Oligo(aryl-triazole)s CH?Cl- interactions guide chloride efficient and selective transmembrane transport

Article abstract of DOI:10.1039/c6cc07792g

A series of oligo(aryl-triazole)s (compounds 1-8) have been synthesized and served as transmembrane anion transporters by only CH?Cl^- interactions. This work confirms their role in the activity of anion transport. By changing the lipophilicity

Full text of DOI:10.1039/c6cc07792g

View Article Online
View Journal
ChemComm
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: S. Chen, S. Zhang,
C. Bao, C. Wang, Q. Lin and L. Zhu, Chem. Commun., 2016, DOI: 10.1039/C6CC07792G.
This is an Accepted Manuscript, which has been through the
Royal Society of Chemistry peer review process and has been
accepted for publication.
Accepted Manuscripts are published online shortly after
acceptance, before technical editing, formatting and proof reading.
Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. We will replace this Accepted Manuscript with the edited
and formatted Advance Article as soon as it is available.
You can find more information about Accepted Manuscripts in the
Information for Authors.
Please note that technical editing may introduce minor changes
to the text and/or graphics, which may alter content. The journal’s
standard Terms & Conditions and the Ethical guidelines still
apply. In no event shall the Royal Society of Chemistry be held
responsible for any errors or omissions in this Accepted Manuscript
or any consequences arising from the use of any information it
contains.
www.rsc.org/chemcomm

Page 1 of 4
ChemComm
View Article Online
DOI: 10.1039/C6CC07792G
ChemComm
COMMUNICATION
Oligo(aryl-triazole)s CH···Cl- Interactions Guide Chloride Efficient
and Selective Transmembrane Transport†
Received 00th January 20xx,
Accepted 00th January 20xx
Sujun Chen, Sitong Zhang, Chunyan Bao,* Chenxi Wang, Qiuning Lin and Linyong Zhu*
DOI: 10.1039/x0xx00000x
www.rsc.org/
A series of oligo(aryl-triazole)s (compounds 1-8) have been
synthesized and served as transmembrane anion transporters by
only CH•••Cl- interactions. The work confirms the roles for the
activity of anion transport. By changing lipophilicity and anion
affinity of the compounds, efficient anion transport with
remarkable Cl- vs HCO3- selectivity was achieved.
As known, ion transport across cell membranes is essential for
the regulation of cellular metabolism, signal transduction,
osmolyte homeostasis and so on.1 Misregulation of ion
transport, especially for anions, leads to a number of diseases
known as “channelopathies”,2 such as cystic fibrosis (CF),
Bartter’s syndrome, Dent’s disease and other diseases.3
Therefore, synthetic molecules and assemblies that can
replicate the activity of faulty anion channels either via a
carrier, relay or channel mechanism present new opportunities
for application as replacement therapy for relative diseases
and tools for the study of transport processes in cells.4
As for nature transporters, ion translocation often involves
high activity and very precise selectivity for specific ions. For
example, Cl- and HCO3- are well known anions in living systems
and dominantly distribute over the cell membrane at
Scheme 1 a) Molecular structures of compounds 1-8; b) the schematic representation
of complexation of compounds;14 and c) the anion transport vesicle assay monitored
using the lucigenin dye, in which aryl-triazoles promote transport of Cl- into the vesicle
millimolar concentrations. Different protein channels are in and NO3- out of the vesicle.
charge of transporting of them, ClC channel family for Cl-
-
selective transport,5 AE1-3 protein for Cl-/HCO3 exchange,6
provide important insights into the biological function and
effects of nature channels.
