Synthesis and characterization of a redox-active ion channel supporting cation flux in lipid bilayers

Article abstract of DOI:10.1039/b907350g

The synthesis, cation binding and transmembrane conductive properties of a novel synthetic ion channel containing a redox-active ferrocene unit are described. Fluorescence spectroscopy was used to demonstrate that the channel supports multiple ion coordin

Full text of DOI:10.1039/b907350g

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Synthesis and characterization of a redox-active ion channel supporting cation
flux in lipid bilayers
b
a
a
a
a
a
Maia Tsikolia,* Adam C. Hall, Cristina Suarez, Zazi O. Nylander, Sarah M. Wardlaw, Molly E. Gibson,
a
a
a
a
a
Kerry L. Valentine, Louisa N. Onyewadume, Deborah A. Ahove, Maya Woodbury, Margaret M. Mongare,
b
b
b
b
C. Dennis Hall, Zuoquan Wang, Bogdan Draghici and Alan R. Katritzky
Received 14th April 2009, Accepted 10th June 2009
First published as an Advance Article on the web 22nd July 2009
DOI: 10.1039/b907350g
The synthesis, cation binding and transmembrane conductive properties of a novel synthetic ion
channel containing a redox-active ferrocene unit are described. Fluorescence spectroscopy was used to
demonstrate that the channel supports multiple ion coordination and association constants for 1:1 and
+
+
1
:2 (channel:cation) coordination for both Na and K were evaluated. Experiments using a black lipid
membrane preparation revealed that this compound functioned effectively as an ion channel for both
+
+
23
+
Na and K . Concomitant Na NMR spectroscopy studies supported this finding and revealed a Na
flux, at least 5 times higher than ion transport rates by monensin. Furthermore, oxidation of the
2
+
3+
redox-active centre (Fe to Fe ) effectively inhibited ion transport.
Introduction
fluorescence spectroscopy was used to study qualitative and quan-
titative aspects of cation binding, by inhibition of photoelectron
transfer from tricoordinate nitrogen to the photoexcited state of
pendant 9-methylanthryl groups. The fluorophores also served to
The passage of ions and other materials across cellular membranes
is a crucial biological process and hence extensive research has
been devoted to understanding mechanisms of ion transport.
During the last two decades, efforts have been made to mimic the
action of biological ion channels by the synthesis of model systems
that span lipid bilayers. Synthetic proteins have been designed to
mimic natural channels, albeit with a single span rather than the
multiple transmembrane domains typical of biological systems.
Other designs include self-assembling cyclic peptide nanotubes
that have been shown to be toxic to bacteria by introducing a pore
1
,2
‘
anchor’ the channel in phospholipid bilayers.
Two techniques were used to characterize conductance across
bilayers mediated by 9. Ion channel activity was first assessed
3
2
using Na NMR spectroscopy in order to measure cumulative
18–20
rates of cation transport across a vesicle membrane.
This
4
method relies on monitoring the rate of exchange of the cation
between the intra- and extra-vesicular environments where the
chemical shift of the extra-vesicular cation is subject to a
5
5c
6
through the plasma membrane and metal-organic frameworks
3+
chemical shift reagent (e.g. Dy ). This technique has been widely
which may offer a new dimension to synthetic ion channels. During
the last decade selective artificial channels have been fashioned
with head groups (e.g. macrocyclic polyethers or carbonyl groups)
to capture specific ions, long aliphatic chains designed to span the
bilayer and‘relay units’, such as other macrocyclicethers, toshuttle
13
exploited, especially by Gokel et al., to examine rates of cation
transport across membranes infused with artificial channels. The
impact of oxidation of 9 on its potential as a conducting ion
channel was also determined using the same methodology. The
second method employed the black lipid membrane technique
to record single (or multiple) open channel events evoked by
the artificial ion channel, enabling calculations of single channel
conductance and current–voltage relationships for the ion channel
activity.
7–12
ions across the membrane. The ‘relay unit’ is seen as a potential
means of stabilizing cations through hydration by hydrogen-
13
9
bonded water or coordination with carbonyl functions. This
multiple ion transport, known as the ‘billiard ball effect’ (in
14
essence, the charge repulsion theory of multiple ion transport )
has been corroborated in channels using other head groups such
The artificial channel used in this study was designed with four
specific features in mind:
15
as the calixarenes. Electrospray mass spectrometry has served to
enhance this view of multiple ion complexation in some artificial
˚
(
i) the channel length was 35–40 A in an attempt to mimic the
dimensions of biological channels;
ii) the terminal nitrogen atoms were equipped with 9-
16
channels.
In the following study we describe a novel synthetic ion channel
(
methylanthryl groups to afford fluorescence detection of cation
coordination and to function as hydrophobic anchors within a
lipid membrane;
1
7
containing a ferrocene unit (9, Scheme 1). As in previous work
a
Program in Biochemistry, Dept. Biological Sciences, Smith College,
Northampton, MA 01063, USA. E-mail: ahall@science.smith.edu;
Fax: 413-585-3786; Tel: 413-585-3467
(iii) the central macrocyclic rings were derived from diaza-15-
+
+
crown-5 in an attempt to achieve selectivity between Na and K ;
iv) the central unit contained ferrocene to act as a redox centre
and therefore a potential switch) and to provide rigidity within
the channel structure.
