Unimolecular artificial transmembrane channel with terminal dihydrogen phosphate groups showing transport selectivity for ammonium

Article abstract of DOI:10.1016/j.cclet.2019.05.009

A new artificial transmembrane channel molecule bearing dihydrogen phosphate groups has been synthesized. The terminal dihydrogen phosphate groups enable the channel to be highly negatively charged at both ends of the channel structures. The artificial channel could incorporate into the lipid bilayer efficiently under low concentration. The channel displays high NH₄⁺/K⁺ selectivity due to the electrostatic interaction and hydrogen bonding between NH₄⁺ and the terminal dihydrogen phosphate groups.

Full text of DOI:10.1016/j.cclet.2019.05.009

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CCLET 4977 No. of Pages 4
Chinese Chemical Letters xxx (2019) xxx–xxx
Contents lists available at ScienceDirect
Chinese Chemical Letters
journal homepage: www.elsevier.com/locate/cclet
Communication
Unimolecular artificial transmembrane channel with terminal
dihydrogen phosphate groups showing transport selectivity for
ammonium
*
Jian-Yu Chen, Qi Xiao, Harekrushna Behera, Jun-Li Hou
Department of Chemistry, Fudan University, Shanghai 200433, China
A R T I C L E I N F O
A B S T R A C T
Article history:
A new artificial transmembrane channel molecule bearing dihydrogen phosphate groups has been
synthesized. The terminal dihydrogen phosphate groups enable the channel to be highly negatively
charged at both ends of the channel structures. The artificial channel could incorporate into the lipid
bilayer efficiently under low concentration. The channel displays high NH4+/K+ selectivity due to the
Received 26 March 2019
Received in revised form 17 April 2019
Accepted 6 May 2019
Available online xxx
+
electrostatic interaction and hydrogen bonding between NH4 and the terminal dihydrogen phosphate
groups.
Keywords:
© 2019 Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences.
Published by Elsevier B.V. All rights reserved.
Artificial transmembrane channel
Ammonium transport
Dihydrogen phosphate
Pillar[5]arene
Transmembrane transport
Transport selectivity
Natural ion channels are a kind of membrane proteins that are
able to mediate the flux of ions to generate resting membrane
potentials and physiological signals, and regulate cell environ-
ments [1]. The channels are able to transport ions in a selective
manner, which are achieved under the aid of specific chemical
groups precisely located within the channel structures. For
example, the selectivity filter decorated with carbonyl oxygen
atoms in the natural KcsA K+ channel allows to conduct K+ ions
while excluding Na+ ions [2]. Inspired by the important function of
channel proteins, chemists have made significant progresses in the
construction of artificial ion channels to mimic the ion transport of
natural channels [3–11] and develop advanced materials [12–16].
During the past decades, significant efforts have been devoted to
achieve the construction of artificial channels with high transport
selectivity by using supramolecular strategy [17,18]. Comparing to
the dynamic structure of supramolecular channels, unimolecular
channels possess more stable structures, which allow for accurate
manipulation of the structure [19–24]. Recently, we have devel-
oped a new strategy to build unimolecualr artificial transmem-
brane channels with confined tubular structures based on
pillararene backbones [25]. We demonstrated that the introduc-
tion of negative charged groups at both ends of the channels not
only enhanced the membrane-incorporation ability of the
channels but also provided filters for achieving cation transport
selectivity [26–28]. It was envisioned that the introduction of
negative charged groups with more density would lead to higher
cation transport selectivity. However, this has not been achieved
yet due to the decreased hydrophobicity of the channel molecules
by introducing negative charged groups, which lead to the weak
membrane-incorporation ability of the channel molecules. Herein
we demonstrated that the unimolecular artificial transmembrane
channel with terminal phosphate groups exhibited not only high
membrane-incorporation ability but also high NH4+/K+ transport
selectivity.
Under physiological conditions, the dihydrogen phosphate
group can ionized to produce two negative charges. Thus, the
channel 1 containing terminal the dihydrogen phosphate groups
were designed (Fig. 1), which should be highly negatively charged
at both ends of the channel molecules under neutral pH. The
channel 1 was prepared by using pillar [5]arene decaacid 2 as
starting material [29–41]. Firstly, the channel precursor 3 was
prepared by coupling the peptide composed of
with 2 in the presence of EDCI. The compound 3 was then
deprotected by TFA to yield channel 1. The alternated - and L-
D-Phe and L-Trp
D
amino acids were designed to ensure the nonpolar side chains of
the amino acid residues outward, which were expected to increase
the membrane-insertion ability of the channel molecules. The
indole group might further enhance the membrane-insertion
ability of the channel due to the formation of hydrogen bonding
between its amide and lipid head-group oxygen atoms [42–44].
* Corresponding author.
E-mail address: houjl@fudan.edu.cn (J.-L. Hou).
https://doi.org/10.1016/j.cclet.2019.05.009
1001-8417/© 2019 Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences. Published by Elsevier B.V. All rights reserved.
Please cite this article in press as: J.-Y. Chen, et al., Unimolecular artificial transmembrane channel with terminal dihydrogen phosphate groups
showing transport selectivity for ammonium, Chin. Chem. Lett. (2019), https://doi.org/10.1016/j.cclet.2019.05.009

