COMMUNICATION

Blockable Zn10L15 ion channels via subcomponent self-assembly Cally J. E. Haynes,+[a] Jinbo Zhu,+[b] Catalin Chimerel,[b] Silvia Hernández-Ainsa,[b] Imogen A. Riddell,ǂ[a] Tanya K. Ronson,[a] Ulrich F. Keyser*[b] and Jonathan R. Nitschke*[a]

Abstract: Metal-organic anion channels based on Zn10L15

pentagonal prisms have been prepared by subcomponent self-assembly. The insertion of these prisms into lipid membranes was investigated by ion-current and fluorescence measurements. The channels were found to mediate the transport of Cl– anions through planar lipid bilayers and into vesicles. Toluenesulfonate anions were observed to bind and plug the central channels of the prisms in the solid state and in solution. In membranes, dodecylsulfate blocked chloride transport through the central channel. Our Zn10L15 prism thus inserts into lipid bilayers to turn on anion transport, which can then be turned off through addition of the blocker dodecylsulfate.

The structure of the cell membrane phospholipid bilayer enables it to function as a selectively permeable barrier. The controlled movement of substances between compartments and the establishment of transmembrane gradients, mediated by membrane bound proteins, is essential to biosynthetic pathways and the maintenance of normal cellular function. [1] The breakdown of ion transport pathways causes an array of undesirable effects at the cellular level, manifesting as a wide variety of disease states, or channelopathies.[2] Research into the design of synthetic anion channels and carriers [3] has thus been motivated by the potential application of these systems as therapeutics, and in sensing and nanotechnology.[4]

Understanding and being able to control compartmentalization is also crucial for the development of complex synthetic systems, with controlled flow of material between compartments providing a means for the interconnection of and communication between system components.[5]

Naturally occurring chloride channels utilize favorable electrostatic environments to promote anion transport and to select anions over cations.[6] Artificial anion channels adopt these principles, incorporating functionalities able to interact non-covalently with anions during transport, thus energetically compensating for the dehydration penalty. A synthetic ion channel must also span the membrane. Although large synthetic molecules can span a bilayer to mediate ion transport, [7] self-assembly has emerged as an attractive strategy to construct complex membrane-spanning structures from smaller, more accessible building blocks. Small organic molecules have been

shown to associate in the bilayer environment to form membrane-spanning ion channels,[8] while DNA and peptides have also served to build transmembrane channels.[8d, 9]

Metal-organic assemblies have been used in biological contexts as therapeutics,[10] probes,[11] and catalysts.[12] Certain complex multimetallic architectures[13] can self-assemble biomimetically,[14] and others have been shown to function as self-assembling anion channels,[15] with the assembly and disassembly of metal-organic channels having been shown to function as a gating mechanism.[16] A challenge in this area is to achieve control over synthetic ion transport systems, either through a ligand-gating “turn on” response,[7e, 16-17] or binding to blockers in a “turn off” response.[7e, 17b, 18] Here we show that appropriately functionalized pentagonal-prismatic Zn10L15

complexes can insert into bilayers so as to act as synthetic ion channels. Ion transport through these prismatic channels was turned off upon addition of blocking dodecylsulfate.

Figure 1. (a) Subcomponent self-assembly of 1a and 1b. (b) Single-crystal X-ray structure of 1b binding two tosylate anions, whose C atoms are shown in purple. H-atoms and disorder are omitted for clarity. ZnII ions are connected with yellow lines to show the overall geometry. Other atoms: gray (C), blue (N), red (O), yellow (S), green (Cl), yellow spheres (Zn), red sphere (Br).

[a] Dr C. J. E. Haynes, Dr I. A. Riddell, Dr T. K. Ronson and Prof. J. R. NitschkeDepartment of Chemistry, University of Cambridge,Lensfield Road, Cambridge CB2 1EW, UK.E-mail: jrn34@cam.ac.uk

[b] Dr J. Zhu, Dr C. Chimerel, Dr S. Hernández-Ainsa and Prof. U. F. Keyser,Cavendish Laboratory, University of Cambridge,JJ Thompson Avenue, Cambridge CB3 0HE, UK.E-mail: ufk20@cam.ac.uk

+ These authors contributed equally to this work.ǂ Current address: School of Chemistry, University of

Manchester, Oxford Road, Manchester, M13 9PL UK.