-
and CFTR for regulated Cl- or HCO3 transport.7 Up to now,
many sophisticated molecules based on prodigiosin,8
calixpyrrole,9,4b and steroidal10 architectures have been
designed for anion transport with high activities, and especially,
some of them have been verified biological activity in
anticancer and antibacterial therapy.4b,8b-c,11 However, the
effort for the control of anion selectivity is still limited.12
Up to date, most commonly applied anion transporters are
based on H-bonding mechanism, in which conventional OH,
NH are always introduced as donors. These kinds of donors can
-
both bind to Cl- and HCO3 and result in co-transport of two
anions.13 Therefore, more specific donors are needed for
achieving selectivity between Cl-/HCO3- anions. Recently, Davis
et al. reported a new class of cyclic biotinuril ester based
anionophores which exhibited chloride-selective transport
employing specific C-H···Cl- interactions.12a Such kind of soft H-
bond favors the transport of softer, more polarizable anions
-
Selectivity between Cl-/HCO3 different anions, albeit not
required for all applications, is of special importance to
-
-
(e.g., Cl-, NO3 ) over harder anions such as HCO3 . However,
due to the high lipophilic character (calculated logp,
clogp≈11.35), the biotinuril esters exhibited moderate
activities only by pre-incorporating to a lipid bilayer. Herein,
This journal is © The Royal Society of Chemistry 2016
Chem.Commun., 2013, 00, 1-3 | 1

ChemComm
Page 2 of 4
COMMUNICATION
ChemComm
we focused on conceptually similar oligo(aryl-triazoles) based
compounds, which have been proven to bind Cl- via specific
CH···Cl- interactions.14,15 Oligo(aryl-triazoles) can be
synthesized in high yields and easy to functionalise, making
them attractive binding motifs for anionic guests. By the
efficient “click” coupling of alkyl azides with alkynes, a series of
aryl-triazoles (compounds 1-8, as illustrated in scheme 1) were
reported in this paper and applied as anion acceptors to
selectively transport Cl- anions through liposomal membranes
by exclusively CH···Cl- interactions. Different substitutions were
applied to optimize the transport activity to achieve both of
high activity and selectivity, mimicking the functionalities as
nature ones, without pre-incorporation into a lipid bilayer.
As for anion transporters, especially for anion carriers, the
trend in transporter efficiency was influenced by 1) the
partition of transporters from the aqueous phase to the lipid
bilayer, 2) mobility within the membrane and 3) “solubilising”
the anion in the lipid membrane (the affinity for binding and
release of anion).16 It suggested that lipophilicity and anion
affinity have great effects on the activity of transporters.
Therefore, different substitutions were introduced in
compounds 1-8 to adjust lipophilicity by changing substituted
alkyloxy side chains (1-5, with clogp from 16.85 to 2.44), and
anion binding affinity by introducing electron-withdrawing
carbonyl ester substitution (6-8).9a,17 All compounds were
prepared by a single click reaction between corresponding
View Article Online
DOI: 10.1039/C6CC07792G
Fig. 1 a) Comparison of chloride transport by compounds 1-8 into vesicles containing
NaNO3 (225 mM) and Lucigenin (1 mM), the concentrations were at 0.25 mol% (molar
ratio of transporter to lipid).
obviously stronger affinities to Cl-, suggesting electron-
withdrawing substitution increased affinities as expected. The
variant in lipophilicity and anion affinity would reveal the
relationships between molecular structures and transport
activity thus provide valuable experience for designing
effective ion transporters.
Chloride transport by compounds 1-8 was explored using
unilamellar
oleoylphosphatidylcholine and cholesterol in a 9:1 ratio with a
mean diameter of 200 nm. NaNO3 (225 mM in 5 mM PB) was
vesicles
composed
of
1-palmitoyl-2-
azide and alkyne derivatives (ESI†, section 3.1-3.2). The binding
-
of the compounds to anions, including Cl- and NO3 , in an
organic medium (CDCl3) was explored using 1H NMR
spectrometer. Job’s Plot method was performed for the
~
used as the suspension solution for internal and external
aqueous solution and a Cl- sensitive dye lucigenin (1 mM) was
enwrapped in the interior of vesicles (ESI†, section 5.1-5.2).
binding stoichiometry at 1:1 ratio (ESI†, Fig. S1-3) as reported
for similar analogs.14 A summary of the results is presented in
Sodium chloride (133 mM) was added to the suspension to
generate Cl- concentration difference. Then compounds 1-8
were added as solutions in THF and the resulting chloride
transport from vesicles monitored through the decay in
lucigenin fluorescence (ESI†, Fig. S4-11). Selected traces from
the experiments are shown in Fig. 1, all compounds showed
activities but with substantial differences depending on the
substitution of side chains. Table 2 summarized the data of
EC50 and kini (ESI†, Fig. S12), two sets of data were reciprocally
consistent except for 8. Considering the complex kinetic
behaviour of transporters, we selected EC50,700 as the key data
Table 2 Quantification analysis of transport assays (Cl-/NO3 ) of compounds 1-8.
