From cholapod to cholaphane transmembrane anion carriers: Accelerated transport through binding site enclosure

Article abstract of DOI:10.1039/b927005a

Cyclosteroidal "cholaphane" anion transporters show increased activities compared to acyclic "cholapod" analogues.

Full text of DOI:10.1039/b927005a

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From cholapod to cholaphane transmembrane anion carriers: accelerated
transport through binding site enclosurew
Luke W. Judd and Anthony P. Davis*
Received (in Cambridge, UK) 22nd December 2009, Accepted 9th February 2010
First published as an Advance Article on the web 25th February 2010
DOI: 10.1039/b927005a
Cyclosteroidal ‘‘cholaphane’’ anion transporters show increased
activities compared to acyclic ‘‘cholapod’’ analogues.
allows them to locate in membranes, extract moderately
hydrophilic anions such as chloride or nitrate, and carry them
through the membrane interior. Activities can be increased
by placing electron-withdrawing groups in the aromatic urea
substituents, by converting ureas to thioureas and by mani-
pulating the group at steroidal C3 (see 1 for numbering).4c
The most powerful examples can effect measureable chloride
transport through vesicle membranes with cholapod/lipid
ratios of 1 : 250 000.
Anion transport across cell membranes is a vital biological
process. Concentration gradients of anions, especially chloride,
must be regulated to control membrane potentials, intra-
cellular pH, cell volume and other transport-related phenomena.1
The dysfunction of chloride channels is associated with impor-
tant human diseases (‘‘channelopathies’’), including cystic
fibrosis, Dent’s disease, myotonia and epilepsy.2 The study
of biological ion transport can be facilitated by small mole-
cules which mimic the action of natural transport systems.
However, while selective cation transporters such as Valinomycin
are readily available and widely used, there is still no equivalent
for anion transport. Progress has been made on synthetic anion
channels and carriers,3 but none is yet established as a prac-
tical research tool. The potential for using these systems to
treat channelopathies, by replacement of missing activities, also
remains unfulfilled.
While the alterations which raise transport activities also
tend to increase H-bond donor effectiveness, the correlation
between activities and affinities is not perfect. Other factors are
clearly at play, and there is therefore potential for increasing
transport rates through other types of structural change. For
example, it is possible that transport could be slowed by the
interaction of bound anions with water at the membrane
boundary. A more enclosed binding site might help to desolvate
the anion and speed the rate of transfer through the membrane.
We now report the synthesis and study of the macrocyclic
cholaphane6 transporters 3 and 4. Cyclisation yields higher
transport rates without greatly affecting affinities. The results
imply that substrate encapsulation can indeed raise performance
in steroid-based anion transporters.
Cholaphanes 3 and 4 were conceived as cyclic analogues of 1
and 2, providing similar arrays of H-bond donors but much
higher levels of steric shielding. Molecular modelling suggested
that chloride ions should be accepted into the binding sites,
and that once bound they should be substantially protected
from interaction with solvent (see Fig. 1). The projected
syntheses involved treatment of steroidal diamines 5 with
the bis-isocyanato strap precursor 6. Bis-isocyanate 6 was
prepared as shown in Scheme 1. Commercially available ester
7 was a-brominated and treated with PPh3 to give phospho-
nium salt 8. Wittig reaction with isophthalaldehyde 9 gave
dienes 10, which were converted to bis-acyl azide 11 via
hydrogenation, ester hydrolysis and coupling to N3À. Thermo-
lysis of 11 in refluxing toluene gave Curtius rearrangement to
6. Bis-isocyanate 6 was converted to cholaphanes 3 and 4 as
shown in Scheme 2. In the former case, the 3a-acetoxy
We have previously shown that cholapods such as 1 and 2 can
serve as effective anion transporters.4 These molecules can
bind anions with high affinities through preorganised arrays of
H-bond donors, while maintaining lipophilicity due to the
steroidal framework.5 It seems this combination of properties
School of Chemistry, University of Bristol, Cantock’s Close, Bristol,
UK BS8 1TS. E-mail: Anthony.Davis@bristol.ac.uk;
Fax: +44 (0)117 929 8611; Tel: +44 (0)117 954 6334
w Electronic supplementary information (ESI) available: Procedures
for the synthesis of 3 and 4, details of molecular modelling and of
transport and binding measurements. See DOI: 10.1039/b927005a
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Scheme 2 (i) TFA, DCM, 16 h; (ii) 6, toluene 16 h; (iii) AcOH, zinc,
12 h, 100%; (iv) DIPEA, trifluoroacetic anhydride, DCM, 16 h, 53%.
