Tris-ureas as transmembrane anion transporters

Article abstract of DOI:10.1039/c6dt02046a

Nine tris-urea receptors (L¹-L⁹) have been synthesised and shown to coordinate to a range of anionic guests both by ¹H NMR titration techniques and single crystal X-ray structural analysis. The compounds have been shown to be capable of mediating the exchange of chloride and nitrate and also chloride and bicarbonate across POPC or POPC:cholesterol 7:3 vesicle bilayer membranes at low transporter loadings. An interesting dependency of anion transport on the nature of the cation is evidence to suggest that a M⁺/Cl^- cotransport process may also contribute to the release of chloride from the vesicles.

Full text of DOI:10.1039/c6dt02046a

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Tris-ureas as transmembrane anion transporters†
Martina Olivari,a Riccardo Montis,a Stuart N. Berry,b Louise E. Karagiannidis,b
Simon J. Coles,b Peter N. Horton,b Lucy K. Mapp,b Philip A. Gale*b and
Claudia Caltagirone*a
Cite this: DOI: 10.1039/c6dt02046a
Nine tris-urea receptors (L1–L9) have been synthesised and shown to coordinate to a range of anionic
guests both by 1H NMR titration techniques and single crystal X-ray structural analysis. The compounds
have been shown to be capable of mediating the exchange of chloride and nitrate and also chloride and
bicarbonate across POPC or POPC : cholesterol 7 : 3 vesicle bilayer membranes at low transporter load-
ings. An interesting dependency of anion transport on the nature of the cation is evidence to suggest that
a M+/Cl− cotransport process may also contribute to the release of chloride from the vesicles.
Received 22nd May 2016,
Accepted 30th June 2016
DOI: 10.1039/c6dt02046a
www.rsc.org/dalton
Gale et al. have also reported the transmembrane anion
Introduction
transport of phosphoric triamide and thiophosphoric tria-
Transmembrane transport of anions across lipid bilayers is an mide-based receptors,7 and tris-urea tripodal receptors.8–10
important biological process that is normally regulated by
Tris-urea receptors can be divided in two main families: tri-
complex membrane spanning proteins. A range of diseases, podal receptors based on flexible linkers such as TREN (tris-
known as “channelopathies”, including cystic fibrosis, are (2-aminoethyl)amine) that are able to preferentially bind oxo-
caused by malfunctioning ion channels.1 There is currently anions5,11–19 and to work as organogelators,20 or rigid spacers
interest in the design of synthetic membrane transporters for such as cyanuric acid,21 benzene,22 or trindane.23
anions that can act as potential future therapeutic substitutes
for these malfunctioning proteins and have other biological new family of tris-ureas24,25 and tris-thioureas26 developed
applications.2–4
mimicking the scaffold of terpyridine as efficient receptors for
Recently Wu and co-workers designed and synthesised a
Gale and co-workers have recently described the anion phosphate and sulfate. Starting from the interesting results
binding properties of a series of ortho-phenylenediamine- obtained by Gale with the ortho-phenylenediamine-based bis-
based bis-ureas.5,6 These compounds are highly effective anion ureas transporters we decided to expand the family of tris-ureas
transporters that function by an anion antiport, and in some reported by Wu and therefore we synthesised nine receptors
cases a HCl symport carrier mechanism. Addition of electron- (L1–L9 in Scheme 1). We investigated the anion binding pro-
withdrawing groups to either the central core or the peripheral perties both in solution and in the solid state of the nine recep-
phenyl groups improved the anion transport ability: the trans- tors and their ability to transport anions across lipid bilayers.
porter activity increased with the electron withdrawing
strength of the substituent with the trend H < F ≈ Cl < CF3 <
CN < NO2. Indeed, the p-nitro functionalised compound was
shown to possess a very high transport activity, facilitating
chloride efflux at concentrations as low as 1 : 1 000 000 trans-
porter to lipid molar ratio.
aDipartimento di Scienze Chimiche e Geologiche, Università degli Studi di Cagliari,
S.S. 554 Bivio per Sestu, 09042 Monserrato (CA), Italy. E-mail: ccaltagirone@unica.it
bChemistry, University of Southampton, Southampton, SO17 1BJ, UK.
