Aggregation behavior and dynamics of synthetic amphiphiles that self-assemble to anion transporters

The amphiphilic heptapeptides-referred to as synthetic anion transporters (SATs)-mediate chloride transport in planar lipid bilayer membranes, synthetic liposomes, and mammalian cells. The SATs described have the general formula R1(2)NCOCH2OCH2CO-(Gly)3-Pro-(Gly)3-OR2. Substitution at R1 and R2 with various aliphatic or aromatic groups alters the ability of SATs to transport chloride through a phospholipid bilayer membrane. Despite extensive structure-activity relationship studies concerning Cl(-)-mediated transport by SATs, relatively little was known about the mechanism of insertion and pore-formation in the membrane. In the current study, the mechanistic behavior of SATs was investigated in aqueous solution and at the air-water interface. In the latter case, Langmuir trough studies and Brewster angle microscopy (BAM) revealed the extent of monolayer stability and organization for SATs. Dynamic light scattering and transmission electron microscopy (TEM) confirmed these results and defined the aggregation behavior of SATs in solution. SAT derivatives that showed low chloride transport activity organized into stable monolayers at the air-water interface, while more active SATs formed less stable monolayers. The relationship between intermolecular organization of SATs and pore-formation in the membrane is discussed along with its implications for chloride transport.     

Transmembrane anion transport by synthetic systems

This highlight examines recent advances in the development of synthetic membrane transporters for anions, highlighting the underlying principles of transporter design and the promising applications of such transporters to biological systems including potential future treatments for diseases like cystic fibrosis which is caused by dysregulation of chloride transport across epithelial cell membranes.     

Amidopyrroles: from anion receptors to membrane transport agents

Amidopyrroles have been employed in a variety of anion receptors and sensors. 2,5-Bisamidopyrroles show selective oxo-anion complexation properties in organic solution whilst bis-amides containing dipyrrolylmethane groups form strong complexes with dihydrogen phosphate anions in mixtures of DMSO-d6 and water. Deprotonation of the 2,5-bisamidopyrrole unit can lead to interesting solid-state structures including the formation of orthogonal hydrogen bonded dimers. Amidopyrrole groups have also been employed in receptors for ion-pairs and in membrane transport agents for HCl.     

Lipid-protein interactions in the membrane: studies with model peptides

We have used fluorescence quenching of tryptophan-containing trans-membrane peptides by bromine-containing phospholipids to study the specificity of peptide-lipid interactions. We have synthesized peptides Ac-K2GLm WLnK2A-amide where m = 7 and n = 9 (L16) and m = 10 and n = 12 (L22). Binding constants of L22 for dioleoylphosphatidylserine [di(C18 : 1)PS] or dioleoylphosphatidic acid [di(C18 : 1)PA] relative to dieoleoylphosphatidylcholine [di(C18 : 1)PC] were close to 1. However, for L16, whilst the bulk of the di(C18 : 1)PA molecules bound with a binding constant relative to di(C18 : 1)PC close to 1, a small number of di(C18 : 1)PA molecules bound much more strongly. Assuming just one high affinity binding site on L16 for anionic lipid, the affinity of the site for di(C18 : 1)PS was calculated to be ca. 8 times that for di (C18 : 1)PC. The relative binding constant was little affected by ionic strength and close contact between the anionic headgroup of di(C18 : 1)PS and a lysine residue on the peptide was suggested. The relative binding constant for di(C18 : 1)PS at this high affinity site was less than for di(C18 : 1)PA. Cholesterol interacts with L22 with an affinity about 0.7 of that of di(C18 : 1)PC. The structure of the peptide itself is important. The peptide Ac-KKGYL6WL8YKKA-amide (Y2L14) incorporated into bilayers of dinervonylphosphatidylcholine [di(C24 : 1)PC] whereas L16 did not incorporate into this lipid. It is suggested that thinning of a lipid bilayer around a peptide to give optimal hydrophobic matching is less energetically unfavourable when a Tyr residue is located in the lipid/water interfacial region.     

