Squaramides as potent transmembrane anion transporters

Article abstract of DOI:10.1002/anie.201200729

Square peg in a round ball: Squaramides are shown to be potent transmembrane anion transporters for both chloride and bicarbonate, performing better than the thiourea and urea analogues. Studies into the nature of this transport point to a mobile carrier mechanism, where the squaramide delivers the anion cargo across the lipid bilayer (see scheme, green sphere=anion). These drug-like molecules provide a platform for the development of a new generation of anion-transport systems. Copyright

Full text of DOI:10.1002/anie.201200729

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Angewandte
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DOI: 10.1002/anie.201200729
Anion Transport
Squaramides as Potent Transmembrane Anion Transporters**
Nathalie Busschaert, Isabelle L. Kirby, Sarah Young, Simon J. Coles, Peter N. Horton,
Mark E. Light, and Philip A. Gale*
The transmembrane transport of anions, particularly chloride
and bicarbonate, is an important biological process that is
normally regulated by transmembrane ion channels. A range
of diseases, known as “channelopathies”,[1] of which cystic
fibrosis is the most common example, are caused by
malfunctioning ion channels.[2] There has therefore been
significant interest recently in the development of small, drug-
like molecules that can facilitate the transport of anions
across lipid bilayers, thereby restoring anion permeability to
membranes containing faulty channel proteins.[3] Supramolec-
ular chemists have developed ion channels (where molecules
form pores in the membrane, through which anions can
diffuse) and mobile carriers (where an anion complex is
formed that diffuses through the membrane) using peptides,[4]
calixarenes,[5] anion p-slides,[6] isophthalamides,[7] calixpyr-
roles[8] and, recently, also many ureas and thioureas.[9,10]
However, to our knowledge, there have not been any reports
of the use of squaramides in transmembrane anion transport.
There are an increasing number of examples of anion
receptors based on the squar-
because the considerations applied to the design of hydro-
gen-bonding anion receptors and hydrogen-bonding organo-
catalysts are closely related.[17] Furthermore, there has been
recent interest by medicinal chemists in introducing squar-
amido functionalities into drug candidates, using them as
isosteres for ureas, guanidines, and other groups.[18] Squar-
amides are believed to be stable to nucleophilic attack, which
could mean they have reduced toxicity[18] and make them
suitable candidates for potential drugs, including for the
treatment of channelopathies. Herein we report the anion-
transport properties of a series of small, fluorinated squar-
amides and compare their transport abilities with analogous
ureas and thioureas.
All of the tested compounds have been previously
reported and can be easily synthesized from adapted liter-
ature procedures (see Supporting Information for details).[19]
Squaramides 1, 4, and 7 were prepared using the zinc triflate-
mediated reaction of squarate esters with the appropriate
aniline, developed by Taylor and co-workers,[13a] whereas
amide motif. Costa and co-
workers have used a range of
computational and experi-
mental techniques to provide
compelling evidence that
squaramide-based molecules
are capable of both cation[11]
and anion binding.[12] More
recently, the groups of
Taylor,[13] Fabbrizzi,[14] and
others[15] have used squar-
amide-based receptors for
the binding of various anions,
including chloride, sulfate and
carboxylates. Squaramides are
also being developed as non-
covalent organocatalysts,[16]
thioureas 2, 5, 8, and ureas 3, 6, and 9 were synthesized by the
reaction of the appropriate anilines with isothiocyanates or
isocyanates, respectively.
[*] N. Busschaert, I. L. Kirby, S. Young, Dr. S. J. Coles, Dr. P. N. Horton,
Dr. M. E. Light, Prof. Dr. P. A. Gale
Chemistry, University of Southampton
Highfield, Southampton, SO17 1BJ (UK)
E-mail: philip.gale@soton.ac.uk
Some of these compounds have been shown to possess
excellent anion binding properties (4 and 6)[14b] or are
attractive organocatalysts (e.g. Schreinerꢀs thiourea 8).[20]
We have previously shown that fluorinated substituents can
greatly improve the transport ability of an anion receptor.[10b]
Compounds 1–9 were therefore considered good candidates
for anion transport studies. To assess the chloride transport
abilities of 1–9, a series of unilamellar 1-palmitoyl-2-oleoyl-
sn-glycero-3-phosphocholine (POPC) liposomes loaded with
NaCl (489 mm) was prepared and suspended in an isotonic,
[**] P.A.G. thanks the EPSRC for access to the crystallographic facilities
at the University of Southampton. Additionally we thank the
University of Southampton for a post-graduate scholarship (I.L.K.)
and the University of Southampton and A*STAR for a post-graduate
scholarship (N.B.).