(
b
Center for Heterocyclic Compounds, Department of Chemistry, 126 Sisler
(
Hall, University of Florida, Gainesville, FL 32611-7200, USA. E-mail:
cdennishall@aol.com; Fax: 352-392-9199; Tel: 352-392-0554
3
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Scheme 1
Results and discussion
2. Cation binding by fluorescence spectroscopy
The 9-anthrylmethyl groups of the channel serve as fluorescent
tags for the detection and possible quantification of coordina-
tion with cations. Fluorescence in the uncomplexed channel is
suppressed by photoelectron transfer (PET) from tricoordinate
nitrogen within the diazamacrocycles. Coordination with cations
suppresses PET and results in various degrees of enhanced
fluorescence dependent on the charge density and association
1
. Synthesis of redox-active ion channels
The ion channel 9 chosen for this particular study was synthesized
by condensation of mono-N-Boc-diaza-18-crown-6 (1) with 9-
chloromethylanthracene, removal of the protecting group from
2
, condensation of the product 3 with 1,10-dibromodecane (1:1
molar ratio), condensation of the product 4 with mono-N-Boc-
21
22
diaza-15-crown-5 (5) , deprotection of the bicyclic product 6 and
condensation of two moles of the resultant amine 7 with 1,1¢-
ferrocenyl diacid chloride (8) (Scheme 1). The compounds were
characterized by high resolution mass spectrometry and H/ C
NMR (see Experimental).
constants of the cations involved. In qualitative terms this is
reflected in the fluorescence intensities recorded at 411 nm (lexc
363 nm) with a cation : ion channel ratio of 50:1, which were
=
1
13
2+
2+
+
shown to decrease in the following order, Mg > Ca > Na >
+
+
+
K (Fig. 1 for Na and K only).
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-
4
◦
Fig. 1 Fluorescence emission intensity of 9 (10 M) as a function of added KSO
3
CF
3
, NaSO
3
CF
3
(0.1M) in CH
3
CN (25 C, lexc = 363 nm). Intensity
+
increasing with molar ratio (9 : M ) increasing from 1:0 to 1:50.
+
+
+
-5
Fig. 2 Plots of 1/I - Io vs 1/M for K and Na at 2 ¥ 10 M of 9.
+
+
The quantitative aspects of cation coordination as determined
by fluorescence spectroscopy are dependent on the initial concen-
tration of the channel. At ca. 10-5 M in ion channel, increasing
concentrations of cation from 5 ¥ 10 to 2 ¥ 10 M gave
fluorescence intensities (I - Io) that fitted a 1:1 coordination ratio
K and Na cations reveal a clear 2:1 cation : ion channel ratio
(Fig. 3).
-
5
-4
(
eqn 1) to give association constant (K
1
) values of ca. 33,000 and
+
+
ca. 19,000 for K and Na respectively (Fig. 2). A similar plot
using Ca gave a K value of 106,000 reflecting association of an
2
+
1
ion with a similar ionic radius to that of sodium but a much greater
charge density.
+
1
/(I - Io) = 1/KDe[IC
0
][M ] + 1/De[IC
0
]
(1)
Where I = fluorescence intensity; K = 1:1 association constant;
De = difference in fluorescence intensity extinction coefficient
between ion channel and complex; [IC
0
] = ion channel initial
+
+
+
concentration and [M ] = concentration of cation. The association
Fig. 3 Job plots using 0.1 M solutions of K (ꢀ) and Na (᭜) cations at
2 ¥ 10 M of 9.
+
-4
constants were obtained by a plot of 1/(I - Io) vs 1/[M ] which,
for 1:1 stoichiometry is linear with slope, S = 1/KDe[IC
0
] and
-
4
intercept, I = 1/De[IC
0
]; thus K = I/S.
At 2 ¥ 10 M and a K
1
value of 33,000 the 1:1 complex is formed
+
+
-2
The higher K
1
value for K over Na is consistent with
to the extent of at least 70%. Beyond 4 ¥ 10 M in cation the 1:1
complex is >90% formed and any further increase in I - Io values
is associated with 2:1 cation : ion channel complex formation.
Thus quantitative aspects of coordination at higher ion channel
concentration were evaluated for K and Na by assuming that
the 1:1 complexes were essentially fully formed before treating the
subsequent data again using eqn. 1 (Fig. 4).
+
+23
coordination by an 18-C-6 system favouring K over Na and
suggests that the initial coordination involves the outside 18-C-6
rings. As the concentration ratio exceeds 10:1 (M : ion channel)
+
+
+
values of I - Io show an increase over the 1:1 complex maximum
indicating the development of a higher degree of coordination.
-
4
At 2 ¥ 10 M in ion channel, Job plots using 0.1 M solutions of
3
864 | Org. Biomol. Chem., 2009, 7, 3862–3870
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+
+
+
-4
Fig. 4 Plots of 1/I - Io vs 1/M for K and Na at 2 ¥ 10 M of 9.
+
+
The results for K and Na are K
2
values of 11,000 and 6,500 re-
spectively, approximately three times lower than the corresponding
values. This is entirely consistent with coordination of a second
K
1
cation responding to cation repulsion, a concept that complements
the so-called “billiard ball effect” as an essential feature of ion
14
-4
channel activity. Beyond 20:1 cation : ion channel at 2 ¥ 10
M
in ion channel, a further increase in I - Io was observed indicating
a 3:1 (or possibly ultimately a 4:1) coordination ratio. This is
1
3
consistent with C NMR and cyclic voltammetry data reported
17
for the analogous ion channel (10, Scheme 1).
In conclusion the fluorescence studies reveal coordination with
+
alkali and alkaline earth cations and in the case of Na and
+
K
multiple complexation of each cation with the ion channel
is observed as expected for a system with four macrocyclic rings.
23
Fig. 5
Na NMR spectra of a single vesicular suspension containing
2
3
3.0 ml of 1 M DyCl
3
. NMR spectra taken prior to, and at different times
3
.
Cation transport by Na NMR spectroscopy
(
10, 60 and 110 minutes) after the addition of 9 (13.2 mM) at 297 K.
