Iodide-Selective Synthetic Ion Channels Based on Shape-Persistent

May 24, 2017 - We report here a synthetic ion channel developed from a shape-persistent porphyrin-based covalent organic cage. The cage was synthesize...
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Iodide-Selective Synthetic Ion Channels Based on Shape-Persistent Organic Cages Bahiru Punja Benke,† Pulakesh Aich,† Younghoon Kim,‡ Kyung Lock Kim,† Md Rumum Rohman,† Soonsang Hong,†,‡ In-Chul Hwang,† Eun Hui Lee,§ Joon Ho Roh,*,† and Kimoon Kim*,†,‡ †

Center for Self-assembly and Complexity (CSC), Institute for Basic Science (IBS), Pohang 37673, Republic of Korea Department of Chemistry, Pohang University of Science and Technology, Pohang 37673, Republic of Korea § Department of Physiology, College of Medicine, The Catholic University of Korea, Seoul 06591, Republic of Korea ‡

S Supporting Information *

to construct such synthetic ion transporters. In a previous study, we used a discrete large metal−organic cage, MOP-18, as an ion transporter; this work demonstrated that a stable single molecule with multiple windows, a large internal cavity, and a hydrophobic exterior can function as an ion channel within a lipid bilayer.20 Taking inspiration from the previous work, we decided to use a 3D organic cage, PB-1A, a new hydrophobic analogue of the porphyrin box PB-1 recently reported by our group21 as an ion transport system because of the molecular characteristics described below (Figure 1). First, the organic cage has a large

ABSTRACT: We report here a synthetic ion channel developed from a shape-persistent porphyrin-based covalent organic cage. The cage was synthesized by employing a synthetically economical dynamic covalent chemistry (DCC) approach. The organic cage selectively transports biologically relevant iodide ions over other inorganic anions by a dehydration-driven, channel mechanism as evidenced by vesicle-based fluorescence assays and planar lipid bilayer-based single channel recordings. Furthermore, the organic cage appears to facilitate iodide transport across the membrane of a living cell, suggesting that the cage could be useful as a biological tool that may replace defective iodide channels in living systems.

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on transport processes across cell membranes by natural ion channels are essential for life.1 Inorganic anions such as chloride, iodide, sulfate, nitrate, and bicarbonate help to regulate various crucial processes in biology.1 Iodine, one of the essential elements in the human body, plays a vital role in hormone biosynthesis in the thyroid gland.2 Sodium iodide symporters (NIS) mediate the transport of iodide from the bloodstream into thyroid cells as well as a number of nonthyroidal tissues, including the mammary gland during lactation, cancerous breast tissue, and lacrimal glands.3 Malfunction of NIS leads to diseases such as goiter, hypothyroidism, and hyperthyroidism.3 More recently, the transport of radioactive iodine 131I by NIS has been exploited for the imaging and treatment of cancer.4,5 However, NIS expression in cells is difficult because it requires gene delivery. Therefore, synthetic iodide channels that can mimic the function of native NIS in the presence of much more abundant physiological anions, in particular, chloride, have great potential for treating advanced forms of thyroid cancer and nonthyroidal malignancies. While chloride-selective synthetic ion channels are well established,6−16 synthetic iodide-selective channels remain largely unexplored, with only a few examples reported in the past decade.17,18 The development of synthetic ion transporters with high transport selectivity for biologically important ions is a fast moving field with a variety of possible approaches.7,9,10,14,17,19 We have been interested in the use of supramolecular chemistry © 2017 American Chemical Society

Figure 1. Structure of PB-1A showing a cavity (diameter ∼1.95 nm), 12 windows (average diameter ∼3.7 Å, including alkyl chains) estimated from the X-ray crystal structure of a closely related compound (a) and overall dimensions (b and c; alkyl chains are omitted for clarity for b and c) (Figures S1 and S2). (d) Schematic diagram for the ion channel formed by PB-1A in lipid bilayer membranes.

internal cavity accessible through molecular-sized windows, which can allow ions to pass through, when it is incorporated into a lipid bilayer membrane. Second, a single molecule of the shape-persistent cage has an outer maximum diameter of 3.64 nm which seems to span a lipid bilayer membrane. Third, the cage is purely organic and easily synthesized from the combination of 3-connected triangular and 4-connected Received: March 18, 2017 Published: May 24, 2017 7432

