2O): d=
162.6 (q, J=35 Hz), 159.2, 149.8, 143.7, 137.4, 129.3, 124.6, 116.4 (q, J=
290 Hz), 116.3, 107.5, 101.5, 64.0, 62.0, 53.6, 48.9, 38.7 ppm; IR (neat): n˜ =
1672 cmÀ1 (C=O); elemental analysis calcd (%) for C175H240N50O30·
(CF3CO2)À
20·ACHTUNGTRENNUNG15H2O (5964.56): C 42.65, H 4.49, N 11.57; found: C 42.60,
H 4.47, N 11.48.
Acknowledgements
Compound 14: Compound 12 (200 mg, 0.019 mmol) was dissolved in
TFA (6 mL). After 5 h, the mixture was evaporated and dried under
vacuum to afford 14 as its trifluoroacetate salt (215 mg, quantitative) as
a colorless solid. 1H NMR (300 MHz, D2O): d=7.67 (br. s, 10H), 6.35
(m, 100H), 5.09 (br. s, 20H), 4.45 (br. s, 40H), 3.91 (m, 100H), 3.42 (br. s,
10H), 3.17 ppm (br. s, 80H); 13C NMR (100 MHz, D2O): d=162.5 (q, J=
35 Hz), 159.7, 158.9, 149.3, 143.9, 139.1, 137.2, 130.2, 128.9, 124.2, 116.4
(q, J=291 Hz), 107.3, 106.5, 101.8, 101.2, 69.3, 63.9, 61.5, 53.6, 51.2,
38.8 ppm; IR (neat): n˜ =1672 cmÀ1 (C=O); elemental analysis cacld (%)
This work was supported by the CNRS (UMR 7509 and 7199). I.N.
thanks the Agence Nationale de la Recherche (ANR) and D.S. the
French Ministry of Research (MENRT) for their fellowships. We further
thank M. Schmitt for NMR measurements.
[1] T. Ogoshi, S. Kanai, S. Fujinami, T.-A. Yamagishi, Y. Nakamoto, J.
Am. Chem. Soc. 2008, 130, 5022–5023.
for C355H480N70O ·
7.87; found: C 41.45, H 4.23, N 7.45.
70 ACHTUNGTRENNUNG H 4.85, N
(CF3CO2)À40·60H2O (12449.59): C41.97,
[2] For reviews on pillar[n]arenes, see: a) P. J. Cragg, K. Sharma, Chem.
Soc. Rev. 2012, 41, 597–607; b) M. Xue, Y. Yang, X. Chi, Z. Zhang,
F. Huang, Acc. Chem. Res. 2012, 45, 1294–1308; c) T. Ogoshi, J. In-
clusion Phenom. Macrocyclic Chem. 2012, 72, 247–262; d) T.
Ogoshi, T.-a. Yamagishi, Eur. J. Org. Chem. 2013, 2961–2975.
[3] a) T. Ogoshi, M. Hashizume, T.-a. Yamagishi, Y. Nakamoto, Chem.
Commun. 2010, 46, 3708–3710; b) Y. Ma, X. Ji, F. Xiang, X. Chi, C.
Han, J. He, Z. Abliz, W. Chen, F. Huang, Chem. Commun. 2011, 47,
12340–12342; c) C. Li, X. Shu, J. Li, S. Chen, K. Han, M. Xu, B.
Hu, Y. Yu, X. Jia, J. Org. Chem. 2011, 76, 8458–8465; d) G. Yu, M.
Agarose gel electrophoresis analysis: Water (30 mL) containing glucose
(5%) or NaCl (150 mm), pCMVLuc (0.4 mg) and increasing amounts of
cationic vectors 13 and 14 were subjected (30 min of complexation time)
to electrophoresis in a 1% agarose gel containing 1 mm EDTA and
40 mm Tris acetate buffer and 0.5 mgmLÀ1 ethidium bromide, for 90 min
at 100 V. DNA was visualized with an UV transilluminator at 254 nm.
Size of particles, & potential: The hydrodynamic radii were determined
on the basis of dynamic light scattering measurements with a Malvern
Chem. Eur. J. 2013, 19, 17552 – 17558
ꢂ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
17557

J.-S. Remy, J.-F. Nierengarten et al.
