- Email: [email protected]
Carriers and channels: current progress and future prospects
711
Carriers and channels: current progress and future prospects Thomas
W Bell
Recent advances in the understanding
of biological transport
and in the design of artificial transport systems have resulted from the structural elucidation of the Kf ion channel and from synthesis of artificial receptors for cations and anions, as well as neutral and zwitterionic organic molecules. Sensors, carriers, and self-assembling
capsules and nanotubes
gated are all
important offsprings of current efforts to mimic natural transport across biomembranes.
Addresses Department of Chemistry, MS21 6, University of Nevada, Reno, NV 89557, USA; e-mail: [email protected] Current Opinion in Chemical Biology 1998, 2:71 l-71 6 http://biomednet.com/elecref/136759310020071
1
0 Current Biology Ltd ISSN 1367-5931
Introduction Membrane transport is a biological function that serves as a challenge to chemists, both to model passive, gated and active transport of ions and small molecules across lipid bilayer membranes, and to invent useful devices inspired by biology. In last year’s Model systems section of Current Opinion in Chemical Biology, Fyles [l’] focused specifically on artificial transporters serving to model mechanisms of natural membrane transport. This review takes a more general approach, including advances in complexation of ions and small molecules in nonpolar media and artificial approaches to gated binding and transport. The intent is not to comprehensively cover this broad subject, but to highlight specific examples of interest published during 1997 and 1998.
Biological transport The X-ray crystallographic solution of the structure of the K+ channel [Z”] was the first for a number of the structurally related family of voltage-gated channels and represents a major advance toward understanding mechanisms of ion-selective transport. Various models have been proposed for K+ versus Na+ selectivity in voltageincluding involvement of cation-x gated channels, interactions [3’] between alkali metal ions and aromatic sidechains of amino acids. The K+ channel from Streptoqces lividam [Z”] consists of four identical polypeptide subunits, creating a cone-shaped structure that is constricted near the extracellular end to form the ‘selectivity filter’; backbone carbonyl oxygen atoms line this site to bind dehydrated K+, but they are held too far apart to coordinate Na+ effectively. MacKinnon et al. [4] also demonstrated that prokaryotic and eukaryotic K+ channels have the same pore structures. Ligand-gated ion channels remain less well characterized, but the glutamate receptor channel has been found to be tetrameric [S] as seen in voltage-gated K+ channels.
The mechanisms of membrane transport mediated by carriers and channel-forming ionophores continue to be active topics of research. Examples include mathematical modeling of carrier-mediated transport [6] and experimental studies of sodium transport by carrier- and channel-type ionophores by means of z3Na-NMR spectroscopy [7]. A system consisting of two artificial membranes containing immobilized enzymes (kinase and phosphatase, respectively), which functions as a pump for glycerol-3-phosphate is of particular interest as a model of active transport [8’]. This result suggests that channel-type structures bearing two different catalytic activities might be used for active transport.
Cation binding Recognition of metal ions by relatively small molecules has been an active area of research for chemists since the discovery that crown ethers can serve as crude mimics of carrier-type ionophores. Structural and thermodynamic studies of lipophilic ion-ionophore complexes are relevant to transport across biomembranes because metal ion selectivities are determined by binding constants within the nonpolar medium. Novel artificial ionophores are also of interest as the essential components of carrier-based ionselective electrodes and optical sensors [9]. Such devices are useful for quantifying physiologically-relevant ions in solution and fluorescent molecules can be tailored as molecular probes for detection of ions and small molecules inside cells and membranes [ 10,111. Novel coordination chemistry and practical uses of relatively well known artificial ionophores continue to be explored. For example, dicyclohexano-18-crown-6 (1, Figure 1) has been found to selectively transport Pb(II), Ga(I1) and Fe(II1) across a plasticized polymer membrane [12]. Calix[4]arenes 2 and 3 (Figure 1) bearing phosphoryl substituents were found to transport alkali metals across liquid membranes in U-tube experiments [13]. (In such experiments, the carrier dissolved in an organic solvent, which simulates the nonpolar interior of a lipid bilayer membrane, facilitates the diffusion of the metal from the higher-concentration aqueous phase to the ‘receiving’ aqueous phase.) Of special interest is a novel calix[4] tube (4, Figure 1) that binds K+ cation with high selectivity [14’]. Designed prior to the structural elucidation of the voltage-gated K+ channel [Z”], this tubular calixarene was intended to mimic cation-x interactions postulated to occur between K+ ions and tyrosine residues in the selectivity filter [3’]. Chemists continually search for synthetically accessible molecular architectures capable of preorganizing metalbinding atoms for selective complexation. A structurally interesting example is the [Z.Z.l]cryptand analog 5
7 12
Model systems
Figure 1
::
2
R=CH,PPh,
3
:: R = CH,CNEt,
8
X = NHCHCO,Me &H,CH(CH,),
9
X=OCH,
6 Current Opinion in Chemical
Structures of synthetic, cation-binding molecules. as cation-binding molecules of novel architecture.
Transport studies have been conducted with l-3, 8 and 9; compounds Et, ethyl; Me, methyl; Ph, phenyl. (See text for full details.)