E-mail: philip.gale@soton.ac.uk
†Electronic supplementary information (ESI) available: Additional information
as noted in the text including synthetic details for the preparation of L1–L9, fit-
tings of 1H-NMR titrations, crystallographic tables, transport studies. CCDC
1481148–1481150 for [L5(Cl−)](TBA+), [L4(Cl−)2](TBA+)2, [L5(AcO−)](TBA+). For ESI
and crystallographic data in CIF or other electronic format see DOI:
10.1039/c6dt02046a
Scheme 1 Representation of receptors L1–L9. Receptor L3 has already
been published.25
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solid state. In order to be consistent with solution studies, all
the crystallization experiments were conducted in DMSO.
Results and discussion
The synthesis of receptor L3 has previously been reported by However, (compatibly with the low solubility of receptors L1–L9
Wu.25 L1–L9 were synthesized via different reaction steps. in common solvents) with the aim of investigating the possible
Firstly 1,3-bis(2-aminophenyl)urea was prepared by reaction of influence of less polar solvents in the anion-binding process,
ortho-phenylenediamine with ortho-nitro-phenylisocyanate in a other solvents (AcOEt, MeOH, EtOH, THF, MeNO2, MeCN)
mixed solvent THF/toluene at 0 °C and subsequent reduction and mixture of solvents (MeOH/MeNO2 and THF/DMF)
of the 1-(2-nitrophenyl)-3-(2-aminophenyl)urea obtained by were employed. Details of the crystallization experiments are
hydrazine and Pd/C 10%. 1,3-Bis(2-aminophenyl)urea was the reported in ESI (Table S1†).
reacted with the appropriate isocyanate (4-(trifluoromethyl)
As shown in Table S1 (see ESI†) only a limited number of
phenyl isocyanate; 3,5-bis(trifluoromethyl)phenyl isocyanate; crystallizations were successful in producing single crystals. In
4-fluorophenyl isocyanate; p-tolyl isocyanate; 2-methoxyphenyl particular, for a total of sixty-six crystallization experiments,
isocyanate; 4-methoxyphenyl isocyanate) in refluxing dichloro- only seven gave samples suitable for X-ray investigation.
methane (DCM) under a N2 atmosphere to obtain L1–L9 in Within these, three gave the crystal structure of the simple
60–90% yield (see ESI† for synthetic details).
tetrabutylammonium salt and the remaining four resulted in
Anion-binding studies were performed by means of crystal structures [L4(Cl−)2](TBA+)2a, [L4(Cl−)2](TBA+)2b (iso-
1H-NMR titrations in DMSO-d6. Stability constants from the structures obtained from MeOH/MeNO2 and MeCN respect-
1H-NMR titration curves obtained (see ESI Fig. S1–S13†) were ively; for the structural description the code [L4(Cl−)2](TBA+)2 is
calculated by fitting the data to a 1 : 1 binding model using used to identify both), [L5(Cl−)](TBA+) and [L5(AcO−)](TBA+)
EQNMR27 as shown in Table 1.
(both obtained from DMSO).
Under the conditions of these experiments, the receptors
In general, these results seem to be consistent with the low
did not interact with nitrate (i.e. no shift of the NH proton anion-binding affinity observed in solution studies (Table 1).
resonances occurred upon addition of tetrabutylammonium This is particularly evident for receptors L1–L3, where the
nitrate). Interestingly, receptors L1–L3 which contain nitro elec- unsuccessful crystallization experiments agree with the negli-
tron-withdrawing groups showed little interaction with chlor- gible interactions observed in solution (Table 1). In the case
ide, while addition of bicarbonate caused the disappearance of of receptor L4, though the compound unexpectedly forms a
the signals attributed to the urea NH groups, evidence in 1 : 2 [L4(Cl−)2](TBA+)2 complex, the affinity toward Cl− is con-
support of a deprotonation or exchange process. Receptor L4, firmed. For the remaining receptors L5-L9, only in the case of
bearing one CF3 substituent on the peripheral phenyl ring receptor L5 it was possible to isolate single crystals of anion
showed some affinity for chloride. On the other hand, recep- complexes with chloride and acetate (not investigated in
tors L6, L7, and L9 were able to bind both chloride and bi- solution).
carbonate with comparable stability constants, while L5 and L8
[L4(Cl−)2](TBA+)2 crystallised in the triclinic crystal system
ˉ
bound bicarbonate preferentially.