Supporting information for this article (experimental details) is
available on the WWW under http://dx.doi.org/10.1002/anie.
201200729.
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chloride-free NaNO3 solution (489 mm), according to pre-
viously reported methodologies (see Supporting Informa-
tion).[21] A small volume of the putative transporter 1–9 in
DMSO was added to these vesicles, and the resulting chloride
efflux was monitored using an ion selective electrode (ISE)
for 300 s. The results for compounds 1–6 are given in Figure 1
and demonstrate that squaramides 1 and 4 are able to
transport chloride out of liposomes much faster than the
analogous thioureas (2, 5) and ureas (3, 6) (additional results
in Supporting Information).
Figure 2. Chloride efflux promoted by receptors 1–6 (1 mol% receptor
to lipid) from unilamellar POPC vesicles loaded with 450 mm NaCl
buffered to pH 7.2 with 20 mm sodium phosphate salts and dispersed
in 162 mm Na2SO4 buffered to pH 7.2 with 20 mm sodium phosphate
salts. At t=120 s, a solution of NaHCO3 was added to give a final
concentration of 40 mm. At the end of the experiment (420 s)
detergent was added to lyse the vesicles and calibrate the ISE to 100%
chloride efflux. Each point represents the average of three trials.
DMSO alone was used as a control experiment.
Figure 2 also shows that for chloride/bicarbonate transport
the squaramide-based compounds 1 and 4 display superior
transport abilities compared to analogous ureas and thioureas
(similar results were obtained for 7–9, Supporting Informa-
tion).
Figure 1. Chloride efflux promoted by receptors 1–6 (1 mol% receptor
to lipid) from unilamellar POPC vesicles loaded with 489 mm NaCl
buffered to pH 7.2 with 5 mm sodium phosphate salts and dispersed
in 489 mm NaNO3 buffered to pH 7.2 with 5 mm sodium phosphate
salts. At the end of the experiment (300 s), detergent was added to
lyse the vesicles and calibrate the ISE to 100% chloride efflux. Each
point represents the average of three trials. DMSO alone was used as
a control experiment.
To quantify the extent to which the squaramides transport
chloride more efficiently than ureas and thioureas, extensive
Hill analyses were performed for both ClÀ/NO3 and ClÀ/
À
À
HCO3 assays (see Supporting Information for details). In
these tests, the vesicle experiments are repeated at various
concentrations of transporter to obtain EC50 values, that is,
the concentration of transporter needed to achieve 50%
chloride efflux in 270 s, and Hill coefficients n that reveal the
stoichiometry of the transport process[23] (Table 1, values for
The transport activity seen in Figure 1 results from either
a ClÀ/NO3À antiport process (anion exchange) or a H+/ClÀ or
Na+/ClÀ symport (co-transport) process. To test which of
these processes is predominant, POPC vesicles containing
NaCl were prepared and suspended in a solution of Na2SO4,
and a sample of 1–6 in DMSO was added to initiate the
experiment (1 mol% transporter to lipid). After 2 min a pulse
of bicarbonate was added to the external solution and the
chloride efflux was monitored for a further 5 min using an ISE
(Figure 2). Under the initial conditions of the experiment (i.e.
before the addition of bicarbonate), no chloride efflux could
be detected, whereas the addition of bicarbonate leads to an
immediate increase in the release of chloride mediated by 1–
6. This strong dependence on the nature of the external anion
is indicative of an anion antiport process. The strong hydro-
philic nature of sulfate precludes lipid bilayer transport of this
anion and therefore no significant chloride efflux is seen when
sulfate is the only external anion. However, when nitrate
(Figure 1) or bicarbonate (Figure 2) are present in the
extravesicular solution, release of chloride from the vesicles
is observed, indicative of a chloride/nitrate and the biologi-
cally more relevant[22] chloride/bicarbonate antiport process.