(Figure l), derived from a functionalized cage compound containing a single ether moiety [15]. This molecule binds Na+ and K+ with high affinity and the X-ray crystal structure of the Ssodium tosylate complex has been reported. Starands are recently discovered, rigid polyether macrocycles, and a theoretical study of Li+ binding to a model for [&]starand (6, Figure 1) has appeared [16]. While C6 (Figure 1) largely encapsulate the metal cation with oxygen or nitrogen atoms, stable complexes of alkali metals can also be formed by highly preorganized, relatively planar arrays of these atoms. For example, fused ring ‘hexagonal-lattice’ host 7 extracts alkali metal salts into organic solvents [17]. It has only five ligand atoms but it binds Na+ and K+ more strongly than do most hexadentate crown ethers. Another cage molecule, adamantane, has been incorporated into the structures of several serine-based cyclodepsipeptides in an effort to conformationally preorganize metal-binding sites [l&19]. The 24-membered macrocycle 8 (Figure 1) transported Na+, Ca2+ and Mg2+ most efficiently, but unselectively, across model membranes [18]. The X-ray structures of three members of this series of macrocycles have been reported [19], and the formation of dimers by association of molecules of 9 in the solid state suggests that 8 may also self-associate during ion transport.
4-7
Biology
are mainly of interest
Anion and molecule binding While many artificial molecular architectures are available for complexation of cations, binding of anions and neutral molecules has proven to be more difficult. Several approaches have been used for anion binding and transport, as described in a recent review [ZO]. Coneshaped molecules, such as calix[4]arenes and resorcinarenes bearing multiple hydrogen-bond donor groups (e.g. urea NH) can bind anions solely by hydrogen bonding, but self-association competes with this hydrogen bonding in nonpolar media. Several thioureaderivatized resorcinarenes have been synthesized and found to transport halide ions through supported liquid membranes [Zl’]. A small preference for chloride over other halides was observed, with the p-fluorophenylthiourea system (10, Figure 2) giving the largest binding constant (4.7 x lo5 M-l in CDCl,). Competing dimerization of 10 is apparently minimized by steric interference between the aryl groups and by the weak tendency of thiourea to form hydrogen-bonded head-to-tail chains, relative to urea. Amino acids are attractive targets for designed binding and transport studies because of their hydrophilicity, chirality and biological significance. The simplest approach to
Carriers
Figure
and channels
Bell
713
2
I NHAc
10
R=p-FC,H,
12 Current Opinion in Chemical
Biology
Structures of artificial receptors for anions and molecules. Thiourea-derivatized resorcinarene enantioselectively complex amino acids. AC, Acetyl; Et, ethyl. (See text for full details.)
amino acid transport is to use commercially available cation-binders, such as crown ethers, azacrowns or cryptands, as carriers in liquid membranes. Such artificial ionophores are known to bind alkylammonium ions, but a recent study of L-tryptophan transport across supported liquid membranes [Z?] showed that crown ethers (l&crown-6 and dibenzo-1%crown-6) were not effective carriers. This suggests that transport mediated by the azacrown Kryptofix 5 (Merck, Darmstadt, Germany) and by [Z.Z.Z]cryptand (Kryptofix 222; Merck, Darmstadt, Germany) are likely to involve ion pairing between the carboxylate group of the amino acid and these protonated carriers. In another study [23], the direction of paminobenzoic acid transport across a liquid membrane appeared to depend on the presence or absence of Na+. Unfortunately, coupled countertransport was not proven because the pH of the receiving phase was varied with the concentration of Na+.
10 transports
halide ions; 11 and 12
Enantioselective binding and transport of amino acids is a particularly challenging problem, and impressive efforts have been made to theoretically model chiral recognition of peptidic ammonium ions by synthetic ionophores, such as 11 (Figure 2) [24]. Instead of binding as an ammonium cation to an ionophore, the amino group of an amino acid can be coordinated to a transition metal in a lipophilic complex. This approach was taken in a thorough study of enantioselective extraction of D-phenylalanine by emulliquid containing sion membranes copper(I1) N-decyl-(L)-hydroxyproline as a chiral carrier [ZS]. Sessler and Andrievsky [26’] designed a lipophilic complexing agent for zwitterionic amino acids by tethering a natural ammonium-binding ionophore (lasalocid) to a cationic carboxylate receptor (sapphyrin). Receptor 12 (Figure 2) was found to be an efficient carrier, showing some selectivity between different amino acids and between enantiomers in U-tube and W-tube experiments.
7 14
Model systems
Figure 3
B(Ot
P I’ /
0
16
x=1
or2
y=l
or2 Current Opinion in Chemical
Structures
of photoswitchable
(13-l
5) and redox-gated
Biology
(16) receptors for alkali and alkaline earth metals (13, 14 and 16) and glucose (15).
Me, methyl; Ph, phenyl. (See text for full details.)
