Enhancement of cation transport in synthetic hydraphile channels having covalently-linked headgroups

Article abstract of DOI:10.1039/b005436o

A novel, pentamacrocyclic host molecule has been prepared in which four symmetry-equivalent diazacrown ethers lead to a dramatic enhancement in Na⁺ transport, across a phospholipid bilayer, relative to open-chained analogs lacking the fourth crown.

Full text of DOI:10.1039/b005436o

Enhancement of cation transport in synthetic hydraphile channels having
covalently-linked headgroups
Hossein Shabany and George W. Gokel*
Bioorganic Chemistry Program and Dept. of Molecular Biology & Pharmacology, Washington University School of
Medicine, 660 South Euclid Ave., Campus Box 8103, St. Louis, MO 63110, USA. E-mail: ggokel@molecool.wustl.edu
Received (in Columbia, MO, USA) 28th June 2000, Accepted 16th October 2000
First published as an Advance Article on the web
A novel, pentamacrocyclic host molecule has been prepared
in which four symmetry-equivalent diazacrown ethers lead
to a dramatic enhancement in Na+ transport, across a
phospholipid bilayer, relative to open-chained analogs
lacking the fourth crown.
diacid, HO2C(CH2)11 < N18N > (CH2)11CO2H, a compound
that was in hand from previous studies11 was converted into the
corresponding dichloride. The cyclization reaction between
H < N18N > (CH2)11CO < N18N > CO(CH2)11 < N18N > H and
ClCO(CH2)11 < N18N > (CH2)11COCl formed tetramide 6. Re-
duction of 6 (BH3/THF) gave tetraamine 7 as a yellow oil (76%
Our strategy1 to design functional, non-peptide cation-conduct-
ing channels2 has two key elements. First, the system was
designed to be flexible so that the structural features could adapt
if their exact physical organization had not been correctly
evaluated. Second, the planned design was modular so that a
compound exhibiting ionophoretic activity in a phospholipid
bilayer membrane could be selectively and readily altered. The
incorporation of flexibility into the framework could permit
minor structural adjustments to compensate for design flaws but
could also lead to a non-optimal structural arrangement. This, in
turn, could result in poorer function than that of which the
system was actually capable.
overall
Ph2CH2 < N18N > H
from
tris(macrocycle).
with ClCOC11O–C6H4–C6H4–O–
Treatment
of
C11COCl gave PhCH2 < N18N > CO(CH2)11O–C6H4–C6H4–
O–(CH2)11CO < N18N > CH2Ph (65%, yellow oil). Hydro-
genolytic debenzylation afforded the biphenyl-bridged
bis(macrocycle). The latter underwent cyclization with ClCO-
C11OC6H4C6H4OC11COCl to give tetraamide 2. Reduction of 2
(BH3/THF) afforded tetraamine 3 (72%, yellow oil). Com-
pounds 4 (40%), 5 (60%) and 8 (62%) were prepared in a
fashion similar to that shown in Scheme 1 and were charac-
terized by standard chemical methods (1H, 13C NMR and FAB-
MS). Dansyl channel 9 has been previously described.5
The first hydraphile structure 1,3 a tris(macrocycle), was
The Na+ transport efficacies of the compounds prepared as
part of this study were assessed in phospholipid bilayers by
using the dynamic 23Na NMR method of Riddell et al.12 This
method involves the formation of liposomes from phosphatidyl
glycerol and phosphatidyl choline in the presence of 100 mM
NaCl. The salt is present within the liposomes and in the
covalently-linked to define the overall transmembrane distance
but the corresponding opposite side pendant chains were
attached only at the distal macrocycles. The conformation
shown for structure 1 has been inferred by changing the
macroring sizes,4 by incorporating fluorescent residues,5 and by
other methods.6 The filled and open circles in the figure are
meant to represent Na+ and H2O, respectively, but details of the
structure within the membrane are not in hand nor are they
implied. Note that we use shorthand developed for the
purpose7
12 < N18N > C12 < N18N > C12 < N18N > C12.
Studies to determine optimal overall length,8 coupled with
to
represent
1
as
follows:
C
the efforts described above, have led to the conviction that we
could connect the two flexible sidechains to form a more rigid
and more efficacious structure. Compound 7 (Scheme 1) was a
target of this approach. The synthetic plan required a strategy
different from that that we had used previously. Compound 1,
which is not cyclic, can be constructed without the requirement
for a cyclization reaction.9 Tetramacrocycle 7, the key com-
pound in this report, was prepared by a sequence that is detailed
in
Scheme
1.
Monobenzyl-4,13-diaza-18-crown-610
(PhCH2 < N18N > H) was alkylated with methyl 12-bromodo-
decanoate. The ester was hydrolyzed, treated with oxalyl
chloride to give PhCH2 < N18N > (CH2)11COCl, and then
Scheme 1 Reagents and conditions: (a) Na2CO3, cat. KI, reflux 24 h, 83%;
(b) 2 M NaOH, reflux, 18 h, 98%; (c) (COCl)2, cat. DMF, 2 h; (d)
diazacrown, Et3N, cat. DMAP, 48 h, 71%; (e) Pd/C, 24 h, 95%; (f) step (c),
HO2C(CH2)11 < N18N > (CH2)11CO2H; (g) Et3N, cat. DMAP, 48 h, 70%;
(h) BH3/THF, 25 °C to rt, 48 h, 65%.
treated
with
diaza-18-crown-6
to
give
PhCH2 < N18N > (CH2)11CO < N18N > -
CO(CH2)11 < N18N > CH2Ph. Debenzylation was accom-
plished by hydrogenolysis to afford the tris(macrocycle). The
DOI: 10.1039/b005436o
Chem. Commun., 2000, 2373–2374
This journal is © The Royal Society of Chemistry 2000
2373

