DOI: 10.1002/chem.201402602
Full Paper
& Ionophores
Molecular Parameters and Transmembrane Transport Mechanism of Imidazolium-Functionalized Binols Marc Vidal and Andreea Schmitzer*[a]
Abstract: We describe the molecular parameters governing the transmembrane activity of imidazolium-functionalized anion transporters and present a detailed mechanistic study. These ionophores adopt a mobile-carrier mechanism for
Introduction
short methyl and butyl chains, a combined mobile-carrier/ transmembrane-pore mechanism for octyl and dodecyl chains, and form transmembrane aggregates for hexadecyl chains.
compounds, a detailed mechanistic study was performed and is presented here.
Cell membranes are real frontiers between the cell interior and the extracellular environment. Their role is to maintain the biochemical equilibrium inside the cell. Cell membranes, composed of lipid bilayers, allow diffusion of small non-polar molecules but are impermeable to ions and polar compounds such as sugars and DNA fragments.[1] To allow transport of such polar species across the membrane, natural mobile transporters or channels are required. Recently, many diseases have been found to be related to an ion-channel dysfunction.[2] Particularly, chloride channels attracted a lot of attention because of their implication in many diseases, such as dystrophiamyotonica, epilepsy, cystic fibrosis, osteopetrosis and myotonia congenital.[3] Synthetic chloride transporters are useful not only to understand the mechanisms behind these disorders, but may have potential therapeutic applications by compensating anion imbalance.[4–8] Regarding the two possible mechanisms, mobile carrier or transmembrane channels, Sessler et al. were the first to develop dipyrroles and tripyrrolesprodigiosin analogues as anion carriers.[8, 9] In terms of transmembrane-pore aggregates with anion-transport properties, a lot of synthetic small molecules have been widely investigated over the last two decades.[10] In our research group, we demonstrated that phenylethylnylbenzimidazolium salts are able to form aggregates and transport chloride anion across phospholipid bilayers.[11] We recently reported imidazolium-functionalised binaphthols (binols) acting as anion transporters and possessing antimicrobial properties. We demonstrated that their biological activity depends on the length of the carbon chain on the imidazolium moieties.[12] In order to understand the molecular parameters governing the transmembrane activity of these
Results and Discussion Synthesis Compounds 1 a–e (Scheme 1) were synthesized following our previously reported procedure.[12] In order to fully understand the role of the imidazolium position and their number, we synthesized compound 2 (possessing only one imidazolium unit in position 3) and 3 (with two octylimidazolium moieties in positions 6 and 6’). The synthesis of compound 3 is similar to the synthesis of compounds 1 a–e, the main difference laying in the first bromination step for positions 6 and 6’. After the protection of the alcohol groups, the bromine substituents allow formylation to be directed to positions 6 and 6’. The formyl groups were then reduced to alcohols with sodium borohydride, and the alcohols groups were chlorinated by using thionyl chloride. The chloride substituents were involved in a sub-
[a] Dr. M. Vidal, Prof. Dr. A. Schmitzer Departement de Chimie, Universit de Montral C. P. 6128 Succursale Centre-Ville, Montral, Qubec, H3C 3J7 (Canada) Fax: (+ 1) 514-343-7586 E-mail: [email protected] Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201402602. Chem. Eur. J. 2014, 20, 9998 – 10004
Scheme 1. Imidazolium-functionalized binols. Tf = trifluoromethanesulfonyl.
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Scheme 2. Synthesis of imidazolium-functionalized binol 3.
sequent nucleophilic substitution with octylimidazole and the synthesis was completed by the deprotection of the alcohol groups and anion metathesis to the NTf2 anion (Scheme 2). Anion transport The chloride-transport properties of compounds 1 a–e, 2 and 3 were first studied using egg yolk phosphatidylcholine large unilamelar vesicles (EYPC LUVs) loaded with the fluorescent dye lucigenin. As lucigenin’s fluorescence is quenched by the presence of chloride anions, the internal solution of liposomes contains NaCl while the external solution contains NaNO3. Using this method, the translocation of chloride anions outside the vesicles can be monitored as an increase of lucigenin’s fluorescence.[13] The more efficient a transporter is, the higher lucigenin’s fluorescence plateau is, and the faster this plateau is reached. At 12.5 mol % (relative to EYPC concentration) compounds 1 a–e were able to transport chloride anions outside the EYPC LUVs, but their transport efficiency depends on the length of the carbon chain on the imidazolium moiety (Figure 1 A). The order of transport activity, as a function of the alkyl chain length is 1 c (C8) > 1 d (C12) > 1 e (C16) > 1 b (C4) > 1 a (C1). This result can be explained by the amphiphilicity (logP shown in Table 1) of the compounds. Indeed, it has already been proven that the efficiency of transporters is linked to their hydrophobicity.[11a] In the case of compounds 1 a–e, the hydrophobicity increases when alkyl chain length increases. The calculated logP values vary between 5.9 for 1 a (C1) to 17.8 for 1 c (C16). Compound 2, which is more hydrophilic and possessing only one imidazolium unit, possesses the lower transport Chem. Eur. J. 2014, 20, 9998 – 10004
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Figure 1. Change in lucigenin fluorescence intensity after addition of 12.5 mol % of 1 a–e, 2 and 3 to EYPC LUVs. Phospholipid concentration: 0.048 mm. Intravesicular: 100 mm NaCl, 10 mm phosphate buffer, lucigenin 1 mm. Extravesicular: 100 mm NaNO3, 10 mm phosphate buffer (pH 6.4). A) Influence of the carbon chain length on the transport activity. B) Influence of the number and the position of imidazolium moieties on the transport activity. Each curve represents the average of three independent trials.
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Full Paper Table 1. Kinetic parameters determined by Hill analysis and hydrophobicity (logP).
