Structure-activity relationships in cholapod anion carriers: Enhanced transmembrane chloride transport through substituent tuning

Article abstract of DOI:10.1002/chem.200801163

Chloride transport by a series of steroid-based "cholapod" receptors/carriers was studied in vesicles. The principal method involved preincorporation of the cholapods in the vesicle membranes, and the use of lucigenin fluorescence quenching to detect inwa

Full text of DOI:10.1002/chem.200801163

FULL PAPER
DOI: 10.1002/chem.200801163
Structure–Activity Relationships in Cholapod Anion Carriers: Enhanced
Transmembrane Chloride Transport through Substituent Tuning
Beth A. McNally,[b] Atanas V. Koulov,[b] Timothy N. Lambert,[b] Bradley D. Smith,*[b]
Jean-Baptiste Joos,[a] Adam L. Sisson,[a] John P. Clare,[a] Valentina Sgarlata,[a]
Luke W. Judd,[a] Germinal Magro,[a] and Anthony P. Davis*[a]
Abstract: Chloride transport by
a
oidal 3-position yielded irregular ef-
fects. Among the new steroids investi-
gated the bis-p-nitrophenylthiourea 3
showed unprecedented activity, giving
measurable transport through mem-
branes with a transporter/lipid ratio of
1:250000 (an average of <2 transporter
molecules per vesicle). Increasing
transporter lipophilicity had no effect,
and positively charged steroids had low
activity. The p-nitrophenyl monourea
25 showed modest but significant activ-
series of steroid-based “cholapod” re-
ceptors/carriers was studied in vesicles.
The principal method involved prein-
corporation of the cholapods in the
vesicle membranes, and the use of luci-
genin fluorescence quenching to detect
inward-transported ClÀ. The results
showed a partial correlation between
anion affinity and transport activity, in
that changes at the steroidal 7 and 12
positions affected both properties in
concert. However, changes at the ster-
ity. Measurements using
a second
method, requiring the addition of
transporters to preformed vesicle sus-
pensions, implied that transporter de-
livery was problematic in some cases.
A series of measurements employing
membranes of different thicknesses
provided further evidence that the
cholapods act as mobile anion carriers.
Keywords: anions · biomembranes ·
receptors · steroids · supramolec-
ular chemistry
Introduction
tion of ion transport underlies the biological activity of
many natural products.
The selective transport of ions through bilayer membranes
is a key process in biology.[1] Nature uses a combination of
gated channels and ATP-driven transporters to establish and
manage ion concentrations in cellular compartments. Dis-
ruption of this system can have major effects. For example,
the malfunction of ion channels leads to a range of medical
problems (channelopathies),[2] while the blocking or promo-
One such family of natural products are the ionophore an-
tibiotics (valinomycin, monensin, gramicidin A etc.).[3] These
compounds provide passages for ions across cell membranes,
either by self-assembling to form channels or by binding and
carrying the ions. Although both cation and anion transport
are important to biology, the naturally-derived ionophores
are strongly biased towards cations. The above-mentioned
are all cationophores, as are nearly all other ion-transporting
secondary metabolites. There are very few anion-transport-
ing natural products,[4] and none which mirror the action
and availability of the major cationophores.[5] Anionophores
would be useful tools for biological research and could have
therapeutic applications. For example a number of channe-
lopathies, including the well-known genetic disorder, cystic
fibrosis, result from defective anion channels.[2,6] It is realis-
tic to hope that anionophores could compensate for the
missing activity, in “channel replacement therapies”.[7]
These possibilities have stimulated recent interest in the
design and study of synthetic anion transporters.[8] The work
has encompassed self-assembling channels,[9] monomeric
channels[10] and receptors acting as mobile carriers.[11]
Among the latter, we have described the “cholapods” 1 and
[a] Dr. J.-B. Joos, A. L. Sisson, J. P. Clare, Dr. V. Sgarlata, L. W. Judd,
Dr. G. Magro, Prof. Dr. A. P. Davis
School of Chemistry, University of Bristol
Cantockꢀs Close, Bristol BS8 1TS (UK)
Fax : (+44)117-9298611
E-mail: Anthony.Davis@bristol.ac.uk
[b] B. A. McNally, A. V. Koulov, T. N. Lambert, Prof. Dr. B. D. Smith
Department of Chemistry and Biochemistry
and Walther Cancer Research Center
University of Notre Dame, Notre Dame IN 46556 (USA)
Fax : (+1)5746316652
E-mail: smith.115@nd.edu
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.200801163.
