Cyclodextrin ion channels

Article abstract of DOI:10.1039/c4ob00480a

Seventeen derivatives of α- and β-cyclodextrins were prepared from the cyclodextrin per-6-azide by click cyclization with terminal alkynes. Sixteen of these half-channel compounds showed significant activity as ion channels in planar bilayer members as assessed by the voltage-clamp technique. Activity ranged from persistent square-top openings to highly erratic conductance; mixed behaviours were evident in virtually all data recorded. Some of the erratic behaviours were shown to follow an apparent power-law distribution of open duration times. The activities observed for the suite were summarized using a model-free activity grid method which displays conductance, duration, and opening behaviour. The overall activity shows the clustering of conductance-duration indicating that activity arises from system properties rather that solely as a property of the compound. The activity grids also support an analysis of structure-activity relationships as they apply to the global behaviour of the compounds and reveal the complexity of a single structure change in controlling the distribution of concurrent conductance behaviours. Transient blockage of channel activity by the hydrophobic guest of the cyclodextrin (1-adamantyl carboxylate) is consistent with the formation of an end-to-end dimer channel among several other competing and interconverting structures. This journal is the Partner Organisations 2014.

Full text of DOI:10.1039/c4ob00480a

Organic &
Biomolecular Chemistry
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Cyclodextrin ion channels†
Jonathan K. W. Chui and T. M. Fyles
Cite this: Org. Biomol. Chem., 2014,
12, 3622
Seventeen derivatives of α- and β-cyclodextrins were prepared from the cyclodextrin per-6-azide by
“click” cyclization with terminal alkynes. Sixteen of these “half-channel” compounds showed significant
activity as ion channels in planar bilayer members as assessed by the voltage-clamp technique. Activity
ranged from persistent square-top openings to highly erratic conductance; mixed behaviours were
evident in virtually all data recorded. Some of the erratic behaviours were shown to follow an apparent
power-law distribution of open duration times. The activities observed for the suite were summarized
using a model-free activity grid method which displays conductance, duration, and opening behaviour.
The overall activity shows the clustering of conductance–duration indicating that activity arises from
system properties rather that solely as a property of the compound. The activity grids also support an
analysis of structure–activity relationships as they apply to the global behaviour of the compounds and
reveal the complexity of a single structure change in controlling the distribution of concurrent conduct-
ance behaviours. Transient blockage of channel activity by the hydrophobic guest of the cyclodextrin
(1-adamantyl carboxylate) is consistent with the formation of an end-to-end dimer channel among
several other competing and interconverting structures.
Received 3rd March 2014,
Accepted 8th April 2014
DOI: 10.1039/c4ob00480a
www.rsc.org/obc
over the channel requires much better structural control,
as has been achieved for example in gramicidin hybrid
Introduction
It has been over 30 years since Tabushi reported the first syn- channels,31–33 crown ether appended helical peptides,9,34–37
thetic ion channel.1 The design was based on a β-cyclodextrin and oligophenyl β-barrels.18,38–42 All these examples involve a
and was the first attempt of de novo design of a structure that design parallel between a tubular channel structure and the
could mimic the ion channels of Nature. Since that seminal ion transport function. The active structures in all these cases
report the field of synthetic ion channels has burgeoned to have high molecular weights – substantially less than the
include active transporters based on a wide range of supra- massive structures of natural proteins – but nonetheless a sig-
molecular structural classes – crown ethers, calixarenes, cyclic nificant challenge for a total synthesis.
and helical peptides, foldamers, metal–organic and hydrogen-
Our experience with simple compounds revealed the advan-
bonded frameworks.2–15 Many of these structures rival the tages of a modular approach with a limited number of opti-
ability of natural ion channels to promote ionic flux through mized reactions to produce
a suite of closely related
bilayer membranes.16 Control of ionic selectivity and overall compounds in a few steps from starting materials.43 We there-
channel function remains a continuing challenge, but there fore sought a design which was amenable to rapid modular
are encouraging reports of synthetic channels as the active assembly based on a macrocyclic portal element to provide the
agents in biological and analytical applications.13,17–24
structural integrity absent in simpler transporters. Although
Over the past decade we have focused on progressively transmembrane spanning structures are appealing, a “half-
simpler compounds in an effort to understand the origins of channel” motif involving end-to-end dimerization of com-
the channel-forming activity of very simple transporters,25–29 ponents in the opposing leaflets of a bilayer membrane, as
but such systems are plagued by “supramolecular polymorph- seen in the gramicidin channel,44–46 offers a clear on-off
ism”30 resulting in a condition where a range of closely related mechanism which could be modulated. The “half channel”
structures of similar activity becomes inevitable. Better control approach reduces the synthetic task two-fold, and a highly
symmetric structure would further streamline the synthesis.
Despite the pioneering Tabushi report1 there have been
Department of Chemistry, University of Victoria, PO Box 3065, Victoria, BC,
relatively few attempts to exploit cyclodextrins as an element in
Canada V8W 3P6. E-mail: tmf@uvic.ca; Fax: +1(250) 721 7147;
ion channel designs.47 In the mid-1990s Lehn reported
Tel: +1(250) 721 7192
“bouquet” molecules including one based on β-cyclodextrin
†Electronic supplementary information (ESI) available: Synthesis procedures
and characterization of new compounds. See DOI: 10.1039/c4ob00480a
where the macrocycle was envisaged to lie at the bilayer
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midplane with oligoethyleneoxy glycolate arms extending from
both the primary and secondary faces; the activity of these
compounds was very low.48 Significant transport activity of a
cyclodextrin channel was reported by Gin49 in which the seven
primary sites of β-cyclodextrin were modified with pentabuty-
lene glycols to produce a compound expected to be sufficiently
long to span a complete bilayer membrane. This compound
supports both cation and anion transport with the balance
dependent upon the pH.50 The channel activity is also light-
modulated by an azobenzene appended to one of the second-
ary sites.51 Channel activity by per-2,3-O-trimethylsilyl per-6-
mercapto β-cyclodextrin has been reported, but the structural
origin of the activity is not clear. A recent report of an ala-
methicin-β-cyclodextrin derivative52 completes the remarkably
short list of synthetic channels built up from cyclodextrins.
