Persistent Organic Nanopores Amenable to Structural and Functional Tuning

Article abstract of DOI:10.1021/jacs.5b12698

Rigid macrocycles 2, which share a hybrid backbone and the same set of side chains while having inner cavities with different inward-pointing functional groups, undergo similar nanotubular assembly as indicated by multiple techniques including ¹H NMR, fluorescence spectroscopy, and atomic force microscopy. The formation of tubular assemblies containing subnanometer pores is also attested by the different transmembrane ion-transport behavior observed for these macrocycles. Vesicle-based stopped-flow kinetic assay and single-channel electrophysiology with planar lipid bilayers show that the presence of an inward-pointing functional (X) group in the inner cavity of a macrocyclic building block exerts a major influence on the transmembrane ion-transporting preference of the corresponding self-assembling pore. Self-assembling pores with inward-pointing amino and methyl groups possess the surprising and remarkable capability of rejecting protons but are conducive to transporting larger ions. The inward-pointing groups also resulted in transmembrane pores with a different extent of positive electrostatic potentials, leading to channels having different preferences for transporting chloride ion. Results from this work demonstrate that synthetic modification at the molecular level can profoundly impact the property of otherwise structurally persistent supramolecular assemblies, with both expected tunability and suprisingly unusual behavior.

Full text of DOI:10.1021/jacs.5b12698

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Article
Persistent Organic Nanopores Amenable to Structural and Functional Tuning
Xiaoxi Wei, Guoqing Zhang, Yi Shen, Yulong Zhong, Rui Liu, Na Yang, Fayez Y. Al-
mkhaizim, Mark Kline, Lan He, Minfeng Li, Zhong-Lin Lu, Zhifeng Shao, and Bing Gong
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.5b12698 • Publication Date (Web): 13 Feb 2016
Downloaded from http://pubs.acs.org on February 19, 2016
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Persistent Organic Nanopores Amenable to Structural and
Functional Tuning
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Xiaoxi Wei,†¶‡ Guoqing Zhang,†‡ Yi Shen,║‡ Yulong Zhong,† Rui Liu,†¶ Na Yang,† Fayez Y. Al-
mkhaizim,¶ Mark Kline,¶ Lan He,§ Minfeng Li,† Zhong-Lin Lu,†* Zhifeng Shao,║* and Bing Gong¶†*
† College of Chemistry, Beijing Normal University, Beijing 100875, China
¶ Department of Chemistry, The State University of New York at Buffalo, Buffalo, New York 14260, United States
║ Bio-ID Center, School of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai 200240, China
§ National Institute for Food and Drug Control, Beijing 100050, China
Supporting Information Placeholder
ABSTRACT: Rigid macrocycles 2, which share a hybrid backbone and the same set of side chains while having inner cavities with
different inward-pointing functional groups, undergo similar nanotubular assembly as indicated by multiple techniques including 1H
NMR, fluorescence spectroscopy, and atomic force microscopy. The formation of tubular assemblies containing sub-nanometer
pores is also attested by the different transmembrane ion-transport behavior observed for these macrocycles. Vesicle-based stopped-
flow kinetic assay and single-channel electrophysiology with planar lipid bilayers show that the presence of an inward-pointing
functional (X) group in the inner cavity of a macrocyclic building block exerts a major influence on the transmembrane ion-
transporting preference of the corresponding self-assembling pore. Self-assembling pores with inward-pointing amino and methyl
groups possess the surprising and remarkable capabibility of rejecting protons but conducive to transporting larger ions. The
inward-pointing groups also resulted in transmembrane pores with different extent of positive electrostatic potentials, leading to
channels having different preferences for transporting chloride ion. Results from this work demonstrate that synthetic modification
at the molecular level can profoundly impact the property of otherwise structurally persistent supramolecular assemblies, with both
expected tunability and suprisingly unusual behavior.
membranes. Our effort had uncovered cation-selective
channels with very high conductance.17
■ INTRODUCTION
Although the inner pores of stacked 1 appear to offer a
Void-containing molecular and supramolecular structures,1,2
prototype based on which different synthetic channels18 might
especially those with small (≤2 nm) and sub-nm pores of a
be developed, unfortunately, tuning the inner pore is not
defined size,4-7 provide confinement under which many
feasible since the cavity of 1 contains few positions that would
fascinating phenomena have been predicted or observed.
