Anion-Selective Cholesterol Decorated Macrocyclic Transmembrane Ion Carriers

Article abstract of DOI:10.1021/jacs.7b07479

Anion transporters play a vital role in cellular processes and their dysregulation leads to a range of diseases such as cystic fibrosis, Bartter's syndrome and epilepsy. Synthetic chloride transporters are known to induce apoptosis in cancer cell lines. Herein, we report triamide macrocycles that are easily synthesized and externally functionalized by pendant membrane-permeable groups. Among a variety of chains appended onto the macrocycle scaffold, cholesterol is found to be the best with an EC₅₀ value of 0.44 μM. The macrocycle is highly anion-selective and transports ions via an OH^-/X^- antiport mechanism. The macrocycle is an interesting scaffold for ion-Transport as it is able to discriminate between various anions and shows a preference for SCN^- and Cl^-. Such anion-selective transporters are highly attractive model systems to study ion-Transport mechanisms and could potentially be of high therapeutic value.

Full text of DOI:10.1021/jacs.7b07479

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Communication
Anion-Selective Cholesterol Decorated
Macrocyclic Transmembrane Ion Carriers
Harekrushna Behera, and Nandita Madhavan
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b07479 • Publication Date (Web): 05 Sep 2017
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Anion-Selective Cholesterol Decorated Macrocyclic Transmembrane
Ion Carriers
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Harekrushna Behera and Nandita Madhavan*
Department of Chemistry, Indian Institute of Technology, Madras, Tamil Nadu, India and Indian Institute of Technology,
Bombay, Maharashtra, India.
Supporting Information Placeholder
ganic cages43 have been utilized as scaffolds for synthetic ion-chan-
ABSTRACT Anion transporters play a vital role in cellular pro-
nels/carriers. Oligoamide based macrocycles are attractive scaf-
folds for anion transport because the amide protons are known to
be good H-bond donors for anions such as halides. Herein, we re-
port triamide macrocycle anion transporters 1-4 appended with lip-
ophilic alkyl, oligoether, cholic acid and cholesterol chains, respec-
tively (Figure 1a). The cholesterol-containing macrocycle 4 (Figure
1b) was found to be the best ion transporter, followed by macrocy-
cle 1. The macrocycle 4 was anion-selective with notably higher
selectivity for SCN¯ and Cl¯. The mechanism of ion-transport was
found to be OH¯/X¯ antiport with the macrocycle behaving as a
carrier.
cesses and their dysregulation leads to a range of diseases such as
cystic fibrosis, Bartter’s syndrome and epilepsy. Synthetic chloride
transporters are known to induce apoptosis in cancer cell lines.
Herein, we report triamide macrocycles that are easily synthesized
and externally functionalized by pendant membrane-permeable
groups. Among a variety of chains appended onto the macrocycle
scaffold, cholesterol is found to be the best with an EC50 value of
0.44 µM. The macrocycle is highly anion-selective and transports
ions via an OH¯/X¯ antiport mechanism. The macrocycle is an in-
teresting scaffold for ion-transport as it is able to discriminate be-
tween various anions and shows a preference for SCN¯ and Cl¯.
Such anion-selective transporters are highly attractive model sys-
tems to study ion-transport mechanisms and could potentially be of
high therapeutic value.
a)
Transmembrane ion-transport is mediated by carriers that ferry
back and forth across the lipid bilayer or by channels that form
pores in the membrane. Selective and regulated ion-transport across
cell membranes is crucial for several biological processes. Particu-
larly, the transport of anions is critical for ion homeostasis, regulat-
ing pH, maintaining osmotic balance and neural signal-transduc-
tion. 1 Several diseases such as cystic fibrosis, Bartter's syndrome,
inherited kidney stone diseases, myotonia and epilepsy have been
associated with dysfunction of chloride channels.2-4 Chloride
transport has also been proposed to be the key mechanism for the
anticancer activity of prodigiosin and its synthetic analog obato-
clax.5-8 The ability of anion transporters to induce apoptosis in can-
cer cell lines has been well studied and validated.7-13 Recently, the
role of chloride transporters in reducing autophagy has also been
demonstrated.14, 15 Therefore, synthetic anion transporters are po-
tentially attractive therapeutics and anticancer agents.