Table 1 Sodium transport by hydraphiles in phospholipid liposomes
surrounding bulk aqueous phase. The 23Na NMR spectrum
observed under these conditions is a singlet. Addition of a Dy3+
shift reagent to the aqueous phase renders the two Na+ ions
magnetically non-equivalent and two signals are observed.
Addition of an ionophore to the bilayer permits exchange of Na+
ions with a concomitant change in linewidth. The exchange rate
constant (k) can be evaluated from the linewidth change
according to the relation k = 1/t = p[(Dn 2 Dn0)]. In this
relationship, t is the half-life and n represents the linewidth. The
experimentally determined values of K are then compared with
An important question is whether the increase in Na+
transport may be attributed to the presence of the crown, to the
higher level of organization present where the two flexible
sidechains were, or to both. We know from the results reported
in the preceding communication that 4,4A-dihydroxybiphenyl is
inactive as a ‘central ion capsule.’ Thus, the fact that
ionophoretic activity of 7 is twice that of 5 confirms that both
variables are important. When only arenes are present as the
central units, no Na+ transport is measured in the NMR
experiment.
The present effort demonstrates that a tetramacrocyclic
hydraphile is more efficacious than its flexible counterpart. This
confirms that distance relationships, headgroup functions, and
ion–capsule interactions occur as previously surmised.
We thank the NIH (GM-36262) and the NSF (CHE-9805840)
for grants that supported this work.
the
value
determined
for
the
dansyl
channel
(Dn < N18N > C12 < N18N > C12 < N18N > Dn 9).13 The com-
pounds that were studied for the present report are recorded in
Table 1. Sodium cation transport by ionophores 1–9 was
investigated at very low concentration (0–20 mM). The rate is
reported as the relative rate krel = 100 kobs/k9. A value of < 2
means that the compound does not transport Na+ at a rate
sufficient to be observed under the experimental conditions.
Dansyl channel 9 is used as the standard rather than gramicidin
because the latter is ‘too robust’. When experiments are not
properly executed, gramicidin will still show transport behavior
but 9 requires a proper experimental environment and is
therefore a better control and standard.
Notes and references
1 G. W. Gokel, Chem. Commun., 2000, 1.
2 D. J. Aidley and P. R. Stanfield, Ion Channels: Molecules in Action,
Cambridge University Press, Cambridge, 1996; G. W. Gokel and
O. Murillo, Acc. Chem. Res., 1996, 29, 425; B. Hille, Ionic Channels of
Excitable Membranes, Sinauer Press, Sunderland, MA, 2nd edn.,
1992.
3 A. Nakano, Q. Xie, J. V. Malle´n, L. Echegoyen and G. W. Gokel, J. Am.
Chem. Soc., 1990, 112, 1287.
4 C. L. Murray, E. S. Meadows, O. Murillo and G. W. Gokel, J. Am.
Chem. Soc., 1997, 119, 7887.
5 E. Abel, G. E. M. Maguire, E. S. Meadows, O. Murillo, T. Jin and G. W.