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2 (see below).[11a,b] These steroid-based compounds possess
preorganised anion-binding sites capable of very high affini-
ties, allied to lipophilic frameworks which promote solubility
ents included OAc, which makes essentially no contribution
to binding,[15] and a number of H-bond donor units which
again promote binding according to their acidities. Addition-
+
ally, receptors 12 and 13 incorporated cationic NMe3
groups, to test the possibility of transport via electroneutral
complexes. Finally cholapods 14 and 15 were employed as
eicosyl ester analogues of 1e and 2 respectively, to explore
the effect of molecular size and lipophilicity.
The syntheses of 1d,e,[11a] 4,[14] 6,[16] 7,[16] 12,[17] 13[17] and
15[18] have been described previously. The remaining com-
pounds were prepared from intermediates 16,[19] 17a,[19] and
17b[20] through straightforward deprotections and derivatisa-
tions.[21]
in apolar media (such as membrane interiors).[12] They can
transport chloride and nitrate ions through vesicle mem-
branes at very low loadings (down to cholapod/lipid ratios
of 1:250000), and are also active in cultured epithelial cells.
The mobile carrier mechanism was supported by the de-
pendence of rates on transporter concentration and mem-
brane fluidity.[11a]
Measurement of transport rates and binding constants—
methodology: Chloride transport by the cholapods was as-
sayed using large unilamellar vesicles (LUVs, 200 nm aver-
age diameter), with back-transport of nitrate to maintain
electroneutrality. The vesicles were prepared from a mixture
Practical applications of these transporters, especially in
medicine, will require high activities so that useful ion fluxes
can be obtained at low doses. Intuitively one might expect a
correlation between anion affinities and transport activities,
and early work suggested that this was the case. Affinities
for Et4N+ClÀ in chloroform (Ka, ClÀ, CHCl3) rose from 3.4”
106 mÀ1 for 1a, via 5.2”108 mÀ1 for 1e, to 1011 mÀ1 for 2, and
transport activities increased accordingly. Compound 2 is
among the strongest of the cholapod anion receptors,[13,14] so
one might conclude that further advances would be difficult.
However, transporters 1 and 2 represent a small fraction of
cholapod structural space, and we were interested to explore
a wider range of molecules. We now describe a study of 16
cholapods which gives a clearer picture of the relationships
between structure, anion affinity and chloride transport ac-
tivity. Our results show that the correlation between affinity
and activity is imperfect, and reveal a new champion among
cholapod anion transporters. We also provide further sup-
port for the mobile carrier mechanism, and show that even
simple ureas can be moderately effective as chloride anion
transporters.
of
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine
(POPC) and cholesterol (7:3), chosen to mimic the outer
leaflet of animal cell plasma
membranes. Two methods were
used to detect the passage of
chloride ions across the mem-
branes. The first involved the
use of lucigenin (18), a chlo-
ride-sensitive fluorescent dye,
to detect inward-flowing chlo-
ride ions.[11b] Briefly, the chola-
pod was mixed with lipid at the
appropriate molar ratio (1:250, 1:2500, 1:25000 or 1:250000).
The mixture was solvated with an aqueous solution of
NaNO3 (225 mm) containing lucigenin (1 mm) and the result-
ing suspension was used to prepare vesicles via freeze-thaw
cycles and extrusion. The vesicle suspension was separated
from external lucigenin by size exclusion chromatography,
diluted with aqueous NaNO3 (225 mm), and placed in the
cuvette of a fluorescence spectrometer. Aqueous NaCl was
added (25 mm final concentration), and chloride influx was
followed by the decrease in fluorescence intensity.
The second method employed a chloride-selective ion-ex-
change electrode (ISE) to detect chloride ions emerging
from the vesicles.[11a] In this case the vesicles were formed
from the POPC/cholesterol mixture without the addition of
transporter. The lipid mixture was solvated with aqueous
NaCl (500 mm), then external NaCl was replaced with
NaNO3 by dialysis. The ISE was placed in the suspension
and the transporter was added as a solution in a small
Results and Discussion
Cholapod structure and synthesis: This study encompassed
the previously-reported transporters 1d, 1e, and 2 along
with the series of cholapods 3–15 shown in Figure 1. All pos-
sess the 7a,12a-bis(thio)ureidyl motif, which tends to afford
G
high anion affinities.[12,14] The choice of 7a,12a-substituent
varied between phenylureidyl, p-nitrophenylureidyl and p-
nitrophenylthioureidyl (for numbering, see 1). The NH acid-
ities of these units increase in the order listed, with corre-
sponding enhancements of anion affinities. The 3a-substitu-
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Anion Carriers
FULL PAPER
Figure 1. Cholapod anion receptors assayed for chloride transport activity in this work.
amount of organic solvent. Chloride efflux was followed
through the increase in signal from the electrode.
