Dissecting the chloride-nitrate anion transport assay

Article abstract of DOI:10.1039/c7cc04912a

A systematic study of chloride vs. nitrate selectivity across six anion transporters has revealed a good correlation between the selectivities of their anion binding and membrane transport properties. This work reveals the limitations of the chloride-nitrate exchange assay and shows how new approaches can be used to measure anion uniport.

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Dissecting the chloride–nitrate anion transport
assay†
Cite this:DOI: 10.1039/c7cc04912a
c
a
b
Yufeng Yang,ab Xin Wu,
Nathalie Busschaert,
Hiroyuki Furuta
and
c
Received 25th June 2017,
Accepted 27th July 2017
Philip A. Gale
*
DOI: 10.1039/c7cc04912a
rsc.li/chemcomm
A systematic study of chloride vs. nitrate selectivity across six anion assays that could measure the rate of anion uniport mediated by
transporters has revealed a good correlation between the selectivities anion transporters without the need for an anion exchange process
of their anion binding and membrane transport properties. This work to occur.10 We here make use of two complementary vesicle-based
reveals the limitations of the chloride–nitrate exchange assay and assays to determine anion transport selectivity, in particular
shows how new approaches can be used to measure anion uniport.
ClÀ/NO3À selectivity, of a library of hydrogen bond-based anion
transporters 1–6 that contain increasing numbers of hydrogen
The design of synthetic anion receptors1 that can carry biological bond donors (Fig. 1). By comparing these results with association
anions, most importantly chloride, across phospholipid bilayers constants for anion complexation determined in acetonitrile,
has been an active area of research in supramolecular chemistry.2 we demonstrate for the first time a strong correlation between
These compounds have future therapeutic potential to replace binding selectivity and transport selectivity across a series of
the function of faulty anion channels in genetic diseases,3 or to structurally diverse anion transporters.
disrupt the ionic and pH gradients in cancer cells.4 Efforts have
Compounds 1, 2, 3, 5, and 6 are examples of anion transporters
been made to improve the potency of anion transporters, allowing previously studied by our group.9,10b,11 These compounds represent
them to function at low concentrations as required for therapeutic two distinct design approaches to highly effective anion transporters.
applications. Typically, chloride–nitrate exchange (antiport) assays Compounds 1–3 contain highly acidic NH groups, leading to
have been used to evaluate the anion transport potency of the high anion binding affinity and the ability to disperse the
transporters.5 In this assay, it is assumed that nitrate transport is negative charge of the bound anion. Compounds 5 and 6 are
unlikely to be the rate-limiting process due to the anion’s high without the electron-withdrawing CF3 groups but contain more
lipophilicity, and therefore this assay is assumed to indicate the hydrogen bond donors that are favourable for binding and
chloride transport activity of the transporters.6 However, in anion transport because of multivalency12 and encapsulation.13 Their
binding studies, chloride is almost always more strongly bound to protonated cationic forms do not participate in anion transport
hydrogen bond donor anion receptors than nitrate due to the as demonstrated previously.10b We synthesised a new receptor 4
higher charge density of ClÀ.7 This could result in faster chloridÀe as a dipodal control of the tripodal thiourea 5. The affinitieÀs
transport than nitrate transport. The study of ClÀ vs. NO3
of these receptors towards anions including ClÀ, BrÀ and NO3
selectivity in membrane transport and its correlation with binding in acetonitrile were determined by UV-vis absorption titra-
selectivity is thus of fundamental importance in addressing the tions using tetrabutylammonium (TBA+) salts of the anions
question whether anion transport selectivity is dominated by anion
lipophilicity8 or anion affinity.9 This information is also practically
useful to unravel the potential limitations of the chloride–nitrate
exchange assay. Recently, we have developed membrane transport
a Chemistry, University of Southampton, Southampton, SO17 1BJ, UK
b Department of Chemistry and Biochemistry, Kyushu University, Fukuoka,
819-0395, Japan
c School of Chemistry, The University of Sydney, NSW 2006, Australia.