(space group P1) with an asymmetric unit consisting of one
A series of crystallization experiments of receptors L1–L9 in receptor L4, two chloride anions and two tetrabutylammonium
presence of an excess of anions such as acetate, chloride, counterions, resulting in a 1 : 2 complex. L4 adopts a closed
bicarbonate and nitrate were carried out with the aim of inves- conformation, with the urea NHs all oriented toward the
tigating the anion-binding properties of the receptors in the centre of a pseudo-cavity. The three urea groups are slightly
tilted to interact via N–H⋯Cl hydrogen bonds (N–H⋯Cl dis-
tances are in the range 2.36(2)–2.51(2) Å, average 2.43 Å) with
the two Cl− anions which respectively lie below (Cl1) and
above (Cl2) the pseudo-cavity (Fig. 1a and b). In particular it is
Table 1 Association constants (Ka/M−1) for the formation of complexes
of L1–L9 with anions added as tetrabutylammonium salts (or tetraethyl-
worth noticing that the shortest distances are observed for Cl2
ammonium in the case of hydrogencarbonate) in DMSO-d6 at 300 K. All
(N1–H1⋯Cl2 2.36(2) Å, N1–H1⋯Cl2 2.34(2) Å).
errors estimated to be ≤14% (see ESI)
[L5(Cl−)](TBA+) also adopts a triclinic crystal system (space
ˉ
Anions
group P1). The asymmetric unit consists of one independent
receptor L5, one independent chloride and one independent
tetrabutylammonium resulting in a 1 : 1 complex. Similarly to
L4 the receptor L5 shows a closed conformation with the two
peripheral urea NHs (Fig. 2a) pointing at the centre of the
pseudo-cavity and interacting with the chloride via four N–
H⋯Cl hydrogen bonds (N–H⋯Cl) distances are in the range
2.32(2)–2.75(3) Å (average distance 2.53 Å). The central urea
NHs are tilted to interact with an adjacent receptor-chloride
unit via a second set of N–H⋯Cl hydrogen bonds (N–H⋯Cl
distances are 2.44(3) Å and 2.74(3) Å respectively) forming a
centrosymmetric dimer (Fig. 2b).
Receptors
Cl−
HCO3
NO3
−
−
L1
L2
L3
L4
L5
L6
L7
L8
L9
<10
<10
<10
262
<10
205
226
28
Deprotonationa
No interaction
No interaction
No interaction
No interaction
No interaction
No interaction
No interaction
No interaction
No interaction
Deprotonationa
Deprotonationa
Deprotonationa
226
203
221
802
240
225
a The NHs signals disappeared after the addition of one equivalent of
anion.
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Fig. 3 Pseudo-cavity and main intermolecular interactions observed in
structure [L5(AcO−)](TBA+); (a) N–H⋯O hydrogen bonds involving peri-
pheral ureas and AcO−. (b) N–H⋯O hydrogen bonds involving central
ureas and AcO− and centro-symmetric dimer. The molecules are
oriented to best show the intermolecular interactions. TBA+ counter
cations are omitted for clarity. N–H⋯O hydrogen bonds are indicated as
black dashed lines, centre of inversion as black circle.
Fig. 1 Pseudo-cavity and main intermolecular interactions observed in
structure L4Cl−, viewed along two orthogonal projections. TBA+ counter
cations are omitted for clarity. N–H⋯Cl hydrogen bonds are indicated
as black dashed lines.
vesicles were suspended at a lipid concentration of 1 mM in
489 mM NaNO3 buffered to pH 7.2 with 5 mM sodium phos-
phate salts.
