Investigating the influence of steric hindrance on selective anion transport

Article abstract of DOI:10.3390/molecules24071278

A series of symmetrical and unsymmetrical alkyl tren based tris-thiourea anion transporters were synthesised and their anion binding and transport properties studied. Overall, increasing the steric bulk of the substituents resulted in improved chloride binding and transport abilities. Including a macrocycle in the scaffold enhanced the selectivity of chloride transport in the presence of fatty acids, by reducing the undesired H⁺ flux facilitated by fatty acid flip-flop. This study demonstrates the benefit of including enforced steric hindrance and encapsulation in the design of more selective anion receptors.

Full text of DOI:10.3390/molecules24071278

molecules
Article
Investigating the Influence of Steric Hindrance on
Selective Anion Transport
Laura A. Jowett 1,2, Angela Ricci 2,3, Xin Wu 1,2, Ethan N. W. Howe 1,2 and Philip A. Gale 1,2,
*
1
School of Chemistry (F11), The University of Sydney, Sydney, NSW 2006, Australia;
ljow8193@uni.sydney.edu.au (L.A.J.); xin.wu@sydney.edu.au (X.W.); ethan.howe@sydney.edu.au (E.N.W.H.)
Chemistry, University of Southampton, Southampton SO17 1BJ, UK; angela.ricci20@gmail.com
Department of Pure and Applied Sciences, Chemistry Section, Universita Degli Studi Di Urbino “Carlo Bo”,
via della Stazione 4, 61029 Urbino PU, Italia
2
3
*
Correspondence: philip.gale@sydney.edu.au
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editor: Jennifer R Hiscock
Received: 21 March 2019; Accepted: 1 April 2019; Published: 2 April 2019
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: A series of symmetrical and unsymmetrical alkyl tren based tris-thiourea anion transporters
were synthesised and their anion binding and transport properties studied. Overall, increasing the
steric bulk of the substituents resulted in improved chloride binding and transport abilities. Including
a macrocycle in the scaffold enhanced the selectivity of chloride transport in the presence of fatty
acids, by reducing the undesired H+ flux facilitated by fatty acid flip-flop. This study demonstrates
the benefit of including enforced steric hindrance and encapsulation in the design of more selective
anion receptors.
Keywords: anion transport; thioureas; lipid bilayer
1. Introduction
Anion transporters that can restore chloride flux through apical epithelial cell membranes have
potential to function as ‘channel replacement therapies’ to ameliorate the symptoms of diseases,
such as cystic fibrosis (CF) [
fibrosis transmembrane conductance regulator (CFTR) protein results in a malfunctioning chloride
channel [
10]. A reduced level of anion (Cl− and HCO3−) transport occurs, leading to a lower
1–7]. CF is a channelopathy, where a genetic mutation to the cystic
8
–
level of osmosis. As a result, sticky mucous secretions occur which results in inflammation and
infections particularly in the lungs [11]. Synthetic chloride transporters have the potential to bypass
the faulty channel and restore activity by independently diffusing across the membrane as a chloride
complex. Our research group has recently focused on designing transporters that are selective for
chloride over proton transport [12], as dissipation of pH gradients in cells has been shown to trigger
apoptosis [13–15], and may disrupt the autophagic process [16,17]. Therefore, the development of
selective chloride transporters that do not facilitate H+/OH− transport is an attractive goal in the
development of putative channel replacement therapies.
Recently, we discovered that anionophores can act as functional mimics of uncoupling proteins,
and facilitate H+ transport via facilitated fatty acid transport [18]. Receptors can complex the negatively
charged fatty acid carboxylate head group and mediate its passage through the lipid bilayer membrane.
Decomplexation occurs, the free fatty acid protonates and diffuses back across the membrane, thus
resulting in proton transport. The rate determining step in the transport cycle is diffusion of the free
receptor, while fatty acid flip-flop provides an alternate pathway through the membrane for anion
transporters, which results in an increased rate of H+/Cl− symport. The increase in H+ transport via
this mechanism is undesirable in the case of chloride selective receptors for the treatment of CF.
Molecules 2019, 24, 1278; doi:10.3390/molecules24071278
www.mdpi.com/journal/molecules