The ion channel cation flux of 9 across unilamellar vesicles was
2
3
17
assessed using Na NMR spectroscopy. Our previous study
the change in NMR signal linewidth was monitored with respect
+
23
showed that a linewidth broadening of the Na IN Na NMR
signal could be used to monitor sodium transport across lipid
bilayers. We set out to use a similar methodology to measure and
afterwards compare the cation flux properties of both synthetic
ion channels. Unilamellar vesicles were prepared by the dialytic
detergent removal technique following the protocol outlined in
the Experimental section. This method is reported to give a spread
of vesicle diameters between 200 and 600 nm. Encapsulation of
intravesicular sodium was found to be consistent with previous
to time after the addition of 13.2 mM 9. The broadening of the
+
Na IN NMR signal in the presence of 9 can be quantitatively
measured with respect to time, reaching equilibrium conditions
after approximately 2 h. Fig. 6 shows the plot obtained from
the linewidth broadening at half height (DLW) versus time for
two separate 13.2 mM and 19.8 mM additions of 9 to vesicular
+
preparations as shown by a consistent integration of the Na IN
NMR signal of ~12% of total sodium available. NMR samples
were prepared in 5 mm NMR tubes using a small volume of
vesicle suspension (0.75 ml) that was treated with 3 ml of a
1
D
M aqueous solution of the shift reagent DyCl and 75 ml of
3
+ +
2
O. Dysprosium induces a separation of the Na IN and Na OUT
+
NMR signals by shifting the extravesicular sodium (Na OUT) signal
~
6–10 ppm upfield. Once separation was confirmed and the system
equilibrated (<5 min), known microliter amounts of 5 mM 9
were added and the resultant broadening of the Na IN signal was
+
monitored over time.
Four different microliter amounts (1 ml, 2 ml, 3 ml and 5 ml) of
the 5 mM solution of 9 were introduced into separate vesicular
suspensions, resulting in concentrations of 9 at 6.6, 13.2, 19.8
and 33.0 mM respectively. Fig. 5 shows an experiment in which
Fig. 6 Sample time dependence of the linewidth broadening (DLW) of
+
the Na NMR signal on addition of 13.2 mM (ꢀ) and 19.8 mM (᭹) of 9
IN
to separate standard vesicle preparations.
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+
Table 1 Observed linewidths (LW) in Hz of the Na IN peak vs [9] together
of an aggregation state pre-incorporation, which had an effect
ultimately on ion release kinetics. As in our case, the addition of
+
with the corresponding rate constants (kobs) for Na exchange
24
the synthetic ion channel as an organic solution to the aqueous
suspension of the vesicles might have enhanced the formation
of aggregates manifesting itself in the variability of the NMR
transport studies.
6
+
+
k
obs/s-1
a
[
9] ¥ 10 /M
Time/min
LW(Na IN
)
LW(Na OUT
)
0
4
120
0
5
120
0
5
213
0
5
120
24
27
28
22
26
36
19
29
42
26
36
82
17
17
18
18
16
18
16
17
18
20
19
20
6
6
.6
.6
13.1
41.2
Finally an interesting feature of 9, as it was of 10, was the
inclusion of a ferrocene redox-active centre to act as a device for
the regulation of ion flux. We followed the same protocol designed
to test the potential gating ability of the redox centre in 9 as
for 10. A fresh sample of vesicles and ion channel were mixed and
1
1
3.2
3.2
1
1
9.8
9.8
53.2
+
monitored for Na IN NMR lineshape broadening. The presence of
3
3
3.0
3.0
sodium transport was established and recorded. Then a separate
aqueous solution of 9 was prepared (5 mM) and treated with an
equimolar amount of cerium (IV) ammonium nitrate. Immediate
addition of the treated ion channel to a fresh sample of vesicles and
immediate NMR analysis showed an average of 90% reduction in
channel activity over three runs. This behavior was also observed
with 10 confirming inhibition of sodium transport in both cases.
The result may be due to cation/cation repulsion, although it
could also be explained by lack of incorporation of the oxidized
channel into the membrane.
169.6
a
+
Normalized to the linewidth of the Na OUT linewidth. In the case of [9] =
-6
-6
+
1
3.2 ¥ 10 M and [9] = 19.8 ¥ 10 M the average LW(Na ) of multiple
runs was evaluated.
suspensions. As expected, the change in linewidth broadening was
markedly larger for the 19.8 mM versus the 13.8 mM addition.
As seen previously with 10, the NMR signal broadening
behaviour is indicative of cation transport and its characterization
yields kinetic information. The broadening also confirms that the
smaller ring size of the inner aza crown-ether macrocycles in 9 in
comparison with 10 does not preclude sodium transport. Table 1
17
4. Cation flux using black lipid membrane preparations
shows the resultant linewidths and calculated rate constants (kobs
p ¥ DLW) for sodium transport via 9 for all given concentrations.
=
Black lipid membrane recordings indicated that 9 was functioning
as an ion channel (Fig. 8a).Voltage steps (10–20 mV increments)
from -80 to +80 mV across the bilayer revealed single (and
multiple) channel open level transitions varying in duration up
to ~200 ms (Fig. 8a) with a mean single channel conductance of
6
A plot of kobs vs [9] was linear (Fig. 7) yielding a k value of 5 ¥ 10
2
l mol- s , which indicates relatively rapid bulk sodium transport,
1
-1
at least five times faster than the rate of transport measured with
19a
monensin, a well-characterized ion transporter.
~
50–55 pS as shown in the amplitude histogram (Fig. 8b). Through
assessment of the current–voltage relationship for amplitudes of
the single channel events (Fig. 8c), it was evident that 9 behaved
as an ohmic resistor in that the conductance did not change
appreciably with varying membrane potentials. By contrast the
frequency of opening of the channels was voltage-dependent in
that the probability of channel open events (Popen) was markedly
enhanced with increasing magnitude of the membrane potential
(Fig. 8d). However, it was noted that this voltage-dependence was
independent of the polarity of the potential increase. This finding
may indicate that when the bilayer is more polarised, the channels
assumed the appropriate orientation in the membrane in order to
sustain an ion flux (regardless of the polarity as may be expected
for a symmetrical ion channel).