Xue, Z. Zhang, J. Li, C. Han, F. Huang, J. Am. Chem. Soc. 2012,
134, 13248–13251.
Forgan, J. M. Spruell, Y. Y. Botros, J. F. Stoddart, J. Am. Chem. Soc.
2011, 133, 5668–5671; c) N. L. Strutt, H. Zhang, M. A. Giesener, J.
Lei, J. F. Stoddart, Chem. Commun. 2012, 48, 1647–1649; d) H.
Deng, X. Shu, X. Hu, J. Li, X. Jia, C. Li, Tetrahedron Lett. 2012, 53,
4609–4612; e) G. Yu, Z. Zhang, J. He, Z. Abliz, F. Huang, Eur. J.
Org. Chem. 2012, 5902–5907.
[4] a) T. Ogoshi, K. Umeda, T.-a. Yamagishi, Y. Nakamoto, Chem.
Commun. 2009, 4874–4876; b) T. Ogoshi, R. Shiga, M. Hashizume,
T.-a. Yamagishi, Chem. Commun. 2011, 47, 6927–6929; c) H. Zhang,
X. Ma, J. Guo, K. T. Nguyen, Q. Zhang, X.-J. Wang, H. Yan, L. Zhu,
Y. Zhao, RSC Adv. 2013, 3, 368–371; d) T. Ogoshi, D. Yamafuji, D.
Kotera, T. Aoki, S. Fujinami, T.-a. Yamagishi, J. Org. Chem. 2012,
77, 11146–11152; e) T. Ogoshi, D. Yamafuji, T.-a. Yamagishi, A. M.
Brouwer, Chem. Commun. 2013, 49, 5468–5470.
[5] a) I. Nierengarten, S. Guerra, M. Holler, J.-F. Nierengarten, R. De-
schenaux, Chem. Commun. 2012, 48, 8072–8074; b) I. Nierengarten,
S. Guerra, M. Holler, L. Karmazin-Brelot, J. Barbera, R. Desche-
naux, J.-F. Nierengarten, Eur. J. Org. Chem. 2013, 3675–3684.
[6] a) W. Si, L. Chen, X. B. Hu, G. Tang, Z. Chen, J.-L. Hou, Z. T. Li,
Angew. Chem. 2011, 123, 12772–12776; Angew. Chem. Int. Ed. 2011,
50, 12564–12568; b) L. Chen, W. Si, L. Zang, G. Tang, Z.-T. Li, J.-L.
Hou, J. Am. Chem. Soc. 2013, 135, 2152–2155.
[15] a) C. Hawker, J. M. J. Frꢀchet, J. Chem. Soc. Chem. Commun. 1990,
1010–1013; b) C. J. Hawker, J. M. J. Frꢀchet, J. Am. Chem. Soc.
1990, 112, 7638–7647.
[16] A. J. Brouwer, S. J. E. Mulders, R. M. J. Liskamp, Eur. J. Org. Chem.
2001, 1903–1915.
[17] H.-K. Shim, D.-H. Hwang, C.-B. Yoon, Macromol. Chem. Phys.
1996, 197, 2393–2401.
[18] T. Ogoshi, T. Aoki, K. Kitajima, S. Fujinami, T.-a. Yamagishi, Y. Na-
kamoto, J. Org. Chem. 2011, 76, 328–331.
[19] The preparation of compound 10 from 9 has already been reported,
but the yield was moderate (17%, see ref. [14e]). By optimizing the
concentration conditions, the amount of BF3·Et2O, and the work-up
procedure, the yield of the cyclization reaction was improved
(40%).
[7] X.-B. Hu, Z. Chen, G. Tang, J.-L. Hou, Z.-T. Li, J. Am. Chem. Soc.
2012, 134, 8384–8387.
[8] G. Yu, X. Zhou, Z. Zhang, C. Han, Z. Mao, C. Gao, F. Huang, J.
Am. Chem. Soc. 2012, 134, 19489–19497.
