"aplosspan:" A bilayer-length, ion-selective ionophore that functions in phospholipid bilayers

Article abstract of DOI:10.1039/b816819a

A structurally simple, novel, membrane-active ionophore has been designed, prepared, characterized, and shown to conduct Na⁺, Cl^-, and carboxyfluorescein anions, probably as a dimer, across liposomal bilayers. The Royal Society of Ch

Full text of DOI:10.1039/b816819a

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‘‘Aplosspan:’’ a bilayer-length, ion-selective ionophore that functions
in phospholipid bilayersw
Wei Wang,a Ruiqiong Lia and George W. Gokel*b
Received (in Austin, TX, USA) 24th September 2008, Accepted 23rd December 2008
First published as an Advance Article on the web 26th January 2009
DOI: 10.1039/b816819a
A structurally simple, novel, membrane-active ionophore has
been designed, prepared, characterized, and shown to conduct
Na+, Clꢀ, and carboxyfluorescein anions, probably as a dimer,
across liposomal bilayers.
Synthetic ion channels, developed originally as biomimetic
entities1 that it was hoped would mimic natural channel
function, have proved themselves to be more than simply
models.2 In fact, synthetic ion channels function in liposomal
and planar phospholipid bilayers and even in vital cells.3 They
exhibit the classic open–close behavior characteristic of
protein channels and natural pores. In many cases, they show
proton, cation, or anion selectivity and even rectification.4 It
seemed clear that for a channel that will span a bilayer, three
minimum elements are required: (1) the channel must be of
sufficient length, (2) a polar central relay element must be
present, and (3) polar headgroups are required to make the
overall assembly amphiphilic. We note that these are necessary
conditions, but their presence does not guarantee either a
membrane-spanning conformation or channel function.
A few relatively simple membrane-spanning transporters or
membrane disruptors have been reported.5 Recent reports
Scheme 1
describe compounds that do not obviously meet these conditions,
but are clearly membrane active.6 Despite confirmed function,
having the size and polarity thought to be required to transport
it is sometimes unclear how the transporters function as a
ions. The first example of the family was prepared, characterized
conductance pore.7 We therefore considered the question of
and assayed for ion transport activity. Such simple structures
how simple a functional, potentially membrane-spanning
that are membrane-active are sometimes referred to by the term
amphiphile could be. There is no fossil or other record from
‘‘disruptor.’’ This name is pejorative and the simple structures
which any early channel structure can be inferred. The earliest
channels must have been simple, poorly selective, and only
discussed here form functional channels. We suggest the name
aplosspan from the Greek aplos (simple) + span for simple,
¨
marginally rectifying. It is unknown if the earliest channels
were even peptides. It should be noted that Fyles and
coworkers have also considered the issue of extremely
simple, membrane-active compounds, although their structures
were designed as leaflet-spanning amphiphiles rather than
membrane-spanning bolaamphiphiles.8
membrane-spanning structures that mediate ion transport.
For simplicity of design and synthesis, the hydroxyl groups
of diethanolamine were chosen as headgroups, and biphenyl
and alkyl chain units composed the hydrophobic tunnels.9
Matile, Sakai, and their coworkers have used arenes exten-
sively and successfully in their family of ‘‘rigid rod’’ channels.10
Acetylated 1,3-diaminobenzene was chosen as the central
relay. The amide residues should serve the water-organizing
role11 of the central relay. Moreover, Kobuke and coworkers
successfully incorporated a bis(aminomethyl)benzene unit
in a synthetic channel that showed rectification properties.4
The compound that was prepared and studied is shown
below as 1 (space-filling model) along with its preparation
(see Scheme 1).