Gokel, J. Am. Chem. Soc., 1997, 119, 9061; E. Abel, G. E. M. Maguire,
O. Murillo, I. Suzuki and G. W. Gokel, J. Am. Chem. Soc., 1999, 121,
9043.
6 O. Murillo, I. Suzuki, E. Abel and G. W. Gokel, J. Am. Chem. Soc.,
1996, 118, 7628.
7 J. C. Hernandez, J. E. Trafton and G. W. Gokel, Tetrahedron Lett., 1991,
6269.
8 C. L. Murray and G. W. Gokel, Chem. Commun., 1998, 2477.
9 O. Murillo, S. Watanabe, A. Nakano and G. W. Gokel, J. Am. Chem.
Soc., 1995, 117, 7665.
10 J. de Mendoza, F. Cuevas, P. Prados, E. S. Meadows and G. W. Gokel,
Angew. Chem., Int. Ed., 1998, 37, 1534.
11 Synthesis of HO2C(CH2)11 < N18N > (CH2)11CO2H was achieved from
the reaction of H < N18N > H with methyl 12-bromododecanoate,
followed by base hydrolysis.
12 F. G. Riddell and M. K. Hayer, Biochem. Biophys. Acta, 1985, 817, 313;
F. G. Riddell, S. Arumugam and B. G. Cox, J. Chem. Soc., Chem.
Commun., 1987, 1890.
13 E. Abel, G. E. M. Maguire, O. Murillo, I. Suzuki and G. W. Gokel,
J. Am. Chem. Soc., 1999, 121, 9043.
14 C. L. Murray, H. Shabany and G. W. Gokel, Chem. Commun., 2000,
2371.
15 S. L. De Wall, E. S. Meadows, L. J. Barbour and G. W. Gokel, Proc.
Natl. Acad. Sci. USA, 2000, 97, 6271.
Channel 1 transports Na+ with a rate 27% that of gramicidin
while its rate is 105% relative to 9, the standard used throughout
this study. The most important finding of the study is that when
an additional crown ether is present as a central unit to organize
water (see preceding communication), joining the flexible
sidechains with a fourth crown produces an ionophore sig-
nificantly (3.5-fold) more active than 1. The very high efficacy
of this structure supports previous conclusions concerning
distance and polarity requirements. The family of structures
also permits us to probe the effects of certain of the modular
subunits.
First, we note that both compounds 2 and 3 are inactive in the
23Na NMR experiment. We attribute this to the lack of a water-
organizing central unit (see preceding communication).14 When
the center of the channel possesses one biaryl unit and an amide-
substituted diazacrown 4, activity is restored but it is modest
(30% of 9). Reduction of the amide residues in 4 affords 5 which
has substantially increased Na+ transport activity (150% of 9).
An increase in transport is expected because the amide residues
conformationally restrict the macrocycle to which they are
attached. The tetraamide precursor to 7, i.e. 6, is considerably
less flexible than 7 but is also 2.5-fold more active than 1. There
may also be a deleterious effect of an arene on cation transport.
In the low polarity environment of the phospholipid bilayer, a
cation–p interaction between Na+ and benzene15 could sub-
stantially diminish the transport rate. Thus, the rate for 8, which
lacks an arene, exceeds that of 5 by a small but significant
amount.
2374
Chem. Commun., 2000, 2373–2374