Switchable
carriers
Carriers that can be switched between high- and low-affinity states by means of an external signal are of interest as minimal models of gated transport across biomembranes. Photoisomerizable azobis(crowns) are among the first such molecules to be synthesized, and lipophilic derivatives of azobis( E-crown-S), including 13 (Figure 3), have been used recently as molecular probes for phase boundary potentials in ion-selective electrode membranes [27]. A photoswitchable extractant for rubidium and cesium has also been developed [28]. The cis isomer 14 (Figure 3), formed by UV irradiation of the tram isomer, can be isomerized back to the low-affinity tram form by heating at 30” C. The Shinkai group, which produced the original ‘photoresponsive’ crown ethers, has now reported a light-gated sugar receptor [29]. The cis form of this bis( boronic acid) (15; Figure 3), formed by UV irradiation of the tram form, binds glucose selective-
ly with reversible formation of a bis(boronic ester). The sugar is released upon isomerization to the tram form, which is induced by visible light irradiation. Photoisomerization often produces subtle changes in the binding abilities of synthetic receptors, while the charge differences caused by redox switching could produce very large differences in affinities toward charged ligands (e.g. cations). The goal of mimicking such effects has been achieved using oxaferrocene cryptands (16, Figure 3), in which oxygen atoms of the cryptand moiety are directly attached to the redox-active ferrocene component [30’]. This conjugation produces excellent communication between the metal centers, and binding constants toward alkali and alkaline earth metal ions are decreased by as much as six orders of magnitude when the ferrocene moiety is oxidized in acetonitrile.
Carriers
Figure
and channels
Bell
715
4
17
R = 4-n-heptylphenyl 18 Current ODinion in Chemical Bioloav
Structure
of an unsymmetrical molecular strip (17) that dimerizes to form a chiral capsule, and the hydrogen-bonding
a peptide nanotube.
(Most sidechains are omitted for clarity.) Dotted lines represent
Self-assembling
capsules and channels
Self-assembly is a technique that is commonly employed by nature to construct large, complicated, functional structures from smaller subunits. Relevant examples are the formation of ion channels by dimerization of gramicidin S and by higher order self-assembly of polyene macrolide antibiotics or proteins [2”,4,31’]. Only relatively recently have chemists exploited the power of self-assembly to produce cavities that are large enough to encapsulate (and therefore transport) small molecules [32”,33’]. For example, calix[4]arenes similar to 10 (Figure 2) bearing urea substituents dimerize to form cavities that encapsulate solvent molecules. Rebek et al. [34] have also produced capsules of various sizes, termed ‘tennis balls’ and ‘soft balls’, by dimerization of bent molecular strips via hydrogen bonding. A recent extension of this work [34] involves the dimerization of an unsymmetrical strip 17 (Figure 4) to form chiral molecular capsules. The ratio of enantiomeric capsules formed was influenced by the presence of chiral ligand molecules, including naturally occurring terpenes. So far, these binding studies have been conducted mainly in solution in organic solvents, but it can be predicted that self-assembling carriers will be developed to transport small molecules across membranes, perhaps enantioselectively.
that the central axes of nanotubes, composed of cyclo([LTrp-D-Leu13-L-Gln-D-Leu), are aligned parallel to the hydrocarbon chains in dimyristoyl phosphatidylcholine membranes, confirming the formation of transmembrane channels.
Conclusions Excellent progress has been made towards understanding selectivity in biological ion transport and in the design of artificial carriers and channels. The structure of the selectivity filter in voltage-gated K+ channels should inspire chemists to tinker with biomimetic versions in order to fully understand the principles of ion transport selectivity. Diverse structures have been synthesized as carriers for cations, anions and small organic molecules. Switchable carriers and self-assembling capsules are particularly important developments in gated ion transport and smallmolecule transport, respectively. Formation of pores in membranes by insertion of peptide nanotubes has been firmly demonstrated. Overall, an enterprise that began as an effort to model and understand biology has blossomed to yield fruits of unimagined beauty and potential utility.
References and recommended
reading
Papers of particular interest, published within the annual period of review, have been highlighted as: l l
Self-assembling carriers hold promise for the future, but good progress has already been made in the formation of ion channels and pores by self-assembly. The engineering of rigid rod molecules as mimics of polyene macrolide ionophores [35] and peptides as mimics of pore-forming proteins [36] are examples of this. The most impressive results in this latter area of research have been obtained in studies of the selfassembly of peptide nanotubes from cyclic D,L-a-peptides and cyclic p peptides [37”]. The hydrogen bonding motif for channels formed by cyclic D,L-a-peptides is shown in Figure 4 (18). These nanotubes have been characterized crystallographically and also form active ion channels in lipid bilayer membranes. Recent spectroscopic studies [38] demonstrated
motif (18) of a cyclic
hydrogen bonds. (See text for full details.)
of special interest * of outstanding interest
1. .
Fyles TM: Bilayer membranes and transporter models. Curr Opin Chem Bioll997, 1:497-505. This is a review of advances over the previous year relating to mechanisms of biomimetic transport, control of bilayer formation and molecular recognition by synthetic lipids. 2. ..
Doyle DA, Cabral JM, Pfuetzner RA, Kuo A., Gulbis JM, Cohen SL, Chait BT, MacKinnon R: The structure of the potassium channel: molecular basis of K+ conduction and selectivity. Science 1998, 280:69-77. This is a full paper describing the X-ray structure of the potassium channel from Streptomyces lividans, including general properties of the ion conduction pore, the cavity and the selectivity filter, which contains two K+ ions. 3. .
Ma JC, Dougherty DA: The cation% interaction. Chem Rev 1997, 97:1303-l 324. A comprehensive review of theoretical and experimental studies of cation-n interactions in artificial receptors and biological structures, including acetylcholine receptors and ion channels.