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The challenge is to design receptors capable of chloride uniport that do not facilitate either the
transport of H+/OH− or fatty acid flip-flop. We have shown with a series of strapped calixpyrroles
that increasing the degree of encapsulation of a receptor results in a high degree of chloride selectivity,
which is not compromised in the presence of fatty acids [19]. To further investigate this phenomenon,
as well as the effects of steric hindrance and preorganisation of anionophores on Cl− > H+/OH−
selective transport, a series of receptors, shown in Figure 1, based on the tren scaffold were developed.
Previously, Wu et. al. [12] reported the transport selectivity of n-pentyl,
tren compounds, and this inspired the study of two additional constitutional isomers,
varying ratios of n-pentyl and t-pentyl substituents. Receptor , with t-butyl groups, was created to
explore whether there is an optimum size for substituents. A macrocycle was incorporated into the
tren scaffold for receptor , and the macrocycle may encapsulate the binding site to a greater extent [20].
1, and t-pentyl,
4, substituted
2
and , with
3
5
6
Furthermore, preorganisation of the thiourea anion binding groups could result in increased anion
binding affinities, which could potentially lead to improved anion transport abilities.
Figure 1. Structure of receptors 1–6.
In this paper, we report the synthesis of symmetrical,
5, and unsymmetrical tren based thiourea
anionophores , and . The anion binding properties of the whole series of receptors were studied
2,
3
6
with a range of biologically relevant anions. Elucidation of the transport mechanism was achieved
using cationophore coupled assays, and a full examination of the chloride transport activity as well as
investigations into the Cl− > H+/OH− transport selectivity were performed.
2. Results and Discussion
2.1. Synthesis
The synthesis of
was synthesised in an 82% yield. Compounds
which is shown in Figure 2. In all cases, mono-Boc protection of tris-(2-aminoethyl)amine was
performed to give , followed by transformation of the two free amines to isothiocyanate groups
in . Intermediate was reacted with either n-pentyl amine, t-pentyl amine, or di-aminoheptane to
obtain the Boc-protected product of receptors , and , intermediates 11, respectively. From here,
Boc-deprotection and subsequent coupling with the appropriate alkyl thiourea gave receptors and
in 55% and 7% yields. However, these conditions were repeatedly unsuccessful in the case of receptor
, therefore, an alternate route was employed. Tris-(2-aminoethyl)amine was reacted directly with
t-pentyl isothiocyanate in a 1:2 molar ratio to favour formation of the di-substituted product. After
1
and
4
was previously reported [12] and using the same method, receptor
5
2,
3
, and were synthesised via a multi-step procedure,
6
7
8
8
2,
3
6
9–
2
6
3

Molecules 2019, 24, 1278
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separation of the desired di-t-pentyl mono-amine tren by column chromatography, coupling with the
n-pentyl thiourea gave receptor in an 83% yield. Full synthetic details and characterisation can be
3
found in the Supplementary Material.
Figure 2. Scheme of the synthetic procedure for unsymmetrical and macrocyclic tren-based
thiourea receptors.
2.2. Anion Binding Studies
The ability of receptors 1–6 to bind biologically relevant anions in solution was investigated using
1H NMR titrations in DMSO-d6/0.5% H2O, with the anion added as either the tetrabutylammonium
(TBA) or tetraethylammonium (TEA) salt. Where possible, the change in the chemical shift of more
than one resonance was followed, allowing global fitting of the titration data to a 1:1 binding model
using Bindfit [21]; summarised in Table 1.
Table 1. Stability constants (Ka) for the complexation of receptors 1–6 with chloride, bicarbonate, and
dihydrogen phosphate (as TBA salts for chloride and dihydrogen phosphate and TEA for bicarbonate)
in DMSO-d6/0.5% H2O at 298 K.
Ka (M−1) for 1:1 Receptor:Anion Complex
Receptor
− a
− b
− b
Ka Cl
Ka HCO3
Ka H2PO4
d
1
2
3
4
5
6
346
770 e
5010 c
5560 f
2460 f
1500
n.d.
n.d.
n.d.
g
g
1080 e
1530
7460
2600
1110
2670
g
2170 b, e
4930 h
n.d.
a
All errors < 15%. Host concentration 5 mM. b Host concentration 1 mM. c Fitted to 7.5 equiv. as peaks broadened
d
and merged at 10 equiv. No association constant determined, peaks broaden up to 1 eq due to strong binding,
then peaks sharpen between 1–4 equiv. from formation of the 1:1 complex, above 4 equiv. peaks broaden signifying
e
f
g
the presence of the 1:2 complex. Two resonances followed and fitted. Three resonances followed and fitted. No
h
association constant determined, peaks broaden up to 1 equiv., then peaks sharpen between above 1 equiv. One
resonance followed and fitted.