In addition to transport rates, the study also required a
comparison of anion affinities. Binding constants are very
sensitive to medium, and the relevant medium in this case is
the apolar membrane interior. It was therefore appropriate
to use an apolar, water-immiscible solvent such as chloro-
form. Cholapod anion affinities in water-saturated chloro-
form were measured using our previously-reported extrac-
tion-based method, with Et4N+ as counterion.[14] The tech-
nique involves the equilibration of cholapods dissolved in
chloroform with salts Et4N+XÀ dissolved in water, followed
The methods differ in two key respects. Firstly, the lucige-
nin-based assay allows preincorporation of the transporter
in the vesicle membranes, while the ISE method requires
that transporter be added from outside to start the experi-
ment. This results from the need to place the chloride inside
the vesicles in the latter case; clearly these vesicles cannot
be prepared from membranes containing transporters. The
ISE-based assay is therefore affected by two factors, i) in-
trinsic transport ability and ii) the ability of the transporter
to partition into the vesicles from an aqueous dispersion.
Secondly the ISE-based assay requires quite high concentra-
tions of chloride (ꢀ0.5m) in the vesicles to ensure an ade-
quate signal. In contrast, the lucigenin method works well
for the lower chloride concentrations typically found in bio-
logical systems.
1
by H NMR analysis to determine the amount of extracted
salt. Binding constants Ka (XÀ, CHCl3) can be calculated
from the extraction constant Ke, and the distribution con-
stant Kd for the salt between chloroform and water in the
absence of receptor (Ka =Ke/Kd). Depending on the anion,
Kd may be determined by direct measurement, or by calibra-
tion using a receptor which can be studied by both extrac-
tion and NMR titration. The method is especially useful for
powerful receptors, such as the cholapods, because of its
high dynamic range; unlike most titration methods, it does
not suffer from an upper limit for measurable Ka values.
Of the two methods, the lucigenin-based assay is thus sim-
pler to interpret (measuring only intrinsic transport ability)
and is also more relevant to biological conditions. It there-
fore served as the mainstay of the present study, while the
ISE method was used to provide supporting and comple-
mentary information.
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A. P. Davis, B. D. Smith et al.
Table 1. Transport activities (lucigenin method) and anion affinities for
electroneutral methyl ester cholapods. Transport rates kobs are for chlo-
Transport by cholapods pre-incorporated in vesicle mem-
branes (lucigenin method): The lucigenin-based assay was
applied to all the cholapods under study at a transporter/
lipid mole ratio of 1:250,[22] and also to the more effective
systems at lower loadings (transporter/lipid 1:2500, 1:25000
and 1:250000). Rates were quantified by fitting the fluores-
cence output to a first-order decay and thus determining an
observed first-order rate constant kobs (sÀ1). Representative
decay curves are shown in Figure 2. The rates were uncor-
ride influx into vesicles with preincorporated cholapods at cholapod/lipid
1:250.[a]
[b]
Cholapod
kobs (1:250) [sÀ1
]
Ka to Et4N+XÀ in CHCl3 [mÀ1
]
X=Cl
X=NO3
3
4
2
1e
5
6
1d
7
8
9
0.82
0.43
0.13
0.19
0.083
0.040
0.017
0.012
0.0087
0.0036
0.0012
0.0011
2.0”109
1.2”1010
1.1”1011[c]
5.2”108[d]
1.5”109
2.8”108
1.5”107[d]
1.8”107
1.0”109
6.3”107
1.5”108
5.9”107
2.5”108
3.2”109
8.5”109
1.7”108[d]
6.6”108
1.1”108
1.0”107[d]
8.5”106
3.0”108
2.1”107
6.1”107
3.7”107
10
11
[a] Lipids were composed of POPC/cholesterol (7:3). Transport rates are
the average of three individual experiments with S.D. Æ 5.0%. [b] Mea-
sured by extraction (see text). Errors are estimated at Æ 15%, assuming
a 1:1 binding model. [c] Ref. [11b]. [d] Ref. [11a].
