Functionalized hydrazide macrocycle ion channels showing pH-sensitive ion selectivities

Article abstract of DOI:10.1039/C6CC08943G

Transmembrane channels formed by functionalized hydrazide macrocycles are reported. The different pH values of buffer solutions have a significant effect on the K⁺/Cl^- selectivity of the macrocycles. This unique transport behavior is

Full text of DOI:10.1039/C6CC08943G

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Marilyn M. Olmstead, Alan L. Balch, Josep M. Poblet, Luis Echegoyenet al.
Reactivity differences of Sc3N@C2n (2n 68 and 80). Synthesis of the
first methanofullerene derivatives of Sc N@D5h-C80
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Functionalized hydrazide macrocycle ion channels showing pH-
sensitive ion selectivities
Received 00th January 20xx,
Accepted 00th January 20xx
Pengyang Xin,* Si Tan, Yaodong Wang, Yonghui Sun, Yan Wang, Yuqing Xu, and Chang-Po Chen*
DOI: 10.1039/x0xx00000x
www.rsc.org/
+
Transmembrane channels formed by functionalized hydrazide capacity and high NH4 /K+ transport selectivity (Scheme 1,
macrocycles are reported. The different pH values of buffer
2
).11 Compare to the helical channel, the macrocyclic channel
solution have a significant effect on the K+/Cl- selectivity of the can be easily synthesized from oneꢀstep macrocyclization. And
macrocycles. This unique transport behavior is mainly induced by the macrocyclic channels have rigid backbone, nanoꢀscaled
the different distribution of charges in the tubular channels under cavity and multiple modifiable sites. All of these make it
various pH values.
possible to modify the backbones of the macrocycles with
inwardꢀpointing functional groups, by which regulable
macrocyclic channels may be developed.
Transmembrane transport of charged species across biological
membranes is crucially important for cellular pH regulation,
metabolite neuronal exchange, nerve and muscle excitation.
Natural ion channels are complex proteins that can form porous
structures in biological membranes to mediate the transport of
ions.1 Inspired by the wideꢀranging functions of natural ion
channels, chemists have made great efforts to constructing
synthetic mimics for gaining insights into the mechanism of
natural channels,2 and developing new technologies in
separation, purification and sensing.3 Currently, nonꢀregulable
artificial channels have been extensively studied.4 In order to
make artificial channels possess more similar qualities to their
natural prototype, several regulable artificial channels have also
been described.5 Variety signals, such as voltage,6 light,7 pH8
and the binding of ligands9 were used to regulate the ionꢀ
transport properties of artificial channels. In spite of important
advances, the design of new unimolecular channel responsive
to external stimulus which can be easily synthesized is still a
challenge.
O
N
O
N
O
N
O
N
n
N
H
N
O
O
H
H
NH
O
O
HN
O
H
RO
H
H
OR
RO
OR
O
RO
H
OR
H
RO
H
OR
H
2
N
O
1
O
N
N
O
O
N
O
N
N
O
O
NH
HN
O
Oligomer:
n = 1, 2, 3, 4
H
H
OMe
MeO
OMe
H
N
MeO
H
N
Polymer:
O
O
n ~
~
9
COOMe
MeOOC
N
H
N
H
O
O
O
R
O
R
O
H
O
NH
O
HN
H
O
RO
N
N
OR
O
O
NH2
3
Ph
O
O
O
RO
H
OR
H
H
H
N
N
R =
N
OH
N
O
N
H
O
O
NH
HN
O
Ph
Ph
NH2
H2N
O
O
O
O
H
N
H
N
N
H
N
H
O
O
O
R
O
R
Scheme 1 Structures of compounds 1–3.
Recently, Li and Hou have demonstrated that hydrogenꢀ
bonded aromatic hydrazide foldamers can efficiently mediate
the transmembrane transport of cations by forming a helical
Herein we have reported the new aromatic hydrazide
macrocycles and their ionꢀtransport properties (Scheme 1, ).
