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The novel chemical platform formed by the branched piperazine-2,5-dione peptide derivatives (2,5-diketopiperazines) for creating non-invasive biologically active peptidomimetics has been developed. A successful application of this approach to orally available hemostimulatory peptidomimetics was demonstrated for all-L cyclopeptide from the Glu-Trp-peptide family. In the 1980s, we have separated and characterized a number of dipeptides from the thymus homogenate. The most active peptide Glu-Trp has been studied and developed into the immunostimulating drug Thymogen. The inversion of the amino acid optical form endows the dipeptides with suppressor properties: D-Glu-D-Trp-OH and D-Glu-(D-Trp)-OH, inhibit proliferation of hemopoietic progenitors in the intact bone marrow. Based on the peptide D-Glu-(D-Trp)-OH, the new immunosuppressive drug Thymodepressin has been prepared. In this work, we applied the platform mentioned above to the design and synthesis of orally active hemosuppressive Thymodepressin® analogs. The novel data for the hemosuppressor activity of the dipeptide D-Glu(D-Trp-OH)-OH and its cyclopeptide analogs are discussed. A new example is presented of a rare phenomenon when enantiomeric molecules demonstrate reciprocal (i.e., opposite) biological activities.

Disaccharide containing unit of peptidoglycan from bacterial cell wall, N-acetyl-d-glucosaminyl-N-acetylmuramyl-l-alanyl-d-glutaminamide (gluсosaminyl-muramyl-dipeptide) registered in Russia as an immunomodulatory drug, is shown to participate in slow equilibrium of α and β anomeric forms. Data of NMR spectra and molecular dynamics indicate that the α-anomer predominantly acquires a folded conformation stabilized by intramolecular hydrogen bond between the alanyl carbonyl and muramyl NH proton. The β-form displays a considerable fraction of extended, non-hydrogen bonded structures. In the standard immunoadjuvant test system, the α-form is practically inactive, and the activity of the equilibrium mixture with α : β = 68 : 32 ratio is due to the presence of β-anomer. Such unique α–β selectivity of biological action must be considered at the design of related immunoactive glycopeptides. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.

A series of linear peptides with the general formula H-Glu(R1)-Glu(R2)-OH was subjected to cyclization under standard conditions. Formation of respective 2,5-diketopiperazines was accompanied by transformation of the N-terminal Glu(R1) to pyroglutamic acid residue. Even in the case R1 is an amino acid residue attached to the N-terminal γ-carboxyl group, lactamization leads to its elimination. The observed reaction has not been reported so far in the literature. Correspondingly, an alternative route to Glu(R1)-Glu(R2)-containing 2,5-diketopiperazines was applied to improve the overall yields.

The linear peptide gramicidin A (gA) forms prototypical ion channels specific for monovalent cations and has been extensively used to study the organization and dynamics of membrane channels. This polymorphic peptide can adopt two different types of structures, the helical dimer β6.3 (‘channel state’) and the double helical structure with two intertwined monomers. The structure of gA in micelles of detergent Triton X-100 has been studied using CD, Fourier transform infrared, and fluorescence spectroscopy. The results obtained demonstrate that only one thermodynamically stable gA structure, the antiparallel left-handed double helix β5.6, is formed in this membrane-mimetic environment. The position of the tryptophan fluorescence maximum at 332 nm is the same as that in phospholipid membranes. The causative factors governing the double helix formation in the micellar medium are discussed on the basis of known physicochemical properties of Triton X-100. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.

The conjugates of a muramyl dipeptide analog GMDP (N-acetylglucosaminyl beta 1-->4 N-acetylmuramyl-L-alanyl-D-isoglutamine) and tuftsin (Thr-Lys-Pro-Arg) were synthesized from unprotected GMDP by mixed anhydride procedure. The identity of the conjugates was confirmed by high resolution NMR and their immunomodulating properties were determined in various tests. It was found that the conjugate in which the GMDP carboxyl group forms an amide bond with the epsilon-amino group of tuftsin lysyl residue exceeds GMDP in all the activities determined. Synergism of GMDP and tuftsin was found in phagocytosis stimulation assay.

A novel chemical platform based on branched piperazine‐2,5‐dione derivatives (2,5‐diketopiperazines) for creating orally available biologically active peptidomimetics has been developed. The platform includes a diketopiperazine scaffold with “built‐in” functionally active peptide fragments covalently attached via linkers. The concept was applied to two hemostimulatory drugs, the dipeptide thymogen (GluTrp) and the tripeptide stemokin (IleGluTrp). Preparation of a series of respective derivatives is described. Of the five synthesized analogues, three demonstrated high hemostimulatory activity in vivo on intact mice and on ex vivo irradiated bone marrow cells. Prospects of further development of the concept are discussed.