Table 2. Chloride transport rates induced by preincorporated cholapods
at lower cholapod/lipid mole ratios.[a]
Cholapod
kobs [sÀ1] for cholapod/lipid 1:x
1:2500
1:25000
1:250000
3
4
2
1e
5
0.34
0.039
0.018
0.012
0.0030
0.0014
0.0016
0.0042
0.0018
0.0012
0.081
0.074
0.022
0.028
0.014
6
[a] Procedures and materials as for Table 1.
Figure 2. Chloride transport into vesicles (POPC/cholesterol 7:3) induced
by preincoporated cholapods, as indicated by the decay in lucigenin fluo-
rescence. a) Cholapod 1e at transporter/lipid 1:250, 1:2500 and 1:25000.
b) Cholapod 3 at transporter/lipid 1:250, 1:2500, 1:25000 and 1:250000.
rected for background influx of chloride, which according to
control experiments occurred at ca. 0.0007 sÀ1 (t = =1430 s).
1
2
Of the 16 cholapods studied, 12 were electroneutral
methyl esters (1d, 1e, 2 and 3–11). The kobs values for these
compounds at transporter/lipid=1:250 are given in Table 1,
where they are related to the binding constants to Et4N+ClÀ
Figure 3. Transport rates kobs from Tables 1 and 2, shown on a logarithmic
scale and colour coded according to transporter/lipid ratio.
À
and Et4N+NO3 in chloroform. Transport rates for the lower
cholapod concentrations are listed in Table 2, and the full
set of kobs is summarised as a bar chart in Figure 3. Results
at the different loadings were mostly, though not fully, self-
consistent. The order of presentation in Table 1 and Table 2,
and Figure 3, represents a consensus activity scale, taking ac-
count of all transporter concentrations.
The first point to note is that activities rise to very high
levels. The most powerful transporter is the 3-acetoxy-7,12-
bis-p-nitrophenylthiourea 3. As shown in Figure 2b, this
compound promotes chloride equilibration in a few seconds,
even at 3/lipid 1:2500. At 3/lipid 1:25000, the half-life is 26
seconds, while at 1:250000 the effect is still measurable. In
this last case, the transporter is operating at the level of a
few molecules per vesicle. A rough calculation[21] suggests
that a 200 nm diameter vesicle should be composed of
ꢀ400000 lipid molecules, so that the average loading at
1:250000 is < 2 cholapods per vesicle.
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Turning to the relationship between structures and activi-
ties, it seems that variation of the urea groups in positions 7
and 12 has predictable, concordant effects on both anion af-
finities and transport effectiveness. For the sequence
(1d;1e;3), mutation from phenylurea to p-nitrophenylurea
to p-nitrophenylthiourea yielded the expected increases in
binding constants and corresponding increases in transport
rates. The same correlation was observed for the pairings
(6;4), (8;2) and (9;5). In contrast, the effect of the substitu-
ent in position 3 is more difficult to understand. From the
series of bis-phenylureas 6, 1d, and 7–11, one concludes that
the 3-substituents promote transport in the order shown in
Figure 4 (top row). However, their effects on anion affinity
are quite different (Figure 4, bottom row). Thus 3a-acetoxy
(as present in 3) is a good substituent for transport but does
not promote binding, 3a-trifluoroacetamido is good for both
functions, and 3a-p-nitrophenylsulfonamido is the most ef-
fective for binding but only moderate for transport. The re-
sults from 7a,12a-bis-p-nitrophenylureas 4, 1e and 5, and
thioureas 3 and 2, are also consistent with Figure 4.
Figure 5. Chloride transport into vesicles (POPC/cholesterol 7:3) by pre-
incorporated eicosyl ester cholapod 14 (c) compared to methyl ester
1e (a). The lucigenin method was used. Results are shown for trans-
porter/lipid=1:250, 1:2500 and 1:25000. Similar results were obtained in
a comparison between eicosyl ester 15 and methyl ester 2.
aqueous interfaces; b) variations in cholapod–anion complex
mobility within the membrane; c) different levels and/or ef-
fects of cholapod aggregation within the membrane;[23] or
d) variations in binding selectivity for the anions involved in
the transport process (chloride and nitrate), and also for the
anionic centres in the membrane phospholipids.[24] Although
further work will be required to clarify the picture, the
trends in Figure 4 should serve as empirical rules for the
design of high performance cholapods.