3
3
tube across lipid bilayers (Scheme 1, 1
).10 To further explore
For these macrocycles, three inwardꢀpointing amino groups
were introduced into the backbone cavity, and the Pheꢀbased
tripeptide chains were used to enhance membraneꢀincorporation
ability of aromatic frameworks. In this paper, we showed that
the new macrocycles can efficiently mediate the transmembrane
transport of ions. The pH of buffer solution has a significant
effect on the ion selectivity of the macrocycles, due to the
presence of the amino groups.
the influence of the backbone cavity on the transporting process,
they have reported several shapeꢀpersistent aromatic hydrazide
macrocycles which exhibit excellent membraneꢀinsertion
We prepared aromatic hydrazide macrocycles
oneꢀstep macrocyclization of corresponding
dialkoxyisophthalohydrazide and diacyl chloride precursors
3
from the
4,6ꢀ
5
4
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(see ESI).11ꢀ12 Amino group deprotection of 3a afforded 6.2), compound 3c possesses a positively charged inner cavity,
compound 3b. Carboxybutyl hydrolysis of 3b yielded the due to the protonation of the inwardꢀpointing amino groups.
compound 3c. We also synthesized Compound
compound.
2
as control This positive charge likely leads to electrostatic interactions
that repulsively with protons but attractively with chloride ions.
O
N
O
N
O
O
H
O
NH
O
O
HN
O
H
O
RO
OR
Cl
NH2
3a
R1
HN
R = L-Phe-L-Phe-L-Phe-COOBut
R1= NHCbz
R1
RO
RO
O
b
c
5
RO
H
OR
H
O
Cl
a
3b
N
3
N
R = L-Phe-L-Phe-L-Phe-COOBut
O
NH
HN
O
O
R1= NH2
R1
O
R1
O
HN
3c
O
O
NH2
R = L-Phe-L-Phe-L-Phe-COOH
H
N
H
N
4
R1= NH2
N
H
N
H
O
O
O
R
O
R
Scheme 2 Synthesis of compound 3 (a) Et3N, DMA, r.t.; (b) H2, Pd/C,
MeOH, r.t.; (c) TFA, DCM, r.t.
Fig. 1 (a) Changes in the fluorescent intensity of HPTS (λex = 460 nm,
λem = 510 nm) in vesicles with time after addition of macrocycles 2
To evaluate the incorporating ability of these macrocycles
into lipid bilayers, the protonꢀtransport activities of 3b, 3c and
2
and 3 (x = 0.1%). (b) Changes in the fluorescent intensity of LG (λex
=
on vesicles were investigated using HPTS assay.9c Briefly, a
suspension of Large Unilamellar Vesicles (LUVs) composed of
egg yolk Lꢀαꢀphosphatidylcholine (EYPC) entrapping pHꢀ
sensitive dye 8ꢀhydroxypyreneꢀ1,3,6ꢀtrisulfonate (HPTS, 0.1
mM, pH = 7.2) was first prepared. Then, a pH gradient across
the membranes was introduced by addition of the vesicle
solution to a buffer (pH = 6.0). By measuring the fluorescent
intensity, the H+ transport activity of these macrocycles was
372 nm, λem = 503 nm) in vesicles with time after addition of
macrocycles 2 and 3 (x = 0.1%).
In addition, for the vesicular transport of proton and
chloride ion texts, the EC50 values of 3c was obviously lower
than that of 3b, which indicated that the hydrophilic terminal
carboxylic acid groups of the 3c might enhance the membraneꢀ
incorporation ability of this compound.
gauged. After a solution of 3b
molar ratio relative to lipid, represented by
,
3c, or
2
in DMSO (
x = 0.1%,
To investigate the ionꢀtransport activity of these macrocycles,
the patchꢀclamp experiments on planar lipid bilayer were
further performed.15 For these experiments, two compartments
containing KCl solution (1.0 M) were separated by a planar
lipid bilayer composed of diphytanoylphosphatidylcholine
x) was injected into
the vesicle solution, the fluorescence intensity of HPTS was
increased significantly and reached 26%, 60%, 84%
respectively (Fig. 1a), which supports the hypothesis that the
macrocycles were involved in the H+ transport across the
membrane of the vesicles.