E-mail: philip.gale@sydney.edu.au
† Electronic supplementary information (ESI) available: Compound synthesis,
anion association constant determination, membrane transport data and other
Fig. 1 Structures of anion transporters 1–6.
experimental details. See DOI: 10.1039/c7cc04912a
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Table 1 Anion binding and transport data for compounds 1–6
Anion transport in lipid bilayers
ISE exchange assayb HPTS assaye
Anion binding in acetonitrilea
Osmotic assayh
ISE
À
HPTS
HPTS
Os
Ka(ClÀ)/
Ka(NO3À)/ Sb(ClÀ/ EC (ClÀ–NO3
EC
(ClÀ)/ EC
(NO3À)/ SHt PTÀS(ClÀ/ EC5O0s(ClÀ)/ EC (NO3À)/ StOs(ClÀ/
50
50
50
50
À
À
Comp. MÀ1
MÀ1
NO3
)
exchange)/mol%
mol%
mol%
NO3
)
mol%
mol%
NO3
)
1
2
3
4
5
6
8.2 Â 105 2.5 Â 103
1.8 Â 104 1.1 Â 103
2.3 Â 104 6.0 Â 102
3.0 Â 104 2.1 Â 102
330
17
38
0.060c
0.30c
0.16c
45
0.0089 f
0.043 f
0.013 f
0.49g
0.0073 f
0.0052 f
0.0014 f
0.045g
0.82
0.12
0.11
0.091
8.1
0.093
0.27
0.12
3.3
0.055
0.037
0.078
0.069
0.031
0.92
0.56
0.18
0.85
0.26
0.26
0.28
10
140
1.7 Â 106 2.7 Â 102 6200
8.3 Â 105 4.9 Â 102 1700
0.31d
0.11
0.0044g
0.0034g
0.036g
0.010g
3.0
4.9
a
Association constants determined by UV-vis titration in acetonitrile at 298 K, usÀing tetrabutylammonium anion salts. Errors were found to be
o10%. ClÀ/NO3 binding selectivity Sb(ClÀ/NO3À) = Ka(ClÀ)/Ka(NO3À). ClÀ–NO3 exchange assay schematically shown in Fig. 2a, using POPC
À
b
LUVs with a mean diameter of B200 nm. EC50 calculated at 270 s. Reported in ref. 9. Reported in ref. 11a. HÀ+–ClÀ cotransport assay
c
d
e
schematically shown in Fig. 2b, using POPC LUVs with a mean diameter of B200 nm. EC50 calculated at 200 s. ClÀ/NO3 transport selectivity in
this assay: SHt PTS(ClÀ/NO3À) = EC
(NO3À)/EC
(ClÀ). Determined in the absence of proton channel gramicidin D. The addition of gramicidin
HPTS
HPTS
f
50
50
D did not affect the overall transport rate because these compounds are by themselves good H+/OHÀ transporters. See ref. 10b. Determined in the
g
presence of gramicidin D to remove the need of anion transporters to facilitate H+/OHÀ transport. Without gramicidin, H+/OHÀ transport
facilitated by these compounds is slow and would rate-limit the overall transport process. See ref. 10b. K+–ÀClÀ cotransport assay schematically
h
shown in Fig. 2c, using POPC LUVs with a mean diameter of B400 nm. EC50 calculated at 600 s. ClÀ/NO3 transport selectivity in this assay:
SOt s(ClÀ/NO3À) = EC (NO3À)/EC (ClÀ).
Os
50
Os
50
(see Table 1 for ClÀ and NO3 binding data, and Table S1 in electrode (ISE). The activity of a transporter was quantified by an
ESI† for BrÀ binding data). For all compounds, the anion effective concentration of the transporter to reach 50% of ion
binding selectivity trend was found to follow the anion charge transport (EC50 value) at 270 s. In this assay, the simple squar-
density (ClÀ 4 BrÀ 4 NO3À). Despite the same anion binding amide 1 was the champion while the two tripodal thioureas 5
À
À
selectivity sequence, the actual extent of ClÀ/NO3 binding and 6 significantly fell behind (Table 1).
selectivity, i.e. the Sb(ClÀ/NO3À) value, did show significant varia-
As the above-mentioned exchange assay provides no informa-
tion among different scaffolds. The two tripodal compounds 5 and tion on ClÀ/NO3À selectivity, we conducted two additional assays
6 have very high (41500-fold) binding selectivities for ClÀ over to directly measure anion selectivity. In the first assay (an HPTS
NO3À, whereas the other ureas and thioureas have a selectivity assay, Fig. 2b), POPC LUVs with a mean diameter of 200 nm were
value of less than 150. A similar trend was found for the BrÀ/NO3
loaded with and suspended in a solution of the N-methyl-D-
À
binding selectivity (Table S1, ESI†). Compared with the dipodal glucamine salt of the anion of interest (NMDG-X, 100 mM,
thiourea 4, the additional thiourea arm in 5 dramatically enhances XÀ = ClÀ or NO3À) buffered at pH 7.0 with HEPES. The external
the binding of ClÀ and BrÀ but does not improve NO3À binding as pH was brought to B8 by addition of a base pulse (5 mM of
much. These binding results give a hint that the tripodal com- N-methyl-D-glucamine), and then the transporter-induced
pounds could behave differently in anion transport selectivity dissipation of the pH gradient across vesicle membranes was
compared with other compounds.