Unmodified β-cyclodextrin lies at the heart of an impressive
system based on the α-hemolysin channel.53–55 In this system,
the protein is modified to accommodate a β-cyclodextrin
‘adapter’ in the throat of a β-barrel pore.56 The conductance of
the pore is modulated as hydrophobic analytes transiently
occupy the cyclodextrin leading to a stochastic sensor capable
of both identifying and quantifying the components of a
mixture.57 The cyclodextrin adapter is itself held dynamically
in the protein channel, so the analytically useful lifetime
depends on the cyclodextrin concentration and kinetics of the
cyclodextrin binding to the protein; durations of seconds are
possible.
Scheme 1 Synthesis and structures of cyclodextrin ion channels pre-
pared. Compound naming lists the components of the structure: [cyclo-
dextrin; α or β] + [alkyne; H 6-hydroxy- or 6-aminohex-1-yne; M 2-
propargylmalonamide; Eth ethisterone; Chol N-propargylcholamide] +
[linkage; E ester; A amide; U urea; O ether] + [total carbon chain length]
+ [F; per-fluoroalkyl].
Here we report the synthesis of cyclodextrin half-channels
derived by per-functionalization of the cyclodextrin primary
positions and their activity as channels assessed by the bilayer
clamp technique. The initial motivations of this study were
threefold: to develop a rapid approach to half channels, to
survey the activity of the compounds in bilayer membranes in
the expectation that key controlling structural variables could
be uncovered, and to discover if transient current blocking
events can be detected for the cyclodextrin alone, in the
absence of a protein scaffold.
required product was achieved. However, purification losses
were very significant in many cases resulting in 20–70% iso-
lated yields.
Previous reports frequently specify near stoichiometric
amounts of copper, and then follow with procedures designed
to scavenge the catalyst from the product, using EDTA extrac-
tion, scavenging resin, and/or chromatography with coordinat-
ing solvents on polar supports. The core problem is that
cyclodextrin-triazole products coordinate copper thereby redu-
cing catalyst reactivity while avidly binding the excess catalyst
during purification. The 2,3-diol fragment of cyclodextrins is
known to bind Cu(II) in basic solution to form multiply che-
lated sandwich structures69 which are implicated as the main
complication in purification.70 Triazoles also bind cupric
ion.71 In simple cases these complexes are known to accelerate
the cycloaddition,72 but this is clearly ineffective in the case of
the oligo-triazoles produced by cyclodextrins.
Results and discussion
Synthesis
The required half channel cyclodextrin derivatives were pre-
pared by Cu(I)-catalysed [3 + 2] Huisgen cycloaddition (“click”
chemistry)58–60 (Scheme 1). The required per-6-azidocyclodex-
trins were prepared following an established procedure.61,62
The required alkynes were either commercially available or
readily prepared by standard reactions; full details are given in
the ESI.† A wide range of reported catalysts and conditions
were screened in order to find an optimal reactivity which
could apply to all required targets. No such magic bullet was
uncovered. This is entirely consistent with prior literature on
click modifications of mono- and per-substituted cyclodextrin,
with or without secondary hydroxyl derivatization.49,52,63–68
Nonetheless, it was possible to find conditions for all alkyne–
cyclodextrin combinations in which 100% conversion to the
Although the purification is tedious and wasteful, product
purity is readily assessed visually by the absence of a greenish
or blue cast, and more reliably by the absence of M+63/M+65
copper adduct ions in the MALDI-MS of products. The six- or
seven-fold symmetry of the required products ensures that
1
incomplete conversion is readily detected in H and 13C-NMR
spectra. Products are therefore identified by sharp, high-sym-
metry NMR spectra and the expected simple MALDI-MS
showing a single molecular ion isotopic cluster.
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A few of the compounds (Scheme 1: α,β-Eth; α,β-Chol; of the activity of some compounds is of the “square-top” on-off
β-HA5F) could be prepared using catalytic CuI (5–10 mol%) variety illustrated in Fig. 1: A for β-HE4F; the portion illus-
with the bound copper removed by silica chromatography trated is approximately 5 minutes of an activity that persisted
using ammonia as a component of the eluent. The majority of in this form for more than an hour. Other square-top beha-
the remaining compounds of Scheme 1 used the CuSO4-ascor- viours such as those in Fig. 1: B (β-HE4) and C (β-HA4) are
bate catalyst system (80 mol% Cu) followed by copper scaven- mixed with other openings of shorter duration superimposed.
ging into Chelex-100 resin. These conditions failed with the This type of superposition of multiple openings within a
malonamides (β-M3O1; β-M3O2; β-M3N6) presumably due to single cluster of activity is well illustrated in Fig. 1: D (β-HA4F)
very efficient copper sequestering under the reaction con- which is an archetypal example of the “multi-opening” type
ditions. Following a screening of several heterogeneous copper previously described.16 More complex conductance events with
catalysts,73,74 the Cu/AlO(OH) system75 eventually resulted in a fewer periods of regularity also occur as in Fig. 1: E (β-HE4)
procedure (Et3N, DMF, sealed vial, 100 °C, 2 h; catalyst and G (β-HE5F). The records also included a few examples of
removal by centrifugation) from which the three malonamides “shark-fin” openings76 (Fig. 1: F βHA4F); these slowly evolving
were isolated.
conductance changes occurred only in the presence of trans-
membrane electrolyte gradients. Prolonged periods of erratic
activity (Fig. 1: H β-HA5F) were common for many of the com-
Overview of activity
The activity of the cyclodextrin derivatives prepared was pounds. To summarize qualitatively: this set of compounds
assessed using the bilayer clamp technique.16 A planar bilayer exhibits the entire set of conductance behaviours ever
membrane composed of diphytanoylphosphatidyl choline observed16 for synthetic ion channels.
