Bis(sulfonamide) transmembrane carriers allow pH-gated inversion of ion selectivity

Article abstract of DOI:10.1039/C7CC00165G

Bis(sulfonamide) based synthetic carriers are reported for inversion of ion selectivity upon deviation of pH within a narrow window. A liposomal membrane potential is also generated when potassium ions are passively transported by these carriers.

Full text of DOI:10.1039/C7CC00165G

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DOI: 10.1039/C7CC00165G
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Bis(sulfonamide) transmembrane carrier allows pH-gated
inversion of ion selectivity
Arundhati Roy, Oindrila Biswas and Pinaki Talukdar*
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
Bis(sulfonamide) based synthetic carriers are reported for the intracellular pH is alkaline.8 On the other hand, natural ion
inversion of ion selectivity upon deviation of pH within a narrow channel such as valinomycin is highly selective towards K+ and
window. A liposomal membrane potential is also generated when consequently generates membrane potential. However, most of the
potassium ion is passively transported by these carriers.
synthetic transporters cannot generate membrane potential due to
lack of selectivity. Therefore, synthetic molecules capable of
perturbing both cation and anion homeostasis in this deviated pH
range and having potential ability to generate membrane potential,
can be applied to improve the combination treatment modality in
addition to the conventional proapoptotic chemotherapy and
radiotherapy.
The bilayer lipid membranes of a cell act as a barrier and protect the
cell from its external environment. They are impermeable to ions
found in the extracellular and intracellular fluids, such as Na+, K+,
Ca2+, Cl‒, etc. Therefore, nature has developed many sophisticated
membrane proteins which facilitate the transport of ions across
membranes. This process is very crucial for maintaining the ionic
and pH homeostasis of cells.1 The ion and pH homeostasis are
fundamental regulators of cellular processes, such as cell volume
regulation, energy transduction, sensory perception, cell adhesion
and movement, as well as, cell signaling. Dysregulated transport of
these ions, occasionally caused by ion channel mutation, are
associated with various “channelopathies”.2
During the past two decades, synthetic systems capable of
mimicking the functions3 of natural ion transporters were focused
extensively to understand the fundamental knowledge of ion
transport mechanism. Subsequently, these synthetic ion transporters
were applied as antibacterial, anticancer, and antiviral agents.4 For
example, cation selective cyclic peptides, reported by Ghadiri and
coworkers, act preferentially on bacterial membranes and these
peptide based tube can collapse transmembrane ion potentials and
cause rapid cell death.5 Recently, few small molecule cation
transport systems with potential antitumor activities have been
reported.6 Compelling evidence is also available which indicates the
regulatory role of K+ and Na+ during both the initial signaling and
the execution phase of apoptosis.7 Interestingly, the extracellular pH
around the tumor microenvironment is typically very acidic while
Inversion of the ion transport selectivity as a result of pH change
can be envisaged as an alternate approach for the transporter
development. However, such approach has been less efficient till
date because the inversion of selectivity is less pronounced within
the pH range narrowly deviated from the physiological value. For
example, the dimeric alamethicin analog with lysine at position 18 in
the sequence (alm-K18) system was shown to form stable anion-
selective channels in membranes at pH 7.0 while cation selectivity
was prominent only at pH > 11.9 Kobuke and co-workers have
reported tetracyanoresorcin[4]arene derivative as a potential K+
channel at higher pH due to deprotonation of hydroxyl groups on the
resorcinarenes.10 Later on, Gin and co-workers have reported a pH-
sensitive aminocyclodextrin based ion channel, which shows faster
anion and cation transport rate at higher pH.11 Therefore, efficient
protonation and deprotonation by marginally altering the pH from
the physiological value can allow a synthetic system to inverse its
selectivity towards either cation or anion. Herein, we report a class
of bis(sulfonamides) as transmembrane ion carrier which displays
Cl selectivity at pH 5.5, while K+ selectivity at pH 7.0.