After addressing the conductance and gating of 9, we then
recorded activity in an asymmetric gradient of 1M NaCl : 1M
KCl in order to explore ion selectivity of the channel. Under
these conditions on occasion (3 of 11 trials) the reversal potential
Fig. 7 Plot showing the linear relationship between kobs and [9] yielding
6
-1 -1
a k
2
value of 5.1 ¥ 10 l mol s .
This k
2
value is similar to that obtained for sodium transport
(Erev) shifted to ~ -15 to -20 mV compared to the Erev
0 mV when recorded in symmetrical conditions (Fig. 9, and
Fig. 8a,b). Using the Goldman–Hodgkin–Katz equation, the
=
+
under the same LUV conditions ([Na ] = 200 mM) using our
6
previous synthetic ion channel 10, which exhibited k
2
= 6 ¥ 10
l mol- s . This indicates that the smaller ring size of the inner aza
crown-ether macrocycle in 9 does not interfere with the overall
transport of, at least, sodium. It is noteworthy that we observed
a substantial variability in rates between NMR trials using 9 that
was not observed for 10, and the figures reported are averages.
In the literature, transport rate variability has been observed with
other synthetic ion transporters. Elliott and co-workers studying
amphiphilic synthetic ionophores pointed out the presence in some
1
-1
relative permeabilities (PNa/P
the channels were impermeable to chloride, it was determined
) were calculated. Assuming that
K
+
+
that 9 has a 2–3-fold selective permeability for Na over K . This
finding is particularly important since equivalent measurements
17
for the previously reported channel showed that 10 demonstrated
+
+
no selective permeability for either Na or K ions (not shown).
Indeed, the activity of 10 in 1M NaCl: 1M KCl asymmetrical
conditions showed Erev ~ 0 mV indicating that this channel was
3
866 | Org. Biomol. Chem., 2009, 7, 3862–3870
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Fig. 8 (a) Typical planar bilayer traces for 9 for membrane held at ±80 mV and recorded in symmetrical 150 mM NaCl conditions. Note there were at
least two channels incorporated in this membrane with closed times indicated by the ‘C’, transitions to one channel open by ‘O1’ and transitions to two
channels open simultaneously by ‘O ’. (b) For the trace in (a) held at 80 mV, all points for 10 s of recording are plotted against current in an amplitude
2
histogram. Note that at least two channels were active in this recording with single channel conductances of ~50 pS. (c) Using symmetrical conditions,
the single channel current amplitudes (Iamp) are plotted against the holding potential for the bilayer (mV). Error bars indicate the range (max–min) of
amplitudes recorded at a given potential for at least two independent recordings. A line of best fit (dotted) is superimposed on the plot indicating that the
channels are essentially ohmic, with a conductance of ~50–55 pS. (d) The probability of opening (Popen) was calculated and plotted against the holding
potential of the bilayer. Error bars indicate the range (max–min) of Popen values recorded at a given potential for at least two independent recordings. Note
how the probability of open channel activity is markedly enhanced with increased potential regardless of polarity.
+
+
non-selective for the flux of monovalent cations. Inspection shows
that 9 differs from 10 due to the replacement of the inner diaza-
between Na and K since the effect of ring size would be minimal.
Clearly therefore, the molecular architecture described here does
not afford consistent selectivity but may, at least, point a way
forward.
1
8 crown-6 rings of 10 by diaza-15-crown-5 units. Thus, the
constriction in the inner rings of the channel may confer a level
+
+
of ion selectivity for Na over a K flux. However, there was
inconsistency in this selectivity result since in 8 of 11 recordings, the
activity in asymmetrical conditions reversed at 0 mV, indicating
no cation selectivity. Furthermore, single channel conductances
recorded in symmetrical 150 mM KCl or LiCl were simliar (~50 pS)
to those recorded in NaCl (data not shown). The lack of consistent
selective behaviour suggests that the channel may adopt different
conformations within the bilayer. Clearly the 18-crown-6 and 15-
crown-5 rings need to align for the ions to pass through both
and hence show selectivity. An alternative conformation, however,
may involve the ferrocene nucleus lying perpendicular, rather than
parallel, to the bilayer thus enabling the 15-crown-5 rings to
act as a “relay” (see ref. 13), rather than a “filter”, by rotation
about the cpd–Fe bond. This would probably not afford selectivity
Finally, to investigate gating by the redox-active ferrocene unit,
we recorded channel activity of 9 and introduced the oxidizing
agent, ceric (IV) ammonium nitrate (1 mM), at a concentration
that was determined to have no effect on bilayer integrity in the
absence of ion channel. Upon introduction of the oxidizing agent
to both recording chambers, ion channel activity was consistently
abolished (Fig. 10).
This activity was not restored through subsequent addition
of a 6-fold excess of the reducing agent, sodium thiosulfate,
possibly due to the size of the thiosulfate ion hindering the
access to the ferrocene unit in the membrane. These data suggest
that channel activity is markedly affected by the redox state.
However, whether oxidation affects channel incorporation into
the membrane, the configuration of 9 in the bilayer or blocks flux
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except Ca(SO
3
CF
3
)
2
were used as obtained from Aldrich. Calcium
17
trifluoromethanesulfonate was prepared as reported previously.
