Curvature-regulated transmembrane anion transport by a trifluoromethylated bisbenzimidazole

Article abstract of DOI:10.1016/j.cclet.2021.01.005

In this paper, we demonstrate that modification of anion-transport active 1,3-bis(benzimidazol-2-yl)benzene with strongly electron-withdrawing trifluoromethyl and nitro groups leads to a dramatic increase in the anionophoric activity, and the activity may

Full text of DOI:10.1016/j.cclet.2021.01.005

Chinese Chemical Letters 32 (2021) 1653–1656
Contents lists available at ScienceDirect
Chinese Chemical Letters
journal homepage: www.elsevier.com/locate/cclet
Communication
Curvature-regulated transmembrane anion transport by a
trifluoromethylated bisbenzimidazole
b
a
a
c
Xiao-Qiao Hong , Yuan-Yuan Xing , Zhong-Kun Wang , Qin-Chao Mao ,
a,d,
Wen-Hua Chen
a
*
School of Biotechnology and Health Sciences, Wuyi University, Jiangmen 529020, China
School of Pharmaceutical Sciences, Tsinghua University, Beijing 100084, China
Guangdong Maoming Health Vocational College, Maoming 525000, China
State Key Laboratory of Chemical Oncogenomics, Key Laboratory of Chemical Genomics, Peking University Shenzhen Graduate School, Shenzhen 518055, China
b
c
d
A R T I C L E I N F O
A B S T R A C T
Article history:
In this paper, we demonstrate that modification of anion-transport active 1,3-bis(benzimidazol-2-yl)
benzene with strongly electron-withdrawing trifluoromethyl and nitro groups leads to a dramatic
increase in the anionophoric activity, and the activity may be greatly regulated by the curvatures of the
liposomes used.
Received 25 November 2020
Received in revised form 18 December 2020
Accepted 4 January 2021
Available online 9 January 2021
©
2021 Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences.
Published by Elsevier B.V. All rights reserved.
Keywords:
Anion transporter
Lysosomal curvature
Anionophoric activity
During the past years, an increasing interest has been attracted
in identifying small-molecule organic compounds that are capable
of facilitating the transport of anions, in particular chloride anions
across phospholipid membranes [1–5]. Literature reports indicate
that such compounds, namely anion transporters are able to
disrupt the homeostasis of cellular anions [6] and as a conse-
quence, they may be developed as a novel class of chemothera-
peutic agents for cancers [7].
Because cellular membranes are rich in complexity and it has
been reported that the bioactivity of anion transporters correlates
with their activity on synthetic cellular models [8], many of the
studies to date have used phospholipid vesicles of varying sizes as
models of biological membranes for investigating anion transport
processes. Transport of anions across phospholipid membranes is
accomplished through the interaction of anion transporters with
the membranes. This is a complex process, in particular for a
mobile carrier, including the recognition of anions by anion
transporters to form supramolecular complexes, diffuse within
the membranes and release the anions on the opposite side of the
membranes [9]. As a consequence, anion transport efficiency may
be affected by many parameters, such as phospholipid compo-
nents and packing. For example, Gale et al. have investigated the
anion transport properties of a range of alkyl-substituted phenyl-
thioureas on vesicles formed from different types of phospholipids
and found that changes in the compositions of lipid bilayers
affected the rate of anion transport for all compounds in the series
[10]. In an earlier study, Regen et al. have reported that the
curvature of lipid bilayers affects the lipid packing and mixing [11].
Specifically, they have found that in cholesterol-poor membranes,
where lipid interactions are relatively weak, curvature has little
influence on sterol–phospholipid association. In sharp contrast,
increased curvature significantly reduces the preference of sterols
and high-melting-point phospholipids to become nearest neigh-
bors in cholesterol-rich membranes, where lipid interactions are
stronger.
