The role of lipophilicity in transmembrane anion transport

Article abstract of DOI:10.1039/c2cc31825c

The transmembrane anion transport activity of a series of synthetic molecules inspired by the structure of tambjamine alkaloids can be tuned by varying the lipophilicity of the receptor, with carriers within a certain log P range performing best.

Full text of DOI:10.1039/c2cc31825c

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COMMUNICATION
The role of lipophilicity in transmembrane anion transportw
b
c
Vittorio Saggiomo,a Sijbren Otto,a Igor Marques,b Vı
´
tor Felix, Tomas Torroba and
´ ´
Roberto Quesada*c
Received 12th March 2012, Accepted 10th April 2012
DOI: 10.1039/c2cc31825c
The transmembrane anion transport activity of a series of synthetic
molecules inspired by the structure of tambjamine alkaloids can be
tuned by varying the lipophilicity of the receptor, with carriers
within a certain log P range performing best.
Development of small molecules capable of facilitating anion
transport through lipid bilayers is an important goal.1 These
molecules could have potential applications in channel replace-
ment therapies, as novel chemotherapeutics or biomembrane
research tools.2 Despite this, little is known regarding the
requirements to produce efficient anion transporters and in
particular the molecular design of anion transporters. It is clear
that ion translocation through a lipid membrane is a complex
event involving many parameters. Anion binding affinity is the
most commonly studied parameter in anion transporters.
Steroid based ‘‘cholapods’’ developed by A. P. Davis and
colleagues are excellent examples of very efficient anion trans-
porters displaying extremely high anion affinities.3 On the
other hand, an important number of efficient nitrate trans-
membrane carriers exhibit negligible nitrate binding affinity.4
Moreover, systems displaying very similar anion binding
affinities can present dramatic differences in anion transport
efficiency.
Fig. 1 Tambjamine derivatives 1–16.
assess the contribution of each factor in the final observed
transport efficiency.
We have recently reported the anion transport properties
of some tambjamine alkaloids and related analogs.8 We
envisaged that these easy-to-make compounds offered a unique
opportunity to investigate the impact of lipophilicity on the
overall efficiency of an anion transporter. We first prepared
compounds 1–13 by systematically varying the substitution of
the enamine alkyl chain (Fig. 1). A number of these compounds
have been identified as naturally occurring bioactive derivatives
(e.g., 11–13).9 Displaying the same bipyrrole–enamine moiety as
anion binding motif, it is expected that these compounds would
have essentially similar anion binding affinity and polar surface
areas, determined by the nitrogen and oxygen atoms. This was
supported by computational studies and the estimation of
chloride binding constants in DMSO solution for representa-
tive derivatives (see ESIw for details).
Log P, the logarithm of the octanol/water partition coeffi-
cient, is the most widely used measure of the lipophilicity. The
log P values for these derivatives were calculated using the
VCCLab software and consensus values for log P were used.10
These values represent the average of the log P values calcu-
lated through different structure-based and property-based
methods (see ESIw for details). The consensus log P value
has been shown to provide accurate results in the prediction of
log P values.11 These calculations were performed for two
different tautomeric forms of the neutral and N-protonated
forms of compounds 1–16 (Table S1, ESIw). There is a linear
Lipophilicity is a parameter of paramount importance in the
pharmaceutical industry.5 However, it has received little atten-
tion in the context of transmembrane anion transport, despite
its widespread use in pharmaceutics, which also involves
membrane translocation steps.6 P. A. Gale and colleagues
have recently introduced lipophilicity calculations, along with
total and polar surface areas assessment for several urea and
thiourea based anion transporters.7 Nevertheless typical modi-
fications introduced in anion receptors, such as replacing urea
by thiourea groups or introducing electron-withdrawing
substituents (such as fluorinated groups), modify both lipo-
philicity and anion binding strength, making it difficult to
a Centre for Systems Chemistry, Stratingh Institute,
University of Groningen, 9747 AG Groningen, The Netherlands
b
´
´
Departamento de Quımica, CICECO and Secc¸a˜o Autonoma de
Cieˆncias da Sau´de, Universidade de Aveiro, 3810-193 Aveiro,
Portugal
c
´
Departamento de Quımica, Facultad de Ciencias, Universidad de
Burgos, 09001 Burgos, Spain. E-mail: rquesada@ubu.es
w Electronic supplementary information (ESI) available: Experimental
details and spectral characterization data, anion binding experiments,
Log P calculations, anion transport experiments, methods for the
computational studies. See DOI: 10.1039/c2cc31825c
c
5274 Chem. Commun., 2012, 48, 5274–5276
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Table 1 Transport activities (%sÀ1) and calculated log P values of
compounds 1–16. Carrier concentration 1 mM, 0.2 mol% carrier
to lipid
Compound
Transport activity/%sÀ1
Log P
1
2
3
4
5
6
7
8
0.034
0.070
0.090
0.193
0.394
0.467
0.682
0.680
0.685
0.530
0.499
0.205
0.319
0.299
0.698
0.248
0.93 Æ 0.46
1.34 Æ 0.51
1.78 Æ 0.52
2.22 Æ 0.57
2.69 Æ 0.63
3.16 Æ 0.71
3.63 Æ 0.79
4.26 Æ 0.69
4.70 Æ 0.74
5.13 Æ 0.81
6.09 Æ 0.92
2.11 Æ 0.56
2.59 Æ 0.64
3.44 Æ 0.69
4.20 Æ 0.78
6.64 Æ 0.96
9
10
11
12
13
14
15
16