Sulfonamides are an important class of compounds, which can act
as receptors for anions,12 as well as cations.13 Therefore, the acidity
(i.e. pKa) of the N−H bond can be considered as one of the dictating
factors behind its binding mode. An optimum pKa value can favor
a Department of Chemistry, Indian Institute of Science Education and Research
Pune, Dr. Homi Bhabha Road, Pashan, Pune 411008, Maharashtra, India.
Email: ptalukdar@iiserpune.ac.in
†
We gratefully acknowledge SERB, Govt. of India (EMR/2014/000873B) for
funding our work. DPC thanks CSIR, Govt. of India for research fellowship.
‡ Electronic Supplementary Information (ESI) available: Synthetic procedures and
experimental methods, additional experimental details and NMR spectra. See
DOI: 10.1039/x0xx00000x
Fig. 1 Structure, estimated pKa and logP values of designed bis(sulfonamides) 1a-1e.
J. Name., 2013, 00, 1-3 | 1
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the neutral state of the sulfonamide at physiological pH, leading to
facilitated anion binding. Further lowering of pKa, based on
structural change, can assist the deprotonation of N−H bond at
physiological pH featuring cation binding. Earlier, we have reported
bis(sulfonamides), formed on the m-xylylenediamine core, which
displayed anionophoric behavior.14 In the present study, we have
introduced o-phenylenediamine as the core to design
bis(sulfonamides) 1a-1e (Fig. 1). Substituents at the para-position of
aryl sulfonyl moieties were varied (R = -CH3, -NO2, -Br, -OCH3 and
-CF3 for 1a-1e respectively) to alter the pKa and logP values.
Estimated pKa1 = 6.85 − 7.39, using the calculator plugins of Marvin
Sketch program,15 indicated an increase in the possibility of
deprotonation upon connecting electron withdrawing R groups and
therefore, signifying better cation recognition. However, the change
of R group also contributed in the predicted logP = 4.01, 3.80, 5.38,
3.57 and 5.83 for 1a-1e, respectively (Fig. 1). According to the
Lipinski’s rule, a logP value close to 5 is crucial to attain improved
membrane permeability, which directly translates into better ion
transport activity.16
View Article Online
DOI: 10.1039/C7CC00165G
Fig.
2
(A) Concentration dependent ion transport activity of 1e with increasing
concentration (0 ‒ 15 µM) and (B) comparison of ion tansport activities of 1a-1e
presented with their normalized emission intensity IF.
gradient in HEPES buffer.19 Variation of NaX salts (X− = F−, Cl−,
Br− and I−) in the extravesicular buffer with iso-osmolar
intravesicular NaCl provided minor differences (i.e. Cl− > F− ≈ Br− ≈
I−) in the ion transport activity of 1e (Fig. 3A). However, the
variation of extravesicular MCl (M+ = Li+, Na+, K+, Rb+ and Cs+)
with iso-osmolar intravesicular NaCl resulted in the transport
activity sequence: K+> Rb+ ≈ Cs+> Na+> Li+ (Fig. 3B) for 1e (2.5
M). The observed data suggested that the ion transport of 1e was
largely influenced by the variation of cations in the extravesicular
buffer. Therefore, preferred cation recognition by 1e, possibly due to
the deprotonation of bis(sulfonamide) N‒H group under the assay
conditions, was predicted. Similarly, 1c also indicated a sharp
influence of cations in transport activity (Fig. S4).
Syntheses of bis(sulfonamide) derivatives 1a-1e were carried out
from o-phenylenediamine 2 following the reported protocol by
Proust et. al.17 The reaction of 2 with aryl sulfonyl chlorides 3a-3e in
dichloromethane, and pyridine as base resulted in the formation of
1a-1e in 78-96% yields (Scheme 1). All compounds were well
1
characterized by H-NMR, 13C-NMR, HRMS, IR and melting point
(see supporting information). Crystal structures of 1c and 1e were
obtained from MeOH/CHCl3 (1:1) solvent system, feature
intramolecular N−H···O hydrogen bond between two arms around
the central aromatic core (Fig. S8-S9).