These findings have raised one fundamental and crucial
concern about how the anionophoric activity of an anion transport
is affected by vesicular curvature. This issue is related to the
assessment of the transport efficiency of anion transport systems
on liposomal membranes of varying curvatures. To clarify this, we
prepared one derivative of anion-transport active 1,3-bis(benzi-
midazol-2-yl)benzene (m-Bimbe,1, Fig.1), that is compound 2, and
investigated the effect of vesicular curvatures on the anion
transport efficiency. Compound 2 bears two trifluoromethyl
groups on each benzimidazolyl subunit and one nitro group at
the central phenyl subunit. This design was rationalized by our
previous findings that introducing strongly electron-withdrawing
groups into the benzimidazolyl and/or central phenyl subunits of
*
Corresponding author at: School of Biotechnology and Health Sciences, Wuyi
University, Jiangmen 529020, China.
E-mail address: whchen@wyu.edu.cn (W.-H. Chen).
https://doi.org/10.1016/j.cclet.2021.01.005
1001-8417/© 2021 Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences. Published by Elsevier B.V. All rights reserved.

X.-Q. Hong, Y.-Y. Xing, Z.-K. Wang et al.
Chinese Chemical Letters 32 (2021) 1653–1656
As anion recognition is the initialstepforaniontransport[17], we
first studied the complexation of compound 2 with chloride anions
as a tetra(n-butyl)ammonium salt (TBACl), by means of ESI MS. As
3
shown in Fig. 2a, mixing of compound 2 with TBACl in CH CN led to
the presence of a new ion peak at m/z 662.63 as well as the ion peak
at m/z 626.68 fromcompound2 itself. This newpeakis assignable to
a 1:1 complexof compound 2 with chloride anions. The binding site
1
and affinity were established by means of H NMR titrations
Fig. 1. m-Bimbe 1 and its trifluoromethylated derivative 2.
(Fig. 2b). AdditionofTBACltoa solutionofcompound2inCD
3
CNled
to large down-field shifts of the C H, indicating that chloride anions
2
are complexed to the 1,3-bis(benzimidazol-2-yl)benzene core.
Non-linearfitting analysis of thesecomplexation-inducedchemical
shifts against the concentrations of TBACl gave the association
a
constant (K ) of compound 2 with chloride anions being
4
(
4
1.45 Æ 0.33) Â 10 L/mol (Fig. S5 in Supporting information), about
-fold greater than that of compound 1 with chloride anions
3
[
K
a
= (3.51 Æ0.09) Â 10 L/mol] under the similar conditions [15].
The anion transport activity of compound 2 was assessed on
liposomal models using a conventional pH discharge assay based
on sodium 8-hydroxypyrene-1,3,6-trisulfonate (HPTS, pK 7.2)
14,15]. In these experiments, vesicles (100 nm, extrusion)
encapsulated with an internal aqueous phase of pH of 7.0 and
HPTS as a reporter for the intravesicular pH change, were prepared
a
[
Scheme 1. Synthesis of compound 2.
m-Bimbe 1 leads to a significant increase in the anion transport
efficiency [12–15].
from egg-yolk L-α-phosphatidylcholine (EYPC) and suspended in
an external aqueous phase of pH of 8.0. As shown in Fig. 3a and
Fig. S6 (Supporting information), addition of compound 2, even at
very low concentrations, led to an increase in the fluorescent
intensity of HPTS, indicating that this compound is very active in
discharging the pH gradient between the exterior and interior of
the EYPC vesicles. The concentration dependent assays gave the
50% efficiency concentration (EC50) value that is defined as the
Compound 2 was prepared from the condensation of 5-nitro-
1,3-benzenedialdehyde [16] with 3,5-bis(trifluoromethyl)-1,2-
benzenediamine (Scheme 1), and fully characterized on the basis
of electrospray ionization mass spectrometry (ESI MS) [low
resolution (LR) and high resolution (HR)] and nuclear magnetic
1
13
resonance (NMR, H and C) data (Supporting information).
À3
CN; (b) 1H NMR titration of compound 2 (1.0 Â 10À3 mol/L) with
Fig. 2. (a) ESI MS spectrum of compound 2 (1.0 Â 10 mol/L) mixed with 50 equivalences of TBACl in CH
3
3
TBACl of varying equivalences in CD CN.