7 16
Model systems
4.
MacKinnon R, Cohen SL, Kuo A, Lee A, Chait BT: Structural conservation in prokaryotic and eukaryotic potassium channels. Science 1998, 280:106-l 09.
22.
Wieczorek P: Factors influencing the transport of tryptophan hydrochloride through supported liquid membranes containing macrocyclic carriers. J Membr Sci 1997, 127:87-92.
5.
Rosenmund C, Stern-Bach Y, Stevens CF: The tetrameric structure of a glutamate receptor channel. Science 1998, 280:1596-l 599.
23.
6.
Hirayama H, Nishimura T, Okita T, Fukuyama Y: A method to evaluate economical carrier-mediated transport across the biological membrane by the optimal control principle. J Membr Sci 1998, 139:109-l 24.
Uglea CV, Croitoru M: Transport of amino acids through liquid membranes Ill. The alkaline ion role. J Membr Sci 1997, 133:127-l 31.
24.
Kimura A, Kuni N, Fujiwara H: Unusual behavior of ion transport mediated by polyene antibiotics. Activation energies for the exchange of Na+ ions through liposomal membranes studied by ssNa-NMR spectroscopy. Chem Pharm Bull 1997, 45:431-436.
Senderowitz H, McDonald DCI, Still WC: A practical method for calculating relative free energies of binding. Chiral recognition of peptidic ammonium ions by synthetic ionophores. J Org Chem 1997, 62:9123-9127.
25.
Pickering PJ, Chaudhuri JB: Enantioselective extraction of CD)phenylalanine from racemic (D/L)-phenylalanine using chiral emulsion liquid membranes. J Membr Sci 1997, 127:l 15-l 30.
Ma’rsterrena B, Nigon C, Michalon P, Couturier R: Active transport of glycerol-3-phosphate with artificial enzyme membranes: a new kinetic model for active transport processes. J Membr Sci 1997, 134:85-99. Experimental and theoretical studies of active transport of glycerol-3-phosphate by artificial enzyme membranes bearing immobilized kinase and phosphatase are described. 9.
Bakker E, Bijhlmann P, Pretsch E: Carrier-based ion-selective electrodes and bulk optodes. 1. General characteristics. Chem Rev 1997,97:3083-3132.
Tohda K, Yoshiyagawa S, Kataoka M, Odashima K, Umezawa Y: Photoswitchable azobiscbenzo-15-crown-5) ionophores as a molecular probe for phase boundary potentials at ion-selective poly(vinyl chloride) liquid membranes. Anal Chem 1997, 69:3360-3369.
28.
Reynier N, Dozol J-F, Saadioui M, Asfari Z, Vicens J: Complexation properties of a new photosensitive calixI4larene crown ether containing azo unit in the lower rim towards alkali cations. Tetrahedron Lett 1998, 39:6461-6464.
Lamb JD, Nazarenko AY: Selective metal ion sorption and transport using polymer inclusion membranes containing dicyclohexano18-crown-6. Separ Sci Techno/ 1997, 32:2749-2764.
29.
Yaftian MR, Burgard M, Matt D, Wieser C, Dieleman Multifunctional calixI4larenes containing pendant phosphoryl groups: their use as extracting agents alkali cations. J lncl Phenom MO/ Ret Chem 1997,
Shinmori H, Takeuchi M, Shinkai S: A novel light-gated sugar receptor, which shows high glucose selectivity. J Chem Sot Tram 2 1998:847-852.
30. .
Desvergne JP, Czarnik AW (Eds): Chemosensors of /on and Molecule Dordrecht: Kluwer; 1997. Recognition. NATO AS/ Series.
11.
Jiwan J-L H, Branger C, Soumillion J-Ph, Valeur B: Ion-responsive fluorescent compounds V. Photophysical and complexing properties of coumarin 343 linked to monoaza-15-crown-5. J Photochem PhotobiolA - Chem 1998, 116:127-l 33.
13.
C: amide and and carriers for 27:127-l 40.
Schmitt P, Beer PD, Drew MGB, Sheen PD: CalixI4ltube: A tubular receptor with remarkable potassium ion selectivity. Angew Chem Int Ed Engll997, 36:1840-l 842. This is a report of a new cage molecule possessing an 8-coordinate binding site and displaying remarkable affinity for K+ over other alkali metals. 14. .
15.
Marchand AP, Alihod_?C Desvergne JP, McKim AS, Kumar KA, MlinariC-Majerski K, Sumanovac T, Bott SG: Synthesis and alkali metal picrate extraction capabilities of a 4-oxahexacyclo[5.4.1.0~~s.03~~c.Os~s.Os~~~ldodecane-derived cryptand. A new ionophore for selective ion complexation. Tetrahedron Lett 1998, 39:1861-l 864.
1 7.
Bell TW, Cragg PJ, Firestone A, Kwok AD-I, Liu J, Ludwig R, Sodoma A: Molecular architecture. 2. Synthesis and metal complexation of heptacyclic terpyridyl molecular clefts. J Org Chem 1998, 6312232.2243.
18.