Fluorescence methods
Fluorescence spectra were recorded using a Fluoromax 3 spec-
trofluorometer (Horiba Jobin Yvon). The fluorescence intensities
-
5
of the ligands (1 ¥ 10 M) excited at 363 nm were measured
in acetonitrile at room temperature. Titrations were conducted
by progressively adding the metal salts, injected as concentrated
solutions, to a cuvette containing the ligand solution (2.5 ml).
Samples were shaken briefly prior to each recording and the added
equivalents of cation were plotted against the emission-intensity
change in the 414–421 nm region.
Preparation of unilamellar vesicles from egg phosphatidyl choline
by detergent dialysis
+
Fig. 9 Ion channel 9 is selectively permeable to Na ions. The above
traces were recorded in an asymmetrical gradient with 1 M NaCl in the cis
chamber and 1 M KCl in the trans. The applied voltages are indicated above
each trace with ‘C’ indicating closed states and ‘O’ open states. Note that at
Unilamellar vesicles were prepared from egg yolk phosphatidyl-
choline using a modified detergent dialysis technique. A sample
of grade 1 lecithin (50 mg) in chloroform–methanol (Lipid
25
0
mV channel openings were clearly evident while at -20 mV there was no
Products, Redhill, Surrey, UK) was evaporated under N and
2
measurable activity, indicating that the Erev under these conditions shifted
to between 0 to -20 mV compared to recordings using a symmetrical
gradient (see Fig. 8a). The above recording was 1 of 3 (from 11 recordings
then dissolved in a solution (3 ml) containing 200 mM NaCl,
5 mM MES (pH: 6.5) and 300 mM, n-octyl-b-D-glucopyranoside
(Calbiochem). The lipid solution was pipetted into a 6 mm
diameter cellulose membrane dialysis tubing, and dialysed against
+
in total) where activity was clearly evident at 0 mV in asymmetrical Na :
+
K conditions.
2
.5 l of the external solution containing 200 mM NaCl, 5 mM
MES (pH: 6.5) at room temperature. External solutions were pre-
equilibrated and continually bubbled with N throughout dialysis,
2
and were changed every 10–12 h with three solution exchanges. In
a final 8 h dialysis, the external solution was exchanged for 2.5 l
of a deoxygenated solution containing 100 mM NaCl, 20 mM
Na-tripolyphosphate, 50 mM choline chloride and 5 mM MES
(
pH: 6.5). The vesicle suspension was then carefully pipetted out
Fig. 10 Channel activity of (a) +70 mV and (b) +70 mV in the presence
of a Ce ion.
of the dialysis bag and used immediately.
4+
2
3
Na NMR measurements
through irreversible oxidation of the ferrocene unit, remains to be
determined.
23
Sodium cation flux was measured following the Na NMR
19
method of Riddell et al. Sodium NMR spectroscopic measure-
ments were performed on a JEOL Eclipse 400 NMR spectrometer
with sodium observation at 105 MHz. A 5 mm tunable probe
Experimental
Melting points were determined on a hot-stage apparatus and are
was used with the following acquisition parameters: tip angle
uncorrected. NMR spectra were recorded in CDCl
3
with TMS
◦
5
7 ; sweep width ±11000 Hz; relaxation delay = 0.5 s; number
1
as the internal standard for H (300 MHz) or CDCl as the
3
of spectral points = 4 K; locked mode (D
2
O). Each spectrum
1
3
internal standard on a Varian Mercury 300 for C (75 MHz).
MALDI mass spectra were recorded on Bruker Reflex II TOF
mass spectrometer retrofitted with Delayed Extraction at the
Department of Chemistry, University of Florida in Gainesville,
Florida, USA. All reactions were carried out under an atmosphere
of nitrogen. Column chromatography was performed with A950–
represents a collection of 200 scans Fourier transformed with
an exponential line broadening of 0.2 Hz. NMR samples were
prepared by mixing 675 ml of vesicular suspension, 75 ml of
1
00 mM NaCl/20 mM Na
DyCl to obtain a sodium spectrum consisting of the intravesicular
resonance separated from the extravesicular resonance by at least
ppm. All spectra were recorded at 291 K. Relaxation times
) for the sodium signals were measured to be ~0.035 s.
5
P
3
O10 in D
2
O and 3.0 ml of 1 M
3
5
00 neutral alumina (60–325 mesh).
6
Thin layer chromatographic (TLC) analyses used aluminium
(
T
1
oxide 60 F254 neutral on aluminium sheets with a 0.2 mm layer
thickness (Merck) with 5% MeOH/CH Cl as eluent. Chromatog-
Linewidth measurements were obtained by deconvolution of the
Na resonance signals by a JEOL Lorentzian bandshape fit.
