Triazole-tailored guanosine dinucleosides as biomimetic ion channels to modulate transmembrane potential

Article abstract of DOI:10.1002/chem.201304530

A click ion channel platform has been established by employing a clickable guanosine azide or alkyne with covalent spacers. The resulting guanosine derivatives modulated the traffic of ions across the phospholipid bilayer, exhibiting a variation in conductance spanning three orders of magnitude (pS to nS). F?rster resonance energy transfer studies of the dansyl fluorophore with the membrane binding fluorophore Nile red revealed that the dansyl fluorophore is deeply embedded in the phospholipid bilayer. Complementary cytosine can inhibit the conductance of the supramolecular guanosine channels in the phospholipid bilayers. Coming through: A click ion-channel platform was established by employing a clickable guanosine azide or alkyne with covalent spacers. The guanosine derivatives modulated the traffic of ions across the phospholipid bilayer. FRET studies of the dansyl fluorophore with the membrane-binding fluorophore Nile red revealed that the dansyl fluorophore is deeply embedded in the phospholipid bilayer (see figure).

Full text of DOI:10.1002/chem.201304530

DOI: 10.1002/chem.201304530
Communication
&
Self-Assembled Nanochannels
Triazole-Tailored Guanosine Dinucleosides as Biomimetic Ion
Channels to Modulate Transmembrane Potential
Y. Pavan Kumar,[a] Rabindra Nath Das,[b] Sonu Kumar,[b] Ole Mathis Schꢀtte,[c]
Claudia Steinem,*[c] and Jyotirmayee Dash*[a, b]
Dedicated to Professor Dr. Goverdhan Mehta on the occasion of his 70th birthday.
yield.[2–4] The development of a facile and modular synthetic
approach would enable the creation of a large array of mem-
brane active structures for studying the structure–activity of
a class of compounds. The key features of synthetic ion chan-
nels that need to be addressed are the pore size, ion selectivi-
ty, voltage and ligand gating and blockage of the channels by
using specific compounds.
Abstract: A “click” ion channel platform has been estab-
lished by employing a clickable guanosine azide or alkyne
with covalent spacers. The resulting guanosine derivatives
modulated the traffic of ions across the phospholipid bi-
layer, exhibiting a variation in conductance spanning
three orders of magnitude (pS to nS). Fçrster resonance
energy transfer studies of the dansyl fluorophore with the
membrane binding fluorophore Nile red revealed that the
dansyl fluorophore is deeply embedded in the phospho-
lipid bilayer. Complementary cytosine can inhibit the con-
ductance of the supramolecular guanosine channels in
the phospholipid bilayers.
Recently lipophilic nucleoside derivatives have been de-
signed to create complex self-assembled structures with desir-
able function.[8] Among the nucleobases, guanine has generat-
ed wide interest in various areas of research ranging from mo-
lecular biology to nanotechnology.[9] Guanosine derivatives can
self-associate to form cyclic tetramers called G-quartets, which
are planar arrangements of four guanine molecules linked to-
gether by Hoogsteen-type hydrogen bonds.[10a,c] These quar-
tets stack on top of one another to give a columnar aggregate,
which is known as a G-quadruplex motif and is stabilized by
certain cations, for example, Na+, K+. These supramolecular
structures are believed to play a key role in the biology of
cancer and ageing.[10d–f] As a hydrogen-bonded macrocyclic ar-
rangement with ionophore properties, G-quartets signify
a promising scaffold for fabricating synthetic ion channels.
Sakai et al. reported that folate dendrimers containing a simi-
lar hydrogen bonding unit like guanine form synthetic trans-
membrane ion channels with a conductance of 21 pS.[11] Davis
and co-workers reported that ditopic guanosine–bile acid con-
jugates can form ion channels in phospholipid membranes
with nanosiemens (nS) conductance.[12] We envisioned that if
a modular synthetic strategy based on CuI-catalyzed 1,3-dipolar
azide–alkyne cycloaddition can be developed, it would signifi-
cantly expand the structure–function relationships of the gua-
nosine-based ion channels through tethering of the covalent
spacers between two guanosine units. Herein, we report the
design and synthesis of diguanosine derivatives using two effi-
cient and modular strategies based on “click chemistry”[13] be-
tween either a clickable guanosine azide or an alkyne with aro-
matic, amphiphilic and lipophilic linkers. The ion channel activi-
ty of these guanosine derivatives have been demonstrated
using voltage-clamp experiments, which show that diguano-
sine derivatives form discrete channels with stable and large
pores with nS conductance in the phospholipid membrane.