(diPhyPC). A solution of 3b
the cis compartment which was grounded, to reach a final
concentration of 0.5 M. After addition of compounds, regular
, 3c, or 2 in DMSO was added to
By fitting the dose
effective concentration needed for 50% activity (EC50) were
determined to be 0.248% (3b), 0.033% (3c) and 0.0037% ( ),
respectively (Fig. S21, ESI).13 The EC50 value of 3c was
remarkably higher than that of . Because of their identical
peripheral structure this result can only be attributed to the
different inwardꢀpointing groups of 3c OCH2CH2NH2) and
OMe). It seems plausible that the decreased protonꢀtransport
−response curves with Hill equation, the
ꢀ
square–like, longꢀlived singleꢀchannel currents were observed,
when a clamped voltage +100 mV was applied across the
planar lipid bilayer (Fig. 2). These results provide strong
evidence that the macrocycles could incorporate into the lipid
bilayer and form ion channels.16
2
2
,
(
−
2
(
−
activity of 3c is due to the steric hindrance of alkylamino
groups, which affected the passage of H+ through the inner pore
of the channel. This possibility has been ruled out by the
additional chlorideꢀtransport expermients. The chlorideꢀ
transport experiments were assessed on LUVs containing
lucigenin (LG, 2 mM, pH = 6.2), a dye whose fluorescence
emission is quenched by chloride ion.14 The results are shown
in Fig. 1b. After a solution of 3b, 3c, or 2 in DMF (x = 0.1%)
was added to the vesicles, the fluorescence intensity of LG was
significantly enhanced and reached 36%, 92%, 87%
respectively. The EC50 values were determined to be 0.134%
Fig. 2 Current traces (15 s) of (a) 3b; (b) 3c and (c) 2 all at 0.5 ꢀM in
the planar lipid bilayer at +100 mV in a symmetrical KCl solution
(1.0 M).
(
3b), 0.0095% (3c) and 0.016% (
2) (Fig. S22, ESI). It was
found that the EC50 value of 3c was lower than that of
2
, which
means 3c has a higher chlorideꢀtransport activity. Thus, the
decreased protonꢀtransport activity of 3c is not caused by the
steric hindrance of alkylamino groups. In buffer solution (pH =
The current–voltage (
clamp experiments at different voltages (Fig. 3). All of these
I–V) plots were obtained from the patch
I
–
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V
+
plots displayed a linear relationship in the range of
100 mV. Their corresponding conductances (γ) for K+ were 4.0, 7.0 and 10.0, there was no significant change in K+/Cl−
calculated to be 11.3 ± 0.2 (3b), 14.6 ± 0.2 (3c) and 16.0 ± 0.3 selectivity of compound 2.
−
100 to which is associated with enhanced Cl− transport activity. At pH
(2) pS. These γ values are comparable to the previously
reported value (γ= 20 ± 0.2 pS) for gramicidin A17, showing the
high efficiency of these macrocycles in transporting K+.
Fig. 4 PK+/PCl− of 2, 3b and 3c under different pH conditions.
In order to explain the pH effect on ion selectivity in this
macrocyclicꢀchannel system, a proposed model was established
(Fig. 5a). This tubular model possesses three inwardꢀpointing
amino groups in the inner cavity, and multiple carboxyl groups
at channel openings. At pH 10.0 (Fig. 5b), the deprotonated
carboxyls of channel 3c created two negatively charged channel
openings. These openings offer an electrostatic barrier for the
entry of Cl− ions into the channel pore, which was preferred to
K+ ions. Under neutral conditions, compound 3c was ionized to
form internal salts. Compound 3c displayed lower selectivity
for K+/Cl−, due to the decrease of negative charge density at
channel openings and positively charged inner cavity. At pH
4.0 complete protonation of the carboxyls and inwardꢀpointing
amino groups of compound 3c further increased the affinity of
the Cl− ions and resulted in the lowest K+/Cl− selectivity. The γ
values for K+ of 3c at pH 4.0 and 10.0 were also support this
proposed model (Fig. S29, ESI).