DOI: 10.1021/jacs.7b02708 J. Am. Chem. Soc. 2017, 139, 7432−7435

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Journal of the American Chemical Society square-shaped building units using a DCC approach, in one pot, in high yield.22 Finally, the organic cage is more stable than metal−organic cages in general, especially in the presence of halide ions or protons over a broad pH range (4.8−13).23 Furthermore, it is sufficiently hydrophobic, due to the presence of multiple alkyl chains, to be incorporated into a lipid bilayer. These characteristic features of PB-1A prompted us to examine the possibility of PB-1A working as an ion channel upon incorporation into lipid bilayer membranes. Herein, we introduce porphyrin-based shape-persistent organic cages as a new class of unimolecular synthetic anion channels (Figure 1). Most interestingly, the organic cage shows dehydration-driven high selectivity for iodide over other physiological anions, in particular chloride which is the most abundant in biological systems. We have also demonstrated that the organic cage can transport iodide across the membrane of a living cell. To enhance the solubility as well as external hydrophobicity for better incorporation to lipid bilayer membranes, we synthesized a new hydrophobic porphyrin box, PB-1A, by introducing octyloxy groups onto the triangular building units and using a slightly modified procedure (Schemes S1 and S2). The X-ray crystal structure of a closely related compound of PB-1A (Zn-PB-DPB; Figure S1) revealed that the windows of PB-1A are lined with the alkyl chains with two different conformations (Figure S2) in a probability ratio of roughly 7:3 (see Supporting Information (SI) for detailed crystallographic analyses).24 A probability-weighted average diameter of the windows was thus estimated to be ∼3.7 Å. However, the actual window size of PB-1A in a lipid bilayer would be slightly different due to the lipophilic and flexible nature of the alkyl chain. With the hydrophobic PB-1A in hand, anion transport by PB-1A across synthetic membranes was investigated using a well-established chloride-sensitive lucigenin assay25 (see SI for detailed experimental procedures). After addition of PB-1A into the vesicle solution, an extravesicular chloride gradient was applied and Cl− influx was monitored as a function of time by measuring the decay in the fluorescence of lucigenin (Figure 2). The linear increase of the transport rate with increasing concentration of PB-1A suggests that the anion transport is through the organic cages, not because of membrane leakage or other artifacts26 (Table S1, Figure S4). To examine the ion transport mechanism, intravesicular NO3− was replaced by the

more hydrophilic sulfate.25b A substantial decrease in transport activity was observed with sulfate (Figure S5). This result indicates that the anion transport via the Cl−/NO3− antiport dominates over the Na+/Cl− symport mechanism. To investigate whether cations contribute to the ion transport process, the standard pH sensitive 8-hydroxypyrene1,3,6-trisulfonic acid (HPTS) assay was performed (see SI for detailed experimental procedures).27 The variation of cations did not generate any significant difference in transport behavior (Figure S6), indicating that the cations have minimal involvement in the transport process.27 To date, the reason for the transport preference of PB-1A for anions over cations remains unclear since 1H NMR studies did not show any noticeable interaction (Figure S7) between the ions and PB1A, which is believed to be neutral (Figure S9) under the conditions used for measuring ion transport activity. Nevertheless, the most plausible driving force for the anion over cation selectivity may be the aliphatic or aromatic, or both, CH···anion interactions near the windows, which have been reported in other macrocycle based anion carriers.28 We further examined the PB-1A-mediated transport behavior of a diverse range of anions to understand its selectivity among the anions by using the pH sensitive HPTS assay. After addition of PB-1A, the vesicles were exposed to an anion gradient by an external addition of anions NaX (X = SO42−, Cl−, Br−, NO3−, or I−). Influx of anion X− was accompanied by OH− efflux (antiport) or H+ influx (symport).29 The resulting intravesicular acidification leads to a decrease in the HPTS emission, the relative extent of which is an indicator of the anion transport activity. Figure 3 shows that the transport kinetics is strongly dependent on the identity of the anion. Interestingly, the anion transport selectivity follows the Hofmeister series, I− > NO3− > Br− > Cl− > SO42− (Figure 3a): the highest transport activity

Figure 2. Normalized emission intensity of chloride-sensitive lucigenin dye (λex = 455 nm, λem = 506 nm) with different concentrations of PB1A (mol % of PB-1A relative to total lipid concentration). Inset: schematic representation of vesicles showing a proposed anion antiport mechanism.