b)
Two very basic design elements for synthesizing anion transport-
ers are a hydrophobic domain to facilitate membrane insertion and
an ion-recognition domain to enhance anion-selectivity. Attractive
electrostatic forces of ammonium species16-18 or less common non-
covalent interactions such as anion- interactions, halogen bonds,
and anion-macrodipole interactions have been utilized to accelerate
anion transport.19-21 Hydrogen-bond donors such as amides,22-25
urea,26-30 -Aminoxy Acids,31 perenosins,13 tambjamines,32 and
hydroxyl groups33, 34 have also been widely utilized as anion-recog-
nition domains. To gain more control over the density and location
of the ion-recognition domains, macrocycles such as calixarenes,35-
Figure 1. a) Macrocycles 1-4; b) Schematic representation of the
cholesterol appended macrocycle 4 – the best transporter.
Synthesis of the macrocycles was highly modular wherein desir-
able pendant groups could be readily appended onto the macrocycle
scaffold 5 as shown in Scheme 1. Macrocycle 5 was easily accessed
in one-pot using a method previously developed in our group.44 Hy-
drogenolysis of the benzyl groups, followed by treatment with the
appropriate R-tosyl group, afforded the requisite functionalized
macrocycles 1-4.
37
cyclic peptides,38, 39 calixpyrroles40-42 and porphyrin-based or-
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Variable temperature HPTS assays with vesicles prepared from
Scheme 1. General method for synthesizing macrocycles
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1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) lipids were
used to distinguish between the carrier and channel mechanisms. 16,
50-52 The fluid-gel transition temperature for DPPC lipid is 41 ºC. A
drastic drop in ion transport activity was observed when the tem-
perature was lowered from 45 ºC to 25 ºC (Figure 3). Such a strong
dependence of activity on membrane rigidity is strongly indicative
of the carrier-mechanism. To rule out expulsion of macrocycle 4
from the vesicle at lower temperatures, the vesicle suspension was
subjected to size exclusion chromatography at 25 ºC prior to carry-
ing out the high temperature HPTS assay. No significant loss in ion
transport activity at 45 ºC was observed (Figure S3).
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Transmembrane ion transport was assessed using vesicles made
from egg yolk phosphatidyl choline (EYPC) containing pH sensi-
tive 8-hydroxypyrene- 1,3,6-trisulfonic acid, trisodium salt (HPTS)
dye as a probe (Figure 2a).45-48 The macrocycles were allowed to
equilibrate with vesicles, following which NaOH was added to in-
crease the external pH by 0.6 units. A gradual increase in the con-
centration of deprotonated HPTS (HPTS¯) dye was observed after
the base pulse (Figure 2b). This increase in internal pH of the ves-
icles suggested that the macrocycles were transporting ions by one
of the mechanisms shown in Figure 2a. At the end of the experi-
ment, Triton X was added to lyse the vesicles to obtain the final
HPTS¯ concentration (to normalize the data). The rate constants
for ion transport obtained by fitting the curves to a first order expo-
nential equation showed that macrocycle 4 was the most active. The
macrocycle 2 containing oligoether chains was found to show only
marginally higher activity than the blank. Dose-response curves
were also obtained to compare the activities of macrocycles 1, 3
and 4. These curves were used to obtain the EC50 value (effective
concentration for 50 % activity) and the Hill coefficient (n) i.e.
number of macrocycles associating with a single ion (Figure S2).49
The EC50 value of macrocycle 4 was found to be very low ca. 0.44
M, almost three times lower than those of macrocycles 1 and 3
(Figure 2c). The Hill coefficients were less than or equal to 1 indic-
ative that the macrocycles were ion-carriers or formed monomolec-
ular channels/stable assemblies.
Figure. 3. Variable temperature HPTS assay with macrocycle 4
(23.1 µM) to illustrate carrier mechanism.
