Imidazolinium-based Multiblock Amphiphile as Transmembrane Anion Transporter

Article abstract of DOI:10.1002/asia.202001106

Transmembrane anion transport is an important biological process in maintaining cellular functions. Thus, synthetic anion transporters are widely developed for their biological applications. Imidazolinium was introduced as anion recognition site to a multiblock amphiphilic structure that consists of octa(ethylene glycol) and aromatic units. Ion transport assay using halide-sensitive lucigenin and pH-sensitive 8-hydroxypyrene-1,3,6-trisulfonate (HPTS) revealed that imidazolinium-based multiblock amphiphile (IMA) transports anions and showed high selectivity for nitrate, which plays crucial roles in many biological events. Temperature-dependent ion transport assay using 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) indicated that IMA works as a mobile carrier. ¹H NMR titration experiments indicated that the C2 proton of the imidazolinium ring recognizes anions via a (C?H)⁺???X^? hydrogen bond. Furthermore, all-atom molecular dynamics simulations revealed a dynamic feature of IMA within the membranes during ion transportation.

Full text of DOI:10.1002/asia.202001106

DOI: 10.1002/asia.202001106
Full Paper
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Imidazolinium-based Multiblock Amphiphile as Transmembrane
Anion Transporter
Miki Mori,[a] Kohei Sato,*[a] Toru Ekimoto,*[b] Shinichi Okumura,[b] Mitsunori Ikeguchi,*[b, c]
Kazuhito V. Tabata,*[d] Hiroyuki Noji,*[d] and Kazushi Kinbara*[a]
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Abstract: Transmembrane anion transport is an important
biological process in maintaining cellular functions. Thus,
synthetic anion transporters are widely developed for their
biological applications. Imidazolinium was introduced as
anion recognition site to a multiblock amphiphilic structure
that consists of octa(ethylene glycol) and aromatic units. Ion
transport assay using halide-sensitive lucigenin and pH-
sensitive 8-hydroxypyrene-1,3,6-trisulfonate (HPTS) revealed
that imidazolinium-based multiblock amphiphile (IMA) trans-
ports anions and showed high selectivity for nitrate, which
plays crucial roles in many biological events. Temperature-
dependent ion transport assay using 1,2-dipalmitoyl-sn-
glycero-3-phosphocholine (DPPC) indicated that IMA works
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as a mobile carrier. H NMR titration experiments indicated
that the C2 proton of the imidazolinium ring recognizes
anions via a (CÀ H)+···XÀ hydrogen bond. Furthermore, all-
atom molecular dynamics simulations revealed a dynamic
feature of IMA within the membranes during ion trans-
portation.
Introduction
nitrate, abundant in our everyday diet, had been believed as a
harmful species to promote gastric cancer, as well as an inert
product of nitrogen oxide (NO) metabolism. Meanwhile, recent
studies indicated that nitrate is a substrate for generating
bioactive NO through the nitrate-nitrite-NO metabolism path-
way, where such a metabolization pathway also mediates cell
signaling and blood flow regulation. It is also considered to play
a therapeutic role in cardiovascular diseases.[3]
Natural membrane proteins have very complex structures
depending on their target ions and transport mechanisms,[4]
and only a few natural anion carriers are known to exist.[5]
Despite lack in structural motifs in natural systems, various
structures have emerged as synthetic anion transporters,
recognizing anions through anion-π interaction, electrostatic
interactions, halogen bonds, chalcogen bonds and hydrogen
bonds.[5,6] Although CÀ H···XÀ hydrogen bond had emerged as
another important noncovalent anion recognition strategy in
developing anion receptors,[7] its role had been supplementary
in transport systems of mobile carriers.[8] Anion transporters
that strongly rely on CÀ H···XÀ hydrogen bonds are still rare
compared to those bearing traditional NÀ H and OÀ H hydrogen
bond donors.[9,10]
Previously, our group has reported series of multiblock
amphiphiles capable of ion transport as channels. They consist
of alternated structure of hydrophilic oligo(ethylene glycol)
chains and hydrophobic aromatic moieties, which self-assemble
by π-stack of aromatic moieties within the lipid bilayer
membranes. The designs of these molecules were derived from
multipass transmembrane proteins, which exhibit their func-
tions by folding within the lipid bilayers.[11–14] Variable designs in
hydrophobic moieties had enabled to develop diverse charac-
teristics in ion transportation. Membrane-tension responsive ion
channel formation was achieved by destabilization of aromatic
stacking by introducing sterically hindered aromatic unit.[12]
Reversible ligand-gating ion transport was realized by incorpo-
Ion homeostasis of the cells is essential element in maintaining
life. Generation and preservation of ion concentration gradient
means regulation of pH, cellular volumes or membrane
potentials, which plays crucial roles in diverse cellular functions
such as apoptosis, differentiation and proliferation.[1] Regulation
of ion concentration is achieved by membrane transporters
embedded within the lipid bilayer membranes, by controlling
transported ionic species and their directions. However, genetic
diseases may trigger disordered ion transport. Synthetic mem-
brane transporters, especially anion transporters, are eagerly
developed for their potentials in biological applications and
therapeutics for diseases related to dysfunction of membrane
transporters, including cystic fibrosis.[2] Among the anions,
[a] M. Mori, Dr. K. Sato, Prof. K. Kinbara
School of Life Science and Technology
Tokyo Institute of Technology
4259 Nagatsuta-cho, Midori-ku, Yokohama, Kanagawa, 226-8501 (Japan)
E-mail: koheisato@bio.titech.ac.jp
kkinbara@bio.titech.ac.jp
[b] Dr. T. Ekimoto, S. Okumura, Prof. M. Ikeguchi
Graduate School of Medical Life Science
Yokohama City University
1-7-29 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa, 230-0045 (Japan)
E-mail: ekimoto@yokohama-cu.ac.jp
ike@yokohama-cu.ac.jp
[c] Prof. M. Ikeguchi
RIKEN Medical Science Innovation Hub Program
1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa, 230-0045 (Japan)
E-mail: ike@yokohama-cu.ac.jp
[d] Prof. K. V. Tabata, Prof. H. Noji
Graduate School of Engineering
University of Tokyo
7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8656 (Japan)
E-mail: kazuhito@nojilab.t.u-tokyo.ac.jp
hnoji@appchem.t.u-tokyo.ac.jp
Supporting information for this article is available on the WWW under
https://doi.org/10.1002/asia.202001106
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Results and Discussion
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Synthesis
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IMA was synthesized following Scheme 2, where hydrophilic
octa(ethylene glycol) derivative (4) and iodinated imidazolinium
salt (9) were synthesized individually. Initially, 2 was synthesized
by Sonogashira coupling[15] of 1 and trimethylsilylacetylene
(TMSA). Ts-OEG-Trt was synthesized following the procedures
reported in our previous paper.[16] 2 and Ts-OEG-Trt were
coupled by Williamson ether synthesis, followed by deprotec-
tion of Trt group to afford 4. Meanwhile, 9 was synthesized
using reductive amination of glyoxal with 6, followed by
cyclization using triethyl orthoformate. Finally, compound 4 and
9 were conjugated via Sonogashira coupling to afford IMA. See
Experimental Section for details on these procedures and
characterizations.
Scheme 1. Molecular structure of IMA. Hydrophobic, hydrophilic, and
imidazolinium moieties are indicated in red, blue, and black, respectively.
ration of ligand-binding phosphoric ester group to the boun-
dary of hydrophilic and hydrophobic units.[13,14] Therefore,
designing suitable hydrophobic unit is necessarily in developing
diverse synthetic ion transport systems by multiblock amphi-
phile.
