Encapsulated binding sites - Synthetically simple receptors for the binding and transport of HCl

Article abstract of DOI:10.1039/b906737j

We report a receptor with an encapsulated amine/amide binding site, which binds HCl with high affinity in organic media - the rate of HCl transport through an apolar phase is controlled by the degree of encapsulation of the HCl binding site.

Full text of DOI:10.1039/b906737j

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Encapsulated binding sites—synthetically simple receptors for the binding
and transport of HClw
Keith J. Winstanley, Sarah J. Allen and David K. Smith*
Received (in Cambridge, UK) 3rd April 2009, Accepted 22nd May 2009
First published as an Advance Article on the web 9th June 2009
DOI: 10.1039/b906737j
We report a receptor with an encapsulated amine/amide binding
site, which binds HCl with high affinity in organic media—the
rate of HCl transport through an apolar phase is controlled by
the degree of encapsulation of the HCl binding site.
X-ray structures of HCl complexes of tren-based amide
N, N0, N00-tris[(2-aminoethyl)-3-nitro-benzamide].10 In the X-ray
structures the amine was protonated, and Clꢀ was bound
to multiple receptors through N–H(amide)ꢁ ꢁ ꢁanion and
ArC–Hꢁ ꢁ ꢁanion hydrogen bond interactions.
We synthesised receptors 1–8 (Fig. 1). We hoped that by
generating binding sites with different degrees of encapsulation,
we should be able to tune the behaviour of the receptors. Full
details of synthesis and characterisation can be found in
the ESIw.
The prodigiosins are a class of natural products with potent
anti-cancer activity and the ability to transport HCl through
cell membranes modulating cellular pH, using a basic amine to
bind H+ and an array of N–H groups to hydrogen bond to the
chloride anion.1 There has therefore been considerable recent
interest in the development of synthetic receptors for HCl. In
2002, Sidorov and co-workers reported a calix[4]arene amide
derivative which formed ion channels and mediated HCl
transport.2 In 2005, Sessler and co-workers made a synthetic
prodigiosin mimic which included a basic imidazole unit to
bind H+ and a bis-pyrrole in order to provide hydrogen bonds
to Clꢀ.3 These derivatives also exhibited anti-cancer activity.
In elegant work, Gale and co-workers have also developed
HCl binding and transport systems based on the combination
of an imidazole and either a pyrrole-amide or a 2,6-dicarbox-
amidopyridine unit.4 Greater pre-organisation and HCl
binding affinity led to a higher membrane-transport flux.
Calixarenes functionalised with spermidine amines have also
recently been used for HCl transport.5
Initially, the ability of these synthetically simple receptors
to bind to anions as their tetrabutylammonium salts was
investigated in CDCl3 : DMSO-d6 (9 : 1) by NMR titration
methods, with the data being fitted to a 1 : 1 model. Receptor
1, which has the most deeply encapsulated binding site was
screened against a range of anions (Table 1). Unsurprisingly,
the receptor showed the strongest binding with fluoride, the
most charge dense anion, with the binding strength decreasing
for chloride and bromide. Surprisingly, receptor 1 bound Clꢀ
more effectively than H2PO4ꢀ. Normally the more basic
ꢀ
H2PO4 anion is more effectively bound than Clꢀ—only in
rare cases has this affinity order been reversed, usually in
sterically congested macrocyclic binding sites.11
Receptors 2 and 3 were studied further to probe this
ꢀ
behaviour (Table 1). Both of these receptors bound H2PO4
ꢀ
For some time, we have been interested in the development of
synthetically simple yet effective anion receptors.6 Inspired by
the work described above, we began to consider whether we
could take an alternative approach towards HCl receptors. Our
design strategy involved the incorporation of a basic amine to
bind H+, along with multiple amides capable of binding Clꢀ.
A large number of anion receptors have been reported based on
‘tren’ (tris(2-aminoethyl)amine), as this commercially available
trigonal scaffold can readily be functionalised to yield three
anion binding hydrogen-bonding amide or urea groups close to
the central core.7 Furthermore, tren receptors have been
demonstrated to show ‘flippase’ activity—translocating anionic
phospholipid analogues through a cell membrane.8 It was
argued that in this case, the tertiary amine unit may play a role
in transporting the countercation through the membrane.