1a 1b 1c 1d 1e 2 3
Alkyl Chain
Position of substitution
EC50,250 [mol %]
n
logP[a]
CH3 C4H9 C8H17 C12H25 C16H33 C8H17 C8H17
3,3’ 3,3’ 3,3’ 3,3’ 3,3’ 3 6,6’
– – 4.62 0.11 9.30 2.57 18.14 4.49 23.33 5.25 11.90 2.24
– – 1.8 1.6 2.0 2.6 3.3
5.9 8.4 11.6 14.7 17.8 8.5 11.8
[a] Calculated with HyperChem 7.5.
activity (Figure 1 B), owing probably to its weaker bilayer insertion. One additional reason could be the weaker complexation of chloride anion, owing to the lack of the second imidazolium cation close to the binding site. Compound 3, possessing the imidazolium units in positions 6 and 6’ presents a weaker chloride-transport activity, probably owing to a less efficient binding site of the chloride anions, but also to an unfavorable repartition of the hydrophilic and hydrophobic moieties in the molecule. Kinetic parameters In an effort to gain more insight into the nature of the transport-active species, a kinetic analysis was performed by quantifying the extent of Cl efflux from EYPC LUVs in the presence of these compounds.[14] In lucigenin-loaded liposomes, the Cl efflux was measured as a function of time and transporter content to obtain the effective concentration, EC50, and Hill coefficient, n, corresponding to the stoechiometry of the transportactive species (Table 1 and Figures S1–S7 in the Supporting Information).[15] Both parameters were calculated using Hill analysis with Origin 8.0. The reported values in Table 1 are the average of three independent measurements. The highest activity in the Cl /NO3 assay was observed for transporter 1 c (EC50 = 4.6 and see the Supporting Information). The found Hill coefficients n 2 for compounds 1 c–e and 2 were consistent with membrane-spanning aggregates formed by stacking of two long molecules. In the case of ionophore 3, the Hill coefficient still suggests that the transport of one chloride anion through the membrane requires more than two molecules of transporter. Mechanism of transport In order to elucidate the transport mechanism of compounds 1 a–e, the efflux of chloride anions was first measured outside dipalmitoylphosphatidylcholine (DPPC) liposomes at temperatures under and above DPPC’s gel/fluid transition temperature (41 8C). This method allows mobile transporters to be differentiated from compounds that form transmembrane aggregates.[16] Indeed, mobile transporters see their diffusion rate restricted when DPPC is in the gel state, while transmembrane channels, once inserted in the membrane, are not affected by Chem. Eur. J. 2014, 20, 9998 – 10004
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Figure 2. Change in lucigenin fluorescence intensity 250 s after addition of transporters to DPPC LUVs. Phospholipid concentration: 0.048 mm. Intravesicular: 100 mm NaCl, 10 mm phosphate buffer, lucigenin 1 mm. Extravesicular: 100 mm NaNO3, 10 mm phosphate buffer (pH 6.4). Each data point represents the average of three trials. Compounds 1 a–b exhibit a mobile transporter mechanism with a characteristic increase of transport activity around 40 8C. Compounds 1 c–e exhibit a channel-formation mechanism, with no major change in activity under and above DPPC gel/fluid transition temperature. [1 a] = 12.5 mol %, [1 b] = 25 mol %, [1 c] = 1.25 mol %, [1 d] = 3.13 mol % and [1 e] = 25 mol %.
the state of the DPPC membrane. The behavior of the transporters 1 a–e in this assay depends on the length of the carbon chain. As shown in Figure 2, compounds 1 a and 1 b have a reduced activity when DPPC is in the gel state, while the behavior of compounds 1 c–e is only slightly influenced by the rigidity of the membrane. This result suggests that compounds 1 a–b act as anion carriers and compounds 1 c–e form transmembrane aggregates spanning across the membrane. Compound 1 c forms the most efficient assembly in terms of chloride transport, but in this case, two different transport mechanisms seem to be operating at the same time. In order to further investigate the behavior of compounds 1 a–e to act as mobile carriers or to form transmembrane aggregates, EYPC and EYPC/cholesterol liposomes were prepared and the chloride transport was compared in the presence of the same amount of transporter (concentrations allowing a reasonable transport activity rate; Figure 3). Cholesterol is often included in the liposome formulation in order to increase the rigidity to the membrane.[17]
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Full Paper served in EYPC/cholesterol liposomes, suggesting a mobile-carrier behavior. For 1 c and 1 d, the transport in EYPC/cholesterol is only slightly lower than the one in EYPC liposomes. For 1 e, the transport activity is identical in both systems, confirming the formation of a stable transmembrane channel. Compounds 1 a–e were also tested by using liposomes loaded with pH-sensitive 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (HPTS; Figure 4).[16, 18] In this assay, the ion-transport ability of the tested compounds is measured indirectly by monitoring the pH changes inside the liposomes. Addition of transporters 1 a–e resulted in an increase of the I1/I0 fluorescence ratio, indicating a transport of protons to the extravesicular solution. Compounds 1 b and 1 c are the most active compounds in the H + /Cl symport process. We previously showed that compounds 1 a–e act as Cl / NO3 antiporters, so with the Figure 3. Comparison of chloride-transport activity of 1 a (50 mol %), 1 b (30 mol %), 1 c (6,25 mol %), 1 d HPTS results in hand, we can (25 mol %) and 1 e (25 mol %) in EYPC and EYPC/cholesterol 7:3 liposomes. Phospholipid concentration: 0.048 mm. affirm that the two H + /Cl and Intravesicular: phosphate buffer 10 mm (pH 6.4), 100 mm NaCl and 1 mm lucigenin. Extravesicular: phosphate Cl /NO3 processes are in combuffer 10 mm (pH 6.4), NaNO3 100 mm. Transporters were added in a methanol solution at t = 50 s and the lyposomes were lysed at t = 300 s by addition of Triton-X. petition. The H + /Cl symport order (1 c > 1 b > 1 d > 1 a = 1 e) is different from the Cl /NO3 one (1 c > 1 d > 1 e > 1 b > 1 a). Compound 1 c possesses both best H + /Cl symport and Cl / NO3 antiport transport properties. Anion selectivity
Figure 4. Chloride-transport activity of 1 a–e in EYPC liposomes containing 0.1 mm HPTS, 100 mm NaCl in phosphate buffer 10 mm (pH 6.4). Phospholipid concentration: 0.048 mm. Extravesicular: 100 mm NaNO3, 10 mm phosphate buffer (pH 6.4). 25 mol % of 1 a–e was added at t = 50 s and the liposomes were lysed at t = 300 s by addition of Triton-X. Each curve represents the average of three independent trials.
