Structurally simple trimesic amides as highly selective anion channels

Article abstract of DOI:10.1039/C9CC00248K

Trimesic amide molecules, which contain simple alkyl chains in their periphery, exhibit interesting anion-transport functions. The most active and highly selective channel TA12 efficiently transports ClO₄^? anions across membranes, with other anions conducted in the order of I^? > NO₃^? > Br^? > Cl^?.

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Structurally simple trimesic amides as highly selective
anion channels†
Cite this: DOI: 10.1039/x0xx00000x
Lin Yuan,a,‡ Jie Shen,b,‡ Ruijuan Ye,c Feng Chenb and Huaqiang Zeng*b
Received 00th January 2012,
Accepted 00th January 2012
DOI: 10.1039/x0xx00000x
www.rsc.org/
(a)
(b)
Trimesic amide molecules, which contain simple alkyl chains
in their periphery, exhibit interesting anion-transporting
functions. The most active and highly selective channel TA12
efficiently transports ClO4 anions across membrane, with
other anions conducted in the order of I- > NO3- > Br- > Cl-.
3.69 Å
-
Cellular ion homeostasis across membrane is precisely
maintained by biological ion transporters, which mostly
transport ions through a channel mechanism. Malfunctioned
protein ion channels often result in dysregulated ion transports
and could lead to various ‘channelopathies’ such as cystic
fibrosis (Cl-), iodide deficiency disorders, paralysis (Na+),
seizure (K+) and malignant hyperthermia (Ca2+). Synthetic ion
channels have thus been actively pursued since 1982,1 aiming
not only to elucidate the possible ion transport mechanisms but
also to promote medical applications in “channel therapies”.2
Considering that protein ion channels often contain a narrow-
sized cavity for ion transport, rationally designed synthetic ion
channel molecules mostly carry either central macrocyclic
units, tubular cavity, or one-dimensionally aligned ion-binding/
transporting units1f,3 as ion permeation pathways.
Top view
H-bonds
1D columnar packing by (TA4)6 in solid state
(c)
Channels
(~ 2 Å across)
On the other hand, some naturally occurring pore-forming
peptides4a-d and their synthetic analogues4e-i readily assemble,
via side chain-side chain interactions, into a barrel stave pore
for mediating killing of bacterial cells. Similar side chain-side
chain interactions are also ubiquitously observed during the
formation of -helix bundles,5a-e protein channels,5f,5g and pore-
forming toxins5h from their constituent units. We believe this
type of side chain-side chain association mode, when used
appropriately, might open a new avenue for designing novel
types of synthetic transmembrane channels, greatly expanding
both the structural and functional repertoires of synthetic
channel systems.6 Nevertheless, this strategy currently still
remains largely unexplored.
Channel-forming packing by (TA4)n in solid state
Fig. 1 (a) Structures of trimesic amide molecules studied in this work.
(b) One-dimensional columnar packing of (TR4)6 supported by a triple
helical H-bonding pattern. (c) Two-dimensional arrangement of 1D
rods that lead to the formation of sizable channels/voids of 2 Å across.
