Thiosquaramides: PH switchable anion transporters

Article abstract of DOI:10.1039/c4sc01629g

The transport of anions across cellular membranes is an important biological function governed by specialised proteins. In recent years, many small molecules have emerged that mimick the anion transport behaviour of these proteins, but only a few of these

Full text of DOI:10.1039/c4sc01629g

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Thiosquaramides: pH switchable anion
transporters†
Cite this: Chem. Sci., 2014, 5, 3617
Nathalie Busschaert,‡a Robert B. P. Elmes,‡b Dawid D. Czech,a Xin Wu,a
Isabelle L. Kirby,a Evan M. Peck,c Kevin D. Hendzel,c Scott K. Shaw,c Bun Chan,b
c
b
ad
*
*
Bradley D. Smith, Katrina A. Jolliffe and Philip A. Gale
The transport of anions across cellular membranes is an important biological function governed by
specialised proteins. In recent years, many small molecules have emerged that mimick the anion
transport behaviour of these proteins, but only a few of these synthetic molecules also display the
gating/switching behaviour seen in biological systems. A small series of thiosquaramides was synthesised
and their pH-dependent chloride binding and anion transport behaviour was investigated using 1H NMR
titrations, single crystal X-ray diffraction and a variety of vesicle-based techniques. Spectrophotometric
titrations and DFT calculations revealed that the thiosquaramides are significantly more acidic than their
oxosquaramide analogues, with pKa values between 4.0 and 9.0. This led to the observation that at pH
7.2 the anion transport ability of the thiosquaramides is fully switched OFF due to deprotonation of the
receptor, but is completely switched ON at lower pH.
Received 2nd June 2014
Accepted 17th June 2014
DOI: 10.1039/c4sc01629g
www.rsc.org/chemicalscience
learn what features of these molecules enable them to effec-
tively mediate the ux of anions through lipid bilayers. Previ-
Introduction
ously, we discovered that thiourea groups, particularly those
with a single aromatic group pendant to the thiourea, are
signicantly more effective anion transporters than analogous
ureas.11 A subsequent QSAR analysis of a series of similar
thioureas showed that for this class of molecule, lipophilicity,
molecular size and the Hammett constant of the substituents
present on the transporter are the key molecular parameters
upon which the transport efficiency of the molecule depends.12
We have also been interested in squaramide based receptors, as
squaramides have been suggested as potent (thio)urea iso-
steres13–19 with potential therapeutic benets against a variety of
diseases (e.g. Chagas disease).20,21 We found that transporters
containing squaramide groups (such as compounds 1–3) could
mediate higher uxes of anions than analogous ureas or thio-
ureas despite having lower lipophilicities.22 In this case the
signicantly higher anion affinities of squaramides (as
compared to urea and thiourea analogues) may be an important
factor. As we had observed with other classes of transporter,23–25
increasing the degree of uorination of the squaramides
resulted in signicantly enhanced transport properties.22
We envisaged that thiosquaramides should have both
enhanced acidity compared to squaramides and higher lip-
ophilicity. To test this hypothesis we have conducted the rst
systematic supramolecular study of thiosquaramides. Speci-
cally, we synthesised thiosquaramides 5–8 and compared their
chloride complexation and transport properties to squaramides
1–4. We nd that the enhanced acidity of the NH groups
results in thiosquaramide deprotonation under physiological
Recent interest in the development of small molecule lipid
bilayer anion transporters has been driven by the potential
application of these compounds to treat diseases caused by
faulty anion transport across cell membranes.1–5 Cystic brosis
is the most well-known example of this type of disease, that are
known collectively as “channelopathies” and are characterised
by faulty chloride transport channels.6 Compounds that can
restore or disrupt such chemical processes in cells also have the
potential to possess anti-cancer properties, where they can
depolarize acidic compartments within cells and trigger
apoptosis.7–10 Therefore one of the goals of our research recently
has been to develop new classes of anion transporters and to
aChemistry, University of Southampton, Southampton, SO17 1BJ, UK. E-mail: philip.
gale@soton.ac.uk; Tel: +44 (0)23 8059 3332
bSchool of Chemistry (F11), The University of Sydney, 2006 NSW, Australia. E-mail:
kate.jolliffe@sydney.edu.au; Fax: +61 2 9351 3329; Tel: +61 2 9351 2297
cDepartment of Chemistry and Biochemistry, University of Notre Dame, Notre Dame,
Indiana, 46556, USA
dDepartment of Chemistry, Faculty of Science, King Abdulaziz University, Jeddah
21589, Saudi Arabia
† Electronic supplementary information (ESI) available: Synthesis and
characterisation of the receptors, details and gures about the X-ray crystal
structures of the receptors and anion complexes, conformational studies,
thiosquaramide stability studies, association constant determination, stack
plots and tplots of the 1H NMR titrations, pH-spectrophotometric titrations,
DFT calculations of pKa values, various vesicle assays methods and Hill plots.
CCDC 1005458–1005461. For ESI and crystallographic data in CIF or other
electronic format see DOI: 10.1039/c4sc01629g
‡ These authors contributed equally to this work.
