Anion Selective Ion Channel Constructed from a Self-Assembly of Bis

Letter Cite This: Org. Lett. 2018, 20, 5991−5994

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Anion Selective Ion Channel Constructed from a Self-Assembly of Bis(cholate)-Substituted Fumaramide Javid Ahmad Malla, Arundhati Roy,† and Pinaki Talukdar* Department of Chemistry, Indian Institute of Science Education and Research Pune, Pune, 411008, India

Org. Lett. 2018.20:5991-5994. Downloaded from pubs.acs.org by UNIV OF SUNDERLAND on 10/05/18. For personal use only.

S Supporting Information *

ABSTRACT: The self-assembled ion channel constructed from bis(cholate)-substituted fumaramide is reported. Such ion channel formation was favored by intermolecular hydrogen bonding interactions among fumaramide moieties. Fluorescence kinetics experiments and planar bilayer conductance measurements confirmed the selective chloride transport through the channel. Theoretical calculations were done to confirm the hydrogen bonded self-assembly of fumaramides and chloride recognition within the cavity.

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due to the presence of terminal steric groups. Such orientation of consecutive molecules allowed each molecule to form hydrogen bonds with four neighboring molecules, and this helped in the formation of a stable supramolecular ion channel in the lipid bilayer membrane.11 It is obvious that if the steric crowding is moved away from the fumaramide core, the flat-ribbon type of consecutive molecules can be regained, and if the steric moiety can provide distinct hydrophobic and hydrophilic faces, supramolecular barrel-stave ion channels can be formed.12 Herein, we demonstrate the formation of anion selective barrel-stave ion channel by a fumaramide system that is bis-functionalized with a cholic acid-derived amine. The cholic acid backbone was used as a side chain, because of its distinct hydrophobic β-face for the van der Waals interactions with the membrane and a hydrophilic α-face (three −OH groups are present on the αface) surface to provide the polar interior of the channel.13 There are multiple reports about ion channel formation utilizing adequately functionalized cholate moieties (with either methylated hydroxyl groups13c−f or free hydroxyl groups14); however, all these channels are reported to be cation selective. We envisaged that the free hydroxyl groups of each cholic acid part would help in the anion recognition, through O−H···anion interactions. In addition, the alkene C C of fumaramide moiety would participate in the anion recognition by anion−π interactions.11 The structure of the designed fumaramide 1a is shown in Figure 1. The corresponding maleamide 1b was also designed as a negative control to appraise the importance of flat-ribbon self-assembly. Syntheses of 1a and 1b were performed from cholic acid involving the conversion of the terminal acid group to the amine by a reported protocol.13g The cholic-acid-derived amine was then coupled to fumaryl chloride 2a to give 1a in

on transport proteins (channels, carriers, and pumps) play pivotal roles in facilitating the controlled movement of ions across lipid membranes.1 The process ensures normal cellular functions, e.g., cell proliferation, cell signaling, transepithelial transport, signal transduction, etc.2 The natural ion channels are very complex from their structural and functional aspects. To solve these mysteries, chemists have developed diverse synthetic molecules, which form either the transmembrane channels or mobile ion carriers3 and mimic both the structure and functions of natural ion transport systems. Moreover, recent findings have suggested potential antibacterial4 and anticancer activities5 of these systems. Such encouraging outcomes have prompted a renewed interest in finding new synthetic systems that can allow selective ion transport across lipid bilayer membranes. Fumaramide is a very captivating chemical moiety for selfassembly, because of its specific conformation and directional intermolecular hydrogen bonding ability.6 The moiety offers two hydrogen bond donors and two hydrogen bond acceptors for efficient self-assembly. In contrast, its maleamide congener is a poor self-assembling moiety, because of intramolecular hydrogen bonding, which locks it in the folded conformation. Therefore, photoisomerization of fumaramide to maleamide and the thermal isomerization of maleamide to fumaramide7 were useful in constructing various types of molecular machines,8 drug delivery systems,9 peptide helicity control,10 etc. However, the intermolecular hydrogen bonding ability of fumaramide has received the least attention in the field of transmembrane ion transport. We have very recently reported that N1,N4-dihexylfumaramide prefers the conventional flatribbon-type hydrogen-bonded self-assembly in the solid state.11 In this assembly, each pair of consecutive molecules is connected by two hydrogen bonds. However, the solid-state structure of N1,N4-dicyclohexylfumaramide reveal that each pair of consecutive molecules are connected by a single hydrogen bond, and these molecules are orthogonally oriented © 2018 American Chemical Society