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The chloride binding affinity increases for the more sterically hindered receptors through the
series. The strongest binding to chloride was observed for the macrocyclic receptor , presumably
due to a preorganisation effect. As expected, the overall binding affinity to bicarbonate was stronger
than chloride with the macrocycle, , again exhibiting the highest binding affinity. However, for
receptors , the opposite trend was evident, and the increased steric bulk of receptors resulted in
6
6
1–5
a decrease in binding affinity to the larger bicarbonate anion through the series. Peak broadening
occurred during titrations with dihydrogen phosphate and therefore association constants could not
be determined for receptors 1–3 and 6. Very high binding affinity was observed between receptors 4
and 5 with dihydrogen phosphate.
2.3. Anion Transport Studies
2.3.1. Transport Mechanism
Complementary cationophore coupled assays have been used to determine the transport
mechanism of several classes of receptors [22 24]. The assay uses naturally occurring cationophores,
–
valinomycin and monensin, that facilitate potassium transport by different mechanisms. Valinomycin
is an electrogenic transporter and this process involves the net flow of charge across a membrane [25].
Selective potassium transport facilitated by valinomycin can dissipate the membrane potential
produced by chloride uniport facilitated by electrogenic anion receptors, leading to an overall KCl
efflux. In contrast, monensin functions as an electroneutral transporter and during the transport
process, the charge is balanced by back transport of a species with the same charge [26]. Deprotonation
of a carboxylic acid group occurs upon metal complexation, allowing K+/H+ exchange, which couples
−
−
−
to the H+/Cl symport (or functionally equivalent Cl /OH antiport) facilitated by an electroneutral
receptor, resulting in overall KCl efflux.
To set up the assay detailed in Figure 3, large unilamellar vesicles (LUVs, 200 nm)
composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC, 200 nm diameter) were
prepared containing potassium chloride (KCl, 300 mM) internal solution, and suspended in
an external solution of potassium gluconate (K-Glu, 300 mM), both buffered to pH 7.2 with
4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) buffer (5 mm). As DMSO solutions,
cationophores were added at t =
−
30 s followed by receptors at t = 0 s, and the resulting chloride efflux
was monitored using a chloride ion selective electrode (ISE). At t = 300 s, the vesicles were lysed with
detergent to give a measurement corresponding to a 100% chloride efflux, allowing calibration of the
results. DMSO was tested as a control and no chloride efflux was detected, therefore the integrity of
the vesicles is not affected by the amount of DMSO used.
Figure 3. Overview of the complementary cationophore coupled assay. POPC vesicles (grey) contain
an internal KCl (300 mM) solution and are suspended in an external K-Glu (300 mM) solution both
buffered to pH 7.2 with HEPES. KCl efflux monitored by a chloride ISE (navy) induced by valinomycin
−
(Vln, blue) facilitated electrogenic K+ transport coupled to compound (C, purple) mediated Cl uniport
(right), and monensin (Mon, red) facilitated electroneutral K+/H+ exchange coupled to compound (C,
−
purple) mediated Cl /H+ symport (left).

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Receptors
1–
6
were tested in the cationophore coupled assay, and the results are shown in Figure 4.
In all cases, ~100% chloride efflux was achieved in the presence of valinomycin. Receptors
displayed a small degree (<20%) of chloride efflux when coupling to monensin. Due to inactivity at the
initial concentration (0.1 mol%, results in Supplementary Material), receptor was tested at a higher
concentration (0.5 mol%). An increase in electroneutral H+/Cl− symport to ~60% was observed for
receptor in the presence of monensin. The results indicated that receptors function predominantly
is capable of some electroneutral
1–5
6
6
1–6
as electrogenic chloride transporters, and macrocyclic receptor
chloride transport at higher receptor concentrations.
6
Figure 4. Results from the cationophore coupled assay. Chloride efflux facilitated by receptor (filled
purple), receptor coupled to valinomycin (0.1 mol%, filled blue), and receptor coupled to monensin
(0.1 mol%, filled red). The cationophore was added at t =
detergent was added at t = 300 s. DMSO (grey), valinomycin (empty blue), and monensin (empty red)
were tested as controls and each data point is the average of two repeats with the error bars showing
−
30 s, the receptor was added at t = 0 s, and
the standard deviation. (a) Receptor 1 (0.1 mol%). (b) Receptor 2 (0.1 mol%). (c) Receptor 3 (0.1 mol%).
(d) Receptor 4 (0.1 mol%). (e) Receptor 5 (0.1 mol%). (f) Receptor 6 (0.5 mol%).
2.3.2. Chloride Transport Selectivity
Quantification of the transport ability and Cl− > H+/OH− selectivity of receptors
achieved using a modified NMDG-Cl assay [12 27]. Three previously optimized conditions [18] were
1–6 was
,
employed. Firstly, (a) in bovine serum albumin (BSA) (1 mol%) treated vesicles (BSA was used to
sequester any fatty acids present in the vesicles giving a fatty acid free lipid bilayer). Secondly, (b) in
the presence of gramicidin (Gra, 0.1 mol%) added to facilitate electrogenic H+ efflux, enabling the
−
study of receptor mediated Cl uniport. Finally, (c) in the presence of the naturally occurring fatty acid,
oleic acid (OA, 2 mol%), which is found in cell membranes and can artificially enhance the overall rate
−
of H+/Cl symport. Anion transporters can accelerate the fatty acid flip-flop mechanism by taking on
the role of a flippase for fatty acid anions. Receptors bind to the anionic carboxylate head group and
mask the charge, allowing rapid passage of the head group through the lipid bilayer. The carboxylate
group can then protonate and diffuse back across the membrane and subsequently deprotonate,
effectively resulting in H+ transport. Dose response studies and subsequent Hill analysis [28] for the
three conditions detailed allowed the calculation of EC50 values to give the effective concentration of
transporter required to achieve 50% chloride efflux from the vesicles. The results are shown in Table 2.