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Herein, we applied this concept of molecular design to
develop a new synthetic anion transporter. Non-ionic, hydro-
philic octa(ethylene glycol) (OEG) chains are located on the one
end of hydrophobic diphenylacetylene units following our
previously developed multiblock amphiphilic compounds.[11–14]
In addition, imidazolinium was introduced to the center of the
hydrophobic moiety, in between two diphenylacetylene units,
as an anion recognition site (Scheme 1). The C2 proton of the
imidazolinium ring is able to act as a (CÀ H)+···XÀ hydrogen
bond donor,[7] which we expected to be the major interaction
for anion recognition. Interestingly, this imidazolinium-based
multiblock amphiphile (IMA) transported ions as a mobile
carrier rather than forming transmembrane channel, with
selectivity for nitrate.
Ion transport activity of IMA
Large unilamellar vesicles (LUVs) of 1,2-dioleyl-sn-glycero-3-
phosphocholine (DOPC) encapsulating pH sensitive 8-hydrox-
ypyrene-1,3,6-trisulfonate (HPTS) were prepared for evaluation
of the ion transport activity of IMA. An aqueous solution of IMA
was added externally to the DOPC LUV suspension in a 4-(2-
hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer
including NaCl (50 mM), with pH gradient (intravesicular pH 7.1
and extravesicular pH 7.9) across the membranes. IMA medi-
ated ion transportation would result in increase of intravesicular
pH, leading to deprotonation of HPTS which enhances its
fluorescence.
First, ion transport activity of IMA was investigated at IMA
concentration of 2 μM. As a result, significant increase in the
fluorescence intensity was observed just after addition of IMA
(Figure 1a, blue plots), indicating that IMA is capable of
Figure 1. (a) Time course change of HPTS fluorescence intensity
(λem =510 nm, λex =460 nm) encapsulated in DOPC LUVs upon addition of
IMA at 0 s, with various IMA concentrations; 0 μM (grey), 0.5 μM (red),
0.75 μM (orange), 1.0 μM (light green), 1.5 μM (green), 2.0 μM (blue), 3.0 μM
(light blue), 5.0 μM (purple) and 7.5 μM (pink). All measurements were
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carried out with 200 μM DOPC at 20 C; 20 mM HEPES, 50 mM NaCl, 30 μM
HPTS, pH 7.1 as an internal buffer; 20 mM HEPES, 50 mM NaCl, pH 7.9 as an
external buffer. Data are averages of three independent measurements,
with error bars indicating standard deviation at every 10 s (extracted for
clarity). (b) Normalized fluorescence intensity of HPTS encapsulated in DOPC
LUVs at 200 s after addition of IMA as a function of IMA concentration. The
solid line represents a curve-fit analysis with the Hill equation.[17]
Scheme 2. Synthetic scheme of IMA. (TMSA=trimethylsilylacetylene, Ts=
(4-methylphenyl)sulfonyl, Trt=triphenylmethyl, BTMA=benzyltrimeth-
ylammonium).
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transmembrane ion transport. In addition, we carried out
conductance measurement with a planar lipid bilayer (Fig-
ure S1, Supporting Information), which also displayed the ion
transportation capability of IMA. This was further confirmed by
changing the concentration of IMA from 0 μM to 7.5 μM, to
result in concentration dependent ion transport activity (Fig-
ure 1a). The ion transportation efficiency of IMA was inves-
tigated by plotting HPTS fluorescence intensity encapsulated in
DOPC LUVs at 200 s against IMA concentration, where the Hill
coefficient (n)[17] and effective concentration (EC50) were
evaluated as n=1.69 (�0.10) and EC50 =1.84 (�0.07) μM (R2 =
0.991) (Figure 1b), respectively.[18]
Next, we explored the species of ions IMA transports.
Therefore, ion transportation assay was carried out by individu-
ally changing the cations and anions of extravesicular buffer
salts, while the intravesicular buffer was fixed to NaCl through-
out the measurements.[19] Indeed, cation selectivity was deter-
mined by using Li+, Na+, K+, Rb+, and Cs+ as extravesicular
cations. In this case, IMA did not show significant dependence
in the change of HPTS fluorescence intensity on cations
(Figure 2a). This suggests that IMA does not facilitate transport
of cations, unlike our previously reported multiblock amphi-
phile, which showed ion transport efficiency depending on the
cationic species.[11] Then, we assumed that anionic species are
responsible for ion transport efficiency by IMA, and anion
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À
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selectivity was examined by using ClÀ , BrÀ , IÀ , NO3 , and ClO4
as extravesicular anions. In sharp contrast to the result on the
cation selectivity investigation, IMA showed significant differ-
ence in the change of HPTS fluorescence intensity, in the order
of NO3À >ClO4À >ClÀ >BrÀ >IÀ (Figure 2b), at 200 s after addi-
tion of IMA. Results from these two experiments strongly
suggest that IMA mediates transport of anions.
Since HPTS based assays allow for only indirect observation
of anion transport, we carried out ion transport assay using a
halide sensitive dye, lucigenin.[19,20] Lucigenin fluorescence
quenches in the presence of ClÀ . We prepared DOPC LUVs
encapsulating lucigenin in a HEPES buffer including NaNO3
(50 mM) without pH gradient across the membranes. ClÀ was
introduced only to the extravesicular buffer solution to create
ClÀ concentration gradient (Figure 2c). If IMA should transport
ClÀ , influx of ClÀ can be detected as quenching of lucigenin
fluorescence encapsulated in DOPC LUVs. In fact, upon addition
of IMA at 2 μM, lucigenin fluorescence quenched drastically
(Figure 2d, green line), indicating the influx of ClÀ . This was
further confirmed by varying IMA concentration from 0 μM to
7.5 μM, which resulted in concentration dependent ClÀ influx
(Figure 2d). These results strongly indicate that Cl– is the
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transported species, thereby suggesting that IMA transports
À
anions. The anion selectivity trend we observed (NO3À >ClO4
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ClÀ >BrÀ >IÀ (Figure 2b) mostly follows hard and soft, acid and
base (HSAB) principle;[21] relatively hard bases are likely being
transported efficiently by a hard cationic proton donor like IMA.
Mechanism of ion transport by IMA
Then, we identified the anionic species transported by IMA. The
electric charge balance of the intravesicular and extravesicular
buffers was kept neutral through the ion transport process.
First, transport rate of H+ and XÀ was compared by performing
HPTS assay in the presence and absence of carbonyl cyanide 4-
(trifluoromethoxy)phenylhydrazone (FCCP), a highly active H+
carrier. When FCCP was incorporated in the membrane with pH
gradient (intravesicular pH 7.1 and extravesicular pH 7.9), FCCP
transports H+ to decrease the H+ gradient, meaning efflux in
this system (Figure 3a). H+ efflux leads to decrease in the
positive charge of the inner aqueous layer. To maintain the
neutral charge balance, IMA would transport either H+ or XÀ .