To check for cation-selectively, the counter cation for OH‾ was
varied (Li+, Na+, K+, Rb+ and Cs+) in the base used to introduce a
pH gradient in the HPTS assay. 21, 53 The ion transport rates were
found to be similar irrespective of the nature of the cation (Figures
4a and 4b). Macrocycles 1-3 gave similar results (Figure S4, Table
S1) suggesting that these compounds either did not transport cati-
ons or transported all cations efficiently. The k values in the HPTS
assay carried out with NaOH were found to be independent of the
extravesicular Na+ ions concentration (Figure S5); ruling out the
Na+ /H+ antiport or Na+/OH‾ symport mechanisms illustrated in
Figure 2a.
a)
This indicated that presumably the OH‾/ Cl‾ antiport mechanism
illustrated in Figure 2a was operative. To further substantiate this
b)
c)
2
hypothesis, replacing Cl‾ with the larger SO4 ‾ in the extra and in-
travesicular solutions in the HPTS assay, showed a complete loss
in activity even with large concentrations of macrocycle 4 (Figure
4c).24 If Na+ transport were occurring by the abovementioned
mechanisms such a drop in activity would not have been observed.
Furthermore, alkalization of the intravesicular solution was also ob-
served in the absence of base pulse when vesicles were prepared in
NaCl buffer and suspended in Na2SO4 buffer (Figure 4d).24 Since,
Macro-
cycle
EC50
n
(µM )[a]
1.33
1.41
0.44
0.8
1.0
0.8
1
3
4
2
the SO4 ‾ ions cannot be transported across the bilayer by the mac-
rocycle, the changes in internal pH could only be explained by a
Cl‾/OH‾ antiport, driven by Cl‾ efflux. Naturally, reversal of the
buffer solutions resulted in acidification of the vesicular compart-
ment due to Cl ‾ influx (Figure S6). To unequivocally rule out the
ability of macrocycle 4 to transport sodium ions across the lipid
bilayer, 23Na NMR measurements were made with vesicles incu-
bated with macrocycle 4.54, 55 A shift reagent (DyCl3 + Na5P3O10)
was added to differentiate the internal and external Na+ ions in the
23Na NMR spectra (Figure 4e). Negligible line broadening was ob-
served for the peaks indicating that no Na+ exchange was occurring
(Table S2).
[a] 0.44 µM = 0.12 mol %
Figure 2. (a) Schematic representation of the HPTS assay for ion
transport, (b) Comparison of activities of macrocycles 1-4 (4.2 µM)
using the HPTS assay. (c) Hill Analyses for macrocycles.
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a)
b)
c)
This would explain the anti-Hofmeister selectivity i.e. Cl¯> Br¯>I¯
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for the halides. The pyridyl groups of the macrocyle will be not be
protonated in the current studies (at ~pH 7) as the pKa of pyri-
dinium ions ranges 5.1 – 5.3 in alcohol based solvents and wa-
ter.56,57 However, the pyridyl groups in the macrocycle can be po-
tentially exploited for pH modulated ion transport. Overall, the an-
ion-selectivity of the macrocycles can be explained by H-bonding
interactions of the anions with the amide NH bonds in the macro-
cycle (Figure 5c). The relative order of ion-transport activity
among macrocycles 1-4 can be rationalized based on the hydropho-
bicity of the appended groups. The cholesterol units being the most
hydrophobic improve membrane permeation and hence ion-
transport activity.
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a)
b)
d)
c)
e)
Figure. 5. a) Schematic and normalised fluorescence time courses
obtained from the HPTS assay used to determine anion selectivity
of macrocycle 4 (0.26 µM of macrocycle used). b) Stacked NMR
plots for titration of macrocycle 4 (0.005 M) with TBACl. c) Sche-
matic illustration for anion-selectivity and high ion transport activ-
ity of macrocycle 4.