Table 1. Despite the different clogp values, compounds 1-5
-
showed similar and low affinities to Cl- or NO3 , and
compounds 6-8 with similar clogp values as those 2-4 exhibited
Table 1 Binding affinities for anions in chloroform, as determined from 1:1 fit mode
from 1H NMR titration data in CDCl3.
Compounds
Ions
Cl-
NO3
K [M-1]
Δδmax[Hz]a
clogpb
6.5 (±0.1)
9.0 (±0.3)
6.6 (±0.3)
5.8 (±0.1)
6.1 (±0.1)
--c, nd
656.4
262.6
546.3
592.2
346.4
--c, nd
1
2
16.80
6.80
-
Cl-
Cl-
-
-
NO3
3
6.33
a
b
-
Compounds
EC50,700s
kini
nc
HCO3
2-
SO4
102 (±12)c
8.7 (±0.6)
7.1 (±0.4)
22 (±2)
144.2c
624.7
645.1
344.1
281.6
350.0
1
2
3
4
5
6
7
8
--nd
0.19
0.31
0.91
0.21
0.15
0.16
2.43
2.65
--nd
--nd
--nd
4
5
6
7
8
Cl-
Cl-
Cl-
Cl-
Cl-
2.97
2.44
6.83
6.43
2.95
0.09±0.02
--nd
0.49
--nd
--nd
--nd
20 (±2)
--nd
--nd
18 (±1)
0.025±0.004
2.22±0.11
0.82
0.75
a, Difference in chemical shift for Hb proton of compounds (ESI†) for free
vs. complex; b, calculated logp, an estimate of lipophilicity where p is the
partition coefficient between octanol and water. Values were obtained
using ALOGPS 2.1; c, titration was processed upon addition of ammonium
salt in DMSO-d6/0.5% water; nd, not determined.
a, EC50,700s defined as concentration (mol% carrier to lipid) needed to obtain 50%
fluorescent quench after 700 s; b, specific initial rate of chloride transport (s-1) is
defined as initial slope of F0/F vs time t, devided by the transporter/lipid ratio and
averaged over a range of experiments at different ratios (ESI†, Fig. S12); c, Hill
coefficient derived from the activity vs concentrations assays, nd, not determined.
2 | Chem.Commun., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 2016

Page 3 of 4
ChemComm
ChemComm
COMMUNICATION
for the comparison of transport activity while taking kini as
reference. It suggested lipophilicity (increased clogp)
promoted Cl- transporter (3>4,5) because of the enhanced
distribution in lipids;18 however, further increase in
lipophilicity significantly decreased the transport activity
View Article Online
DOI: 10.1039/C6CC07792G
(
1
,
2<3) attributed to the solubility problems (precipitation
appeared in the system),19 despite similar anion affinities for 1-
. As expected, electron-withdrawing substitutions induced
enhanced transport activity, especially for compound with
5
7
very low EC50 value at ~0.025 mol%. It is evident that
lipophilicity and anion affinity have profound impacts on the
transport abilities of these compounds, as also illustrated by
the chloride selective electrode assay (ESI†, Fig. S13-15).
The transmembrane transport in Fig. 1 is a passive process,
-
with charge balance across the membrane by a Cl-/NO3
antiport process or a Na+/Cl- symport process. To test which of
-
these process is predominant, lucigenin assay with NO3
2-
replaced by SO42-, a significantly hydrophilic anion (ΔGh(SO4
)
= -1080 kJ mol-1) that cannot pass through the lipid bilayer
membrane, was explored. As implied in Fig. 2a, taking
compound
7 as example, the fluorescence decay was
negligible under this condition, indicating that the inward flow
of charge couldn’t be balanced and chloride transport stopped
by the developing electrical potential. Same result was
monitored using a chloride selective electrode (Fig. 2b). All
-
these suggested the Cl-/NO3 antiport, not Na+/Cl- symport,
mechanism, in which compound
7 acted as antiporter to
exchange chloride with intravesicular nitrate.