Ranganathan D, Haridas V, Madhusudanan KP, Roy R, Nagaraj R, John GB: Serine-based cyclodepsipeptides on an adamantane building block: design, synthesis, and characterization of a novel family of macrocyclic membrane ion-transporting depsipeptides. J Am Chem Sot 1997,119:11578-l 1584.
20.
.
31. .
Eisenberg B: Ionic channels in biological membranes: natural nanotubes. Accounts Chem Res 1998, 31 :117-l 23. This is a thorough review of experimental and theoretical studies relating to biological ion channels.
de Mendoza J: Self-assembling cavities: present and future. Chem Eur J 1998,4:1373-l 377. This is a shorter review, which focuses on hydrogen-bonding dimerization of substituted calixarenes and molecular strips, including chiral capsules (which are described in greater detail in [34]). 33. .
34.
Schmidtchen FP, Berger M: Artificial organic host molecules for anions. Chem Rev 1997, 97:1609-l 646.
Rivera JM, Martin T, Rebek J Jr: Chiral spaces: dissymmetric capsules through self-assembly. Science 1998, 279:1021-l
023.
35.
Sakai N, Brennan KC, Weiss LA, Matile S: Toward biomimetic ion channels formed by rigid-rod molecules: length-dependent iontransport activity of substituted oligo(p-phenylenejs. J Am Chem Sot 1997,119:8726-8727.
36.
Seth S, Balaram P, Mathew MK: Characterization of a 22-residue peptide derived from a designed ion channel. Biochim Siophys Acta 1997, 1328:177-l 84.
novel
Boerrigter H, Grave L, Nissink JWM, Chrisstoffels LAJ, van der Maas JH, VerboomW, de Jong F, Reinhoudt DN: (Thio)urea resorcinarene cavitands. Complexation and membrane transport of halide anions. J Org Chem 1998, 63:4174-4180. This is the first report of facilitated membrane transport of halide anions through supported liquid membranes; the artificial receptors acted as carriers with a small preference for chloride over other halides. 21.
Plenio H, Aberle C: Oxaferrocene cryptands as efficient molecular switches for alkali and alkaline earth metal ions. Organometallics 1997,16:5950-5957. Novel ferrocene-containing cryptands are described for which binding of alkali and alkaline earth metals is effectively switched off by oxidation of the ferrocene moiety.
Conn MM, Rebek J Jr: Self-assembling capsules. Chem Rev 1997, 97:1647-l 668. This is a complete review of artificial self-assembling cavities, including hydrogen-bonding systems, hydrophobic assemblies and metal-templated rings and cages.
Cui C, Cho SJ, Kim KS: Cation affinities of 11sl starand model. Comparison with 12-crown-4: crucial role of dipolar moiety orientations. J Phys Chem A 1998, 102:1119-l 123.
Karle IL, Ranganathan D, Haridas V: Adamantane-constrained cyclodepsipeptides: crystal structure and self-assembling properties of cyclo(Adm-Ser), and cyclo(Adm-Ser-Xaa),, Xaa =Val/Ser. J Am Chem Sot 1998,120:6903-6908.
Perkin
32. ..
16.
19.
Sessler JL, Andrievsky A: Efficient transport of aromatic amino acids by sapphyrin-lasalocid conjugates. Chem Eur J 1998, 4:159-l 67. This paper reports the synthesis and characterization of several sapphyrin-lasalocid conjugates that transport aromatic a amino acids through liquid membranes; a key carrier showed an intrinsic preference for phenylalanine over tryptophan and tryptophan over tyrosine. 27.
10.
12.
26. .
37. ..
Hartgerink JD, ClarkTD, Ghadiri MR: Peptide nanotubes and beyond. Chem Eur J 1998,4:1367-l 372. This is a review of recent advances in self-assembly of nanotubes from cyclic D,L-a-peptides and cyclic p peptides, including applications in biological chemistry and materials science. 38.
Kim HS, Hartgerink JD, Ghadiri MR: Oriented self-assembly cyclic peptide nanotubes in lipid membranes. JAm Chem 1998, 120:4417-4424.
of Sot
Carriers and channels: current progress and future prospects Thomas
W Bell
Recent advances in the understanding
of biological transport
and in the design of artificial transport systems have resulted from the structural elucidation of the Kf ion channel and from synthesis of artificial receptors for cations and anions, as well as neutral and zwitterionic organic molecules. Sensors, carriers, and self-assembling
capsules and nanotubes
gated are all
important offsprings of current efforts to mimic natural transport across biomembranes.
Addresses Department of Chemistry, MS21 6, University of Nevada, Reno, NV 89557, USA; e-mail: [email protected] Current Opinion in Chemical Biology 1998, 2:71 l-71 6 http://biomednet.com/elecref/136759310020071
1
0 Current Biology Ltd ISSN 1367-5931
Introduction Membrane transport is a biological function that serves as a challenge to chemists, both to model passive, gated and active transport of ions and small molecules across lipid bilayer membranes, and to invent useful devices inspired by biology. In last year’s Model systems section of Current Opinion in Chemical Biology, Fyles [l’] focused specifically on artificial transporters serving to model mechanisms of natural membrane transport. This review takes a more general approach, including advances in complexation of ions and small molecules in nonpolar media and artificial approaches to gated binding and transport. The intent is not to comprehensively cover this broad subject, but to highlight specific examples of interest published during 1997 and 1998.