Supporting information for this article is given via a link at the end of the document.

COMMUNICATION Subcomponent self-assembly, in which aldehyde and

amine subcomponents come together around metal-ion templates, has been used to generate complex structures in a modular and hence tunable fashion by other groups[19] and ourselves.[20] Examples of such structures have been shown to be water- and buffer-stable.[21] We previously reported that the pentagonal-prismatic Zn10L15 architecture (1) is templated by five perchlorate anions that within the peripheral binding pockets, housing a sixth halide anion in a central, channel-like cavity that is lined with a spherical array of ten C-H hydrogen bonds.[20a, 20b]

In the present study, we observed 1 also to bind tosylate anions above and below the halide anion at the entrance to the central channel, shown in the crystal structure of Figure 1c, [22] in solution and in the solid state. In the crystal, 1 contains a central channel-like cavity with a diameter of ca. 2.3 Å. 1H NMR titrations (SI Section 12) support a 2:1 tosylate: Zn10L15 binding mode, with the imine and 5,5’-bipyridyl signals assigned to those protons closest to the solid-state tosylate binding site exhibiting the greatest change in chemical shifts. Prism 1 thus is able to bind three different types of anions in distinct binding pockets.

The toroidal structure of 1, with its central tubular hole, evokes naturally occurring ion channels. We therefore synthesized and characterized an analog of 1 incorporating long alkyl chains on its aniline residues, reasoning that the alkyl substituents would enhance the lipophilicity of the Zn10L15

complex and increase its dimensions to match the thickness of a lipid bilayer. We also hypothesized that the binding of sulfonate anions above and below the central pocket could lead to blocking behavior, as observed in natural anion channels.[8h]

The reaction of 4-nonylaniline with A, Zn(ClO4)2 and KBr in CD3CN generated Br-Zn10L15 (1a) as the uniquely-observed product by NMR and ESI-MS (SI Figures S2-S4). Including the flexible nonyl chains, we estimate the axial length of 1a to be up to 42 Å (see SI Section 15), congruent with the thickness of the lipid bilayers used in this work.[23]

Ionic current measurements indicated that 1a inserted into planar lipid bilayers[24] composed of 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC).[24] A planar DPhPC lipid bilayer was prepared to span a hole in a PTFE film, which separated the two electrolyte chambers (0.2 M KCl) of our experimental cell. Following the addition of 1a in MeCN (10 L of 0.2 mM) from the cis side with gentle stirring, distinct single-channel currents were observed at an applied voltage of 50 mV across the membrane (Figure 2b).

We also performed negative control experiments (Figure S12). We tested pure acetonitrile, a solution of just the organic ligand subcomponents, a Zn6L8 extended grid structure formed from the same organic ligands with Zn(OTf)2,[20b] and a mixture of the organic ligands with Zn(ClO4)2 in the absence of a templating bromide anion. No ionic current flow was observed in any of these cases.

A histogram of the conductance steps observed, with Gaussian maximum of 0.19 nS, is shown in Figure 2c. We hypothesize that the range of conductance states and fluctuations in ionic current observed could be caused by aggregation and tumbling of the cages. The possibility of dynamic reconfiguration into larger (cf. Zn6L18) pores also cannot be excluded based on the available analytical techniques, although such structures have never been observed for zinc, only cadmium.[25]

When a stable insertion of 1a was observed during the current recording, a voltage ramp from -150 mV to +150 mV was applied to obtain a current-voltage (I-V) curve (Figure 2d),

showing that the ion channel exhibits ohmic behavior. The slope of the I-V curve gives a conductance of 0.15 nS.