Simple primordial channels must have existed or the
evolution of boundary membranes would have been inimical
to survival. Using the three criteria noted above, we designed
compounds that incorporated synthetically accessible subunits
a Department of Chemistry, Washington University, St. Louis,
MO 63130, USA
b Departments of Chemistry & Biochemistry and Biology, Center for
Nanoscience, University of Missouri – Saint Louis, One University
Boulevard, Saint Louis, MO 63121, USA. E-mail: gokelg@umsl.edu;
Tel: +1 314-516-5321
w Electronic supplementary information (ESI) available: Carboxy-
fluorescein release, Hill plot of compound 1, cation vs. anion
selectivity, synthesis of 1, sodium release experiments. See DOI:
10.1039/b816819a
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The preparation of 1 is shown in Scheme 1. 12-Bromodo-
decanoic acid was converted to the acid chloride ((ClCO)2)
and then coupled with 1,3-phenylenediamine (84%) to afford
Br(CH2)11CONHC6H4NHCO(CH2)11Br (i). Diethanolamine’s
hydroxyl groups were protected (ii, t-butyldimethylsilyl
ethers, 74%) and N-acylated (iii, ClCH2COCl, CH2Cl2,
74%).
4,40-Dihydroxybiphenyl
was
coupled
to
ClCH2CON(CH2CH2OTBDMS)2 (iv, Na2CO3, KI, D, PrCN,
55%) to give HOC6H4C6H4OCH2CON(CH2CH2OTBDMS)2.
The synthesis was completed by reaction of the biphenol
fragment with the 1,3-diamide (v, Na2CO3, KI, D, PrCN,
19%) followed by hydroxyl group deprotection (vi, HCl,
EtOH, 93%). Tetraol 1 is a slightly brown solid, mp B189 1C
(dec).12 Full experimental and spectral details are included in
the ESI.w
It seemed possible that either cations or anions or both
could be transported by 1. The activity of 1 was surveyed by
recording ion release from liposomes. This method is more
convenient and less time consuming than using the planar
bilayer voltage clamp (BLM) method. The latter method was
also used as described below because ‘‘this is the sole method
to unequivocally establish the presence of channel activity, in
contrast to detergent effects that might be expected for
amphiphilic compounds’’.8 Initially, sodium ion release from
dioleolylphosphatidylcholine (DOPC, 0.4 mM) vesicles
mediated by 1 was assayed by use of a sodium ion selective
electrode (ISE). The vesicles (200 nm) were suspended in a
sodium-free buffer consisting of 750 mM choline chloride and
15 mM HEPES at pH = 7.0. The internal buffer was 750 mM
NaCl and 15 mM HEPES at pH = 7.0. Compound 1 was
dissolved in DMSO and added to an aqueous suspension of
liposomes. Sodium cation release was monitored by using
a sodium ISE; vesicle preparation and ISE methodology
have been reported.13 Cation release from vesicles showed
predictable behavior, increasing proportionally over a nine-fold
concentration range (Fig. 1).
Fig. 2 Current–time records showing the channel gating behavior of
1 (asolectin, 1 mM) (A) gating current 0.80 pA, applied voltage 60 mV,
3 min into experiments; (B) gating current 1.44 pA, applied voltage
60 mV, 9 min into experiments; (C) gating current 1.86 pA, applied
voltage 70 mV, 25 min; (D) gating current 5.5 pA, applied voltage
40 mV, 2 h. Buffer: 450 mM KCl, 10 mM HEPES, pH = 7.0.
concentration range, suggests that more than one monomer is
involved in the conducting pores. We note that transport
activities assayed by ISE or by fluorescence methods are not
directly comparable owing to differences in the analytical
methods and conditions.16
Channel behavior was confirmed for 1 by a planar bilayer
conductance study. Compound 1 (1 mM) was added to the cis
chamber in a planar bilayer voltage clamp apparatus. The
buffer was 450 mM KCl, 10 mM HEPES, pH = 7 and the
bilayer was formed from asolectin (soybean). Traces obtained
during the experiments are shown in Fig. 2. Ion selectivity
experiments were conducted using an asymmetric buffer
solution (trans: 450 mM KCl, 10 mM HEPES, pH = 7; cis:
3 M KCl, 10 mM HEPES, pH = 7). A current–voltage (I–V)
plot revealed a Vr (reversal membrane potential) value of
ꢀ14.95 mV. The Goldman–Hodgin–Katz equation was used
to calculate a selectivity of P(K+)/P(Clꢀ) = 2.3, indicating
that 1 is a modestly selective cation channel (experimental
details in ESIw).