allow structural alterations without compromising the integrity
Among known systems,2,3 the stacking of ring-like molecules
of the H-bond-constrained19 macrocyclic backbone.
leads to tubular assemblies that can be modified by varying the
constituent molecular components.3,4 Of particular interest is
We report herein the formation of nanotubular stacks of
macrocycles 2 (Figure 1) and their ion-transport properties.
the stacking of rigid macrocycles, which leads to nanotubes
containing non-deformable inner pores with precisely defined
The inner pores of the tubular stacks, structurally modified by
incorporating macrocycles sharing the same periphery but
diameters. Such self-assembling nanopores, if amenable to
having cavities with inward-pointing functional groups, were
structural modifications, may afford systems with novel
molecular recognitions, mass-transport, and many other
found to exhibit different ion-transporting preferences.
capabilities.
Macrocycles 2 can be regarded as being derived from
merging the backbones of and oligo(m-phenylene
Since their discovery8a and the elucidation of the mechanism
of their formation,8b aromatic oligoamide macrocycles 1
(Figure 1) have served as the basis for creating several classes
of rigid macrocycles9 that have greatly expanded the inventory
of such shape-persistent molecular objects.8-13 The stacking of
1,14 along with the discrete15 or extremely strong16 tubular
assembly of derivatives of 1, have demonstrated the persistent
assembly of these macrocycles.
1
ethynylene) (or m-PE) macrocycles, another class of well
studied rigid macrocycles.10 Thus, replacing part (~1/3), i. e., a
1, 3-benzenedicarboxamido residue, of
1
with
a
diethynylbenzene unit (Figure 1a), results in
a
hybrid
backbone containing a segment that allows inward-pointing
functional groups (X) to be placed. Results based on simple
modeling indicate that the introduced diethynylbenzene
moiety (Figure 1b, blue) closely matches the shape of the
replaced residue (Figure 1b, red). With their overall flat shape
(Figure S41), macrocycles 2 are expected to aggregate
anisotropically,16 i. e., to stack into tubular assemblies. A
The tubular assemblies of 1 had prompted us to probe their
functions, one of which involves ion transport across
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secondary amide side chain is included in 2 to reinforce the
directionality of the stacking and to also facilitate the
characterization of the supramolecular assemblies.
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Me
O
Me
O
Me
O
Me
O
(a)
Me
O
Me
O
5
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R
N
H
O
R
H
O
H
R
N
H
R
R
N
H
O
O
N
N
O
O
N
H
R
O
O
N
N
O
O
O
O
O
O
O
R
O
R
O
R
O
R
O
R
O
H
O
H
O
H
R
O
O
H
O
H
O
H
N
O
2
O
N
O
O
1
9
N
O
N
O
O
Me
O
Me
Me
Me
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Me
O
O
Me
X
N
H
N
H
Me
Me
Me
O
R
O
R
Me
O
NH
R'
(b)
7.28 Å
124o
5.43 Å
142o
6.95 Å
120o
O
X
O
O
O
N
O
N
N
H
N
H
H
H
O
R
O
R
O
O
NH
R'
Me
Me
1, 3-diethynylbenzene
residue
1, 3-benzenedicarboxamide
residue
N,N'-1, 3-phenylenebis(carboxamide)
residue
Figure 1. (a) Design of macrocycle 2 by integrating the backbone of 1 and that of m-PE macrocycles. (b) Comparison of the two different
monomeric residues of 1 with 1, 3-diethynylbenzene residue.
■ RESULTS AND DISCUSSION
Synthesis. The synthesis of macrocycles
2 involves
coupling trimer diacid chloride 3 with the corresponding
trimer diamines 4 (Scheme 1). Thus, a mixture of 3, prepared
from the corresponding diacid (0.37 mmol), and 4-H (0.304
o
mmol) in CH2Cl2 was stirred at 0 C for 3 hours. The reaction
mixture was then washed with water, saturated brine, and
dried over anhydrous Na2SO4. Removing CH2Cl2 led to a
residue that was purified by column chromatography (silica
gel), giving 2-H as a white solid (185 mg, 41%). Macrocycles
2-Me, 2-F and 2-NH2 were prepared under the same condition,
with un-optimized yields ranging from 11% to 24% after
extensive purification (see Supporting Information).