Dialytic transport of carboxylic acids through an anion exchange membrane

The transport behavior of five carboxylic acids of relevance in biotechnology (acetic, propionic, lactic, oxalic, citric) in diffusion dialysis and neutralization dialysis through an anion exchange membrane is investigated. The dependence of acid anion flux on base concentration in neutralization dialysis is analyzed in terms of two limiting situations (boundary layer control and membrane control) by an empirical two-parameter flux equation in formal analogy to a Langmuir function. When coupled to a life fermenter, neutralization dialysis is a means to control the pH of the fermentation medium. By removing biotoxic acids, it improves microbial productivity, as exemplified with the Propioni system producing vitamin B₁₂ and propionic acid.     

Carboxylate anion diminishes chloride transport through a synthetic, self-assembled transmembrane pore

Six amphiphilic heptapeptides with the structure (C18H37)2NCOCH2OCH2CO-(Gly)3-Pro-(Gly)n-(Glx)-(Gly)m-O(CH2)6CH3, in which Glx represents glutamic acid or its benzyl ester and n+m=2, have been studied. In addition, the glutamate residue in the GGGPGGE sequence was esterified by fluorescent 1-pyrenemethanol. These compounds insert into phospholipid bilayers and form anion-conducting pores. Hill plots based on carboxyfluorescein release indicate that the pores are at least dimeric. Studies that involved ion-selective electrode techniques showed that transport of chloride varied with the position of glutamate within the peptide chain and whether glutamic acid was present as the free acid or its benzyl ester. Chloride transport activity was significantly higher for the glutamate esters than for free carboxylates irrespective of the glutamate position. Activity was highest when the glutamate residue in approximately (Gly)3-Pro-(Xxx)3 approximately was closest to the C terminus of the peptide. A fluorescent pyrene residue was introduced to probe the aggregation state of the amphiphile. The selectivity of the pore for Cl(-) over K+ was maintained even when the carboxylate anion was present within it. Complexation of Cl(-) by the ionophoric peptides was confirmed by negative ion mass spectrometry. Planar bilayer voltage clamp experiments confirmed that pores with more than one conductance state may form in these dynamic, self-assembled pores.     

Aggregation and supramolecular chirality of achiral amphiphilic metalloporphyrins

Metalloporphyrin derivatives with three hydrophobic dodecyl chains and a hydrophilic ester or carboxylic acid substituent were designed in order to clarify the effect of the central metal ions on the aggregation as well as the supramolecular chirality in the Langmuir-Schaefer films. All the metalloporphyrins showed good spreading behavior on water surface and can be transferred onto solid substrates. The transferred films were characterized by a variety of methods including UV-visible spectroscopy, circular dichroism (CD) spectroscopy, FTIR spectroscopy, atomic force microscopy (AFM) and scanning electron microscope (SEM) measurements. It has been found that the copper derivative forms J-aggregates as well as H-aggregates in the film. Moreover, the film showed strong CD signals. Change from the ester substitution to carboxylic acid caused the decrease of the supramolecular chirality. On the contrary, the zinc derivative showed only a negligible CD signal although the corresponding free base could assemble into a chiral assembly. A possible mechanism for the subtle relationship between supramolecular chirality and molecular structures has been proposed.     

Hydrazide-functionalized magnetic microspheres for the selective enrichment of digested tryptophan-containing peptides in serum

The extreme complexity of protein samples is becoming a great challenge for proteomic analysis, especially for those having large dynamic range of protein abundance. To solve this problem, and to overcome the limitation of the current proteomic technologies, a new method using hydrazide-functionalized magnetic microspheres was established in this study. With this method, tryptophan (Trp)-containing peptides can be selectively and sensitively enriched from complex and low-volume samples. Furthermore, combined with 1D-LC-MS/MS analysis, the strategy was successfully applied to the proteomic study of mouse serum. The proportion of Trp-containing peptides was increased from 19.4% to 80.2% through enrichment, and the complexity of the sample was reduced more than two times. An additional 113 Trp-containing peptides and 48 novel proteins were detected compared to the conventional method. This enrichment method provides a means for identifying more proteins as potential biomarkers in serum and other complex samples.     

