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1. The synthesis of seven topochemical analogs of the antibiotic valinomycin has been effected. 2. The complex-formation and antimicrobial properties of the analogs obtained have been studied. 3. The synthesis of valinomycin enriched with the isotope15N in one L-valine residue has been effected.

1. An empirical method for the quantitative determination of the number of NH groups of different types in peptides based on measurements of the integral intensity of the amide A bands in the IR spectra has been developed. 2. The IR spectra of the diastereomeric cyclohexaalanyls in CHCl3 solutions have been studied; they indicate the participation of an average of four NH groups in IMBHs. 3. On the basis of a theoretical conformational analysis and of dipole-moment measurements, the system of IMHBs and the type of dominant conformation of the cyclohexapeptides in nonpolar solvents have been established.

1. The UV spectra of aqueous solutions of cyclohexapeptides constructed of L(D)-alanine and glycine residues exhibit a considerable (about 45%) hypochromism of the band of the π→π* transition which has hitherto been observed among peptide-protein systems only in α-helical polypeptides.

The first total synthesis of natural cyclodepsipeptide, namely sporidesmolide I, has been achieved and thus an unambiguous proof of its structure has been given.

1. The synthesis of five valinomycin analogs differing by the configurations of the amino-acid and hydroxy-acid residues and also by the volumes of the radicals of the hydroxy-acid residues has been effected.

It has been shown that the formula (1) proposed by the Japanese workers Ito and Ogawa does not correspond to the structure of the antibiotic esperine.

1. The concentration dependences of the molecular weights and dipole moments of methylamides of N-acetyl α-amino acids and their N-methyl derivatives in chloroform have been studied.

The spatial conformation of the “polar” form of enniatin B has been established. A conformation of the N1 type has been proposed for the “nonpolar” form.

The fragmentation of cyclodidepsipeptides during electron impact was investigated. The nature of the hydroxy and amino acid residues contained in the 2,5-diketomorpholines can be established on the basis of the mass spectra.

1. The NMR spectra of cyclohexapeptides constructed of L(D)-alanine and glycine residues have been studied in (CD3)2SO, CF3COOH, and H2O solutions.

1. The dispersion of the optical rotation and UV absorption spectra of derivatives of gramicidin S with heavy atoms in solutions of ethanol was studied. 2. All the derivatives studied have a similar and rather rigid conformation. 3. A lack of correspondence between the data on the dispersion of the optical rotation and the structure of gramicidin S in the form of fragments of anα-helical conformation, proposed by certain authors, was established.

1. A new method has been proposed for studying the conformational states of peptides in solutions which is based on a consideration of the ESR spectra of their spin-labeled derivatives. 2. The synthesis of derivatives of gramicidin S containing one and two iminoxyl radicals in the2Orn and7Orn side chains has been effected. 3. On the basis of an analysis of the ESR spectra of the compounds obtained at −196°C and 30–62°C, conformations of the side chains of modified ornithine residues in chloroform and ethanol have been proposed.

1. Enniatins A and B have the cyclohexadepsipeptide structures (XX) and (VIII) respectively, and these were confirmed by the total synthesis of these antibiotics. 2. Two analogs of enniatins A and B were synthesized: the cyclohexadepsipeptide (XXI) and the cyclooctadepsipeptide (XII). 3. Among the depsipeptide cyclic polymer -homologs investigated the highest antimicrobic activity is possessed by cyclohexadepsipeptides, and the activity of the latter depends relatively little on the structures of the methylamino acid residues of which they are composed.

1. The IR spectra of methylamides of N-acetyl α-amino acids and their N-methyl derivatives in CCl4 and CHCl3 solutions have been investigated. An assignment of the bands of the NH and CO stretching vibrations in the folded (with an intramolecular H bond) and the extended conformations has been made. 2. It has been shown that the relative amounts of the folded forms depend on the nature of the amino acid residue and the conditions of the medium. With an increase in the polarity of the solvent, the proportion of folded conformations decreases.