Scheme 1. Synthesis of Macrocycles 2.
Me
O
Me
O
Me
O
Me
O
R1
R1
H
H
O
N
N
O
R1
R1
H
O
H
O
N
N
O
O
O
R1
R1
O
O
O
O
O
R1
R1
O
O
O
H
3
Et3N, CH2Cl2
0 °C
O
Cl
O
Cl
H
2
N
O
N
+
O
NH2
NH2
Me
Me
X
O
Me
Me
X
R2
O
N
H
4
R2
Figure 2. (a) Normalized emission spectra of 2-H (1 µM) in DMF
(red) and CCl4/CHCl3 (7/3, v/v) (blue) excited at 325 nm (slitex
8 nm, slitem = 12 nm). (b) Normalized excitation spectra of 2-H (1
O
N
H
=
O
2-H and 4-H:
X = H
X = CH3
X = F
R1
R2
=
=
2-Me and 4-Me:
2-F and 4-F:
µM) in CCl4/CHCl3 (7/3, v/v) monitored at 385 nm (purple) and
O
2-NH2 and 4-NH2: X = NH2
O
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455 nm (green) with background subtraction (slitex = 8 nm, slitem
12 nm).
=
S34) in DMF and CCl4/CHCl3 (7/3, v/v), suggesting that all
four macrocycles 2 undergo the same assembly by stacking
into tubular stacks that are chiral as indicated by their circular
dichroism spectra (Figure S35).
1
2
3
4
5
6
7
8
Macrocycles 2 Undergo Anisotropic Aggregation in Non-
1
Polar Solvents. In CDCl3, the H NMR signals of 2-H are
The Tubular Stacking of 2 is Confirmed by Atomic
Force Microscopy (AFM). Additional evidence showing the
tubular stacking of 2 is provided by AFM images (Figures 3
and S36-39), which reveal very similar fine nanofilaments.
The measured distances between adjacent nanofilaments are
2.8 ± 0.2 nm for 2-H and 2-Me, 2.8 ± 0.3 nm for 2-F, and 2.7
± 0.2 nm for 2-NH2. These values are consistent with the
diameter of the macrocycles, confirming that 2-H, 2-Me, 2-F
and 2-NH2, with the same periphery, indeed superpose into
well-aligned nanotubes.
considerably broadened, which indicates that the molecules of
2-H undergo decreased molecular motion due to aggregation;
in DMSO-d6, the spectrum of 2-H contains well-dispersed
peaks, suggesting that the individual molecules were fully
solvated (Figures S23 and S24). Similarly, the 1H NMR
spectra of 2-Me, 2-F and 2-NH2 contain signals that are
broadened in CDCl3 but are well-resolved in DMSO-d6
(Figures S25-S30), indicating that, in CDCl3, these
macrocycles undergo aggregation that is interrupted in the
much more polar DMSO.
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Macrocycles 2 Show Different Behavior for Proton
Transport. The tubular assemblies of 2, consisting of
macrocyclic molecules stacked directly on top of one another,
should contain cylindrical pores defined by the cavities of the
macrocycles. A transmembrane pore, consisting of about ten
stacked macrocyclic molecules7,17 and thus having about ten X
groups on its inner surface, may exhibit mass-transport
behavior that is influenced by the X groups.
In comparison to their noncyclic precursors (Figure S31),
macrocycles 2 exhibit much stronger absorption and emission,
which allows their aggregation to be examined at significantly
reduced concentrations (1-5 µM). In DMF in which 2-H is
fully solvated, an emission band at 385 nm is observed; in
CCl4/CHCl3 (7/3, v/v), a broad band at 455 nm, which can be
attributed to π-stacked aromatic rings21a that exist in the
ground state and give “excimer-like” emissions, appears
(Figure 2a).21b Consistent with ground-state association,
monitoring the emission of 2-H at 385 nm and 455 nm reveals
two bands around 300 nm and 280 nm, respectively, in the
excitation spectra (Figure 2b). The excitation band at 280 nm,
being monitored at 455 nm that corresponds to the aggregated
state of 2-H, is blue-shifted relative to the 300-nm excitation
band due to the free monomers of 2-H. This observation
suggests that macrocycle 2-H most likely formed H-type
aggregates,21a i. e., the macrocylic molecules stack face-to-face
into tubular stacks.
