Small neutral molecular carriers for selective carboxylate transport

Article abstract of DOI:10.1039/c2cc37468d

A series of neutral thiourea receptors were found to mediate the antiport of chloride with a range of biologically relevant carboxylate anions across phospholipid bilayers. Simple structural modification of the carriers resulted in a change in the lactate/pyruvate transport selectivity.

Full text of DOI:10.1039/c2cc37468d

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Small neutral molecular carriers for selective
carboxylate transport†
Cite this: DOI: 10.1039/c2cc37468d
Cally J. E. Haynes, Stuart N. Berry, Joachim Garric, Julie Herniman,
Jennifer R. Hiscock, Isabelle L. Kirby, Mark E. Light, Gregory Perkes and
Philip A. Gale*
Received 12th October 2012,
Accepted 14th November 2012
DOI: 10.1039/c2cc37468d
www.rsc.org/chemcomm
A series of neutral thiourea receptors were found to mediate the it was produced, or transported elsewhere for oxidation.8 Some
antiport of chloride with a range of biologically relevant carboxylate organs, including the heart, brain and slow twitch muscle fibres
anions across phospholipid bilayers. Simple structural modification preferentially take up lactate rather than glucose as an energy
of the carriers resulted in a change in the lactate/pyruvate transport source.9 The majority of lactate is transported by monocarboxylate
selectivity.
carrier proteins, which can also transport other carboxylates
including pyruvate and propionate.6 A recent study has suggested
The design of molecular carriers for the transport of chloride and that disrupting the expression of the neuronal lactate transporter
bicarbonate across phospholipid bilayers has progressed rapidly in MCT2 leads to amnesia in rats, suggesting that lactate transport in
recent years.1 However, there have been few reports to date of the neurones is necessary for long-term memory formation.10 The
lipid bilayer transport of carboxylate anions. The transmembrane production of small molecule carboxylate transporters may useful
transport of N-protected amino acids through U-tubes is known, provide biological tools for the study of such processes.
although the receptors that are reported to mediate this process by
Our group11 and others3,12 have shown that thioureas can
selectively binding to the carboxylate component are often charged function as excellent anion carriers. For example, we have pre-
and relatively hydrophilic,2 which can be an undesirable viously reported that thioureas 1 and 2 can facilitate ClÀ/NO3À and
À
characteristic for a lipid bilayer anion carrier.3 The transport ClÀ/HCO3 exchange,11d,13 whilst indole substituted thioureas
of lactate across liquid4 and polymeric5 membranes has been show enhanced transport rates.11b,13 We decided to investigate
reported using simple amines and ammonium salts as mobile whether thioureas could facilitate the selective transmembrane
carriers, with applications in industrial lactate extraction.
transport of L-lactate, herein referred to as lactate, and pyruvate,
Carboxylates are common in biological systems and their in addition to propionate.
transmembrane transport is crucial for cell function.6 There are a
large number of small molecule carboxylates within the body,
which are often extremely similar structurally; as such it is critical
that a prospective carboxylate transporter should be inherently
selective for a target species over highly similar analogues. For
example, pyruvate and lactate differ only in the keto- or hydroxyl-
Using receptors 1 and 2 as a starting scaffold, we incorporated
functionality in the 2-position as they are directly interconvertible additional hydrogen bond donors and acceptors into the design
metabolites. Pyruvic acid is the end product of glycolysis and is the in order to allow the formation of hydrogen bonds between the
starting point for the Krebs cycle. In both the absence and hydroxyl or keto groups of lactate and pyruvate. Receptor 3
presence7 of oxygen, pyruvic acid is converted to lactic acid that contains a methylimidazole group to potentially offer a basic
can later be re-oxidised to pyruvic acid to feed into the respiratory nitrogen hydrogen bond acceptor in a position which may be
process. Lactate may be oxidised within the muscle tissue in which able to bind to the hydroxyl O–H of lactate but offer no
enhancement to pyruvate binding. Receptor 4 contains an amido
Chemistry, University of Southampton, Southampton SO17 1BJ, UK.
E-mail: philip.gale@soton.ac.uk; Fax: +44 (0)23 8059 6805;
functionality in the 2-position of the indole scaffold, which could
function as either a hydrogen bond donor (NH) or acceptor
(CQO) depending on the conformation of the receptor.
Anion binding strength has previously been correlated with
transport activity.3,14 In order to investigate the solution phase
binding properties of receptors 3–4, we performed 1H NMR
Tel: +44 (0)23 8059 3332
† Electronic supplementary information (ESI) available: Details of synthesis, the X-ray
crystal structure of the lactate complex of receptor 2, stability constant determination
and membrane transport studies. CCDC 905379. For ESI and crystallographic data in
CIF or other electronic format see DOI: 10.1039/c2cc37468d
c
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hydrogen bond formation and possibly a change in receptor
conformation (see Fig. S29 in the ESI†).
In order to assess the carboxylate transport activity of these
receptors we adapted the pulse assay which has previously been
reported. We prepared a sample of POPC vesicles containing
NaCl, which were suspended in Na2SO4. A sample of the
receptor in a DMSO (2 mol% w.r.t. lipid) was added, and any
resulting chloride efflux was monitored using a chloride selective