three amide groups do not stay coplanar with the central
benzene ring, but they are not perpendicular to the central ring
either. This interplanar tilting angel of the amide groups with
Trimesic amides (TAs) are an interesting class of molecules
that have been investigated quite intensively in crystal design
and growth7a,7b as well as materials science.7c,7d In TAs, the
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(b)
(a)
pH = 8
100 mM NaCl
Conc = 20 uM
20
10
0
Cl-
TA8
OH-
TA10
TA6
TA12
6 Å
pH = 7
TA14
100 mM NaCl
Blank
HPTS
Channel with a larger pore of 6 Å by
0
100
200
300
(TA12)2
(TA12)12
Time (s)
(c)
(d)
pH = 7
Blank
0
-20
200 mM NaCl
Cl-
200 mM K2SO4
Conc = 20 uM
200 mM Na2SO4
Conc = 20 uM
200 mM Na2SO4
-
60
40
20
0
NO3
60
40
20
0
or K2SO4
gA (1 nM)
gA (1 nM)
TA14
TA12
M+
H+
-40
TA6
Blank
TA8
TA10
TA14
TA12
TA6
TA12
Blank
TA14
TA6
TA8
TA10
-60
TA6
200 mM
NaNO3
pH = 7
-80
TA8
HPTS
SPQ
Conc = 20 uM
-100
0
100
Time (s)
200
300
0
100
200
Time (s)
300
0
100
200
300
Time (s)
(e)
(f)
Current (fA)
60
140 mv
60
40
20
0
pH = 8
120 fA
[VA] = 3 pM
[TA8] = 20 uM
100 mM KCl
-
3 s
OH-
Cl
Cl
-
40
20
0
120 mv
100 mv
TA8
31.5%
27.9%
TA8+VA
TA8
K+
-150
-100
-50
0
50
100
150
pH = 7
100 mM NaCl
0 mv
Voltage (mV)
VA 12.7%
9.8%
Blank
-20
-40
-60
VA
-100 mv
-140 mv
HPTS
γCl= 396.5 ± 10.3 fS
0
100
200
Time (s)
300
Fig. 2 (a) A schematic illustration of TA12, forming a larger pore of 6 Å obtained using PM6D3. (b) pH-sensitive HPTS assay for evaluating and comparing
the ion transport activities of ion channels; RCl- = (ICl- – I0)/(ITriton– I0) whereas ICl- and I0 are the ratiometric values of I460/I403 at t = 300 s before addition of
triton, and ITriton is the ratiometric value of I460/I403 at t = 300 s right after addition of triton. (c) Chloride-sensitive SPQ assay for evaluating chloride-
transporting functions. (d) Sulphate-containing LUV assay for assessing applicability of Na+-based ion exchange as the ion transport mechanism. (e)
Valinomycin-based LUV assay for comparing relative transport rates between OH- and Cl-. (f) Single channel current traces for chloride transport recorded at
various voltages in symmetric baths (cis chamber = trans chamber = 1 M KCl) as well as the determined chloride conductance value for TA8.
respect to the aromatic ring plane highly depends on the
Although the enclosed channels found in (TA4)n are likely
peripheral substituents.7b With the use of peripheral either too narrow for ion conduction or of low activity in ion
butylbenzene groups as in TA4 (Fig. 1a), the amide groups, transport, existence of such channels is highly interesting and
being perfectly self-complementary to each other in H-bond- inspiring from which we could quickly envision a novel ion
forming character, generate
a triple helical network of channel system. More specifically, we anticipated that a simple
intermolecular H-bonds(Fig. 1b).7b This H-bonding network extension of n-butyl chains to longer alkyl chains from hexyl to
subsequently links TA4 molecules into -stacked discotic 1D tetradecyl chains (Fig. 1a) might result in the formation of a
rods in solid state, with peripheral butylbenzene groups larger pore as schematically exemplified by (TA12)6 (Fig. 2a).
organized into a hexagonal arrangement. Further associations In the fortunate scenarios, these larger pores may enable ions to
among these 1D rods similarly proceed in the form of get transported across membrane, with ion transport activities
hexagonal packing, creating numerous sizeable voids/channels further tunable via tuning length and type of the alkyl chains as
of 2 Å across (Fig. 1c).
well as other functional groups. We further anticipated that
anions should be transported more easily than cations as the
2 | J. Name., 2012, 00, 1-3
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(a)
(b)
pH = 8
X
‖
-
Br-
Cl-
I-
NO3
-
ClO4
100 mM NaX
Cl-
Br-
I-
X-
OH-
-
ClO4
NO3
-
pH = 7
100 mM NaX
HPTS
(c)
(d)
100
Initial rate
const (S-1)
0.0117
100
80
60
40
20
0
[TA12] = 20 uM
TA12 for NaClO
TA12 for NaClO4
4
7 uM
6 uM
5 uM
4 uM
160
120
80
NaClO4
NaI
0.0123
50
3 uM
EC = 3.0 0.2 uM
±
50
2 uM
1 uM
n = 2.0
NaNO3
NaBr
0.0021
0.0006
40
0.5 uM
Blank
0
NaCl 0.0003
0
0
100
200
300
0
100
200
300
0
2
4
6
8
Time (s)
Conc (uM)
Time (s)
Fig. 3 (a) LUV scheme for determining anion selectivity. (b) Fractional transport activities measured for five types of anions by all five TA channels. (c)
-
Initial rate constants determined for TA12-mediated anion transports. (d) Determination of EC50 value for TA12-mediated transport of ClO4 using the Hill
analysis.