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conditions, with little chloride transport across lipid bilayers.
However, under acidic conditions some thiosquaramides Scheme 1 Synthesis of thiosquaramide receptors 5–8. Yields are
given between brackets.
become active transporters. This unusual pH-switch mecha-
nism has potential to become a new way to activate chloride
transport within acidic environments in biological systems.
˚
˚
distances of 2.774–2.815 A for 5$DMSO and 2.755–2.776 A for
8$DMSO and N–H/O angles of 166–170ꢀ for 5$DMSO and 160–
164ꢀ for 8$DMSO). These structures are similar to the crystal
Results and discussion
Synthesis and characterisation
Oxosquaramides 1–4 were prepared using the zinc triate
mediated reaction of diethyl squarate with the appropriately
substituted aniline, developed by Taylor and co-workers.26
Initial attempts to form the thiosquaramide derivatives 5–8
using Lawesson's reagent proved unsuccessful, while reaction
with tetraphosphorus decasulde (P4S10) gave thiosquaramide
products in low yield as part of a complex mixture of products.
However we found that the zwitterionic P4S10$pyridine complex,
formed by reaction of P4S10 in reuxing pyridine,27 allowed the
conversion of oxosquaramides 1–4 into thiosquaramides 5–8 in
66%, 48%, 37% and 60% yields respectively (Scheme 1). This
strategy afforded each of the thiosquaramides in reasonable
yields and 6–8 were easily puried by ash chromatography,
while thiosquaramide 5 needed no further purication. Crystals
suitable for single crystal X-ray analysis were obtained for thi-
osquaramides 5 and 8 by recrystallization from a concentrated
DMSO solution allowing the elucidation of their solid state
structure (see Fig. 1b for 8$DMSO). Tables of hydrogen bonds,
data collection and renement details can be found in the ESI.† Fig. 1 Front and side views of the X-ray crystal structure of 4$DMSO
(a) and 8$DMSO (b). Ligands and DMSO molecules are shown in ball-
and-stick. Non-interacting hydrogen atoms and non-interacting
DMSO molecules are omitted for clarity. Hydrogen bonds are repre-
sented by dashed lines and atoms are colour coded as follows: C
Both 5 and 8 were found to form a 1 : 1 complex with DMSO in
the solid state. In both cases the thiosquaramide moiety acts as
a hydrogen bond donor to a single DMSO molecule with two
hydrogen bonds formed between both available thiosquar-
(black), H (white), F (green), N (blue), S (yellow) and O (red). Relevant
amide NH groups and the oxygen atom of DMSO (N/O torsion angles are given in green.
3618 | Chem. Sci., 2014, 5, 3617–3626
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structures of the DMSO solvates of analogous squaramides 1 considerably lower affinities for Clꢁ than 2 and 3. The associ-
(previously published)22 and 4 (Fig. 1a) and also to those ation constants (Ka) of 1–8 in 0.5% H2O in DMSO-d6 with Clꢁ are
reported recently in a related study of luminescent squaramide summarised in Table 1 and demonstrate an unexpected trend
based receptors.28 However, unlike the structures of oxosquar- whereby increasing the number of electron withdrawing
amides 1 and 4 the thiosquaramide structures are not fully substituents on the thiosquaramide unit results in lower affin-
planar, with the aromatic group being twisted out of the plane ities for Clꢁ. This is opposite to the trend seen for squaramides
of the cyclobutene ring (see Fig. 1, torsion angles between the 1–4 and suggests that 6 and 7 may exist in deprotonated form in
planes dened by the aromatic rings and the cyclobutene ring DMSO. Further support of this hypothesis is the recent report by
ranging between 2–6ꢀ and 15–19ꢀ for oxosquaramides Taylor et al. that a nitro-substituted squaramide exists in DMSO
1$DMSO22 and 4$DMSO respectively and between 26–32ꢀ and in a partially deprotonated form.26
1
32–42ꢀ for analogous thiosquaramides 5$DMSO and 8$DMSO
To clarify this effect, H NMR titrations with 6 and 7 were
respectively). This twisted conformation likely results in a also performed in CD3CN with TBACl. In this solvent the 1H
diminished contribution from the aromatic C–H hydrogen NMR spectrum of 6 undergoes signicant changes whereby the
bonds that are oen observed in complexes of oxosquar- NH signals attributed to the thiosquaramide are shied
amides.22,29–31 The crystal structures reveal that steric hindrance downeld upon the addition of TBACl (Fig. 2). The addition of
between the large sulfur atoms and the ortho-CH hydrogen Clꢁ initially resulted in peak broadening of all signals but when
atoms is the cause of the non-planar geometry of the thio- more than 1 equivalent of anion was added, well resolved
squaramides and it is reasonable to assume that a fully planar signals were observed that were shied signicantly downeld
anti/anti conformation can not be adopted in solution. In order from their original positions. This observation along with the
to examine whether there is a difference in conformation considerable shis exhibited by the aromatic protons strongly
between the oxosquaramides and thiosquaramides in solution,
dynamic 1H NMR studies at various temperatures were per-
formed on the most soluble compounds 4 and 8 (details can be
found in the ESI†). It was found that in 1 : 1 CDCl3 : DMSO-d6
oxosquaramide 4 is locked in the anti/anti conformation at
Table 1 Summary of the chloride association constants Ka (Mꢁ1) of
receptors 1–8 in 0.5% H2O in DMSO-d6 and CD3CN at 298 Ka
Ka (Mꢁ1
)
temperatures between 273–333 K, whilst thiosquaramide 8
appears to be present in both syn/anti and anti/anti confor-
mations with fast inter-conversion between each conformation.