Fig. 3. (a) Typical plots of the relative fluorescent intensity (FI, ex/em 460/510 nm) of HPTS against time in the presence of compound 2 of varying concentrations in EYPC-
based liposomes (100 nm), under the conditions of intravesicular 0.1 mmol/L HPTS in 25 mmol/L HEPES buffer (50 mmol/L NaCl, pH 7.0) and extravesicular 25 mmol/L HEPES
buffer (50 mmol/L NaCl, pH 8.0). (b) Relative efflux of chloride anions out of EYPC liposomes (100 nm) enhanced by compound 2 of varying concentrations, under the
3
conditions of intravesicular 500 mmol/L NaCl in 25 mmol/L HEPES buffer (pH 7.0) and extravesicular 500 mmol/L NaNO in 25 mmol/L HEPES buffer (pH 7.0).
1654

X.-Q. Hong, Y.-Y. Xing, Z.-K. Wang et al.
Chinese Chemical Letters 32 (2021) 1653–1656
Fig. 4. (a) Relative efflux of chloride anions out of EYPC liposomes (100 nm), enhanced by compound 2 (0.1 mol%), under the conditions of intravesicular 500 mmol/L NaCl in
5 mmol/L HEPES buffer (pH 7.0) and extravesicular 500 mmol/L NaNO , 500 mmol/L NaHCO or 250 mmol/L Na SO in 25 mmol/L HEPES buffer (pH 7.0). (b) Typical U-tube
experiments in the presence of compound 2 (1.0 mmol/L) in nitrobenzene containing 2 mmol/L TBAPF . Measuring conditions: U-tube chloride-receiving phase, 500 mmol/L
NaNO , 25 mmol/L HEPES buffer (pH 7.0); and U-tube chloride-donating phase, 500 mmol/L NaCl, 25 mmol/L HEPES buffer (pH 7.0).
2
3
3
2
4
6
3
effective transporter loading in molar percentage (mol%) of
2, we measured the efflux of chloride anions in the presence of
different alkali metal cations or anions. As shown in Figs. 4a and
Fig. S7b (Supporting information), the efflux rate of chloride anions
was regulated not by the alkali metal cations but by the anions,
which suggests that compound 2 mediates the transport of
chloride anions in an anion exchange fashion [22,23]. U-tube
experiments (Fig. 4b) suggest that compound 2 functions as a
mobile carrier [24], like a “ferryboat” diffusing back and forth
within the interior of the membranes [25].
compound 2 to lipid when 50% of the maximum rate is reached,
À3
being (2.76 Æ 0.36) Â 10 mol% (or 4.86 nmol/L). Under the same
conditions, this EC50 value was 11.08 Æ 1.36 mol% (or 19.5
mmol/L)
3
for compound 1. These results represent 4.0 Â 10 -fold enhance-
ment in the transport efficiency upon the modification of
compound 1 with trifluoromethyl and nitro groups (to give
compound 2). Calcein leakage assay confirmed that the observed
powerful activity of compound 2 was authentic rather than due to
the disruption of the EYPC liposomal membranes (Fig. S7a in
Supporting information) [18].
To probe the effect of liposomal curvature on the anion transport
efficiencyofcompound 2, we measured the effluxof chloride anions
out of the EYPC vesicles with average liposome diameters from
51 nmto603 nm(Fig.S8andTableS1inSupportinginformation),by
means of chloride ion selective electrode technique. These
liposomes were prepared by extrusion methods. As shown in
Fig. 5a, the vesicular curvature has an obvious effect on the chloride
efflux rate. To quantitatively characterize this effect, we measured
the EC50 values of compound 2 on EYPC liposomes of those
diameters (Figs. S9–S13 in Supporting information and Table 1).
As a result, the EC50 values decrease with the decrease in the
liposomal sizes, that is, increased curvature (i.e., liposomes of
smaller diameters) led to an increase in the activity of compound 2.
This effect becomes more evident when the diameters of the
liposomes are reduced to ca 140 nm and below (Fig. 5b and Table 1).
Studies to date have indicated that the anion binding affinity
and lipophilicity of an anion transporter are two key parameters
that play a critical role in the anion transport process [19]. The
above-mentioned finding that compound 2 exhibits ca. 4-fold
greater anion binding affinity toward chloride anions than
compound 1, may rule out the possibility that the remarkably
enhanced anionophoric activity of compound 2 was mainly due to
the increase in the anion-binding affinity. As compound 2 (clog
P = 8.05) is much more lipophilic than compound 1 (clog P = 4.60),
one may consider that the increased lipophilicity of compound 2
induced by the four trifluoromethyl substituents, is mainly
responsible for the increase in the anion transport efficiency
[
20,21].