ClÀ/HCO3 assays can be found in the Supporting Informa-
À
tion). It can be seen from the EC50 values in Table 1 that the
squaramide-based compounds (1, 4, and 7) are nearly one
order of magnitude better than the analogous thioureas and
ureas, allowing detectable chloride efflux at much lower
concentrations of transporter.[24]
Because 1–9 have planar, aromatic substituents, it can be
postulated that anion transport occurs through channels
formed by stacking of these molecules, although this would
be entropically unfavorable. The Hill coefficients n (Table 1)
suggest that the transport of one chloride anion through the
membrane requires only one or two molecules, which are not
enough to span a POPC lipid bilayer. To test this idea, the
liposome experiments were repeated using vesicles made of
7:3 POPC:cholesterol. It has often been suggested that
cholesterol can increase the viscosity of a lipid bilayer,[25]
thereby decreasing the ability of a mobile carrier to diffuse
through the membrane, whereas the transport ability of ion
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Table 1: Summary of the chloride binding (Ka) and anion transport (EC50
and n) properties of compounds 1–9. Calculated logP and TPSA [ꢀ2] are
also presented.
without high lipophilicity is useful in the development of
drug-like transport systems with therapeutic potential. This
potential is because the lower lipophilicity of squaramides
keeps them within the boundaries defined by Lipinskiꢀs rule
of five, which dictates that logP < 5 for drug-like systems.[29]
It could also be that squaramide-based compounds are
better anion transporters because the two oxygen atoms
provide a metal binding site (complexes of squaramides with
alkylammonium cations have been reported)[11] and there-
fore, transport could possibly occur through the binding of
a neutral ion pair (for example as NaCl/NaNO3 exchange or
NaCl/NaHCO3 exchange). However, when the vesicle studies
were repeated with K+ salts, or when a Cs+ gradient was used,
the same transport rates were found as with Na+ salts
(Supporting Information). Hence, it is unlikely that the
metal ions play an important role in the transport of anions
by squaramides 1, 4, and 7.
[b]
Compound
Ka[a]
EC50
n[c]
clogP[d]
TPSA[e]
[ꢀ2]
[mÀ1
]
[mol%]
1
2
3
4
5
6
7
8
9
260
15
31
458
43
75
643
41
88
1.38
1.7
2.02
3.66
2.51
3.87
5.50
4.35
5.71
7.34
6.20
48.03
21.30
32.74
47.61
21.04
32.28
47.94
21.61
32.69
[f]
[f]
–
–
–
–
[f]
[f]
0.06
0.22
0.42
0.01
0.16
0.30
1.2
1.6
2.2
1.1
2.0
1.4
[a] Association constant [mÀ1] for 1–9 with Bu4NCl in [D6]DMSO/0.5%
water at 298 K. [b] Concentration of transporter (mol% carrier to lipid)
needed to obtain 50% chloride efflux in 270 s during the ClÀ/NO3
À
experiments. [c] Hill coefficient for ClÀ/NO3À experiments. [d] clogP
calculated using Spartan ’10 for Macintosh (Ghose–Crippen model).
[e] Total polar surface area (TPSA) calculated using Spartan ’10 for
Macintosh. The receptors were minimized using AM1 semi-empirical
methods. Values are given for the cis/cis conformer, which is believed the
most stable conformer for N-phenyl squaramides.[30] [f] Transporter was
not active enough to perform Hill analysis.
Therefore, the most likely explanation for the superior
transport abilities of the squaramides is due to the enhanced
anion-binding properties of squaramide-based compounds. It
has been suggested that squaramides possess exceptional
anion-binding abilities, because ion binding increases the
aromaticity in the four-membered ring.[12b] Fabbrizzi and co-
workers have shown that squaramides (including 4) display
higher anion binding constants in acetonitrile than the
analogous ureas, which was attributed to the more convergent
hydrogen-bond array and a larger participation of the
ortho CH protons in hydrogen bonding to the anion.[14] To
show that the same is true for all of the transporters, 1H NMR
spectroscopic titrations at 298 K in [D6]DMSO containing
0.5% water were performed for all anions relevant in the
transport studies (using the tetrabutylammonium (Bu4N) or
channels should remain unaffected. However, the cholesterol
test gave inconclusive results, with some compounds (e.g. 1)
displaying slower transport activity, indicative of a mobile
carrier mechanism, whereas most other compounds showed
significantly faster transport (Supporting Information). This
increase might be due to increased partitioning of the
receptors into a lipid bilayer containing cholesterol, but this
could not be verified. We then performed U-tube experiments
using nitrobenzene as the organic phase to discriminate
between ion channel and mobile carrier behavior. Significant
chloride transport through the U-tube could be detected for
most of the compounds, which can only be the result of
a mobile carrier mechanism, because ion channel formation is
impossible owing to the sheer size of the U-tube organic
phase. Once again, it was the fluorinated squaramide
compounds 4 and 7 that proved to be the most efficient
transporters. These experiments provide supporting evidence
that the mobile-carrier activity seen in the U-tube experiment
is also present in liposomes (Supporting Information).