+
2
2
raphy columns were packed with alumina [neutral, type 507C,
Fluka (0.05–0.15 mm; pH 7.0 ± 0.5)], or silica gel 60 (F254,
Ion channel recordings: black lipid membrane experiments
0
.035–0.070 mm, Merck). Solvents were purified by standard
procedures and were stored under nitrogen over molecular sieves
4–8 mesh). All the metal trifluoromethanesulfonates (triflates)
Standard black lipid membrane techniques were employed to
record synthetic ion channel activity in planar lipid bilayers
(
3
868 | Org. Biomol. Chem., 2009, 7, 3862–3870
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with phospholipid membranes formed through a painted bilayer
method. Phosphatidylcholine (DOPC) and phosphatidylglycerol
CH
(MgSO
2
Cl
2
(5 ml), washed with water (10% w/v, 3 ¥ 5 ml), dried
) and concentrated in vacuo. The residue was purified by
25
4
(
DOPG, Avanti Polar Lipids, Birmingham, AL, USA) were stored
column chromatography (alumina, MeOH/CH
2
Cl
2
) to give 6 as
◦
1
as chloroform dispersions at -80 C in sealed glass vials. Channel 9
was stored in methanol at -80 C prior to use. Electrolyte solutions
in distilled water consisted of (i) for symmetrical solutions:
1
KCl, with 10 mM HEPES, titrated to pH 7.2 with NaOH or
KOH and stored at 4 C. A lipid/ion channel solution, 85:15%
an orange oil (0.10 g, 63%). H NMR (CDCl
3
) d 8.53 (d, J =
◦
8.7 Hz, 2H), 8.40 (s, 1H), 8.0 (d, J = 8.5 Hz, 2H), 7.53–7.43 (m,
4H), 4.56 (s, 2H), 3.84–3.75 (m, 4H), 3.74–3.30 (m, 28H), 3.31–
3.12 (m, 4H), 3.11–2.80 (m, 10H), 2.71–2.48 (m, 2H), 1.75–1.40 (m,
50 mM NaCl, (ii) for asymmetrical solutions, 1 M NaCl/1M
1
3
13H), 1.39–1.18 (12H); C NMR (CDCl ) d 155.1, 131.3, 131.3,
3
◦
130.2, 128.9, 127.4, 125.5, 125.1, 124.7, 79.5, 70.8, 70.5, 70.3, 70.2,
DOPC/DOPG including 0.5–1 mg of 9/mg of lipid, was prepared
by evaporating off the chloroform/methanol from the stock
solutions under a stream of nitrogen, before dispersal in n-decane
70.0, 69.8, 69.4, 69.2, 67.9, 67.4, 55.8, 54.8, 53.8, 53.4, 52.8, 52.6,
51.6, 49.2, 49.0, 48.8, 29.4, 29.2, 28.4, 27.2, 27.1. HRMS (MALDI-
+
TOF): m/z calculated for C52
H
84
N
4
O
9
+
[M - C15
H
10 + H] 719.5529,
(
50 mg/ml lipid). This lipid/ion channel solution was used to
found 719.5629; [M - C15H10 + Na] calculated 741.5348, found
pre-treat the aperture in a delrin bilayer cup. The dispersion was
spread using a ‘painting stick’ fashioned from a glass Pasteur
pipette. The stick was dipped in the decane solution and a small
quantity painted across the 200 mm-diameter hole in the cup and
allowed to dry. The electrolyte solutions were added to the delrin
cup (referred to as cis, volume 3 ml) and to the block chamber
741.5453.
N-(9-Anthrylmethyl)-N¢-[10-(diaza-18-crown-6)decyl]-diaza-15-
crown-5 (7). N-(9-Anthrylmethyl)-N¢[10-(N¢-t-butoxycarbonyl-
diaza-15-crown-5)decyl]-diaza-18-crown-6 (6, 0.09 g, 0.10 mM)
was dissolved in a mixture of TFA (0.35 g, 3.09 mM) and CH
2
Cl
2
◦
(
1 ml) and stirred at 20 C for 16 h. The solution was evaporated
(
trans, volume 3 ml). Silver-silver chloride electrodes linked the
to dryness and the residue was dissolved in CH
with Na CO solution (10% w/v, 3 ¥ 3 ml), dried (MgSO
concentrated in vacuo to give 7 as an orange oil (77 mg, 96%).
2
Cl
2
(3 ml), washed
electrometer head to the cell via agar bridges (2% agar in 3 M KCl)
with the reference electrode placed in the cis chamber. Electrodes
2
3
4
) and
◦
were stored in bleach and the bridges were stored at 4 C in 3 M
1
H NMR (CDCl
3
) d 8.46 (d, J = 8.8 Hz, 2H), 8.31 (s, 1H), 7.90
KCl. The whole apparatus was enclosed in a grounded Faraday
cage positioned on a metal base plate resting on an air-cushioned
table (Warner Instrument Corporation, Hamden, CT).
To form a bilayer, the painting stick was again dipped in
the lipid/decane solution, and the stick was submerged in the
electrolyte solution and drawn across the aperture in the cup.
Lipid bilayer formation through thinning usually occurred within
(
(
(
d, J = 8.5 Hz, 2H), 7.45–7.33 (m, 4H), 4.50 (s, 2H), 3.58–3.45
m, 28H), 2.82 (t, J = 5.7 Hz, 4H), 2.71–2.61 (m, 12H), 2.39
t, J = 7.7 Hz, 4H), 1.35 (brq, 4H), 1.28–1.06 (pseudo-s, 12H);
1
3
C NMR (CDCl ) d 131.3, 131.3, 130.4, 128.8, 127.3, 125.5,
3
125.2, 124.7, 70.7, 70.5, 70.2, 70.1, 70.0, 69.9, 69.2, 56.6, 56.0,
54.4, 54.3, 53.9, 53.7, 51.8, 48.9, 48.7, 29.5, 27.4, 27.0, 26.9. 809.
HRMS (MALDI-TOF): m/z calculated for C47
H
76
N
4
O [M +
7
1
–10 min resulting in a final capacitance of the bilayers ~80–
+
+
H] 809.5787, Found: 809.5882; calculated [M + Na] 831.5606,
found 831.5720.
1
20 pF. Currents were recorded using a Warner amplifier (model
BC-525D) using variable holding potentials, filtered at 1 kHz with
a low-pass 8-pole Bessel filter (Model LPF-8, Warner Instruments)
and then acquired using Clampex 9.2 software (Axon Instruments)
with a sampling rate of 4 kHz. In experiments involving the
oxidizing agent, 1 mM ammonium cerium (IV) nitrate was
added to both chambers and the solutions were stirred using a
magnetic stirrer. This concentration of the oxidizing agent was
determined not to disrupt the integrity of the membrane bilayers.
All experiments were carried out at room temperature.
1,1¢-Ferrocenedicarboxylic acid was prepared as described
26
previously.