[9] I. Nierengarten, K. Buffet, M. Holler, S. P. Vincent, J.-F. Nierengart-
en, Tetrahedron Lett. 2013, 54, 2398–2402.
[10] G. Yu, Y. Ma, C. Han, Y. Yao, G. Tang, Z. Mao, C. Gao, F. Huang,
J. Am. Chem. Soc. 2013, 135, 10310–10313.
[11] The present paper describes the first examples of transfection ex-
periments with pillar[5]arene derivatives, however, synthetic vectors
constructed on macrocyclic scaffolds have been described; for
a review, see: C. Ortiz Mellet, J. M. Benito, J. M. Garcia Fernandez,
Chem. Eur. J. 2010, 16, 6728–6742.
[20] a) J. Iehl, J.-F. Nierengarten, Chem. Eur. J. 2009, 15, 7306–7309;
b) J. Iehl, J.-F. Nierengarten, Chem. Commun. 2010, 46, 4160–4162;
c) J.-F. Nierengarten, J. Iehl, V. Oerthel, M. Holler, B. M. Illescas, A.
MuÇoz, N. Martꢆn, J. Rojo, M. Sꢉnchez-Navarro, S. Cecioni, S.
Vidal, K. Buffet, M. Durka, S. P. Vincent, Chem. Commun. 2010, 46,
3860–3862; d) P. Compain, C. Decroocq, J. Iehl, M. Holler, D. Ha-
zelard, T. Mena Barragꢉn, C. Ortiz Mellet, J.-F. Nierengarten,
Angew. Chem. 2010, 122, 5889–5892; Angew. Chem. Int. Ed. 2010,
49, 5753–5756; e) M. Durka, K. Buffet, J. Iehl, M. Holler, J.-F. Nier-
engarten, J. Taganna, J. Bouckaert, S. P. Vincent, Chem. Commun.
2011, 47, 1321–1323; f) S. Cecioni, V. Oerthel, J. Iehl, M. Holler, D.
Goyard, J.-P. Praly, A. Imberty, J.-F. Nierengarten, S. Vidal, Chem.
Eur. J. 2011, 17, 3252–3261; g) M. Durka, K. Buffet, J. Iehl, M.
Holler, J.-F. Nierengarten, S. P. Vincent, Chem. Eur. J. 2012, 18,
641–651.
[12] For selected examples of synthetic gene vectors constructed on mac-
rocyclic scaffolds, see: a) F. Sansone, M. Dudic, G. Donofrio, C. Riv-
etti, L. Baldini, A. Casnati, S. Cellai, R. Ungaro, J. Am. Chem. Soc.
2006, 128, 14528–14536; b) V. Bagnacani, F. Sansone, G. Donofrio,
L. Baldini, A. Casnati, R. Ungaro, Org. Lett. 2008, 10, 3953–3956;
c) S.-A. Cryan, A. Holohan, R. Donohue, R. Darcy, C. M. OꢄDris-
coll, Eur. J. Pharm. Sci. 2004, 21, 625–633; d) N. Mourtzis, M. Para-
vatou, I. M. Mavridis, M. L. Roberts, K. Yannakopoulou, Chem.
Eur. J. 2008, 14, 4188–4200; e) S. Srinivasachari, K. M. Fichter,
T. M. Reineke, J. Am. Chem. Soc. 2008, 130, 4618–4627; f) S. A.
Cryan, R. Donohue, B. J. Ravoo, R. Darcy, C. M. OꢄDriscoll, J.
Drug Delivery Sci. Technol. 2004, 14, 57–62; g) A. Dꢆaz-Moscoso, L.
Le Gourriꢀrec, M. Gꢇmez-Garcꢆa, J. M. Benito, P. Balbuena, F.
Ortega-Caballero, N. Guilloteau, C. Di Giorgio, P. Vierling, J.
Defaye, C. Ortiz Mellet, J. M. Garcꢆa Fernandez, Chem. Eur. J. 2009,
15, 12871–12888; h) A. Mꢀndez-Ardoy, M. Gꢇmez-Garcꢆa, C. Ortiz
Mellet, N. Sevillano, M. D. Girꢇn, R. Salto, F. Santoyo-Gonzalez,
J. M. Garcꢆa Fernandez, Org. Biomol. Chem. 2009, 7, 2681–2684;
i) C. Bienvenu, ꢈ. Martꢆnez, J. L. Jimꢀnez Blanco, C. Di Giorgio, P.