Following extensive optimization, we have developed two
general approaches for synthesizing the acetylene and azide
building blocks (Figure 1, Scheme 1 and Scheme 2). We have
incorporated: 1) the azide unit in guanosine 1, and 2) the acet-
Biological membranes consisting of a lipid bilayer play funda-
mental roles in partitioning cells and organelles in all living or-
ganisms. Transmembrane proteins embedded in the bilayer,
that is, natural ion channels, facilitate the transport of ions
across these highly insulating barriers.[1] Inspired by these
channels in living systems, the creation of natural and non-nat-
ural molecules that can mimic structural aspects of natural
transmembrane ion channel proteins in lipid bilayers have re-
ceived much attention in recent years.[2,3] Biomimetic nano-
channels have been developed to understand mechanistic de-
tails of channel proteins on the molecular level[4] and have
been found useful as drug delivery systems,[5] antimicrobial
agents[6] and biosensors.[7] Most nonpeptidic ion channels have
been prepared using multistep linear synthesis with low overall
[a] Y. P. Kumar, Prof. Dr. J. Dash
Department of Organic Chemistry
Indian Association for the Cultivation of Science
Jadavpur, Kolkata-700 032 (India)
E-mail: ocjd@iacs.res.in
[b] R. N. Das, S. Kumar, Prof. Dr. J. Dash
Department of Chemical Sciences
Indian Institute of Science Education and Research Kolkata
Mohanpur Campus, Mohanpur, - 741 252 (India)
[c] O. M. Schꢀtte, Prof. Dr. C. Steinem
Institute for Organic and Biomolecular Chemistry
Georg August University Gçttingen
Tammannstr. 2, 37077 Gçttingen (Germany)
E-mail: csteine@gwdg.de
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201304530.
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The fluorescent dinucleoside 5a can self-assemble to pro-
vide a G-quartet-based fluorescent ion channel, and the ion
channels can be stabilized due to the p–p interactions be-
tween the naphthalene rings of the dansyl derivative of each
G-quartet layer. Then, we introduced a polyethylene glycol
(PEG) linker between two guanosine units by ligation of PEG
diacetylene 3b (n=9) with the guanosine azide 1 under stan-
dard “click” condition. The ditopic guanosine 5b containing
biocompatible PEG as a spacer can provide hydrophilicity to
the hydrophobic guanosine nucleoside and may lead to the
formation of G-quartet-based amphiphilic ion channels.
The second approach was then followed to access lipophilic
and carboxamide-containing guanosine derivatives (Scheme 2).
Click reaction was performed between two lipophilic guano-
sine alkyne 2 with a lipophilic polyalkane diazide 4a to obtain
the bis-guanosine triazole derivative 6a in high yield
(Scheme 2). An aromatic bis-carboxamide azide 4b was suc-
cessfully employed as the spacer with the guanosine alkyne 2
under identical reaction condition to afford 6b in good yield
(Scheme 2).
To demonstrate that these dinucleosides form ion channels
for K+ ions, voltage-clamp experiments on planar solvent-free
bilayers were performed in
Figure 1. Azide and alkyne building blocks.
a buffer containing KCl (1m) and
NaH2PO4 (2 mm) at pH 7.4.
Planar solvent-free bilayers were
generated by spreading giant
unilamellar vesicles (GUVs) con-
sisting of 1,2-diphytanoyl-sn-
glycero-3-phosphocholine
(DPhPC) and cholesterol (9:1;
Figure S1 in the Supporting In-
formation) on a small aperture in
glass.[16] Current traces were re-
corded after adding the dinu-
cleoside 5 and 6 (20 mm) to
the cis-side of the chamber at
an applied voltage of À100 mV
(Figure 2, Figures S2–S4 in the
Supporting Information).
The insertion of nucleosides 5
and 6 into the lipid membrane
resulted in characteristic current
steps suggesting the formation
Scheme 1. Synthesis of bis-guanosine derivatives from azido guanosine.
of
a
defined ion channel
(Figure 2) that opens and closes.
