Synthetic chloride transporters with the binding mode observed in a ClC chloride channel

Article abstract of DOI:10.1039/c2cc35743g

A series of synthetic molecules bearing the same hydrogen bonding mode observed in StClC were prepared and their transport ability of chloride ion across a lipid membrane was systematically optimized.

Full text of DOI:10.1039/c2cc35743g

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COMMUNICATION
Synthetic chloride transporters with the binding mode observed in a
ClC chloride channelw
Ye Rin Choi,a Min Kyung Chae,a Dongwook Kim,b Myoung Soo Lah*b and
Kyu-Sung Jeong*a
Received 8th August 2012, Accepted 30th August 2012
DOI: 10.1039/c2cc35743g
A series of synthetic molecules bearing the same hydrogen
bonding mode observed in StClC were prepared and their
transport ability of chloride ion across a lipid membrane was
systematically optimized.
as unambiguously determined by the crystal structure of a
complex between 4f and tetrabutylammonium chloride
(TBACl). In particular, transport experiments reveal that 4h
greatly facilitates the transport of chloride ion across a lipid
(POPC) bilayer membrane.
In the design of synthetic molecules capable of transporting
anions across a lipid membrane should be considered several
factors, including the binding affinity with a target anion,
lipophilicity and hydrophilicity for effective partitioning in
the lipid-water interphase, and mobility inside the lipid
bilayer. Despite much progress for the past several years, the
rational design of synthetic anion transporter still remains
highly challenging. Our approach therefore relies on the
systematic modification of the side chains (R1, R2 and R3) of
an initially designed molecule 4a. Syntheses are outlined in
Scheme 1. Ureas 3a–3h were synthesized by iodination of appro-
priate anilines,11 followed by the treatment with triphosgene.12
Then, Sonogashira reaction13 of 3a–3h with the corresponding
alcohols gave compounds 4a–4h.
The transmembrane transport of anions across biological
membranes is a crucial process to regulate ionic concentra-
tions, maintain cellular volume and pH, transmit electrical
signals, etc.1 Dysfunction of anion transport, e.g. chloride ion,
is therefore associated with serious diseases, such as cystic
fibrosis and Bartter’s syndrome.2 There are two distinct path-
ways, transmembrane channels and mobile carriers, for anions
to pass through the lipid membrane. Much effort has been
devoted to the synthesis and characterization of small syn-
thetic molecules capable of forming anion channels or func-
tioning as anion carriers,1,3 based on a variety of molecular
scaffolds, such as oligoamides,4 electron-deficient aryls,5
steroids,6 macrocycles,7 acyclic and cyclic pyrroles,8 and
others.9 The development of synthetic anion transporters may
provide us with an opportunity help to gain insight into the
underlying principles of anion transport in biological systems
and to find suitable compounds for biomedical applications.
In 2002, MacKinnon and coworkers reported the crystal
structure of a ClC chloride channel from S. typhimurium
(StClC) wherein chloride ion was held by four hydrogen
bonds; two with main-chain amide NHs (Ile 356 and Phe 357)
and two with side-chain OHs (Ser 107 and Tyr 445) (Fig. 1).10
Stabilized further by helix dipole interactions, the chloride ion
was surrounded by hydrophobic side chains of amino acids.
By mimicking the hydrogen-binding mode observed here, we
have prepared a series of anion receptors 4a–4h with different
side chains, each of which possesses a well-defined cavity with
convergent urea and hydroxyl groups. The receptors strongly
bind chloride ion by four hydrogen bonds like in StClC,
The binding mode of these receptors with chloride ion was
first confirmed by the X-ray analysis. Single crystals of
complex 4fÁTBACl were obtained by slow diffusion of heptane
into a toluene/CH2Cl2 solution containing 4f and excess
TBACl. As anticipated, the chloride ion is held by four
hydrogen bonds, two with urea NHs and two with terminal
OHs (Fig. 2). Two phenyl rings in the complex are significantly
tilted, and a dihedral angle is approximately 661 between two
phenyl planes. As the result, two hydroxyls are located back
and forth to create a three-dimensional cavity wherein the
chloride ion is completely entrapped with hydrogen bond
a Department of Chemistry, Yonsei University, Seoul 120-749, Korea.