The difference in conformational equilibria is attributed to the
larger steric size of the sulfur atoms in 8 which destabilize the
anti/anti conformation.
Receptor
DMSO-d6
CD3CN
c
1
2
3
4
5
6
7
8
260b
458b
643b
145
270
60
—
c
—
c
—
c
—
c
—
>104
114
Anion binding
d
—
c
It has been observed that squaramides exhibit signicantly
higher anion affinities compared to their urea and thiourea
analogues28–31 and that this results in superior anion transport
abilities.22,32 A recent computational study concluded that thi-
osquaramides should have high acidity and hydrogen bond
ability.33 Thus, we initially expected that thiosquaramides 5–8
would exhibit higher anion affinities than squaramide
analogues 1–4. In order to evaluate the anion binding abilities
of thiosquaramides 5–8, 1H NMR spectroscopic titrations at 298
402
—
a
b
Data tted to a 1 : 1 model with errors <15%. Value taken from ref.
c
22.
Receptor not sufficiently soluble to allow accurate Ka
determination. d No measurable anion binding.
34
K in 0.5% H2O in DMSO-d6 were performed by addition of
tetrabutylammonium chloride (TBACl). Where possible, the
data obtained from these titrations were plotted as the cumu-
lative changes in chemical shi (Dd) against the equivalents of
anion added and the resulting plots were analysed using
Hyperquad©.35 The best t in all cases was to a 1 : 1 binding
model (see ESI† for tted data). The changes observed in the 1H
NMR spectra of 5 and 8 upon the addition of TBACl showed that
the NH signals associated with the thiosquaramide NHs were
signicantly shied downeld. Unexpectedly however, the CF3
substituted receptors 6 and 7 did not exhibit such large changes
and showed lower binding affinities for Clꢁ than both 5 and 8.
Moreover, while 5 and 8 showed similar or higher affinities for
Fig. 2 Stack plot of the 1H NMR spectrum of receptor 6 (2.5 ꢂ 10ꢁ3 M)
upon titration with chloride (0–12 eq.) added as its tertabutylammo-
nium salt in CD3CN at 25 ꢀC. From bottom to top: 0, 0.5, 1.0, 1.5, 2, 3, 4,
Clꢁ than their squaramide counterparts 1 and 4, 6 and 7 showed 6, 8, 10, 12 equiv. chloride.
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suggests the formation of a tightly bound host–guest complex in (Fig. 3). Once again, a non-planar twisted conformation is
solution. Subsequent additions induced no further changes to observed that diminishes the potential contributions of the
the spectra. Interestingly, under the same conditions, the ortho-CH hydrogen atoms. Furthermore, the 2 : 1 stoichiometry
stability constant obtained for Clꢁ with 7 was found to be observed here is different to the 1 : 1 stoichiometry seen in
signicantly lower than the value obtained with 6, indicating solution and in the crystal structures of analogous squaramides
that this receptor is also partially deprotonated in CD3CN (Table and it is not clear whether this is a packing effect resulting from
1). However, while considerations of NH acidity and deproto- the smaller TMA counter cation. Nevertheless, this crystal
nation can explain the behaviour observed for thiosquaramides structure shows that a single chloride anion can accommodate
6 and 7, they do not explain why the chloride affinities of 1 and 5 multiple thiosquaramide receptors, which is relevant for the
are almost identical in DMSO with 0.5% water, while the chlo- transmembrane anion transport ability of the thiosquaramides
ride affinity of thiosquaramide 8 is twice as high than analogous as the hydrophilic chloride anion needs to be screened from the
squaramide 4. Due to their higher NH acidity it is expected that apolar interior of the membrane in order to allow transport.
thiosquaramides will display higher anion binding affinity than
squaramides, but it appears that the higher conformational
freedom of thiosquaramides (see above) can require a structure-
dependent penalty to be paid upon anion binding. The
magnitude of this penalty is difficult to predict but is expected
by theoretical and experimental means. Computational pKa
to be different for simple thiosquaramide 5 and sterically bulky
pKa determination
In order to gain an insight into the acid–base behaviour of
receptors 1–8 in aqueous solution pKa values were determined
values were obtained using density functional theory (DFT) at
thiosquaramide 8.