The results are consistent with those obtained in DPPC liposomes, as for 1 a and 1 b, only a small rate of transport was obChem. Eur. J. 2014, 20, 9998 – 10004
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The rate of chloride transport in EYPC liposomes is dependent on the extravesicular anions (Figure 5), suggesting an antiport mechanism for these transport processes.[19] However, different observations can be made: first, as a general trend, the more hydrophilic compounds favour the antiport of the more hydrophilic anions and the more hydrophobic compounds favour the antiport of less hydrophilic anions. Compound 1 b, possessing a reasonable level of chloride transport shows a selectivity towards SO42 : DGh(SO42 ), 1080 kJ mol 1 > DGh (HCO3 ), 1 335 kJ mol > DGh (NO3 ), 300 kJ mol 1.[20] This selectivity towards sulfate is of particular interest as sulfate is one of the most hydrophilic anions, having shown a lot of difficulty to cross phopholipid bilayers, only a few examples of Cl /SO42 antiport processes having been described in the literature.[21] Compound 1 c, which is more hydrophobic than 1 b, favours
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Figure 5. Influence of the extravesicular anion. Intravesicular phosphate buffer 10 mm (pH 6.4), 100 mm NaCl and 1 mm lucigenine. Phospholipid concentration: 0.048 mm. Extravesicular 100 mm NaNO3 or NaHCO3 or Na2SO4 in phosphate buffer 10 mm (pH 6.4). [1 a] = 50 mol %, [1 b] = 20 mol %, [1 c] = 5 mol %, [1 d] = 10 mol %, [1 e] = 20 mol %. Transporters were injected at t = 50 s. Triton-X was injected at t = 300 s. Each curve represents the average of three trials.
Figure 6. Efflux of CF out of EYPC LUVs. Phospholipid concentration: 0.02 mm. Intravesicular: 100 mm KCl, 10 mm HEPES buffer, carboxyfluorescein (20 mm). Extravesicular: 100 mm KCl, 10 mm HEPES buffer (pH 7). Transporters at 25 % mol were injected at t = 50 s. Triton-X was injected at t = 300 s. Each curve represents the average of three trials.
the transmembrane transport of carbonate. Again, this is of particular interest for biological applications as carbonate transport, in general, and Cl /HCO3 antiport in particular, is very important for pH and bicarbonate regulation in many organs, such as lungs, heart, kidney and brain.[22] For long alkyl chains, less selectivity was observed. However, the most hydrophobic compound, 1 e, showed a certain unfavoured Cl /SO42 antiport.
Transport of carboxyfluoresceine across the phospholipid bilayer We also investigated the ability of transporters 1 a–e to transport organic compounds across the phospholipid bilayer. For these studies, EYPC LUVs loaded with a 20 mm carboxyfluorescein (CF) solution were used. CF is an anionic fluorescent dye of 1 nm 1 nm size[16] that autoquenches its fluorescence when concentrated. In the presence of the transporters, the CF efflux can be monitored by following the increase of fluorescence when CF is diluted outside the liposome.[23] As shown in Figure 6, at 300 s after the addition of the transporter to the liposomes solution, 60 % of the CF was transported outside the liposomes by compound 1 c, while only 17 % of CF was transported by mobile transporter 1 b. This result suggests again that compound 1 c forms transmembrane pores able to transport organic compounds of nanometric sizes. Compound 1 a was completely inactive, but surprisingly compounds 1 b, 1 d and 1 e showed, although small, a certain level of carboxyfluorescein transport. However, the observed activity in the presence of these compounds could also be the result of a H + /Cl co-transport process inside the liposomes, resulting in an increased intravesicular pH. Intra- and extravesicular NaCl concentrations were the same (100 mm), but because an alkaline step, followed by an HCl reequilibration, were required to solubilize CF inside the liposomes, a small pH imbalance could occur. In this case, transChem. Eur. J. 2014, 20, 9998 – 10004
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Figure 7. A) Efflux of CF after addition of 0.1–50 mol % of 1 c. B) Dose-response curve and adjustment to the Hill equation. Phospholipid concentration: 0.02 mm. Intravesicular: HEPES 10 mm (pH 7) and carboxyfluorescein (20 mm). Extravesicular: 100 mm NaCl, 10 mm HEPES buffer (pH 7). Transporters were injected at t = 50 s. Triton-X was injected at t = 300 s. Each curve represents the average of three trials.
porters 1 b, 1 d and 1 e may transport Cl /H + inside the liposomes. Transporter 1 c being the only effective transporter for CF efflux, a detailed kinetic study was performed for this compound. As shown in Figure 7, two different types of behaviour can be observed. At concentrations below 1.56 mol %, transporter 1 c only acts as a H + /Cl transporter in order to equilibrate the intra-/extravesicular pH imbalance. Beyond this con-
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Full Paper centration, CF is transported across the phospholipid bilayer. At concentrations below 6.25 mol %, the transport process is slow and the slow increase in CF’s fluorescence is probably compensated by the extinction of fluorescence provoked by the influx of protons into the liposomes. At 12.5 mol % of 1 c, CF is rapidly transported outside the liposomes. The EC50, 250 value of 21.2 % was also calculated, as previously described, as well as the Hill coefficient, n 2. These results suggest the formation of a transmembrane pore by the assembly of two monomers, able to allow the translocation of nanometric objects. An alternative hypothesis could be membrane destabilization and CF leakage. However, if a leakage was involved, a more pronounced effect would be observed for longer alkyl chains, that is, compounds 1 d and 1 e. Stability of the liposomes in the presence of transporters The integrity of the liposomes after the addition of the imidazolium-functionalized binols, was verified by dynamic light scattering (DLS) measurements. The size of the liposomes was measured 5 min after the addition of 50 mol % of transporter, this concentration being the highest used in all our studies (we can assume that at lower concentrations, the effect of the transporter on the membrane is less important). As shown in Figure 8 and Table 2, all the liposomes were monodisperse after addition of the transporters. This first observation suggests that the liposomes are intact, eliminating the hypothesis of liposome destruction. However, variation of the liposome’s diameter was observed in the presence of all the transporters. The increase of the liposomes size was only 10 % for transport-
ers 1 a and 1 b, but it was 40 % for compound 1 c. For compounds 1 d and 1 e, 160 and 180 % size increases, respectively, were observed. These results suggest, again, that 1 a and 1 b act as mobile transporters, not altering at all the size of the liposomes. In the case of compounds 1 c–e, the formation of transmembrane pores results in the swelling of the liposomes, but not their destruction.