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Fig. 1. Chemical structure and synthetic procedures of the channel 1 and its
schematic representation for the formation of unimolecular transmembrane
channel in lipid bilayers.
The length of the channel molecule was determined to be 3.5 nm,
which matches well with the thickness of the hydrophobic area of
the lipid bilayer.
The possibility of forming transmembrane channel in lipid
bilayers of 1 was firstly investigated by assessing their proton
transport activity with large unilamellar vesicles (LUVs) [45–47].
LUVs were made from egg yolk phosphatidyl choline (EYPC)
containing pH sensitive 8-hydroxypyrene-1,3,6-trisulfonic acid
Fig. 2. The proton transport experiments of 1 on LUV by using HPTS assay. (a)
Transport activity under different concentrations of 1. (b) The relationship of the
transport activity vs. the concentration of 1. The solid red line is the fitting plot by
using Hill equation.
trisodium salt (HPTS) as
a fluorescence probe (pH 7.2). A
suspension of LUV entrapped with HPTS was added to a buffer
(pH 6.0) to produce a higher H+ concentration outside the vesicles.
The flux of H+ into the vesicles was assessed by monitoring the
fluorescence intensity of HPTS. With addition of pure DMSO to the
vesicle suspension, little fluorescence intensity change was
observed, suggesting that the bilayer is un-permeable for H+.
However, upon addition of the solution of 1 in DMSO to the vesicle
suspension under the same conditions, the fluorescence intensity
of HPTS increased significantly during 5 min (Fig. 2a). These results
demonstrated that the compound 1 was able to insert into bilayers
and form transmembrane channels to mediate the transport of H+.
It was found that the transport activity, as indicated by the final
fluorescence intensity reached, was strongly dependent on the
concentration of 1 [represented by the molar ratio relative to lipid
(x)]. As x increased from 0 to 0.75%, the fluorescence intensity
increased significantly (from 13% to 75%) (Fig. 2b). Further
increasing x caused only a slight increase in fluorescence intensity.
The effective concentration required for 50% transport activity
(EC50) and the Hill coefficient (n) were calculated to be 0.17% and
0.6 by fitting the plot with Hill equation [48]. The low EC50
indicates that the new channel with terminal negative charged
phosphate groups is very effective in incorporation into the lipid
bilayers, while the small n value shows that the channel works in a
unimolecular manner [49].
To further confirm the formation of transmembrane channels in
the lipid bilayers, the conductance measurements on a planar lipid
bilayer were also performed [50]. For the experiments, two
compartments containing KCl solution (1.0 mol/L) were separated
by a planar lipid bilayer composed of diphytanoyl-phosphatidyl-
choline (diPhyPC). The solution of 1 in DMSO was added to the cis
compartment, which was grounded. A clamped voltage of +100 mV
was applied across the bilayer, and the conductance traces were
recorded. It was observed that the traces displayed regular square-
like signals (Fig. 3a). The presence of the conductance signals
strongly supports the formation of transmembrane channel of 1 in
Fig. 3. Conductance measurements of 1 on planar lipid bilayers. Current traces
(20 s) at 100 mV in (a) KCl (1.0 mol/L) and (b) NaCl (1.0 mol/L); (c) I–V plots in KCl
and NaCl solutions (1.0 mol/L).
the bilayer. For the conductance measurements, the number of the
current levels in the trace reflects the amount of the channel
molecules in the bilayer. The variation in the channel conforma-
tions would lead to different currents of the levels. In our
experiments, two main levels of currents were observed in
Please cite this article in press as: J.-Y. Chen, et al., Unimolecular artificial transmembrane channel with terminal dihydrogen phosphate groups
showing transport selectivity for ammonium, Chin. Chem. Lett. (2019), https://doi.org/10.1016/j.cclet.2019.05.009

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3
conductance trace (Fig. 3a), indicating that two channels formed in
the bilayer under the experimental conditions. By further
analyzing the conductance trace, it was found that the current
values at each level were identical. This result clearly demonstrat-
ed that the channels had only one stable channel conformation in
the bilayer [51]. The current-voltage (I-V) plot of the channel was
further measured by performing the experiments at different
voltages (Fig. 3c). It was found that the plot exhibited a linear I–V
relationship in the range of -100 mV to +100 mV. The correspond-
ing conductance (gK+) of the channel for K+ was calculated to be
16 Æ 0.5 pS. The conductance measurements were also performed
in NaCl (1.0 mol/L) solution by using the same method. It was found
that the single channel current was obvious weaker than that in
KCl solution (Fig. 3b). The corresponding conductance (gNa+) for
Na+ was determined from the I–V plot to be 2.6 Æ 0.3 pS (Fig. 3c).
This small conductance value suggested the weak transport
activity for Na+, which might be result of strong coordination of
the phosphate groups with Na+.
phosphate group could be hydrolyzed by alkaline phosphatase. The
transport activity of channel developed herein may be regulated by
the enzyme, which is currently under investigations.
Acknowledgments
We are grateful to the National Natural Science Foundation of
China (Nos. 21725202, 21572035), the National R&D Program of
China (No. 2017YFA0206901), and STCSM (Nos. 18XD1400800,
18JC1411600) for financial support.
Appendix A. Supplementary data
Supplementary material related to this article can be
found, in the online version, at doi:https://doi.org/10.1016/j.
cclet.2019.05.009.
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Please cite this article in press as: J.-Y. Chen, et al., Unimolecular artificial transmembrane channel with terminal dihydrogen phosphate groups
showing transport selectivity for ammonium, Chin. Chem. Lett. (2019), https://doi.org/10.1016/j.cclet.2019.05.009