Ion transport activities of 1a-1e across large unilamellar vesicles
(LUVs) composed of egg-yolk phosphatidylcholine (EYPC) with
entrapped 8-hydroxypyrene-1,3,6-trisulfonate dye (HPTS, 1 mM) in
HEPES buffer (pH = 7.0) were evaluated by applying a pH gradient
∆pH = 0.8 (pHin = 7.0 and pHout = 7.8) (Fig. S1).18 Ion transport
activity of 1a-1e indicated compound 1e as the most efficient ion
transporter (Fig. 2A, S2). These studies also provided the transport
activity order: 1d < 1b < 1a < 1c <1e, based on the EC50 = 40, 43,
14, 211 and 10 μM for 1a-1e, respectively (Fig. 2B, S3). Lowest
activity of 1d explained based on its lowest N−H bond acidity (pKa1
= 7.39) as well membrane permeability (logP = 3.57). Although, the
pKa1 = 6.85 was estimated for 1b, its lower activity compared to 1c
and 1e was corroborated the lower logP = 3.80. Obtained data
indicated that the compounds 1c and 1e having logP value higher
than 5, displayed higher ion transport ability as compared to other
bis(sulfonamides). The higher activity of 1e as compared to 1c, can
be corroborated to higher lipophilicity which is due to the presence
of two –CF3 groups (as fluoride atom increases lipophilicity of
molecule). Based on this outcome, the effect of either extravesicular
cation or anion on the transport activity of 1e across EYPC-LUVs
with entrapped HPTS was determined by applying a similar pH
In the next stage, the carrier induced passive influx of cations
were studied.20 EYPC-LUVs were prepared by trapping NaCl (100
mM) and HPTS (1 mM) in HEPES buffer (pH = 7.0). The
extravesicular salts in the same buffer were varied from NaCl to
KCl. Without applying any initial pH gradient, the addition of 1e (c
= 5 µM) resulted in the steady increase of HPTS fluorescence (Fig.
4A) when the extravesicular buffer contains NaCl. Further
application of the pH gradient provided a sharper increase of the
fluorescence. In the presence of extravesicular KCl, the addition of
1e led to the sudden initial increase influorescence followed by a
slow diminution with time (Fig. 4B). The faster increase in initial
fluorescence clearly corroborates to a very fast influx of K+ resulting
in the generation of transmembrane potential due to the
Scheme 1 Synthesis of bis(sulfonamides) 1a-1e.
2 | J. Name., 2012, 00, 1-3
Fig. 3 (A) Anion selectivity of 1e (10 M) and (B) cation selectivity of 1e (2.5 M)
determined from HPTS assay.
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accumulation of more positive charge inside, and negative charge
outside the bilayer membrane. The creation of transmembrane
potential further induces efflux of H+ synergistically to restore the
electrical balance. Subsequent decrease of fluorescence was
associated with the slow H+ influx to equilibrate pH gradient caused
during K+/H+ antiport. Further addition of NaOH resulted in an
abrupt increase in the internal HPTS fluorescence suggesting a rapid
H+ efflux for equilibrating the pH.
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DOI: 10.1039/C7CC00165G
To obtain the direct proof of membrane polarization as a result of
K+ transport, safranin O (a membrane potential sensitive fluorescent
dye) was used during ion transport studies.20-21 EYPC-LUVs were
prepared with filled KCl (100 mM) in HEPES buffer (pH = 7.0) and
dispersed in isoosmolar NaCl buffer and safranin O (60 nM). K+
efflux, upon addition of 1e (5 µM), resulted in the rise of positive
charge outside bilayer membrane which in turn generated
transmembrane potential. The process led to a sharp increase in the
safranin O fluorescence (at λem = 581 nm with λex = 522 nm),
confirming the generation of membrane potential due to selectivity
towards K+ (Fig. 4C). Fluorescence intensity was normalized by
taking intensity at t = 300 s as maximum point.
Fig. 5 (A) Cation selectivity of 1e determined from HPTS assay and (B) anion selectivity
of 1e determined at different pH without applying base pulse.