Finally, the two cationic cholapods 12 and 13 were found
to have little or no transport activity. Again, this could be
due in principle to the cholapods partitioning into the aque-
ous phase. However, previous work has shown that 12 can
mediate the translocation of phosphatidylserine head groups
in vesicles, and that the corresponding eicosyl ester is equal-
ly active.[17] By the argument used previously, this strongly
suggests that 12 and (presumably) 13 locate within the mem-
brane. The most likely explanation for their low chloride
transport activity is the formation of extremely stable elec-
troneutral complexes[25] which only slowly release anions
into the aqueous phase.
Figure 4. Relative effects of cholapod 3a substituents on chloride trans-
port (top row) and anion binding in chloroform (bottom row).
This irregular correlation between affinities and transport
rates cannot yet be fully explained. Two of the simpler hy-
potheses seem unlikely. Firstly, an increase in receptor
strength beyond a certain point can be counterproductive
for transport, because high affinities inhibit anion release.
However, in this case the transport rates should pass
through a maximum as affinities are raised, and no such re-
lationship is observed for the cholapods. Secondly, the re-
sults could be affected by differential partitioning of the
cholapods between membrane and aqueous phase. Howev-
er, experiments employing eicosyl esters 14 and 15 demon-
strated that increasing hydrophobicity made very little dif-
ference to transport activity. Results for 14 are shown in
Figure 5, superimposed upon the almost identical data ob-
tained for methyl ester analogue 1e. Such similarities are
only likely if both methyl and eicosyl esters are fully concen-
trated in the membrane. If this is true for both 14/1e and
15/2, it is probably also true for the remaining cholapods.
Other factors affecting transport activities could be; a)
variations in the kinetics of anion binding at the membrane/
Transport by externally added cholapods (ISE method): A
majority of the cholapods were also studied using the ISE-
based method, in which the transporter was added external-
ly to preformed vesicles. The results are gathered in Table 3.
In this case, initial rates were determined from the slope of
the ISE output, expressed as % of total signal variance per
second. The relative activities are significantly different
from those obtained by the lucigenin method. For example,
while 4 and 1e retain their positions near the top of the
table, 2 and 3 are among the least effective. The major
source of difference is presumed to be the mode of trans-
porter addition. While some cholapods disperse readily in
the aqueous medium and rapidly partition into the mem-
branes, others appear to form aggregates or precipitates
which are not available to the vesicles. Biological applica-
tions will require delivery to cell membranes, so the data
highlight an issue which should be addressed in future work.
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A. P. Davis, B. D. Smith et al.
In the mean time cholapods 1e, 6 and especially 4, provide
good levels of activity with both modes of addition.
Table 3. Transport activities for externally added cholapods (ISE
method).[a]
Cholapod
Initial transport
Cholapod
Initial transport
rate [% sÀ1
]
rate [% sÀ1
]
4
6
1e
1d
8
0.61
0.54
0.52
0.085
0.084
0.059
10
11
9
13
2
0.033
0.026
0.017
0.010
0.010
0.008
5
3
[a] Cholapod/lipid mole ratio 1:250. Lipids were composed of POPC/cho-
lesterol 7:3. Rates are the average of three individual experiments with
S.D. Æ 5.0%.
Effect of bilayer thickness on transport rate—Further evi-
dence for the carrier mechanism: Mechanistic understanding
is critical for the optimisation of cholapod transport activi-
ties. A key question is that of mobile carrier vs. stationary
channel. Thus far, two lines of evidence have suggested that
the cholapods act as carriers.[11a] Firstly, concentration–activi-
ty studies have shown no sign of cooperative behaviour.
More than one cholapod would be required to form a sta-
tionary channel and, unless self-association is exceptionally
strong, this should lead to a second- or higher-order concen-
tration dependence. In fact, the concentration dependence
for 1d was found to be less than first-order.[11a] Secondly,
transport rates were sensitive to the fluidity of the mem-
brane. In dipalmitoylphosphatidylcholine (DPPC) vesicles,
which show a gel/liquid crystalline phase transition at 418C,
the cholapods were inactive in the less fluid gel phase. How-
ever they transported chloride effectively above the transi-
tion temperature, implying that mobility within the mem-
brane was required for activity.[11a]
Given the importance of this issue, we have sought further
confirmation that the carrier mechanism is indeed correct.