Conclusion By introducing imidazolium moieties in positions 3 and 3’ of 1,1’-binaphtol, a new family of imidazolium-based transporters emerged. The transport properties of these imidazolium-functionalized binols depend on the length of the imidazolium alkyl chain, the position and the number of imidazolium units on the binol scaffold. The transport mechanism also depends on the length of the alkyl chain, allowing the transporter to act either as a mobile transporter in the case of methyl and butyl chains and forming transmembrane anion pores for octyl, dodecyl and hexadecyl chains. The selectivity for the transported anion is dependent on the molecular structure of the transporter. The transmembrane-active aggregates seem to be formed by two monomers. Again, the length of the alkyl chain dictates the stability of the formed aggregates, compound 1 e forming the more stable hydrophobic channels (Figure 9). However, these hydrophobic channels are not efficient for anion transport, the distance between two imidazolium cations in the dimer being probably too important. Transporter 1 c is the most efficient transporter, possessing a dual behavior, of mobile carrier and forming transmembrane pores. In this case, the dimer is probably too small to cross the bilayer from one side to the other and the aggregate too instable to remain in the phospholipid environment. This dimer could disassemble and the monomer may then act as a mobile carrier. Finally, the particular geometry of the binol unit may be the reason for the opening of transmembrane pore to a size large enough to allow the transport of carboxyfluorescein, in the case of 1 c.
Figure 8. Size of the liposomes after addition of methanol, Triton X-100 or a methanol solution of transporters 1 a–e.
Table 2. Size of the liposomes determined by DLS after addition of methanol, Triton X-100 or 50 mol % of 1 a–e.
MeOH Triton 1a 1b 1c 1d 1e
Alkyl group
Diameter [nm]
Variation of diameter [%]
– – CH3 C4 H 9 C8H17 C12H25 C16H33
141 6 92 154 13 153 13 197 15 364 36 395 45
– 93 10 9 40 159 181
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Figure 9. Schematic representation of the active transmembrane dimers formed by 1 c-e.
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Full Paper Acknowledgements This work was supported by the Natural Sciences and Engineering Research Council of Canada, the Fonds Qubcois de la Recherche sur la Nature et les Technologies, the Centre of Green Chemistry and Catalysis, the Canada Foundation for Innovationand the Universit de Montreal. We thank colleagues for careful discussions of this manuscript. Keywords: anion binding · binol · cell membranes · imidazolium salts · transmembrane transporters [1] H. Nikaido, Microbiol. Mol. Biol. Rev. 2003, 67, 593 – 656. [2] a) C. A. Hubner, Hum. Mol. Genet. 2002, 11, 2435 – 2445; b) E. C. Emery, G. T. Young, E. M. Berrocoso, L. Chen, P. A. McNaughton, Science 2011, 333, 1462 – 1466; c) F. M. Ashcroft, Nature 2006, 440, 440 – 447. [3] a) L. Puljak, G. Kilic, Biochim. Biophys. Acta. 2006, 1762, 404 – 413; b) A. S. Verkman, L. J. Galietta, Nat. Rev. Drug. Discov. 2009, 8, 153 – 171. [4] a) C. J. Haynes, N. Busschaert, I. L. Kirby, J. Herniman, M. E. Light, N. J. Wells, I. Marques, V. Felix, P. A. Gale, Org. Biomol. Chem. 2014, 12, 62 – 72; b) P. B. Cranwell, J. R. Hiscock, C. J. Haynes, M. E. Light, N. J. Wells, P. A. Gale, Chem. Commun. 2013, 49, 874 – 876; c) C. J. Haynes, S. N. Berry, J. Garric, J. Herniman, J. R. Hiscock, I. L. Kirby, M. E. Light, G. Perkes, P. A. Gale, Chem. Commun. 2013, 49, 246 – 248; d) H. Itoh, S. Matsuoka, M. Kreir, M. Inoue, J. Am. Chem. Soc. 2012, 134, 14011 – 14018. [5] G. W. Gokel, N. Barkey, New J. Chem. 2009, 33, 947 – 963. [6] J. Rennolds, S. Butler, K. Maloney, P. N. Boyaka, I. C. Davis, D. L. Knoell, N. L. Parinandi, E. Cormet-Boyaka, Toxicol. Sci. 2010, 116, 349 – 358. [7] D. Fialho, S. Schorge, U. Pucovska, N. P. Davies, R. Labrum, A. Haworth, E. Stanley, R. Sud, W. Wakeling, M. B. Davis, D. M. Kullmann, M. G. Hanna, Brain 2007, 130, 3265 – 3274. [8] C. R. Yamnitz, S. Negin, I. A. Carasel, R. K. Winter, G. W. Gokel, Chem. Commun. 2010, 46, 2838 – 2840.
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[9] J. L. Sessler, L. R. Eller, W. S. Cho, S. Nicolaou, A. Aguilar, J. T. Lee, V. M. Lynch, D. J. Magda, Angew. Chem. 2005, 117, 6143 – 6146; Angew. Chem. Int. Ed. 2005, 44, 5989 – 5992. [10] P. A. Gale, R. Perez-Tomas, R. Quesada, Acc. Chem. Res. 2013, 46, 2801 – 2813. [11] a) C.-R. Elie, N. Noujeim, C. Pardin, A. R. Schmitzer, Chem. Commun. 2011, 47, 1788 – 1790; b) C.-R. Elie, M. Charbonneau, A. R. Schmitzer, MedChemComm 2012, 3, 1231; c) C. R. Elie, A. Hebert, M. Charbonneau, A. Haiun, A. R. Schmitzer, Org. Biomol. Chem. 2013, 11, 923 – 928. [12] M. Vidal, C.-R. Elie, S. Campbell, A. Claing, A. R. Schmitzer, Med. Chem. Commun. 2014, 5, 436 – 440. [13] B. A. McNally, A. V. Koulov, B. D. Smith, J. B. Joos, A. P. Davis, Chem. Commun. 2005, 1087 – 1089. [14] S. Matile, N. Sakai in Analytical Methods in Supramolecular Chemistry (Ed.: C. Schalley), Wiley-VCH, Weinheim, 2007, pp. 391 – 418. [15] a) A. V. Hill, Biochem. J. 1913, 7, 471 – 480; b) S. Bhosale, S. Matile, Chirality 2006, 18, 849 – 856. [16] J. T. Davis, O. Okunola, R. Quesada, Chem. Soc. Rev. 2010, 39, 3843 – 3862. [17] W. F. Bennett, J. L. MacCallum, D. P. Tieleman, J Am Chem. Soc 2009, 131, 1972 – 1978. [18] D. B. Spry, A. Goun, C. B. Bell 3rd, M. D. Fayer, J. Chem. Phys. 2006, 125, 144514. [19] P. A. Gale, R. Perez-Tomas, R. Quesada, Acc Chem. Res. 2013, 46, 2801 – 2813. [20] N. Busschaert, P. A. Gale, C. J. Haynes, M. E. Light, S. J. Moore, C. C. Tong, J. T. Davis, W. A. Harrell, Jr., Chem. Commun. 2010, 46, 6252 – 6254. [21] N. Busschaert, M. Wenzel, M. E. Light, P. Iglesias-Hernandez, R. PerezTomas, P. A. Gale, J. Am. Chem. Soc. 2011, 133, 14136 – 14148. [22] E. Cordat, J. R. Casey, Biochem. J. 2009, 417, 423 – 439. [23] G. W. Gokel, I. A. Carasel, Chem. Soc. Rev. 2007, 36, 378 – 389.
