Fluorinated Bambusurils as Highly Effective and Selective Transmembrane Cl−/HCO3− Antiporters

Abstract

•Fluorinated bambusuril macrocycles function as effective Cl−/HCO3− antiporters•Very high anion affinities can have detrimental effects on transport•Two anions can be complexed simultaneously if they can interact with each other via H bonding•The ability to bind two anions simultaneously allows their efficient exchange In biology, the transport of ions across lipid membranes is crucial and is generally performed by membrane proteins. Deficiencies in transport are at the origin of various diseases, such as cystic fibrosis. In this context, synthetic anion carriers incorporated within the lipid bilayer could play a remedial role. They extract ions from one side of the membrane, move across, and release the ions on the other side. To function as an effective carrier, a balance must be found between their ability to extract and release. Here we demonstrate that bambusuril macrocycles bind nitrate so strongly that it limits the rate of transport. However, the fact that bambusurils can simultaneously bind chloride and bicarbonate leads to an efficient exchange of these ions. The mechanistic understanding provided here will help the development of anion transporters for biomedical applications. The exchange of chloride and bicarbonate across lipid bilayers is an important biological process. Synthetic molecules can act as mobile carriers for these anions, although most show little selectivity. Here we report on three bambus[6]uril macrocycles functionalized with fluorinated benzyl groups, which are able to exchange Cl− and HCO3− efficiently. Remarkably, rates for Cl−/NO3− exchange are two orders of magnitude lower. The higher rates of Cl−/HCO3− transport can be explained by the ability of the bambusurils to complex Cl− and HCO3− simultaneously, facilitating their exchange at the bilayer interface. Furthermore, the exceptionally high affinity and selectivity of these systems for NO3− appear to contribute to the poor Cl−/NO3− exchange. This work not only demonstrates the importance of anion binding characteristics on anion transport but also the potential relevance of bambusurils for anion transport applications considering the high rate observed for Cl−/HCO3− exchange. The exchange of chloride and bicarbonate across lipid bilayers is an important biological process. Synthetic molecules can act as mobile carriers for these anions, although most show little selectivity. Here we report on three bambus[6]uril macrocycles functionalized with fluorinated benzyl groups, which are able to exchange Cl− and HCO3− efficiently. Remarkably, rates for Cl−/NO3− exchange are two orders of magnitude lower. The higher rates of Cl−/HCO3− transport can be explained by the ability of the bambusurils to complex Cl− and HCO3− simultaneously, facilitating their exchange at the bilayer interface. 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Fluorinated groups were introduced with the aim of enhancing rates of transport, yielding three new fluorinated bambus[6]urils (Scheme 1, 2–4). We present the anion binding studies for macrocycles 2–4 and report on their ability to act as Cl−/NO3− and Cl−/HCO3− transporters across liposome membranes. We explain how the particular anion binding characteristics result in exchange of Cl− and HCO3− at unprecedented rates. We designed three derivatives of bambusuril 1 to investigate the effect of the number and position of fluorine atoms or fluorine-containing groups on the benzyl substituents on anion binding and transport (Scheme 1). Computed electrostatic surface potentials and cLogP values suggest that macrocycles 2, 3, and 4 have a lower electron density inside their cavities compared with parent bambusuril 1 (see Figure 1A) and increased lipophilicity (cLogP values increased from 15 for 1 to 36–40 for 2–448Calculated logP values are an estimate of lipophilicity and were obtained using TorchLite 10.5.0.). Both are important factors reported to affect the transmembrane transport