A useful probe is the dependence of transport rates on
membrane thickness. Research by Läuger and co-workers
on the cation carrier valinomycin showed a simple correla-
tion between transport rates and membrane hydrocarbon
chain lengths.[26] For a stationary channel, this type of rela-
tionship is not expected. Rates should be similar for a range
of membrane thicknesses, but should fall off dramatically
once the channel can no longer span the bilayer.[27] We
therefore tested cholapods for chloride transport in vesicles
formed from the six phosphatidylcholines (PCs) 19–24 (acyl
chain lengths C14–C24). Unsaturated lipids were employed to
maintain the fluid phase. One set of experiments employed
the ISE method with cholapod 6, and membranes formed
from pure PC and also from PC/cholesterol 7:3. A second
series employed the lucigenin method with cholapod 1e and
the PC/cholesterol vesicles.
Figure 6. Chloride transport by cholapod 1e into vesicles formed from
lipids 19–24, assayed by the lucigenin method. a) Fluorescence decay
curves. Traces are labelled according to the number of carbons in the
lipid acyl chains. b) Dependence of kobs (logarithmic scale) on lipid acyl
chain length.
creased. The scale of the change was roughly similar to that
for valinomycin. Moving from PC 20 (C16 acyl) to PC 23
(C22 acyl), kobs decreased by a factor of 16, while the corre-
sponding figure for valinomycin was 10.[26,28] The results
from the ISE experiments were essentially similar to those
obtained by the lucigenin method. The presence of choles-
terol in the membranes slowed transport by a small amount
in most cases, but did not affect the general trend. These ex-
periments provide a third argument for favouring the carri-
er, as opposed to channel, mechanism for anion transport by
cholapods.
Results from the lucigenin experiments are shown in
Figure 6. As expected for the carrier mechanism, the trans-
port rates decreased steadily as the lipid chain lengths in-
Chloride transport by a monourea—A remarkably simple
anionophore: All the cholapod transporters discussed in this
paper feature two axially directed urea groups, positioned
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Anion Carriers
FULL PAPER
such that they can cooperate in anion binding. As a final
step in our structure–activity studies, we were interested to
determine how a single urea group might perform. p-Nitro-
phenylurea 25 was chosen as a lipophilic monourea with rel-
atively powerful H-bond donors. The extraction method was
used to determine an apparent Ka of ꢀ8000mÀ1 for 25 +
Et4N+ClÀ in water-saturated chloroform.[29] At relatively
high concentrations, 25 was found to be a fairly effective
chloride transporter. At a trans-
1:250000 (less than two transporter molecules per vesicle).
We have also shown that increased lipophilicity confers no
advantage, and that a positive charge is strongly deleterious.
The comparison with simple monourea 25 confirms the ben-
efit of the preorganized cholapod architecture. Results with
a second measurement method, involving the addition of
cholapod to preformed vesicles, demonstrate that transport-
er delivery to the vesicle membranes occurs with varying ef-
fectiveness. Finally, experiments with membranes of differ-
ent thicknesses have provided further support for the
mobile carrier mechanism.
In future work, we will improve our understanding of
cholapod anion transport by applying further techniques, es-
pecially conductivity measurements in membrane patches.
This new knowledge will aid the optimisation of transport
properties. We will also seek to improve transporter delivery
to preformed synthetic and biological membranes. Opti-
mised cholapod anionophores should be valuable as tools
for membrane transport research, and may uncover new
modes of biological activity.
porter/lipid ratio of 1:150, it
promoted chloride efflux from
vesicles with
k
obs =0.0056 sÀ1
(see Figure 7). Comparison with
Table 1, and considering the dif-
ference in loadings, suggests
that it is roughly equivalent to
cholapod
9 (which is a far
stronger receptor). Although it is interesting that such a
simple structure can show significant activity, the results
confirm the value of the cholapod design. The monourea 25
is about fifty times less efficient than cholapod 1e, in which
two p-nitrophenylurea units are preorganised on the steroi-
dal scaffold.