Figure 3. Comparison of anion transport activity of PB-1A determined by HPTS assay. (a) Normalized fluorescence intensity ratio (I460/I403) of the HPTS emission at λem of 510 nm. (b) The plot of dehydration free energy30 vs initial rate constants of anion transport (see SI for detailed analysis). 7433

DOI: 10.1021/jacs.7b02708 J. Am. Chem. Soc. 2017, 139, 7432−7435

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Journal of the American Chemical Society

yellow fluorescent protein (YFP-H148Q/I152L) gene32 (hereafter termed YFP-HEK-293T cells). Figure 5a shows the

was observed from the weakly hydrated iodide and the lowest from the strongly hydrated sulfate. We emphasize that PB-1A transports the iodide about 60 times more efficiently than chloride, the most abundant biological anion (Figure 3b and Table S2). The correlation between the anion hydrophobicity and transport activity implies that the dehydration of the anions is crucial for their transport through PB-1A. The inverse linear relationship between dehydration free energy and initial transport rate constants (Figures 3b and S8) supports that dehydration of the anions controls the anion transport activity through PB-1A. The differences in the interactions between the anions and the alkyl chains lining the windows of PB-1A may also contribute to the different anion transport activities. Voltage clamp measurements were performed to gain a better understanding of the ion transport process of PB-1A in a planar lipid bilayer. Figure 4a shows a long-lived single-channel

Figure 5. (a) Schematic representation of iodide transport in YFPHEK-293T cells, and (b) YFP-HEK-293T cell images (A−D). Untreated cells in PBS buffer (A), after 1 h incubation with PB-1A (B), after 10 min treatment with NaI (C), and after the removal of extracellular NaI (D). Scale bar is 50 μm.

outline of the cell experiment (see SI for detailed experimental procedures). YFP-HEK-293T cells were treated with PBS buffer solution containing channel PB-1A (in THF) (Figure 5b, B). An extracellular iodide gradient was applied to the incubated live cells by addition of NaI (10 mM). Quenching of the YFP fluorescence observed in the PB-1A treated cells indicated iodide influx across the membrane of YFP-HEK-298T cells (Figure 5b, C). The control experiments without PB-1A and only in the presence of THF showed no quenching after the addition of NaI (Figure S14). Recovery of YFP fluorescence by replacement of extracellular iodide with chloride suggests that the iodide transport was reversible (Figure 5b, D). Taken together, these results demonstrate that PB-1A can be stably incorporated into cell membranes and can then mediate the transport of iodide across the cell membrane. In summary, we have introduced a new class of unimolecular synthetic ion channels from a 3D, shape-persistent organic cage, PB-1A, which was synthesized by employing a synthetically economical DCC approach. PB-1A not only displays a transport preference for anions over cations but also discriminates among the inorganic anions, showing the most pronounced transport selectivity for iodide. The correlation observed between anion transport activity and hydrophobicity demonstrates that the dehydration of the anions is crucial for their transport through PB-1A. Distinct open and closed states of PB-1A were observed by single channel electrophysiological measurements, which suggests that the transport occurs by a channel mechanism. The stable incorporation of PB-1A into a cellular membrane allows the efficient transport of iodide across the membranes of live cells. This result suggests that this material may have biological applications such as diagnosis and treatment of iodide transport disorders, including some cancers. Taking advantage of the synthetic tractability of the porphyrin units may allow the development of stimuli-responsive iodide transport systems. Work along this line is currently underway in our laboratory.

Figure 4. (a) Typical single-channel current profile measured with PB1A at 180 mV in symmetrical KCl (2 M) solution. (b) A histogram of the currents at 180 mV.