A vesicle-based stopped-flow kinetic assay was adopted to
monitor proton transport across lipid bilayers. Large
unilamellar vesicles (LUVs) of the phospholipid 1-palmitoyl-
2-oleoyl-sn-glycero-3-phosphocholine (POPC) were prepared
with encapsulated 8-hydroxypyrene-1, 3, 6-trisulfonate
(HPTS, 0.1 mM) a pH-sensitive fluorescent dye,22 in 100 mM
HEPES, 145 mM KCl, at pH 7.0. Each of compounds 2 was
mixed the lipid by dissolving in chloroform before the LUVs
were prepared, with a molar ratio of lipid to compound at 263
to 1 and a final concentration of 10 µM in the solution used for
the stopped-flow assay.
control
2-H
2-NH2
2-Me
2-F
2-NH2
2-H
Figure 4. Results from stopped-flow measurements of the
emission intensity of HPTS (0.1 mM) entrapped in 100-nm POPC
large unilamellar vesicles (LUVs) in the presence of macrocycles
2 (10 µM in a pH-7.0 buffer). The LUVs were pre-incubated with
valinomycin (5 µM) for 5 min and then exposed to a proton
gradient by rapidly mixing with an equal volume of another
solution (buffer with 80 mM HCl).
Figure 3. The AFM images of 2-H (a), 2-Me (b), 2-F (c), and 2-
NH2 (d). Each compound (10 µM in CHCl3) was deposited on
freshly cleaved mica, then incubated in saturated CCl4 vapor over
night. AFM imaging was performed in air with the tapping mode
(scale bar = 300 nm).
Stopped-flow kinetic assay was performed by rapidly
mixing a solution (80 µL) of the LUVs with one of
compounds 2 incorporated (in 100 mM HEPES, 145 mM KCl,
The same difference in emission and exciation bands also
exists in the spectra of 2-Me, 2-F and 2-NH2 (Figures S32-
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pH 7.0) and a solution of a more acidic buffer (100 mM of 2-NH2. Being placed para to the electron-withdrawing
1
2
3
4
5
6
7
8
HEPES, 145 mM KCl, 80 mM HCl) at an equal volume in a
stopped-flow spectrometer at 12 oC. The time-dependent
change in the fluorescence emission of the encapsulated HPTS
was monitored at 510-550 nm with the excitation wavelength
being set at 450 nm. A control experiment was also performed
under the same condition without incorporating any of
compounds 2. Before each fluorescence measurement, the
vesicles were pre-incubated for 5 min with 5 µM of
valinomycin, a potassium carrier that serves to counter the
charge buildup caused by proton influx and thus retains the
membrane potential to near zero, which eliminates a limiting
factor in the kinetic measurements. 23
carbonyl group of the amide side chain and electronically
similar to that of a primary amide, the NH2 group of 2-NH2
has Hs with a significant partial positive charge that interacts
repusively with proton.
control
2-H
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2-F
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2-Me
The emission intensity of the pH-sensitive HPTS
encapsulated in the LUVS serves as a direct indicator for the
pH value inside the vesicles. With a higher extravesicular
proton concentration, proton influx leads to an increase of
acidity inside the vesicles and thus quenching of the
fluorescence emsission of HPTS.
2-NH2
As shown in Figure 4, while significant enhancement of
proton transport is observed in the presence of 2-H and 2-F,
no transport above background is detected with 2-Me or 2-
NH2. Results from the stopped-flow kinetic measurements
provide a background proton permeability of about 2.1×10-9
m/s for the vesicles in the control experiment, while the proton
permeability in the presence of 2-H is 8.5 × 10-9 m/s, which is
four times that of the control. The values of proton
permeability in the presence of the other three macrocycles are
2.1 × 10-9 m/s (2-Me), 4.6 × 10-9 m/s (2-F) and 2.0 × 10-9 m/s
(2-NH2), respecitvely. Thus, the presence of macrocycles 2-
Me or 2-NH2 does not lead to proton transport that is higher
than that of the background, while adding 2-H or 2-F results in
proton transport that is noticeably above that of the
background.
