Letter pubs.acs.org/OrgLett
Bis-15-crown-5-ether-pillar[5]arene K+‑Responsive Channels Wei-Xu Feng,†,‡ Zhanhu Sun,‡ Yan Zhang,‡ Yves-Marie Legrand,‡ Eddy Petit,‡ Cheng-Yong Su,† and Mihail Barboiu*,†,‡ †
Lehn Institute of Functional Materials, School of Chemistry and Chemical Engineering, Sun Yat-Sen University, 135 Xingang West Road, Guangzhou 510275, China ‡ Adaptive Supramolecular Nanosystems Group, Institut Europeen des Membranes, ENSCM/UMII/UMR-CNRS 5635, Place Eugene Bataillon, CC 047, 34095 Montpellier, Cedex 5, France S Supporting Information *
ABSTRACT: An artificial selective K+ channel is formed from the supramolecular organization on bis(benzo-15-crown-5ether-ureido)-pillar[5]arene compound. This channel achieves a selectivity of SK+/Na+ = 5 for an initial transport rate of kK+ = 3.2 × 10−3 s−1. The cation-file diffusion occurs via selective macrocyclic-filters anchored on inactive supporting pillar[5]arene relays. The sandwich-type binding geometry of the K+ cation by two 15-crown-5 moieties sites is a key feature influencing channel efficiency.
T
diffusion via site-to-site jumping along the crown sites. We recently discovered that stacked benzo-15-crown-5 sandwichtype binding sites can induce the formation of highly selective K+ channels, recovering the hydration sphere of the K+ cations against the imperfectly coordinated Na+ cations. Several biomimetic K+ responsive channels have been designed to contain a macrocyclic unit, a H-bonding self-assembling moiety, and a hydrophobic tail at the interface with the bilayer membrane.20−22 The pillar[5]arene molecular platform attracts significant attention because of its predictable and rigid conformation and easy functionalization.23 Recently, pillar[5]arene has been used as a central relay for unimolecular constructs for water,24,25 protons,26 or amino-acid channels.27 Cation translocation via pillar[5]arene-based channels, to the best of our knowledge, have been considered a secondary process in the construction of highly permeable water channels.24,25,28 Herein, we report the synthesis of bis(benzo-15-crown-5ether-ureido)-pillar[5]arene compound 5. It combines a central pillar[5]arene scaffolding relay/platform with two benzo-15crown-5 cation binding sites. Compound 5 is spontaneously inserted into lipid bilayers and forms ion-channels showing well-defined ionic conductance behaviors. These channels are able to slowly transport smaller Li+, fit Na+, and bigger Cs+ cations, while the medium-sized K+ and Rb+ cations with optimal diameters for forming sandwich complexes with two benzo-15-crown-5 macrocycles are selectively transported with higher transport rates. It is important to note the significant transport activity of K+ cations, which are highly preferred over Na+ cations. This is a novel interesting example of a selective
ransmembrane ionic transport is a vital process for living cells.1 It controls cell membrane potentials, underlying many biological functions such as the transmission of nerve influx, muscular contraction, or metabolite neuronal exchanges.2,3 Natural systems have solved the selectivity issues by combining optimal coordination with adaptive functional dynamics of the binding sites around the cation.5 Because the structure of the KcsA K+ natural channel was elucidated, we know that the translocation of K+ is regulated by the selectivity filter of KcsA, providing closely spaced carbonyl sites for perfect coordination of the K+, whereas the smaller Na+ cations are incompletely coordinated.4 Inspired by the development of interesting applications in blood and biological fluids dialysis, synthetic artificial K+ carrier or channel structures have been designed and reported.6−11 Highly selective molecular receptors like cryptands strongly bind the cations. They are usually less effective transporters but reduce the dynamics needed for cation translocation.6 Flexible lariat-crown-ether,7 barrel-stave,8 or calixarene9 systems have used to overcome these limits by combining selective binding and dynamic behaviors for effective functional transport. “Chundle”,10 “unimolecular”,11 and “hydraphile” channels have been designed to contain polar crown-ether12 or calixarene13 relays in the middle of the bilayer that act as a central scaffold for the various termini arms, spanning the membrane toward the polar ends of the bilayers and playing the role of gatekeepers at the entry and exit of the channel. Another strategy involves macrocyclic molecules that can be self-assembled into oligomeric channels spanning the bilayer membrane structure. Peptide-appended14 or ureido-crown ethers 15−19 may form such active membrane-spanning channel-like superstructures, acting as the directional polar cation-binding relays in the hydrophobic bilayer environment. The translocation mechanism occurs through cation-file © XXXX American Chemical Society