E-mail: ksjeong@yonsei.ac.kr; Fax: +82-2-364-7050;
Tel: +82-2-2123-2643
b Interdisciplinary School of Green Energy, Ulsan National Institute of
Science & Technology, Ulsan, 689-798, Korea
w Electronic supplementary information (ESI) available: Characteri-
zation data of new compound, binding studies and details of transport
experiments. CCDC 875574. For ESI and crystallographic data in CIF
or other electronic format see DOI: 10.1039/c2cc35743g
Fig. 1 Schematic representation of the hydrogen-bonding mode
observed in the X-ray structure of StClC chloride channel.10
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Table 1 Association constants (Ka Æ 12%, M–1) and Gibbs energy
(kcal molÀ1) of complex for 4a–4h with tetrabutylammonium chloride
in 1% (v/v) H2O/CD3CN at 24 Æ 1 1C
Entry
Receptor
Ka (MÀ1
)
DG (kcal molÀ1
)
1
2
3
4
5
6
7
8
4a
4b
4c
4d
4e
4f
5100
8500
8000
9200
8700
17 000
13 000
17 000
À5.05
À5.35
À5.32
À5.40
À5.37
À5.76
À5.60
À5.76
4g
4h
(entry 2–5) and the steric bulkiness of substitents (R2 and R3)
at the propargylic positions (entry 6–8).
Next, the chloride transport abilities of 4a–4h across a lipid
bilayer membrane were compared by a fluorescence assay in
large unilamellar vesicles (LUVs) comprising 1-palmitoyl-
2-oleoyl-sn-glycero-3-phosphocholine (POPC).15 The LUVs
were loaded with NaNO3 (200 mM) and a chloride-sensitive
dye lucigenin (1 mM), and suspended in a phosphate buffer
solution (pH = 7.2) containing NaNO3 (200 mM) and NaCl
(30 mM). The flow of chloride ion into the vesicle was
monitored by addition of a DMSO solution of 4a–4h (1 mol%
relative to POPC), based on the gradual decrease of fluorescence
intensity of the internal lucigenin. After 400 s, detergent was
added to lyse the vesicles and final fluorescence intensity was set
as If. The results were shown in Fig. 3.
Scheme 1 Syntheses of receptors 4a–4h.
The activity of the chloride transport is negligible without
any anion receptor or in the presence of an initially prepared
compound 4a with R1 = t-butyl. We first attempted to
optimize the transport activity using a series of the esters
4b–4e which bear different types of alkyl groups; straight
(butyl, octyl), a branched (dimethyloctyl), and a more hydro-
philic chain (butoxyethoxyethyl). Although there were small
differences in the transport efficiency between the esters, the
improvement was not satisfactory at all. Then, the ester
functionality was replaced by chloro substituent to give com-
pound 4f which delightfully showed noticeable improvement
of the transport activity. In recent years, Gale et. al. demon-
strated nicely that the fluorinated substituents (e.g. CF3)
greatly increased the transport ability of an anion receptor
Fig. 2 (a) Front view and (b) side view of the crystal structure of 4f
with tetrabutylammonium chloride. The CH hydrogen atoms and
tetrabutylammonium are omitted for clarity, and hydrogen bonds
are shown as dashed lines.
distances of 3.32–3.36 A for NÁ Á ÁCl and 3.12–3.22 A for
OÁ Á ÁCl (see ESIw, Table S2).