the M06-2X/6-311+G(3df,2p) level, in conjunction with the SMD
Attempts were also made to investigate the anion binding
continuum solvation model. Two water molecules were
behaviour of the thiosquaramides in the solid state. Previous X-
included in the model in order to take into account explicit
interactions (details can be found in the ESI†). The results of the
calculations are presented in Table 2. Experimental pKa values
were obtained through pH-spectrophotometric titrations in a
ray crystal studies of oxosquaramides have shown that they
form 1 : 1 complexes with tetrabutylammonium chloride in the
solid state and that the squaramide adopts a fully planar anti/
anti conformation in which the chloride anion is bound via
mixture of acetonitrile–water (9/1 v/v; in the presence of 0.1 M
hydrogen bonds from both NH hydrogen atoms and ortho-CH
TBAPF6) as described by Fabbrizzi and co-workers.31 During the
spectrophotometric measurements, each of the oxosquar-
amides was seen to undergo a single deprotonation event,
characterised by a hypochromism at ꢃ370 nm and a concomi-
tant hyperchromism at ꢃ340 nm. Similarly, the thiosquaramide
deprotonation was signalled by a hypochromism at ꢃ405 nm
and a concomitant hyperchromism at ꢃ335 nm and was
accompanied by a change in the colour of the solution from
bright yellow to dull orange. In Fig. 4, the titration proles
comparing squaramide 2 and the analogous thiosquaramide 6
are shown. A four parameter sigmoid curve was tted through
the data points using Sigma Plot (Systat Soware Inc., Chicago,
IL, USA) with the point of inexion corresponding to the pKa.
hydrogen atoms.22,26,30 As a comparison, single crystals of a
chloride complex of thiosquaramide 6 were obtained by the
slow evaporation of a 1 : 9 water–acetonitrile solution of 6 in the
presence of excess tetramethylammonium (TMA) chloride
(attempts to grow single crystals in the presence of TBACl
failed). The X-ray crystal structure showed that thiosquaramide
6 can form a 2 : 1 receptor–chloride complex in the solid state,
whereby a single chloride anion is coordinated by two thio-
squaramide receptors via a total of four hydrogen bonds (N/Cl
ꢀ
˚
distances of 3.163–3.209 A and N–H/Cl angles of 163–171 )
Table 2 Comparison of pKa values of receptors 1–8 (6 ꢂ 10ꢁ5 M)
measured by spectrophotometric titrations in acetonitrile–water (9/1
v/v; in presence of 0.1 M TBAPF6) and calculated using DFT (M06-2X/
6-311+G(3df,2p) with SMD continuum solvation)
pKa
Receptor
Experimental
Computational
1
2
3
4
5
6
7
8
11.7a
9.8
11.3
9.1
7.9
12.0
9.0
6.6
4.5
9.6
8.5b
12.1
7.3
Fig. 3 X-ray crystal structure of 6 with TMA chloride. Ligands are
shown in ball-and-stick and bound chloride is shown in spacefill (0.6
times van der Waals radius). TMA counterions and non-interacting
hydrogen atoms are omitted for clarity. Hydrogen bonds are repre-
sented by dashed lines and atoms are colour coded as follows: C
(black), H (white), F (green), N (blue), S (yellow) and Cl (dark green).
5.3
4.9
7.7
a
Comparable to pKa obtained by Ni et al. in DMSO: pKa(1) ¼ 12.48.
Comparable to pKa obtained by Ni et al. in DMSO: pKa(3) ¼ 8.37.
b
3620 | Chem. Sci., 2014, 5, 3617–3626
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that thioureas are better anion transporters than ureas due to
their higher NH acidity and due to the higher lipophilicity of the
sulfur atom.11,24 It was therefore envisaged that thiosquar-
amides have the potential to exceed any of the afore-mentioned
functional groups in their intrinsic anion transport ability. To
test this hypothesis vesicle-based experiments were performed
according to standard literature procedures.37,38 Initially, uni-
lamellar 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)
liposomes loaded with a NaCl solution (489 mM) buffered to pH
7.2 were prepared and suspended in an isotonic NaNO3 solu-
tion (489 mM) buffered to pH 7.2. A small volume of a DMSO
solution34 of 1–8 was added to these liposomes and the
resulting chloride efflux was monitored using an ion selective
electrode (ISE). The results for thiosquaramides 5–8 are given
in Fig. 5 (empty symbols) and show that the thiosquaramides
possess only limited anion transport ability at this pH (the
results for oxosquaramides 1–3 have been previously repor-
ted).22 Spectrophotometric pKa determinations, however,
indicate that the pKa values of the thiosquaramides are in the
range 4.0–8.0 (Table 2). This implies that at pH 7.2 a large
proportion of the thiosquaramides will be present in their
deprotonated, negatively charged form and will be unable to
transport anions across lipid bilayers due to charge repulsion.