Received: March 13, 2014 Published online on July 7, 2014
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& Ionophores
Molecular Parameters and Transmembrane Transport Mechanism of Imidazolium-Functionalized Binols Marc Vidal and Andreea Schmitzer*[a]
Abstract: We describe the molecular parameters governing the transmembrane activity of imidazolium-functionalized anion transporters and present a detailed mechanistic study. These ionophores adopt a mobile-carrier mechanism for
Introduction
short methyl and butyl chains, a combined mobile-carrier/ transmembrane-pore mechanism for octyl and dodecyl chains, and form transmembrane aggregates for hexadecyl chains.
compounds, a detailed mechanistic study was performed and is presented here.
Cell membranes are real frontiers between the cell interior and the extracellular environment. Their role is to maintain the biochemical equilibrium inside the cell. Cell membranes, composed of lipid bilayers, allow diffusion of small non-polar molecules but are impermeable to ions and polar compounds such as sugars and DNA fragments.[1] To allow transport of such polar species across the membrane, natural mobile transporters or channels are required. Recently, many diseases have been found to be related to an ion-channel dysfunction.[2] Particularly, chloride channels attracted a lot of attention because of their implication in many diseases, such as dystrophiamyotonica, epilepsy, cystic fibrosis, osteopetrosis and myotonia congenital.[3] Synthetic chloride transporters are useful not only to understand the mechanisms behind these disorders, but may have potential therapeutic applications by compensating anion imbalance.[4–8] Regarding the two possible mechanisms, mobile carrier or transmembrane channels, Sessler et al. were the first to develop dipyrroles and tripyrrolesprodigiosin analogues as anion carriers.[8, 9] In terms of transmembrane-pore aggregates with anion-transport properties, a lot of synthetic small molecules have been widely investigated over the last two decades.[10] In our research group, we demonstrated that phenylethylnylbenzimidazolium salts are able to form aggregates and transport chloride anion across phospholipid bilayers.[11] We recently reported imidazolium-functionalised binaphthols (binols) acting as anion transporters and possessing antimicrobial properties. We demonstrated that their biological activity depends on the length of the carbon chain on the imidazolium moieties.[12] In order to understand the molecular parameters governing the transmembrane activity of these
Results and Discussion Synthesis Compounds 1 a–e (Scheme 1) were synthesized following our previously reported procedure.[12] In order to fully understand the role of the imidazolium position and their number, we synthesized compound 2 (possessing only one imidazolium unit in position 3) and 3 (with two octylimidazolium moieties in positions 6 and 6’). The synthesis of compound 3 is similar to the synthesis of compounds 1 a–e, the main difference laying in the first bromination step for positions 6 and 6’. After the protection of the alcohol groups, the bromine substituents allow formylation to be directed to positions 6 and 6’. The formyl groups were then reduced to alcohols with sodium borohydride, and the alcohols groups were chlorinated by using thionyl chloride. The chloride substituents were involved in a sub-
[a] Dr. M. Vidal, Prof. Dr. A. Schmitzer Departement de Chimie, Universit de Montral C. P. 6128 Succursale Centre-Ville, Montral, Qubec, H3C 3J7 (Canada) Fax: (+ 1) 514-343-7586 E-mail: [email protected] Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201402602. Chem. Eur. J. 2014, 20, 9998 – 10004
Scheme 1. Imidazolium-functionalized binols. Tf = trifluoromethanesulfonyl.
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Scheme 2. Synthesis of imidazolium-functionalized binol 3.
sequent nucleophilic substitution with octylimidazole and the synthesis was completed by the deprotection of the alcohol groups and anion metathesis to the NTf2 anion (Scheme 2). Anion transport The chloride-transport properties of compounds 1 a–e, 2 and 3 were first studied using egg yolk phosphatidylcholine large unilamelar vesicles (EYPC LUVs) loaded with the fluorescent dye lucigenin. As lucigenin’s fluorescence is quenched by the presence of chloride anions, the internal solution of liposomes contains NaCl while the external solution contains NaNO3. Using this method, the translocation of chloride anions outside the vesicles can be monitored as an increase of lucigenin’s fluorescence.[13] The more efficient a transporter is, the higher lucigenin’s fluorescence plateau is, and the faster this plateau is reached. At 12.5 mol % (relative to EYPC concentration) compounds 1 a–e were able to transport chloride anions outside the EYPC LUVs, but their transport efficiency depends on the length of the carbon chain on the imidazolium moiety (Figure 1 A). The order of transport activity, as a function of the alkyl chain length is 1 c (C8) > 1 d (C12) > 1 e (C16) > 1 b (C4) > 1 a (C1). This result can be explained by the amphiphilicity (logP shown in Table 1) of the compounds. Indeed, it has already been proven that the efficiency of transporters is linked to their hydrophobicity.[11a] In the case of compounds 1 a–e, the hydrophobicity increases when alkyl chain length increases. The calculated logP values vary between 5.9 for 1 a (C1) to 17.8 for 1 c (C16). Compound 2, which is more hydrophilic and possessing only one imidazolium unit, possesses the lower transport Chem. Eur. J. 2014, 20, 9998 – 10004
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Figure 1. Change in lucigenin fluorescence intensity after addition of 12.5 mol % of 1 a–e, 2 and 3 to EYPC LUVs. Phospholipid concentration: 0.048 mm. Intravesicular: 100 mm NaCl, 10 mm phosphate buffer, lucigenin 1 mm. Extravesicular: 100 mm NaNO3, 10 mm phosphate buffer (pH 6.4). A) Influence of the carbon chain length on the transport activity. B) Influence of the number and the position of imidazolium moieties on the transport activity. Each curve represents the average of three independent trials.