Actually, transport activity of IMA showed a large enhancement
(72%–41%–(4%–1%)=28%) in the presence of FCCP (Figure 3b,
red line). Such cooperativity in transport indicates that transport
rate of XÀ by IMA is higher than that of H+.[22–24]
Figure 2. (a) and (b) Time course change of HPTS fluorescence intensity
(λem =510 nm, λex =460 nm) encapsulated in DOPC LUVs in a HEPES buffer
upon addition of IMA with different cations (Li+, Na+, K+, Rb+, Cs+, XÀ =ClÀ
(a)) and different anions (ClÀ , BrÀ , IÀ , NO3À , ClO4À , M+ =Na+ (b)). Measure-
°
ments were carried out at [DOPC]=200 μM, [IMA]=2 μM at 20 C, where an
intravesicular buffer is 20 mM HEPES, 50 mM NaCl, 30 μM HPTS, pH 7.1 and
an extravesicular buffer is 20 mM HEPES, 50 mM MX, pH 7.9. (c) Schematic
illustration of lucigenin assay. (d) Time course change of lucigenin
fluorescence intensity (λem =535 nm, λex =450 nm) encapsulated in DOPC
LUVs at various IMA concentrations; 0 μM (grey), 0.75 μM (red), 1.0 μM
(orange), 1.5 μM (light green), 2.0 μM (green), 3.0 μM (blue), 5.0 μM (light
blue) and 7.5 μM (purple). An intravesicular buffer (pH 7.1) contains 300 μM
lucigenin, 20 mM HEPES, 50 mM NaNO3, and an extravesicular buffer
(pH 7.1) contains 20 mM HEPES, 50 mM NaNO3, and NaCl aq. was added to
reach final concentration of 10 mM. Data are averages of three measure-
ments, with error bars indicating standard deviation at every 10 s (extracted
for clarity).
Then, the transported XÀ was further determined by
employing valinomycin, a K+ selective carrier. In this assay, K+
was included in the extravesicular buffer, instead of Na+ used
in the FCCP assay (Figure 3c). Here, we confirmed that 12.5 pM
valinomycin itself induced a slight increase in the intravesicular
HPTS fluorescence (6%–1%=5%, Figure 3d, green line), mean-
ing that anion transporter is also necessary for transport of K+
by valinomycin. In other words, valinomycin induced K+ influx
leads to increase of the positive charge at the inner aqueous
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layer, which also requires transport of anions so as to maintain
the electric charge balance neutral. Under the conditions of this
valinomycin assay, IMA is considered to transport either OHÀ or
ClÀ . Therefore, only the case of faster transport of OHÀ than ClÀ
should result in the larger enhancement of HPTS fluorescence
than that in the case IMA transports the anion alone. Actually,
the enhancement of HPTS fluorescence triggered by IMA
addition in the presence and the absence of valinomycin were
almost identical (Figure 3d, red and blue lines, 39% in both
conditions), implying no cooperativity of ion transportation by
valinomycin and IMA. Such lack in the cooperativity strongly
indicates that ClÀ transport is dominant while OHÀ transport is
negligible.
Another possibility we need to consider in valinomycin
assay is H+/M+ antiport, where H+ efflux in exchange to K+
influx by valinomycin. However, taking the lack in the cation
selectivity of IMA into account (Figure 2a), IMA is not likely to
transport cations, suggesting that H+/M+ antiport is also
negligible.[23]
1H NMR titration
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The anion binding property of IMA was evaluated by H NMR
titration in CDCl3, using tetrabutylammonium chloride (TBACl)
as ClÀ source. IMA concentration was fixed to 1 mM throughout
the measurements whereas TBACl concentration was varied.
Upon increasing the TBACl concentration, chemical shift of Ha
exhibited down field shift of Δδ=1.48 ppm, which is indicative
of the interaction between ClÀ and Ha via (CÀ H)+···XÀ hydrogen
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bond (Figure 4b). In addition, H chemical shifts of the protons
around the aromatic ring also changed, exhibiting up field shifts
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Influence of membrane fluidity on ion transport by IMA
We then had interest in how IMA transports anions through the
lipid bilayer membranes at the molecular level. There are two
possible mechanisms of transmembrane ion transport by
molecular species, i.e. transporting ions through the formation
of an ion channel or as a mobile carrier. Ion channels form
pores for ions to pass through the membranes, whereas mobile
carriers bind with anions and diffuse across the membranes.
Therefore, ion transport efficiency of mobile carriers strongly
depends on the membrane fluidity since diffusion is restricted
in the membranes with low fluidity, while ion channels are not
significantly affected, as transmembrane diffusion is less
important.[25–27] Thus influence of the membrane fluidity was
investigated using 1,2-dipalmitoyl-sn-glycero-3-phosphocholine
(DPPC) instead of DOPC for HPTS assay. DPPC adopts highly
fluid liquid phase above the phase transition temperature (Tm =
°
41 C), and much less fluid gel phase below Tm. We prepared
DPPC LUVs encapsulating HPTS at pH 7.1, and added a IMA
Figure 3. (a) Schematic illustration of HPTS assay in the presence of FCCP.
(b) Comparison of ion transport in the presence and absence of FCCP (1 μM)
with or without IMA (2 μM). Measurements were carried out at [DOPC]
°
solution at 50 C (liquid phase) to enhance the incorporation of
IMA to the membranes. Then, temperature was changed to
°
=200 μM, 20 C, with an intravesicular buffer (pH 7.1) containing 20 mM
°
°
either 50 C or 20 C (gel phase), followed by addition of NaOH
aq. for generation of pH gradient across the membranes to
initiate the ion transport by IMA incorporated within the
membranes (Figure 3e). As a result, significant increase of HPTS
HEPES, 50 mM NaCl, 30 μM HPTS, and an extravesicular buffer (pH 7.9)
containing 20 mM HEPES, 50 mM NaCl. (c) Schematic illustration of HPTS
assay in the presence of valinomycin. (d) Comparison of ion transport in the
presence and absence of valinomycin (VA, 12.5 pM) with or without IMA
°
(2 μM). Measurements were carried out at [DOPC]=200 μM, 20 C, with an
°
fluorescence was observed at 50 C (Figure 3f, red line)
intravesicular buffer (pH 7.1) containing 20 mM HEPES, 50 mM NaCl, 30 μM
HPTS, and an extravesicular buffer (pH 7.9) containing 20 mM HEPES, 50 mM
KCl. (e) Schematic illustrations of the assay using DPPC to investigate the
influence of membrane fluidity on ion transport by IMA. IMA was
°
compared to the blank measurement at 50 C (Figure 3f, grey
°
line), whereas increase of HPTS fluorescence at 20 C (Figure 3f,
blue line) was almost identical to the blank measurement at
20 C (Figure 3f, black line). These results indicate that IMA is
°
introduced into the membranes at 50 C (liquid phase), followed by i)
°
°
equilibration of measurement temperature to 50 C (liquid phase) or 20 C
(gel phase) and ii) pH gradient generation upon addition of NaOH aq. to the
extravesicular buffer for initiation of ion transport. (f) Time course change of
°
capable of ion transport only through the liquid phase
membranes, thereby indicating IMA acts as a mobile carrier.
Mobile carrier mechanism is a unique property of IMA, since
our previously reported multiblock amphiphiles were all
channel-forming molecules.[11–14]
°
HPTS fluorescence encapsulated in DPPC LUVs with or without IMA at 50 C
°
or 20 C. Measurements were carried out at [IMA]=0.25 μM, [DPPC]
=200 μM, 20 mM HEPES, 50 mM NaCl, 30 μM HPTS as an intravesicular
buffer, and 20 mM HEPES, 50 mM NaCl as an extravesicular buffer. Data are
averages of three measurements, with error bars indicating standard
deviation at every 10 s (extracted for clarity).
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for Hd and He (Figure 4c), and down field shift for Hc (Figure 4e).
These indicate the presence of the interaction between ClÀ and
aromatic rings (e.g. anion-π interaction or hydrogen bond) in
addition to (CÀ H)+···XÀ hydrogen bond of Ha. Curve fitting-
analysis of the chemical shifts of Hb by BindFit v0.5[28,29] suggest
the stoichiometry of IMA and ClÀ to be 2:1, where the
association constants were evaluated as K11 =1.69×102 MÀ 1
(error: �9.35%) and K21 =2.02×102 MÀ 1 (error: �4.83%).[30,31]
membranes, since the quenching efficiency depends on the
distance of the diphenylacetylene units from the spin labels.