alkyl chains, which decorate the pore interior, should interact Na+ ions and efflux of H+ ions. Similar results were seen when
more favourably with hydrated anions than hydrated cations. 200 mM K2SO4 was used (Fig. 2d). These three sets of data
Here, we confirm our such hypotheses and show that TAs, shown in Fig. 2b-2d unambiguously establish chloride anions
when modified to carry longer alkyl chains, do can function as as one of the main transport species. In other words, either Cl-
good anion-transporting channels.
/OH- antiport or Cl-/H+ symport should account for the ion
A well-established HPTS-based LUV assay (Fig. 2b) was transport mediated by the five TA channels.
then adopted for evaluating ion transporting activities of five Valinomycin (VA), a potent K+ carrier, was then introduced
TA channels (TAn, n = 6, 8, 10, 12 and 14). The principle into the LUV-based assay to compare the transport rates
behind this assay is that ion exchange, occurring in one of the between Cl- and OH- (Fig. 2e). To maintain charge neutrality,
four possible modes (Na+/H+ antiport, Na+/OH- symport, Cl- VA-mediated influx of K+ ions will be accompanied by influx
/OH- antiport or Cl-/H+ symport), will result in an increase in of either Cl- or OH- anions (or efflux of H+ ions), which
intravesicular pH, which in turn increases the fluorescence respectively cause either negligible or significant increases in
intensity of pH-sensitive HPTS dye. Plotting changes in fluorescence intensity. Experimentally, addition of VA at 3 pM
fluorescence intensity vs time at the same channel concentration leads to increases of 3.6% (e.g., 31.5% - 37.9%) in the presence
therefore allows one to compare the relative ion transport of TA8 and of 2.9% (e.g., 12.7% - 9.8%) in the absence of
abilities of ion channels.
TA8. The relative change of 0.7% (e.g., 3.6% - 2.9%) is
At 20 M, TA8 displays the highest fractional ion transport obviously insignificant, thereby supporting Cl- transport rate to
activity, which is followed by TA10 > TA6 > TA12 > TA14 be much faster than that of OH- or H+ ions. This conclusion is
(Fig. 2b). This trend in ion transport activity persists when also consistent with that drawn from FCCP-based assay (Fig.
chloride-sensitive SPQ assay was carried out to evaluate the S1).
chloride-transporting abilities of these channels (Fig. 2c).
Additionally, given the highly hydrophobic nature of the
Further, using the LUV assay containing 200 mM Na2SO4 (Fig. pore, hydrated OH- anions should be transported more
2d), all TA channels at 20 M produce smaller increases in preferentially than hydrated H+ ions. In other words, the above
fluorescence intensity than the background signal, indicating results collectively establish that TA8 predominantly mediates
influx of H2SO4 to some varying extents, rather than influx of exchanges of Cl- and OH- anions across the membrane.
Na+ ions, under a high ionic concentration gradient in the
The chloride-transporting behaviour by TA8 was then
presence of TA channels. In sharp contrast, gramicidin A (gA) examined in the planar lipid bilayer. The evolved single
results in fluorescence increase by 60% at the extremely low channel current traces for chloride at various voltages (Fig. 2f)
concentration of 1 nM (Fig. 2d) as a result of rapid influx of conclusively demonstrate that TA8-mediated chloride transport
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b
Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, The
Nanos, Singapore 138669. E-mail: hqzeng@ibn.a-star.edu.sg; Tel: +65-
6824-7115
occurs through a channel, rather than a carrier, mechanism.
Fitting of a linear current-voltage (I-V) curve yields the Cl−
conductance (γCl-) of 396.5 ± 10.3 fS for TA8
.
c
Department of Chemical and Biomolecular Engineering, National
University of Singapore, Singapore 117585
† Electronic Supplementary Information (ESI) available: Experimental
procedures and ion transport activities. See DOI 10.1039/b000000x/
‡These authors contributed equally.