1
,1¢-Bis-(chlorocarbonyl)ferrocene (8). Oxalyl chloride (8.13 g,
4.08 mM) was added over 1 min to a suspension of 1,1¢-
ferrocenedicarboxylic acid (2.11 g, 7.70 mM) in dry CH Cl (37 ml)
6
2
2
under nitrogen. The mixture was heated under reflux for 9 h and
stirred at 20 C for a further 1.5 h. The solution was concentrated
in vacuo and the residue recrystallized from n-pentane to give 8
◦
◦
1
as red crystals (1.90 g, 79%), m.p.= 106.0–109.0 C, H NMR
Synthesis of ion channel
1
3
(
(
CDCl
CDCl
3
) d 5.05 (t, J = 2.0, 4H), 4.77 (t, J = 2.0 Hz, 4H); C NMR
) d 168.7, 77.3, 76.1, 74.1.
4
,13-Diaza-18-crown-6 was purchased from IBC Advanced
3
Technologies, Inc. N-t-Butoxycarbonyl-diaza-18-crown-6 (1),
N-t-butoxycarbonyl-diaza-15-crown-5 (5), N-(9-anthrylmethyl)-
N¢-t-butoxycarbonyl-diaza-18-crown-6 (2), N-(9-anthrylmethyl)-
diaza-18-crown-6 (3), and N-(9-anthrylmethyl)-N¢-(10-bromo-
decyl)diaza-18-crown-6 (4) were all prepared as described
1
,1¢-Bis-[N-carbonyl-N¢-[-10-(N¢-(9-anthrylmethyl)-diaza-18-
crown-6)decyl]-diaza-15-crown-5]ferrocene (9). 1,1¢-Bis(chloro-
carbonyl)ferrocene (8, 22 mg, 0.07 mM) in dry toluene
2.67 ml) was added dropwise over
lution of N-(9-anthrylmethyl)-N¢-[10-(diaza-18-crown-6)decyl]-
diaza-18-crown-6 (7, 0.11 g, 0.14 mM) and dry Et N (14 mg,
0.14 mM) in dry toluene (2.67 ml). The mixture was stirred
at 20 C for 24 h under nitrogen, filtered and concentrated in
vacuo to give an orange residue. The product 9 was obtained by
(
5 min to a so-
17
previously.
N-(9-Anthrylmethyl)-N¢[10-(N¢-t-butoxycarbonyl-diaza-15-
crown-5)decyl]-diaza-18-crown-6 (6). solution of N-(9-
anthrylmethyl)-N¢-(10-bromodecyl)-diaza-18-crown-6 (4, 0.11 g
3
◦
A
0
0
.16 mM) and N-t-butoxycarbonyl-diaza-15-crown-5 (5, 0.05 g
.16 mmol) in propionitrile (2 ml) was heated under reflux over
column chromatography (alumina, 4–5% MeOH/ethyl acetate) as
1
an orange oil (0.12 g, 86%). H NMR (CDCl
3
) d 8.54 (d, J = 8.5 Hz,
anhydrous Na
2
CO
3
(0.33 g, 3.19 mM) and KI (3 mg, 0.02 mM)
4H), 8.40 (s, 2H), 7.99 (d, J = 8.0 Hz, 4H), 7.54–7.41 (m, 8H),
4.73–4.66 (m, 4H), 4.58 (s, 4H), 4.48–4.46 (m, 4H), 3.80–3.40 (m,
64H), 3.10–2.85 (m, 8H), 2.84–2.56 (m, 16H), 2.52–2.45 (m 8H),
for 20 h under nitrogen. The mixture was cooled, filtered and
concentrated in vacuo. The yellow residue was redissolved in
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1
3
1
.64–1.37 (m, 8H), 1.36–1.05 (pseudo-s, 24H); C NMR (CDCl
3
)
4 (a) F. Otis, N. Voyer, A. Polidori and B. Pucci, New J. Chem., 2006, 30,
1
85; (b) G. G. Kochendoerfer, D. Clayton and C. Becker, Protein Pept.
d 172.1, 131.3, 131.3, 130.3, 128.9, 127.4, 125.5, 125.1, 124.7, 77.2,
1.9, 71.8, 70.6, 70.4, 70.0, 53.8, 53.4, 51.7, 29.6, 29.5, 29.4, 27.4,
7.3. HRMS (MALDI-TOF): m/z calculated for C106
M + H] 1857.1252, Found: 1857.1288, calculated [M + Na]
Lett., 2005, 12, 737; (c) E. Biron, F. Otis, J.-C. Meillon, M. Robitaille, J.
Lamothe, P. Van Hove, M.-E. Cormier and N. Voyer, Bioorg. Med.
Chem., 2004, 12, 1279; (d) G. W. Gokel, P. H. Schlesinger, N. K.
Djedovic, R. Ferdani, E. C. Harder, J. Hu, W. M. Leevy, J. Pajewska,
R. Pajewski and M. E. Weber, Bioorg. Med. Chem., 2004, 12, 1291;
7
2
[
H
158FeN
8
O
16
+
+
1879.1071, found 1879.1356.
(
9
e) J.-C. Meillon and N. Voyer, Angew. Chem., Int. Ed. Engl., 1997, 36,
67.
5
6
(a) D. T. Bong, T. D. Clark, J. R. Granja and M. R. Ghadiri, Angew.
Chem., Int. Ed., 2001, 40, 988; (b) M. Amorin, L. Castedo and
J. R. Granja, Chem. Eur. J., 2008, 14, 2100; (c) S. Fernandez-Lopez,
H. S. Kim, E. C. Choi, M. Delgado, J. R. Granja, A. Khasanov, K.
Kraehenbuehl, G. Long, D. A. Weinberger, K. M. Wilcoxen and M. R.
Ghadiri, Nature, 2001, 412, 452.