More quantitative information on binding between anion
receptors 4a–4h and chloride ion were found in 1H NMR
spectroscopy in 1% (v/v) H2O/CD3CN. Upon addition of
TBACl, the NH and OH signals of 4a–4h were largely shifted
downfield by Dd = 0.83–0.95 and 0.92–1.06 ppm, respectively,
indicative of the formation of strong hydrogen bonds. Based
on changes in the chemical shifts of these protons, associa-
tion constants were determined by nonlinear squares fitting
analysis,14 and the results are summarized in Table 1. Two
trends are apparent. First, the magnitudes of the association
constants depend on the nature of substituents (R1) on the
phenyl ring. As expected, electron-withdrawing substituents
such as ester and chloro groups enhance the association
constant because of the increased hydrogen-bonding ability
of urea NHs by direct resonance. Second, the binding affinities
are essentially identical, regardless of the chain length of esters
Fig. 3 Chloride influx through vesicles containing 200 mM NaNO3,
1 mM lucigenin and 10 mM phosphate buffer pH 7.2.
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across lipid membranes.16 Further dramatic improvement was
observed when dimethyl side chains (R2 and R3) were changed
to more lipophilic groups, diethyl (4g) and methyl isobutyl
unit (4h). The binding affinities of chloro substituents 4f–4h
are essentially identical, and therefore the large enhancement
of the transport activity of 4g and 4h is possibly attributed to
the increased lipophilicity and mobility; that is, longer alkyl
chains such as isobutyl may wrap around chloride ion more
completely, thus facilitating the diffusion of the complex in the
lipid membrane.17
5 (a) V. Sidorov, F. W. Kotch, G. Abdrakhmanova, R. Mizani,
J. C. Fettinger and J. T. Davis, J. Am. Chem. Soc., 2002, 124, 2267;
(b) V. Gortaeu, G. Bollot, J. Mareda, A. Perez-Velasco and
S. Matile, J. Am. Chem. Soc., 2006, 128, 14788; (c) V. Sidorov,
F. W. Kotch, J. L. Kuebler, Y.-F. Lam and J. T. Davis, J. Am.
Chem. Soc., 2003, 125, 2840; (d) J. T. Davis, Nat. Chem., 2010,
2, 516.
6 (a) G. Deng, T. Dewa and S. L. Regen, J. Am. Chem. Soc., 1996,
188, 8975; (b) A. V. Koulov, T. N. Lambert, R. Shukla, M. Jain,
J. M. Boon, B. D. Smith, H. Li, D. N. Sheppard, J.-B. Joos,
J. P. Clare and A. P. Davis, Angew. Chem., Int. Ed., 2003, 42, 4931;
(c) B. A. McNally, A. V. Koulov, T. N. Lambert, B. D. Smith,
J.-B. Joos, G. Magro and A. P. Davis, Chem.–Eur. J., 2008,
14, 9599.
7 (a) N. Madhavan, E. C. Robert and M. S. Gin, Angew. Chem.,
Int. Ed., 2005, 44, 7584; (b) S. Licen, C. Coppola, J. D’Onofrio,
D. Montesarchio and P. Tecilla, Org. Biomol. Chem., 2009, 7, 1060;
(c) K. Hagiwara, Y. Sei, M. Akita and M. Yoshizawa, Chem.
Commun., 2012, 48, 7678.
8 (a) J. L. Sessler, L. R. Eller, W.-S. Cho, S. Nicolau, A. Aguilar,
J. T. Lee, V. M. Lynch and D. J. Magda, Angew. Chem., Int. Ed.,
2005, 44, 5989; (b) P. A. Gale, Chem. Commun., 2005, 3761;
(c) C. C. Tong, R. Quesada, J. L. Sessler and P. A. Gale, Chem.
Commun., 2008, 6321; (d) R. I. S. Diaz, J. Regourd,
P. V. Santacroce, J. T. Davis, D. L. Jakeman and A. Rhompson,
Chem. Commun., 2007, 2701.
9 (a) M. Wenzel, M. E. Light, A. P. Davis and P. A. Gale, Chem.