There could be several possible reasons why the squar-
amides outperform the ureas and thioureas in the anion-
transport studies. We have reported previously that thioureas
are better transporters than ureas and that fluorinated
compounds are better than unfluorinated ones because of
their higher lipophilicity and hence the enhanced partitioning
into a lipid bilayer.[10] Although it has been suggested that
squaramides are more lipophilic than ureas,[26] our calcula-
tions of the Ghose–Crippen logP and TPSA (total polar
surface area) values[27] of compounds 1–9 (Table 1) suggest
that the squaramide-based compounds are less lipophilic than
their urea and thiourea analogues, which has also been
proposed by Storer et al.,[18] and others.[28] This observation
makes it unlikely that the enhanced transport behavior of the
squaramides is due to their lipophilicity. On the other hand,
the fact that squaramides have enhanced transport abilities
À
tetraethylammonium (Et4N) salts). No interaction with NO3
was found in this solvent mixture, whereas the interaction
À
with HCO3 proved to be difficult to interpret because of
À
deprotonation of the squaramides and thioureas by HCO3
(as confirmed by the addition of aliquots of strong base
Bu4NOH, which resulted in similar NMR spectra as the
addition of Et4NHCO3, Supporting Information). The titra-
tion data with Bu4NCl could be fitted to a 1:1 model using the
WinEQNMR2 computer program,[31] and the results are
summarized in Table 1. It is clear from Table 1 that the
squaramide-based compounds 1, 4, and 7 all display associ-
ation constants with chloride that are one order of magnitude
larger than the association constants of the ureas and
thioureas. These data imply that the enhanced anion binding
is the most likely reason for the order of magnitude better
transport activity displayed by the squaramides.
The ability of these compounds to bind anions was also
confirmed by single-crystal X-ray diffraction (Figure 3;
details in the Supporting Information). Crystals of the
chloride complexes could be obtained for all squaramide-
based compounds ([1-Cl]À, [4-Cl]À, and [7-Cl]À) by the slow
evaporation of a DMSO solution containing the receptor and
excess Bu4NCl (at 508C). All chloride complexes show a 1:1
À
stoichiometry and have two, near linear N H···Cl hydrogen
À
bonds (all N H···Cl angles are > 1608 and N···Cl distances
vary from 3.029(19) ꢁ to 3.201(18) ꢁ), providing evidence for
a convergent hydrogen-bond array of the squaramides that is
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Figure 3. An ORTEP view of [4-Cl][Bu4N] with thermal ellipsoids set at
50% probability. Hydrogen bonds are represented by dashed lines.
The counterion (Bu4N) is omitted for clarity.
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suitable for interaction with halide anions (Figure 3). Even
though the interactions with Bu4NNO3 and Et4NHCO3 could
not be detected in DMSO solutions, we were able to obtain
crystal structures of complexes with these anions ([3-HCO3]À,
[(6)3-CO3]2À, [(9)3-CO3]2À, and [3-NO3]À; Supporting Infor-
mation). This result provides evidence that the urea com-
pounds can interact with nitrate and (bi)carbonate and
thereby supports the idea that chloride/nitrate and chloride/
bicarbonate exchange occurs across lipid bilayers.
In summary, we have presented the first examples of
squaramide-based compounds that can transport anions
across lipid bilayers, presumably via mobile-carrier and
anion-exchange mechanisms. It was shown that the squar-
amides possess better anion-transport activities than analo-
gous ureas and thioureas, and this was mainly because of the
enhanced anion-binding properties of the squaramide-based
receptors. Since the squaramide functionality provides a way
to increase the transport ability of a receptor without
significantly increasing the lipophilicity, it offers an ideal
platform for designing future anion transporters. More
complex squaramide-containing transporters are currently
being investigated in our laboratory and will be published in
due course.
Received: January 26, 2012
Published online: March 27, 2012
Keywords: anion transport · anions · fluorinated ligands ·
.
squaramides · supramolecular chemistry
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See Supporting Information for details and discussion. However,
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lower concentrations, the transport mediated by the squar-
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and thioureas.
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[19] The urea and thiourea compounds herein have been used
extensively before in various subjects. For a list of the exact
references used during synthesis, see Supporting Information.
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