The aforestated outcome of cation selectivity of 1e encouraged us
to investigate the effects of pH on its ion selectivity. LUVs were
prepared with entrapped HPTS dye. In the extravesicular MX salt,
either anions (X− = F−, Cl−, Br− and I−) or cations (M+ = Li+, Na+,
K+, Rb+ and Cs+) were varied with iso-osmolar intravesicular NaCl.
For each experiment, the intravesicular and extravesicular pHs were
kept same (i.e. either 5.5 or 7.0 or 8.0). Cation selectivity (mostly
towards K+) was observed when compound 1e (7.5 M) was added
to the cuvette containing LUVs at pH = 7.0 and 8.0 (Fig. 5A).
However, no significant anion selectivity was observed at these pHs.
When same experiments were carried out at pH = 5.5, significant
anion selectivity (mostly towards Cl) was observed, and selectivity
among cations became insignificant (Fig. 5B). All these results
suggest that 1e can bind to a cation at pH ≥ 7.0, and at acidic pH, i.e.
pH = 5.5, the selectivity of the system gets reversed towards anions.
To establish further the pH dependent ion selectivity of 1e,
chloride efflux across bilayer lipid membranes were studied at
different pHs by chloride selective electrodes (ISEs).22 Chloride ion
efflux by 1e (20 µM) across EYPC-LUVs at different pH (i.e.
intravesicular and extravesicular pH = 5.8, 7.0 and 8.0) with
intravesicular NaCl (500 mM), and iso-osmolar extravesicular
NaNO3 indicated maximum activity at pH = 5.8 as compared to that
observed at pH = 7.0 and 8.0 (Fig. 6A). These data reconfirms that
1e is a preferential cation transporter at high pH, i.e. in its
deprotonated form, and an anion transporter at low pH, i.e. in its
protonated form, establishing the pronounced selectivity switching
effect by pH as the signal. Concentration dependent Cltransport
activity profile of 1e (Fig. 6B) at pH = 5.8 provided EC50 =14 µM
(Fig. S6) and initial rate (IR) was found to be 5.27 × 10-3 s‒1 at c = 15
µM (Fig. S7).
To attain theoretical insight about the geometry of 1e with K+ ion
complex, initial structures were generated by using CONFLEX 7
program.23 A [(1e)2K+] complex was considered based on the Hill
coefficient value obtained from EYPC-LUVs⊃HPTS assay. Initial
geometries were predicted by using MMFF94s force field of the
software and the conformation with highest Boltzmann population
(85%) was selected for the geometry optimization study. The most
populated structure was optimized by Gaussian 09 program
package24 using B3LYP functional and 6-311G (d,p) basis set25 and
most stable conformation of [(1e)2K+] complex was obtained. The
optimized structure shows binding with K+ ion with two molecules
of 1e through the electrostatic bond between deprotonated N‒H
group of bis(sulfonamide) 1e and one molecule of K+ ion (Fig. 7)
validating the obtained cation selectivity and mode of binding.
Fig. 4 Normalized fluorescence intensity with time in extravesicular (A) NaCl buffer, (B)
KCl buffer with compound 1e (5 µM) and (C) fluorescence based membrane potential
experiment with Safranin O dye.
Fig. 6 (A) Chloride ion selectivity of 1e (20 µM) at different pHs and (B) chloride Ion
efflux of 1e with increasing concentration (0 ‒ 25 µM) determined by using ISE.
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K. Sahlholm, T. Knöpfel, M. García-Valverde, A.VMiew. ARroticdleilOlan,linEe.
Jantus-Lewintre, R. Farràs, F. Ciruela,DRO.I:P1é0r.e10z3-T9o/Cm7CásC0a0n1d65RG.
Quesada, J. Am. Chem. Soc., 2015, 137, 15892; (b) S.-K. Ko, S.
K. Kim, A. Share, V. M. Lynch, J. Park, W. Namkung, W. Van
Rossom, N. Busschaert, P. A. Gale, J. L. Sessler and I. Shin, Nat.
Chem., 2014, 6, 885.