Molecules 2019, 24, 1278
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Table 2. EC50 values shown for the NMDG-Cl assay with receptors 1–6 in BSA treated vesicles, in the
presence of gramicidin (Gra) and in the presence of oleic acid (OA).
EC50 (mol%) a
e
g
f
Receptor
FGra
FOA/Gra
FOA
(b) Gra c
(a) BSA b
(c) OA d
1
2
3
4
5
6
0.236
0.218
0.175
0.164
0.295
1.900
0.0113
0.00748
0.00420
0.00223
0.00483
0.0238
0.0214
0.0195
0.00834
0.00970
0.0121
0.126
21
29
42
74
61
80
11
11
21
17
24
15
1.9
2.6
1.9
4.3
2.5
5.4
Values obtained are concentration dependent and can change with different assay conditions, e.g., EC50 values of
a
previously reported receptors
1
and
4
are lower due to the higher volume of DMSO solutions used [12
,
18]. Values
reported in transporter to lipid molar ratio (mol %). Measures Cl−/H+ symport (Cl−/OH− antiport) after vesicle
b
treatment with BSA (1 mol%). Measures total Cl−/H+ symport (Cl−/OH− antiport), rate limiting H+ transport is
c
facilitated by Gra (0.1 mol%). Measures Cl−/H+ symport (Cl−/OH− antiport), with H+ transport facilitated by
d
the fatty acid flip-flop of OA (2 mol%). Factor of enhancement in the Cl− uniport in the presence of Gra, FGra is
e
calculated by dividing the EC50 (BSA) by the EC50 (Gra). FGra > 1 indicates Cl− transport enhancement. Factor of
f
enhancement in the overall rate of Cl−/H+ cotransport in the presence of OA, FOA is calculated by dividing the
EC50 by the EC50 (OA). FOA > 1 indicates the receptor can assist the flip-flop of OA−, increasing pH dissipation.
g
Factor of chloride transport selectivity retention in the presence of oleic acid, FGra/OA is calculated by dividing the
EC50 (OA) by the EC50 (Gra). FGra/OA > 1 indicates Cl− selectivity retention.
POPC LUVs (200 nm) were loaded with N-methyl-D-glucamine chloride (NMDG-Cl, 100 mM)
and 8-hydroxypyrene-1, 3, 6-trisulfonic acid (HPTS, a pH sensitive dye, 1 mM), and suspended in an
external solution of NMDG-Cl. All solutions were buffered to pH 7 with HEPES (10 mM). Receptors
were added as a DMSO solution at t =
NMDG (0.5 M) base pulse at t = 0 s, which increased the external pH to 8. The fluorescence ratio of
8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (HPTS) (basic form, = 460 nm, = 510 nm,
−
15 s, and transport was initiated by the addition of an
λ
ex
λ
em
divided by the acidic form,
λ
= 403 nm,
λ
em = 510 nm) was mon−itored as the pH gradient across the
ex
membrane was dissipated by either: (a) Receptor mediated H+/Cl symport (or OH /Cl exchange),
(b) a combination of receptor mediated Cl− uniport coupled to H+ efflux facilitated by Gra, and
receptor mediated H+/Cl− symport, or (c) a combination of receptor mediated Cl− symport coupled
to H+ efflux facilitated by fatty acid flip-flop, and receptor mediated H+/Cl− symport. The assay
conditions and transport pathways are shown in Figure 5.
−
−
Receptors 1–6
display poor H+/Cl− symport abilities in BSA treated vesicles in the NMDG-Cl
assay (Table 2). The low activity was expected, as the cationophore coupled assay showed these
compounds predominantly function as electrogenic chloride transporters rather than electroneutral
H+/Cl− co-transporters. However, mirroring the trend in the chloride binding affinities, the EC50
values for receptors
from additional t-pentyl substitution. Receptor
EC50 of 0.164 mol% in the BSA treated vesicles. t-Butyl substitution in receptor
1
–
4
decreased, indicating better transport abilities, as the encapsulation increases
is the best transporter of this series with the lowest
results in the second
4
5
worst transport ability in this series, suggesting that the more lipophilic pentyl substitution is favoured
over butyl substitution. Although it displays the strongest chloride binding affinity, macrocyclic
receptor 6 has the highest EC50 of the series. The poorer chloride transport is potentially due to the
more rigid structure of the macrocycle, reducing the deliverability, and hindering passage of the free
receptor as well as the receptor:chloride complex across the lipid bilayer.