Received: July 6, 2018 Published: September 27, 2018 5991

DOI: 10.1021/acs.orglett.8b02115 Org. Lett. 2018, 20, 5991−5994

Letter

Organic Letters

fluorescent dye, 8-hydroxy-1,3,6-pyrenetrisulfonate (HPTS) and then applying a pH gradient of 0.8 unit (pHin 7.0 and pHout 7.8) with NaOH (see the SI).15 Under comparable conditions, the activity of compound 1a (6 μM) was much higher than that of 1b (6 μM) (see Figure 3A). The dose-

Figure 1. Structures and self-assembly processes of (A) fumaramide 1a and (B) maleamide 1b. (C) Description of the R group.

41% yield (see Scheme 1). The reaction of the same amine with maleic acid 2b in the presence of EDC·HCl provided 1b in 36% yield.

Figure 3. (A) Comparison of activities of 1a and 1b (6 μM concentration) and photoisomerization of 1a to 1b (6 μM concentration). (B) Anion selectivity of 1a 3 μM.

Scheme 1. Synthesis of Fumaramide 1a and Maleamide 1b

responsive ion transport activity of 1a and 1b provided the half maximal activity (EC50) values of 3.1 μM, i.e., 4.6 mol %, relative to lipid concentration (see Figure S3A in the SI) and 9.8 μM, i.e., 14.6 mol %, relative to lipid concentration (see Figure S3B in the SI), respectively. The fumaramide derivative showed much better activity than the corresponding maleamide derivative. The Hill coefficient n = 4 for 1a indicated that the ion channel is formed by the self-assembly of monomers in the lipid bilayer membrane.16 Furthermore, the compound 1a was subjected to the photoisomerization of the CC bond (see the SI) and ion transport activity was checked. The activity of the resulting sample (6 μM) was comparable to that of 1b (see Figure 3A). This information further indicates that the trans conformation of the CC bond provides efficient self-assembly to form the active ion transport system. When the conformation of the CC bond was changed to the cis form, the self-assembly was disturbed because of the intramolecular hydrogen bonding of each isomerized molecule. The ion transport activity of the fumaramide derivative encouraged us to evaluate the ion selectivity and mechanism of ion transport. To evaluate the preferential transport of cations or anions through the channel, HPTS assay was used and the transport activity was studied by varying the extravesicular ions.15,17 At first, ion transport of 1a was evaluated across EYPC-LUVs with entrapped HPTS in the presence of intravesicular NaCl and extravesicular isoosmolar MCl (M+ = Li+, K+, Rb+, and Cs+). In each case, the variation of external cations did not show any significant differences in the transport activity of 1a (see Figure S5 in the SI). This result rules out the involvement of any cation (i.e., H+/M+ antiport or M+/OH− symport) in the ion transport process. Next, the anion selectivity was checked by varying the intravesicular as well as extravesicular anions,18 and changes in the ion transport rate were examined when a pH gradient of 0.8 (pHin 7.0 and pHout 7.8) was applied. Considerable changes in the ion transport rate were observed for different NaX salts (X− = Cl−, Br−, I−, NO3−, and ClO4−) (see Figure 3B, as well as Figure S6 in the SI). The observed ion transport activity sequence corroborated to the Cl− ion selectivity of compound 1a. The anion selectivity order seems to be

At first, the morphological characterization of 1a was performed using atomic force microscopy (AFM). The sample of the compound, when prepared in THF (100 μM), showed self-assembled fibril formation (see Figure 2A, as well as Figure

Figure 2. (A) Atomic force microscopy (AFM) image of 1a in THF at 100 μM concentration. (B) Possible modes of self-assembly in solution phase by 1a.