Figure 4. Assays to determine ion-selectivity. a) Schematic of
HPTS assay with macrocycle 4 and normalised fluorescence vs.
time plots to determine ion-transport mechanism by varying b) ex-
ternal cations in MOH pulse; 0.26 µM macrocycle used and c) the
anion in the buffer (NaCl was replaced by Na2SO4); 22.2 µM mac-
rocycle used; d) Schematic of assay and normalized fluorescence
versus time plots for assay without base pulse to prove OH‾/ Cl‾
antiport mechanism (2.1 µM of macrocycle used). e) Schematic of
23Na NMR assay and stacked NMR plots obtained assay with dif-
ferent macrocycle concentrations to rule out Na+ transport.
In summary, triamide macrocycles 1-4 appended with membrane
permeable alkyl, oligoether, cholic acid and cholesterol chains, re-
spectively, were synthesized using a modular synthetic approach.
This approach allows for easy modification of the macrocycle pe-
riphery with desirable pendant groups. Macrocycle 4 containing
cholesterol was the most active and had a very low EC50 value of
0.44 µM. The higher activity of macrocycle 4 has been attributed
to the high membrane permeability of the hydrophobic cholesterol
side chains. A carrier mechanism has been proposed for ion
transport based on the high dependence of ion transport activity on
membrane rigidity. The macrocycle was highly anion-selective and
transported anions via an X¯/OH¯ antiport mechanism. The mac-
rocycle could discriminate among anions and showed a preference
for SCN¯ and Cl¯. The pyridyl units in macrocycle provide excit-
ing opportunities for modulating the ion selectivity as well as
transport activity using pH. Current efforts are focussed on regulat-
ing the ion-transport activity of the macrocycles using external
stimuli such as pH and light.
The selectivity studies above confirmed that the predominant
ion-transport mechanism was OH‾/ Cl‾ antiport. Therefore, it was
not surprising that the ion transport rates were dependent on the
nature of the extravesicular anions in the HPTS assay with macro-
cycle 4 (Figures 5a and S7). The macrocycle showed minimal
transport of NO2¯and CH3COO¯. For the other anions the ion
transport activity was found to be SCN¯ > Cl¯ > Br¯, ClO4¯, I¯.
The order of anion-selectivity does not directly follow the Hofmeis-
ter series. A combined effect of hydrophobicity and hydrogen
bonding interactions appears to dictate the selectivity of these ani-
ons. The highest selectivity for the linear SCN¯ can be explained
based on the Hofmeister series. However, in the case of the other
ions the order of selectivity is the reverse of the Hofmeister series.
The un-functionalized macrocycle was found to bind with Cl¯.44
A
downfield shift as well as sharpening of the amide proton for mac-
rocycle 4 was also observed upon addition of TBACl (Figure 5b).
The selectivity order for the other ions is presumably dominated by
the hydrogen bonding ability of the ions to the macrocyclic amides.
ASSOCIATED CONTENT
Supporting Information
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22. Cook, G. A.; Pajewski, R.; Aburi, M.; Smith, P. E.; Prakash, O.;
Detailed procedures, characterization of compounds, raw plots for
vesicle assays, DLS data, and NMR titration data has been provided
in the Supporting information. This material is available free of
charge on the ACS Publications website at http://pubs.acs.org
Tomich, J. M.; Gokel, G. W. J. Am. Chem. Soc. 2006, 128, 1633-1638.
23. Busschaert, N.; Kirby, I. L.; Young, S.; Coles, S. J.; Horton, P. N.;
Light, M. E.; Gale, P. A. Angew. Chem. Int. Ed. 2012, 51, 4426-4430.
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25. Saha, T.; Hossain, M. S.; Saha, D.; Lahiri, M.; Talukdar, P. J. Am.
Chem. Soc. 2016, 138, 7558-7567.
26. McNally, B. A.; Koulov, A. V.; Lambert, T. N.; Smith, B. D.; Joos, J.-
B.; Sisson, A. L.; Clare, J. P.; Sgarlata, V.; Judd, L. W.; Magro, G.;
Davis, A. P. Chem. -Eur. J. 2008, 14, 9599-9606.
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AUTHOR INFORMATION
Corresponding Author
*E-mail: nanditam@chem.iitb.ac.in
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27. Valkenier, H.; Davis, A. P. Acc. Chem. Res. 2013, 46, 2898-2909.