Fig. 1 Structure of 4ÁClÀ as predicted by modelling (ESIw). Also
shown is the solvent-accessible surface, calculated using a probe of
radius 1.4 A.
(25 mM final concentration) was added and the chloride influx
was monitored via decay in fluorescence. The initial rate of
fluorescence decay was converted to the initial rate of chloride
influx using the method of Pope and Leigh,9 applied to the
lucigenin assay by Wissing and Smith (ESIw).10 Transport
is assumed to occur via an antiport mechanism, whereby
electroneutrality is maintained by counter-transport of nitrate
anions.11
steroidal precursor 127 was treated with TFA to remove the
Boc protecting groups and, after basic workup, the resulting
diamine was allowed to react with 6 to give macrocycle 3. A
similar procedure was applied to 13 to give 14, after which
azide reduction and trifluoroacetylation gave cholaphane À4.
The affinities of 3 and 4 for Et4N+ClÀ and Et4N+NO3 in
water-saturated chloroform were measured by a modified
version of Cram’s extraction method, as described previously.8
Anion transport was studied in vesicles, using a method based
on the ability of chloride ions to quench the emission of the
fluorescent dye lucigenin.4b The method employed large
unilamellar vesicles (0.4 mM, 200 nm mean diameter) which
were composed of 1-palmitoyl-2-oleoylphosphatidylcholine
(POPC) and cholesterol in a 7 : 3 ratio. The receptors were
mixed with the lipid at the ratio of 1 : 2500. The vesicles
were prepared with internal and external aqueous NaNO3
(225 mM) and internal lucigenin (1 mM) (ESIw). NaCl
Results for both affinity and transport measurements are
given in Table 1, while traces for typical transport experiments
are shown in Fig. 2. In terms of binding constants, it appears
that cyclisation has mixed effects. Affinities for Et4N+ClÀ
are slightly increased, by factors of 1.6 (for 3 vs. 1) andÀ1.5
(for 4 vs. 2). On the other hand, binding to Et4N+NO3 is
reduced by factors of 2.4 (for 3 vs. 1) and 2.5 (for 4 vs. 2).
Table 1 Initial rates of ClÀ influx to vesicles promoted by 1–4, and
binding constants to Et4N+ salts in water-saturated chloroform
Initial ClÀ
influx rate/
mM minÀ1
Ka
(Et4N+ClÀ)/MÀ1
Ka
(Et4N+NO3À)/MÀ1
Receptor
1
2
3
4
0.36
2.06
6.46
8.13
1.5 Â 107
2.8 Â 108
2.4 Â 107
4.3 Â 108
1.0 Â 107
1.1 Â 108
4.2 Â 106
4.4 Â 107
Scheme 1 (i) NBS, methyl acetate, hn, reflux 16 h, 100%; (ii) PPh3,
acetone, 16 h, 72%; (iii) NaOMe, reflux 16 h, 73%; (iv) Pd/C. EtOH,
H2 (4 atm), rt, 48 h, 99%; (v) NaOH, MeOH, H2O, reflux, 1 h, 84%;
(vi) SOCl2, Et3N, DCM, reflux, 2 h; (vii) NaN3, THF, H2O, 2 h;
(viii) toluene, reflux, 2 h.
Fig. 2 Chloride transport into vesicles (7 : 3 POPC/cholesterol)
facilitated by cholapods 1 and 2 and cholaphanes 3 and 4 at a
1 : 2500 receptor to lipid ratio. Each line is a representative data set.