Figure 2. (a) Schematic representation of 1a within a lipid bilayer; equatorial chains are omitted for clarity. (b) Ionic current recording across a planar DPhPC bilayer containing 1a in 0.2 M KCl, 5 mM Tris, pH 7.5, demonstrating the insertion and gating of 1a at 50 mV. (c) Histogram of conductance obtained from the insertions and closures of the channel and a Gaussian fit to the data. (d) Current (I) vs. voltage (V) curve obtained during the stable insertions of 1a, averaged from four measurements of distinct insertions. Error bars represent standard deviations.

We next assessed the ion transport activity of 1a using the established HPTS (8-hydroxypyrene-1,3,6-trisulfonic acid) fluorescence assay (SI Scheme S14).[8b, 26] Large unilamellar vesicles (LUVs) with ~200 nm diameter that encapsulated pH-sensitive HPTS were prepared from POPC (2-oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine). The fluorescence intensity of HPTS at 510 nm increased with pH, enabling the transport of H+

or OH- through the LUV membranes to be readily monitored. Before the addition of 1a, a pH gradient (ΔpH = 0.8) across the vesicle membrane was generated by adding NaOH to the extravesicular buffer, raising the pH to 7.5. HPTS fluorescence enhancement was observed once 1a was added (Figure 3a), consistent with 1a-mediated transport of H+ or OH- across the membrane. The LUVs were lysed following completion of the measurement by addition of the detergent Triton X-100, giving the final HPTS fluorescence intensity for normalization.

Figure 3. a) Change in fluorescence intensity (FI) of HPTS (λex = 450 nm, λem = 510 nm) on the addition of increasing concentrations of 1a to POPC-LUVs HPTS. Solution inside liposomes: 1 mM HPTS, 10 mM HEPES, 100⊃ mM NaCl, pH = 6.7; solution outside liposomes: 10 mM HEPES, 100 mM NaCl, pH = 7.5; b) Hill analysis showing the change in fluorescence intensity as a function of 1a concentration.

COMMUNICATION The increase in HTPS fluorescence intensity was shown to

depend upon the concentration of 1a, increasing six-fold within the range 0.8 – 12 µM. Based on the dose-response curve shown in Figure 3b, an EC50 (the effective concentration required to give half of the maximum response) for 1a of 5.02 μM was obtained. We observed that 1a selectively transports halide anions over larger anions (NO3

-, SO42-, ClO4

-, tosylate) (SI section 9). A halide transport preference was observed from Cl-

< Br- < I-, suggesting that the channel also discriminates based on the hydration penalty. Sulfate anions were not observed to interact with 1a in the same way as organic sulfonate guests (SI Figures S23-S27), which we infer to result from the larger sulfate dehydration penalty, which also commonly inhibits its transport.[27] In contrast, changing the cation from Na+ to Li+, K+ or Rb+ did not alter the observed transport, (SI Figure S15b).

The transport of Cl– into POPC-LUVs was also investigated by encapsulating the fluorescent indicator lucigenin, whose fluorescence emission at 505 nm is quenched by halide ions inside the vesicles (SI Scheme S18).[28] In order to avoid a false positive signal arising from the small amount of Br– template, 1a was premixed with the POPC before formation of the LUVs. Following the addition of NaCl in the extravesicular buffer to induce a 24 mM chloride gradient across the lipid membrane, a more rapid decrease in fluorescence was observed in the presence of 1a, (“group 0” in Figure 4b and SI Figure S19) as compared with the blank group. This observation is consistent with the insertion of 1a gating the influx of Cl– into vesicles.

Based on the crystal structure of 1 (Figure 1c) we hypothesized that transport could be controlled by blocking the entrance and exit of ions to membrane embedded channels using non-transported anions that bound in the manner of tosylate. Although pre-mixing 1a with n-tetrabutylammonium (TBA) tosylate in acetonitrile led to a turning off of ion transport in planar lipid bilayer experiments, functioning 1a was not blocked following the addition of tosylate to the buffer once 1a had partitioned into the lipid bilayer. This observation suggested that tosylate was not amphiphilic enough to interact with membrane-bound 1a. We thus turned to amphiphilic dodecylsulfate, which we hypothesized would be more likely to partition into the bilayer in order to interact with 1a.

