Uncovering new properties of imidazolium salts: Cl- transport and supramolecular regulation of their transmembrane activity

Article abstract of DOI:10.1039/c0cc04280c

We report the Cl^- transport activity for three imidazolium-based transporters. We present significant findings regarding the use of α-cyclodextrin and cucurbit[7]uril macrocycles to form inclusion complexes with these salts and to inhibit their

Full text of DOI:10.1039/c0cc04280c

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Uncovering new properties of imidazolium salts: Clꢀ transport and
supramolecular regulation of their transmembrane activityw
Claude-Rosny Elie,z Nadim Noujeim,z Christophe Pardin and Andreea R. Schmitzer*
Received 7th October 2010, Accepted 10th November 2010
DOI: 10.1039/c0cc04280c
We report the Clꢀ transport activity for three imidazolium-
based transporters. We present significant findings regarding the
use of a-cyclodextrin and cucurbit[7]uril macrocycles to form
inclusion complexes with these salts and to inhibit their
membrane activity.
Chloride, an inorganic anion that is completely dissociated
and highly solvated in solution,1 is one of the most abundant
anions found in physiological environments. The transport of
Clꢀ in biological systems is crucial to myriad biological
processes such as signal transduction, regulation of cell volume,
stabilization of resting membrane potential or fluid transport
in epithelia.2 Chloride is transported across cellular
membranes by different natural anion transport proteins that
act as anion exchangers or cation-dependent co-transporters.3
Understanding processes involved in the natural anion transport
and discovering new membrane-active anion compounds have
encouraged the development of synthetic transmembrane ion
transporters.4 For example cystic fibrosis is caused by a
mutation in the cystic fibrosis transmembrane conductance
regulator protein (CFTR) that facilitates transmembrane Clꢀ
transport. A synthetic sterol analog with polyamine side-chain
functions developed by Regen et al. has been used to
completely restore Clꢀ transport in cystic fibrosis cells.5 We
report here the Clꢀ transport activity for three imidazolium-
based Clꢀ transporters. We present significant findings
regarding the use of water-soluble a-cyclodextrin (a-CD) and
cucurbit[7]uril (CB7) to form inclusion complexes with these salts
in order to inhibit their membrane activity. To the best of our
knowledge this is the first example of imidazolium cations that
induce anion transport across a biomembrane, where activity can
be controlled by the formation of inclusion complexes.
Scheme
1
Imidazolium salts studied for transmembrane Clꢀ
transport activity.
supramolecular interactions developed by imidazolium salts are
p-stacking interactions, aliphatic interactions, and dipolarizability/
polarizability.11 We have recently designed and synthesized
imidazolium salts to maximize stacking interactions by the
introduction of aromatic groups on the imidazolium moiety.12
The imidazolium salts we studied as anion transporters are
shown in Scheme 1. They differ in the substitution pattern of
the two nitrogen atoms of the imidazolium ring, but more
importantly, in their self-association properties in aqueous
solution and capacity to partition in the bilayer. Recently,
Matile et al. elaborated the concept to exploit anion–p interactions
for this purpose and suggested that p-acidic aromatics can be
linked together to produce unbendable scaffolds with multiple
binding sites for anions to move along, across a lipid bilayer
membrane.13 In the case of compounds 2 and 3 the intrinsic
anion–p interaction is even stronger, due to the imidazolium
positive charge close to the aromatic system.
Compounds 1–3 were tested for Clꢀ transport activity using
liposome assays previously reported.14 Liposomes loaded with
the Clꢀ sensitive dye, lucigenin, were subjected to a Clꢀ gradient
in the intravesicular solution. A cartoon representation of the
liposome assay sequence is shown in the inset of Fig. 1. Direct
measurement of Clꢀ efflux by using lucigenin depicted in Fig. 1
ꢀ
confirms that only compound 3 induces Clꢀ/NO3 exchange in
The biocompatibility of the imidazole ring provides a
scaffold for biomimetic applications.6 Among the imidazolium
salt’s most promising attributes are antimicrobial action7 and
molecular self-assembly into liquid crystals.8 While the imidazole
ring affords a wealth of biophysical-related applications, it has
previously been incorporated in the structure of transport carriers
by Gale et al.9 Imidazolium salts represent a new and completely
unexplored class of potential anion transporters. They adopt
self-organized structures mainly through H-bonds that induce
structural directionality, contrary to classical salts which mainly
form aggregates through electrostatic interactions.10 Further
lipid vesicles while analogs 1 and 2 are completely inactive.