Biological transport The X-ray crystallographic solution of the structure of the K+ channel [Z”] was the first for a number of the structurally related family of voltage-gated channels and represents a major advance toward understanding mechanisms of ion-selective transport. Various models have been proposed for K+ versus Na+ selectivity in voltageincluding involvement of cation-x gated channels, interactions [3’] between alkali metal ions and aromatic sidechains of amino acids. The K+ channel from Streptoqces lividam [Z”] consists of four identical polypeptide subunits, creating a cone-shaped structure that is constricted near the extracellular end to form the ‘selectivity filter’; backbone carbonyl oxygen atoms line this site to bind dehydrated K+, but they are held too far apart to coordinate Na+ effectively. MacKinnon et al. [4] also demonstrated that prokaryotic and eukaryotic K+ channels have the same pore structures. Ligand-gated ion channels remain less well characterized, but the glutamate receptor channel has been found to be tetrameric [S] as seen in voltage-gated K+ channels.
The mechanisms of membrane transport mediated by carriers and channel-forming ionophores continue to be active topics of research. Examples include mathematical modeling of carrier-mediated transport [6] and experimental studies of sodium transport by carrier- and channel-type ionophores by means of z3Na-NMR spectroscopy [7]. A system consisting of two artificial membranes containing immobilized enzymes (kinase and phosphatase, respectively), which functions as a pump for glycerol-3-phosphate is of particular interest as a model of active transport [8’]. This result suggests that channel-type structures bearing two different catalytic activities might be used for active transport.
Cation binding Recognition of metal ions by relatively small molecules has been an active area of research for chemists since the discovery that crown ethers can serve as crude mimics of carrier-type ionophores. Structural and thermodynamic studies of lipophilic ion-ionophore complexes are relevant to transport across biomembranes because metal ion selectivities are determined by binding constants within the nonpolar medium. Novel artificial ionophores are also of interest as the essential components of carrier-based ionselective electrodes and optical sensors [9]. Such devices are useful for quantifying physiologically-relevant ions in solution and fluorescent molecules can be tailored as molecular probes for detection of ions and small molecules inside cells and membranes [ 10,111. Novel coordination chemistry and practical uses of relatively well known artificial ionophores continue to be explored. For example, dicyclohexano-18-crown-6 (1, Figure 1) has been found to selectively transport Pb(II), Ga(I1) and Fe(II1) across a plasticized polymer membrane [12]. Calix[4]arenes 2 and 3 (Figure 1) bearing phosphoryl substituents were found to transport alkali metals across liquid membranes in U-tube experiments [13]. (In such experiments, the carrier dissolved in an organic solvent, which simulates the nonpolar interior of a lipid bilayer membrane, facilitates the diffusion of the metal from the higher-concentration aqueous phase to the ‘receiving’ aqueous phase.) Of special interest is a novel calix[4] tube (4, Figure 1) that binds K+ cation with high selectivity [14’]. Designed prior to the structural elucidation of the voltage-gated K+ channel [Z”], this tubular calixarene was intended to mimic cation-x interactions postulated to occur between K+ ions and tyrosine residues in the selectivity filter [3’]. Chemists continually search for synthetically accessible molecular architectures capable of preorganizing metalbinding atoms for selective complexation. A structurally interesting example is the [Z.Z.l]cryptand analog 5
7 12
Model systems
Figure 1
::
2
R=CH,PPh,
3
:: R = CH,CNEt,
8
X = NHCHCO,Me &H,CH(CH,),
9
X=OCH,
6 Current Opinion in Chemical
Structures of synthetic, cation-binding molecules. as cation-binding molecules of novel architecture.
Transport studies have been conducted with l-3, 8 and 9; compounds Et, ethyl; Me, methyl; Ph, phenyl. (See text for full details.)
(Figure l), derived from a functionalized cage compound containing a single ether moiety [15]. This molecule binds Na+ and K+ with high affinity and the X-ray crystal structure of the Ssodium tosylate complex has been reported. Starands are recently discovered, rigid polyether macrocycles, and a theoretical study of Li+ binding to a model for [&]starand (6, Figure 1) has appeared [16]. While C6 (Figure 1) largely encapsulate the metal cation with oxygen or nitrogen atoms, stable complexes of alkali metals can also be formed by highly preorganized, relatively planar arrays of these atoms. For example, fused ring ‘hexagonal-lattice’ host 7 extracts alkali metal salts into organic solvents [17]. It has only five ligand atoms but it binds Na+ and K+ more strongly than do most hexadentate crown ethers. Another cage molecule, adamantane, has been incorporated into the structures of several serine-based cyclodepsipeptides in an effort to conformationally preorganize metal-binding sites [l&19]. The 24-membered macrocycle 8 (Figure 1) transported Na+, Ca2+ and Mg2+ most efficiently, but unselectively, across model membranes [18]. The X-ray structures of three members of this series of macrocycles have been reported [19], and the formation of dimers by association of molecules of 9 in the solid state suggests that 8 may also self-associate during ion transport.