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In contrast, the influence of cyclisation on transport rates is
consistently positive, and can be very substantial. Thus, the
3a-acetoxy cholaphane 3 promotes chloride–nitrate exchange
18 times more effectively than the analogous cholapod 1. In
the case of the 3a-trifluoroacetamides 2 and 4 the cyclic
structure is 4 times more active, a smaller but significant
enhancement.
Notes and references
1 Chloride Movements Across Cellular Membranes, Advances in
Molecular and Cell Biology, ed. M. Pusch, Elsevier, San Diego,
2007, vol. 38; T. J. Jentsch, V. Stein, F. Weinreich and
A. A. Zdebik, Phys. Rev., 2002, 82, 503.
2 T. J. Jentsch, C. A. Hubner and J. C. Fuhrmann, Nat. Cell Biol.,
2004, 6, 1039; F. M. Ashcroft, Ion Channels and Disease, Academic
Press, London, 2000.
3 Reviews: A. P. Davis, D. N. Sheppard and B. D. Smith, Chem. Soc.
Rev., 2007, 36, 348; X. Li, Y. D. Wu and D. Yang, Acc. Chem.
Res., 2008, 41, 1428; J. Mareda and S. Matile, Chem.–Eur. J., 2009,
15, 28; G. W. Gokel and N. Barkey, New J. Chem., 2009, 33, 947;
Recent examples: P. V. Santacroce, J. T. Davis, M. E. Light,
P. A. Gale, J. C. Iglesias-Sanchez, P. Prados and R. Quesada,
J. Am. Chem. Soc., 2007, 129, 1886; B. A. McNally, E. J. O’Neil,
A. Nguyen and B. D. Smith, J. Am. Chem. Soc., 2008, 130, 17274;
P. V. Jog and M. S. Gin, Org. Lett., 2008, 10, 3693; I. Izzo,
S. Licen, N. Maulucci, G. Autore, S. Marzocco, P. Tecilla and
F. De Riccardis, Chem. Commun., 2008, 2986; 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.
The data imply that cyclisation to a cholaphane architecture
is a useful strategy for improving the transport properties of
cholapods. The effect does not seem to be related to chloride
binding affinities. Although the cholaphanes bind chloride
more strongly than the cholapods, the difference is very small.
Previous studies suggest that, when cholapods are closely
related, a 40-fold increase in binding power will result in a
roughly 10-fold increase in transport rates.4c On this basis, the
affinity increase observed here (factor of B1.5) should have a
minimal effect.
Two alternative explanations may be considered. Firstly,
the binding results suggest that cyclisation promotes selectivity
for chloride. For example, in the case of 2 and 4, chloride/
nitrate selectivities are 3 and 10 respectively. Results from our
earlier work on cationic cholaphanes point in the same
direction.6d Chloride/nitrate selectivity brings no advantage
(indeed, it could be counterproductive in our antiport system),
but other selectivities could be beneficial. In particular, selecti-
vity for chloride vs. phosphodiester monoanions could be
critical. It is possible that transport could be inhibited by the
binding of receptor molecules to lipid head-groups at the
surface of the membrane.12 Cholaphanes 3 and 4 might show
selectivity against the head-groups and might thus be less
affected than the corresponding cholapods.
4 (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 and
A. P. Davis, Angew. Chem., Int. Ed., 2003, 42, 4931;
(b) B. A. McNally, A. V. Koulov, B. D. Smith, J. B. Joos and
A. P. Davis, Chem. Commun., 2005, 1087; (c) B. A. McNally,
A. V. Koulov, T. N. Lambert, B. D. Smith, J. B. Joos, A. L. Sisson,
J. P. Clare, V. Sgarlata, L. W. Judd, G. Magro and A. P. Davis,
Chem.–Eur. J., 2008, 14, 9599.