The synthesis of antamanide, retroantamanide, and perhydroantamanide has been effected. It has been shown that the inversion of the direction of acylation — the equilibrium interchange of the valine and alanine residues (retroantamanide) — lowers the stability of the complexes with sodium and potassium in ethanolic solutions, while the replacement of the phenyl groups by cyclohexyl groups has practically no effect on complex formation.

1. By means of physical methods (IR and NMR spectra, ORD curves, dipole moments) and a theoretical conformational analysis, a study has been made of the conformational states of three topochemical analogs of valinomycin: cyclo-(D-Val-D-HyIv-L-Val-L-Lac)3 (II), cyclo-(D-HyIv-L-Ala-L-HyIv-D-Val)3 (III), and cyclo-(D-HyIv-D-Val-L-HyIv-L-Ala)3 (IV). 2. In compounds (II) and (III) steric interactions prevent the formation of “bracelet” conformations and the formation of complexes with alkali-metal ions. 3. In nonpolar media, compound (IV) assumes a “bracelet” conformation similar to that of valinomycin. 4. In view of steric hindrance, compound (IV) is incapable of adopting the “propeller” conformation that is characteristic for valinomycin, which leads to a reduced surface activity. 5. The conformation of the depsipeptide chain of the K+ complex of compound (IV) is similar to the conformation of the K+ complex of valinomycin. However, in it the cation is more feebly shielded from interaction with the solvent, which explains the higher stability and surface activity of the complex.

A synthetic peptide corresponding to the transmembrane segment M2 (residues 236–267) of the α-subunit of the nicotinic acetylcholine receptor from Torpedo californica has been studied by two dimensional 1H-NMR spectroscopy in a chloroform-methanol (1:1) mixture containing 0.1 M LiClO4. Reconstruction of the spatial structure of M2 from the NMR data resulted in an α-helix formed by residues 241–263. Distribution of the molecular hydrophobicity potential on the helix surface is very similar to that in five-helix bundles of proteins with a known three dimensional structure: two hydrophilic bands located on the opposite helix sides separated by strong hydrophobic zones.

The conformation of species 3 of Val-gramicidin A in dioxane has been determined by two-dimensional NMR spectroscopy. It is presented by the left handed ⇅ππ5.6LD double helix, a suitable model of an ion permeable pore across the membrane matrix.

It is demonstrated that the partial photoionization cross sections of outer subshells of atomic Eu in the giant resonance region are determined by the action of the 4d-electron excitations. The cross section for photoionization of the semifilled 4f7 subshell is also entirely dominated by the interaction with 4d10 electrons.

The photoionization cross sections of outer and intermediate shells including 4d of La and its ions are calculated in the region of the “giant” resonance. The prominent effects of both intershell correlational effects and rearrangement are demonstrated.

A series of methylamides and dimethylamides of N-acetyl amino acids, which are the simplest models of peptides, have been synthesized by known methods of peptide chemistry.

1. Free hydroxyacylamino acids and their methyl esters, and also free aminoacyloxy acids, morpholinize under mass spectrometry conditions.

Using flow cytometry and fluorescence polarization analysis, specific muramyl peptide-binding sites were shown to be located inside T-lymphocytes, macrophages and neuroblastoma cells, but not inside B-cells. No binding sites were found on the cell surface. The number of binding sites for each cell type was determined. Two types of binding sites were observed for myelomonocytic WEH1-3 cells with K fd values of 21 and 540 nM. Inhibition analysis demonstrated that for effective binding, an intact glycopeptide molecule and D-configuration of isoglutamine residue are important.