The observed opening and clos-
ylene unit in guanosine 2 (Scheme 1 and Scheme 2). The click-
able lipophilic guanosine azide 1 and guanosine alkyne 2
building blocks were prepared from guanosine using two to
three step protocols (see the Supporting Information). The flu-
orescent dansyl dialkyne 3a was prepared from the commer-
cially available dansyl amide in high yield (see the Supporting
Information). The azido guanosine derivative 1 was treated
with the dansyl containing dialkyne 3a in the presence of Na-
ascorbate and CuSO4·5H2O in tBuOH/H2O (1:1) as solvent[14] to
give fluorescent diguanosine nucleoside 5a in high yield.[15]
ing can be attributed to the dynamic self-assembly and disas-
sembly of the chiral supramolecular structures formed by the
guanosine derivatives. Statistical data analysis of the current
traces showed that there is a collection of conductance values
spanning three orders of magnitude from <0.1 to 10 nS. Gua-
nosine–dansyl conjugate 5a exhibited 61.3% of the <0.1 nS
channels and 36.1% of the 0.1–0.5 nS ones with lifetimes of up
to 1 s. The diguanosine 5b with the flexible PEG linker pro-
duced considerably larger conductance values within seconds,
which is consistent with larger pores (Figure 2, Table 1).
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the species formed by 6a. The
diguanosine
6b
containing
phenylene dicarboxamide unit
as the spacer most frequently
exhibited channels with conduc-
tance levels of 1–2 nS (87.6%).
We then investigated the ion
channel activity of diguanosine–
PEG conjugate 5b using dif-
ferent ionic species passing
through the membrane (Table 2).
Since various monovalent cat-
ions of different size can tem-
plate a wide variety of supra-
molecular structures, we hy-
pothesized that the channel’s
conductance could alter in the
presence of such monovalent
cations.[9,10] The conductance de-
Scheme 2. CuI catalyzed Huisgen 1,3-dipolar cycloaddition with a guanosine alkyne.
pends on the pore size as well
as on the size of the ions. A suf-
ficiently large channel does not require full dehydration of the
ion. It is interesting to note that small ion channel conduc-
Table 1. Frequency of events for channels formed by 5 and 6.[a]
Conductance
5a
5b
6a
6b
[nS]
events [%]
events [%]
events [%]
events [%]
<0.1
0.1–0.5
0.5–0.8
0.8–1
1–2
2–5
5–10
>10
428 (61.3)
252 (36.1)
18 (2.6)
–
–
–
–
–
4 (0.6)
49 (7.9)
50 (8.0)
35 (5.6)
152 (24.4)
299 (48.0)
33 (5.3)
1 (0.2)
2 (0.3)
106 (15.1)
52 (7.4)
81 (11.5)
384 (54.8)
70 (10.0)
6 (0.9)
–
8 (0.9)
30 (3.3)
67 (7.4)
789 (87.6)
6 (0.7)
1 (0.1)
–
–
[a] The number and frequency of ion channel open events were taken
from patch-clamp experiments (Figure 2, Figures S2–S4 in the Supporting
Information). Numbers in brackets indicate the relative percentage be-
tween the boundaries.
Figure 2. a) Results of voltage-clamp experiments showing representative
states recorded after the addition of 5b (20 mm) to the cis-side of the cham-
ber after the planar bilayer was formed. The experiment was performed at
À100 mV in a buffer containing KCl (1m) and Na2HPO4 (2 mm) at pH 7.4.
b) Distribution of the observed conductance states (628 events) of com-
pound 5b. c) Open time distribution (628 events) of 5b.
Table 2. Frequency of events for channels formed by 5b in the presence
of different ions.[a]
Conductance
[nS]
Na+
events [%]
K+
Cs+
events [%]
Remarkably, about 48% of these channels formed by 5b
showed conductance values of 2–5 nS and are “open” up to
5 s in the planar membranes. Diguanosine 5b also showed
channels with conductance values of 5–10 nS (5.3%). It is note-
worthy that crown ethers usually exhibit pS range conduc-
tance, whereas a PEG–diguanosine forms channels with nS
conductance. The putative ion channel formed by 6a contain-
ing lipophilic alkyl groups provided a conductance of 1–2 nS
(54.8%). It was also observed that the channels formed by 6a
were more stable and “open” for a longer time compared to
the hydrophilic polyether 5b (Table S1 in the Supporting Infor-
mation). This may be attributed to the higher lipophilicity of
events [%]
<0.1
0.1–0.5
0.5–0.8
0.8–1
1–2
2–5
5–10
>10
–
–
–
–
4 (0.6)
49 (7.9)
50 (8.0)
8 (1.3)
85 (14.1)
8 (1.3)
35 (5.6)
1 (0.2)
469 (22.6)
1591 (76.8)
12 (0.6)
–
152 (24.4)
299 (48.60)
33 (5.3)
401 (66.4)
98 (16.2)
3 (0.5)
1 (0.2)
–
[a] The number and frequency of ion channel open events were taken
from patch-clamp experiments (Figure 2, Figures S5 and S6 in the Sup-
porting Information). Numbers in brackets indicate the relative percent-
age between the boundaries.