In our effort to elucidate the anion selectivity of TA8 using the
LUV scheme shown in Fig. 3a, we were surprised to find that both I-
-
and ClO4 are transported much faster than Cl- at the same
concentration of 20 M after subtracting background signals and
further normalization (Fig. 3b). These findings prompted us to carry
out a more systematic examination of anion transport involving all
five types of anions by the remaining four TA channels. Our
summative results in Fig. 3b show that, except for the chloride
anions for which TA8 acts as the best channel, TA12 induces the
1. I. Tabushi, Y. Kuroda and K. Yokota, Tetrahedron Lett., 1982, 23,
4601.
2. (a) J. T. Davis, O. Okunola and R. Quesada, Chem. Soc. Rev., 2010, 39,
3843; (b) P. R. Brotherhood and A. P. Davis, Chem. Soc. Rev., 2010, 39,
3633; (c) S. Matile, A. Vargas Jentzsch, J. Montenegro and A. Fin,
Chem. Soc. Rev., 2011, 40, 2453; (d) J. Montenegro, M. R. Ghadiri and
J. R. Granja, Acc. Chem. Res., 2013, 46, 2955; (e) T. M. Fyles, Acc.
Chem. Res., 2013, 46, 2847; (f) F. Otis, M. Auger and N. Voyer, Acc.
Chem. Res., 2013, 46, 2934; (g) G. W. Gokel and S. Negin, Acc. Chem.
Res., 2013, 46, 2824; (h) F. De Riccardis, I. Izzo, D. Montesarchio and
P. Tecilla, Acc. Chem. Res., 2013, 46, 2781; (i) L. D. Mosgaard and T.
Heimburg, Acc. Chem. Res., 2013, 46, 2966; (j) B. Gong and Z. Shao,
Acc. Chem. Res., 2013, 46, 2856; (k) D. S. Kim and J. L. Sessler, Chem.
Soc. Rev., 2015, 44, 532; (l) J.-Y. Chen and J.-L. Hou, Org. Chem.
Front., 2018, 5, 1728.
3. (a) A. Vargas Jentzsch and S. Matile, J. Am. Chem. Soc., 2013, 135,
5302; (b) A. Gilles and M. Barboiu, J. Am. Chem. Soc., 2016, 138, 426;
(c) C. L. Ren, J. Shen and H. Q. Zeng, J. Am. Chem. Soc., 2017, 139,
12338; (d) C. Ren, X. Ding, A. Roy, J. Shen, S. Zhou, F. Chen, S. F.
Yau Li, H. Ren, Y. Y. Yang and H. Zeng, Chem. Sci., 2018, 9, 4044.
4. (a) Y. Lai and R. L. Gallo, Trends Immunol., 2009, 30, 131; (b) R. E.
W. Hancock, E. F. Haney and E. E. Gill, Nat. Rev. Immunol., 2016, 16,
321; (c) S. M. Mandal, S. Sharma, A. K. Pinnaka, A. Kumari and S.
Korpole, BMC Microbiol., 2013, 13, 152; (d) F. S. Tareq, M. A. Lee,
H.-S. Lee, Y.-J. Lee, J. S. Lee, C. M. Hasan, M. T. Islam and H. J. Shin,
Org. Lett., 2014, 16, 928; (e) A. Makovitzki, D. Avrahami and Y. Shai,
Proc. Natl. Acad. Sci. U S A., 2006, 103, 15997; (f) G. Wiedman, S. Y.
Kim, E. Zapata-Mercado, W. C. Wimley and K. Hristova, J. Am. Chem.
Soc., 2016, DOI: 10.1021/jacs.6b11447; (g) A. J. Krauson, O. M. Hall,
T. Fuselier, C. G. Starr, W. B. Kauffman and W. C. Wimley, J. Am.
Chem. Soc., 2015, 137, 16144; (h) R. Rathinakumar and W. C. Wimley,
J. Am. Chem. Soc., 2008, 130, 9849; (i) Z. Y. Ong, N. Wiradharma and
Y. Y. Yang, Adv. Drug Deliv. Rev., 2014, 78, 28.
-
-
fastest transports for all other four anions (Br-, I-, ClO4 and NO3 ),
while TA14 displays very high selectivity toward I- anions relative
to both Br- and Cl- anions. In comparison, fractional ion transport
activities of Cl-, I- or ClO4- were measured to be 3.5, 6.1 and 10.0%
-
for TA4, suggesting that highly active transports of I- or ClO4 by
TA12 are mediated by a channel, rather than a carrier mechanism,
which is also consistent with single channel current traces obtained
for chloride transport by TA8 (Fig. 2f).