The ion selective electrode experiments were therefore
repeated with unilamellar POPC vesicles loaded with NaCl
solution (489 mM) buffered to pH 4.0 and suspended in NaNO3
solution (489 mM) buffered to pH 4.0. At this pH the majority
of the compounds should be present in their neutral form and
anion transport should be ‘switched on’. The results shown in
Fig. 5 (lled symbols) reveal that the anion transport ability of
thiosquaramides 5 and 6 is indeed signicantly enhanced at
Fig. 4 (a) Absorption spectra taken over the course of a pH-spec-
trophotometric titration of 2 (6 ꢂ 10ꢁ5 M) in an acetonitrile–water
mixture (9/1 v/v; in presence of 0.1 M TBAPF6). Inset: Comparison plots
of absorbance at 268 nm ( ), 340 nm ( ) and 370 nm ( ) vs. pH. (b)
Absorption spectra taken over the course of a pH-spectrophotometric
titration of 6 (5 ꢂ 10ꢁ5 M) in an acetonitrile–water mixture (9/1 v/v; in
presence of 0.1 M TBAPF6). Inset: comparison plots of absorbance at
288 nm ( ), 331 nm ( ) and 408 nm ( ) vs. pH.
The obtained pKa values corresponding to a single deprotona-
tion event are shown in Table 2 and range from 4.9–12.1.
Encouragingly, pKa values for receptors 1 and 3 have also
recently been determined in DMSO by Ni et al. using the
Bordwell method and show values that are in close agreement
with the values presented in this study (pKa(1) ¼ 11.7 (found),
12.48 (literature); pKa(3) ¼ 8.5 (found), 8.37(literature)).36 In
general, there are reasonable agreements between the theoret-
ically computed and the experimentally determined pKa values.
As expected, both methods indicate that the thiosquaramides
have lower pKa values than their analogous oxosquaramides
and that the addition of electron withdrawing substituents
results in a further lowering of the pKa values. The most acidic
receptor was shown to be 7 (pKa ¼ 4.9 (expt), 4.5 (DFT)). These
values support the observation that electron decient thio-
squaramides exist in their deprotonated form at neutral pH and
can explain some of the anion binding behaviour observed for
thiosquaramides 6–7. Furthermore, pKa values below or around
7 indicate that thiosquaramides might be useful as pH-switch-
able anion transporters.
Fig. 5 Chloride efflux from POPC vesicles at pH 7.2 (empty symbols)
and pH 4.0 (filled symbols) mediated by thiosquaramides 5–8 (1 mol%
with respect to lipid). POPC vesicles were loaded with a 489 mM NaCl
solution buffered to pH 7.2 with 5 mM phosphate salts or to pH 4.0
with 5 mM citrate salts, and were suspended in a 489 mM NaNO3
solution buffered to pH 7.2 with 5 mM phosphate salts or to pH 4.0
with 5 mM citrate 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 a minimum of 6 inde-
Anion transport
Previous studies have shown that squaramides are potent
transmembrane anion transport agents that can outperform
analogous ureas and thioureas.22,32 It has also been suggested pendent trials. DMSO was used as a control.
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lower pH (the results for the oxo-squaramides can be found in a problem when aggregates or higher stoichiometries are
the ESI†). needed for chloride transport (such as a 2 : 1 stoichiometry seen
In order to quantify this effect, Hill analyses39 of the anion in the solid state, Fig. 3). This was corroborated by the high Hill
transport facilitated by 1–8 were performed at both pH 7.2 and coefficients (n-values) that were observed during the Hill anal-
pH 4.0, whereby the vesicle-based experiments were repeated at yses of 5 and 6 at pH 4.0 (see ESI†), indicating that higher order
various concentrations of receptor to obtain EC50 values, i.e. the aggregates are indeed required for anion transport by these
concentration of transporter (in mol% with respect to lipid) simple thiosquaramides.24,40 Another reason for the low anion
required to obtain 50% chloride efflux in 270 s (Table 3). It is transport activity of 4, 7 and 8 could be their lipophilicity. It has
clear from Table 3 that the oxosquaramides are powerful anion oen been suggested that ion transport across lipid bilayers is
transporters at both pH 7.2 and pH 4.0, with little difference in diminished when receptors become too lipophilic to interact
activity between the two pHs. In contrast, all of the thiosquar- with the ions at the lipid-water interphase.12,24,41,42 In order to
amides are unable to effectively transport chloride out of POPC obtain an estimate of the lipophilicity of compounds 1–8,
vesicles buffered to pH 7.2 at low carrier concentrations, but experimental retention times on reversed-phase HPLC43 were
thiosquaramides 5 and 6 are signicantly better transporters determined (Table 3). It is clear that the three inactive trans-
than their analogous oxosquaramides at pH 4.0. These Hill porters are also the receptors with the highest lipophilicity
analyses further conrm the hypothesis that the trans- (highest retention time), suggesting that lipophilicity may also
membrane anion transport activity of the thiosquaramides can contribute to the inactivity of 4, 7 and 8.
be switched on by lowering the pH. The fact that thiosquar-
In principle, the chloride transport behaviour of the thio-
amides 5 and 6 display higher anion transport activity at pH 4.0 squaramides could be due to either the formation of
than the analogous oxosquaramides 1 and 2 is presumably due membrane-spanning channels or a mobile carrier mechanism.