Fig. 2 Representation of transport activity, measured as initial chloride
efflux (%sÀ1) vs. calculated average log P for compounds 1–16.
Compounds 1–13 feature aliphatic hydrophobic side
groups. In order to assess whether the relationship between
hydrophobicity and transport activity also holds for aromatic
side groups we prepared compounds 14 and 16 in which the
O–Me fragment was replaced by an O–Bn substituent (Fig. 1).
This modification impacts significantly on the overall lipo-
philicity of the molecule without modifying the anion binding
cleft. The transport activity of 14 and 16 matched fairly well
with the trend shown by 1–13. The n-propyl substituted
derivative 3 induced very limited chloride efflux, being too
hydrophilic. Replacing the O–Me fragment by a O–Bn sub-
stituent as in 14 increases the log P value of the molecule
significantly, resulting in an improved transport activity
(Table 1, Fig. 3). Likewise, modifying the lipophilicity of
compound 10 (R = n-decyl) as in compound 16 had a
detrimental effect as a result of the increase in the already
higher than optimal log P value. Since the maximum transport
activity was found for compounds having log P values around
4.2 we decided to prepare an analog displaying this log P
value. Theoretical calculations indicated an optimal value for
the R = n-pentyl O–Bn derivative 15. This compound was
synthesized and was found to be among the most active of the
series, confirming that log P values may be used to predict
transport activity.
relationship between the calculated set of log P values for all
tautomers. At physiological pH values it is expected that the
tambjamine derivatives are protonated and therefore this is
likely the active form of these molecules. We also checked the
reliability of the calculated values using experimental data. To
obtain a measure of the lipophilicity of the compounds we
used reverse phase HPLC.12 Retention times are directly
proportional to the lipophilicity of the compounds. These
results correlated well with the predicted log P values (see
Fig. S9, ESIw).
The anion transport properties of these molecules were
assayed in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine
(POPC) vesicles (see ESIw for details). Chloride efflux from
chloride loaded vesicles in the presence of external bicarbonate
was monitored using a chloride selective electrode.13 Direct
evidence of bicarbonate transport was obtained by 13C NMR
(see Fig. S16, ESIw). We chose this assay due to the biological
relevance of the chloride/bicarbonate exchange process. For
comparative purposes the relative transport activity was
expressed as the initial rate of chloride efflux (%sÀ1).14 This
value results from the fitting of the traces representing the
chloride efflux (see ESIw for details). Hill analysis suggested a
discrete carrier mechanism, with EC50 values (concentration
needed for inducing 50% of chloride efflux after five minutes)
in the submicromolar range for the most active derivatives.15
There are substantial differences in the transmembrane
anion transport activity of these compounds, ranging from
limited to excellent at the concentration screened (Table 1).
A plot of the transport activity vs. log P showed a maximum
activity for compounds having log P values close to 4.2 Æ 0.5
(Fig. 2). The transport activity diminished for compounds
having log P values above or below this value. Compounds
with log P values lower than 2 displayed very limited activity.
It should be noted that diminished activity as a result of an
increase in the lipophilicity probably also reflected the poorer
deliverability from the aqueous phase to the lipid bilayer
and/or the lower mobility of these compounds within the
membranes. These results showed that carrier activity is
influenced by ionophore lipophilicity with an optimal value
of this parameter giving a maximum in transmembrane trans-
port activity.
Fig. 3 Comparison of chloride efflux mediated by –OMe substituted
compounds 3, 5, 10 (square symbols) and the parent –OBn substituted
derivatives 14, 15, 16 (triangle symbols) in phospholipid vesicles
(1 mM, 0.2 mol% carrier to lipid concentration). Each trace represents
the average of three trials.
c
This journal is The Royal Society of Chemistry 2012
Chem. Commun., 2012, 48, 5274–5276 5275

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In conclusion, we have studied the transmembrane trans-
port activity of anion transporters inspired by the structure of
the tambjamine natural products. We have shown that lipo-
philicity is correlated to this activity, which is tuned by varying
the lipophilicity of these compounds, being optimal for a
log P = 4.2. This information was used to design new
derivatives with optimal activity. These findings may be helpful
in the analysis of structure–activity relationships of other
ionophoric compounds.
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The authors thank financial support from Consejerı
Educacion de la Junta de Castilla y Leon (project BU005B09),
the Ministerio de Ciencia e Innovacion of Spain (project
CTQ2009-12631-BQU and Ramon y Cajal contract (R.Q.))
´
a de
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and EU (COST Action CM1005 ‘‘Supramolecular Chemistry
in Water’’). V. S. thanks ‘‘NWO, The Netherlands Organi-
sation for Scientific Research’’ for a Rubicon fellowship. The
computational studies were funded by FEDER through the
Operational Program Competitiveness Factors – COMPETE
´
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5276 Chem. Commun., 2012, 48, 5274–5276
This journal is The Royal Society of Chemistry 2012