Since HCO3- is the another dominant anions existed in living
-
systems as Cl-, the selectivity between Cl-/HCO3 was explored
-
by using HCO3 as the background anion. Chloride transport
was also monitored by using both a lucigenin fluorescence Figure 3 Effect of amount of cholesterol in EYPC/cholesterol membrane on transport by
a) compound 7 at 0.125 mol% and b-c) compound 3 at different concentrations (0.125
mol% for b and 2.0 mol% for c).
assay and a chloride selective electrode. As shown in Fig. 2a-b,
the transport by 7 was almost negligible, implying that the
-
membrane is impermeable to HCO3 . Combining the bad and
-
almost no binding interactions for receptor- HCO3 , the high
-
selectivity for Cl- vs HCO3 should be resulted from the specific
CH···Cl- interactions and was quite different as those
transporters by traditional NH H-bonding interactions.13
Finally, the transport mechanism of the compounds, by
either a mobile carrier or a channel formation was explored.
Addition of cholesterol, to increase bilayer viscosity, is one
method to test mobile carrier activity, since the increase of
cholesterol in a membrane decreases the fluidity and thereby
hampers the movement of carriers; in contrast, channels
should be unaffected.13 Therefore, the lucigenin assay (Cl-
-
/NO3 exchange) was applied to
7 using vesicles prepared with
different levels of cholesterol. As illustrated in Fig. 3a, the
results clearly indicated a significant reduction in the transport
activity of
more cholesterol, suggesting a carrier mechanism was likely
for chloride transport. Further evidence for carrier
mechanism was obtained through a classic U-tube experiment
on compound (ESI , Fig. S16). The increased chloride
concentration in the receiving phase indicated that compound
acted as mobile carriers for Cl- transport.
7 when the vesicle bilayer composition included
a
7
†
Fig. 2 Exchange of chloride for different anions by 7: a) Lucigenin vesicle fluorescence
assay at 0.25 mol%, b) Chloride efflux determined by Cl- selective electrode assay at
0.50 mol%.
7
This journal is © The Royal Society of Chemistry 2016
Chem.Commun., 2013, 00, 1-3 | 3

ChemComm
Page 4 of 4
COMMUNICATION
ChemComm
6
7
8
S. L. Alper, R. B. Darman, M. N. Chernova and VNie.wKA.rtiDcleaOhnl,linJe.
Nephrol., 2002, 15, 41-53.
Unlike compound
7, it was noticed that most of other
P. M. Q. MallaReddy M Reddy, JOP. J. DPaOnI:c1r0e.1a0s3,92/0C06C1,C07792G
2
, 212-
compounds with moderate transport ability exhibited different
dynamic decay processes. Taking compound as example
(ESI , Fig. S6), the fluorescence was gradually reduce after
3
218.
†
(a) J. T. Davis, Top. Heterocycl. Chem., 2010, 24, 145-176; (b)
R. I. S. Diaz, J. Regourd, P. V. Santacroce, J. T. Davis, D. L.
Jakeman and A. Thompson, Chem. Commun., 2007, 2701-
2703; (c) J. L. Sessler, L. R. Eller, W.-S. Cho, S. Nicolaou, A.
Aguilar, J. T. Lee, V. M. Lynch and D. J. Magda, Angew. Chem.,
Int. Ed., 2005, 44, 5989-5992.
addition of transporter at low concentrations, however, the
fluorescence appeared a rapid drop followed by gradually
decrease when the concentration was up to 1.25 mol%. And
the drop was enhanced with concentrations. All these guided
us to suggest that different mechanism maybe appear for
9
(a) P. A. Gale, C. C. Tong, C. J. E. Haynes, O. Adeosun, D. E.
Gross, E. Karnas, E. M. Sedenberg, R. Quesada and J. L.