Indeed, membranes with 5-doxyl PC showed the highest
efficiency in IMA fluorescence (λex =300 nm, λmax =468 nm)
quenching of 34% (Figure 5, red line), while those of 12-doxyl
PC and 16-doxyl PC were 22% and 19%, respectively (Figure 5,
blue and green lines). These comparisons suggest that IMA
tends to locate close to the surface of the membrane rather
than its hydrophobic center. We assume that the imidazolinium
moiety prefers aqueous environment to the hydrophobic
environment of lipid bilayer membrane to allow recognition of
anions. Anion binding leads to neutralization of the plus charge
of the imidazolinium moiety, which enables IMA to cross the
hydrophobic layer of the membranes.
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Fluorescence depth quenching
In our previous work, we reported that the hydrophobic units
of channel-forming multiblock amphiphiles prefer to be located
at the hydrophobic environment of the lipid bilayers, rather
than its interface.[11] Since IMA behaved as a mobile carrier
differently from the precedent multiblock amphiphiles which
formed ion channels, we also investigated the location of IMA
within the lipid bilayers by fluorescence depth quenching
experiment.[32] We prepared DOPC LUVs at pH 7.1 in the
presence of any of the following spin-labeled lipids individually:
1-palmitoyl-2-stearoyl-(5-doxyl)-sn-glycero-3-phosphocholine
(5-doxyl PC), 1-palmitoyl-2-stearoyl-(12-doxyl)-sn-glycero-3-
phosphocholine (12-doxyl PC), or 1-palmitoyl-2-stearoyl-(16-
doxyl)-sn-glycero-3-phosphocholine (16-doxyl PC), which bear
spin probes at the different positions of the alkyl chains. IMA
gives a fluorescence emission due to the diphenylacetylene
units, which is possibly quenched by spin probes. The efficiency
of fluorescence quenching within the membrane would be
informative of the location of the IMA molecule inside the
Molecular Modeling
To obtain explicit molecular models of IMA in the membrane-
water system, all-atom molecular dynamics (MD) simulations
were performed. As the initial structures for the MD simulations,
IMA was embedded in the DOPC lipid bilayer membrane: The
imidazolinium ring was placed in the center of the membrane,
and the orientation of the hydrophobic moiety was set to the
perpendicular or parallel to the membrane (Figure S3). To mimic
a situation during the anion transport, one ClÀ ion was placed
near the C2 proton of the imidazolinium ring. Three independ-
ent 1-μs simulations were carried out from 4 initial models
(12 μs in total). During a 1 μs simulation, the hydrophobic
moiety moved to the surface of the membrane near the
phosphorus atoms (black, red, and blue in Figure 6a), and, the
L-shape conformation was observed at the surface: One OEG
chain (magenta in Figure 6a) was buried in the membrane and
the other OEG chain (cyan in Figure 6a) exposed to the water-
membrane interface (Figure 6b). In all the simulations, the
hydrophobic moiety of IMA was located at the surface of the
Figure 5. Fluorescence spectra of IMA in LUVs at ratio of [IMA]/[total PC]
=5/200 with excitation at 300 nm. All measurements were carried out at
Figure 4. (a) Identification of protons. 1H NMR spectra of [IMA]=1 mM with
various equivalents of TBACl in CDCl3 corresponding to (b) Ha, (c) He and Hd,
(d) Hb, and (e) Hc. The equivalents of TBACl are denoted at the left side of
[total PC]=200 μM in 20 mM HEPES, 50 mM NaCl, pH 7.1 at 20 C. As
°
membrane constitutes, DOPC (black line) and DOPC containing 10 mol% of
5-doxyl PC (red line), 12-doxyl PC (blue line), or 16-doxyl PC (green line)
were used.
°
spectra. All measurements were carried out at 25 C.
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membrane rather than the center of membrane (Figures S4–6),
which was in good agreement with the experimental results of
the fluorescence depth quenching. Interestingly, the L-shape
conformation was observed in the last stage of the most MD
simulations, even though the simulations were started from
various initial models (Figures S4, S5).
proton, and the other IMA (magenta in Figure S11) was placed
so as to sandwich the ClÀ ion between the two IMAs. The
orientation of the hydrophobic moieties of the IMA dimer was
set to the perpendicular or parallel to the membrane. Three
independent 1-μs simulations were carried out from the two
initial models (6 μs in total). Except for one simulation
(described later), the hydrophobic moieties of the IMA dimer
moved to the membrane surface as was so for the 1:1
stoichiometry model described above (Figures S12–15). In the
simulations starting from the perpendicular model, both the
two IMAs formed the L-shape conformation at the surface. By
contrast, in the simulations starting from the parallel model,
one IMA formed the L-shape conformation, and the other IMA
formed the V-shape conformation in which both OEG chains
exposed to the water-membrane surface. In addition, regardless
of the initial models, the two IMAs formed a dimer during the
transport of the ClÀ ion from the center of the membrane to
the membrane-water surface. However, after the ClÀ ion
dissociated from the dimer, the two IMAs did not form a stable
dimer and were sometimes separated from each other (Fig-
ure S13).
1
2
3
4
5
6
7
8
9
The interaction between the ClÀ ion and the imidazolinium
ring was stable during the movement of IMA from the center to
the surface of the membrane (Figures 6a, S7). During the
movement of IMA, the ClÀ ion was located near the C2 proton
(Figures 6c, S7), and the ClÀ ion held its position without
exceeding the methyl group of the diphenylacetylene unit.
These interaction manners are consistent with those revealed
by 1H NMR experiments. At the membrane surface, water
molecules came to be close to the ClÀ ion, and, due to the
weakening of the interaction, the ClÀ ion dissociated from IMA
(Figure S8).
Since the experimental Hill coefficient (n=1.69) suggested
that the number of transport molecules involved in the anion
transport process is possibly two, the 2:1 binding-stoichiometry
model for IMA and the ClÀ ion was further examined. As the
initial model of the dimer IMA simulation, the imidazolinium
ring of one IMA was placed in the center of the membrane
(green in Figure S11), one ClÀ ion was placed near the C2
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In one simulation starting from the parallel model, the IMA
dimer was stably present in the membrane with the ClÀ ion
sandwiched between the two IMAs during 1 μs (Figure 7a). At
the first stage, the two IMAs formed a dimer structure with the
V-shape conformations facing each other (Figure 7b), and, in
Figure 7. Structure models of IMA dimer in the membrane-water system
from all-atom molecular dynamics simulations. (a) The time evolution of the
z-coordinate of the two IMAs and a ClÀ ion (orange). The trajectories of
three carbon atoms for each IMA (green, magenta in the schematic figure)
at the imidazolinium (black solid line, black triangle plot) and the edges of
the OEG chains (magenta and cyan, red and blue) are plotted, and the
carbon atoms are shown as spheres in the schematic figure. The average
coordinates of the phosphorus atoms of the upper and lower membrane
molecules are shown in green. Snapshots at (b) 300 ns and (c) 1 μs are
illustrated.
Figure 6. Structure models of IMA in the membrane-water system from all-
atom molecular dynamics simulations. (a) The time evolution of the z-
coordinate of the IMA and a ClÀ ion (orange). The trajectories of five carbon
atoms at the imidazolinium (black), the edges of the diphenylacetylene units
(red and blue) and OEG chains (magenta and cyan) are plotted, and the
carbon atoms are shown as spheres in the schematic figure. The average
coordinates of the phosphorus atoms of the upper and lower membrane
molecules are shown in green. Snapshots at (b) 1 μs and (c) 54.4 ns are
illustrated.