Received: February 6, 2017
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DOI: 10.1021/acs.orglett.7b00352 Org. Lett. XXXX, XXX, XXX−XXX
Letter
Organic Letters artificial channel with a very simple structure that is responsive to K+ cations. The syntheses of channel-forming bis(benzo-15-crown-ether5-ureido)-pillar[5]arene 5 and of the reference nonmacrocyclic compound bis(benzo-ureido)-pillar[5]arene 6 are presented in Scheme 1. 1,4-Bis(2-bromo-ethoxy)benzene 129 was reacted Scheme 1. Synthesis Protocols for Compounds 2−6
with 1,4-dimethoxybenzene to provide bis(2-bromethoxy)pillar[5]arene 2 and then converted to bis(2-azidoethoxy)pillar[5]arene 3 and bis(2-aminoethoxy)-pillar[5]arene 4, which was finally reacted in situ with 4-amino-benzo-15crown-5-ether to provide compound 5. Reference control compound 6 was synthesized by the reaction of 4 with isothiocyanatobenzene. The spectroscopic and analytical data of 1−6 are in accordance with the proposed structures (Figures S1−S10). 1 H NMR dilution experiments were carried out to analyze the self-assembly behavior of 5 in solution. The chemical shift of Ha of the urea group shows upfield chemical shifts at low concentration until reaching a plateau, and then the signal shifts back to lower field (Figure 1a). We assume that the process occurring at the plateau concentrations is the formation of a dimer from monomers. Then, the dimer would be essentially fully formed below 12 mM, where oligomeric species emerge with increasing concentration of 5. We have conducted the same experiments in the presence of excess KTf or NaTf (Tf = CF3SO3−) (Figure 1b, Figure S11 and S12). We observe only downfield shifts of NHa protons with increasing concentration of 5, indicating self-association via intermolecular urea-urea or urea-anion H-bonding.16 Higher values of downfield chemical shifts of proton NHa observed in the presence of KTf when compared with the presence NaTf indicate that the oligomeric species 5-KTf with K+ are more stable than that with 5-NaTf. Furthermore, to gain more insight into the self-assembly process, we compared the chemical shifts of aromatic Hb and Hc protons next to the crown ethers: for 5KTf, the chemical shifts were observed at 6.84 ppm as a single peak at 32 mM, whereas they are upfield at 6.76 ppm upon dilution at 9.6 mM (Figures S11 and S12). On the contrary, for 5-NaTf, no chemical shifts were observed for Hb and Hc at different concentrations. This is reminiscent of the formation of the oligomeric structures in solution resulting from synergetic self-assembly of K+(15crown-5)2 sandwich aggregates and anion-urea H-bonding.30
Figure 1. Chemical shifts of the N−Ha protons plotted against increasing concentration of compound 5 in the (a) absence or (b) presence of 125 mM of NaTf or KTf in CD3CN at 293 K.
The close proximity of 15-crown-5 macrocycles favors crownether-cation contacts recovering the hydration sphere of the cation and favoring the selective binding of K+ cations against the equatorially coordinated Na+ cations. This results in less connectivity, suggesting less effective self-assembly processes of 5-NaTf oligomers. Subsequently, we use pH gradient assays to investigate the bilayer transport activities of compounds 5 and 6 that were codissolved together with L-α-phosphatidyl-choline before the formation of the vesicles (LUV).31−34 The transport data show that compound 5 is highly active toward K+ and Rb+, slightly active toward Cs+ (Figure 2a, Figures S18 and S21), and practically inactive for the Na+ and Li+ (Figure 2b, Figure S15). For example, at 50 μM of compound 5, 50% of K+ and only 11% of Na+ were transported at 500 s. Figure 3 shows the activities of 5 toward different alkali metal cations, which clearly emphasizes a selectivity of SK+/Na+ = 5 with an initial transport rate for K+ of kK+ = 3.2 × 10−3 s−1. This rate is the on the same order of magnitude measured under the same conditions for cholesteryl-thioureido-15-crown-5-ether20 (kK+ = 3.9 × 10−3 s−1) and one order of magnitude lower than squalyl-amido-15-crown-5-ether21 (kK+ = 87.5 × 10−3 s−1) or B
DOI: 10.1021/acs.orglett.7b00352 Org. Lett. XXXX, XXX, XXX−XXX
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Organic Letters
Table 1. EC50, Hill Factors n, Percent Transport, and Initial Transport Rates of Compound 5 (110 μM) towards Alkali Cations EC50 [μM] n transport [%] k × 103 [s−1] a
Li+
Na+
K+
Rb+
Cs+
a 0.48 14 0.6
a 0.57 19 0.5
38.4 1.25 66 3.1
41.2 1.01 51 2.6
a 0.61 29 0.8
No EC50 for these cations due to low transport activity.