S1 in the Supporting Information (SI)). The observation can be rationalized based on CO···H−N (among fumaramides) and C−O···H−O (among hydroxyl groups present on the αface of cholates) hydrogen bond interactions, and van der Waals interactions (among β-faces of cholates) resulting in the fibrils (Figure 2B). These fibrils further self-assemble to form hierarchical self-assembled structures. This interesting result encouraged us to evaluate the ion transport activity of the compound. The ion transport activities of 1a and 1b were evaluated across egg-yolk phosphatidylcholine-based large unilamellar vesicles (EYPC-LUVs), prepared by entrapping pH-sensitive 5992

DOI: 10.1021/acs.orglett.8b02115 Org. Lett. 2018, 20, 5991−5994

Letter

Organic Letters

Cl− ion (3.6 Å). The variation of current with voltage (I−V) showed a linear relation in the range of −90 mV to +90 mV in both symmetrical as well as unsymmetrical concentrations of KCl (see Figure 4C), with the reversal potential to be −15 ± 2 mV. Thus, the channel shows ohmic behavior, which is expected due to the non-dipolar nature of channel forming molecules. These data encouraged us to construct a theoretical model of the proposed channel. At first, the cholate moieties were replaced with the propyl groups and an equivalent all-atom model such a channel was constructed using four fumaramide molecules.16 The presumed structure was optimized using MOPAC201223 software with the PM6-DH+24 method. The results indicate that fumaramide molecules are interlocked by intermolecular CO···H−N hydrogen bonding interactions and recognition of Cl− ion within the cavity is through anion−π interactions25 (see Figure S11 in the SI). Subsequently, a model of the full channel was constructed involving four molecules of 1a and three Cl− ions were placed within the channel (one Cl− near the fumaramide moieties and two Cl− either side, near the cholates). The rationale for placing more than one Cl− simultaneously within the channel was motivated by the literature reports, which demonstrate the presence of multiple ions within natural channels26 when more than one ion binding site is present. Moreover, it was done to reduce the time of optimization. The all-atom model was also optimized using the same method (see Figure 5, as well as

controlled by both the radius and hydration energy of anions (see Figure S7 in the SI).19 The ion selectivity studies confirmed the participation of anions in the ion transport mechanism. The hydrogen bonding interactions of an anion with the hydroxyl groups of cholate and anion−π interactions of the ion with the fumaramide moieties can be responsible for its recognition within the lumen of the channel. To confirm further the Cl− ion selectivity of the fumaramide derivative 1a, the influx of Cl− ions into vesicles was monitored by lucigenin fluorescence assay following a reported protocol.20 Ion transporter structure formed by compound 1a showed a concentration-dependent quenching of lucigenin fluorescence, confirming the Cl− influx during transport (see Figure S8A in the SI). To gain more insight into the mechanism of ion transport, a modified method of the lucigenin assay was used. The EYPC liposomes were prepared by entrapping NaCl (200 mM) and 1 mM of lucigenin dye.21 Then, isoosmolar Na2SO4 was added to the extravesicular solution and Cl− efflux was monitored. A similar experiment was also performed with extravesicular isoosmolar NaNO3. The results indicate that the SO42− ion being highly hydrophilic is not transported easily by 1a (see Figure S8B in the SI). However, the less polar NO3− ion is easily transported into the vesicles by 1a with the concomitant efflux of Cl−, suggesting that the antiport mechanism of ion transport is operative. The effect of 1a on the lipid bilayer membrane was checked by using vesicles with entrapped ANTS-DPX dye. The EYPCLUV⊃ANTS-DPX (λem = 520 nm with λex = 353 nm) were prepared according to the known procedure (see the SI). No significant leakage of the dye from vesicles confirmed that the compound neither destroys the integrity of the lipid bilayer membrane nor large transmembrane pores are formed by the compound (see Figure S9B in the SI).22 To understand whether the ion transport is facilitated by either transmembrane channel or mobile carrier formed by 1a, the ionic conductance across the planar lipid bilayer membrane was measured. In this experiment, two compartments (cis and trans chambers) containing KCl solution (1 M) were separated by a planar lipid bilayer composed of diphytanoyl phosphatidylcholine (diPhyPC) lipid.3b,19b When compound 1a (20 μM) was added to the trans-chamber, distinct single channel opening and closing events were observed at different holding potentials, indicating the formation of ion channels in the lipid bilayer membrane (see Figures 4A and 4B). The single-channel conductance of this pore was determined to be 40.06 ± 0.9 pS in 1 M KCl solution. The diameter of the channel was also determined as 4.1 ± 0.12 Å, which is close to the diameter of

Figure 5. Side view of computationally optimized full-channel noncovalent interactions among fumaramides and anion−π with two Cl− ions (green) placed near the end and one at center of the channel. Cyan-colored dots represent the centroids of CC bonds.