Molecules 2019, 24, 1278
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Figure 5. Top: Overview of the NMDG-Cl assay. POPC vesicles (grey) contain an internal NMDG-Cl
(100 mM) and HPTS (1 mM) solution and are suspended in an external NMDG-Cl (100 mM) solution
both buffered to pH 7.2 with HEPES (10 mM). HCl efflux, monitored by pH sensitive HPTS, induced
by (
electrogenic H+ efflux coupled to compound (C, purple) mediated Cl uniport, and (
a b) gramicidin (Gra, green) facilitated
) compound (C, purple) mediated Cl−/H+ symport, (
−
b
) oleic acid (OA,
−
orange) flip-flop facilitated H+ transport coupled to compound (C, purple) mediated Cl /H+ symport.
−
Bottom: (
a
) Compound mediated Cl /H+ symport via receptor deprotonation. The neutral receptor (T,
−
purple) binds an anion (Cl , green). The negatively charged complex moves across the membrane, and
the anion dissociates. The receptor is deprotonated (H+, blue), and the negatively charged receptor
moves across the membrane. The receptor is protonated to complete the cycle. (b) Compound mediated
Cl− uniport. The neutral receptor (T, purple) binds an anion (Cl−, green). The negatively charged
complex moves across the membrane, and the anion dissociates. Gramicidin (Gra, green) facilitates
electrogenic H+ efflux to balance the charge gradient build up. The −neutral receptor diffuses back
across the membrane to complete the cycle. (
acid flip-flop pathway. The neutral receptor (T, purple) binds an anion (Cl , green) and transports it
through the membrane as the negatively charged complex. The anion dissociates on the exterior of the
c
) Compound mediated Cl /H+ symport coupled to fatty
−
−
membrane. The neutral receptor binds to a negatively charged deprotonated fatty acid (COO , orange)
and moves across the membrane as the negatively charged complex. Fatty acids can translocate across
the membrane when protonated (H+, blue) to complete the cycle.
Including the proton channel, Gra, in the NMDG-Cl assay accelerates H+ efflux across the
membrane. Therefore, the rate-limiting H+ transport step for electrogenic receptors
1–6 is removed,
and the rate of chloride uniport is increased. A decrease in EC50 values by over one order of magnitude
in all cases indicated an increase in chloride transport. A factor of enhancement in chloride transport
(FGra) was then quantified, by dividing (a) EC50 (BSA) by (b) EC50 (Gra). Values > 1 demonstrate an
enhancement of the chloride transport, and the results are shown in Table 2. A bar chart is also included
for clarity in Figure 6. Calculated FGra values showed impressive enhancement in chloride transport for
all receptors. The most encapsulating receptors,
receptor showed the largest enhancement in chloride transport of the series. The same trend was
repeated for receptors . Increasing encapsulation from additional t-pentyl substituents resulted in
higher FGra values, corresponding to increased chloride uniport.
4–6, displayed the highest FGra values, and macrocyclic
6
1–4