4-7
Biology
are mainly of interest
Anion and molecule binding While many artificial molecular architectures are available for complexation of cations, binding of anions and neutral molecules has proven to be more difficult. Several approaches have been used for anion binding and transport, as described in a recent review [ZO]. Coneshaped molecules, such as calix[4]arenes and resorcinarenes bearing multiple hydrogen-bond donor groups (e.g. urea NH) can bind anions solely by hydrogen bonding, but self-association competes with this hydrogen bonding in nonpolar media. Several thioureaderivatized resorcinarenes have been synthesized and found to transport halide ions through supported liquid membranes [Zl’]. A small preference for chloride over other halides was observed, with the p-fluorophenylthiourea system (10, Figure 2) giving the largest binding constant (4.7 x lo5 M-l in CDCl,). Competing dimerization of 10 is apparently minimized by steric interference between the aryl groups and by the weak tendency of thiourea to form hydrogen-bonded head-to-tail chains, relative to urea. Amino acids are attractive targets for designed binding and transport studies because of their hydrophilicity, chirality and biological significance. The simplest approach to
Carriers
Figure
and channels
Bell
713
2
I NHAc
10
R=p-FC,H,
12 Current Opinion in Chemical
Biology
Structures of artificial receptors for anions and molecules. Thiourea-derivatized resorcinarene enantioselectively complex amino acids. AC, Acetyl; Et, ethyl. (See text for full details.)
amino acid transport is to use commercially available cation-binders, such as crown ethers, azacrowns or cryptands, as carriers in liquid membranes. Such artificial ionophores are known to bind alkylammonium ions, but a recent study of L-tryptophan transport across supported liquid membranes [Z?] showed that crown ethers (l&crown-6 and dibenzo-1%crown-6) were not effective carriers. This suggests that transport mediated by the azacrown Kryptofix 5 (Merck, Darmstadt, Germany) and by [Z.Z.Z]cryptand (Kryptofix 222; Merck, Darmstadt, Germany) are likely to involve ion pairing between the carboxylate group of the amino acid and these protonated carriers. In another study [23], the direction of paminobenzoic acid transport across a liquid membrane appeared to depend on the presence or absence of Na+. Unfortunately, coupled countertransport was not proven because the pH of the receiving phase was varied with the concentration of Na+.
10 transports
halide ions; 11 and 12
Enantioselective binding and transport of amino acids is a particularly challenging problem, and impressive efforts have been made to theoretically model chiral recognition of peptidic ammonium ions by synthetic ionophores, such as 11 (Figure 2) [24]. Instead of binding as an ammonium cation to an ionophore, the amino group of an amino acid can be coordinated to a transition metal in a lipophilic complex. This approach was taken in a thorough study of enantioselective extraction of D-phenylalanine by emulliquid containing sion membranes copper(I1) N-decyl-(L)-hydroxyproline as a chiral carrier [ZS]. Sessler and Andrievsky [26’] designed a lipophilic complexing agent for zwitterionic amino acids by tethering a natural ammonium-binding ionophore (lasalocid) to a cationic carboxylate receptor (sapphyrin). Receptor 12 (Figure 2) was found to be an efficient carrier, showing some selectivity between different amino acids and between enantiomers in U-tube and W-tube experiments.
7 14
Model systems
Figure 3
B(Ot
P I’ /
0
16
x=1
or2
y=l
or2 Current Opinion in Chemical
Structures
of photoswitchable
(13-l
5) and redox-gated
Biology
(16) receptors for alkali and alkaline earth metals (13, 14 and 16) and glucose (15).
Me, methyl; Ph, phenyl. (See text for full details.)
Switchable
carriers
Carriers that can be switched between high- and low-affinity states by means of an external signal are of interest as minimal models of gated transport across biomembranes. Photoisomerizable azobis(crowns) are among the first such molecules to be synthesized, and lipophilic derivatives of azobis( E-crown-S), including 13 (Figure 3), have been used recently as molecular probes for phase boundary potentials in ion-selective electrode membranes [27]. A photoswitchable extractant for rubidium and cesium has also been developed [28]. The cis isomer 14 (Figure 3), formed by UV irradiation of the tram isomer, can be isomerized back to the low-affinity tram form by heating at 30” C. The Shinkai group, which produced the original ‘photoresponsive’ crown ethers, has now reported a light-gated sugar receptor [29]. The cis form of this bis( boronic acid) (15; Figure 3), formed by UV irradiation of the tram form, binds glucose selective-
ly with reversible formation of a bis(boronic ester). The sugar is released upon isomerization to the tram form, which is induced by visible light irradiation. Photoisomerization often produces subtle changes in the binding abilities of synthetic receptors, while the charge differences caused by redox switching could produce very large differences in affinities toward charged ligands (e.g. cations). The goal of mimicking such effects has been achieved using oxaferrocene cryptands (16, Figure 3), in which oxygen atoms of the cryptand moiety are directly attached to the redox-active ferrocene component [30’]. This conjugation produces excellent communication between the metal centers, and binding constants toward alkali and alkaline earth metal ions are decreased by as much as six orders of magnitude when the ferrocene moiety is oxidized in acetonitrile.
Carriers
Figure
and channels
Bell
715
4
17
R = 4-n-heptylphenyl 18 Current ODinion in Chemical Bioloav
Structure
of an unsymmetrical molecular strip (17) that dimerizes to form a chiral capsule, and the hydrogen-bonding
a peptide nanotube.