Figure S12 in the SI). The resulting supramolecular structure showed that many of the hydroxyl groups of the cholate moieties are participating in the inter-stave hydrogen bonding interactions, (O···H−O distance = 1.7−1.9 Å), thus, stabilizing the channel structure. Some of the hydroxyl groups form hydrogen bonds with the Cl− ions (i.e., OH···Cl− bond distances of 2.0−2.5 Å). The interactions of the Cl− with surrounding fumaramides were also evident. In conclusion, we have designed a bis(cholate)-substituted fumaramide for barrel-stave supramolecular anion channel formation. The positioning of the steric and rigid cholate moiety away from the fumaramide core favored their hydrogen-bonded barrel-stave self-assembly. The hydroxyl groups of cholate moieties and alkene π-electron cloud of fumaramides provided the polar channel interior, which is suitable for anion binding. Fluorescence-based ion transport experiments indicated the formation of a supramolecular ion channel by fumaramide molecules. Such channel allowed the antiport of anions with the highest selectivity toward chloride.

Figure 4. Single-channel conductance of 1a (20 μM) recorded (A) at +30 mV and (B) at −100 mV under symmetrical KCl solutions. (C) I−V plots of 1a under symmetrical and unsymmetrical concentrations of KCl. 5993

DOI: 10.1021/acs.orglett.8b02115 Org. Lett. 2018, 20, 5991−5994

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Organic Letters

Chem. 2014, 6, 885−892. (d) Soto-Cerrato, V.; Manuel-Manresa, P.; Hernando, E.; Calabuig-Fariñas, S.; Martínez-Romero, A.; FernándezDueñas, V.; Sahlholm, K.; Knöpfel, T.; García-Valverde, M.; Rodilla, A. M.; Jantus-Lewintre, E.; Farràs, R.; Ciruela, F.; Pérez-Tomás, R.; Quesada, R. J. Am. Chem. Soc. 2015, 137, 15892−15898. (e) CastilloÁ vila, W.; Abal, M.; Robine, S.; Pérez-Tomás, R. Life Sci. 2005, 78, 121−127. (6) Biscarini, F.; Cavallini, M.; Leigh, D. A.; León, S.; Teat, S. J.; Wong, J. K. Y.; Zerbetto, F. J. Am. Chem. Soc. 2002, 124, 225−233. (7) Leigh, D. A.; Perez, E. M. Chem. Commun. 2004, 2262−2263. (8) (a) Kay, E. R.; Leigh, D. A.; Zerbetto, F. Angew. Chem., Int. Ed. 2007, 46, 72−191. (b) Hernandez, J. V.; Kay, E. R.; Leigh, D. A. Science 2004, 306, 1532−1537. (9) Martinez-Cuezva, A.; Valero-Moya, S.; Alajarin, M.; Berna, J. Chem. Commun. 2015, 51, 14501−14504. (10) Mazzier, D.; Crisma, M.; De Poli, M.; Marafon, G.; Peggion, C.; Clayden, J.; Moretto, A. J. Am. Chem. Soc. 2016, 138, 8007−8018. (11) Roy, A.; Gautam, A.; Malla, J. A.; Sarkar, S.; Mukherjee, A.; Talukdar, P. Chem. Commun. 2018, 54, 2024−2027. (12) (a) Matile, S. Chem. Soc. Rev. 2001, 30, 158−167. (b) Gilles, A.; Barboiu, M. J. Am. Chem. Soc. 2016, 138, 426−432. (c) Hu, X.-B.; Chen, Z.; Tang, G.; Hou, J.-L.; Li, Z.-T. J. Am. Chem. Soc. 2012, 134, 8384−8387. (d) Ronan, D.; Jeannerat, D.; Pinto, A.; Sakai, N.; Matile, S. New J. Chem. 2006, 30, 168−176. (13) (a) 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. (b) Ryu, E.-H.; Zhao, Y. J. Org. Chem. 2006, 71, 9491−9494. (c) Kobuke, Y.; Nagatani, T. J. Org. Chem. 2001, 66, 5094−5101. (d) Ma, L.; Harrell, W. A.; Davis, J. T. Org. Lett. 2009, 11, 1599−1602. (e) Bandyopadhyay, P.; Janout, V.; Zhang, L.