Sessler, J. Am. Chem. Soc., 2010, 132, 3240-3241; (b) C. C.
Tong, R. Quesada, J. L. Sessler and P. A. Gale, Chem.
Commun., 2008, 6321-6323.
compound
3 at high concentrations. To confirm the hypothesis,
both low (0.125 mol%) and high concentrations (2.0 mol%) of
3
were applied for variant cholesterol assay. As illustrated in Fig.
3b, the transport showed typical carrier mechanism at 0.125
mol% by the obviously decreased activity with higher ratio of
10 (a) H. Valkenier, L. W. Judd, H. Li, S. Hussain, D. N. Sheppard
and A. P. Davis, J. Am. Chem. Soc., 2014, 136, 12507-12512;
(b) D. S. Kim and J. L. Sessler, Chem. Soc. Rev., 2015, 44, 532-
546; (c) H. Valkenier and A. P. Davis, Acc. Chem. Res., 2013,
46, 2898-2909.
11 (a) P. A. Gale, R. Pérez-Tomás and R. Quesada, Acc. Chem.
Res., 2013, 46, 2801-2813; (b) V. Soto-Cerrato, P. Manuel-
Manresa, E. Hernando, S. Calabuig-Fariñas, A. Martínez-
Romero, V. Fernández-Dueñas, K. Sahlholm, T. Knöpfel, M.
García-Valverde, A. M. Rodilla, E. Jantus-Lewintre, R. Farràs,
F. Ciruela, R. Pérez-Tomás and R. Quesada, J. Am. Chem. Soc.,
2015, 137, 15892-15898.
cholesterol, coinciding with the U-tube experiment (ESI†, Fig.
S16). However, at high concentration of 2.0 mol% as shown in
Fig. 3c, the drop value kept constant despite the change of
cholesterol content, which guided us to speculate the channel
contribution by the stacking of compound
Inferential evidence for channel stacking in membrane was
supported by the self-assembly of compound in CHCl3 in the
presence of anions (ESI , Fig. S17).
3
in membrane.20
3
†
In conclusion, we have demonstrated that aryl-triazole
based oligomers can mediate the transmembrane transport by
anion recognition. The distinctive soft CH···Cl- interactions
12 (a) M. Lisbjerg, H. Valkenier, B. M. Jessen, H. Al-Kerdi, A. P.
Davis and M. Pittelkow, J. Am. Chem. Soc., 2015, 137, 4948-
4951; (b) P. V. Santacroce, O. A. Okunola, P. Y. Zavalij and J. T.
Davis, Chem. Commun., 2006, 3246-3248.
exhibited excellent Cl-/HCO3 selectivity and the adjustment of
-
13 (a) A. V. Koulou , T. N. Lambert, R. Shukla, M. Jain, J. M. Boon,
B. D. Smith, H. Y. Li, D. N. Sheppard, J. B. Joos, J. P. Clare, A. P.
Davis, Angew. Chem. Int. Ed., 2003, 42, 4931-4933; (b) S.
Hussain, P. R. Brotherhood, L. W. Judd, A. P. Davis, J. Am.
Chem. Soc., 2011, 133, 1614-1617; (c) S. J. Moore, C. J. E.
Haynes, J. Gonzalez, J. L. Sutton, S. J. Brooks, M. E. Light, J.
Herniman, G. J. Langley, V. Soto-Cerrato, R. Perez-Tomas, I.
lipophilicity and affinity optimized an efficient activity at very
low transporter/lipid molar ratio (EC50 of
7 at ~0.025 mol%).
With the facile click-based synthesis, we believe the
oligotriazoles provide new opportunities for developing more
potent anion transporters with high activity and selectivity.
This work was supported by NSFC (21472044, 21425311,
and 21373084) and The Program for Professor of Special
Appointment (Eastern Scholar) at Shanghai Institutions of
Higher Learning.
Marques, P. J. Costa, V. Felix, P. A. Gale, Chem. Sci., 2013, 4,
103-117; d) N. J. Andrews, C. J. E. Haynes, M. E. Light, S. J.