(diPhyPC) was formed across an orifice of 250 µm diameter in
The activity arises by symmetrical addition of compound to
a Delrin cup interposed between two pools of electrolyte, typi- the bilayer, and it is therefore not surprising that only Ohmic
cally 1 M alkali metal chloride. Potential applied between two behaviours were observed. Many of the conducting openings
Ag/AgCl electrodes produced transmembrane currents which carry significant ionic current but there is little selectivity
were amplified, filtered and digitized. With the exception of between cations which follows the usual dehydration Eisen-
β-HEPh (very poor solubility), all compounds of Scheme 1 gave man I sequence5 in which more easily dehydrated cations are
significant conductance activity by a number of methods of transported more readily (Cs+ > K+ > Na+).
compound introduction in different electrolytes. Activity
Some of the erratic conductance behaviour (Fig. 1: H)
required the compound to be added to both faces of the appears to possess an underlying structure. This is illustrated
bilayer, either through initial mixing with lipid, or by addition qualitatively in Fig. 2A (β-Eth; Table 1 entry 5) in which data
of solutions to both sides of the bilayer.
are plotted for different time spans of a single file filtered to
Fig. 1 shows examples of the types of conductance–time ensure the same density of points across the panel; the visual
records produced by these compounds. A significant proportion “self-similarity” of the three traces over several time decades
Fig. 1 Examples of conductance behaviours of cyclodextrin ion channels. DiPhyPC, 1 M CsCl except as indicated. (A) βHE4F, −100 mV; (B) βHE4,
+150 mV; (C) βHA4, +100 mV; (D) βHA4F, +80 mV; (E) βHE4, +80 mV; (F) βHA4F, −120 mV, gradient 1 M CsCl–0.2 M CsCl; (G) βHA5F, +100 mV, 1 M
NMe4NO3; (H) βHA5F, +50 mV.
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analysis selects a threshold current and counts an “event” as
the duration that the current exceeds the threshold. The list of
open-durations is then fit to a power-law function of the form:
pðxÞ ¼ Cxꢀα
ð1Þ
p(x) is some property of the system, e.g. the “open duration”
times, α is the exponent of the power-law, and C is a scaling
constant related to the number of observations. A statistically
robust approach to fitting experimental data has been
reported78 and implemented79 to find the value of α and the
minimum value of x (xmin) above which the power-law applies.
Fitting results are displayed on a log–log cumulative distri-
bution plot. An example of this type of data and its analysis is
given in Fig. 2B. The implementation also provides a statistical
test of the power-law hypothesis as a p-value with a cutoff of
p > 0.1 indicating greater than 90% probability that the power-
law hypothesis is supported. Confidence in the power-law
hypothesis increases as p tends to one.
Four of the compounds show this type of behaviour sus-
tained for long enough in the absence of other types of behav-
iour to produce data suited to the analysis. Results are given in
Table 1. Not all “erratic” data acquired support the power-law
hypothesis as strongly as the set of observations in Table 1 but
this is principally due to limitations on the periods where the
erratic behaviour is dominant. Several hundred events are
required and the analysis relies heavily on long-duration
events. The values of α range from 1.86 to 2.72; they are a
system property, as opposed to an inherent property of a
compound.
What is the physical origin of this underlying structure in
these data? Newman78 discusses several possibilities which
have been proposed in other contexts. Of these – a “random
walker” mechanism – offers a plausible scenario. In this
mechanism the “walker” would be a minimal conducting state
Fig. 2 Erratic conductance of β-Eth. (A) Current–time record (diPhyPC;
1 M CsCl; −100 mV) over three time spans of the same section processed
to show 1000 data points in the width displayed. (B) Cumulative distri-
bution function of “open durations” above a threshold of −60 mV for the
same data.
suggests the data have an underlying fractal structure. We pre- formed from some combination of a few of the appended
viously reported that the duration of excursions across a groups on the cyclodextrin. Thermally driven random align-
threshold (“open duration”) could be fitted to a power-law ments and disturbances of the appended groups would alter
relationship for two of the compounds in this report.77 The the conductance and the state would exist only so long as there
Table 1 Power-law fitting of open durationsa
Comp.
Duration (s)
Pot. (mV)
Threshold (pA)
xmin (ms)
n > xmin
α
p-Value
1 β-Ethb
600
1800
250
2100
3600
300
600
600
300
900
125
125
−100
−25
50
100
−50
−50
50
5
5
−60
−35
30
31
153
55
319
26
23
72
200
164
35
40
64
511
571
161
485
4725
538
568
295
282
1752
111
180
186
2.10
2.35
1.84
2.51
1.88
2.06
1.93
2.10
2.34
2.10
2.45
2.16
2.72
0.35
0.10
0.53
0.75
0.84
0.37
0.43
0.64
0.15
0.45
0.89
0.36
0.42
2 β-Ethb
3 β-Ethb
4 β-Ethc
5 β-Ethd
6 β-Ethd
30
7 β-Ethd
−75
−60
75
25
25
60
50
8 β-Ethd
9 β-HE4b
10 β-M3O2b
11 β-M3O2b
12 β-M3O2b
13 β-HA5Fb
50
120
120
530
100
125
50
89
a A file of the duration indicated was processed for events exceeding the threshold current and longer than 20 ms to give n events with associated
“open duration” that were fit to a power-law to return the value of xmin, the number of events that exceed this value, the value of α and the
p-value. b 1 M CsCl. c 1 M Et4NCl. d 1 M Me4NCl.