Conclusions
Spectroscopic and cyclic voltammetry studies suggest that the
synthetic, redox-active ion channel 9 coordinates cations to afford
a ‘billiard ball’ mechanism of ion transport analogous to that
found with natural protein channels. This study has also shown
that 9 conducts Na ions through lipid bilayers at a rate that
is approximately five times faster than the ionophore, monensin.
N. Sakai and S. Matile, Angew. Chem., Int. Ed., 2008, 47, 9603.
7 Y. J. Jeon, H. Kim, S. Jon, N. Selvapalam, D. H. Oh, I. Seo, C. S. Park,
+
S. R. Jung, D. S. Koh and K. Kim, J. Am. Chem. Soc., 2004, 126, 15944.
M. E. Weber, W. Wang, S. E. Steinhardt, M. R. Gokel, W. M. Leevy
and G. W. Gokel, New J. Chem., 2006, 30, 177.
8
Although the data were inconsistent, 9 also demonstrated some
9 W. Wang, C. R. Yamnitz and G. W. Gokel, Heterocycles, 2007, 73, 825.
10 J. Carlos Iglesias-Sanchez, W. Wang, R. Ferdani, P. Prados, J. de
Mendoza and G. W. Gokel, New J. Chem., 2008, 32, 878.
1 G. W. Gokel and M. M. Daschbach, Coord. Chem. Rev., 2008, 252, 886.
2 W. Wang, R. Li and G. W. Gokel, Chem. Commun., 2009, 911.
13 C. L. Murray, H. Shabany and G. W. Gokel, Chem. Commun., 2000,
+
+
selectivity for supporting a Na flux over a K flux, possibly
dependent on the appropriate orientation of the channel in the
membrane. Oxidation of the ferrocene unit within the channel
inhibits the ion transport, demonstrating the potential role of a
redox-active center as a switch in regulating transmembrane cation
flux.
1
1
2
371.
1
4 (a) D. A. Dougherty and H. Lester, Angew. Chem., Int. Ed., 1998, 37,
2
1
329; (b) P. J. Cragg, M. C. Allen and J. W. Steed, Chem. Commun.,
999, 553 .
1
1
1
5 P. Schmitt, P. D. Beer, M. G. B. Drew and P. Sheen, Angew. Chem., Int.
Acknowledgements
Ed. Engl., 1997, 35, 1840.
6 H. Shabany, C. L. Murray, C. A. Gloeckner, M. A. Grayson, M. L.
Gross and G. W. Gokel, Chem. Commun., 2000, 2375.
We would like to acknowledge: Dr Charles Amass, Chemistry De-
partment, Smith College for helpful advice on the sodium NMR
spectroscopy, the National Science Foundation (NSF grant#
CHE-0513445), and Merck/AAAs Undergraduate Science Re-
search program for supporting the work of all the undergraduates
coauthors (Nylander, Wardlaw, Gibson, Valentine, Onyewadume,
Ahove, Woodbury and Mongare) and undergraduates: Angela
Saquibal, Mary Banks, Anu Maharjan, Monica Wang and Jill
Flynn.
7 A. C. Hall, C. Suarez, A. Hom-Choudhury, A. N. A. Manu, C. Dennis
Hall, G. J. Kirkovits and I. Ghiriviga, Org. Biomol. Chem., 2003, 1,
2
973.
18 M. M. Pike, S. R. Simon, J. A. Balshi and C. S. Springer, Proc. Natl.
Acad. Sci. U. S. A., 1982, 79, 810.
1
9 (a) F. G. Riddell and M. K. Hayer, Biochim. Biophys. Acta, 1985, 817,
13; (b) F. G. Riddell and S. J. Tompsett, Biochim. Biophys. Acta, 1990,
3
1024, 193; (c) D. Duval, F. G. Riddell, S. Rebuffat, N. Platzer and B.
Bodo, Biochim. Biophys. Acta, 1998, 1372, 370.
2
2
0 G. R. A. Hunt and J. A. Veiro, Biochem. Soc. Trans., 1986, 14, 602.
1 K. Ashwani, S. Mogeswaran and O. Sutherland, Tetrahedron, 1986, 42,
3
291.
2
2 (a) A. P. de Silva and S. A. de Silva, J. Chem. Soc., Chem. Commun.,
Notes and references
1986, 1709; (b) K. Kubo, R. Ishige and T. Sakurai, Talanta, 1999, 49,
3
39.
1
2
3
D. J. Aidley and P. R. Stanfield, Ion channels : molecules in action,
University Press, Cambridge, 1996.
23 C. Hamamci, H. Ho s¸ g o¨ ren and S. Erdo g˘ an, Talanta, 1998, 47, 229.
24 E. K. Elliott, M. M. Daschbach and G. W. Gokel, Chem. Eur. J., 2008,
14(19), 5871.
25 A. J. Williams, in Microelectrode Techniques–The Plymouth Workshop
Handbook, ed. D. Ogden, The Company of Biologists Ltd., Cambridge,
2nd edn, 1994, pp. 79–101.
W. D. Stein, Channels, Carriers and Pumps, Academic Press, New York,
1
990.
(a) T. M. Fyles, Chem. Soc. Rev., 2007, 36, 335; (b) G. W. Gokel and
I. A. Carasel, Chem. Soc. Rev., 2007, 36, 378; (c) U. Koert and P. Reiss,
Supramol. Chem., 2002, 2, 29; (d) G. W. Gokel and A. Mukhopadhyay,
Chem. Soc. Rev., 2001, 30, 274.
26 V. G. Albano, L. Busetto, F. Marchetti, M. Monari, S. Zacchini and V.
Zanotti, J. Organomet. Chem., 2005, 690, 4666.
3
870 | Org. Biomol. Chem., 2009, 7, 3862–3870
This journal is © The Royal Society of Chemistry 2009