measured using an intravesicular fluorescent pH indicator
All compounds have been initially subject to a traditional 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS). The overall process
ClÀ–NO3À exchange assay (Fig. 2a). Briefly, 1-palmitoyl-2-oleoyl- leading to pH gradient dissipation is H+–XÀ symport or OHÀ–XÀ
sn-glycero-3-phosphocholine (POPC) large unilamellar vesicles antiport, which may be rate-limited by H+ or OHÀ transport.
(LUVs) were loaded with NaCl (490 mM) buffered at pH 7.2, and However the need for an anion transporter to facilitate H+ or
suspended in NaNO3 (490 mM) buffered at pH 7.2. The anion OHÀ transport can be eliminated by using the proton channel
transporter was added to the vesicle suspension as a DMSO gramicidin,10b allowing this assay to reveal the ability of an anion
solution. Chloride efflux (due to ClÀ–NO3 exchange induced transporter to facilitate XÀ uniport (Fig. 2b).14 In the second
À
by the anion transporters) was monitored via appearance of ClÀ assay (osmotic assay), larger POPC LUVs (mean diameter
in the external solution, measured by a chloride ion-selective 400 nm) were loaded with a buffered KX (300 mM) solution
Fig. 2 Schematic representation of three membrane transport assays used. See ESI,† for Experimental details.
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and suspended in a buffered K+ gluconate (300 mM) solution. ionophore) but also on the ability of the complex to translocate
A combination of valinomycin (for transporting K+) and an anion across lipid bilayers.16
transporter give overall K+–XÀ cotransport (Fig. 2c), leading to
It is interesting to compare the activity determined in the three
water efflux to balance the osmotic difference and the resultant membrane transport assays. The data from HPTS and osmotic
shrinkage of vesicles, which can be monitored by following the assays agreed well with each other, with the EC50 value in the
light scattering of vesicles using a fluorometer.15 This provides a osmotic assay consistently being about an order of magnitude higher
method of quantifying the rate of anion uniport facilitated by than that in the HPTS assay for the same compound transporting
anion transporters.14 For all compounds, we determined the EC50 the same anion.17 Both assays have revealed the ClÀ transport
value at 200 s in the HPTS assÀay and at 600 s in the osmotic assay. activity in the sequence of 6 4 5 4 1 4 3 4 2 4 4, whereas
À
In both assays, the ClÀ/NO3 transport selectivity St(ClÀ/NO3
)
NO3À transport activity in the sequence of 3 4 2 4 1 4 6 4 5 4 4.
was quantified by the ratio between EC50 of NO3À transport and The ClÀ–NO3 exchange assay showed the activity sequence
À
EC50 of ClÀ transport, where an St(ClÀ/NO3À) value 4 1 indicates of 1 4 6 4 3 4 2 E 5 4 4, which reflected the activity of
ClÀ selectivity (Table 1).
transporting the ‘‘slower’’ anion (ClÀ in the cases of 1–4, and
The results from both HPTS and osmotic assays demon- NO3À in the cases of 5 and 6). By converting the EC50 values to
strate that NO3 transport proceeds faster than ClÀ transport normalised activities, a reasonable agreement between the
À
for monopodal transporters 1–3 and dipodal transporter 4. This three transport assays can be observed (Table S2 in ESI†). It is
À
À
is consistent with the higher lipoÀphilicity of NO3 than ClÀ evident that the ClÀ–NO3 exchange assay gives a fair assess-
while contrary to the ClÀ 4 NO3 binding selectivity of the ment of ClÀ transport activity of compounds 1–4 because the
compounds in acetonitrile. The results suggest that in these overall process is rate-limited by ClÀ transport in these cases.
cases transport selectivity is governed by the ease of anion However, the ClÀ transport activity of 5 and 6 is clearly under-
dehydration. In contrast to 1–4, tripodal compounds 5 and 6 estimated in the exchange assay. Both compouÀnds are in fact
transported ClÀ faster than NO3À, with 5 showing an 8-fold better ClÀ transporters than the best ClÀ–NO3 exchanger 1.
ClÀ/NO3À selectivity in the HPTS assay and a 10-fold selectivity Compound 1 turned out to be neither the best ClÀ transporter
À
in the osmotic assay. Therefore, 5 and 6 are unusual examples nor the best NO3 transporter (Fig. 4).