The anion-selective transport ability of compound 2 was
2
Specifically, the activity increases by ca.10 -fold when the vesicular
confirmed by use of chloride selective electrode technique. As
shown in Fig. 3b, addition of compound 2 to vesicles containing
intravesicular NaCl and extravesicular isotonic NaNO led to the
3
efflux of chloride anions out of the intravesicular media, indicating
that compound 2 is able to mediate the transport of chloride
anions. To clarify the probable transport mechanism of compound
diameters decrease from 603 nm to 51 nm. This effect is considered
due to the less efficient packing of the lipids as a result of increased
curvature [11], which may make compound 2 more ready to be
inserted into the membranes to function as an anion carrier.
As high anion transport efficiency was observed on the EYPC
liposomes that are comparable to cancer cells in sizes [26], we
Fig. 5. (a) Relative efflux of chloride anions out of EYPC liposomes of varying sizes enhanced by compound 2 (1 mol%), under the conditions of intravesicular 500 mmol/L NaCl
in 25 mmol/L HEPES buffer (pH 7.0) and extravesicular 500 mmol/L NaNO in 25 mmol/L HEPES buffer (pH 7.0). The experiments conducted on EYPC liposomes of 100 nm with
DMSO were used as a control. (b) Plot of the relative transport efficiency against the average liposome diameters.
3
1655

X.-Q. Hong, Y.-Y. Xing, Z.-K. Wang et al.
Chinese Chemical Letters 32 (2021) 1653–1656
Table 1
Declaration of competing interest
Chloride transport efficiency (EC50, mol%) of compound 2 on EYPC liposomes of
different sizes.a
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
Liposomal size (nm)
Chloride transport efficiency
EC50 (mol%)
RA b
À3
5
1.2 Æ 4.2
(9.71 Æ 1.08) Â 10
95.8
37.8
18.8
2.8
À2
Acknowledgments
9
7.4 Æ 3.8
(2.46 Æ 0.25) Â 10
À2
142.9 Æ 3.6
(4.95 Æ 1.17) Â 10
271.8 Æ 6.6
0.33 Æ 0.08
0.93 Æ 0.38
Financial support from the National Natural Science Foundation of
China (No. 21877057) and Jiangmen Program for Innovative Research
Team, China (No. 2018630100180019806) is acknowledged.
6
02.5 Æ 21.3
1.0
a
Measured under the conditions of intravesicular 500 mmol/L NaCl in 25 mmol/L
3
HEPES buffer (pH 7.0) and extravesicular 500 mmol/L NaNO in 25 mmol/L HEPES
buffer (pH 7.0).
Appendix A. Supplementary data
b
RA denotes the chloride transport efficiency relative to that on vesicles with the
diameter of 602.5 nm.
Supplementarymaterialrelatedtothisarticlecanbefound, inthe
online version, at doi:https://doi.org/10.1016/j.cclet.2021.01.005.
hypothesized that compound 2 may exhibit equally potent
anionophoric activity in vitro and disrupt the homeostasis of
anions in cancer cells, leading to cell death. To address this issue,
we firstly measured the capability of compound 2 to facilitate the
influx of chloride anions into HeLa cervical and A549 lung
adenocarcinoma cancer cell lines, using a fluorescent assay
based on a chloride anion sensitive fluorescent dye, N-(ethox-
ycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE) [14].
As shown in Fig. S14 (Supporting information), addition of
compound 2 to HeLa and A549 cell lines led to a significant
decrease in the fluorescence of MQAE, revealing the strong influx of
chloride anions into the cells. MTTassays indicate that compound 2
exhibits potent cytotoxicity with the 50% inhibition concentration
References
[1] J.T. Davis, P.A. Gale, R. Quesada, Chem. Soc. Rev. 49 (2020) 6056–6086.
[2] X.H. Yu, X.Q. Hong, Q.C. Mao, W.H. Chen, Eur. J. Med. Chem. 184 (2019) 111782.
[3] P.A. Gale, J.T. Davis, R. Quesada, Chem. Soc. Rev. 46 (2017) 2497–2519.