current profile with distinct open and closed states. This result indicates that PB-1A acts as a dynamic ion channel. The single channel conductance obtained at high voltage was found to be about 18 pS (Figure 4), which seems reasonable considering the window size of PB-1A (averaged diameter ∼3.7 Å, including alkyl chains) estimated from the X-ray crystal structure of the closely related compound (see SI, Figures S1 and S2). This conductance value is two times smaller than that of MOP-18 (36 pS) which has two different sized windows (3.8 and 6.6 Å for 8 triangular and 6 square windows, respectively) with a Hille diameter of 5.4 Å.20 The single channel conductance data are highly reproducible, as we obtained similar results from multiple experiments performed on different days. Interestingly, the current−voltage profile of PB-1A presents a nonlinear relationship (Figure S13). Although unusual, such nonohmic behavior of ion transporters is not unprecedented for ion channels or pores.31 The outward rectification at high voltages could be due to reduction of energetic barriers for opening the molecular windows. Further studies including investigation of the apparent nonohmic behavior of the PB-1A mediated ion transport are currently underway. Finally, we inserted PB-1A into the membrane of live cells to see if it could facilitate transmembrane iodide transport in the living systems. Human embryonic kidney (HEK-293T) cells were transfected with a plasmid containing iodide-sensitive 7434

DOI: 10.1021/jacs.7b02708 J. Am. Chem. Soc. 2017, 139, 7432−7435

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(20) Jung, M.; Kim, H.; Baek, K.; Kim, K. Angew. Chem., Int. Ed. 2008, 47, 5755. (21) Hong, S.; Rohman, M.; Jia, J.; Kim, Y.; Moon, D.; Kim, Y.; Ko, Y. H.; Lee, E.; Kim, K. Angew. Chem., Int. Ed. 2015, 54, 13241. (22) (a) Hasell, T.; Cooper, A. I. Nat. Rev. Mater. 2016, 1, 16053. (b) Zhang, G.; Mastalerz, M. Chem. Soc. Rev. 2014, 43, 1934. (23) Metal−organic complexes made from Pd metal are kinetically labile. See: (a) Wilson, C. P.; Boglio, C.; Ma, L.; Cockroft, S. L.; Webb, S. J. Chem. - Eur. J. 2011, 17, 3465. (b) Fujita, M.; Umemoto, K.; Yoshizawa, M.; Fujita, N.; Kusukawa, T.; Biradha, K. Chem. Commun. 2001, 6, 509. (24) Despite numerous attempts, we have not been able to obtain high quality single crystal X-ray diffraction data for PB-1A; however, the overall structure of PB-1A including the cavity and window sizes is similar to that of the corresponding porphyrin box unit in Zn-PBDPB. (25) (a) McNally, B. A.; Koulov, A. V.; Smith, B. D.; Joos, J.; Davis, A. P. Chem. Commun. 2005, 8, 1087. (b) McNally, B. A.; O’Neil, E. J.; Nguyen, A.; Smith, B. D. J. Am. Chem. Soc. 2008, 130, 17274. (26) After finishing the lucigenin and HPTS assays, PB-1A was recovered from aqueous buffer solution by precipitation and its structure was still intact as confirmed by NMR spectroscopy (Figures S16 and S17). (27) Sakai, N.; Matile, S. J. Phys. Org. Chem. 2006, 19, 452. (28) Lisbjerg, M.; Valkenier, H.; Jessen, B. M.; Al-Kerdi, H.; Davis, A. P.; Pittelkow, M. J. Am. Chem. Soc. 2015, 137, 4948. (29) Milano, D.; Benedetti, B.; Boccalon, M.; Brugnara, A.; Iengo, E.; Tecilla, P. Chem. Commun. 2014, 50, 9157. (30) (a) Nightingale, E. R. J. Phys. Chem. 1959, 63, 1381. (b) Tansel, B. Sep. Purif. Technol. 2012, 86, 119. (31) Jones, J. E.; Diemer, V.; Adam, C.; Raftery, J.; Ruscoe, R. E.; Sengel, J. T.; Wallace, M. I.; Bader, A.; Cockroft, S. L.; Clayden, J.; Webb, S. J. J. Am. Chem. Soc. 2016, 138, 688. (32) 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. 2015, 8, 24.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/jacs.7b02708. Experimental details, single-channel conductance data, Xray single-crystal structure (PDF) Crystallographic data (CIF)

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AUTHOR INFORMATION

Corresponding Authors

*[email protected] *[email protected] ORCID

Joon Ho Roh: 0000-0002-5564-6779 Kimoon Kim: 0000-0001-9418-3909 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Drs. Kyeng Min Park and James Murray for helpful discussions. This work was supported by the Institute for Basic Science (IBS) [IBS-R007-D1].

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DOI: 10.1021/jacs.7b02708 J. Am. Chem. Soc. 2017, 139, 7432−7435