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was enough structure to support ionic conduction. A “combi-
nation of exponentials” mechanism also produces power-law
behaviour.78 We do not know how many concurrent active
states we observe, but it would not require many transmem-
brane pairs of seven-armed units to produce a complex con-
ductance profile. The power-law fits we report might simply
reflect the inherent complexity of a few embedded cyclodex-
trins capable of producing closely related conductance states
via thermal motions which would be individually decaying
exponentially.
These conductance records appear erratic and challenging,
but the underlying structures carry ion currents that are vastly
larger than those carried by conventional square-top channels,
even very active ones such as those produced by βHE4F.
Activity grids
Whatever the physical origin of the erratic events, it is clear
that they are an inherent part of the behaviour of this class of
compounds, as are the other behaviours illustrated in Fig. 1.
Although we could focus on the subset of behaviours amen-
able to analysis of specific conductance and event lifetime, the
raw data appear much richer. The uncovering of the structure
in erratic events therefore prompted us to look for a means to
summarize individual experiments in a way that would permit
us to compare a range of experiments on a single compound
and to survey all compounds in a model-free context. These
considerations prompted our development of activity grids
and their exploration with literature data16 and a limited set of
experimental data.43
Fig. 3 Processing of experimental data to produce an activity grid. (A)
Experimental current–time record (β-HE4F, 3.3 µM in
1 M CsCl,
diPhyPC); (B) applied potential; (C) computed conductance–time
record; (D) expanded sections showing additional conductance features;
(E) activity grids derived from the expanded sections and their sum-
mation to produce the overall grid for this experimental record.
Activity grids summarize experimental observations by the
type, the duration, and the conductance of events observed on
a coarse-grained scale (Fig. 3). The type of event observed is
encoded using a colour: green for the “square top” events such
as Fig. 1A, yellow for flickering between two open states
(Fig. 1C), blue for multi-level openings (Fig. 1D), red for spikes
and “shark fins” (Fig. 1F), and purple for erratic events, includ-
ing both those that can be fitted to a power-law and those
which simply appear to be similarly erratic. Experimental vari-
ables (filtering, digitizer resolution, bilayer quality) may make
some coordinates of the grids unobservable; these are indi-
cated in grey. An experimental file (current–time) may contain
data at a number of applied potentials which makes it challen-
ging to relate different portions of the record directly. The
initial processing converts current–time to conductance–time
using the known applied potentials (Fig. 3C from 3A and B). A
single experimental file may contain several types of events
(colours) over several orders of magnitude in duration and
conductance. The experimental record is therefore inspected
at various expansions (Fig. 3D) to produce a series of grids that
are summed to give the overall record (Fig. 3E). Finally, the
range of observables is established. The baseline in Fig. 3D
gives the noise at approximately 2 ps, thus the 1–3 ps row is
indicated in grey as unobservable. The filtering of the signal
similarly made events shorter than 10 ms unobservable in this
experiment.
How reliable is the activity grid methodology? The grids are
generated by human observation and require the observer to
rescale in both dimensions of the conductance–time curve.
Replicates by independent observers shown in Fig. 4A and B
show overall good agreement but do highlight a couple of
areas where disagreements occur. From a larger set of such
replicates, three areas of confusion are evident. One relates to
Fig. 4 Activity grid methodology. (A) Initial coding of five experimental
files; (B) independent re-coding of the same files (conditions: diPhyPC,
1 M CsCl).
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the determination of the duration of an erratic “event” as it is separate data files comprising a total of 2–3 hours of obser-
difficult to tell if the event returns to a quiescent baseline. vation for each experiment. This compound is highly active
This is an inherent property of such signals. A second area with a dominant square-top opening behaviour (Fig. 1A), and
concerns short duration spikes, some of which are associated gives similar levels of activity via a number of different
with capacitive discharging of the bilayer as the potential methods of compound introduction: mixed in lipid prior to
changes are applied. The third area is the detection of low con- bilayer formation (Fig. 5A), from electrolyte solution (25 µM;
ductance events, particularly those of long duration. These fre- Fig. 5B), by “contact injection” in which a stock solution in
quently are missed by one observer or another. The grids MeOH is injected in close proximity to the bilayer (Fig. 5C,D),
generated by a single trained observer are highly repeatable or by physical transfer to the membrane by brushing (Fig. 5E).
and the majority of the comparisons discussed below are of Contact injection is the usual method employed in our prior
this type.
work and generally in the literature as it is simple to do and
If the activity grid methodology accurately summarizes an usually produces activity fairly quickly. These individual experi-
experimental record it can be used to probe one of the funda- ments are clearly related to one another but they are not iden-
mental questions of the bilayer-clamp technique – how repro- tical in the view they give of the distribution of the different
ducible is the experiment? Fig. 5 gives activity grids separated types of activity, and the conductance and duration of each
by the type of activity observed for five independent experi- type of activity under a given set of conditions. Clearly the
ments with the same compound (β-HE4F), membrane lipid reproducibility of the experiment is moderate at best. The
(diPhyPC) and electrolyte (1 M CsCl). The grids combine 3–4 generation of activity grids requires a significant duration of
experimental observation, but it is also clear that several obser-
vations of the same system in independent experiments is also
required.