7
8
F. Lefranc and R. Kiss, Neoplasia, 2008, 10, 198.
B. A. Webb, M. Chimenti, M. P. Jacobson and D. L. Barber, Nat.
Rev. Cancer 2011, 11, 671.
9
V. Borisenko, M. S. P. Sansom and G. Andrew Woolley,
Biophys. J., 2000, 78, 1335.
Fig. 7 Geometry optimized structure of [(1e)2K+]. Color codes: green = potassium, blue 10 W.-H. Chen, M. Nishikawa, S.-D. Tan, M. Yamamura, A. Satake
= nitrogen, yellow = sulfur, maroon = oxygen, blackish grey = carbon, cyan = fluorine.
and Y. Kobuke, Chem. Commun., 2004, 872.
11 N. Madhavan and M. S. Gin, ChemBioChem, 2007, 8, 1834.
12 (a) S. V. Bhosale, S. V. Bhosale, M. B. Kalyankar and S. J.
Langford, Org. Lett., 2009, 11, 5418; (b) O. Mammoliti, S.
Allasia, S. Dixon and J. D. Kilburn, Tetrahedron, 2009, 65, 2184.
13 (a) M. K. Richter, M. Sander, M. Krauss, I. Christl, M. G.
Dahinden, M. K. Schneider and R. P. Schwarzenbach, Environ.
Sci. Tech., 2009, 43, 6632; (b) T. Koike, E. Kimura, I. Nakamura,
Y. Hashimoto and M. Shiro, J. Am. Chem. Soc., 1992, 114, 7338;
(c) J. P. Malval, R. Lapouyade, J.-M. Leger and C. Jarry,
Photochem. Photobiol. Sci., 2003, 2, 259; (d) L. Prodi, F.
Bolletta, M. Montalti and N. Zaccheroni, Eur. J. Inorg. Chem.,
1999, 1999, 455.
14 T. Saha, M. S. Hossain, D. Saha, M. Lahiri and P. Talukdar, J.
Am. Chem. Soc., 2016, 138, 7558.
15 Marvin 5.8.0, 2012, ChemAxon(http://www.chemaxon.com).
16 C. A. Lipinski, F. Lombardo, B. W. Dominy and P. J. Feeney, Adv.
Drug Deliv. Rev., 2001, 46, 3.
In conclusion, we demonstrated the design and syntheses of
bis(sulfonamides) bearing five different groups to tune their N−H
acidity and lipophilicity attributes. Ion transport activity and
selectivity were extensively studied by several fluorescence based
assays with EYPC liposomes. They displayed strong cation
selectivity as compared to anion selectivity due to binding of the
cation with deprotonated derivative at physiological pH. This cation
selectivity was found to be dependent on pH and it switched to
anions electivity at acidic pH. This ion selectivity flipping behavior
could be realized within very narrow pH range. This observation was
further reiterated by monitoring chloride efflux at different pH by
chloride selective electrode. Passive transport of K+ and generation
of transmembrane potential for selective transport of K+ was also
demonstrated by monitoring fluorescence of Safranin O. The mode
of binding with K+ was also showed by theoretical calculations.
AR thanks University Grants Commission (UGC), Govt. of India for
research fellowships. We gratefully acknowledge SERB, Govt. of
India (EMR/2014/000873B) and the Director, IISER Pune for
funding our work.
17 N. Proust, J. C. Gallucci and L. A. Paquette, J. Org. Chem., 2009,
74, 2897.
18 (a) P. Talukdar, G. Bollot, J. Mareda, N. Sakai and S. Matile,
Chem. Eur. J., 2005, 11, 6525; (b) N.-T. Lin, A. Vargas Jentzsch,
L. Guenee, J.-M. Neudorfl, S. Aziz, A. Berkessel, E. Orentas, N.
Sakai and S. Matile, Chem. Sci., 2012, 3, 1121; (c) T. Saha, A.
Roy, M. L. Gening, D. V. Titov, A. G. Gerbst, Y. E. Tsvetkov, N. E.
Nifantiev and P. Talukdar, Chem. Commun., 2014, 50, 5514.