5 A. P. Davis and J.-B. Joos, Coord. Chem. Rev., 2003, 240, 143;
A. P. Davis, Coord. Chem. Rev., 2006, 250, 2939.
6 The term ‘‘cholaphane’’ is used for macrocycles formed from bile
acid and aromatic units. See: (a) A. P. Davis, R. P. Bonar-Law and
J. K. M. Sanders, in Comprehensive Supramolecular Chemistry,
ed. Y. Murakami, Pergamon, Oxford, 1996, vol. 4 (Supramolecular
Reactivity and Transport: Bioorganic Systems), p. 257;
(b) E. Virtanen and E. Kolehmainen, Eur. J. Org. Chem., 2004,
3385; (c) A. P. Davis, Molecules, 2007, 12, 2106. We have
previously described two anion-binding cholaphanes containing
quaternary ammonium groups; see: (d) A. L. Sisson, J. P. Clare
and A. P. Davis, Chem. Commun., 2005, 5263. Although these
systems showed interesting anion phase transfer properties, the
positive charge renders them unsuitable for membrane transport
(see discussion in ref. 4c).
Secondly, as originally hypothesised, the effect may relate to
anion desolvation. Although the cholapods bind anions very
strongly, their substrates remain relatively exposed to the
external environment. For example, modelling suggests that
a chloride ion bound to 2 can be solvated by at least 3 water
molecules. In contrast, a chloride ion bound to 4 is shielded
more effectively, with room for perhaps one water molecule to
access the anion (see Fig. 1). This difference may be significant
because bound water molecules can link to bulk water through
hydrogen bonding at the membrane interface. These bonds
may need to be broken before the complex can traverse the
membrane, and this may provide a barrier to transport.
Substrate encapsulation is a common feature of cation carriers
such as Valinomycin and Nonactin.13
7 V. del Amo, L. Siracusa, T. Markidis, B. Baragana,
K. M. Bhattarai, M. Galobardes, G. Naredo, M. N. Perez-Payan
´ ´
and A. P. Davis, Org. Biomol. Chem., 2004, 2, 3320.
8 (a) D. J. Cram and J. M. Cram, Science, 1974, 183, 803;
(b) A. J. Ayling, S. Broderick, J. P. Clare, A. P. Davis,
M. N. Pe
Chem.–Eur. J., 2002, 8, 2197; (c) J. P. Clare, A. J. Ayling, J. B. Joos,
A. L. Sisson, G. Magro, M. N. Perez-Payan, T. N. Lambert,
rez-Payan, M. Lahtinen, M. J. Nissinen and K. Rissanen,
´ ´
´
´
R. Shukla, B. D. Smith and A. P. Davis, J. Am. Chem. Soc., 2005,
127, 10739.
9 A. J. Pope and R. A. Leigh, Planta, 1988, 176, 451.
10 F. Wissing and J. A. C. Smith, J. Membr. Biol., 2000, 177, 199.
11 As described in ref. 4a, the antiport mechanism has been
conclusively demonstrated for cholapod 1.
12 It has been shown that cholapods can carry phospholipid
head-groups through membrane interiors. This implies that the
cholapod-head-group interaction is significant, and could well
interfere with chloride transport. See: T. N. Lambert,
´ ´
J. M. Boon, B. D. Smith, M. N. Perez-Payan and A. P. Davis,
J. Am. Chem. Soc., 2002, 124, 5276.
13 B. T. Kilbourn, J. D. Dunitz, L. A. R. Pioda and W. Simon,
J. Mol. Biol., 1967, 30, 559; K. Neupert-Laves and M. Dobler,
Helv. Chim. Acta, 1975, 58, 432.
Although we cannot distinguish between these options
at present, the results suggest that the cyclic cholaphane
architecture is especially promising for transmembrane anion
transport. In future research we hope to prepare cholaphanes
with higher anion affinities, and the potential to set new
standards as anion carriers.
Financial support from the BBSRC (BBS/S/E/2006/13241),
and the EPSRC (EP/F03623X/1) is gratefully acknowledged.
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Chem. Commun., 2010, 46, 2227–2229 | 2229