Figure
5.
Normalized
emission
intensity
of
N-
(ethoxycarbonylmethyl)-6-methoxyquinolinium
bromide
(MQAE) encapsulated inside POPC LUVs. Solutions of chloride-
free LUVs with MQAE (10 mM) were first incubated in an
isotonic buffer (pH 7.4) containing KCl (100 mM) for 1 minute.
Aliquots of THF and solutions (1 mM) of each of 2 in THF were
added (final concentratuon of a compound = 10 µM in solution).
Emission intensity at 460 nm (λex = 354 nm) was monitored.
Triton X-100 was added to rupture the LUVs.
Macrocycles 2-H and 2-NH2 Form Single Channels with
Different Cation/Anion Preferences. Conclusive evidence
for the existence of transmembrane ion channels was provided
by single-channel electrophysiology, which involves the
measurement of electric current across a planar lipid bilayer
that separates two, i. e., cis and trans, chambers, each of which
holds a 1-mL saline solution. Details of preparing planar lipid
bilayers can be found in the Supporting Information and in our
previous reports.7,17 Examining 2-H and 2-NH2 in 0.5 M KCl
revealed long-lasting single conductance steps (Figures 6a and
6b) with single-channel conductance of 238 ± 60 pS and 231 ±
44 pS, respectively.
Given their identical periphery, the different behavior of 2-
H, 2-Me, 2-F and 2-NH2 toward proton transport can only be
attributed to the membrane-spanning inner pores offered by
the tubular assemblies of these macrocycles. Due to their
different X groups, the self-assembling pores of 2 have
different properties and thus behave differently when
mediating transmembrane proton transport.
Macrocycles
2
Have Different Propensities for
Transporting Chloride Ion. The lack of enhanced proton
transport with 2-Me or 2-NH2 may be due to a simple reason:
the methyl or amino groups sealed off the self-assembling
pores to keep any ions from passing through. This possibility
was examined with the likely transport of other ions. Rapid
monitoring of chloride transport can be conveniently
performed with LUVs containing N-(ethoxycarbonylmethyl)-
The cation/anion preferences of 2-H and 2-NH2 channels
were compared by determining reversal potentials (ΔVs),25
which involved creating a salt gradient and measuring currents
across lipid bilayers (Supporting Information). The x-
intercepts of the I/V plots (Figures 6c and 6d) reveal ΔVs of -
14.09 mV and -9.68 mV for 2-H and 2-NH2, respectively. The
ΔV of 2-H is closer to, and that of 2-NH2 is much smaller than
the Nernst potential of K+ (-17.81 mV). Calculations based on
the Goldman–Hodgkin–Katz (GHK) flux equation,25 which
describes the ionic flux across a cell membrane as a function
of the transmembrane potential and the concentrations of the
ion inside and outside of the cell, revealed K+/Cl- permeability
ratios (PK+/PCl-) of 9 for the 2-H channel, and of 3.5 for the 2-
NH2 channel. Thus, with similar conductance, the 2-H channel
mainly transports K+ while the 2-NH2 channel shows a
signficantly lowered preference for K+, which is accompanied
by enhanced Cl- conductance.
6-methoxyquinolinium bromide (MQAE),22
a
chloride-
sensitive dye (Figure 5). Contrary to the control experiment
which did not include any of macrocycles 2 and showed little
chloride transport, the presence of macrocycles 2 facilitated
chloride transport to different extents. While 2-H was found to
lead to a small decrease in fluorescence emission, a significant
reduction in emission intensity was observed with 2-NH2. The
chloride transport activities of 2-Me and 2-F are between
those of 2-H and 2-NH2. Thus, the pores of 2-Me and 2-NH2,
being impermeable to proton but still conducting chloride, are
not physically blocked. The differential enhancement of
chloride transport in the presence of 2-NH2 could be due to the
numerous amino Hs in the inner pore of the tubular assembly
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(b)
(a)
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-1.911
-2.243 (or -2.471**)
(d)
(c)
(c)
-2.713
-1.654
Figure 7. Electrostatic potential maps of (a) 2-H, (b) 2-Me, (c) 2-
F, and (d) 2-NH2. Geometry optimization and energy
minimization were performed with density function B3LYP 6-
31G** in vacuum. Both GAMESS and Spartan were used for the
calculation of the electrostatic potential maps, along with the local
ionization potential maps. The overall electrostatic charge of the
inner cavity of each macrocycle, which was calculated by adding
the charges of the atoms that define the cavity, is shown under
each map. For 2-Me, 2-F, and 2-NH2, the charges of all the
carbonyl oxygen atoms, along with F, or the methyl or amino Hs,
are included. *Value obtained by including the charges of the two
triple bonds flanking the aromatic H atom, based on the
expectation that the inward-pointing H atom of 2-H, with its small
size, does not exclude the π electrons of the triple bonds from
being part of the edge of the inner cavity of 2-H.