19 (a) V. Gorteau, G. Bollot, J. Mareda, A. Perez-Velasco and S.
Matile,J. Am. Chem. Soc., 2006, 128, 14788; (b) A. Roy, T. Saha,
M. L. Gening, D. V. Titov, A. G. Gerbst, Y. E. Tsvetkov, N. E.
Nifantiev and P. Talukdar, Chem. Eur. J., 2015, 21, 17445;
20 A. Gilles and M. Barboiu, J. Am. Chem. Soc., 2016, 138, 426.
21 (a) H.-Y. Zha, B. Shen, K.-H. Yau, S.-T. Li, X.-Q. Yao and D. Yang,
Org. Biomol. Chem., 2014, 12, 8174;(b) J.-Y. Winum and S.
Matile, J. Am. Chem. Soc., 1999, 121, 7961.
22 (a) N. Busschaert, M. Wenzel, M. E. Light, P. Iglesias-
Hernández, R. Pérez-Tomás and P. A. Gale, J. Am. Chem. Soc.,
2011, 133, 14136; (b) 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;(c) J. H.
Lee, J. H. Lee, Y. R. Choi, P. Kang, M.-G. Choi and K.-S. Jeong, J.
Org. Chem., 2014, 79, 6403.
Notes and references
1
(a) T. J. Jentsch, V. Stein, F. Weinreich and A. A. Zdebik, Physiol.
Rev., 2002, 82, 503; (b) J. I. Vandenberg, M. D. Perry, M. J.
Perrin, S. A. Mann, Y. Ke and A. P. Hill, Physiol. Rev., 2012, 92,
1393; (c) H. Misonou, D. P. Mohapatra and J. S. Trimmer,
NeuroToxicol., 2005, 26, 743; (d) P. A. Gale, M. E. Light, B.
McNally, K. Navakhun, K. E. Sliwinski and B. D. Smith, Chem.
Commun., 2005, 3773; (e) P. R. Brotherhood and A. P. Davis,
Chem. Soc. Rev., 2010, 39, 3633.
2
3
J. R. Broughman, L. P. Shank, W. Takeguchi, B. D. Schultz, T.
Iwamoto, K. E. Mitchell and J. M. Tomich, Biochem., 2002, 41,
7350.
(a) G. W. Gokel and S. Negin, Acc. Chem. Res., 2013, 46, 2824;
(b) N. Busschaert and P. A. Gale, Angew. Chem. Int. Ed., 2013,
52, 1374; (c) J. W. F. Robertson, J. J. Kasianowicz and S.
Banerjee, Chem. Rev., 2012, 112, 6227; (d) A. L. Sisson, M. R.
Shah, S. Bhosale and S. Matile, Chem. Soc. Rev., 2006, 35, 1269.
(a) B. C. Pressman, Annu. Rev. Biochem., 1976, 45, 501; (b) B.
Jing, V. Janout, B. C. Herold, M. E. Klotman, T. Heald and S. L.
Regen, J. Am. Chem. Soc., 2004, 126, 15930.
S. Fernandez-Lopez, H.-S. Kim, E. C. Choi, M. Delgado, J. R.
Granja, A. Khasanov, K. Kraehenbuehl, G. Long, D. A.
Weinberger, K. M. Wilcoxen and M. R. Ghadiri, Nature, 2001,
412, 452.
4
5
23 CONFLEX 7, H. Goto, S. Obata, N. Nakayama, and K. Ohta,
CONFLEX Corporation, Tokyo, Japan, 2012.
24 M. J. Frisch, et. al. Gaussian 09, Vol. Gaussian, Inc.,
Wallingford, CT, USA, 2009.
25 (a) A. D. McLean and G. S. Chandler, J. Chem. Phys., 1980, 72,
5639; (b) M. P. Andersson and P. Uvdal, J. Phys. Chem., 2005,
109, 2937.
6
(a) V. Soto-Cerrato, P. Manuel-Manresa, E. Hernando, S.
Calabuig-Fariñas, A. Martínez-Romero, V. Fernández-Dueñas,
4 | J. Name., 2012, 00, 1-3
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