Fig. 3 I-V plots of macrocycles (0.5 ꢀM) in the planar lipid bilayer in
a symmetrical KCl solution (1.0 M).
Having established that macrocycles 3b, 3c and 2 mediates
the efficient transport of K+ ions through a channel mechanism,
the patchꢀclamp experiments were further used to probe their
ion selectivity. Alkaline cations selectivities were first tested by
repetition of the patchꢀclamp experiment with different M+/K+
gradients under neutral conditions. From the corresponding
curves, we could determine their reversal potentials (εrev) for
the respective cations and the permeability ( ) ratios using the
I−V
P
Goldman−Hodgkin−Katz equation.18 All the channels exhibited
an EisenmanꢀI selectivity towards alkaline cations (Cs+ > Rb+ >
K+ > Na+) (Fig. S24ꢀ25, ESI). These findings indicated that the
cations moved across the cavity of the macrocyclic channels
after being dehydrated.19
In these new macrocyclic channels, multiple amino and
carboxyl groups were introduced at backbone cavity and
channel opening, respectively. We postulated that the pH value
of electrolyte may influence the ion selectivity of the
macrocyclic channels. To inspect and verify this possibility, the
K+/Cl− selectivity of these macrocycles were confirmed by
measuring the I–V curves under different pH values. Briefly,
the saline solutions (0.3M and 1M) were adjusted to pH 4.0, 7.0,
or 10.0. Then, the saline solutions were added to the both side
of the bilayer (diPhyPC), trans chamber: KCl (1.0 M), cis
chamber: KCl (0.3 M). The PK
+
/
PCl values were obtained from
−
Goldman−Hodgkin−Katz equation as described before. The
results are shown in Fig. 4 (see ESI Fig. S26ꢀ28 for the details
of experiment). For compound 3b and 3c, the K+/Cl− selectivity
decreased considerably with the decrease of the pH value of
electrolyte. At pH 10.0, the PK
+
/
PCl values of 3b and 3c was
−
determined to be 9.3 and 7.4, which revealed that the two
macrocyclic samples were mainly transport K+ under alkaline
condition. At pH 7.0, the PK
macrocycles were reduced to 4.9 and 3.7. At pH 4.0, 3b and 3c
displayed lower PK PCl values (2.8 and 2.3). This result
implies that the macrocyclic channels have a signficantly
reduced transport activity toward K+ under acidity condition,
+
/
PCl
−
values of the two
+
/
−
Fig. 5 (a) A schematic diagram for the transmembrane
channels formed by 3c in the lipid bilayer. (b) Illustration of
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Bao, H. Wang, Y. Lin, H. Jia, L. Zhu, Chem. Commun., 2013,
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variations of pH value.
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In summary, we have developed a class of pHꢀsensitive ion
channels by introducing three inwardꢀpointing amino groups
into the cavity of the macrocyclic channels. These highly
efficient unimolecular channels could be readily constructed
from oneꢀstep macrocyclization. The protonation and
deprotonation of multiple amines and carboxyls in the channels
upon pH variation change the charge distribution, which
contributes to the pHꢀsensitive ion selectivity. This finding
illustrated that the design of new smart artificial channels may
be easily achieved by introducing multiple inwardꢀpointing
functional groups into the backbone cavity of aromatic
hydrazide macrocycles. Currently we are modifying backbone
cavity with multiple mercapto groups, by which redoxꢀ
responsive macrocyclic channels may be developed.
This work was financially supported by National Science
Foundation of China (21602044), Henan Science and
Technology Program (162300410022), Henan Normal
University Research Foundation for the Doctoral Program
(qd15110), Henan Normal University Science Foundation for
Young Scholars (2015QK10) and Innovative Talents Program
of Henan Province (174100510025).
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