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the latter half, they formed an LV-shape conformation in which Experimental Section
1
2
3
4
5
6
7
8
9
one was L-shape conformation and the other was V-shape
conformation (Figure 7c). The relative position between the two
imidazolinium rings was maintained in the perpendicular
direction of the membrane, and the fact that one IMA forming
the L-shape conformation acted as a lid seemed to be the
reason why the ClÀ ion could be retained structurally (Fig-
ure S16).
Materials
DOPC, DPPC, 5-doxyl PC, 12-doxyl PC and 16-doxyl PC were
purchased from Avanti Polar Lipids. TMSA, triisopropylsilane,
glyoxal, Pd(PPh3)2Cl2, CH(OEt)3, 4-iodo-phenol, 2,6-dimethylaniline,
BTMA·ICl2, NaBH4, HPTS, FCCP and TBANO3 were purchased from
Tokyo Chemical Industry. TBACl, CuI, TsOH·H2O, K2CO3, CaCO3,
NaHSO3, anhydrous Na2SO4, HEPES and salts used for buffers were
purchased from Nacalai Tesque. HCOOH, NH4BF4, and NaClO4 were
purchased from Wako Pure Chemical Industries. Lucigenin and Et3N
were purchased from Sigma-Aldrich. Valinomycin was purchased
from abcam. Anhydrous DMF and THF were purchased from Kanto
Chemical and passed through two sequential drying columns on a
Glass-Contour system immediately prior to use. Deionized water
(filtered throught a 0.22 μm membrane filter, >18.2 MΩcm) was
Compared with the 1:1 binding-stoichiometry model, the
2:1 model had more structural variations that could stably
transport the ClÀ ion in the membrane. In both the stoichiom-
etry models, the interaction manner between one IMA and the
ClÀ ion was essentially identical. However, even with the L-
shape conformation, the IMA dimer could sandwich the ClÀ ion
at the membrane surface (Figure S17), and, therefore, the
interaction between IMA and the ClÀ ion was retained for a
long time. The V-shape conformation of one IMA held the ClÀ
ion stably for a short time, and however, the LV-shape
conformation of the IMA dimer allowed the ClÀ ion to be held
for a long time (Figure 7). In addition, according to the
interaction energy between IMA and the ClÀ ion during the
anion transport, the IMA dimer showed more stable interaction
energy than the IMA monomer (Figure S18). Taken together, for
the anion transport in the membrane, the 2:1 binding-
stoichiometry model was preferable, which was in good agree-
ment with the experimental Hill analysis.
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purified in
a Milli-Q system of Millipore. Silica gel column
chromatography was carried out with Silica Gel 60 (spherical,
neutral, particle size: 63–210 μm) purchased from Kanto Chemical.
Analytical TLC was performed on precoated, glass-backed silica gel,
Merck 60 F254. Visualization of the developed chromatogram was
performed by UV absorbance or iodine. Gel permeation chromatog-
raphy (GPC) was performed on JAI LaboACE LC-5060. CHCl3 used
for GPC was purchased from Nacalai Tesque. Spectra/Por Dialysis
Membrane (MWCO 3500) was purchased from Funakoshi.
Measurements
1H and 13C NMR spectra were recorded on Bruker biospin AVANCE
III 400 or Bruker biospin AVANCE III HD500. The chemical shifts
were determined with respect to tetramethylsilane (TMS), or a
residual non-deuterated solvent as an internal standard. Matrix-
assisted laser desorption/ionization time-of-flight mass (MALDI-TOF
MS) spectrometry was performed on Bruker UltrafleXtreme in
reflector mode, using either α-cyano-4-hydroxycinamic acid (CHCA)
or dithnaol as matrices, and electrospray ionization time-of-flight
(ESI-TOF) MS was performed on Bruker Daltonics micrOTOF II.
Fluorescence spectra and time course change of fluorescence were
recorded on JASCO FP-6500 spectrometer using quartz cell of
10 mm optical path length. Large unilamellar vesicles were
prepared using Avanti Mini Extruder with 100 nm polycarbonate
membranes.
Conclusion
In conclusion, transmembrane anion transport properties of
imidazolinium-based multiblock amphiphile (IMA) were re-
ported. The imidazolinium ring, which was introduced at the
center of the hydrophobic unit, played crucial role in anion
transport. It recognizes anions via a (CÀ H)+···XÀ hydrogen bond,
as demonstrated in NMR experiments. Interestingly, the ion
transport activity of IMA depended on the membrane fluidity,
which is a unique property of IMA working as a mobile carrier,
though alternated structure of hydrophilic and hydrophobic
units is structural motif of transmembrane proteins. Further-
more, fluorescence depth quenching experiments revealed that
the hydrophobic unit of IMA is located at the surface of the
lipid bilayer membranes. MD simulations were consistent with
the experimental results, where the hydrophobic unit of IMA
was located near the surface of the bilayer membranes and
anions remained near the C2 proton of the imidazolinium ring
during anion transportation. In addition, IMA is suggested to
form dimeric complexes with anions within the lipid bilayer
membranes. Upon changing the anions and cations of the
buffer solution in ion transport assays, IMA most efficiently
Synthesis
Synthesis of 3: To a dry acetone (55 mL) solution of Trt-PEG8-Ts[16]
(5.06 g, 6.59 mmol) was added 2 (1.88 g, 9.87 mmol) and K2CO3
(2.95 g, 21.3 mmol) at room temperature under Ar, and the
resulting mixture was refluxed for 9 h. Then, the reaction mixture
was filtered, and the resulting solution was evaporated to dryness
under reduced pressure. Brine was added to the residue, and the
resulting mixture was then extracted with CHCl3 for four times. The
organic extract was dried over anhydrous Na2SO4 and evaporated
to dryness under reduced pressure. The crude product was purified
by silica gel column chromatography with hexane and EtOAc (3/7
to 0/10) to afford a mixture of 3 and its TMS protected derivative
(4.00 g). To a dry MeOH (40 mL) solution of this mixture (4.00 g)
was added K2CO3 (2.05 g, 14.83 mmol), at room temperature under
Ar, and the resulting mixture was stirred for 5 h. The solution was
evaporated to dryness under reduced pressure. Sat. NaHCO3 aq.
was added to the residue, and the resulting mixture was then
extracted with CHCl3 for three times. The organic extract was dried
over anhydrous Na2SO4 and evaporated to dryness under reduced
pressure. The crude product was purified by silica gel column
À
transported NO3 , an anion considered to play important roles
in several biological events.[3] We believe that the imidazoli-
nium-based multiblock structural motif would contribute to the
development of synthetic anion transporters.