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tance in the range of 0.1–0.8 nS was observed in K+- and Cs+
-containing buffer, whereas a larger conductance of 1–5 nS
with more events was observed in the presence of Na+. Chan-
nels with conductance levels of 1–2 nS (22.6%), 2–5 nS (76.8%)
and 5–10 nS (0.6%) were formed by 5b in Na+-containing
buffer. The conductance decreased to 1–2 nS in Cs+-containing
buffer. These results indicate the presence of multiple pore
structures in the membrane and the selectivity is controlled by
the self-organized dynamic structure in the presence of differ-
ent monovalent ions of different size. No measurable conduc-
tance was observed for 5b in MgCl2 and CaCl2 in planar bilayer
membranes indicating high selectivity for monovalent cations.
Circular dichroism spectroscopic analysis of 5b provides evi-
dence in support of G-quadruplex formation in the phospho-
lipid bilayer (Figure S7 in the Supporting Information).[17] Fluo-
rescence spectroscopy was then used to investigate the loca-
tion of the dansyl group of 5a in the phospholipid bilayer.[18,19]
The fluorescence emission spectra of 5a were recorded with
an increasing concentration of liposomes (lex =350 nm, Fig-
ure S8 in the Supporting Information). In the absence of lipo-
somes, the dansyl–guanosine conjugate 5a showed two emis-
sion bands at 430 and 554 nm, which were assigned to the
guanosine and the dansyl group, respectively. The fluorescence
intensity of guanosine band of 5a increased after stepwise ad-
dition of liposome suspension, indicating binding of the iono-
phore 5a to the membrane (Figure S7 in the Supporting Infor-
mation).[20d] Further, the emission maximum of the dansyl
group showed an about 19 nm blue shift with increasing lipo-
some concentration (0–1 mm lipid).[20] This blue shift in emis-
sion maximum is characteristic for a less polar and less hydrat-
ed microenvironment of the dansyl group and can be inter-
preted as a localization of the dansyl fluorophore of 5a in the
bilayer.
Figure 3. Fluorescence spectra (lex =350 nm) of dansyl–guanosine conjugate
5a (500 nm), Nile red (5 mm), 5a and Nile red in the presence of LUVs in PBS
(2.7 mm KCl, 136.9 mm NaCl, 1.5 mm KH2PO4, 8.1 mm NaH2PO4, pH 7.4).
Fçrster resonance energy transfer (FRET) studies further sug-
gest that the dansyl fluorophore is localized in the phospholip-
id bilayer. Nile red is a membrane binding dye, the photophy-
sics of which in lipid bilayers has widely been studied.[18,20] As
the absorption maxima (551 nm) of Nile red overlaps with the
emission of 5a, these two can be considered as a donor–ac-
ceptor FRET pair, in which 5a acts as a donor moiety, while
Nile red is the acceptor. The results show that upon addition
of 5a to a Nile red containing vesicle suspension, the intensity
of Nile red at 620 nm increases, whereas the intensity of 5a at
535 nm decreases (Figure 3). Here, efficient FRET indicates that
the dansyl moiety must be in close proximity to the Nile red
molecule, which is well known to specifically bind to the lipid
membrane.[20] The ion current steps (Figure 2, Figures S5 and
S6 in the Supporting Information) can thus be attributed to an
assembly and disassembly of stably inserted G-quartet aggre-
gates and are most likely not related to transient insertions of
molecules into the membrane.
Figure 4. Time-course of CF leakage from liposomes (DPhPC/cholesterol, 9:1)
caused by 5b in PBS (2.7 mm KCl, 136.9 mm NaCl, 1.5 mm KH2PO4, 8.1 mm
NaH2PO4, pH 7.4). At t=70 min, the liposomes were treated with Triton-X.
ed with CF and the fluorescence intensity was monitored over
time at 520 nm (lex =492 nm). No release of CF was observed
in the absence of 5b or upon addition of DMSO. Addition of
5b led to a small increase in fluorescence intensity, indicating
that 5b does not significantly alter the integrity of the mem-
brane (Figure 4). The percentage of CF release was determined
by monitoring the fluorescence intensity of released CF and
compared to a 100% release by destroying the liposomes
using Triton-X. The percentage release of CF from vesicles into
the extravesicular solution was calculated to be 12.3% after
1 h.