We note as well that the activity grids as now employed do
not embody the clear impression of human observers that the
square-top activity is dominant in the experiments of Fig. 5. A
single observation of
a particular activity–conductance–
duration is sufficient to colour a grid coordinate; subsequent
observations of the same type are not counted. Thus the
weight of repeated observations of a particular type is not rep-
resented. We chose this approach for practical reasons at the
outset when it was not clear if there was useful information to
be encoded in the metadata. That early decision effectively
reduces the information content of the grids generated in this
study. A more nuanced approach to include frequency of
observation would emphasize greater similarities in the
different experiments given in Fig. 5.
There is statistical power in the dataset as a whole. A total
of about 100 hours of observations of the 16 active compounds
was analysed by the activity grid method. As previously demon-
strated for literature data,16 the grid coordinates of each type
can be summed and the proportion of observations of a par-
ticular type and grid coordinate can be represented as a colour
density scale as shown in Fig. 6. Nearly half the recorded meta-
data are of the erratic type (46%). Multi-level (20%), spike
(16%), and square-top (14%) activities are similarly prevalent.
The flicker activity is rare (4%) in the dataset.
Fig. 5 Activity grids for different methods of introduction of β-HE4F
(diPhyPC, 1 M CsCl). (A) Compound mixed in lipid prior to bilayer for-
mation; (B) compound dissolved in the contacting electrolyte; (C, D)
compound introduced by “contact injection” injection of a MeOH solu-
tion in close proximity to the bilayer; (E) compound physically trans-
ferred via brushing the bilayer.
Fig. 6 Summary of activity for the entire dataset.
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The notable feature of Fig. 6 is the clustering around two cyclodextrins as portals, a conductance of 600 ps is
specific conductance–duration coordinates. With the exception expected (1 M CsCl). It is evident from Fig. 6 that some obser-
of short-duration spikes, all other types of activity cluster in vations fall into this range, but the clustering in the data
the region of 10–100 ps conductance and 0.1–10 seconds dur- occurs at lower conductance. The specific characteristics of the
ation events. The spikes are shorter duration but are predomi- expected dimer of half-channels will be superimposed upon
nantly of 10–100 ps conductance. A very similar range of the clustering of the system background.
clustering values was evident in the survey of literature data,16
Structure–activity relationships
and we previously argued that clustering is the signature of the
energetic consequences of the insertion of a small-molecule A goal of this study is to explore the relationship between half-
additive into a bilayer. The similarity between Fig. 6 and the channel structure and transport activity. The complexity of the
literature dataset strongly supports this view. The proportions data does not preclude that analysis, but it does alter how this
of the various types of activities differ between this dataset and type of question is approached. The set of compounds pre-
the dataset derived from published data, but their conduc- pared (Scheme 1) was created with implicit structural vari-
tance–duration characteristics do not.
ations. One main variable is the extended length of the
Clustering of activities on a global scale also allows criteria appended groups, which we naively viewed as a way to probe
to be established to identify those observations in the dataset the location of the cyclodextrin. If the cyclodextrin is relatively
that relate to the structures of the compounds. If a particular deep into the bilayer with the secondary face at the polar head
type of event, well removed from the cluster, is observed for a group region, then shorter derivatives (such as β-HE4; fully
particular compound, then the likelihood that the structure is extended length from the 6′O of the cyclodextrin ∼1.4 nm;
related to the activity is greatly enhanced. This places special overall length including the cyclodextrin ∼1.9 nm) would more
value on events of long duration and high conductance, and readily form channels than longer derivatives (e.g. β-HE8;
on events that occur relatively rarely, such as flickering. The chain length ∼1.9 nm; overall length ∼2.4 nm). Conversely, if
duration is related to the stability of the conducting species; a the triazole polarity required the cyclodextrin to sit relatively
longer duration opening implies a more stable structure. The high in the polar and mid-polar region, then the converse
conductance is related to the pore size of the conducting would be true. Such an analysis incorrectly assumes a static
species, typically via an approximate equation derived by bilayer thickness,82 but whatever our naïve expectation, the
Hille:80
activity of the series shows that length alone does not control
activity.
A second factor relates to the commonly asserted require-
ment for overall columnar shape of the channel-forming com-
pound in order to be compatible with lamellar phase lipids.5
ꢀ
ꢁꢀ
ꢁ
1
g
l þ πr
ρ
πr2
¼
ð2Þ
2
g is the conductance of the channel of length l and radius r. The cyclodextrin represents a very large head group with a
The equation is derived from the conductance of an equivalent large area in projection at the bilayer head group plane. The
volume and dimension of the electrolyte solution of specific tail cross section area of six or seven polymethylene chains, or
macroscopic conductance (ρ). The Hille equation is known to even triazoles, is substantially less in cross sectional area. The
give physically unrealistic dimensions for low conductance malonate series of compounds (such as β-M3O2), and the
channels,16 and has systematic bias evident from experiments steroidal derivatives were intended to more effectively fill the
on protein channels of known dimensions, for which more volume behind the cyclodextrin to impart a more columnar
sophisticated analyses are available.81 Two uses are common shape to the half-channel. The appendages of the esters lack
for synthetic ion channels: the derivation of r from an experi- specific sites for shape-controlling secondary interactions,
mental g which requires an assumption of l, or the prediction whereas the amide and urea derivatives were expected to
of an expected value of g from dimensions of a presumed provide sites of intra- and inter-molecular hydrogen bonding.