activity of receptors, as discussed above.29Busschaert N. Wenzel M. Light M.E. Iglesias-Hernández P. Pérez-Tomás R. Gale P.A. Structure-activity relationships in tripodal transmembrane anion transporters: the effect of fluorination.J. Am. Chem. Soc. 2011; 133: 14136-14148Crossref PubMed Scopus (257) Google Scholar, 30Valkenier H. Judd L.W. Li H. Hussain S. Sheppard D.N. Davis A.P. Preorganized Bis-thioureas as powerful anion carriers: chloride transport by single molecules in large unilamellar vesicles.J. Am. Chem. Soc. 2014; 136: 12507-12512Crossref PubMed Scopus (76) Google Scholar, 31Valkenier H. Dias C.M. Porter Goff K.L. Jurček O. Puttreddy R. Rissanen K. Davis A.P. Sterically geared tris-thioureas; transmembrane chloride transporters with unusual activity and accessibility.Chem. Commun. (Camb.). 2015; 51: 14235-14238Crossref PubMed Google Scholar, 32Cooper J.A. Street S.T.G. Davis A.P. A flexible solution to anion transport: powerful anionophores based on a cyclohexane scaffold.Angew. Chem. Int. Ed. 2014; 53: 5609-5613Crossref PubMed Scopus (48) Google Scholar Calculated logP values are an estimate of lipophilicity and were obtained using TorchLite 10.5.0. The synthesis of the new bambusurils followed the traditional approach,43Lizal T. Sindelar V. Bambusuril anion receptors.Isr. J. Chem. 2018; 58: 326-333Crossref Scopus (53) Google Scholar which is based on the preparation of glycoluril derivatives bearing the corresponding substituents and their acid-catalyzed macrocyclization with paraformaldehyde (Scheme 1; see Supplemental Information [Section 2] for details). The X-ray crystal structure of macrocycle 4 (Figure 1B) revealed alternating arrangements of six glycoluril units connected via six methylene bridges, which is the typical structural feature of bambusurils. Methine hydrogen atoms of the glycoluril units arrange into two cycles inside the macrocycle cavity and thus define the binding pocket in which an anion can be stabilized by C-H···anion interactions. Formation of host-guest complexes between bambusurils 2, 3, and 4 and Cl−, NO3−, and HCO3− was investigated by 1H NMR spectroscopy in acetonitrile. Binding studies of 1 in acetonitrile were not possible as this macrocycle has no significant solubility in the absence of anions. With less than 1 equiv of Cl− or NO3− present in the solution of 2, 3, or 4, two sets of signals are observed for the macrocycle in the 1H NMR spectra, indicating that host-guest exchange is slow on the NMR timescale (see Figures 2A–2C as illustrative examples). Only one set of signals, corresponding to the bambusuril with an included anion, are observed with 1 equiv and these signals do not shift upon addition of more than 1 equiv of Cl− or NO3− and no new signals are observed, indicating the exclusive formation of 1:1 complexes with these anions. Signals corresponding to free macrocycle were absent when equimolar mixtures of host and guest were analyzed, which precluded determination of association constants for these 1:1 host-guest complexes by direct NMR titrations. Series of competition experiments with weakly binding anions were therefore designed and monitored using 1H NMR and isothermal titration calorimetry (ITC), for which the determined binding affinities are given in Table 1 (see Supplemental Information [Section 3] for details).Table 1Affinity Constants and Anion Transport Data for Bambusurils 1–4BambusurilAffinity Constants in AcetonitrileaAffinities were determined for the tetrabutylammonium salts (tetraethylammonium for HCO3−) in acetonitrile at 30°C (unless stated otherwise) by competition binding experiments using ITC (for 3), 1H NMR (for 4) or combination of both methods (for 2), see Supplemental Information for details. Experimental errors were calculated from two or three independent experiments.Cl−/NO3− TransportCl−/HCO3− TransportKa NO3− (M−1)Ka Cl− (M−1)Ka1 HCO3− (M−1)Ka2 HCO3− (M−1)t1/2 (1:200)bHalf-lives were obtained from single exponential fits of the inverse fluorescence curves F0/F, at bambusuril/lipid ratio 1:200 for