The C′= O and Cα signals in the 13C nuclear magnetic resonance (NMR) spectra of valinomycin have been assigned and the vicinal 1H…13C coupling constants have been determined by double and triple heteronuclear resonance. In conjunction with the vicinal H-NCα-H and H-CαCβ-H proton-proton constants, the results led to unequivocal determination of the torsion angles φ and of the population distribution of the Cα-Cβ rotational states. The Φ torsion angles for the hydroxy acid residues were estimated from the vicinal 1H-CαC′-15N constants. The combined data permitted refinement of the conformational states of valinomycin in different solvents. For the KS+ complex of valinomycin the observed couplings are in complete accord with the conformations we had earlier proposed for solutions and that had also been established by X-ray analysis. In the 13C spectra of the valinomycin-Tl+ complex 13C…203,205Tl+ spin-spin couplings were observed for the l and d-valine carbonyls, unequivocal proof of the donor-acceptor interaction with the cation. In media of weak polarity (cyclohexane, chloroform) the conformation of the valinomycin molecule is similar to that of the K+ complex. In such a ‘bracelet’ structure formed by six fused β-turns of type II and II′, the amino acid carbonyls are axial with certain inclination towards the symmetry axis. On formation of a 1:1 complex with a cation the carbonyl orientation changes, now bending towards the center of the molecular cavity. In the ‘propeller’ conformation, predominant in solvents of medium polarity (for instance CCl4/(C2H3)2SO, 3/1) the three β-turns are of type II. The 1H and 13C chemical shifts are interpreted in terms of conformational changes in the valinomycin molecule, intermolecular and intramolecular hydrogen bonds and interaction with the metal cation.

The binding and uptake of native low-density lipoproteins and malondialdehyde-treated low density lipoproteins by human hepatocytes in primary culture has been analyzed. Experiments with 125I-labeled malondialdehyde-treated low-density lipoproteins showed that cultured liver cells took up and degraded malondialdehyde-treated low-density lipoproteins, but the cell type(s) responsible for this action remain unclear. Immunofluorescent visualization of receptor-bound low-density lipoproteins revealed that low-density lipoprotein binding sites were distributed on the surface of nearly all cells of the culture. Binding sites for malondialdehyde-treated low-density lipoproteins were found in only 5% of the cultured cells, and these cells differed from hepatocytes in shape and size. Cultured hepatocytes internalized and native low-density lipoproteins, but not malondialdehyde-treated low-density lipoproteins, labeled with the fluorescent dye 3',3'-dioctadecylindocarbocyanine. About 15% of the cells that take up 3',3'-dioctadecylindocarbocyanine-labeled malondialdehyde-treated low-density lipoproteins could be identified as liver endothelial cells and macrophages, since they internalized formaldehyde-treated human albumin and fluorescent carboxylated microspheres. Our results indicate that human hepatocytes in primary culture express surface receptors for native low-density lipoproteins but not for modified low-density lipoproteins.

The capability of immunoactive muramyl peptides to bind specifically to rat brain membranes has been discovered. The reaction of an N-acetylglucosaminylmuramyl dipeptide analog having an additional C-terminal lysine residue (GMDP-Lys) with Bolton-Hunter reagent or N-hydroxysuccinimidyl-4-azidosalicylate afforded two acylated derivatives, GMDP-Lys-(Hp) and GMDP-Lys(Azs). Their iodination resulted in radioactive derivatives (spec. act. approximately 2000 Ci/mmol) whose binding to rat brain membranes is characterized by Kd approximately 3 nM and Bmax approximately 10 fmol/mg membrane protein. Binding could be inhibited by muramyl dipeptide (MDP), GMDP-Lys, and GMDP, while fragments of the latter, N-acetylglucosamine dipeptide and disaccharide, were ineffective.

Mouse macrophages (line J 774) were incubated with monospecific goat anti-low density lipoprotein antibodies, which were conjugated to horseradish peroxidase (AB-HRP). Addition of low density lipoprotein (LDL) modified by treatment with malondialdehyde to cultures of these cells resulted in a dose-dependent increase in the amount of cell-associated enzyme activity. The concentration curve was hyperbolic with half-saturation of modified LDL at a concentration of about 3 μg/ml. This effect was completely blocked by polyinosic acid and was not observed in experiments with human fibroblasts, which do not exhibit high affinity binding sites that recognize chemically modified LDL. Our data indicate that receptor-mediated endocytosis of AB-HRP in the presence of native or modified LDL may be used as very simple, efficient, and sensitive assay for investigation of the scavenger receptors for modified LDL.