respectively, reminiscent of class (I) channels,33,34 which means one or more molecules are responsible for the formation of the channel. For the other cations Li+, Na+, and Cs+ showing low transport activity, the Hill coeficients are Rb+ > Cs+ > Na+ > Li+, corresponding to Eisenman sequence IV,35 which is indicative of the binding to the crown ether playing a more prominent role as compared to that of dehydration. Conversely, the control reference compound 6 free of macrocylic binding moieties shows slight transport activities and can even be considered as inactive toward all of the cations (Figures S26−S30). In conclusion, the bis(benzo-15-crown-5-ether-ureido)pillar[5]arene self-assembled in the lipid bilayer membranes to form active channels that are responsive to K+ cations. The 15-crown-5-ether head groups can undoubtedly be recognized as contributing on the selective cation-binding function of the channel, and the pillar[5]arene relays are playing the role of supporting groups that must be considered in the design of a synthetic channel. This behavior is consistent with the probable formation of a range of channel-type oligomers within the bilayer membrane that can be considered as active directional relays. The cation-file diffusion is occurring via successive siteto-site jumping along the optimally disposed macrocyclic crown sites spatially disposed by inactive pillar[5]arene relays. The benzo-15-crown-5 macrocycles act as exceptional hydrophilic lubricants, replacing the hydration sphere of K+ cations in the hydrophobic bilayer membrane environment. In contrast, for the smaller Na+ cation, the dehydration is not completely compensated meanwhile for forming a stable channel. Even the exact transport mechanism is difficult to completely describe by these data, we presume that the sandwich coordination of 15-crown-5 ether moieties toward K+ cations may again be considered an important structural behavior strongly determining the observed K+/Na+ selectivity.20−22 The transport activity detected with compound 5 is actually novel direct evidence of a paradigm in which a sandwich complex between crown-binding sites and the cations does not inhibit or lower the conductance of a stacked macrocyclic channel.
Figure 2. Transport of (a) K+ and (b) Na+ cations trough bilayer membranes containing compound 5 channels as determined in a pH gradient assay as a function of time.
Figure 3. Pseudo first-order rate constants k for the transport of alkali cations through LUVs containing 50 μm of 5 in the vesicle stock solution.
hexyl-amidobenzo-15-crown-5-ether22 (kK+ = 175 × 10−3 s−1). This indicates relatively low fluidity, which in turn indicates the formation of robust channel-type superstructures of 5 in the bilayer membrane. It is worth noting that the macrocyclic crown ether moieties are the only possible sites that can interact with the cations. For further insights into the transport mechanism to be obtained, Hill analysis was performed (Table 1). The Hill coefficient n towards K+ and Rb+ cations is 1.25 and 1.01,
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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.7b00352. Synthesis, NMR, and bilayer transport data (PDF) C
DOI: 10.1021/acs.orglett.7b00352 Org. Lett. XXXX, XXX, XXX−XXX
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(30) Cazacu, A.; Legrand, Y. M.; Pasc, A.; Nasr, G.; Van der Lee, A.; Mahon, E.; Barboiu, M. Proc. Natl. Acad. Sci. U. S. A. 2009, 106, 8117− 8122. (31) Barboiu, M.; Le Duc, Y.; Gilles, A.; Cazade, P. A.; Michau, M.; Legrand, Y.-M.; van der Lee, A.; Coasne, B.; Parvizi, P.; Post, J.; Fyles, T. Nat. Commun. 2014, 5, 4142. (32) Haynes, C. J.; Busschaert, N.; Kirby, I. L.; Herniman, J.; Light, M. E.; Wells, N. J.; Marques, I.; Felix, V.; Gale, P. A. Org. Biomol. Chem. 2014, 12, 62−72. (33) Matile, S.; Sakai, N. In Analytical Methods in Supramolecular Chemistry; Schalley, C. A., Ed.; Wiley-VCH: Weinheim, Germany, 2007; pp 381−418. (34) Bhosale, S.; Matile, S. Chirality 2006, 18, 849−856. (35) Eisenman, G.; Horn, R. J. Membr. Biol. 1983, 76, 197−225.
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Zhanhu Sun: 0000-0002-1207-781X Cheng-Yong Su: 0000-0003-3604-7858 Mihail Barboiu: 0000-0003-0042-9483 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was conducted within ANR-15-CE29-0009 DYNAFUN and the Recruitment Program of Global Experts (1000 Talent Plan, WQ20144400255) of SAFEA, China.
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DOI: 10.1021/acs.orglett.7b00352 Org. Lett. XXXX, XXX, XXX−XXX