structure. The systematic bias of eqn (1) can be assessed using The per-fluoroalkyl derivatives (such as βHE4F) were expected
known protein channel dimensions and experimental conduc- to act similarly, based on a tendency for fluorous phases to
tance data.81 Bilayer “thickness” is not a static parameter, but separate from hydrocarbon phases. In all cases the expectation
if we assume a channel length of 3.5 nm,30 r is underestimated was that channel-forming activity would correlate with struc-
by eqn (2) by about 33% for a known g; conversely g is propor- tural factors that would stabilize columnar shapes.
tionally overestimated starting with a known r. These correc-
The expectation that some specific structural variable
tion factors apply well to the experimental conductance of an would turn on channel activity and that compounds lacking
unmodified α-haemolysin pore; a length of 5 nm and a radius this element would be inactive is not supported by the data.
of 0.45 nm gives a calculated conductance of 720 ps in good All the compounds of Scheme 1, except βEPh, show some type
agreement with 750 ps from experiment (1 M NaCl).57 Inser- of activity. This is not to say that structure plays no role in con-
tion of β-cyclodextrin into the α-haemolysin channel reduces trolling activity, only that the question needs to be reframed.
the conductance to 280 ps, consistent with a restriction to a
Fig. 7 shows an approach to exploring this question, in
pore of ∼3 Å radius in the middle of the channel. If the chan- which the activity grids for the single-chained compounds are
nels of this report involve passage over a 3.5 nm thickness via displayed with the incremental structural changes, relating
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Fig. 7 Activity grids of single-chain derivatives related by incremental structural changes.
pairs of compounds, indicated with arrows. In general the relate to experiments with additives discussed below with
chain length increases down the figure and functional group respect to Fig. 8.) A similar set of changes is seen in the shift
changes occur left to right. The question is whether any of the of ester to amide in βHE4 to βHA4; the latter has a diminished
obvious differences are significant in terms of the structural range of multi-opening events as well. These are the expected
changes. We reiterate that “significance” in the statistical changes in which more structured columnar channels are
sense is low due to the very limited number of observations formed with intramolecular hydrogen bonding or fluorous
underpinning some elements of the metadata. In addition, association. However, this is conclusion is not general as the
grid coordinates where the clustering noted above occurs must next higher homolog βHA5F shows substantial erratic behav-
have low significance as well. The clustering means that many iour over a range of conductance–duration coordinates and
compounds produce openings of a particular type, duration, this is one of the compounds that has been shown to fit the
and conductance, so these are of regions high similarity and power-law behaviour discussed above.
cannot address differences between compounds, whether
The length of the appended group plays some role as the
structurally constrained or not. As a guide to the eye, these longer derivatives are notably less prone to exhibit square-top
areas have been partially screened to reduce their visual and multi-level events compared with a similar distribution of
impact. If there are differences between compounds, they will conductance–duration for erratic events (compare βHE4/βHE8
most readily be recognized if they lie outside these screened and βHA4/βHA8). In the longer derivatives, the potential for
areas.
hydrogen-bonding plays very little role as the overall behaviour
An example of the type of difference that may be structu- is essentially unaltered in the ester-amide-urea sequence
rally controlled is the comparison of βHE4 and βHE4F. These (compare βHE8, βHA8, and βHU6). The role of “length” as a
two compounds are similar with the most significant differ- structural variable therefore appears to influence the distri-
ence of the reduction of the range of erratic events for the bution of conducting behaviours occurring in parallel, rather
fluorinated derivative. (There are differences in multi-level and than suppressing or enhancing activity. There is no “optimum
flickering events as well, but the underlying data for these length” signature in the data.
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Fig. 8 Additives alter the activity of cyclodextrin ion channels. (A) Activity grids of membrane conductance before and after addition of adamantane
carboxylic acid (grids sum several experiments with various concentrations and electrolytes). (B) Example of high-conductance openings by β-Chol
(2 mol%) with added adamantane carboxylic acid (2 mol% in diPhyPC; 1 M CsCl). (C) Example of high-conductance flicker openings by β-HE4F
(3.3 µM in electrolyte) with added adamantane carboxylic acid (0.28 mM) and β-cyclodextrin (7 mM; 1 M CsCl; diPhyPc). (D) Example of low-
conductance events by β-HE4F (3.3 µM in electrolyte) with added adamantane carboxylic acid (0.28 mM; 1 M CsCl; diPhyPc). (E) Example of low-
conductance events by β-HE4F (12 µM in electrolyte) with added adamantane carboxylic acid (0.33 mM; 1 M NaCl; diPhyPc).
Structure does influence activity, but the observed activities in the presence of adamantane carboxylic acid. Additional
do not operate exclusively under the control of the structure of examples of this type of experiment are given in Fig. 8.
the cyclodextrin derivative in the system. This is another mani-
The activity grids of the three compounds given in Fig. 8A
festation of the clustering in the overall dataset. If some pro- show that the addition of a potential guest for the cyclodextrin
portion of the activity relates to the general properties of any significantly alters the collection of observed behaviours.
compound in the bilayer environment, then that subset of There is a general increase in the conductance and duration of
activities is effectively independent of structural changes in a square-top and flicker events relative to smaller changes in the
pairwise comparison.