Department of Chemistry, Universite´ de Montre´al, 2900 Edouard
´ ´
Montpetit CP 6128 Succursalle Centre-ville, Montreal, Quebec,
Canada. E-mail: ar.schmitzer@umontreal.ca
w Electronic supplementary information (ESI) available: Experimental
details, synthetic procedure, NMR, fluorescence and molecular
modeling data. See DOI: 10.1039/c0cc04280c
z Equal contribution.
Fig. 1 Relative activity of compounds 1–3 in the lucigenin-based Clꢀ
transport assay. Intravesicular 100 mM NaCl, 10 mM phosphate
buffer, extravesicular 100 mM NaNO3, 10 mM phosphate buffer
(pH 6.4). 0.25 mM solutions of 1, 2, 3 were injected at t = 35 s; the
Blank: aqueous 10% Triton-X was injected at t = 500 s.
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Fig. 3 Relative activity of compound 3 (0.25 mM) in the presence of
a-CD and CB7: intravesicular 100 mM NaCl, 10 mM phosphate
buffer, extravesicular 100 mM NaNO3, 10 mM phosphate buffer
(pH 6.4).
Fig. 2 Self-assembly of 2 and 3 modeled by PM3 geometry optimization
and proposed anion–p interactions favouring Clꢀ transport across the
bilayer in 3. The p–p interactions and anion–p interactions are shown in
green, the anion–cation interactions are shown in light blue (Brꢀ are
shown in yellow and Clꢀ in red).
cavity. As can be seen in Fig. 3, 1 or 2 equivalents of CB7
partially inhibit the transport properties of compound 3.
However, 2 equivalents of a-CD completely inhibit the Clꢀ
transport. These results suggest that the nature of the inclusion
complex may play a fundamental role on the transport properties.
To the best of our knowledge this is the first example of simple
anion transporter across a model biomembrane whose activity
can be controlled by the formation of inclusion complexes.
The formation of the inclusion complexes with a-CD or CB7
may result in the assembly of more highly water-soluble
complexes and in the displacement of the partition equilibrium
of 3 towards the aqueous phase. As less self-assembled
aggregates of 3 are present in the bilayer, transport inhibition
is observed. This affirmation allows us to emphasize the
importance of the hydrophobicity of the anion transporter,
as the more hydrophilic compounds 1 and 2 do not transport.
The shape of the transporter as well as its self-assembly
properties are other parameters to consider for this new class
of imidazolium-based synthetic anion transporters.
Fluorescence spectra recorded under the same conditions as the
anion transport assays provide strong evidence of the partition of
compounds 2 and 3 into the less polar liposomal bilayer (ESIw).
Emission spectra of 2 and 3 indicate the existence of the excimers
of the aromatic rings, suggesting that the aromatic–aromatic
interactions are pronounced in the presence of liposomes. The
self-association of 1 in the solid state has been well described,15
and its solubility in aqueous solution does not suggest any self-
assembly under the transport assay conditions. To gain better
insight into the geometrical parameters of more hydrophobic
compounds 2 and 3 in aqueous solution, we performed a
computational study using the PM6/SCF-MO method, at the
restricted Hartree–Fock level, applying COSMO parameters in
MOPAC2009TM 16
As shown in Fig. 2, the effective p–p inter-
.
The formation of the inclusion complexes was observed by
1H NMR and UV spectroscopy studies of compound 3 in the
presence of different concentrations of CB7 (Fig. 4) and a-CD
(ESIw). The 1H NMR spectrum in the presence of CB7 shows
complexed and uncomplexed broad peaks for the aromatic
protons, suggesting rapid association rate or the presence of
multiple complexes. The titration isotherms and Jobs’ plots
(see ESIw) obtained by UV spectroscopy confirmed that the
complexation involves multiple equilibria, where more than
two species are present at equilibrium.