Although it is known that amines can encourage the binding
of acidic anions,9 there have been few other attempts to harness
the basicity of the central tertiary amine of tren-amides. In one
crystallographic study, Suresh and co-workers reported the
in preference to Clꢀ. It is notable that H2PO4 binding
remained fairly constant across the three receptors, whereas
chloride binding became less favourable for receptors 2 and 3
than it was for receptor 1. This suggests that the internal cavity
of receptor 1 is actually particularly well suited for chloride
Department of Chemistry, University of York, Heslington,
YO10 5DD, York, UK. E-mail: dks3@york.ac.uk;
Fax: +44 (0)1904 432516
w Electronic supplementary information (ESI) available: Synthesis
and characterisation of HCl binders. See DOI: 10.1039/b906737j
Fig. 1 Receptors 1–8 investigated in this paper.
Chem. Commun., 2009, 4299–4301 | 4299
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Table 1 Binding affinities, K, for some of the synthetic receptors with
tetrabutylammonium anion salts in CD3CN : DMSO-d6 (9 : 1).
Titration data were fitted to 1 : 1 stoichiometry, K values determined
at 298 K with units of molꢀ1 dm3. Missing data were not determined.
All data are averaged over multiple runs with errors ꢃ15%
groups). To probe this further, we investigated the binding
of HBF4. In this case, the binding stoichiometry was closer
to 1 : 1, with the maximum in the Job plot being observed
at 0.55 (Fig. S1, ESIw). This suggests that protonation of the
tertiary amine still occurs, but BF4ꢀ is less able to interact with
the amide groups in the ‘cavity’ of the receptor. We also
investigated the ability of these receptors to bind benzoic
acid. No binding was observed, proving that the acid must
be sufficiently strong for recognition to take place.
ꢀ
ꢀ
Receptor
Clꢀ
H2PO4
Fꢀ
BzOꢀ
Brꢀ
NO3
1
2
3
6
8
550
150
130
120
75
280
305
260
—
1090
—
—
—
—
250
—
—
—
35
—
—
—
—
o1
—
—
—
—
We analysed the HCl binding of these receptors by
integration of the NMR spectra in order to yield apparent
equilibrium constants (Kapp) for the 1 : 2 binding event
(Table 2). It should be noted that these Kapp values include
multiple binding events, and only give a general indication of
the relative strength of interaction. Receptor 1 binds HCl most
effectively, while the less deeply encapsulated receptors 2 and 3
are less effective—in this case by an order of magnitude.
Receptor 8, with longer alkyl substituents than receptor 3, is
a less effective HCl receptor. These studies indicate that similar
factors influence the binding of HCl as for the binding of
tetrabutylammonium chloride: i.e., receptor 1 has high HCl
affinity, potentially due to specific anionꢁ ꢁ ꢁshell interactions,
or the development of a unique microenvironment.
—
—
binding. It is possible that ArC–Hꢁ ꢁ ꢁanion interactions10 or
anion–p interactions12 also contribute to chloride binding.
Alternatively the enhanced binding may be a consequence of
the aromatic-ether organic shell providing a more polar local
microenvironment, better able to stabilise bound Clꢀ.13
We also determined the chloride binding affinities for simple
aliphatic chain functionalised receptors 6 and 8. We observed
that as the receptor became more heavily encapsulated, the
affinity for Clꢀ decreased—receptor 8 only had about half the
binding affinity of receptor 3. The flexible substituents in these
receptors therefore appear to hinder chloride binding—this
supports the hypothesis that the specific nature/structure of
the organic shell in receptor 1 plays a key role in enhancing
chloride anion binding.
We screened the ability of these receptors to transport HCl
through an apolar phase using a simple U-tube. The receptor
was dissolved in dichloromethane (10 mM), and placed at the
base of the U-tube. In the left-hand-arm of the tube
was aqueous HCl (pH 0.9) and in the right-hand-arm was
deionised water (Fig. S3, ESIw). The transport of HCl was
monitored by following the pH change in the right-hand-arm
over four hours. In the absence of receptor, there was no
transport of HCl, and pH was constant (Fig. 3 and 4, circles).
On using triethylamine (Et3N) as a control ‘receptor’, there
was once again no transport of HCl (Fig. 3, triangles). Indeed,
conversely, the pH in the right-hand-arm increased—indicating
that Et3N partly partitions from CH2Cl2 into the aqueous
phase but is unable to transport HCl. Receptors 1 and 2,
however, gave effective transport of HCl—as observed by the
significant decrease in pH (Fig. 3, squares and diamonds).
Within error, both receptors exhibited similar rates of HCl
transport. This is interesting, as their affinities for HCl were
different by an order of magnitude. This demonstrates that
binding strength is not the only factor which controls ion
transport.