Experimental Section
General: Where not previously reported, cholapods were prepared and
characterised as described in the Supporting Information. Other materi-
als and equipment were sourced as follows: lipids, Avanti Polar Lipids,
Inc.; POPC, Genzyme; cholesterol, Sigma; lucigenin, Molecular Probes;
Lipofast hand held extruder, Avestin; polycarbonate membranes
(200 nm), Avestin; quartz cuvettes, Fischer-Scientific; chloride selective
electrode, Fisher-Scientific. Fluorescence spectra were obtained using a
Jobin-Yvon Fluoromax-3 fluorimeter with FT WinLab software and ex-
ternal water bath cooling unit.
Preparation of unilamellar vesicles: All lipids and cholesterol were
stored in a À208C freezer as solids or as chloroform solutions (10 or
20 mgmLÀ1). The chloroform solutions were combined with a solution of
cholapod as appropriate to give the desired lipid or lipid–cholapod mix-
ture in a ten mL round bottom flask. The chloroform was removed using
a rotary evaporator followed by evacuation on a high vacuum pump line
for 1–3 h. The aqueous solution to be placed inside the vesicles (1 mL)
was added and the lipid film was resolvated by vortexing in the presence
of a glass ring. The lipid solution subsequently underwent nine freeze-
thaw cycles and was extruded twenty-nine times through a 200 nm poly-
carbonate membrane to give the vesicle suspension.
Figure 7. Chloride transport into vesicles (POPC/cholesterol, 7:3) by p-
nitrophenylurea 25 at transporter/lipid 1:150, assayed by the lucigenin
method.
Conclusion
Lucigenin assay (typical procedure): A suspension of unilamellar vesicles
(200 nm mean diameter, 20 mm lipid concentration) composed of POPC/
Cholesterol (7:3 molar ratio) and cholapod, and containing aqueous
NaNO3 (225 mm) and lucigenin (1 mm), was prepared. A portion of this
suspension (500 mL) was loaded onto a Sephadex G-50 column and
eluted with aqueous NaNO3 (225 mm) to remove the un-encapsulated lu-
cigenin. The elution process was monitored with a hand-held UV/Vis
lamp. The vesicles were then diluted with aqueous NaNO3 (225 mm) to a
final 25 mL total volume (0.4 mm lipid). An aliquot of this suspension
(3 mL) was placed in a cuvette, and aqueous NaCl (25 mm) was added.
The lucigenin fluorescence was monitored at 450/505 ex/em with a 3 nm
slit width, with the temperature maintained at 258C. The results shown in
the Figures are representative traces of three individual experiments. The
In previous work we have shown that cholapods can be ex-
tremely effective as chloride transporters. Herein we present
a study encompassing an extended and varied range of these
anionophores. Structure–activity relationships have been in-
vestigated using a method in which transporters are prein-
corporated in vesicle membranes. The results are puzzling in
some respects; some imply that binding strength and trans-
port rates are correlated, while others show that additional
factors are also significant (see Figure 4). However, impor-
tantly, we have found that very high transport activities can
be achieved. Our new champion, 3, achieves a transport half
life of 26 seconds at the very low transporter/lipid ratio of
1:25000. It is measurably effective even at transporter/lipid
lucigenin fluorescence quenching curves were fitted by non-linear com-
obst
puter methods to the first order decay equation (IoÀIt)/
(IoÀIf)=1ÀeÀk
A
where Io is initial fluorescence intensity, It is intensity at time t, If is inten-
sity at time final. The results for kobs in Tables 1 and 2 are the average of
three individual experiments with S.D. Æ 5.0%.
Chem. Eur. J. 2008, 14, 9599 – 9606
ꢁ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
9605

A. P. Davis, B. D. Smith et al.
Ion-selective electrode (ISE) assay (typical procedure): A suspension of
unilamellar vesicles (1 mL, 30 mm in lipid) composed of POPC/cholester-
ol (7:3 molar ratio) prepared using aqueous NaCl (500 mm) was dialysed
against aqueous NaNO3 (500 mm) using spectra/pore 12–14000 MWCO
dialysis tubing (Fisher Scientific) for ten to twelve hours. The resulting
suspension was diluted to 1 mm lipid concentration with aqueous NaNO3
(500 mm). A solution of the appropriate cholapod (5 mm) was prepared
in THF. Typically, 4 mL of this solution was added to 5 mL of the vesicle
suspension for a final transporter concentration of 4 mm. Chloride release
was monitored for six minutes using a chloride selective electrode with
the temperature maintained at 258C. At the end of the experiment the
vesicles were lysed with polyoxyethylene 8 lauryl ether detergent to
obtain 100 percent chloride release. The results in Table 3 are the average
of three individual experiments with S.D. Æ5.0%. Additional experimen-
tal details and typical transport data are described in ref. [11a].