The dansyl–guanosine conjugate 5a most frequently exhibit-
ed channels with small conductance values in the range
<0.1 nS (Table 1), which can be attributed to the size of a G-
quartet diameter similar to that reported for the folate dendri-
mer, which produced a conductance of 21 pS.[12] The p–p inter-
actions and the hydrophobic interactions of the dansyl central
unit may stabilize the supramolecular architecture of the quar-
tets within the bilayer. Since the insertion of 5b and 6 into
The carboxyfluorescein (CF) release assay was used to moni-
tor whether large pores are formed in the vesicles upon inter-
action with 5a that might cause the observed large conduc-
tance values.[21] CF-encapsulating vesicles were prepared com-
posed of DPhPC/cholesterol (9:1). The PEG–guanosine conju-
gate 5b (100 mm in DMSO) was added to the vesicles preload-
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membranes produced large conductance in the nS range, the
functional pores are expected to be larger than a quartet
formed from each of 5 and 6.[12–14] The voltage clamp experi-
ments of dinucleosides 6a and 6b indicated the formation of
thermodynamically favorable ion channels with a conductance
range of 1–2 nS. Guanosine derivative 5b containing the long
chain spacer like PEG gave higher conductance. The self-associ-
ation of the guanosine moieties into a G-quartet would result
in a columnar supramolecular architecture to form barrel-
stave-type ion channels (Figure 5).
sociate and the channels disappear. We observed that ion
channels formed from compound 5b disappear upon addition
of excess cytosine (5 mm) to the lipid bilayers containing 5b,
which may be attributed to a lack of any stable (long-lasting)
three-dimensional structure (Figure 6). CD spectra of a solution
Figure 6. Inhibition of ion channel formed from 5b using complementary
cytosine.
containing 5b (10 mm) and LUVs at pH 7.4 in PBS were record-
ed in the absence and presence of cytosine. Addition of
7 equiv of cytosine led to a decrease of the band intensity at
246 nm and formation of several new positive peaks at 261,
276 and 282 nm (Figure S9 in the Supporting Information).
This suggests a structural transition upon addition of cyto-
sine.[22]
In summary, a self-assembled “click” ion channel platform
has been established using a one-pot azide–alkyne cycloaddi-
tion of lipophilic guanosine azide and guanosine alkyne build-
ing blocks with a variety of covalent spacers. Our results show
that not only bile-acid units can be used as a spacer between
guanosine units for transport of ions, but the pore size and
conductance can be modulated to give more regular openings
by changing simplified click spacers. The noteworthy aspects
of our approach include expansion of guanosine-based ion
channels, monitoring the formation of ion channels using fluo-
rescent guanosine in the lipid bilayer and cytosine induced in-
hibition of the conductance formed from the PEG-linked gua-
nosine. These investigations should enable a new modular ap-
proach for the preparation of analogues and decoration of ex-
isting ion channels leading to increased diversity and in turn,
pore size and ionic selectivity.
Figure 5. Proposed model of: a) barrel-stave-type cation channels, and
b) formed by diguanosine derivatives in a phospholipid bilayer.
A cation-filled G-quadruplex presumably directs the assem-
bly of these dynamic structures within the membrane. Since
the size of the monovalent cations can lead to variations in
the supramolecular organization of guanosine derivatives, dif-
ferent cations found to alter pore size and opening and closing
of the channels. Since we did not observe significant leakage
of CF (~10 ꢂ long and 6.5 ꢂ wide) from liposomes using the
PEG derivative 5b, we hypothesize the formation of barrel-
stave-type of ion channels (Figure 5a). The channels formed by
5b with conductance values of 5–10 nS (5.3%) are expected to
form barrel-stave-type ion channels (Figure 5b) which can pro-
mote the transport of CF (12.3% after1h).
Acknowledgements
The authors thank Professor Kankan Bhattacharyya, Sirshendu
Ghosh and Manish Debnath of IACS Kolkata for helpful discus-
sions. This work was supported by the Department of Atomic
Energy (BNRS) and Department of Biotechnology (DBT), India.
Y.P.K. and R.N.D. thank CSIR India; R.N.D. thanks the DAAD Ex-
change Programme for a research fellowship.
Our next effort was to study the inhibition of the ion chan-
nels formed by the insertion of guanosine derivatives into the
lipid bilayers. We envisaged that, since the ion channels are
formed from the spontaneous self-assembly of guanosine, the
addition of cytosine to the lipid bilayer would break the self-
assembly of guanosine and inhibit the ion flow. Due to the
complementary geometric arrangement of hydrogen-bond
donors and acceptors in guanosine and cytosine, they can as-
Keywords: amphiphilic guanosine · click chemistry · ion
channels · inhibition · phospholipid bilayers
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