Membrane permeability14 was further assayed by carboxy-
fluorescein (CF) release (fluorescence dequenching) from
DOPC liposomes (see ESIw). The log–log plot of v/Vm ꢀ v
(ordinate) vs. concentration gave a straight-line relationship
(r2 = 0.89) with a slope of 1.5 for the CF transport data for 1.
This Hill plot,15 although evaluated over a limited (6-fold)
The planar bilayer conductance data revealed two facts.
First, the channels or pores formed from 1 exhibited classic
open–close behaviour. Second, the initial conductance was
B13 picoSiemens (pS, trace A). However, during the next
several hours, the conductance pore gradually enlarged,
suggesting a higher aggregation state and larger pore. After
2 h, the conductance had increased from 0.013 nS (13 pS) by
about tenfold to B0.14 nS. Using the Hille equation, we
calculated the corrected Hille diameter,17 dHille 4 2.6 A and
enlarged over time to dHille 4 9.3 A. A dimeric or trimeric pore
of diameter 2.6 A that enjoys some flexibility is of sufficient
size to pass Na+ (ionic diameter: 1.9 A) or K+ (2.7 A) ions if
they are not fully solvated. Zhou et al.18 have calculated the
hydrated radii of Na+ and K+ to be 5.98 A and 5.50 A,
respectively, and they would presumably be too large to be
transported in that form. The calculated hydrated diameter of
Clꢀ is 6.48 A from the same source and the ionic diameter is
Fig. 1 Fractional sodium release from liposomes (DOPC, 0.4 mM)
mediated by 1. External buffer (750 mM choline chloride, 15 mM
HEPES, pH=7.0), internal buffer (750 mM NaCl, 15 mM HEPES,
pH = 7.0).
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3.6 A. The conductance pathway of the ClC protein chloride
transporter is thought to pass chloride ions in a chain bridged
by dynamic (disordered) water molecules.19
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This work demonstrates several important principles. First,
exceptionally simple monomers form pores that are functional
channels. Compound 1 was designed to be long enough to
span the bilayer. It forms oligomeric pores that exhibit classic
open–close behavior, although the aggregation state and pore
size appear to increase over time. Nevertheless, this extremely
simple, membrane-length compound is both functional and
selective for K+ over Clꢀ. These compounds were designed
to demonstrate that complex design criteria such as the
incorporation of, for example, crown ethers20 or guanine21
residues, while potentially beneficial, may not be required for
transport function. Compound 1 appears to fulfil the three
criteria noted in the beginning of this report, although detailed
evidence for bilayer conformation such as we reported for
hydraphiles20 is laborious to obtain. Our studies show that
compounds that were designed to incorporate elements
expected to impart specific properties should be compared
with much simpler analogs to confirm that the design element
is functioning as imagined.
12 1H-NMR (DMSO-d6): 1.31–1.61 (36H, m), 2.29–2.32 (4H, m),
3.46–3.63 (16H, m), 4.01 (4H, m), 4.95 (4H, s), 6.97–7.02 (8H, m),
7.20–7.31 (3H, m), 7.51–7.56 (8H, m), 7.96 (1H, s), 9.88 (2H, s).
13C-NMR: 25.20, 25.27, 36.42, 47.92, 49.49, 58.64, 58.78, 65.66,
67.51, 110.19, 113.97, 114.85, 114.96, 127.03, 127.256, 128.68
132.24, 132.53, 139.60, 157.43, 157.83, 167.71, 171.36. FAB-MS
for silyl protected 1, m/z calcd.: (M + Na) 1609.9912, found:
1609.9950.
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We thank the NIH for support of this work through grant
GM 36262.
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