Commun., 2011, 47, 7641; (b) X. Li, B. Shen, X.-Q. Yao and
D. Yang, J. Am. Chem. Soc., 2007, 129, 7264; (c) B. A. McNally,
E. J. O’Neil, A. Nguyen and B. D. Smith, J. Am. Chem. Soc.,
2008, 130, 17274; (d) T. M. Fyles and H. Luong, Org. Biomol.
Chem., 2009, 7, 733; (e) W. Wang, R. Li and G. W. Gokel,
Chem.–Eur. J., 2009, 15, 10543; (f) S. K. Berezin and J. T. Davis,
J. Am. Chem. Soc., 2009, 131, 2458; (g) W. A. Harrell, Jr.,
M. L. Bergmeyer, P. Y. Zavalij and J. T. Davis, Chem. Commun.,
2010, 46, 3950; (h) N. J. Andrews, C. J. E. Haynes, M. E. Light,
S. J. Moore, C. C. Tong, J. T. Davis, W. A. Harrell, Jr. and
P. A. Gale, Chem. Sci., 2011, 2, 256; (i) S. Bahmanjah, N. Zhang
and J. T. Davis, Chem. Commun., 2012, 48, 4432; (j) C. Chhun and
A. R. Schmitzer, Med. Chem. Commun., 2011, 2, 987;
(k) C. J. E. Haynes, S. J. Moore, J. R. Hiscock, I. Marques,
P. J. Costa, V. Felix and P. A. Gale, Chem. Sci., 2012, 3,
1436; (l) J. T. Davis, P. A. Gale, O. A. Okunola, P. Prados,
J. C. Iglesias-Sanchez, T. Torroba and R. Quesada, Nat. Chem.,
2009, 1, 138.
To reveal the transport mechanism, the transport experi-
ment was also carried out under identical conditions except
using vesicles loaded with Na2SO4, instead of NaNO3. Even
compound 4h showed no activity of transporting chloride ion
into the vesicles containing Na2SO4 (EÀSIw, Fig. S10), implying
that the transport occurs by ClÀ/NO3 exchange mechanism.
In addition, no difference in the transport rate was found when
potassium chloride was used instead of sodium chloride (see
ESIw, Fig. S11), indicative of an antiport mechanism. Finally,
the transport rate of chloride ion was found to be linearly
proportional to the concentration of 4h (see ESIw, Fig. S12
and S13), suggesting that 4h function as a molecular carrier for
chloride ion, not forming a channel.
In conclusion, we have demonstrated that the chloride
transport across a lipid membrane can be drastically improved
by the systematic modification of synthetic receptors with the
bonding motif observed in a ClC chloride channel. In parti-
cular, the chloro substituent greatly enhances the efficiency of
the chloride transport, which can be further improved by the
incorporation of longer alkyl chains around the binding site
possibly due to the increased lipophilicity and mobility of the
chloride complex in the lipid membrane.
This work was supported by the National Research
Foundation of Korea (NRF) grand funded by the Korea
government (MEST) (2012-0004966). Y.R.C. acknowledges
the fellowship of the BK21 program from the Ministry of
Education and Human Resources Development. We thank
P. A. Gale (University of Southampton) for helpful comments
on the transport experiment.
10 R. Dutzler, E. B. Campbell, M. Cadene, B. T. Chait and
R. MacKinnon, Nature, 2002, 415, 287.
11 (a) W.-W. Sy, Synth. Commun., 1992, 22, 3215; (b) D. M. Lindsay,
W. Dohle, A. E. Jensen, F. Kopp and P. Knochel, Org. Lett., 2002,
4, 1819.
12 J. R. Hiscock, C. Caltagirone, M. E. Light, M. B. Hursthouse and
P. A. Gale, Org. Biomol. Chem., 2009, 7, 1781.
13 (a) K. Sonogashira, in Metal-Catalyzed Cross-Coupling Reactions,
ed. F. Diederigh and P. J. Stang, Wiley-VCH, Weinheim,
Germany, 1998, p. 203; (b) R. Chinchilla and C. Najera, Chem.