A
small amount of DMSO solution of the receptor
(0.02–2 mol% with respect to lipid) was added to the vesicles
suspension, and the resulting chloride efflux was monitored
using a chloride ion selective electrode (ISE) for 300 s. At the
end of the experiment, the vesicles were lysed by the addition
of detergent, and the final electrode reading was used to cali-
brate 100% chloride release. We found that all the compounds
except L1 (i.e. L2–L9) (at 2 mol% with respect to lipid) were
capable of mediating chloride transport.
Under these experimental conditions the most active com-
pounds are to be L2, L3, L4 and L6 (Fig. 4 and S14–S29 in ESI†)
In order to determine the mechanism of chloride release by
receptors L2–L9, the transport assays were repeated suspending
Fig. 2 Pseudo-cavity and main intermolecular interactions observed in
structure [L5(Cl−)](TBA+), (a) N–H⋯Cl hydrogen bonds involving peri-
pheral ureas and Cl−. (b) N–H⋯Cl hydrogen bonds involving central
ureas and Cl− and centro-symmetric dimer. The molecules are oriented
to best show the intermolecular interactions. TBA+ counter cations and
positional disorder in the CF3 groups are omitted for clarity. N–H⋯O
hydrogen bonds are indicated as black dashed lines, centre of inversion
as black circle.
Similarly to the previous two structures, [L5(AcO−)](TBA+)
ˉ
crystallises in the triclinic crystal system (space group P1).
The asymmetric unit consists of one independent receptor L5,
one independent acetate and one independent tetrabutyl-
ammonium. The receptor molecule adopts a closed confor-
mation (Fig. 3a) to form a pseudo-cavity similar to those
observed for [L4(Cl−)2](TBA+)2 and [L5(Cl−)](TBA+). The peri-
pheral NHs are oriented toward the centre of the pseudo-cavity
interacting with the AcO− via N–H⋯O hydrogen bonds (N–
H⋯O distances are in the range 1.89(2)–2.34 (2) Å, average dis-
tance 2.13 Å). This is oriented perpendicularly to the plane of
the pseudo-cavity and interacts via two N–H⋯O hydrogen
bonds (N–H⋯O distances are 1.97(2) Å and 1.99(2) Å respect-
ively) with the central urea NHs of an adjacent receptor mole-
Fig. 4 Chloride efflux promoted by a DMSO solution of compounds
cule (Fig. 3b) to form a centrosymmetric dimer similar to that L1–L9 (2 mol% carrier to lipid) from unilamellar POPC vesicles loaded
observed for [L5(Cl−)](TBA+).
with 489 mM NaCl buffered to pH 7.2 with 5 mM sodium phosphate
salts. The vesicles were dispersed in 489 mM NaNO3 buffered to pH 7.2
with 5 mM sodium phosphate salts. At the end of the experiment deter-
gent was added to lyse the vesicles and calibrate the ISE to 100% chlor-
The anion transport properties of receptors L1–L9 were
studied using vesicle-based methods.28 A sample of uni-
lamellar POPC vesicles was prepared containing 489 mM NaCl
ide efflux. Each point represents an average of three trials. DMSO was
buffered to pH 7.2 with 5 mM sodium phosphate salts. The used as a control.
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POPC vesicles loaded with NaCl (451 mM with 20 mM phos-
phate buffer at pH 7.2) in a solution of Na2SO4 (150 mM with
20 mM phosphate buffer at pH 7.2). DMSO suspensions of
compounds L2–L9 were then added to the suspension. Usually
2−
during this experiment the antiport mechanism (2Cl−/SO4
exchange) would not be expected to be observed due to the
2−
high hydrophilicity of the SO4 anion29 which would inhibit
the chloride efflux from the liposomes.9 However, as shown in
Fig. 5, during the first two minutes of the experiment signifi-
cant chloride release was observed for compounds L3, L2 and
L6. There is also some chloride efflux mediated by the other
compounds except for L4 and L5 under these conditions. After
120 s, a pulse of NaHCO3 solution was added, and we observed
an increase of chloride efflux (Fig. 5 and S31–S46 in ESI†), evi-
dence in support of a chloride/bicarbonate exchange process.
