Aggregation and supramolecular membrane interactions that influence anion transport in tryptophan-containing synthetic peptides.

Self-assembly is a desired property in supramolecular chemistry, but extensive aggregation may be counterproductive. Rigid systems typically have better organization, but are inherently less dynamic. This work shows that ion transport by amphiphilic heptapeptides (synthetic anion transporters or SATs) is affected by aggregation of the monomers in the bulk aqueous phase to which they are added and within the bilayer. Ion transport was assessed for all compounds by assay of Cl(-) release from liposomes. The mechanism of ion transport was confirmed by planar bilayer conductance studies for two compounds at opposite ends of the efficacy scale. Dynamic light scattering, the Langmuir trough, transmission electron microscopy, ion release from liposomes, and planar bilayer conductance studies were used to assess the importance of self-assembly versus aggregation in ion transport. Generally, greater aggregation was has an adverse effect on the transport, although at least dimerization is required for amphiphilic heptapeptides to readily transport Cl(-). Anion transport in these systems was found to be sensitive to changes in the C-terminal portion of the (Gly)(3)Pro(Gly)(3) sequence. Moreover, a significant difference in transport efficacy was apparent when L-Trp was replaced by D-Trp in the same position.

UV resonance Raman study of cation-π interactions in an indole crown ether.

UV resonance Raman (UVRR) spectroscopy is used to probe changes in vibrational structure associated with cation-π interactions for the most prevalent amino acid π -donor, tryptophan. The model compound studied here is a diaza crown ether with two indole substituents. In the presence of sodium or potassium sequestered in the crown ether, or a protonated diaza group on the compound, the indole moieties participate in a cation-π interaction in which the pyrrolo group acts as the primary π-donor. Systematic shifts in relative intensity in the 760-780 cm-1 region are observed upon formation of this cation-π interaction; we propose that these modifications reflect shifts of the delocalized, ring-breathing W18 and hydrogen-out-of-plane (HOOP) vibrational modes in this spectral region. The observed changes are attributed to perturbations of the π-electron density as well as of normal modes that involve large displacement of the hydrogen atom on the C2 position of the pyrrole ring. Modest variations in the UVRR spectra for the three complexes studied here are correlated to differences in cation-π strength. Specifically, the UVRR spectrum of the sodium-bound complex differs from those of the potassium-bound or protonated-diaza complexes, and may reflect the observation that the C2 hydrogen atom in the sodium-bound complex exhibits the greatest perturbation relative to the other species. Normal modes sensitive to hydrogen-bonding, such as the tryptophan W10, W9, and W8 modes, also undergo shifts in the presence of the salts. These shifts reflect the strength of interaction of the indole N-H group with the iodide or hexafluorophosphate counteranion. The current observation that the W18 and HOOP normal mode regions of the indole crown ether compound are sensitive to cation-pyrrolo π interactions suggests that this region may provide reliable spectroscopic evidence of these important interactions in proteins.

Anion transport properties of amine and amide-sidechained peptides are affected by charge and phospholipid composition.

Four synthetic anion transporters (SATs) having the general formula (n-C(18)H(37))(2)N-COCH(2)OCH(2)CO-(Gly)(3)Pro-Lys(epsilon-N-R)-(Gly)(2)-O-n-C(7)H(15) were prepared and studied. The group R was Cbz, H (TFA salt), t-Boc, and dansyl in peptides 1, 2, 3, and 4 respectively. The glutamine analog (GGGPQAG sequence) was also included. A dansyl-substituted fluorescent SAT was used to probe peptide insertion; the dansyl sidechain resides in an environment near the bilayer's midpolar regime. When the lysine sidechain was free or protected amine, little effect was noted on final Cl(-) transport rate in DOPC : DOPA (7 : 3) liposomes. This stands in contrast to the significant retardation of transport previously observed when a negative glutamate residue was present in the peptide sequence. It was also found that Cl(-) release from liposomes depended on the phospholipid composition of the vesicles. Chloride transport diminished significantly for the free lysine containing SAT, 2, when the lipid was altered from DOPC : DOPA to pure DOPC. Amide-sidechained SATs 1 and 5 showed a relatively small decrease in Cl(-) transport. The effect of lipid composition on Cl(-) transport was explained by differences in electrostatic interaction between amino acid sidechain and lipid headgroup, which was modeled by computation.

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.

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.

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.

Transport of chloride and carboxyfluorescein through phospholipid vesicle membranes by heptapeptide amphiphiles.

Seven synthetic anion transporters (SAT) of the general form R(2)N-COCH(2)OCH(2)CO-(Gly)(3)-Pro-(Gly)(3)-OR' were prepared. Three pairs of compounds each contained twin n-hexyl, n-decyl, and n-octadecyl (R) groups at the N-terminus and one contained twin n-tetradecyl groups. Three of the compounds were C-terminated by benzyl and three by heptyl (R') residues. The ability of these compounds to mediate ion release from phospholipid vesicles was assessed. Chloride release was measured by ion selective electrode measurements and by chloride quenching of the fluorescent dye lucigenin. Transport of the anion carboxyfluorescein (CF) was measured by fluorescence dequenching. Differences in both the C- (R') and N-terminal (R) residues within the ionophores affected anion transport. The chloride release data acquired by ion selective electrode and fluorescence methods were similar but not identical. A possible carrier mechanism for Cl(-) transport was discredited. Both Cl(-) and CF anions were released from vesicles by these compounds. The results of CF and Cl(-) transport showed good consistency when the ionophore's N-terminal chains were either decyl or octadecyl but not when they were hexyl. The transport of CF and Cl(-) appears to be fundamentally different when R is C(6) compared to C(10) or C(18). Differences between the behavior of SATs with Cl(-) and CF were also reflected in negative ion mass spectrometric studies.