Vierling, C. Ortiz Mellet, J. Defayec, J. M. Garcꢆa Fernꢉndez, Org.
Biomol. Chem. 2012, 10, 5570–5581; j) R. Lalor, J. L. DiGesso, A.
Mueller, S. E. Matthews, Chem. Commun. 2007, 4907–4909; k) R. V.
Rodik, A. S. Klymchenko, N. Jain, S. I. Miroshnichenko, L. Richert,
V. I. Kalchenko, Y. Mꢀly, Chem. Eur. J. 2011, 17, 5526–5538; l) V.
Bagnacani, V. franceschi, M. Bassi, M. Lomazzi, G. Donofrio, F.
Sansone, A. Casnati, R. Ungaro, Nature Comm. 2013, 4, 1721.
[13] a) H. C. Kolb, M. G. Finn, K. B. Sharpless, Angew. Chem. 2001, 113,
2056–2075; Angew. Chem. Int. Ed. 2001, 40, 2004–2021; b) C. Re-
mzi Becer, R. Hoogenboom, U. S. Schubert, Angew. Chem. 2009,
121, 4998–5006; Angew. Chem. Int. Ed. 2009, 48, 4900–4908.
[14] a) H. Zhang, N. L. Strutt, R. S. Stoll, H. Li, Z. Zhu, J. F. Stoddart,
Chem. Commun. 2011, 47, 11420–11422; b) N. L. Strutt, R. S.
[21] Such interactions have been reported for bolaamphiphilic pillar[5]ar-
ene derivatives, see: L. Gao, B. Zheng, Y. Yao, F. Huang, Soft
Matter 2013, 9, 7314–7319.
[22] P. L. Felgner, Y. Barenholz, J.-P. Behr, S. H. Cheng, P. Cullis, L.
Huang, J. A. Jessee, L. Seymour, F. Szoka, A. R. Thierry, E. Wagner,
G. Wu, Hum. Gene Ther. 1997, 8, 511–512.
[23] D. Goula, J.-S. Remy, P. Erbacher, M. Wasowicz, G. Levi, B. Abdal-
lah, B. A. Demeneix, Gene Ther. 1998, 5, 712–717.
[24] a) M. Guillot-Nieckowski, S. Eisler, F. Diederich, New J. Chem.
2007, 31, 1111–1127, and references therein; b) A.-M. Caminade,
C.-O. Turrin, J.-P. Majoral, Chem. Eur. J. 2008, 14, 7422–7432; c) A.-
M. Caminade, J.-P. Majoral, New J. Chem. 2013, 37, 3358–3373.
[25] H. Isobe, W. Nakanishi, N. Tomita, S. Jinno, H. Okayama, E. Naka-
mura, Chem. Asian J. 2006, 1, 167–175.
[26] D. Sigwalt, M. Holler, J. Iehl, J.-F. Nierengarten, M. Nothisen, E.
Morin, J.-S. Remy, Chem. Commun. 2011, 47, 4640–4642.
[27] a) T. Ogoshi, Y. Nishida, T.-a. Yamagishi, Y. Makamoto, Macromole-
cules 2010, 43, 3145–3147; b) T. Ogoshi, R. Shiga, T.-a. Yamagishi, J.
Am. Chem. Soc. 2012, 134, 4577–4580; c) S. Dong, C. Han, B.
Zheng, M. Zhang, F. Huang, Tetrahedron Lett. 2012, 53, 3668–3671;
d) T. Ogoshi, D. Yamafuji, T. Aoki, K. Kitajima, T.-a. Yamagishi, Y.
Hayashi, S. Kawauchi, Chem. Eur. J. 2012, 18, 7493–7500; e) P. Wei,
X. Yan, J. Li, Y. Ma, Y. Yao, F. Huang, Tetrahedron 2012, 68, 9179–
9185.
Received: July 31, 2013
Published online: November 11, 2013
17558
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