On the basis of initial rate constants (Figs. 3c and S2), I- anions are
transported by TA12 at a speed that is about 20 and 41 times those
for Br- and Cl- anions, respectively. Following the Hill analysis, the
EC50 values at which channels reach 50% ion transport activity were
determined to be 3.0 and 12.1 M for ClO4- (Fig. 3d) and I- (Fig. S3)
anions, respectively. On this basis, TA12-mediated transport of
-
ClO4 anions is four times as fast as I- anions. It might be worth
pointing out that initial rate constants for I- transport decrease much
more rapidly upon decreasing the channel concentration than those
for ClO4- transport (Figs. S4 vs S5).
Lastly, with respect to a typical thickness of 34 Å for the
hydrophobic membrane region, an inter-planar separation distance of
3.69 Å observed for TA4 in the crystal structure (Fig. 1b)7b suggests
10 molecules of TA12 would be required to form a rod-like structure
that can fully span the hydrophobic membrane region. In this regard,
-
the EC50 value in terms of effective channel concentration for ClO4
is 0.3 M. This value corresponds to a channel:lipid molar ratio of
1:104 or 0.96 mol%, certainly pointing to a highly efficient transport
of ClO4- anions by TA12.
5. (a) E. Wolf, P. S. Kim and B. Berger, Protein Sci., 1997, 6, 1179; (b) S.
Y. Lau, A. K. Taneja and R. S. Hodges, J. Biol. Chem., 1984, 259,
13253; (c) T. Sasaki and E. T. Kaiser, J. Am. Chem. Soc., 1989, 111,
380; (d) C. P. Hill, D. H. Anderson, L. Wesson, W. F. DeGrado and D.
Eisenberg, Science, 1990, 249, 543; (e) C. E. Schafmeister, S. L.
LaPorte, L. J. W. Miercke and R. M. Stroud, Nat. Struct. Biol., 1997, 4,
1039; (f) R. E. Hibbs and E. Gouaux, Nature, 2011, 474, 54; (g) X.
Hou, L. Pedi, M. M. Diver and S. B. Long, Science, 2012, 338, 1308;
(h) M. D. Peraro and F. G. van der Goot, Nat. Rev. Microbiol., 2015, 14,
77.
To summarize, stimulated by the sizable channels/voids created
via a hexagonal arrangement of 1D rod-like structures that were
assembled from TA4 molecules, we have successfully uncovered a
new class of synthetic anion channels with unusual selectivties.
Based on the EC50 values, the most active and highly selective anion
-
channel TA12 transports ClO4 anions three times faster than I-
anions, which, on the basis of initial rate constants, are transported
drastically much faster than Br- and Cl- anions by 19 and 40 times,
respectively. Further exquisite optimization of the peripheral
substituents may lead to improved activities with interesting anion
selectivities.
This work is funded by the Institute of Bioengineering and
Nanotechnology (Biomedical Research Council, Agency for
Science, Technology and Research, Singapore) and Natural Science
Foundation of Hunan Province of China (2018JJ3193).
6. C. L. Ren, F. Zeng, J. Shen, F. Chen, A. Roy, S. Y. Zhou, H. S. Ren and
H. Q. Zeng, J. Am. Chem. Soc., 2018, 140, 8817.
7. (a) M. P. Lightfoot, F. S. Mair, R. G. Pritchard and J. E. Warren, Chem.
Commun., 1999, 19, 1945; (b) V. Nagarajan and V. R. Pedireddi, Cryst.
Growth Des., 2014, 14, 1895; (c) S. Hasegawa, S. Horike, R. Matsuda,
S. Furukawa, K. Mochizuki, Y. Kinoshita and S. Kitagawa, J. Am.
Chem. Soc., 2007, 129, 2607; (d) S. Cantekin, T. F. A. de Greef and A.
R. A. Palmans, Chem. Soc. Rev., 2012, 41, 6125.
Notes and references
a College of Chemistry and Bioengineering, Hunan University of Science and
Engineering, Yongzhou, Hunan, China 425100
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