other types of events. Examples of the high-conductance
Fig. 7 does highlight the existence of some anomalous events observed are given in Fig. 8B and C; both show a domi-
behaviours. The long-duration/high conductance flicker event nant long-lived conductance state (1.7–1.8 ns) with both posi-
observations in βHA4F appear to be substantially different tive and negative excursions in conductance. This state closes
than in the related βHE4F. Similarly, the long duration/high to the baseline in Fig. 8B or to a long-lived state of lower but
conductance square-top and flicker events in βHE8 appear still significant conductance (750 ps) in Fig. 8C. The flickering
almost in isolation and therefore take some significance by of Fig. 8C is reminiscent of the flickering observed with
their rarity. This latter example was observed in an experiment β-cyclodextrin in α-haemolysin.54,56 Indeed the lifetime of the
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lower conductance state is 11 3 ms in fortuitous agreement
with a 14 ms lifetime reported for adamantane carboxylic acid
blockage of the β-cyclodextrin–α-haemolysin hybrid.54,56
It is very unlikely that the flickering in Fig. 8C is the transi-
ent blockage of a cyclodextrin portal in a dimeric channel of
the type envisaged in the design as the conductance is simply
too large. A Hille analysis of the 1.7 ns upper state gives an
apparent radius of 6 Å, a factor of two larger than expected for
a β-cyclodextrin. The long-lived stable state is potentially a can-
didate for a dimeric cyclodextrin channel but this shows no
“blockage” behaviour. Note as well that these high conduc-
tance states show even larger conductance openings on top of
them. This suggests conductance via an aggregate that adds
an additional component. The structure of that aggregate
might well depend on a hydrophobic guest, but is not what the
design implied.
Fig. 9 Candidate structures of potential conducting states.
Better candidates for partial blockage events are found
among lower conductance flicker events, such as those given compound also shows flickering consistent with transient
blockage by a hydrophobic guest, presumably of a channel
in Fig. 8D and E. These examples show a high proportion of
square-top openings that appear to underlie more rapid flick- involving an ion trajectory through a cyclodextrin (Fig. 8D and
ering. In both cases the flickering dies off to leave the square- E). The implication is that the unblocked square-top state in
these records is itself the signature of a channel with a cyclo-
approximately at the filtering frequency (5 kHz; 200 µs); flicker- dextrin portal.
top behaviour (see ESI†). The lifetime of these events is
ing is detected but the lifetime cannot be quantified. This type
of flickering superimposed on regular square-top events has only
been observed in the presence of adamantane carboxylic acid.
Some structural possibilities for active structures are
sketched in Fig. 9. Activity arises from addition to both sides
of the bilayer, so an inserted monomer (Fig. 9A) is not likely to
be active for the shorter derivatives (e.g. β-HE4). As the
appended group length increases the same type of inserted
Mechanistic implications
This study was based on a premise that half-channels with monomer would interfere in the opposed bilayer leaflet
cyclodextrin portals would provide a channel-like structure (Fig. 9B). This type of structure is a candidate for the activity
observed in the longer derivatives (e.g. β-HE8) and the steroid
and thereby limit the diversity of channel activities found for
simple acyclic oligoester channel-forming compounds. derivatives (e.g. β-Eth) as additional disorder in the opposed
Although good activity was found with essentially all com- leaflet could provide water defects for the channel. This type of
channel was previously proposed for the activity of sugar-
pounds prepared, there was no corresponding decrease in the
complexity of the conductance behaviours observed. Indeed, based CyPLOS channels.83 This structure is consistent with the
the observation of apparent power-law behaviour suggests shift in the distribution of activities as the “length” of the com-
pound is varied as discussed above.
increased complexity rather than simplification. However, it is
clear that even the erratic conductance reflects a specific group
The end-to-end dimer (Fig. 9C) is the design expectation.
of structures. Using activity grids as a means to abstract the Larger aggregates (Fig. 9D) offer a possibility of ion trajectories
outside the cyclodextrin portal in the contact regions between
activity type–conductance–duration information revealed a
clustering that is very similar to that derived from literature two cyclodextrins. The Eisenman I sequence indicates signifi-
data.16 The clustering observed via the activity grids implies cant dehydration of the ions as they traverse the membrane,
consistent with regions in contact with alkyl chains as encoun-
structures of similar properties can be formed by different
compounds. The activity grids also allowed an overall struc- tered in Fig. 9B and D. The same condition potentially would
ture–activity analysis which showed that the composition of be encountered within the dimer channel if the appended
groups collapsed on one another. In such a case the conduc-
tance expected would be much reduced from that of an open
the channel-forming compound did directly control the
activity.
The principal mechanistic implication in the face of this cylindrical channel of 3.5 nm length and the radius of the
cyclodextrin. The square-top channels of β-HE4F noted above
contradictory set of observations is that there can be no single
mechanism. Active structures of a variety of types must co-exist have a conductance of 30–50 ps depending on the experiment.
and interconvert leading to the complex behavioural patterns In the absence of more detailed modelling we cannot say if
this conductance value aligns with the proposed structure. The
observed. Even though the covalent architecture tethers many
components together around a portal, the potential for supra- larger conductance observed is expected for transport via
molecular polymorphism persists in this design.
aggregated states as observed in other systems.25,43,84 Of the
structures of Fig. 9, all would be capable of binding a suitable
One potential exception to this general conclusion is found
in the very abundant square-top behaviour of βHE4F. This hydrophobic guest, but only the dimer channel shows the
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potential to lead to transient blocking events of the type shielded by a grounded aluminium Faraday cage. Agar salt
observed.
bridges (2 M KNO3 in 1% Agar) were used to stabilize junction
potentials and were employed between the electrolyte in each
well of the cell and Ag/AgCl electrodes. Electrolyte solutions
were prepared from high purity salts and nanopure water.
Conclusions
A
stock solution of diphytanoyl phosphatidylcholine
Tabushi1 was right from the outset – half-channel derivatives
of cyclodextrins provide easy access to active ion channels.