We have investigated effects of native low-density lipoproteins (LDL) and malondialdehyde-treated LDL on the interaction of 5(6)-carboxyfluorescein-labeled liposomes bearing antibodies to LDL with cultured J774 macrophages. It was found that an addition of modified LDL to the incubation medium resulted in 15-20-fold increase of carboxyfluorescein binding to cells, whereas native LDL did not produce such effect. The increase of carboxyfluorescein binding to macrophages in the presence of modified LDL was not due to an enhanced leakage of the label from liposomes. The modified-LDL-mediated binding of carboxyfluorescein to cells was reduced to 20-30% of the initial level in the presence of cell-respiration inhibitors (NaF and antimycin A). Fluorescent microscopy data also indicate the modified-LDL-induced incorporation of liposome contents into cells. The results obtained in this study make it possible to assume that in the presence of malondialdehyde-treated LDL, liposomes with antibodies to LDL may be incorporated into macrophages via the receptor-mediated pathway for modified LDL.

Fifteen Naja naja oxiana neurotoxin II derivatives mono and di spin-labeled on Lys-15, Lys-25, Lys-26, Lys-44 and Lys-46 have been prepared and the position of the label(s) identified. Spin label distances to a number of the side chains were determined by 1H NMR for the mono-labeled species and interlabel distances were determined by EPR for the di-labeled residues. Binding of the mono spin-labeled derivatives to solubilized acetylcholine receptor protein (AChR) from Torpedo marmorata was followed by EPR. By means of a spin probe technique, it was found that spin labels on Lys-15, Lys-25, Lys-46 and, especially Lys-26, are less accessible in the AChR complexes than in the free toxins. Involvement of the dansyl labels on Lys-26 and Lys-46 in the binding was demonstrated by fluorescence spectroscopy. The data obtained yielded evidence of a β-structure of the toxin and allowed preliminary characterization of the neurotoxin binding surface.

A new modification of enzyme immunoassay-enzyme-linked-immunoreceptor assay (ELIRA) was used to study low-density lipoprotein (LDL) binding by cultured human skin fibroblasts. Time-course and concentration curves obtained by this method were typical of LDL binding by cultured fibroblasts. According to ELIRA, fibroblasts bound native LDL 10-20 times more effectively than reductively methylated LDL or native LDL in the presence of heparin. Receptor-specific Km and maximum binding capacity calculated from these data were 5.4 micrograms/ml and 177.5 ng/mg of cell protein, respectively. Receptor-specific Km and maximum binding capacity for surface 125I-LDL binding measured in the same experiment were 11.0 micrograms/ml and 138.0 ng/mg of cell protein. Incubation of cells with isolated LDL or with unfractionated serum containing the same amount of apoB yielded similar concentration curves for lipoprotein binding. These data indicate that the ELIRA can be used for investigation of receptor-mediated lipoprotein binding without purification of lipoproteins.

Structure, ion binding properties and mechanisms of induced ion transport are considered for various groups of ion carriers. Contribution of geometrical and electronic factors to the experimentally observed ionic selectivity is discussed for selected ionophores and in general terms. Molecular mechanisms underlying the channel mediated ion transport in amphotericin B, gramicidin A and alamethicin I families are presented and analyzed in view of their ionic selectivity.

The last decade witnessed the birth and tempestuous development of a new field in bioorganic chemistry. Its objects of study are membrane-active complexones (see Ovchinnikov et al., 1974b and references therein), often called ionophores because of their ability to bind metal ions in solution and to transport them in one or the other stage of the transmembrane ion transporting process. It is this property that has secured a place for the ionophores among modern biochemical and biophysical techniques as highly effective tools for studying ion movements across membranes.