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Full Paper Table 1. Kinetic parameters determined by Hill analysis and hydrophobicity (logP).
1a 1b 1c 1d 1e 2 3
Alkyl Chain
Position of substitution
EC50,250 [mol %]
n
logP[a]
CH3 C4H9 C8H17 C12H25 C16H33 C8H17 C8H17
3,3’ 3,3’ 3,3’ 3,3’ 3,3’ 3 6,6’
– – 4.62 0.11 9.30 2.57 18.14 4.49 23.33 5.25 11.90 2.24
– – 1.8 1.6 2.0 2.6 3.3
5.9 8.4 11.6 14.7 17.8 8.5 11.8
[a] Calculated with HyperChem 7.5.
activity (Figure 1 B), owing probably to its weaker bilayer insertion. One additional reason could be the weaker complexation of chloride anion, owing to the lack of the second imidazolium cation close to the binding site. Compound 3, possessing the imidazolium units in positions 6 and 6’ presents a weaker chloride-transport activity, probably owing to a less efficient binding site of the chloride anions, but also to an unfavorable repartition of the hydrophilic and hydrophobic moieties in the molecule. Kinetic parameters In an effort to gain more insight into the nature of the transport-active species, a kinetic analysis was performed by quantifying the extent of Cl efflux from EYPC LUVs in the presence of these compounds.[14] In lucigenin-loaded liposomes, the Cl efflux was measured as a function of time and transporter content to obtain the effective concentration, EC50, and Hill coefficient, n, corresponding to the stoechiometry of the transportactive species (Table 1 and Figures S1–S7 in the Supporting Information).[15] Both parameters were calculated using Hill analysis with Origin 8.0. The reported values in Table 1 are the average of three independent measurements. The highest activity in the Cl /NO3 assay was observed for transporter 1 c (EC50 = 4.6 and see the Supporting Information). The found Hill coefficients n 2 for compounds 1 c–e and 2 were consistent with membrane-spanning aggregates formed by stacking of two long molecules. In the case of ionophore 3, the Hill coefficient still suggests that the transport of one chloride anion through the membrane requires more than two molecules of transporter. Mechanism of transport In order to elucidate the transport mechanism of compounds 1 a–e, the efflux of chloride anions was first measured outside dipalmitoylphosphatidylcholine (DPPC) liposomes at temperatures under and above DPPC’s gel/fluid transition temperature (41 8C). This method allows mobile transporters to be differentiated from compounds that form transmembrane aggregates.[16] Indeed, mobile transporters see their diffusion rate restricted when DPPC is in the gel state, while transmembrane channels, once inserted in the membrane, are not affected by Chem. Eur. J. 2014, 20, 9998 – 10004
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Figure 2. Change in lucigenin fluorescence intensity 250 s after addition of transporters to DPPC LUVs. Phospholipid concentration: 0.048 mm. Intravesicular: 100 mm NaCl, 10 mm phosphate buffer, lucigenin 1 mm. Extravesicular: 100 mm NaNO3, 10 mm phosphate buffer (pH 6.4). Each data point represents the average of three trials. Compounds 1 a–b exhibit a mobile transporter mechanism with a characteristic increase of transport activity around 40 8C. Compounds 1 c–e exhibit a channel-formation mechanism, with no major change in activity under and above DPPC gel/fluid transition temperature. [1 a] = 12.5 mol %, [1 b] = 25 mol %, [1 c] = 1.25 mol %, [1 d] = 3.13 mol % and [1 e] = 25 mol %.
the state of the DPPC membrane. The behavior of the transporters 1 a–e in this assay depends on the length of the carbon chain. As shown in Figure 2, compounds 1 a and 1 b have a reduced activity when DPPC is in the gel state, while the behavior of compounds 1 c–e is only slightly influenced by the rigidity of the membrane. This result suggests that compounds 1 a–b act as anion carriers and compounds 1 c–e form transmembrane aggregates spanning across the membrane. Compound 1 c forms the most efficient assembly in terms of chloride transport, but in this case, two different transport mechanisms seem to be operating at the same time. In order to further investigate the behavior of compounds 1 a–e to act as mobile carriers or to form transmembrane aggregates, EYPC and EYPC/cholesterol liposomes were prepared and the chloride transport was compared in the presence of the same amount of transporter (concentrations allowing a reasonable transport activity rate; Figure 3). Cholesterol is often included in the liposome formulation in order to increase the rigidity to the membrane.[17]
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Full Paper served in EYPC/cholesterol liposomes, suggesting a mobile-carrier behavior. For 1 c and 1 d, the transport in EYPC/cholesterol is only slightly lower than the one in EYPC liposomes. For 1 e, the transport activity is identical in both systems, confirming the formation of a stable transmembrane channel. Compounds 1 a–e were also tested by using liposomes loaded with pH-sensitive 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (HPTS; Figure 4).[16, 18] In this assay, the ion-transport ability of the tested compounds is measured indirectly by monitoring the pH changes inside the liposomes. Addition of transporters 1 a–e resulted in an increase of the I1/I0 fluorescence ratio, indicating a transport of protons to the extravesicular solution. Compounds 1 b and 1 c are the most active compounds in the H + /Cl symport process. We previously showed that compounds 1 a–e act as Cl / NO3 antiporters, so with the Figure 3. Comparison of chloride-transport activity of 1 a (50 mol %), 1 b (30 mol %), 1 c (6,25 mol %), 1 d HPTS results in hand, we can (25 mol %) and 1 e (25 mol %) in EYPC and EYPC/cholesterol 7:3 liposomes. Phospholipid concentration: 0.048 mm. affirm that the two H + /Cl and Intravesicular: phosphate buffer 10 mm (pH 6.4), 100 mm NaCl and 1 mm lucigenin. Extravesicular: phosphate Cl /NO3 processes are in combuffer 10 mm (pH 6.4), NaNO3 100 mm. Transporters were added in a methanol solution at t = 50 s and the lyposomes were lysed at t = 300 s by addition of Triton-X. petition. The H + /Cl symport order (1 c > 1 b > 1 d > 1 a = 1 e) is different from the Cl /NO3 one (1 c > 1 d > 1 e > 1 b > 1 a). Compound 1 c possesses both best H + /Cl symport and Cl / NO3 antiport transport properties. Anion selectivity
Figure 4. Chloride-transport activity of 1 a–e in EYPC liposomes containing 0.1 mm HPTS, 100 mm NaCl in phosphate buffer 10 mm (pH 6.4). Phospholipid concentration: 0.048 mm. Extravesicular: 100 mm NaNO3, 10 mm phosphate buffer (pH 6.4). 25 mol % of 1 a–e was added at t = 50 s and the liposomes were lysed at t = 300 s by addition of Triton-X. Each curve represents the average of three independent trials.