-h.; Regen, S. L. J. Am. Chem. Soc. 2001, 123, 7691−7696. (f) Zhou, J.; Lu, Y.-M.; Deng, L.-Q.; Zhou, Z.-Z.; Chen, W.-H. Bioorg. Med. Chem. Lett. 2013, 23, 3145−3148. (g) Zhou, Y.; Ryu, E.-H.; Zhao, Y.; Woo, L. K. Organometallics 2007, 26, 358−364. (14) (a) Di Filippo, M.; Izzo, I.; Savignano, L.; Tecilla, P.; De Riccardis, F. Tetrahedron 2003, 59, 1711−1717. (b) Yoshii, M.; Yamamura, M.; Satake, A.; Kobuke, Y. Org. Biomol. Chem. 2004, 2, 2619−2623. (15) Roy, A.; Saha, D.; Mandal, P. S.; Mukherjee, A.; Talukdar, P. Chem. - Eur. J. 2017, 23, 1241−1247. (16) Litvinchuk, S.; Bollot, G.; Mareda, J.; Som, A.; Ronan, D.; Shah, M. R.; Perrottet, P.; Sakai, N.; Matile, S. J. Am. Chem. Soc. 2004, 126, 10067−10075. (17) Saha, T.; Roy, A.; Gening, M. L.; Titov, D. V.; Gerbst, A. G.; Tsvetkov, Y. E.; Nifantiev, N. E.; Talukdar, P. Chem. Commun. 2014, 50, 5514−5516. (18) (a) Ren, C.; Ding, X.; Roy, A.; Shen, J.; Zhou, S.; Chen, F.; Yau Li, S. F.; Ren, H.; Yang, Y. Y.; Zeng, H. Chem. Sci. 2018, 9, 4044− 4051. (b) Benke, B. P.; Aich, P.; Kim, Y.; Kim, K. L.; Rohman, M. R.; Hong, S.; Hwang, I.-C.; Lee, E. H.; Roh, J. H.; Kim, K. J. Am. Chem. Soc. 2017, 139, 7432−7435. (19) (a) Marcus, Y. J. Chem. Soc., Faraday Trans. 1991, 87, 2995− 2999. (b) 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. (20) McNally, B. A.; Koulov, A. V.; Smith, B. D.; Joos, J.-B.; Davis, A. P. Chem. Commun. 2005, 1087−1089. (21) Roy, A.; Saha, D.; Mukherjee, A.; Talukdar, P. Org. Lett. 2016, 18, 5864−5867. (22) Sordé, N.; Matile, S. J. Supramol. Chem. 2002, 2, 191−199. (23) Stewart, J. J. P. MOPAC2012; Stewart Computational Chemistry: Colorado Springs, CO, USA, 2012. (24) Korth, M. J. Chem. Theory Comput. 2010, 6, 3808−3816. (25) Gorteau, V.; Bollot, G.; Mareda, J.; Matile, S. Org. Biomol. Chem. 2007, 5, 3000−3012. (26) (a) Tao, X.; Avalos, J. L.; Chen, J.; MacKinnon, R. Science 2009, 326, 1668−1674. (b) Mindell, J. A.; Maduke, M.; Miller, C.; Grigorieff, N. Nature 2001, 409, 219−223.

Planar bilayer conductance measurements confirmed the formation of ion channels 4.1 ± 0.12 Å in diameter and provided an additional support to chloride selectivity. Geometry optimized structure of the proposed channel with internal chloride ions confirmed the formation of CO···H− N hydrogen-bonded channel and anion recognition by anion-π interactions from electron-deficient CC moieties and hydrogen bonding from the hydroxyl groups.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.8b02115. General methods and experimental measurements (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Pinaki Talukdar: 0000-0003-3951-4335 Present Address †

Institute of Bioengineering and Nanotechnology, 31 Bioplis Way, The Nanos, Singapore 138669. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank SERB, Govt. of India (Nos. EMR/2014/000873B and EMR/2016/001897) for funding. A.R. thanks UGC, India for research fellowship. The authors are thankful to Dr. Pankaj Mandal and his student, Dr. Sohini Sarakar, for their help during the photoisomerization experiment.

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REFERENCES

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DOI: 10.1021/acs.orglett.8b02115 Org. Lett. 2018, 20, 5991−5994