[4] N. Busschaert, A.P. Gale, Angew. Chem. Int. Ed. 52 (2013) 1374–1382.
[5] S. Zhang, I. Boussouar, H. Li, Chin. Chem. Lett. 32 (2021) 642–648.
[6] N. Busschaert, S.H. Park, K.H. Baek, et al., Nat. Chem. 9 (2017) 667–675.
[7] I. Alfonso, R. Quesada, Chem. Sci. 4 (2013) 3009–3019.
[8] J.L. Sessler, L.R. Eller, W.S. Cho, et al., Angew. Chem. Int. Ed. 44 (2005) 5989–5992.
[9] M. Mehiri,W.H. Chen, V. Janout, S.L. Regen, J. Am. Chem. Soc.131 (2009)1338–1339.
[10] M.J. Spooner, P.A. Gale, Chem. Commun. 51 (2015) 4883–4886.
[11] H. Mitomo, W.H. Chen, S.L. Regen, Langmuir 25 (2009) 4328–4330.
[
12] C.C. Peng, Z. Li, L.Q. Deng, Z.F. Ke, W.H. Chen, Bioorg. Med. Chem. Lett. 26 (2016)
442–2445.
13] C.C. Peng, M.J. Zhang, X.X. Sun, et al., Org. Biomol. Chem. 14 (2016) 8232–8236.
2
(
1
IC50) values being 9.67 Æ 1.80
mmol/L toward HeLa cells and
mol/L toward A549 cells, respectively (Fig. S15 in
[
0.48 Æ 1.20
m
[14] X.H. Yu, C.C. Peng, X.X. Sun, W.H. Chen, Eur. J. Med. Chem. 152 (2018) 115–125.
[15] X.H. Yu, X.Q. Hong, W.H. Chen, Org. Biomol. Chem. 17 (2019) 1558–1571.
Supporting information).
[16] A. Company, L. Gomez, R. Mas-Balleste, et al., Inorg. Chem. 46 (2007) 4997–
In conclusion, we have shown that modification of 1,3-bis
5
012.
(
benzimidazol-2-yl)benzene with strongly electron-withdrawing
[17] H. Valkenier, C.M. Dias, K.L.P. Goff, et al., Chem. Commun. 51 (2015) 14235–
trifluoromethyl and nitro groups leads to a dramatic increase in the
anionophoric activity and the activity is highly dependent on the
curvature of the liposomes used, generally on liposomes of smaller
sizes exhibiting better transport efficiency. The present finding
suggests that particular cautions should be taken when one
evaluates the transport efficiency of different anion transporters
on liposomal membranes of varying curvatures. The potent
anionophoric activity and in vitro cytotoxicity suggests that this
compound is exploitable as an apoptotic agent, in particular for the
cancer cells at early stages.
14238.
[
[
[
18] S.J. Moore, M. Wenzel, M.E. Light, et al., Chem. Sci. 3 (2012) 2501–2509.
19] V. Saggiomo, S. Otto, I. Marques, et al., Chem. Commun. 48 (2012) 5274–5276.
20] Z. Li, Y. Chen, D.Q. Yuan, W.H. Chen, Org. Biomol. Chem. 15 (2017) 2831–2840.
[21] H. Valkenier, C.J.E. Haynes, J. Herniman, P.A. Gale, A.P. Davis, Chem. Sci. 5 (2014)
128–1134.
1
[
[
22] P.A. Gale, C.C. Tong, C.J.E. Haynes, et al., J. Am. Chem. Soc. 132 (2010) 3240–3241.
23] Z. Li, X.H. Yu, Y. Chen, D.Q. Yuan, W.H. Chen, J. Org. Chem. 82 (2017)13368–13375.
[24] T. Saha, M.S. Hossain, D. Saha, M. Lahiri, P. Talukdar, J. Am. Chem. Soc. 138
2016) 7558–7567.
(
[
[
25] G.W. Gokel, A. Mukhopadhyay, Chem. Soc. Rev. 30 (2001) 274–286.
26] J. Zhou, A. Kulasinghe, A. Bogseth, et al., Microsyst. Nanoeng. 5 (2019) 8.
1656