Lanthanide-mediated supramolecular cages and host-guest interactions

The structure and thermodynamic properties of lanthanide complexes with a new tripodal ligand L2 have been elucidated using different physicochemical methods. At stoichiometric ratios, the tetrahedral three-dimensional complexes with lanthanide cations are formed in acetonitrile with good stabilities. Despite minor structural changes comparing to previously investigated tripodal ligands, the resulting assembly exhibits different features revealed with the crystal structure of [Eu(4)L2(4)](OH)(ClO(4))(11) (orthorhombic, Pbcn). Interestingly, the highly charged edifice contains an inner cage encapsulating a perchlorate anion. Such lanthanide mediated cage-like assemblies are rare, and may be of interest for different sensing applications. Indeed, the anionic guest can be exchanged with different anions. The related host-guest equilibria were investigated with NMR techniques. Various aspects of these reactions are qualitatively discussed.     

Fluorescent, synthetic amphiphilic heptapeptide anion transporters: evidence for self-assembly and membrane localization in liposomes

Synthetic anion transporters (SATs) of the general type (n-C18H37)2N-COCH2OCH2CO-(Gly)3-Pro-(Gly)3-O-n-C7H15, 1, are amphiphilic peptides that form anion-conducting pores in bilayer membranes. To better understand membrane insertion, assembly and aggregation dynamics, and membrane penetration, four novel fluorescent structures were prepared for use in both aqueous buffer and phospholipid bilayers. The fluorescent residues pyrene, indole, dansyl, and NBD were incorporated into 1 to give 2, 3, 4, and 5, respectively. Assembly of peptide amphiphiles in buffer was confirmed by monitoring changes in the pyrene monomer/excimer peaks observed for 2. Solvent-dependent fluorescence changes that were observed for indole (3) and dansyl (4) side-chained SATs in bilayers showed that these residues experienced an environment between epsilon=9 (CH2Cl2) and epsilon=24 (EtOH) in polarity. Fluorescence resonance energy transfer (FRET) between 2 and 3 demonstrated aggregation of SAT monomers within the bilayer. This self-assembly led to pore formation, which was detected as Cl(-) release from the liposomes. The results of acrylamide quenching of fluorescent SATs supported membrane insertion. Studies with NBD-labeled SAT 5 showed that peptide partition into the bilayer is relatively slow. Dithionite quenching of NBD-SATs suggests that the amphiphilic peptides are primarily in the bilayer's outer leaflet. Images obtained by using a fluorescence microscope revealed membrane localization of a fluorescent SAT. Taken together, this study helps define the insertion, membrane localization, and aggregation behavior of this family of synthetic anion transporters in liposomal bilayers.     

Chromium(VI) transport through the Raipore 1030 anion exchange membrane

The flat sheet Raipore R1030 anion exchange membrane has been evaluated as a sample interface in an optical sensor for Cr(VI) monitoring. The R1030 is an anion exchange membrane containing quaternary ammonium groups. The Donnan dialysis (DD) that takes place has been enhanced with facilitated transport of Cr(VI) anions by using a 1,5-diphenylcarbazide (DPC) solution as stripping phase. The DPC acts as a reducing reagent for Cr(VI), and as a complexing reagent for the generated Cr(III). The Cr(III) complex is a strongly absorbing species, and this is the basis of the optical detection. The effect of chemical parameters on Cr(VI) transport has been evaluated. Experiments with UV-VIS detection have shown that the membrane R1030-DPC system exhibits features suitable for Cr(VI) optical sensing. A simplified model based on a kinetic approach is reported describing the transport mechanism of the chemically facilitated DD process.     

The C-terminal ester of membrane anchored peptide ion channels affects anion transport

Five heptapeptide derivatives, [CH3(CH2)17]2NCOCH2OCH2CO-Gly-Gly-Gly-Pro-Gly-Gly-Gly-OR, in which R = ethyl, 2-propyl, heptyl, benzyl, and cyclohexylmethyl, were found to transport chloride anion through a phospholipid bilayer to varying extents dependent on the identity of R. It was concluded that the R group is a membrane anchor for the synthetic chloride channels.     