Initial 1H NMR titrations of TBA dodecylsulfate into an CD3CN solution of 1a suggested binding in a similar fashion to tosylate (SI Figures S23-24). Subsequent UV-Vis titrations were used to measure the binding between 1a and the TBA salts of dodecylsulfate and tosylate in octanol. Analysis of the data based on a 1:2 non-cooperative binding model using Bindfit [29] indicated that dodecylsulfate bound twice as strongly (K11 = 1.02 × 105 M-1 ± 2.7%) as tosylate (K11 = 4.8 × 104 M-1 ± 1.1%) (see SI Section 14), possibly due to favorable interactions between the alkyl chains of 1a and dodecylsulfate.

Crucially, when sodium dodecylsulfate (SDS) was added to systems in which 1a had partitioned into a planar lipid bilayer, anion transport was impeded. Blocking of the ion-channel function of 1a by SDS was measured using both the lucigenin assay and ionic current recordings (Figure 4b, c). Since SDS is known to act as a surfactant, we also checked the effect of SDS on the stability of the lipid membranes. We found that adding SDS (114 M) did not disrupt the planar lipid bilayers and LUVs used in this work (SI Figure S20).

Blocking of 1a as an ion channel was studied by premixing different concentrations of SDS with lucigenin POPC-⊃ 1a-LUVs and incubating the solutions for 10 min before recording fluorescence intensities.

As shown in Figure 4b, chloride transport was blocked by SDS in a dose-dependent manner. During current recordings, 10 to 20 µL of 4 mM SDS were added to both sides of the membrane when a stable insertion was obtained. Example current traces are given in Figure 4c and SI Figure S22. The ion channel was observed to close within 20 seconds in response to the addition of SDS.[30]

Figure 4 a) Schematic representation of the proposed interaction of dodecylsulfate anions with 1a (equatorial chains have been omitted for clarity). Color scheme: gray (C), red (O), yellow (S), green (Cl), yellow spheres (Zn), dark red spheres (Br), Dodecylsulfate C-atoms are shown in purple; (b) bar graph of the relative fluorescence change of lucigenin encapsulated in POPC-1a-LUVs mediated by 0.75 mol% 1a (no 1a was preset in the blank group) with increasing concentrations of SDS. I is the fluorescence change 5 minutes after the addition of NaCl, and I0 is the fluorescence change of the vesicles with no SDS after 5 minutes; (c) an example current trace showing the “switching off” of the channel activity of 1a after the addition of sodium dodecylsulfate.

In conclusion, we have constructed metal-organic cage ion channels from simple organic precursors and metal salts through subcomponent self-assembly. The channels were found to mediate the passage of chloride across POPC and DPhPC bilayers. The transport activity was modulated by the addition of SDS, which is inferred to bind and block the channel.

Larger prismatic channels produced through the same method[31] may serve to gate more complex substrates between membrane-separated spaces. These channels could thus serve as crucial building blocks for the construction of complex and functional abiological chemical systems, in which subsystems are spatially distinct, as with living cells.

Additional research data supporting this publication are available as Supplementary Information at the journal's website.

Acknowledgements

This work was supported by the UK Engineering and Physical Sciences Research Council (EPSRC, EP/M008258/1). Mass spectra were provided by the EPSRC UK National Mass Spectrometry Facility at Swansea University.

COMMUNICATION Keywords: synthetic ion channel • lipid membrane • subcomponent self-assembly • supramolecular chemistry • metal-organic cages

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COMMUNICATION

Entry for the Table of Contents

COMMUNICATION

Zn10L15 pentagonal prisms prepared by subcomponent self-assembly can function as anion channels in phospholipid bilayers. The channels are blocked by amphiphilic dodecylsulfate anions, which bind and plug the central cavities of the channels.

Cally J. E. Haynes, Jinbo Zhu, Catalin Chimerel, Silvia Hernández-Ainsa Imogen A. Riddell Tanya K. Ronson, Ulrich F. Keyser* and Jonathan R. Nitschke*

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Blockable Zn10L15 ion channels via subcomponent self-assembly