A lucigenin assay for chloride/sulfate exchange was run
with compounds L2–L9 which showed that these compounds
do not mediate sulfate transport (see ESI, Fig. S56† in the case
of L6, as a representative compound).10
Fig. 6 Percentage chloride efflux at 270 s mediated by L2, L3, and L6
(0.1 mol% carrier to lipid) from unilamellar POPC vesicles loaded with
either 489 mM NaCl (red) or 489 mM CsCl (blue) buffered to pH 7.2 with
5 mM sodium phosphate salts. The vesicles were dispersed in 489 mM
NaNO3 buffered to pH 7.2 with 5 mM sodium phosphate salts.
Although it has been recently reported that ureas and
thioureas can facilitate proton or hydroxide transport,30
a
rate of chloride release to be different in the presence of CsCl.
HPTS assay to test for H+/Cl− co-transport resulted in inconclu- As shown in Fig. 6 in the case of L2, L3, and L6 (and Fig. S47–
sive results for the class of molecules presented herein (see S55 in the ESI†) the release of chloride is dependent on the
ESI, Fig. S57† in the case of L6 as a representative compound).
nature of metal cation and it is reduced when the internal
To further examine the origin of the chloride transport solution was replaced by CsCl.
during the first two minutes of this assay, we examined the
The results described above suggest, from the first assay,
possibility of a mechanism involving sodium/chloride co- that the receptors can facilitate Cl−/NO3− exchange; the second
−
transport. The Cl−/NO3 transport assays were repeated using assay, supports the hypothesis of Cl−/HCO3 antiport mecha-
vesicles containing caesium chloride (489 mM NaCl buffered nism although in the first two minutes in the presence of
to pH 7.2 with 5 mM sodium phosphate salts) instead of sulfate for all the compounds except L4 and L5 a small amount
sodium chloride, suspended in an isotonic sodium nitrate of chloride efflux was observed. The caesium chloride assay
solution. In the event of NaCl co-transport we would expect the demonstrates that for L2, L3, L6–L9 the counter cation is
involved in transport possibly via a M+/Cl− co-transport
−
process.
We tested the Cl−/NO3−antiport activity of L2–L9 in vesicles
composed of POPC–cholesterol (7 : 3). This mixture is a closer
mimic of biological membranes than pure POPC lipid bilayers.
The presence of cholesterol is known to increase the order in
the bilayer and its viscosity. All the receptors tested showed a
reduced rate of transport in the POPC–cholesterol system (see
Fig. 7 for L2 and Fig. S58–S64 in ESI† for L3–L9), providing evi-
dence that for this class of receptor diffusion through the
interior of the bilayer may be the rate determining step.31
To quantify the transport activity of compounds L2–L9 Hill
analyses32,33 for the chloride/nitrate and chloride/bicarbonate
antiport assays were performed (see Fig. S14–S29, S31–S46 in
ESI†). Hill analysis allows the calculation of the EC50,270 s
which is a measure of transporter efficiency, defined as the
required receptor concentration to mediate 50% of the total
chloride efflux 270 s after the addition of the carrier (or after
Fig. 5 Chloride efflux promoted by a DMSO solution of compounds
L2–L9 (2 mol% carrier to lipid) from unilamellar POPC vesicles loaded
with 451 mM NaCl buffered to pH 7.2 with 20 mM sodium phosphate the bicarbonate ‘pulse’). This allows us to compare the trans-
salts. The vesicles were dispersed in 150 mM Na2SO4 buffered to pH 7.2
with 20 mM sodium phosphate salts. At t = 120 s a solution of sodium
bicarbonate was added such that the external concentration of bi-
carbonate was 40 mM. At the end of the experiment, detergent was added
port activity of the compounds. These values are summarised
in Table 2, together with the Hill coefficients, which can be
correlated to the number of transporter molecules required to
transport a single anion and can provide further evidence for a
mobile carrier mechanism.
to lyse the vesicles and calibrate the ISE to 100% chloride efflux. Each
point represents an average of three trials. DMSO was used as a control.