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chromatography with hexane and EtOAc (2/8 to 0/10) to allow
to dryness under reduced pressure. The crude product was purified
by silica gel column chromatography with hexane and EtOAc (6/1)
as an eluent, followed by recrystallization in hexane to yield 67% of
8 as colorless plate-like crystals (859 mg, 1.65 mmol). 1H NMR
1
2
3
4
5
6
7
8
9
isolation of 3 (3.71 g, 5.20 mmol) as yellow oil in 78% yield (for two
steps). 1H NMR (400 MHz, CDCl3 containing 0.03% TMS, 23 C): δ
°
7.46 (d, J=7.3 Hz, 6H), 7.41 (d, J=8.8 Hz, 2H), 7.29 (t, J=7.6 Hz, 6H),
7.22 (t, J=7.1 Hz, 3H), 6.85 (d, J=8.8 Hz, 2H), 4.12 (t, J=4.8 Hz, 2H),
3.84 (t, J=4.9 Hz, 2H), 3.72–3.62 (m, 26H), 3.23 (t, J=5.2 Hz, 2H),
2.98 (s, 1H) ppm; 13C NMR(125 MHz, CDCl3 containing 0.03% TMS,
°
(400 MHz, CDCl3 containing 0.03% TMS, 24 C): δ 7.31 (s, 4H), 3.29
(br, 2H), 3.15 (s, 4H), 2.23 (s, 12H) ppm; 13C NMR (125 MHz, CDCl3
°
containing 0.03% TMS, 25 C): δ 149.9, 137.6, 132.0, 85.3, 48.8,
°
25 C): δ 159.3, 144.3, 133.7, 128.9, 127.9, 127.1, 114.7, 114.5, 86.7,
18.4 ppm; ESI-TOF MS (MeOH, positive mode): m/z: calculated for
83.8, 76.0, 71.0, 70.9, 70.8, 70.7, 69.8, 67.6, 63.5 ppm; ESI-TOF MS
(MeOH, positive mode): m/z: calculated for C43H52O9: 712.36; found:
735.60 [M+Na]+, 751.57 [M+K]+.
C18H22I2N2: 519.98; found: 521.07 [M+H]+, 543.06 [M+Na]+.
Synthesis of 9: A mixture of 8 (537 mg, 1.03 mmol), NH4BF4
(107 mg, 1.02 mmol) and triethyl orthoformate (13 mL) was stirred
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°
Synthesis of 4: To a MeOH/THF=14/1 (1.5 mL) solution of 3
(527 mg, 0.739 mmol) was added triisopropylsilane (117 mg,
0.741 mmol) and TsOH·H2O (6 mg, 0.031 mmol) at room temper-
ature, and the reaction mixture was stirred in dark for 9 h. Sat.
NaHCO3 aq. was added to the mixture, and the resulting mixture
was stirred for 5 min and evaporated under reduced pressure to
remove organic solvents. Then, the resulting mixture was extracted
with CHCl3 for three times. The organic extract was washed with
brine, dried over anhydrous Na2SO4 and evaporated to dryness
under reduced pressure. The crude product was purified by silica
gel column chromatography with EtOAc and MeOH (100/0 to
at 120 C for overnight. After the mixture was cooled down to room
temperature, Et2O was added to the mixture. The resulting
precipitate was filtered off, and purified by repeated recrystalliza-
tion in MeOH to allow isolation of 9 as pale orange solids in 50%
1
°
yield (321 mg, 0.52 mmol). H NMR (400 MHz, DMSO, 23 C): δ 8.97
(s, 1H), 7.70 (s, 4H), 4.44 (s, 4H), 2.34 (s, 12H) ppm; 13C NMR
°
(125 MHz, DMSO, 25 C): δ 160.2, 138.2, 137.3, 133.2, 96.8, 50.7,
16.7 ppm; ESI-TOF MS (MeOH, positive mode): m/z: calculated for
C19H21I2N2+: 530.97; found: 530.96 [M]+.
Synthesis of IMA: 4 was freeze dried, and Et3N was degassed prior
to use. To a solution of 4 (89 mg, 0.19 mmol) in Et3N (0.6 mL) and
dry DMF (0.25 mL) were added Pd(PPh3)2Cl2 (17 mg, 0.025 mmol),
CuI (4 mg, 0.021 mmol), 9 (47 mg, 0.076 mmol) and dry DMF
(0.2 mL), and the resulting mixture was stirred for 1.5 h at room
temperature in dark. The resulting mixture was dried under reduced
pressure, and the residue was purified by silica gel column
chromatography with CH2Cl2 and MeOH (95/5 to 93/7), followed by
gel permeation chromatography with CHCl3 to allow isolation of
IMA as yellow oil in 44% yield (43 mg, 0.033 mmol). 1H NMR
90/10) to allow isolation of 4 (208 mg, 0.442 mmol) as colorless oil
1
°
in 59% yield. H NMR (400 MHz, CDCl3 containing 0.03% TMS, 24 C):
δ 7.41 (d, J=8.9 Hz, 2H), 6.85 (d, J=8.9 Hz, 2H), 4.13 (t, J=4.8 Hz,
2H), 3.85 (t, J=4.9 Hz, 2H), 3.73–3.59 (m, 28H), 2.99 (s, 1H) ppm; 13C
°
NMR(125 MHz, CDCl3 containing 0.03% TMS, 25 C): δ 159.3, 133.7,
114.7, 114.5, 83.8, 76.0, 72.7, 71.0, 70.8, 70.7, 70.5, 69.8, 67.6,
61.9 ppm; ESI-TOF MS (MeOH, positive mode): m/z: calculated for
+
C24H38O9: 470.25; found: 493.25 [M+Na]
.
°
Synthesis of 6: To
a
solution of 2,6-dimethylaniline (2.95 g,
(500 MHz, CDCl3 containing 0.03% TMS, 25 C): δ 8.33 (s, 1H), 7.45
24.3 mmol) in CH2Cl2/MeOH=5/2 (415 mL) were added BTMA·ICl2
(9.35 g, 26.8 mmol) and CaCO3 (3.96 g, 39.6 mmol) at room temper-
ature, and the resulting mixture was stirred for 1.5 h. The excess
CaCO3 was filtered off, and the filtrate was evaporated to remove
organic solvents. 10% NaHSO3 aq was added to the residue, and
the resulting mixture was extracted with t-butylmethyl ether for
four times. The organic extract was dried over anhydrous Na2SO4,
evaporated and vacuumed to afford crude brown solids, which
were purified by repeated recrystallization in hexane to afford 6 as
(d, J=8.5 Hz, 4H), 7.36 (s, 4H), 6.91 (d, J=8.5 Hz, 4H), 4.69 (s, 4H),
4.16 (t, J=4.7 Hz, 4H), 3.87 (t, J=4.7 Hz, 4H), 3.65-3.74 (m, 52H),
3.60 (t, J=4.6 Hz, 4H), 2.62 (t, J=6.3 Hz, 2H), 2.47 (s, 12H) ppm; 13C
NMR (125 MHz, CDCl3 containing 0.03% TMS, 25 C): δ 159.25,
159.02, 135.61, 133.23, 132.17, 131.69, 126.27, 114.77, 114.69, 91.76,
°
86.74, 72.50, 70.84, 70.60, 70.54, 70.29, 69.61, 67.51, 61.68, 52.14,
18.24 ppm; HRMS (ESI+): m/z: calculated for C67H95N2O18
:
+
1215.6574 ;found: 1215.6546 [M]+. See also Figures S19-S21 for
NMR and MS spectra.
1
pale yellow needle-like crystals in 92% yield (5.55 g, 22.4 mmol). H
Preparation of DOPC LUVs for HPTS assay. A CHCl3 solution of
DOPC (10 mM) was evaporated in a glass tube to form a thin lipid
film. The film was dried for at least 1 h under vacuum and hydrated
with a HEPES buffer (20 mM HEPES, 50 mM NaCl, 30 μM HPTS,
pH 7.1, the same volume as CHCl3, the final concentration of DOPC:
10 mM) at 37 C. The resulting mixture was vortexed, followed by
freezing and thawing (5 cycles), incubated at 37 C for at least 1 h,
and then extruded through a 100-nm membrane for 21 times at
room temperature. The obtained suspension was dialyzed by a
HEPES buffer (20 mM HEPES, 50 mM NaCl, pH 7.1) at room temper-
ature using Spectra/Por Dialysis Membrane (MWCO 3500).