actions of the aromatic arms result in the formation of a planar
bilayer in the aggregates. In compounds 2 and 3, in addition to
forming electrostatic interactions with the bromide anions, the
imidazolium rings form a localized positive charge close to this
aromatic part. However, in the case of 2, the aromatic interactions
are predicted to form in only one direction. For compound 3, the
imidazolium units, separated by the aromatic spacers, do
not provide an enclosed binding site for the anions, but the
aggregation process is predicted to achieve aggregates with the
right length for hydrophobic matching with the lipid bilayer
membrane. Compound 3 does not bind the inorganic anion but
either forms transient channels with a positively charged and
aromatic interior allowing permeation of the inorganic anion and
transport through anion–p interactions. Since the imidazolium
unit is positively charged, independently of the pH, the anion–p
interactions may be strong enough to allow Clꢀ to cross the
aromatic channel. Even if compounds 1–3 look similar to the
carriers previously reported by Sessler and Gale et al.,17 they act
rather as channel forming transporters than carriers, as
compound 2 is completely inactive.
For the specific application as chloride transmembrane
transport, the active compounds must be sufficiently water-
soluble for delivery, but also be sufficiently lipophilic for
insertion into the biomembrane. Therefore, our strategy to
We previously reported that we can control the equilibria of
different imidazolium aggregates in aqueous solution in the
presence of CDs.12a In this study we set out to determine if the
inclusion complexes formed by these salts can be used to block
the transport of the active compound. It was not unexpected
that both a-CD and CB7 form inclusion complexes with 3, by
accommodating the aromatic arms into their hydrophobic
Fig. 4 Partial NMR 1H spectra of 3 in the presence of different
amounts of CB7 in D2O/CD3OD (50/50 V/V), [3] = 0.25 mM.
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the transport activity of imidazolium-based compounds by the
formation of different inclusion complexes with water-soluble
macrocycles. The imidazolium-based compounds obviously
exhibit a great potential as anion transporters, which provide
a pathway for the design of novel transporters with high
efficiency and good opportunities for the development of
imidazolium-based compounds for biomedical applications.
Further studies on the mechanism and the anion transport as
well as the synthesis of other imidazolium-based compounds
for anion transport are currently underway in our group.
We are grateful to the Natural Sciences and Engineering
Research Council of Canada, the Centre of Green Chemistry
Fig. 5 Fluorescence spectra of 3 recorded in the same conditions as
the anion transport experiment. Without liposomes after 1, 10, 20 min;
in the presence of liposomes after 1, 10 and 20 min. Two equivalents of
CB7 or a-CD were added after 10 min and the spectra were recorded at
t = 20 min (excitation at 290 nm).
and Catalysis and Universite de Montreal for financial
´ ´
support. We thank Prof. Lafleur and Prof. Keillor, University
of Montreal, for access to extruder and fluorimeter.
use amphiphilic molecules that are only partially soluble in
water, but are also able to form inclusion complexes with non-
toxic macrocycles, allows us to control their partition and
diffusion across membranes. Self-assembly of 3 cannot be
demonstrated in aqueous solution by 1H NMR study at
0.25 mM. At this low concentration aggregates are already
formed. The self-association results in the formation of water-
insoluble aggregates that precipitate. Even if the self-aggregation
of compound 3 is too complex to determine the thermo-
dynamic parameters and the binding constants with a-CD
and CB7, we demonstrate here that we can regulate the Clꢀ
transport by the formation of inclusion complexes at only
2 equivalents of a-CD. The insertion of 3 in the phopholipid
bilayer and its extraction by the macrocycle towards the
aqueous phase can be observed by fluorescence (Fig. 5). The
fluorescence spectra in the presence of 2 equivalents of a-CD
and CB7 are completely different and confirm the hypothesis
that a-CD act more rapidly on the disruption of the aggregate.
Depending on the size of its cavity, the macrocycle can bind
and accommodate the aggregate without disrupting it, as in
the case of CB7. a-CD can accommodate only the monomer in
its cavity, which results in the disruption of the aggregate
and the complete disappearance of the conductive channels
(Fig. 6 and ESIw for the molecular modeling results).
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the imidazolium salt 3 transports Clꢀ make it an attractive
candidate for developing therapies for Clꢀ transport-related
deficiencies such as cystic fibrosis. We present here a new
property of imidazolium salts which can act as powerful anion
transporters. We also demonstrate the possibility to regulate
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6 Schematic representation of the formation of inclusion
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transporter from the bilayer.
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1790 Chem. Commun., 2011, 47, 1788–1790
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