to their higher NH acidity and higher lipophilicity. It must be Oxosquaramides 1–3 have previously been shown to function
noted, however, that thiosquaramides 7 and 8 (and oxosquar- primarily via a mobile carrier mechanism22 and it is likely that
amide 4) do not show any signicant anion transport at either the same holds true for the thiosquaramides. Although the high
pH 7.2 or pH 4.0. There could be several explanations for this Hill coefficients observed for 5 and 6 could indicate channel
behaviour. The experimental pKa value of receptor 7 (Table 2), formation (see ESI†), they are more likely the result of aggre-
suggests that even at pH 4.0 a signicant amount of the receptor gation (or precipitation) of the receptors, or multiple receptors
will be present in its inactive, deprotonated form (pure POPC complexing one chloride anion for optimal screening of the
vesicles were found to be unstable at pH < 4.0 and it was thus highly hydrophilic anion from the lipid environment.24,40
not possible to test whether this receptor could also be Evidence for a mobile carrier mechanism by thiosquaramides
‘switched on’ at even lower pH). While pKa considerations can 58 was obtained by repeating the transport experiments with
explain the inactivity of compound 7, they cannot explain the vesicles consisting of 3 : 7 cholesterol : POPC. It has oen been
behaviour seen for tert-butyl substituted receptors 4 and 8, reported that cholesterol increases the viscosity of a lipid bilayer
which have much higher pKa values (Table 2). On the other and thereby exerts a negative effect on processes that depend on
hand, 4 and 8 are the most bulky receptors and this might cause diffusion through the bilayer (such as a mobile carrier mecha-
nism).44–47 A decrease in chloride transport mediated by 1–8 was
observed at both pH 7.2 and pH 4.0 for cholesterol-containing
liposomes, evidence in support of a mobile carrier mechanism
Table 3 Summary of the anion transport behaviour of receptors 1–8
(see ESI†). Furthermore, calcein leakage assays48–50 revealed that
EC50 at pH 7.2a
(mol%)
EC50 at pH 4.0a
(mol%)
Retention
timeb (s)
Apparent
pKa
the large highly anionic dye calcein did not leak from POPC
vesicles at pH 7.2 or pH 4.0 in the presence of 1–8 (see ESI†),
excluding the formation of pores or channels with large internal
diameter and vesicle disruption induced by the transporters.
The most plausible anion transport mechanism displayed by
thiosquaramides 1–8 is therefore a mobile carrier process.
The charge imbalance created by the transmembrane anion
transport promoted by synthetic molecules can be compensated
via either an antiport process, where two anions are transported
across the bilayer in opposite directions, or a symport process,
where both an anion and a cation are transported in the same
direction. The chloride efflux shown in Fig. 5 is therefore most
c
1
2
3
4
5
6
7
8
1.38d
0.06d
0.01d
1.42
0.08
0.01
20.5
26.0
29.2
33.8
22.7
26.6
31.2
35.0
10.9 (ꢄ0.5)
11.2 (ꢄ0.2)
9.8 (ꢄ0.3)
e
e
e
—
—
—
—
e
0.0125f
6.3 (ꢄ0.1)
0.68
0.0125f
6.0 (ꢄ0.2)
e
e
e
—
—
—
e
e
e
—
—
—
a
Concentration of transporter (mol% with respect to lipid) needed to
achieve 50% chloride efflux in 270 s from POPC vesicles lled with
b
NaCl and buffered to pH 7.2 or pH 4.0. Retention time of the
c
ꢁ
likely the result of either a pure ‘switch-on’ Clꢁ/NO3 antiport
compounds on a reversed-phase HPLC column. pKa value obtained
by performing anion transport studies at various pH (see main text for
mechanism or the occurrence of a H+/Clꢁ symport process at
lower pH (or a combination of both). To investigate which
mechanism is dominant for the thiosquaramide based recep-
tors, POPC liposomes containing a NaCl solution and sus-
pended in a Na2SO4 solution were prepared at pH 7.2, pH 4.0
and in the presence of a pH gradient (pH 4.0 inside and pH 7.2
d
e
details). Value taken from ref. 22. Receptors are not active enough
f
to allow EC50 or pKa determination. Hill analysis revealed high n-
values (n
> 5), rendering the EC50 values inaccurate, however,
loadings of 0.0125 mol% and higher consistently gave chloride
effluxes greater than 50% in 270 s and EC50 values can thus be
estimated as 0.0125 mol% or lower (lower loadings of transporters
gave inconsistent results – see ESI†).