The results are consistent with those obtained in DPPC liposomes, as for 1 a and 1 b, only a small rate of transport was obChem. Eur. J. 2014, 20, 9998 – 10004
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The rate of chloride transport in EYPC liposomes is dependent on the extravesicular anions (Figure 5), suggesting an antiport mechanism for these transport processes.[19] However, different observations can be made: first, as a general trend, the more hydrophilic compounds favour the antiport of the more hydrophilic anions and the more hydrophobic compounds favour the antiport of less hydrophilic anions. Compound 1 b, possessing a reasonable level of chloride transport shows a selectivity towards SO42 : DGh(SO42 ), 1080 kJ mol 1 > DGh (HCO3 ), 1 335 kJ mol > DGh (NO3 ), 300 kJ mol 1.[20] This selectivity towards sulfate is of particular interest as sulfate is one of the most hydrophilic anions, having shown a lot of difficulty to cross phopholipid bilayers, only a few examples of Cl /SO42 antiport processes having been described in the literature.[21] Compound 1 c, which is more hydrophobic than 1 b, favours
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Figure 5. Influence of the extravesicular anion. Intravesicular phosphate buffer 10 mm (pH 6.4), 100 mm NaCl and 1 mm lucigenine. Phospholipid concentration: 0.048 mm. Extravesicular 100 mm NaNO3 or NaHCO3 or Na2SO4 in phosphate buffer 10 mm (pH 6.4). [1 a] = 50 mol %, [1 b] = 20 mol %, [1 c] = 5 mol %, [1 d] = 10 mol %, [1 e] = 20 mol %. Transporters were injected at t = 50 s. Triton-X was injected at t = 300 s. Each curve represents the average of three trials.
Figure 6. Efflux of CF out of EYPC LUVs. Phospholipid concentration: 0.02 mm. Intravesicular: 100 mm KCl, 10 mm HEPES buffer, carboxyfluorescein (20 mm). Extravesicular: 100 mm KCl, 10 mm HEPES buffer (pH 7). Transporters at 25 % mol were injected at t = 50 s. Triton-X was injected at t = 300 s. Each curve represents the average of three trials.
the transmembrane transport of carbonate. Again, this is of particular interest for biological applications as carbonate transport, in general, and Cl /HCO3 antiport in particular, is very important for pH and bicarbonate regulation in many organs, such as lungs, heart, kidney and brain.[22] For long alkyl chains, less selectivity was observed. However, the most hydrophobic compound, 1 e, showed a certain unfavoured Cl /SO42 antiport.
Transport of carboxyfluoresceine across the phospholipid bilayer We also investigated the ability of transporters 1 a–e to transport organic compounds across the phospholipid bilayer. For these studies, EYPC LUVs loaded with a 20 mm carboxyfluorescein (CF) solution were used. CF is an anionic fluorescent dye of 1 nm 1 nm size[16] that autoquenches its fluorescence when concentrated. In the presence of the transporters, the CF efflux can be monitored by following the increase of fluorescence when CF is diluted outside the liposome.[23] As shown in Figure 6, at 300 s after the addition of the transporter to the liposomes solution, 60 % of the CF was transported outside the liposomes by compound 1 c, while only 17 % of CF was transported by mobile transporter 1 b. This result suggests again that compound 1 c forms transmembrane pores able to transport organic compounds of nanometric sizes. Compound 1 a was completely inactive, but surprisingly compounds 1 b, 1 d and 1 e showed, although small, a certain level of carboxyfluorescein transport. However, the observed activity in the presence of these compounds could also be the result of a H + /Cl co-transport process inside the liposomes, resulting in an increased intravesicular pH. Intra- and extravesicular NaCl concentrations were the same (100 mm), but because an alkaline step, followed by an HCl reequilibration, were required to solubilize CF inside the liposomes, a small pH imbalance could occur. In this case, transChem. Eur. J. 2014, 20, 9998 – 10004
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Figure 7. A) Efflux of CF after addition of 0.1–50 mol % of 1 c. B) Dose-response curve and adjustment to the Hill equation. Phospholipid concentration: 0.02 mm. Intravesicular: HEPES 10 mm (pH 7) and carboxyfluorescein (20 mm). Extravesicular: 100 mm NaCl, 10 mm HEPES buffer (pH 7). Transporters were injected at t = 50 s. Triton-X was injected at t = 300 s. Each curve represents the average of three trials.