Cl−/NO3− transport and at 1:5,000 for Cl−/HCO3− transport (see Supplemental Information for details). (s)[I]cThe specific initial rate [I] is determined by dividing the initial rates (obtained from the fit of F0/F with a double exponential fit) by the bambusuril/lipid ratio and averaging for the different ratios. (s−1)t1/2 (1:5000)bHalf-lives were obtained from single exponential fits of the inverse fluorescence curves F0/F, at bambusuril/lipid ratio 1:200 for Cl−/NO3− transport and at 1:5,000 for Cl−/HCO3− transport (see Supplemental Information for details). (s)[I]cThe specific initial rate [I] is determined by dividing the initial rates (obtained from the fit of F0/F with a double exponential fit) by the bambusuril/lipid ratio and averaging for the different ratios. (s−1)1NDdNot determined because bambusuril 1 is not soluble in acetonitrile. Affinities of 1 for various anions in CHCl3 are reported in Havel et al.45 and Havel and Sindelar.46NDdNot determined because bambusuril 1 is not soluble in acetonitrile. Affinities of 1 for various anions in CHCl3 are reported in Havel et al.45 and Havel and Sindelar.46NDdNot determined because bambusuril 1 is not soluble in acetonitrile. Affinities of 1 for various anions in CHCl3 are reported in Havel et al.45 and Havel and Sindelar.46––eNo significant Cl−/NO3− transport observed.–eNo significant Cl−/NO3− transport observed.17092(1.4 ± 0.2) × 109(8.7 ± 0.7) × 107(1.4 ± 0.3) × 107–fNo binding observed.–eNo significant Cl−/NO3− transport observed.–eNo significant Cl−/NO3− transport observed.431003(1.0 ± 0.2) × 1010(2.2 ± 0.4) × 109>4 × 108(1.8 ± 0.2) × 104gDetermined at 20°C.4000.337904(5 ± 1) × 1011(6 ± 1) × 1010>2 × 109(1.8 ± 0.1) × 104gDetermined at 20°C.491641,600a Affinities were determined for the tetrabutylammonium salts (tetraethylammonium for HCO3−) in acetonitrile at 30°C (unless stated otherwise) by competition binding experiments using ITC (for 3), 1H NMR (for 4) or combination of both methods (for 2), see Supplemental Information for details. Experimental errors were calculated from two or three independent experiments.b Half-lives were obtained from single exponential fits of the inverse fluorescence curves F0/F, at bambusuril/lipid ratio 1:200 for Cl−/NO3− transport and at 1:5,000 for Cl−/HCO3− transport (see Supplemental Information for details).c The specific initial rate [I] is determined by dividing the initial rates (obtained from the fit of F0/F with a double exponential fit) by the bambusuril/lipid ratio and averaging for the different ratios.d Not determined because bambusuril 1 is not soluble in acetonitrile. Affinities of 1 for various anions in CHCl3 are reported in Havel et al.45Havel V. Svec J. Wimmerova M. Dusek M. Pojarova M. Sindelar V. Bambus[n]urils: a new family of macrocyclic anion receptors.Org. Lett. 2011; 13: 4000-4003Crossref PubMed Scopus (98) Google Scholar and Havel and Sindelar.46Havel V. Sindelar V. Anion binding inside a bambus[6]uril macrocycle in chloroform.ChemPlusChem. 2015; 80: 1601-1606Crossref Scopus (41) Google Scholare No significant Cl−/NO3− transport observed.f No binding observed.g Determined at 20°C. Open table in a new tab As with Cl− and NO3−, bambusuril 2 forms exclusively a 1:1 complex with HCO3− (Figure S54). The association constant of 2·HCO3− was determined by a direct ITC titration (Table 1). The determination of the association constants between bicarbonate and bambusurils 3 and 4 was, however, complicated by unusual NMR binding features that differed from those observed in the case of Cl− and NO3−. Addition of less than 1 equiv of HCO3− also leads to two sets of signals (Figure 3B) and only signals corresponding to macrocycles with encapsulated anions were visible when equimolar concentrations of host and guest were present, suggesting the formation of very stable 1:1 complexes (Figure 3C). However, addition of more than 1 equiv of the anions resulted in a downfield chemical shift of the methine signals of more than 0.2 ppm for both hosts (Figures 3D–3K and S77). These complexati