Simulations of DNA coiling around a synthetic supramolecular cylinder that binds in the DNA major groove

In this work we present the results of a molecular simulation study of the interaction between a tetracationic bis iron(II) supramolecular cylinder, [Fe2(C25H20N4)3]4+, and DNA. This supramolecular cylinder has been shown to bind in the major groove of DNA and to induce dramatic coiling of the DNA. The simulations have been designed to elucidate the interactions that lead the cylinder to target the major groove and that drive the subsequent DNA conformational changes. Three sets of multi-nanosecond simulations have been performed: one of the uncomplexed d(CCCCCTTTTTCC) d(GGAAAAAGGGGG) dodecamer; one of this DNA complexed with the cylinder molecule; and one of this DNA complexed with a neutralised version of the cylinder. Coiling of the DNA was observed in the DNA-cylinder simulations, giving insight into the molecular level nature of the supramolecular coiling observed experimentally. The cylinder charge was found not to be essential for the DNA coiling, which implies that the DNA response is moderated by the short range interactions that define the molecular shape. Cylinder charge did, however, affect the integrity of the DNA duplex, to the extent that, under some circumstances, the tetracationic cylinder induced defects in the DNA base pairing at locations adjacent to the cylinder binding site.     

A synthetic, chloride-selective channel that alters chloride transport in epithelial cells

An Ussing chamber was used to demonstrate that synthetic amphiphilic anion transporters function as chloride transporters in mammalian airway epithelial cells.     

Liquid membrane transport of supramolecular complexes of some amines and amino acids with macrocyclic ligands

The transport of some amines in protonated form was studied (viz. methylamine, dimethylamine, diethylamine andn-propylamine) and -amino acids (l-leucine,l-methionine,l-isoleucine,l-phenylalanine,l-valine,l--alanine andl-cysteine). The following macrocyclic ligands were used as carriers throughout the experiments: 15-crown-5 (15C5), 18-crown-6 (18C6), benzo-18-crown-6 (B18C6), dibenzo-18-crown-6 (DB18C6), diazacrown ether [2.2] (1,7,10,16-tetraoxa-4,13-diazacyclooctadecane) and cryptand [2.2.2] (4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo [8.8.8] hexacosane). The active transport, assisted by pH gradient, of amino acids and amines in protonated form as ion pairs in the presence of picrate anion was performed. The experiments suggested the influence of the ligand size, the donor atom type, and the substituents on the transport phenomena.     

Construction and activity of a synthetic basement membrane with active laminin peptides and polysaccharides

Cell-adhesive peptides derived from extracellular matrix (ECM) proteins are potential candidates for incorporating cell-binding activities into materials for tissue engineering. We have identified a number of cell adhesive peptides from laminins, which are major components of basement membrane ECM. Our goal is the development of synthetic basement membranes using the peptides on scaffolds. We review peptide–polysaccharide complexes, which were prepared by conjugation of the peptides to chitosan and alginate, and the biological activities of the resulting matrices. The peptide–polysaccharide matrices can also be used as a biomaterial for cell transplantation. These studies suggest that the peptide–polysaccharide complexes have the potential to mimic the multifunctional basement membrane and may be useful for tissue engineering.     

A carbamoyl-protective group for tyrosine that facilitates purification of hydrophobic synthetic peptides

The Boc-N-methyl-N-[2-(methylamino)ethyl]carbamoyl group (Boc-Nmec) is reported as a new side chain-protective group for tyrosine in Fmoc solid-phase peptide synthesis. Tyrosine is incorporated into the peptide as Fmoc-Tyr(Boc-Nmec)-OH by standard coupling methods. During the cleavage of the peptide from the resin with TFA the Boc group is simultaneously cleaved while the cationic N-methyl-N-[2-(methylamino)ethyl]carbamoyl group remains attached to the tyrosine residue, thereby increasing the solubility of the peptide. After purification of the peptide, the Nmec protective group can be cleaved under neutral or mild alkaline conditions via an intramolecular cyclization reaction.     

Non-covalent interactions exert extraordinary influence over conformation and properties of a well-known supramolecular building block

At low pH, and in the presence of 4,4'-bipyridine, p-sulfonatocalix[4]arene crystallizes in the 1,3-alternate conformation rather than the expected cone conformation and exhibits remarkable stability.