Rev., 2007, 107, 874.
14 (a) K. A. Connors, Binding Constants, John Wiley & Sons,
New York, 1987; (b) R. S. Macomber, J. Chem. Educ., 1992,
69, 375; (c) P. Thordarson, Chem. Soc. Rev., 2011, 40, 1305.
15 B. A. McNally, A. V. Koulov, B. D. Smith, J. B. Joos and
A. P. Davis, Chem. Commun., 2005, 1087.
16 (a) N. Busschaert, M. Wenzel, M. E. Light, P. Iglesias-Hernandez,
R. Perez-Tomas and P. A. Gale, J. Am. Chem. Soc., 2011,
133, 14136; (b) N. Busschaert, I. L. Kirby, S. Young, S. J. Coles,
P. N. Horton, M. E. Light and P. A. Gale, Angew. Chem., Int. Ed.,
2012, 51, 1; (c) S. J. Moore, M. Wenzel, M. E. Light, R. Morley,
S. J. Bradberry, P. Gomez-Iglesias, V. Soto-Cerrato, R. Perez-
Tomas and P. A. Gale, Chem. Sci., 2012, 3, 2501.
Notes and references
1 A. P. Davis, D. N. Sheppard and B. D. Smith, Chem. Soc. Rev.,
2007, 36, 348.
2 (a) D. N. Sheppard, D. P. Rich, L. S. Ostedgaard, R. J. Gregory,
A. E. Smith and M. J. Welsh, Nature, 1993, 362, 160;
(b) T. J. Jentsch, T. Maritzen and A. A. Zdebik, J. Clin. Invest.,
2005, 115, 2039.
3 (a) A. P. Davis, Coord. Chem. Rev., 2006, 250, 2939;
(b) B. A. McNally, W. M. Leevy and B. D. Smith, Supramol.
Chem., 2007, 19, 29; (c) J. T. Davis, O. A. Okunola and
R. Quesada, Chem. Soc. Rev., 2010, 39, 3843; (d) G. W. Gokel
and N. Barkey, New J. Chem., 2009, 33, 947; (e) S. Matile,
A. V. Jentzsch, J. Montenegro and A. Fin, Chem. Soc. Rev.,
2011, 40, 2453; (f) C. J. E. Haynes and P. A. Gale, Chem.
Commun., 2011, 47, 8203; (g) P. A. Gale, Acc. Chem. Res., 2011,
44, 216; (h) J. M. Boon and B. D. Smith, Curr. Opin. Chem. Biol.,
2002, 6, 749.
4 (a) P. H. Schlesinger, R. Ferdani, J. Liu, J. Pajewska, R. Pajewski,
M. Saito, H. Shabany and G. W. Gokel, J. Am. Chem. Soc., 2002,
124, 1848; (b) A. Hennig, L. Fischer, G. Guichard and S. Matile,
J. Am. Chem. Soc., 2009, 131, 16889; (c) T. Rezai, B. Yu,
G. L. Millhauser, M. P. Jacobson and R. S. Lokey, J. Am. Chem.
Soc., 2006, 128, 2510.
17 (a) L. W. Judd and A. P. Davis, Chem. Commun., 2010, 46, 2227;
(b) M. Yano, C. C. Tong, M. E. Light, F. P. Schmidtchen and
P. A. Gale, Org. Biomol. Chem., 2010, 8, 4356; (c) S. Hussain,
P. R. Brotherhood, L. W. Judd and A. P. Davis, J. Am. Chem.
Soc., 2011, 133, 1614; (d) V. Saggiomo, S. Otto, I. Marques,
V. Felix, T. Torroba and R. Quesada, Chem. Commun., 2012,
48, 5274.
c
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