Moore, C. C. Tong, J. T. Davis, W. A. Harrell Jr, P. A. Gale,
Chem. Sci., 2011, 2, 256-260.
14 (a) H. Juwarker, J. M. Lenhardt, D. M. Pham and S. L. Craig,
Angew. Chem., Int. Ed., 2008, 47, 3740-3743; (b) R. M.
Meudtner and S. Hecht, Angew. Chem., Int. Ed., 2008, 47
4926-4930.
15 (a) Y. Li and A. H. Flood, Angew. Chem. Int. Ed., 2008, 47
,
Notes and references
1
(a) S. Y. Chiu and G. F. Wilson, J. Physiol., 1989, 408, 199-222;
(b) T. E. DeCoursey, K. G. Chandy, S. Gupta and M. D. Cahalan,
Nature, 1984, 307, 465-468; (c) B. Legutko, M. Staufenbiel
and K. Krieglstein, Int. J. Dev. Neurosci., 1998, 16, 347-352.
F. M. Ashcroft, Ion Channels and Disease, Academic Press,
San Diego, 2000.
,
2649-2652; (b) Y. Hua, Y. Liu, C.-H. Chen and A. H. Flood, J.
Am. Chem. Soc., 2013, 135, 14401-14412; (c) Y. Li and A. H.
Flood, J. Am. Chem. Soc., 2008, 130, 12111-12122.
2
3
16 (a) A. P. Davis, D. N. Sheppard and B. D. Smith, Chem. Soc.
Rev., 2007, 36, 348-357; (b) P. A. Gale, Acc. Chem. Res., 2011,
44, 216-226.
(a) A. Fürstner, Angew. Chem., Int. Ed., 2003, 42, 3582-3603;
(b) C. A. Hübner and T. J. Jentsch, Hum. Mol. Genet., 2002, 11
,
17 J. Cai and J. L. Sessler, Chem. Soc. Rev., 2014, 43, 6198-6213.
18 (a) C. J. E. Haynes, N. Busschaert, I. L. Kirby, J. Herniman, M. E.
Light, N. J. Wells, I. Marques, V. Felix and P. A. Gale, Org.
Biomol. Chem., 2014, 12, 62-72; (b) V. Saggiomo, S. Otto, I.
Marques, V. Felix, T. Torroba and R. Quesada, Chem.
Commun., 2012, 48, 5274-5276.
2435-2445; (c) B. Dworakowska and K. Dołowy, Acta Biochim.
Pol., 2000, 47,685–703.
(a) J. T. Davis, O. Okunola and R. Quesada, Chem. Soc. Rev.,
2010, 39, 3843-3862; (b) S.-K. Ko, S. K. Kim, A. Share, V. M.
Lynch, J. Park, W. Namkung, W. Van Rossom, N. Busschaert,
4
P. A. Gale, J. L. Sessler and I. Shin, Nat. Chem., 2014,
892; (c) G. W. Gokel and S. Negin, Acc. Chem. Res., 2013, 46
6, 885-
19 N. Busschaert, M. Wenzel, M. E. Light, P. Iglesias-Hernandez,
,
R. Perez-Tomas and P. A. Gale, J. Am. Chem. Soc., 2011, 133
14136-14148.
,
2824-2833; (d) P. R. Brotherhood and A. P. Davis, Chem. Soc.
Rev., 2010, 39, 3633-3647; (e) H. Li, H. Valkenier, L. W. Judd,
P. R. Brotherhood, S. Hussain, J. A. Cooper, O. Jurček, H. A.
20 (a) C. R. Yamnitz, S. Negin, I. A. Carasel, R. K. Winter and G.
W. Gokel, Chem. Commun., 2010, 46, 2838-2840; (b) T. Liu, C.
Bao, H. Wang, Y. Lin, H. Jia and L. Zhu, Chem. Commun., 2013,
49, 10311-10313.
Sparkes, D. N. Sheppard and A. P. Davis, Nat. Chem., 2016, 8,
24-32.
5
R. Dutzler, E. B. Campbell, M. Cadene, B. T. Chait and R.
MacKinnon, Nature, 2002, 415, 287-294.
4 | Chem.Commun., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 2016