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Solution studies demonstrate that these receptors bind anions
with moderate stability constants (as reported in Table 1).
Solid state studies confirm the results observed in solution
and indeed, despite many attempts, only three crystal struc-
tures were obtained. In particular, the crystal structure of L4 in
the presence of chloride suggests that the receptor has a good
degree of pre-organization and binds chloride via the urea NH
groups with a 1 : 2 stoichiometry. We also demonstrated that
these systems are able to mediate transmembrane chloride
transport as mobile carriers with different mechanisms,
−
−
Cl−/NO3 and Cl−/HCO3 antiport, and metal-dependent
cation co-transport. Hill-plot analysis demonstrates that the
most active compound of the series is L4.34
Fig. 7 Chloride efflux promoted by a DMSO solution of compound L2
(0.2 mol% carrier to lipid) from unilamellar vesicles comprising of either
POPC or POPC–cholesterol (7 : 3 molar ratio, POPC : Chol), loaded with
489 mM NaCl buffered to pH 7.2 with 5 mM sodium phosphate salts.
The vesicles were dispersed in 489 mM NaNO3 buffered to pH 7.2 with
5 mM sodium phosphate salts. At the end of the experiment detergent
was added to lyse the vesicles and calibrate the ISE to 100% chloride
efflux. Each point represents an average of three trials. DMSO was used
as a control.
Acknowledgements
CC would like to thank Fondazione Banco di Sardegna for
financial support. PAG thanks the University of Southampton
and the A*STAR ARAP programme for a studentship (SNB), the
Royal Society and the Wolfson Foundation for a Research
Merit Award and the EPSRC (EP/K039466/1) (Core Capability
for Chemistry Research in Southampton). We also thank the
EPSRC for access to the crystallographic facilities at the Univer-
sity of Southampton.35
Table 2 The EC50 is the concentration (mol% carrier to lipid) needed to
obtain 50% efflux after 270 s and n is the Hill coefficient that represents
an estimate of the number of transporter molecules required to trans-
port a single anion
Notes and references
1 F. M. Ashcroft, Ion Channels and Disease, Academic Press,
San Diego, 2000.
EC50 at 270 s
EC50 at 270 s
(Cl−/NO3
)
n
(Cl−/HCO3
)
n
−
−
Compound
2 P. A. Gale, R. Pérez-Tomás and R. Quesada, Acc. Chem. Res.,
2013, 46, 2801–2813; N. Busschaert and P. A. Gale, Angew.
Chem., Int. Ed., 2013, 52, 1374–1382.
3 S. Matile, A. Vargas Jentzsch, J. Montenegro and A. Fin,
Chem. Soc. Rev., 2011, 40, 2453–2474.
L2
L3
L4
L5
L6
L7
L8
L9
0.095
0.047
0.039
0.066
0.10
0.6
4.98
1.207
1.67
1.07
0.88
1.39
0.96
1.29
0.88
0.50
0.21
0.14
0.25
0.76
4.27
21.93
8.43
0.76
0.77
0.98
0.97
0.80
0.88
0.86
0.84
4 (a) V. Soto-Cerrato, P. Manuel-Manresa, E. Hernando,
S. Calabuig-Fariñas, A. Martínez-Romero, V. Fernández-
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1.96
From the EC50,270 values reported in Table 2 the most
s
active transporter among the series appears to be L4 (EC50,270 s
0.039 mol% and 0.14 mol% with respect to lipid for nitrate
and bicarbonate antiport, respectively).
Further, Hill coefficients of ∼1 provides further evidence
that these compounds are functioning via a mobile carrier
mechanism and are not forming membrane spanning chan-
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Conclusions
In conclusion we have synthesised nine tris-urea receptors
bearing a range of substituents attached to the pendant arms.
We have studied the anion binding properties of the receptors
both in solution and in the solid state and we have tested their
ability to mediate chloride transport through membranes.
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