À
of anion transporters that can overrule the normally observed
For a better understanding of the ClÀ 4 NO3 selectivity
À
NO3 4 ClÀ lipophilicity bias. This cannot be interpreted as a of the tripodal scaffold, we performed PM6-optimisation of
À
general ‘‘anti-Hofmeister’’ selectivity as 5 and 6 transported the the structures of the ClÀ and NO3 complexes of 5. Fig. 5 (see
more lipophilic anion BrÀ faster than ClÀ (Fig. S50 in ESI†). It is Fig. S53 in ESI† for ball-and-stick models) shows that the
interesting to note that the transport selectivity St(ClÀ/NO3À) in spherical anion ClÀ fits well and is well-encapsulated inside the
general shows a good correlation with the binding selectivity cavity of the tripodal scaffold, whereas binding of the planar
Sb(ClÀ/NO3À) (Fig. 3 and Fig. S52 in ESI†). Both selectivities anion NO3 forced the scaffold to adopt a more opÀen conforma-
À
follow the trend of 5 4 6 4 1 4 2–4. Chloride 4 nitrate selectivity tion leaving a significant part of the bound NO3 exposed to
is only observed for 5 and 6 that have the largest binding preference the solvent. Encapsulation of the anion is crucial for effective
for ClÀ over NO3À among the library. Squaramide 1, despite being a membrane transport as demonstrated by previous work by
NO3À-selective transporter, has a significantly larger St(ClÀ/NO3
)
Davis13 and us,10b and therefore the poor encapsulation of
À
value compared with 2–4, consistent with 1 being the third NO3À could contributes to the ClÀ 4 NO3À transport selectivity
most ClÀ 4 NO3À selective binder. The correlation, however, is of the tripodal compounds.
not perfect with compounds 2–4. This may be rationalised by
the idea that the rate of ionophore-catalysed ion transport
depends not only on the ion binding affinity (which determines
the amount of ion–ionophore complex with respect to the free
Fig. 4 Histogram showing activities of 1–6 in facilitating uniport of ClÀ
(green bars) and NO3À (blue bars). Activities are expressed as reciprocal of
EC50 values (at 200 s) determined in the HPTS assay. The red dashed line
Fig. 3 Correlation between transport selectivity log SHt PTS(ClÀ/NO3À) and
binding selectivity log Sb(ClÀ/NO3À). Compound numbers are shown next
to the data points.
shows that compound 1 has the highest activity in facilitating the slower
anion uniport process, which explains its highest activity in the ClÀ–NO3
À
exchange assay.
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Fig. 5 PM6-optimised structures of ClÀ (a) and NO3 (b) complexes of 5
shown in space-filling models.
À
In summary, we have examined the chloride vs. nitrate
selectivity of representative hydrogen bond-based anion transpor-
ters in both binding and transport. A strong correlation between
transport selectivity and binding selectivity has been found. Only
À
the tripodal transporters 5 and 6 that have a large ClÀ/NO3
binding preference can overcome the Hofmeister lipophilicity bias
to transport ClÀ selectively over NO3À. We have compared the
anion transport potency determined from different assays, showing
À
that the commonly used ClÀ–NO3 exchange assay is valid in
most cases for evaluating ClÀ transport activity but sometimes
gives an underestimation in the case of a poor NO3À transporter.
Importantly, we have also demonstrated that compound 5 func-
tions as a highly ClÀ-selective transporter showing a B10-fold
9 N. Busschaert, I. L. Kirby, S. Young, S. J. Coles, P. N. Horton,
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À
ClÀ over NO3 selectivity in membrane transport, because of its
complementary fit for ClÀ. We believe that the structure-selectivity
and binding-transport relationships demonstrated here, and
the different assays provided in this work will provide valuable
tools for future development of highly potent and selective
anion transporters for biomedical applications.
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activity in) mediating anion uniport. Examples include prodigiosin
and an analogue of 5 with cyano substituents. In those cases, the
HPTS assay showed no gramicidin-induced enhancement of trans-
port rate and the valinomycin-coupled assay showed little or no
activity. See ref. 10b.
PAG thanks the University of Sydney and the ARC (DP170100118)
for funding and the JSPS for an Invitation Fellowship to the University
of Kyushu. We thank the Kyushu University Leading Program for
‘‘Molecular System for Devices’’ from MEXT Japan (Y. Yang).
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Chem. Commun.
This journal is ©The Royal Society of Chemistry 2017