3622 | Chem. Sci., 2014, 5, 3617–3626
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outside the vesicle). The double negatively charged sulfate antiport processes that occur at all pHs. However, when a pH
anion is considerably more hydrophilic than nitrate gradient is applied across the phospholipid bilayer, a fast
(DGhydr(SO42ꢁ) ¼ ꢁ1080 kJ molꢁ1; DGhydr(NO3ꢁ) ¼ ꢁ300 kJ dissipation of the gradient is observed upon the addition of
molꢁ1 51 and is therefore more challenging to transport across a both oxosquaramides and thiosquaramides 1–8 (Fig. 6), sug-
)
lipid bilayer. It was found that in these circumstances only a gesting that H+/Clꢁ symport (or OHꢁ/Clꢁ antiport) can occur
minimal amount of chloride was transported out of the lipo- under these conditions. Similar HPTS uorescence based
somes at both pH 7.2 and pH 4.0 (see ESI†). Furthermore, even studies were also conducted on vesicles loaded with NaCl and
in the presence of a pH gradient no signicant chloride efflux suspended in Na2SO4 (see ESI†). As with the previous experi-
occurred upon the addition of transporters 1–8 (see ESI†). While ments with external nitrate, only a small increase in internal pH
these results strongly suggest that an antiport mechanism is the was observed when both the intravesicular and extravesicular
dominant process for the chloride transport facilitated by 1–8, it solution were buffered to pH 7.2, while a large increase in
must be noted that a pH gradient of pH 4.0 to pH 7.2 corre- internal pH was seen when a pH gradient was applied. However,
sponds to a gradient in [H+] of ꢃ10ꢁ4 M. This is a very small unlike the nitrate tests, a large increase in internal pH was also
fraction of the nearly 500 mM [Clꢁ] gradient that is applied to observed when both intra- and extravesicular solution were
the system and HCl symport mechanisms are therefore buffered to pH 6.0, hinting that H+/Clꢁ symport (or OHꢁ/Clꢁ
extremely hard to detect using a chloride selective electrode.
antiport) can occur without a pH gradient providing that sulfate
In order to study the possibility of HCl symport in more is the external anion. In conclusion, it appears that the chloride
detail, a series of vesicle-based experiments were conducted transporꢁt ability of the thiosquaramides is mainly the result of a
using the pH-dependent uorescence properties of 8-hydroxy- Clꢁ/NO3 antiport mechanism that can be switched on by
1,3,6-pyrenetrisulfonate (HPTS) (Fig. 6).49,52,53 Initially, POPC lowering the pH, while H+/Clꢁ symport will occur only when a
liposomes were prepared containing NaCl (489 mM) and HPTS favourable [H+] gradient is applied or in the absence of an
(1 mM) buffered to pH 7.2 or pH 6.0, and suspended in a NaNO3 external anion suitable for antiport.
(489 mM) solution buffered to pH 7.2 or pH 6.0. The results
The pH-switchable transport behaviour displayed by the
shown in Fig. 6 for 2 and 6 reveal that in the absence of a pH thiosquaramides renders this class of compound interesting for
gradient the intravesicular pH does not change upon the addi- potential biological activity, as the careful control of ion trans-
tion of thiosquaramide based transporters (results for the other port processes is of utmost importance in a biological setting.
receptors can be found in the ESI†), indicating that the chloridꢁe For example, many of the naturally occurring chloride trans-
effluxes shown in Fig. 5 are mainly the result of Clꢁ/NO3
porting proteins exhibit a gating or switching mechanism where
the ion transport is activated by a specic biological trigger (e.g.
pH, Ca2+, voltage or ligand gating).54–56 Further-more, it has
oen been suggested that HCl symport can result in anti-cancer
activity7–10 and as the internal and external pH of cancer cells is
thought to be different to that of healthy cells,57 pH switchable
chloride transporters could become promising leads for anti-
cancer drugs. In order to be biologically relevant, this pH-switch
should occur around the physiological pH of 7.4 (in the range
pH 6–8). To probe whether the pH-switch of the thiosquar-
amides falls into this pH range, the Clꢁ/NO3 experiments
ꢁ
depicted in Fig. 5 were repeated at a range of pHs (see ESI† for
details on the different pHs and buffers used). The results for 2
and 6 are shown in Fig. 7 and clearly show that the chloride
transport ability of thiosquaramide 6 can be switched on at pH <
7, with the major switch occurring in the region pH 5.5–7.0.
Fig. 7 also conrms that chloride transport by the analogous
oxosquaramide 2 is relatively pH-independent and that thio-
squaramide 6 displays more potent transmembrane anion
Fig. 6 Investigation of HCl transport facilitated by 1–8 using the
fluorescent dye HPTS to estimate the intravesicular pH. For the transport activity than oxosquaramide 2 at pH < 6.0. In addition,
experiments without pH gradient, POPC vesicles were loaded with
489 mM NaCl, 1 mM HPTS, buffered to pH 7.2 with phosphate buffer or
to pH 6.0 with citrate buffer, and suspended in a solution of 489 mM
NaNO3 buffered to pH 7.2 with phosphate buffer or to pH 6.0 with
the exact position of the switch should correspond to the pKa
value of the receptor in the conditions of the anion transport
experiments (but should be more accurately referred to as an
‘apparent pKa’ as it is a combination of the NH pKa and the
citrate buffer. For the experiments with pH gradient, POPC vesicles
were loaded with 489 mM NaCl, 1 mM HPTS, buffered to pH 6.0 with transport ability of the receptor). In order to calculate the
citrate buffer, and suspended in a solution of 489 mM NaNO3 buffered
apparent pKa values it is theoretically possible to plot the
to pH 6.0 with citrate buffer, and at time t ¼ 0 s a NaOH solution was
chloride efflux (%) aer a certain amount of time versus pH and
added to achieve an external pH of 7.0. At time t ¼ 50 s a DMSO
t the data to a sigmoidal function. However, this single-point
solution of the transporter was added (1 mol% with respect to lipid) and
approach can articially raise the value of the calculated pKa,
at time t ¼ 350 s detergent was added. Each line represents the
average of 3 independent trials and DMSO was used as a control.