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Figure 6. Chloride transport enhancement results. In the presence of Gra, a factor of chloride
transport enhancement, FGra, (green) is calculated by dividing the EC50 (BSA) by the EC50 (Gra). Factor
of enhancement in the overall rate of Cl−/H+ cotransport in the presence of OA, FOA (orange), is
calculated by dividing the EC50 by the EC50 (OA). Values over the threshold (>1) indicate an increase in
the rate of chloride transport in the conditions described.
The addition of OA to the NMDG-Cl assay allowed an evaluation of the receptors’ ability to
contribute to the naturally occurring fatty acid transmembrane proton shuttling pathway, by assisting
the flip-flop of the negatively charged fatty acid head group. The pathway is depicted in Figure 4.
Dividing (a) EC50 (BSA) by (c) EC50 (OA) allowed a factor of enhancement (FOA) in the overall rate of
H+/Cl− transport in the presence of OA to be calculated. The results shown in Table 2 and Figure 6,
give FOA values > 1 for all receptors, indicating that the overall rate of H+/Cl− symport is increased
due to receptor coupled fatty acid flip-flop. Enhancement factors are greater in the presence of
Gra compared to OA. Therefore, receptors 1–6 preferentially facilitate chloride uniport compared to
H+/Cl− symport, reinforcing the electrogenic transport mechanism shown by receptors
cationophore coupled assay.
1–6 in the
In the presence of OA, chloride uniport is often diminished, because receptors can display some
affinity towards the fatty acid headgroup [18]. As free fatty acids are abundant in cell membranes,
reducing affinity for fatty acid headgroups is an important consideration in the development of
anionophores for the potential treatment of CF. Therefore, an assessment of the ability of receptors
1
–
6
to retain chloride selective transport in the presence of OA was calculated. Assuming the chloride
uniport achieved in the presence of Gra (i.e., EC50 (Gra)) corresponds to the chloride selective transport
by receptors , a factor of the chloride transport selectivity retention in the presence of OA (FOA/Gra)
1–
6
can be calculated. Dividing (c) EC50 (OA) by (b) EC50 (Gra) gives FOA/Gra values, which can be found in
Table 2. Values > 1 indicate a retention in chloride selective transport in the presence of OA. FOA/Gra
values of ~2 were obtained for receptors
transport retention. Macrocyclic receptor
in the presence of OA, FOA/Gra = 5.4, with the most encapsulating receptor
1
6
–
3
and
retained the highest degree of chloride selective transport
also retaining chloride
5, indicating a slight amount of chloride selective
4
selective transport, FOA/Gra = 4.3. The results demonstrate that it is advantageous to include enforced
steric hindrance and encapsulation in the design of anion receptors to reduce affinity towards the fatty
acid headgroup and retain chloride selective transport.
3. Conclusions
We have shown that increasing the steric hindrance of simple tren based receptors results
in an increase in binding affinity towards chloride. However, the reduced structural freedom of
these receptors results in a decrease in binding affinity towards the bicarbonate anion. From the
cationophore coupled assay, it was evident that this series of tren-based tris-thiourea receptors function

Molecules 2019, 24, 1278
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predominantly as electrogenic chloride transporters. The H+/Cl− symport activity of this series was
poor as to be expected, but chloride uniport in the presence of the proton channel, Gra, showed high
activity. Enhancement of chloride transport in the presence of Gra was impressive over the whole
series, however, all receptors were able to facilitate the undesirable fatty acid flip-flop proton transport
pathway at different degrees. This unwanted effect was less apparent for the more sterically hindered
receptors,
4 and 6, demonstrating the benefit of encapsulating receptors. Overall, this study shows
that an elaborated receptor design is crucial to minimise H+/OH− transport and to obtain chloride
selective transporters.
Supplementary Materials: Synthetic procedures and compound characterizations, methodology and results for
anion binding experiments, and methodology and results of anion transport experiments are available online at
http://www.mdpi.com/1420-3049/24/7/1278/s1. Figures S1–S104.
Author Contributions: Conceptualization, L.A.J., A.R., X.W., E.N.W.H. and P.A.G; methodology, L.A.J., A.R., X.W.,
E.N.W.H. and P.A.G.; investigation, L.A.J. and A.R.; resources, P.A.G.; data curation, L.A.J.; writing—original draft
preparation, L.A.J.; writing—review and editing, L.A.J., X.W., E.N.W.H, and P.A.G.; supervision, X.W., E.N.W.H.
and P.A.G.; funding acquisition, P.A.G.
Funding: This research was funded by the ARC (DP180100612), the EPSRC (EP/J009687/1) and The University
of Sydney.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the
study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to
publish the results.
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Sample Availability: Samples of the compounds are not available from the authors.
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