We then carried out NMR titrations of the receptors with
HCl (as a solution in Et2O). On the addition of HCl, the amide
protons of the receptors flattened into the baseline, and new
peaks corresponding to the amide protons in the HCl complex
appeared further downfield—indicative of kinetically slow
binding. Furthermore, a peak assigned to the protonated
tertiary amine appeared at ca. 10 ppm. Job plot analysis was
performed, and surprisingly, a 1 : 2 (host : guest) stoichiometry
was observed (Fig. S1, ESIw). We hypothesise that the
tetrahedral geometry of the central amine means that N–H+
is directed away from the three amide N–H groups (in agreement
with X-ray studies).10 This gives rise to two different chloride
binding sites in solution (Fig. 2). This proposal is in agreement
with previously published crystallographic studies on the
interactions between protonated tren-amine derivatives and
anionic guests—in which, protonation of the tertiary amine led
to the formation of complexes with a range of anion binding
sites.14 We propose that in the first binding site, H+ protonates
the nitrogen, and an associated chloride ion is held in close
proximity by electrostatic attraction. In the second binding
site, the chloride binds to the amide protons, (again with an
associated proton, perhaps also interacting with the CQO
The ability of receptors 3–8 to transport HCl through the
U-tube was studied (Fig. 4). Only the longer chain receptors 7
and 8 were able to rapidly transport HCl through CH2Cl2
(Fig. 4, squares and triangles). Receptor 6 was also able to
Table 2 Apparent binding affinities, Kapp, for some of the synthetic
receptors with HCl (dissolved in Et2O) in CD3CN : DMSO-d6 (9 : 1).
Titration data were fitted to 1 : 2 (receptor : HCl) stoichiometry,
Kapp values determined by spectral integration at 298 K with units of
molꢀ2 dm6. All data are averaged over multiple runs with errors ꢃ15%
Receptor
HCl
1
2
3
8
1.3 ꢄ 106
2.3 ꢄ 105
1.4 ꢄ 105
9.2 ꢄ 104
Fig. 2 Schematic 1 : 2 (host : guest) binding model for receptor 1
with HCl.
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the encapsulating shell playing an active role in assisting HCl
transport. We suggest that such simple compounds may
have interesting membrane-transport abilities and intriguing
biological activities.
We thank EPSRC and The University of York for financial
support of this research.
Notes and references
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Fig. 3 U-tube transport results for HCl transport illustrating the pH
change in the right-hand-arm of the apparatus over time. Circles
represent no receptor, triangles represent Et3N. Squares represent
receptor 1 and diamonds receptor 2.
3 J. L. Sessler, L. R. Eller, W.-S. Cho, S. Nicolaou, A. Aguilar,
J. T. Lee, V. M. Lynch and D. J. Magda, Angew. Chem., Int. Ed.,
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4 (a) P. A. Gale, M. E. Light, B. McNally, K. Navakhun,
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3773–3775; (b) P. A. Gale, J. Garric, M. E. Light,
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Fig. 4 U-tube transport results for HCl transport illustrating the pH
change in the right-hand-arm of the apparatus over time. Circles
represent no receptor. Receptors are represented by plus signs (3),
crosses (4), dashes (5), diamonds (6), squares (7) and triangles (8).
7 See for example: (a) S. Valiyaveettil, J. F. L. Engbersen,
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slowly transport HCl, although the rate was significantly lower
(Fig. 4, diamonds). This clearly demonstrates that those HCl
receptors with a greater degree of core encapsulation are
more effective transporters—irrespective of the inherent HCl
binding affinity. This is in agreement with the observation that
both receptors 1 and 2 transport HCl, even though they have
different HCl affinities. We propose that the enhanced HCl
transport exhibited by the more encapsulated receptors is a
consequence of their ability to effectively shield the bound HCl
from the apolar environment, coupled with the preference of
these less polar receptors to remain within the apolar phase.
These observations are in agreement with the hypothesis
that the best transport agents do not necessarily have the
highest affinities for the target—a degree of binding is required
in order to facilitate transport, but most importantly, the
receptor must screen the ions being transported from the
surrounding apolar phase and then release the ions once
transport is complete.15
10 P. S. Lakshminarayanan, E. Suresh and P. Ghosh, Inorg. Chem.,
2006, 45, 4372–4380.
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In summary, this paper reports synthetically simple
receptors with encapsulated binding sites, which have high
affinities for HCl. These compounds can be considered as
synthetic prodigiosin mimics. Receptor 1, with its binding site
encapsulated within an aromatic ether shell, has the highest
affinity both for TBACl and HCl—we propose that this
is a consequence of favourable interactions/environment
between the organic shell and the bound chloride anion. We
demonstrate that well-encapsulated tren-amide binding sites
can be used to transport HCl through an apolar phase, with
12 P. de Hoog, P. Gamez, I. Mutikainen, U. Turpeinen and
J. Reedijk, Angew. Chem., Int. Ed., 2004, 43, 5815–5817.
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