electrode (ISE). After 2 minutes, a spike of the sodium salt of the
anion of interest was added such that the external concentration
of this salt was 40 mM. In this way, by studying the efflux of
titrations with tetrabutylammonium or tetraethylammonium chloride after the pulse of the anion salt it is possible to
salts of the anions in DMSO-d6/H2O (0.5%). The data was fitted indirectly monitor the antiport of the anion of interest. For
to a 1 : 1 binding model using WinEQN MR2.15 The binding comparison to previously reported receptors, we also included
constants calculated are shown in Table 1 and show that the nitrate and bicarbonate as antiport substrates. At the end of the
receptors have low affinity for chloride and do not detectably experiment, the vesicles were lysed with detergent to calibrate
interact with nitrate under these experimental conditions. 100% chloride efflux. The results are shown in Fig. 2–4 and
Higher affinities are observed with bicarbonate and carboxyl- Fig. S32 in the ESI.†
ates with the highest affinity observed with compound 4 and
The relative lipophilicity of receptors 1–4 was investigated by
propionate.
using HPLC to examine their retention time on a reverse phase
The amide NH resonance of receptor 4 underwent only a column (see the ESI† Table S1).18 These results indicate that
very small downfield shift (o0.5 ppm) during the titration with receptor 3 is less lipophilic than parent compounds 1, while
lactate, implying that it is not directly involved in the binding receptor 4 is more lipophilic than receptor 2. This parameter
(see Fig. S28 in the ESI†). Bates et al. have proposed a carbox- does not appear to be a determining factor in the relative
ylate binding mode for receptors of this type as shown in Fig. 1, transport activity of these receptors; however, given the structural
in which the amide NH faces away from the binding cleft.16 By variation across the series this is perhaps unsurprising
extension, such a binding mode could facilitate hydrogen bond
Prior to the spike of anion at 120 s, no transport is observed,
formation with the amide carbonyl as an acceptor. An inter- confirming an anion antiport mechanism. No chloride efflux
action of this type has also been previously reported.17 How- was observed in the absence of a receptor (Fig. S31 in the ESI†).
ever, no evidence of this interaction could be observed by NMR For every receptor the most efficient antiport process was the
techniques. In contrast, the amide NH resonance was observed exchange of chloride with propionate, due to the hydrophobicity
to undergo a small but detectable downfield shift during the of this anion. Receptor 1 showed only very poor transport
titrations with pyruvate and propionate, which implies some properties (Fig. S32 in the ESI†).
Receptor 2 is an excellent anion antiporter,13 and is the most
efficient antiporter for every transport process in this study
(Fig. 2). Close to 100% of the intra-vesicular chloride was
Table 1 Stability constants (Ka) of 1 : 1 complexes in DMSO-d6/H2O 0.5% at
25 1C, calculated following the resonance of the aromatic adjacent thiourea NH.
Errors are estimated to be o15%a,b
Anion
1
2
3
4
Chloride
Nitrate
Bicarbonate
Propionate
Lactate
14b
17b
12
12
o10b
262b
434
66
o10b
414b
771
61
o10
o10
389
1735
180
90
c
—
362
38
42
Pyruvate
42
28
a
Anions were added as the tetrabutylammonium salt except for bicar-
b
bonate, which was added as the tetraethylammonium salt. Binding
constants for 1 and 2 with chloride, nitrate and bicarbonate have been
previously reported.13 c Binding constant could not be calculated due to
broadening of the NH resonances.
Fig. 2 Chloride efflux mediated by receptor 2 (2 mol% w.r.t. lipid) from POPC
vesicles containing NaCl buffered to pH 7.2 with sodium phosphate salts. The
vesicles were suspended in Na2SO4 buffered to pH 7.2 with sodium phosphate
salts. At t = 120 s, a pulse of sodium salt was added such that the final
concentration was 40 mM.
Fig. 1 The acetate binding mode proposed by Bates et al. and a possible binding
mode of receptor 4 with lactate.16
c
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In summary, we have reported that a series of thioureas can
mediate the selective transport of monocarboxylates across
lipid bilayers, and that simple structural modification of the
receptor scaffold results in modulation of anion selectivity. We
are currently working to expand these motifs for the capture
and transport of more complex species, and these results will
be published in due course.
We thank the EPSRC for a doctoral prize award (CJEH) and a
postdoctoral fellowship (JG).
Notes and references
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Fig. 4 Chloride efflux mediated by receptor 4 (2 mol% w.r.t. lipid) from POPC
vesicles containing NaCl buffered to pH 7.2 with sodium phosphate salts. The
vesicles were suspended in Na2SO4 buffered to pH 7.2 with sodium phosphate
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released by the end of each experiment regardless of the
external anion. Analysis of the initial transport rate indicates
that pyruvate is transported faster than lactate.
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more efficiently than pyruvate (Fig. 3 and 4), a reversal of the trend
observed for receptor 2. This suggests that the synthetic modifica-
tion of the thiourea core has resulted in greater selectivity for
lactate transport. This may be due to hydrogen bond formation
between the hydroxyl group of lactate with the methylimidazole
group of receptor 3 and the amide CQO of receptor 4, providing
stronger binding and better shielding of the anion from the apolar
interior of the bilayer. This work emphasises the importance of
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c
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