(d)
Figure 6. Single channel recordings of (a) 2-H and (b) 2-NH2 (0.5
µM, 50 mV in 0.5 M KCl). Current-voltage plots of the channels
formed by (c) 2-H (0.5 µM) and (d) 2-NH2 (0.5 µM) from
reversal potential experiments (25 oC; cis chamber: 1 M KCl;
trans chamber: 0.5 M KCl).
In conclusion, with a hybrid backbone and the same set of
side chains, macrocycles 2 undergo the same nanotubular
assembly that is demonstrated by analysis using multiple
techniques. The formation of tubular assemblies with the same
outer surface but different inner pores defined by stacked
macrocycles 2 is also supported by the distinctly different ion-
transporting behavior of these molecules. That the properties
of the self-assembling pores directly influence ion transport
behavior is shown by the different preferences of macrocycles
2 for transporting protons and chloride ions, due to the
presence of the X groups. The rejection of proton by the 2-Me
The overall electrostatic charges of the cavities of 2 were
estimated computationally (Figure 7), results from which
largely reflect the observed ion transport behavior of these
molecules by showing a rough correlation to the observed
cation/anion transporting preferences. With cavities that are
more negative than those of 2-Me and 2-NH2, macrocycles 2-
H and 2-F form channels that enhance proton, but not chloride
transport. In contrast, the 2-Me and 2-NH2 channels exhibit
the opposite trend.
The rejection of the smallest cation, i.e., H+, but not K+, by
2-Me and 2-NH2 is remarkable, given that proton rejection is
an ability observed with biological channels such as
aquaporins26 but realized with few synthetic channels.27
Whether such an effect involves the so-called orientational H-
bonding defect in which structured water28 in a nanopore is
physically and/or electrostatically interrupted by methyl or
amino group remains to be elucidated.
and 2-NH2 channels is
capability rarely observed with synthetic channels. The
different propensities of the four macrocycles for
a surprising and extraordinary
2
transporting chloride ions point to the possibility of
performing systematic structural and functional tuning of the
corresponding self-assembling nanopores and the channels
based on them. Understanding the mechanisms governing the
observed ion transport is fundamentally important and will
undoubtedly facilitate further refined design of functional
nanopores, leading to a host of synthetic channels and devices
with tailored selectivity and specificity.
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V.; He, L.; Zeng, X. C.; Shao, Z. F.; Gong, B. Nature Commun. 2012,
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ASSOCIATED CONTENT
1
2
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Supporting Information. Synthetic and experimental details,
AFM images, NMR, mass, fluorescence and CD spectra. This
material is available free of charge via the Internet at
http://pubs.acs.org.
5
AUTHOR INFORMATION
Corresponding Authors
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12142. (b) He, L.; An, Y.; Yuan, L. H.; Feng, W.; Li, M. F.; Zhang,
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Liu, R.; He, L.; Zeng, X. C.; Gong, B. Angew. Chem. Int. Ed. 2009,
48, 3150.
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*bgong@buffalo.edu
*zs9q@virginia.edu
*luzl@bnu.edu.cn
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‡These authors contributed equally.
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The authors declare no competing financial interest.
ACKNOWLEDGMENT
We thank the Natural Science Foundation of China (91227109,
11374207, 21303104, 21273148) and the US National Science
Foundation (CHE-1306326 and CBET-1512124) for support.
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