What was missing in early analyses was an understanding of
how many different ways there are to generate transmembrane
ion currents via half-channel structures that have character-
istics recognized as “channels” (or at very least “channel-like”).
Our principle finding is that addition of compounds of this
type to bilayers will always open a number of parallel
pathways.
More generally, the activities inherent in this class of com-
pounds mirror the activities found in all other synthetic ion
channels. Using the activity grid analysis, we can detect the
signal of design expectations, overlain with the complexities
inherent to small-molecule transporters. In keeping with other
synthetic transporters, the background activity relates to
defects inherent in the emplacement of molecules in a bilayer.
This leaves as an unresolved challenge to heal defects so the
characteristics of defined structures increase in probability in
the overall activity. Whether we should seal off the defect path-
ways that are responsible for irregular conductance traces to
leave “pure” square-top ion channels is an academic question;
from the practical perspective of moving ions, defect-related
channels are vastly superior.
(diPhyPC) in chloroform (Avanti Polar Lipids; shipped on dry
ice) was divided into sealed glass vials under an argon atmos-
phere and stored at −12 °C. For use in an experiment, a stream
of dry nitrogen was passed through the vial for 1 hour. The
dried lipid was diluted with decane to give a solution concen-
tration of 25 mg mL−1 in lipid.
Bilayers were formed by either brushing or dipping: after
lipid in decane had been introduced by brushing, a lipid/
decane film formed on the surface of the electrolyte, and
bilayers could then be formed by withdrawal of 2–3 mL of elec-
trolyte from the cell holder by syringe to expose one face of the
aperture to the air–water interface held in the cell holder, fol-
lowed by reintroduction of the electrolyte to oppose mono-
layers across the aperture in the cuvette. Bilayer quality was
monitored via the capacitance and stability under applied
potential, using the criteria previously described.85 The
measured voltage was applied with respect to the trans
(cuvette) side of the bilayer, making the trans side the relative
ground. Digitized data files were analysed using the pClamp10
suite of programs.
The compounds are introduced to the membrane in two
ways, depending on the solvent in which the compound can
be dissolved: direct injection – all injection experiments uti-
lized bilayers that were apparently stable at 100 mV for periods
of 20 minutes or more. Aliquots (1–5 µL of transporter solu-
tions in MeOH were injected with a microliter syringe as close
as possible to the bilayer in the free well of the cuvette holder
(cis side), and gently stirred with a stream of nitrogen for
5 minutes. Pre-mixed into lipid – in this method, 1 mol% of
compound (in CDCl3 or MeOH-d4) was added to the diPhyPC/
CHCl3 solution, and solvent removed with a stream of N2, and
bilayer membrane prepared by brushing/dipping as described
above. Most of the bilayers formed with this method gave
bilayers with good quality.
From a methodological perspective the activity grids work
as designed. They provide a high-level view of the transport
activities that is useful in the structure–activity analysis under-
taken. At the same time, some deficiencies are obvious. In the
next iteration, the representation should be redesigned to
show the frequency or proportion of observations of a given
type. However, accurately extracting this metadata will necessi-
tate software and automation to assist the human observer
with this daunting task.
Of the two methods, direct injection is preferable, as it
allows monitoring of pristine bilayer prior to compound intro-
duction. Following direct injection, channel behaviour typi-
cally appears within 20 minutes of compound introduction,
and persists over period of hours. Once stabilized, continuous
data acquisition of at least 30–60 minutes is required to
Experimental
Complete synthetic details for all new compounds are available
in the ESI.†
Voltage clamp data acquisition
A model BC-525A bilayer clamp (Warner Instrument Corp.) provide sufficient statistical power for the power-law analysis;
was used for planar bilayer experiments. The analogue output shorter acquisition periods (10 minutes) are sufficient to
was filtered with an 8-pole Bessel filter (Frequency Devices, characterize other types of behaviours.
model 902) and digitized with a 330 kHz digitizer (Axon Instru-
The activities of all compounds examined are summarized
ments, Digidata 1200A). Data acquisition was controlled by the in a standard format in the ESI† showing activity grid deri-
pClamp8 software package (Axon Instruments). Data were col- vation for individual data files acquired.
lected at 10 Hz, analogue filtered at 1 Hz, and digitally filtered
Power-law fitting procedure
at 50 Hz. The headstage and the bilayer chamber (3 mL poly-
styrene cuvette with 250 µm diameter aperture held in a 5 mL Fitting experimental data to a power-law requires two distinct
PVC holder) were placed on a floating table and electrically steps. The first step transforms the irregular current trace into
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a list of opening times; this list is then fitted to a power-law 17 A. Lorin, M. Noel, M. E. Provencher, V. Turcotte,
distribution. (1) Manipulation of the digitally filtered traces
was carried out using Clampfit 10 of the pClamp suite. A cus-
S. Cardinal, P. Lague, N. Voyer and M. Auger, Biophys. J.,
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tomized threshold search was used to generate the list of 18 N. Sakai, J. Mareda and S. Matile, Acc. Chem. Res., 2008, 41,
events. The threshold was set across the fluctuating section of 1354–1365.
the trace to maximize the number of events. Within that 19 F. Mora, D.-H. Tran, N. Oudry, G. Hopfgartner,
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to the fitting program. The list of opening durations, obtained 21 P. L. Boudreault and N. Voyer, Org. Biomol. Chem., 2007, 5,
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Acknowledgements
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Research Council of Canada and the University of Victoria is 26 H. Luong and T. M. Fyles, Org. Biomol. Chem., 2009, 7,
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