Synthetic, biologically active amphiphilic peptides.

Amphiphilic peptides typically consist of a peptide portion that may be 5-25 (or more) amino acids in length. The hydrophobic portion may be a single fatty acid residue, but can also be more elaborate. The main focus of this article lies on the family of synthetic anion binders (SATs) of the general structure (R(1))(2)N-COCH(2)OCH(2)CO-(Aaa)(n)-OR(3). The most-common R(1) group is the octadecyl (C(18)H(37)) group. The most studied peptide sequence in this family is (Gly)(3)-Pro-(Gly)(3), although different sequences (and longer and shorter peptides) have been prepared as well. The C-terminal ester residue providing the most effective anion release from liposomes is heptyl (C(7)H(15)), although many others have been examined. The compound (C(18)H(37))(2)N-COCH(2)OCH(2)CO-(Gly)(3)-Pro-(Gly)(3)-OBn (Bn=benzyl) was found to mediate Cl(-) transport in mouse epithelial cells.

Biologically active, synthetic ion transporters.

The compelling chemical goal of modeling protein channel behavior has led to synthetic compounds that are true ion channels. Although they largely lack the selectivity and sophistication of highly evolved proteins, they successfully perform a variety of biological functions. This tutorial review describes these novel structures and their activity in living systems. Different channel structures show antibacterial to anticancer activity when tested against a variety of cell types.

Solid-state 23Na NMR study of sodium lariat ether receptors exhibiting cation-pi interactions.

Noncovalent cation-pi interactions are important in a variety of supramolecular and biochemical systems. We present a 23Na solid-state nuclear magnetic resonance (SSNMR) study of two sodium lariat ether complexes, 1 and 2, in which a sodium cation interacts with an indolyl group that models the side chain of tryptophan. Sodium-23 SSNMR spectra of magic-angle spinning (MAS) and stationary powdered samples have been acquired at three magnetic field strengths (9.4, 11.75, 21.1 T) and analyzed to provide key information on the sodium electric field gradient and chemical shift (CS) tensors which are representative of the cation-pi binding environment. Triple-quantum MAS NMR spectra acquired at 21.1 T clearly reveal two crystallographically distinct sites in both 1 and 2. The quadrupolar coupling constants, CQ(23Na), range from 2.92 +/- 0.05 MHz for site A of 1 to 3.33 +/- 0.05 MHz for site B of 2; these values are somewhat larger than those reported previously by Wong et al. (Wong, A.; Whitehead, R. D.; Gan, Z.; Wu, G. J. Phys. Chem. A 2004, 108, 10551) for NaBPh4, but very similar to the values obtained for sodium metallocenes by Willans and Schurko (Willans, M. J.; Schurko, R. W. J. Phys. Chem. B 2003, 107, 5144). We conclude from the 21.1 T data that the spans of the sodium CS tensors are less than 20 ppm for 1 and 2 and that the largest components of the EFG and CS tensors are non-coincident. Quantum chemical calculations of the NMR parameters substantiate the experimental findings and provide additional insight into the dependence of CQ(23Na) on the proximity of the indole ring to Na+. Taken together, this work has provided novel information on the NMR interaction tensors characteristic of a sodium cation interacting with a biologically important arene.

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.

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.

Lariat ether receptor systems show experimental evidence for alkali metal cation-pi interactions.

Cation-pi interactions occur between cations and electron-rich species such as double bonds, triple bonds, and arenes. The pi-electron system may be neutral or anionic, but the latter are less relevant to biology, at least so far as is currently known. Among the 20 essential amino acids, there are four aromatic residues. These are benzene, phenol, indole, and imidazole, on the side chains of phenylalanine, tyrosine, tryptophan, and histidine, respectively. Of these, imidazole is expected to be a sigma-donor, and benzene, phenol, and indole are antipicated to serve as pi-donors. Sodium and potassium are the most abundant cations in living systems. This Account describes an experimental system that has been developed to probe, especially by X-ray crystallography, the interactions that occur between Na(+) or K(+) and the neutral arenes of particular biological significance.

Calcium cation complexation by lariat ether receptors having arene-terminated sidearms.

The first reported calcium azalariat complex has an arene terminated sidearm that behaves differently from an otherwise identical indole-sidearmed complex; twin phenolic sidearms on a diaza-18-crown-6 lead to an infinite, H-bonded network.

Correlation of Solution and Gas Phase Complexation Assessed by Electrospray Ionization Mass Spectrometry: Application to One-, Two-, and Three-Ring Macrocycles.

Electrospray ionization mass spectrometry (ESI-MS) was used to probe multiple cation complexation by C(12)H(25)(CH(2))(12)(CH(2))(12)C(12)H(25), 2, and <18N>CH(2)C(6)H(4)CH(2), 3. Complexation of two cations (2Na(+), 2 K(+), or Na(+) and K(+)) by 3 and three cations by 2 (3 Na(+), 3 K(+), and mixtures) as well as mixed proton-metallic cation complexes of both were observed. The K(+)/Na(+) cation-binding selectivity of 18-crown-6 was studied by ESI-MS of a methanol solution, and the selectivity profile was favorably compared with data obtained previously by ion-selective electrode techniques in the same solvent.