28. Li, H.; Valkenier, H.; Judd, L. W.; Brotherhood, P. R.; Hussain, S.;
Cooper, J. A.; Jurček, O.; Sparkes, H. A.; Sheppard, D. N.; Davis, A.
P. Nat. Chem. 2016, 8, 24-32.
Notes
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The authors declare no competing financial interests.
29. Roy, A.; Saha, D.; Mukherjee, A.; Talukdar, P. Org. Lett. 2016, 18,
5864-5867.
ACKNOWLEDGMENT
30. Lisbjerg, M.; Valkenier, H.; Jessen, B. M.; Al-Kerdi, H.; Davis, A. P.;
Pittelkow, M. J. Am. Chem. Soc. 2015, 137, 4948-4951.
31. Li, X.; Wu, Y.-D.; Yang, D. Acc. Chem. Res. 2008, 41, 1428-1438.
32. Knight, N. J.; Hernando, E.; Haynes, C. J. E.; Busschaert, N.; Clarke,
H. J.; Takimoto, K.; Garcia-Valverde, M.; Frey, J. G.; Quesada, R.;
Gale, P. A. Chem. Sci. 2016, 7, 1600-1608.
33. Berezin, S. K.; Davis, J. T. J. Am. Chem. Soc. 2009, 131, 2458-2459.
34. Saha, T.; Dasari, S.; Tewari, D.; Prathap, A.; Sureshan, K. M.; Bera, A.
K.; Mukherjee, A.; Talukdar, P. J. Am. Chem. Soc. 2014, 136, 14128-
14135.
This research was supported by DST (SR/S1/OC-41/2009), New
Delhi, India. H.B. thanks IIT Madras for fellowship. We thank Dr.
E. Prasad for the use of his DLS instrument.
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Tecilla, P. Chem. Commun. 2014, 50, 9157-9160.
56. Chmurzyński, L. J. Heterocycl. Chem. 2000, 37, 71-74.
57. Yan, Y.; Zhao, W.; Bhagavathy, G. V.; Faurie, A.; Mosey, N. J.;
Petitjean, A. Chem. Commun. 2012, 48, 7829-7831.
19. Jentzsch, A. V.; Hennig, A.; Mareda, J.; Matile, S. Acc. Chem. Res.
2013, 46, 2791-2800.
20. Busschaert, N.; Caltagirone, C.; Van Rossom, W.; Gale, P. A. Chem.
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21. Gorteau, V.; Bollot, G.; Mareda, J.; Matile, S. Org. Biomol. Chem.
2007, 5, 3000-3012.
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Figure 1a: Macrocycles 1-4
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Figure 1b: Schematic representation of the cholesterol appended macrocycle 4 – the best transporter
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Figure 2a: Schematic representation of the HPTS assay for ion transport
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b) Comparison of activities of macrocycles 1ꢀ4 (4.2 µM) using the HPTS assay
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Figure 3: Variable temperature HPTS assay with macrocycle 4 (23.1 µM) to illustrate carrier mechanism.
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Assays to determine ion-selectivity. a) Schematic of HPTS assay with macrocycle 4 and normalised
fluorescence vs. time plots to determine ion-transport mechanism by varying
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Figure 4b: Normalised fluorescence vs. time plots to determine ionꢀtransport mechanism by
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Figure 4c: Normalised fluorescence vs. time plots to determine ionꢀtransport mechanism by varying the
anion in the buffer (NaCl was replaced by Na2SO4); 22.2 µM macrocycle used;
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Figure 4d: Schematic of assay and normalized fluorescence versus time plots for assay without base pulse to
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Figure 4e: Schematic of 23Na NMR assay and stacked NMR plots obtained assay with different macrocycle
concentrations to rule out Na+ transport.
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a) Schematic and normalised fluorescence time courses obtained from the HPTS assay used to determine
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a) Schematic and normalised fluorescence time courses obtained from the HPTS assay used to determine
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b) Stacked NMR plots for titration of macrocycle 4 (0.005 M) with TBACl.
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c) Schematic illustration for anion-selectivity and high ion transport activity of macrocycle 4.
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