°
NMR (400 MHz, CDCl3 containing 0.03% TMS, 25 C): δ 7.27 (s, 2H),
3.58 (br 2H), 2.13 (s, 6H) ppm; 13C NMR (125 MHz, CDCl3 containing
°
0.03% TMS, 25 C): δ 142.7, 136.6, 124.3, 79.3, 17.4 ppm; ESI-TOF MS
(MeOH, positive mode): m/z: calculated for C8H10IN: 246.98; found:
247.99 [M+H]+.
°
°
Synthesis of 7: To a solution of 6 (1.99 g, 8.05 mmol) in EtOH
(16 mL) were added 39% aqeous solution of glyoxal (602 μL,
4.04 mmol) and catalytic amount of HCOOH (ca. 42 μL) at room
temperature. After the reaction mixture was stirred for 17 h, the
resulting yellow precipitate was collected by filtration and washed
with cold MeOH to allow isolation of 7 as yellow solids in 60% yield
(1.27 g, 2.46 mmol). 1H NMR (400 MHz, CDCl3 containing 0.03%
Concentration dependency on ion transport. To a HEPES buffer
(1.96 mL, 20 mM HEPES, 50 mM NaCl, pH 7.9) in a clean quartz cell
of 10 mm optical path length was added DOPC LUVs suspension
containing HPTS (40 μL) prepared above. The cell was set to the
fluorescence spectrometer equipped with a magnetic stirrer at
TMS, 24 C): δ 8.05 (s, 2H), 7.43 (s, 4H), 2.12 (s, 12H) ppm; 13C NMR
°
°
(125 MHz, CDCl3 containing 0.03% TMS, 25 C): δ163.7, 149.6, 137.1,
129.0, 89.2, 18.0 ppm; ESI-TOF MS (MeOH, positive mode): m/z:
calculated for C18H18I2N2: 515.95; found 538.94 [M+Na]+.
°
20 C (t=0). Time course change of the fluorescence intensity was
Synthesis of 8. To a solution of 7 (1.27 g, 2.46 mmol) in THF/
measured at λem =510 nm (λex =460 nm). An aqueous solution of
IMA (0–1.5 mM, 10 μL) was added at t=10 s, followed by addition
of 10%wt TritonX-100 (15 μL) at t=210 s to cause lysis for complete
disruption of the pH gradient. Normalized fluorescence intensities
in Figures 1–3 were calculated following the equation 1, where It, I0,
and Ilysed represent fluorescence intensity at t (s) after the addition
°
MeOH=5/1 (14 mL) was added NaBH4 (210 mg, 5.55 mmol) at 0 C
in ice bath. Then the reaction mixture was stirred for 4 h at room
temperature, and the resulting mixture was evaporated to remove
organic solvents. Sat. NH4Cl aq. was added to the residue, and the
resulting mixture was then extracted with Et2O for three times. The
organic extract was dried over anhydrous Na2SO4 and evaporated
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of IMA, before the addition of IMA (t=0) and after the lysis by the
addition of 10%wt Triton X-100, respectively.
and finally, 10%wt TritonX-100 (15 μL) was added at t=250 to
induce lysis for complete disruption of chloride gradient.
1
2
Preparation of DPPC LUVs for HPTS assays. A CHCl3 solution of
DPPC (10 mM) was evaporated in a glass tube to form a thin lipid
film. The film was dried for at least 1 h under vacuum and hydrated
with a HEPES buffer (20 mM HEPES, 50 mM NaCl, 30 μM HPTS,
pH 7.1, the same volume as CHCl3, the final concentration of DPPC:
3
4
5
6
7
8
9
I ¼ It À I0=Ilysed À I0
(1)
Hill analysis. Hill coefficient (n) and effective concentration (EC50)
were calculated using the equation 2,[17] where Y and [c] represent
fluorescence intensity as transmembrane ion transport activity and
concentration of IMA, respectively.
°
10 mM) at 50 C. The resulting mixture was vortexed, followed by
°
freezing and thawing (5 cycles), incubated at 50 C for at least 1 h,
and then extruded through a 100 nm membrane for 21 times at
n
°
60 C. The obtained suspension was dialyzed by a HEPES buffer
(20 mM HEPES, 50 mM NaCl, pH 7.1) at room temperature using
Spectra/Por Dialysis Membrane (MWCO 3500).
Y ¼ Y1 þ ðY0 À Y1Þ=ð1 þ ðc=EC50Þ Þ
(2)
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Ion selectivity assay. To a HEPES buffer (1.96 mL, 20 mM HEPES, pH
7.9) including 50 mM MCl (M=Li+, Na+, K+, Rb+, Cs+ for cation
Investigation of carrier mechanism. To a HEPES buffer (1.96 mL,
20 mM HEPES, 50 mM NaCl, pH 7.1) in a clean quartz cell of 10 mm
optical path was added a DPPC LUV suspension encapsulating
HPTS (40 μL) prepared above. The cell was set to the fluorescence
selectivity assay) or 50 mM NaX (X=ClÀ , BrÀ , IÀ , NO3À , ClO4 for
À
anion selectivity assay) in a clean quartz cell of 10 mm optical path
length, was added a DOPC LUVs suspension containing HPTS
(40 μL) prepared above. The cell was set to the fluorescence
spectrometer equipped with magnetic stirrer at 20 C (t=0). Time
course change of fluorescence intensity It was measured at λem
510 nm (λex =460 nm). An aqueous solution of IMA (400 μM, 10 μL)
was added to the cuvette at t=10 s, and 10%wt TritonX-100
(15 μL) was added at t=210 s to induce lysis for complete
disruption of pH gradient.
°
spectrometer equipped with a magnetic stirrer, and stirred at 50 C
°
for 2 min. Then an aqueous solution of IMA (50 μM, 10 μL) was
=
added to the resulting solution. For the measurement at the liquid
°
°
phase (50 C), the sample solution was stirred at 50 C for 30 s prior
to measurement (t=0). As for the measurement at the gel phase
°
°
(20 C), the sample solution was stirred at 50 C for 30 s, and then
°
cooled down to 20 C for 4 min to equilibrate the sample solution
(t=0). A NaOH aq. solution (0.6 M, 32 μL) was added to create pH
gradient at t=30, followed by addition of 10%wt TritonX-100
(20 μL) at t=230 to induce lysis for complete disruption of pH
gradient. The data shown in Figure 3f were normalized following
the eq. (1), where I0 is the fluorescence intensity just before the
addition of NaOH aq.
FCCP assay. To a HEPES buffer (1.96 mL, 20 mM HEPES, 50 mM
NaCl, pH 7.9) in a clean quartz cell of 10 mm optical length was
added a DOPC LUVs suspension encapsulating HPTS (40 μL). The
cell was set to the fluorescence spectrometer equipped with a
°
magnetic stirrer at 20 C (t=0). Time course change of fluorescence
intensity was measured at λem =510 nm (λex =460 nm). A DMSO
solution of FCCP (400 μM, 5 μL) was added at t=10 and an
aqueous solution of IMA (400 μM, 10 μL) was added at t=50,
followed by addition of 10%wt TritonX-100 (15 μL) at t=250 to
induce lysis for complete disruption of pH gradient. The data shown
in Figure 3b were normalized following the eq. (1), where I0 is the
fluorescence intensity just before the addition of IMA.
1H NMR titration. IMA, TBAX (X=Cl or NO3) was dried under
vacuum before preparing the stock solutions. Then stock solutions
of 2 mM IMA, and 40 mM, 20 mM, 10 mM or 5 mM TBAX in CDCl3
were prepared individually. An IMA solution (250 μL) and the TBAX
solutions were mixed in a NMR tube, and CDCl3 was added to
adjust a total volume of the sample solution to 500 μL where the
concentration of IMA was fixed to 1 mM. The NMR tubes were
sealed tightly through the measurement. NMR measurements were
Valinomycin assay. To a HEPES buffer (1.96 mL, 20 mM HEPES,
50 mM KCl, pH 7.9) in a clean quartz cell of 10 mm optical length
was added a DOPC LUVs suspension encapsulating HPTS (40 μL).