porters 1 b, 1 d and 1 e may transport Cl /H + inside the liposomes. Transporter 1 c being the only effective transporter for CF efflux, a detailed kinetic study was performed for this compound. As shown in Figure 7, two different types of behaviour can be observed. At concentrations below 1.56 mol %, transporter 1 c only acts as a H + /Cl transporter in order to equilibrate the intra-/extravesicular pH imbalance. Beyond this con-
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Full Paper centration, CF is transported across the phospholipid bilayer. At concentrations below 6.25 mol %, the transport process is slow and the slow increase in CF’s fluorescence is probably compensated by the extinction of fluorescence provoked by the influx of protons into the liposomes. At 12.5 mol % of 1 c, CF is rapidly transported outside the liposomes. The EC50, 250 value of 21.2 % was also calculated, as previously described, as well as the Hill coefficient, n 2. These results suggest the formation of a transmembrane pore by the assembly of two monomers, able to allow the translocation of nanometric objects. An alternative hypothesis could be membrane destabilization and CF leakage. However, if a leakage was involved, a more pronounced effect would be observed for longer alkyl chains, that is, compounds 1 d and 1 e. Stability of the liposomes in the presence of transporters The integrity of the liposomes after the addition of the imidazolium-functionalized binols, was verified by dynamic light scattering (DLS) measurements. The size of the liposomes was measured 5 min after the addition of 50 mol % of transporter, this concentration being the highest used in all our studies (we can assume that at lower concentrations, the effect of the transporter on the membrane is less important). As shown in Figure 8 and Table 2, all the liposomes were monodisperse after addition of the transporters. This first observation suggests that the liposomes are intact, eliminating the hypothesis of liposome destruction. However, variation of the liposome’s diameter was observed in the presence of all the transporters. The increase of the liposomes size was only 10 % for transport-
ers 1 a and 1 b, but it was 40 % for compound 1 c. For compounds 1 d and 1 e, 160 and 180 % size increases, respectively, were observed. These results suggest, again, that 1 a and 1 b act as mobile transporters, not altering at all the size of the liposomes. In the case of compounds 1 c–e, the formation of transmembrane pores results in the swelling of the liposomes, but not their destruction.
Conclusion By introducing imidazolium moieties in positions 3 and 3’ of 1,1’-binaphtol, a new family of imidazolium-based transporters emerged. The transport properties of these imidazolium-functionalized binols depend on the length of the imidazolium alkyl chain, the position and the number of imidazolium units on the binol scaffold. The transport mechanism also depends on the length of the alkyl chain, allowing the transporter to act either as a mobile transporter in the case of methyl and butyl chains and forming transmembrane anion pores for octyl, dodecyl and hexadecyl chains. The selectivity for the transported anion is dependent on the molecular structure of the transporter. The transmembrane-active aggregates seem to be formed by two monomers. Again, the length of the alkyl chain dictates the stability of the formed aggregates, compound 1 e forming the more stable hydrophobic channels (Figure 9). However, these hydrophobic channels are not efficient for anion transport, the distance between two imidazolium cations in the dimer being probably too important. Transporter 1 c is the most efficient transporter, possessing a dual behavior, of mobile carrier and forming transmembrane pores. In this case, the dimer is probably too small to cross the bilayer from one side to the other and the aggregate too instable to remain in the phospholipid environment. This dimer could disassemble and the monomer may then act as a mobile carrier. Finally, the particular geometry of the binol unit may be the reason for the opening of transmembrane pore to a size large enough to allow the transport of carboxyfluorescein, in the case of 1 c.
Figure 8. Size of the liposomes after addition of methanol, Triton X-100 or a methanol solution of transporters 1 a–e.
Table 2. Size of the liposomes determined by DLS after addition of methanol, Triton X-100 or 50 mol % of 1 a–e.
MeOH Triton 1a 1b 1c 1d 1e
Alkyl group
Diameter [nm]
Variation of diameter [%]
– – CH3 C4 H 9 C8H17 C12H25 C16H33
141 6 92 154 13 153 13 197 15 364 36 395 45
– 93 10 9 40 159 181
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Figure 9. Schematic representation of the active transmembrane dimers formed by 1 c-e.
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Full Paper Acknowledgements This work was supported by the Natural Sciences and Engineering Research Council of Canada, the Fonds Qubcois de la Recherche sur la Nature et les Technologies, the Centre of Green Chemistry and Catalysis, the Canada Foundation for Innovationand the Universit de Montreal. We thank colleagues for careful discussions of this manuscript. Keywords: anion binding · binol · cell membranes · imidazolium salts · transmembrane transporters [1] H. Nikaido, Microbiol. Mol. Biol. Rev. 2003, 67, 593 – 656. [2] a) C. A. Hubner, Hum. Mol. Genet. 2002, 11, 2435 – 2445; b) E. C. Emery, G. T. Young, E. M. Berrocoso, L. Chen, P. A. McNaughton, Science 2011, 333, 1462 – 1466; c) F. M. Ashcroft, Nature 2006, 440, 440 – 447. [3] a) L. Puljak, G. Kilic, Biochim. Biophys. Acta. 2006, 1762, 404 – 413; b) A. S. Verkman, L. J. Galietta, Nat. Rev. Drug. Discov. 2009, 8, 153 – 171. [4] a) C. J. Haynes, N. Busschaert, I. L. Kirby, J. Herniman, M. E. Light, N. J. Wells, I. Marques, V. Felix, P. A. Gale, Org. Biomol. Chem. 2014, 12, 62 – 72; b) P. B. Cranwell, J. R. Hiscock, C. J. Haynes, M. E. Light, N. J. Wells, P. A. Gale, Chem. Commun. 2013, 49, 874 – 876; c) C. J. Haynes, S. N. Berry, J. Garric, J. Herniman, J. R. Hiscock, I. L. Kirby, M. E. Light, G. Perkes, P. A. Gale, Chem. Commun. 2013, 49, 246 – 248; d) H. Itoh, S. Matsuoka, M. Kreir, M. Inoue, J. Am. Chem. Soc. 2012, 134, 14011 – 14018. [5] G. W. Gokel, N. Barkey, New J. Chem. 2009, 33, 947 – 963. [6] J. Rennolds, S. Butler, K. Maloney, P. N. Boyaka, I. C. Davis, D. L. Knoell, N. L. Parinandi, E. Cormet-Boyaka, Toxicol. Sci. 2010, 116, 349 – 358. [7] D. Fialho, S. Schorge, U. Pucovska, N. P. Davies, R. Labrum, A. Haworth, E. Stanley, R. Sud, W. Wakeling, M. B. Davis, D. M. Kullmann, M. G. Hanna, Brain 2007, 130, 3265 – 3274. [8] C. R. Yamnitz, S. Negin, I. A. Carasel, R. K. Winter, G. W. Gokel, Chem. Commun. 2010, 46, 2838 – 2840.
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Received: March 13, 2014 Published online on July 7, 2014
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