Angew. Chem. 2003, 115, 5081; Angew. Chem. Int. Ed. 2003, 42,
4931; b) B. A. McNally, A. V. Koulov, B. D. Smith, J. B. Joos, A. P.
Davis, Chem. Commun. 2005, 1087; c) J. L. Sessler, L. R. Eller, W. S.
Cho, S. Nicolaou, A. Aguilar, J. T. Lee, V. M. Lynch, D. J. Magda,
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5989; d) P. A. Gale, M. E. Light, B. McNally, K. Navakhun, K. E.
Sliwinski, B. D. Smith, Chem. Commun. 2005, 3773; e) P. V. Santa-
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Prados, R. Quesada, J. Am. Chem. Soc. 2007, 129, 1886.
[12] A. P. Davis, J.-B. Joos, Coord. Chem. Rev. 2003, 240, 143; A. P.
Davis, Coord. Chem. Rev. 2006, 250, 2939.
[13] Just one relative, a tris-p-nitrophenylthiourea, is slightly more pow-
erful. See ref. [14].
[14] J. P. Clare, A. J. Ayling, J. B. Joos, A. L. Sisson, G. Magro, M. N.
PØrez-Payµn, T. N. Lambert, R. Shukla, B. D. Smith, A. P. Davis, J.
Am. Chem. Soc. 2005, 127, 10739.
[15] In a separate work we have studied cholapods with 3a-H, and have
found that their chloride affinities are similar to analogues with 3a-
OAc.
[16] A. L. Sisson, V. del Amo Sanchez, G. Magro, A. M. E. Griffin, S.
Shah, J. P. H. Charmant, A. P. Davis, Angew. Chem. 2005, 117, 7038;
Angew. Chem. Int. Ed. 2005, 44, 6878.
Acknowledgements
Financial support from the BBSRC (BBS/B/11044 and BBS/S/E/2006/
13241), the EPSRC (GR/R04584/01 and EP/F03623X/1), NIH (USA)
and the University of Bristol is gratefully acknowledged.
[17] J. M. Boon, T. N. Lambert, A. L. Sisson, A. P. Davis, B. D. Smith, J.
Am. Chem. Soc. 2003, 125, 8195.
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[21] For more details see Supporting Information.
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cholesterol are included in the lipid molecule count.
[23] Aggregation effects might explain some of the variations in trans-
porter performance at different loadings. For example, cholapod 2
performs relatively poorly at the high loading of receptor/cholapod
1:250, and this might reflect self-association to form inactive assem-
blies.
[24] A possible role for chloride/nitrate selectivity was suggested by a
referee. With relatively simple liquid organic membranes it is known
that, if the rate determining step is membrane diffusion, then trans-
port rate is a maximum when the ion and counter-ion have the same
carrier affinities (T. M. Fyles, in Inclusion Aspects of Membrane
Chemistry, Vol. 2 (Eds.: T. Osa, J. Atwood), Kluwer, Dordrecht,
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However, this is not surprising because diffusion through thin vesi-
cle membranes may not be fully rate-determining, and the associa-
tion kinetics may be affected by additional factors such as carrier–
phospholipid and ion–phospholipid interactions.
[5] Prodigiosins (ref. [4a]) are relatively well-studied, but transport H+
along with ClÀ. They do not therefore serve as counterparts to iono-
phores like valinomycin, with purely cation-transporting properties.
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[28] The measurements on valinomycin employed monoglyceride lipids,
as opposed to the PCs used in the present work.
[29] Very recently, Pescatori et al. have measured the association con-
stant for another lipophilic p-nitrophenylurea to Bu4NCl in (dry)
CHCl3, by UV titration. Their value of 14000mÀ1 is consistent with
our result for 25. See: L. Pescatori, A. Arduini, A. Pochini, F. Ugoz-
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[11] a) A. V. Koulov, T. N. Lambert, R. Shukla, M. Jain, J. M. Boon, B. D.
Smith, H. Y. Li, D. N. Sheppard, J. B. Joos, J. P. Clare, A. P. Davis,
Received: June 13, 2008
Published online: September 4, 2008
9606
www.chemeurj.org
ꢁ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2008, 14, 9599 – 9606