especially in the case of highly active anion transporters (see
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neutral species and consequently provide a true ON–OFF switch
for anion transport. So far, there have only been two other
reports concerning switching-off anion transport by deproto-
nation, but these involve deprotonation of phenolic OH groups
which have high pKa values and the switch consequently occurs
at pHs that are not physiologically relevant (pH > 7.4).65,66 On the
other hand, the ON–OFF switch in the region pH 6–7 gives the
thiosquaramide based receptors more potential in the biolog-
ical arena and an investigation of the biological activity of thi-
osquaramides is currently ongoing in our laboratories.
Conclusions
In this paper we have reported the rst use of thiosquaramides
as anion receptors and pH-switchable anion transporters.
Proton NMR titrations in DMSO-d6 and CD3CN, as well as single
crystal X-ray crystallography, revealed that thiosquaramides are
able to form 1 : 1 and 2 : 1 complexes with chloride anions. It
was also found that thiosquaramides containing electron with-
drawing substituents are present in their deprotonated forms in
DMSO and water. Vesicle studies using ion-selective electrodes
and pH sensitive uorescent dyes indicated that the trans-
membrane anion transport abilities of the thiosquaramides is
switched OFF at pH > 7 and switched ON at pH < 7. It was also
shown that in the ON mode, the thiosquaramides function as
mobile carriers that can promote chloride efflux mainly via a
chloride/nitrate antiport process, although HCl symport also
occurs in the presence of a pH gradient. This paper provides one
of the few examples of truly controllable and switchable anion
transport by synthetic molecules and this nding renders thi-
osquaramide based receptors interesting targets for developing
future biologically active anion transporters.
Fig. 7 pH dependence of the chloride transport ability of compounds
2 and 6 (1 mol% with respect to lipid). Chloride transport experiments
were conducted as described in Fig. 5 but at various different pHs. The
obtained data was fitted to an asymptotic function and the initial rate
of chloride efflux was calculated (see ESI† for details) and plotted as a
function of pH. Apparent pKa values could subsequently be estimated
by fitting the obtained plot to the following sigmoidal function: y ¼
max + ((min ꢁ max)/(1 + 10(pK ꢁx))).
a
ESI†). A more accurate approach is therefore to use the nor-
malised initial rate of chloride efflux to calculate apparent pKa
values. The values obtained using this method are summarised
in Table 3 and largely agree with the pKa values obtained
through spectrophotometric titrations and DFT calculations
(Table 2). The apparent pKa values of the oxosquaramides are in
the region pH 10–11, while the apparent pKa thiosquaramides 5
and 6 is in the region pH 6–7. The major discrepancy between
the spectrophotometric pKa values and the apparent pKa values
is that a less pronounced effect of the addition of electron Acknowledgements
withdrawing substituents is observed for the apparent (trans-
We thank the EPSRC (EP/J009687/1) and the ARC
port) pKa compared to the spectrophoto-metric pKa. It is
possible that, in the case of apparent pKa values, higher anion
transport activity compensates for the stronger NH acidity of
receptors containing electron with-drawing substituents.
(DP140100227) for funding and the EPSRC for access to the
crystallographic facilities at the University of Southampton. XW
thanks the China Scholarship Council and the University of
Southampton for nancial support. ILK thanks the University of
Southampton for a teaching assistantship. BC acknowledges
the provision of computing time from the NCI. BDS thanks the
NSF (USA). PAG thanks the Royal Society and the Wolfson
Foundation for a Royal Society Wolfson Research Merit Award.
PAG and KAJ thank the European Cooperation in Science and
Technology (COST) action CM1005 “Supramolecular Chemistry
in Water” for support.
In summary, it was found that thiosquaramides 5 and 6 are
able to transport chloride across phospholipid bilayers in a pH-
dependent manner via an acidic ‘switch-on’ antiport mecha-
nism. At high pH (pH > 7), the thiosquaramides are present as
deprotonated, negatively charged species that are unable to
complex and transport anions, while at lower pH (pH < 7) they
are present as neutral species capable of anion binding. This is
a signicantly different mechanism than the behaviour of other
pH-dependent anion transporters or HCl symporters, such as
the prodigiosins.58–64 In these cases the anion transport of a
neutral compound is enhanced by protonation of a basic
functional group to form a positively charged species that can
coordinate and subsequently transport anions. However, the
neutral species is usually still an active transporter and so the
anion transport cannot be fully ‘switched off’ using this
approach. The thiosquaramides on the other hand can alternate
between an inactive negatively charged species and an active
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