(Most sidechains are omitted for clarity.) Dotted lines represent
Self-assembling
capsules and channels
Self-assembly is a technique that is commonly employed by nature to construct large, complicated, functional structures from smaller subunits. Relevant examples are the formation of ion channels by dimerization of gramicidin S and by higher order self-assembly of polyene macrolide antibiotics or proteins [2”,4,31’]. Only relatively recently have chemists exploited the power of self-assembly to produce cavities that are large enough to encapsulate (and therefore transport) small molecules [32”,33’]. For example, calix[4]arenes similar to 10 (Figure 2) bearing urea substituents dimerize to form cavities that encapsulate solvent molecules. Rebek et al. [34] have also produced capsules of various sizes, termed ‘tennis balls’ and ‘soft balls’, by dimerization of bent molecular strips via hydrogen bonding. A recent extension of this work [34] involves the dimerization of an unsymmetrical strip 17 (Figure 4) to form chiral molecular capsules. The ratio of enantiomeric capsules formed was influenced by the presence of chiral ligand molecules, including naturally occurring terpenes. So far, these binding studies have been conducted mainly in solution in organic solvents, but it can be predicted that self-assembling carriers will be developed to transport small molecules across membranes, perhaps enantioselectively.
that the central axes of nanotubes, composed of cyclo([LTrp-D-Leu13-L-Gln-D-Leu), are aligned parallel to the hydrocarbon chains in dimyristoyl phosphatidylcholine membranes, confirming the formation of transmembrane channels.
Conclusions Excellent progress has been made towards understanding selectivity in biological ion transport and in the design of artificial carriers and channels. The structure of the selectivity filter in voltage-gated K+ channels should inspire chemists to tinker with biomimetic versions in order to fully understand the principles of ion transport selectivity. Diverse structures have been synthesized as carriers for cations, anions and small organic molecules. Switchable carriers and self-assembling capsules are particularly important developments in gated ion transport and smallmolecule transport, respectively. Formation of pores in membranes by insertion of peptide nanotubes has been firmly demonstrated. Overall, an enterprise that began as an effort to model and understand biology has blossomed to yield fruits of unimagined beauty and potential utility.
References and recommended
reading
Papers of particular interest, published within the annual period of review, have been highlighted as: l l
Self-assembling carriers hold promise for the future, but good progress has already been made in the formation of ion channels and pores by self-assembly. The engineering of rigid rod molecules as mimics of polyene macrolide ionophores [35] and peptides as mimics of pore-forming proteins [36] are examples of this. The most impressive results in this latter area of research have been obtained in studies of the selfassembly of peptide nanotubes from cyclic D,L-a-peptides and cyclic p peptides [37”]. The hydrogen bonding motif for channels formed by cyclic D,L-a-peptides is shown in Figure 4 (18). These nanotubes have been characterized crystallographically and also form active ion channels in lipid bilayer membranes. Recent spectroscopic studies [38] demonstrated
motif (18) of a cyclic
hydrogen bonds. (See text for full details.)
of special interest * of outstanding interest
1. .
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Model systems
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Pickering PJ, Chaudhuri JB: Enantioselective extraction of CD)phenylalanine from racemic (D/L)-phenylalanine using chiral emulsion liquid membranes. J Membr Sci 1997, 127:l 15-l 30.
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Reynier N, Dozol J-F, Saadioui M, Asfari Z, Vicens J: Complexation properties of a new photosensitive calixI4larene crown ether containing azo unit in the lower rim towards alkali cations. Tetrahedron Lett 1998, 39:6461-6464.
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Yaftian MR, Burgard M, Matt D, Wieser C, Dieleman Multifunctional calixI4larenes containing pendant phosphoryl groups: their use as extracting agents alkali cations. J lncl Phenom MO/ Ret Chem 1997,
Shinmori H, Takeuchi M, Shinkai S: A novel light-gated sugar receptor, which shows high glucose selectivity. J Chem Sot Tram 2 1998:847-852.
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15.
Marchand AP, Alihod_?C Desvergne JP, McKim AS, Kumar KA, MlinariC-Majerski K, Sumanovac T, Bott SG: Synthesis and alkali metal picrate extraction capabilities of a 4-oxahexacyclo[5.4.1.0~~s.03~~c.Os~s.Os~~~ldodecane-derived cryptand. A new ionophore for selective ion complexation. Tetrahedron Lett 1998, 39:1861-l 864.
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Sakai N, Brennan KC, Weiss LA, Matile S: Toward biomimetic ion channels formed by rigid-rod molecules: length-dependent iontransport activity of substituted oligo(p-phenylenejs. J Am Chem Sot 1997,119:8726-8727.
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novel
Boerrigter H, Grave L, Nissink JWM, Chrisstoffels LAJ, van der Maas JH, VerboomW, de Jong F, Reinhoudt DN: (Thio)urea resorcinarene cavitands. Complexation and membrane transport of halide anions. J Org Chem 1998, 63:4174-4180. This is the first report of facilitated membrane transport of halide anions through supported liquid membranes; the artificial receptors acted as carriers with a small preference for chloride over other halides. 21.
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Perkin
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16.
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10.
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26. .
37. ..
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of Sot