The cell was set to the fluorescence spectrometer equipped with a
°
carried out at 25 C using Bruker biospin AVANCE III HD500.
Fluorescence depth quenching. A CHCl3 solution of DOPC (10 mM,
150 μL) or a CHCl3 solution of a mixture of DOPC (10 mM, 135 μL)
and 5-, 12-, or 16-doxyl PC (1 mM, 135 μL) ([DOPC/doxyl PC]=9/1)
in a glass tube was evaporated to form a thin lipid film. The
resulting films were dried under vacuum for 1 h and hydrated with
a HEPES buffer (20 mM HEPES, 50 mM NaCl, pH 7.1, 1.5 mL, the final
°
magnetic stirrer at 20 C (t=0). Time course change of fluorescence
intensity was measured at λem =510 nm (λex =460 nm). A DMSO
solution of valinomycin (5 nM, 5 μL) was added at t=10 and an
aqueous solution of IMA (400 μM, 10 μL) was added at t=50,
followed by addition of 10%wt TritonX-100 (15 μL) at t=250 to
induce lysis for complete disruption of pH gradient. The data shown
in Figure 3d were normalized following the eq. (1), where I0 is the
fluorescence intensity just before the addition of IMA.
°
concentration of phospholipids: 1 mM) at 37 C. The resulting
mixture was vortexed followed by freezing and thawing (5 cycles),
°
incubated at 37 C for 1 h, and then extruded through 100 nm
membrane for 21 times. The phospholipids suspension (400 μL) was
diluted with a HEPES buffer (20 mM HEPES, 50 mM NaCl, pH 7.1,
1.6 mL, the final concentration of phospholipids: 200 μM), followed
by addition of an aqueous solution of IMA (1 mM, 10 μL), and
Preparation of DOPC LUVs for lucigenin assay. DOPC LUVs
encapsulating lucigenin dye was prepared following the same
procedures as the HPTS assay, using a HEPES buffer (20 mM HEPES,
50 mM NaNO3, 300 μM lucigenin, pH 7.1). Dialysis was carried out
with a HEPES buffer (20 mM HEPES, 50 mM NaNO3, pH 7.1) at room
temperature using Spectra/Por Dialysis Membrane (MW3500).
°
stirred for 30 s at 20 C for the fluorescent measurements at λex =
300 nm.
Modelling of IMA. A three dimensional structure of an IMA was
built by 2D Sketcher and Minimize-Selected-Atoms modules in
MAESTRO (Shrödinger release 2019-1). One IMA was embedded in
DOPC membrane and water molecules using the Membrane Builder
implemented in CHARMM-GUI.[33–38] The imidazolinium ring was
placed at the center of the lipid bilayer, and the four different initial
structures were prepared depending on the orientation of the
hydrophobic moiety of the IMA and the presence or absence of ClÀ :
The orientation was set so that the hydrophobic moiety was
Lucigenin assay. To a HEPES buffer (1.96 mL, 20 mM HEPES, 50 mM
NaNO3, pH 7.1) in a clean quartz cell of 10 mm optical path length
was added a DOPC LUVs suspension encapsulating lucigenin
(40 μL) prepared above. The cell was set to the fluorescence
spectrometer equipped with a magnetic stirrer at 20 C (t=0). Time
course change of fluorescence intensity was measured at λem
°
=
535 nm (λex =450 nm). NaCl aq. (2 M, 10 μL) was added at t=10, an
aqueous solution of IMA (0–1.5 mM, 10 μL) was added at t=50,
Chem Asian J. 2020, 15, 1–12
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perpendicular or parallel to the membrane (Figure S3a-d), and a ClÀ
was placed near the C2 proton of the imidazolinium ring (Fig-
ure S3e,f). The unit cell was set to a rectangular cell, and the IMA
was embedded in a 50 Å×50 Å DOPC bilayer at the x-y plane in the
center of the cell, and the number of DOPC molecules at the upper
and lower leaflets was ~33 and ~34 for the perpendicular model,
or ~34 and ~36 for the parallel model, respectively. Along the z-
axis direction, the cell was filled with water molecules (TIP3P water
model[39]), and the water thickness was set to 22.5 Å. In addition to
the water molecules, counterions (ClÀ ) and 50 mM NaCl were
added.
Project for Supporting Drug Discovery and Life Science Research
(Basis for Supporting Innovative Drug Discovery and Life Science
Research (BINDS)) from AMED under Grant Number
JP20an0101009 to M.I., and RIKEN Dynamic Structural Biology
Project (M.I.).
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Conflict of Interest
The authors declare no conflict of interest.
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All-atom molecular dynamics (MD) simulations of the IMA em-
bedded in the membrane-water system were carried out. All the
MD simulations were performed using the MD program package
GROMACS ver. 2016.3.[40–42] The force field for the membranes and
the solvent molecules was the CHARMM36 m force field,[43–47] and
that for the IMA was the CHARMM General Force Field (CGenFF).[48]
The electrostatic interaction was handled by the smooth particle
mesh Ewald method,[49] and the van del Waals interaction was
truncated by the switching function with the range of 10–12 Å. The
bond lengths involving hydrogen atoms were constrained by the P-
LINKS algorithm.[50] Before the production runs, according to the
default setups of the CHARMM-GUI, an energy minimization and
equilibration runs were executed sequentially. The setups of the
equilibration runs were the same as those of our previous
simulations.[14] The production runs were performed with the
isothermal-isobaric ensemble and the 2 fs timestep. The temper-
ature and pressure were set to 300 K and 1 atm. The thermostat
and the barostat were the Nosé-Hoover scheme[51,52] and the semi-
isotropic Parrinello-Rahman approach,[53,54] respectively. After the
equilibration runs, three independent production runs were
performed for each initial structural model. The length of the
production run was 1 μs (3 μs for each model, and 12 μs in total).
The snapshot was saved every 1 ns.
Keywords: Anion transport
amphiphile · imidazolinium · hydrogen bond
· mobile carrier · multiblock
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The authors thank Materials Analysis Division, Open Facility,
1
Tokyo Institute of Technology, for ESI-TOF MS, MALDI-TOF MS, H
and 13C NMR spectroscopy, and the Open Research Facilities for
Life Science and Technology, Tokyo Institute of Technology, for
fluorescence spectroscopy. This work was supported by Grand-in-
Aid for Scientific Research on Innovative Areas “Molecular Engine
(No.8006)” (18H05419 and 18H05418 to KK, 18H05426 to M.I.),
Grant-in-Aid for Scientific Research B (19H02831 to KK), “Program
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Manuscript received: September 16, 2020
Revised manuscript received: November 21, 2020
Accepted manuscript online: November 28, 2020
Version of record online: ■■■, ■■■■
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FULL PAPER
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An imidazolinium-based multiblock
amphiphile, a mobile carrier capable
of transmembrane anion transport
with selectivity for nitrate, was
developed. It recognizes anions via
(CÀ H)+···XÀ hydrogen bond of proton
at C2 position of the imidazolinium
ring.
M. Mori, Dr. K. Sato*, Dr. T. Ekimoto*,
S. Okumura, Prof. M. Ikeguchi*,
Prof. K. V. Tabata*, Prof. H. Noji*,
Prof. K. Kinbara*
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Imidazolinium-based Multiblock
Amphiphile as Transmembrane
Anion Transporter