A number of protected proline-containing dipeptides Boc-Xaa-Pro-OBu(t) were converted via epimerization-free oxidation with RuO4 to dipeptides with an internal pyroglutamic acid residue, Boc-Xaa-Glp-OBu(t). The latter were subjected to oxidative Hoffman-type rearrangement induced by PhI[OC(O)CF3]2 to give N-(aminoacyl)-pyroglutamates. The behavior of these derivatives under basic conditions was studied, and for two such a derivatives an aminoacyl incorporation reaction was observed, producing otherwise poorly accessible 10-membered-ring dilactams derived from 1,4-diaminobutyric and glutamic acids in practicable yields.

Twenty-four bifunctional diketopiperazines, cyclo(-Aax-Bbx-) consisting of glutamic or aspartic acid (Aax) and lysine, ornithine or diaminobutyric (Dab) acid (Bbx) were synthesized. d -Dab moiety was incorporated as d -Gln followed by Hoffman-type rearrangement induced by PhI[OC(O)CF 3 ] 2 . Cyclization conditions for the related linear dipeptide precursors allowing racemization to be kept at a minimum were determined.

Azidobenzoyl (AzBz) and benzoylbenzoyl (BzBz) derivatives of α-conotoxin MI and l-benzoylphenylalanine (Bpa) analogs of α-conotoxin GI were synthesized. All these compounds, similarly to native α-conotoxins, completely displaced the radioiodinated MI or GI from the membrane-bound nicotinic acetylcholine receptor (AChR) of Torpedo californica. However, the GI(Bpa11) analog was considerably less potent than GI in competing with radioiodinated α-bungarotoxin (αBgt). Irradiation of iodinated AzBz derivatives bound to AChR resulted in labeling of all AChR subunits. The BzBz and Bpa derivatives gave lower levels of specific cross-linking but considerable labeling at additional sites that was enhanced, rather than suppressed, by an excess of native α-conotoxins or αBgt. Both equilibrium binding of benzophenone-derivatized α-conotoxins and their cross-linking could be totally abolished by physostigmine. The results obtained demonstrate that (a) specific binding sites for α-conotoxins and αBgt are overlapping but not identical, (b) each of the AChR subunits can be labeled with photoactivatable α-conotoxins and (c) enhancement of benzophenone-derivatized α-conotoxins cross-linking at additional (physostigmine-related) sites by αBgt or GI indicates that these antagonists induce structural alterations in the AChR outside their binding sites.

An efficient scheme for the synthesis of α-conotoxins, containing 12–18 amino acid residues and two disulfide bridges, was proposed. Its advantages are: (1) the avoidance of orthogonal protections of Cys residues; (2) a lower number of stages in a cycle of the peptide chain elongation by the method of solid phase synthesis; (3) the linear product is sufficiently pure for being used at the next stage of the disulfide bond formation without additional purification; and (4) a substantially reduced time of oxidation to disulfides at pH 10, which led to the target product in a high yield. A number of natural α-conotoxins (GI, ImI, EI, MII, and SIA), affecting the muscle and neuronal nicotinic acetylcholine receptors of various types, and several new analogues of these conotoxins (in particular, [Tyr10]ImI, [Gln12]GI, and [Ser1]GI) were synthesized by this scheme. They were used for elucidating the spatial structure of α-conotoxins by 1H NMR spectroscopy and for studying the ligand-binding sites of their receptors.

A 600 MHz NMR study of α-conotoxin ImI from Conus imperialis, targeting the α7 neuronal nicotinic acetylcholine receptor (nAChR), is presented. ImI backbone spatial structure is well defined basing on the NOEs, spin-spin coupling constants, and amide protons hydrogen-deuterium exchange data: rmsd of the backbone atom coordinates at the 2–12 region is 0.28 A in the 20 best structures. The structure is described as a type I β-turn (positions 2–5) followed bya distorted helix (positions 5–11). Similar structural psattern can be found in all neuronal-specific α-conotoxins. Highly mobile side chains of the Asp-5, Arg-7 and Trp-10 residues form a single site for ImI binding to the α7 receptor. When depicted with opposite directions of the polypeptide chains, the ImI helix and the tip of the central loop of long chain snake neurotoxins demonstrate a common scaffold and similar positioning of the functional side chains, both of these structural elements appearing essential for binding to the neuronal nAChRs.

The crystal and molecular structure of the valinomycin analogue, cyclo [(D-Val-L-Lac-L-Ala-D-Hyi)2 (D-Val-L-Lac-L-Val-D-Hyi)] has been solved by x-ray direct methods using the “Shake and Bake” procedure. The crystals, grown from a mixture of octane/CH2Cl2, belong to space group P21 (Z = 4 ) with cell parameters a = 10.29, b = 32.08, c = 18.73 A β = 97.05°, and contain two molecules per asymmetric unit. After anisotropic refinement the standard reliability factor was R1 = 0.058. The conformations of both independent molecules is similar to that observed for isoleucinomycin, cyclo [-(D-Ile-L-Lac-L-Ile-D-Hyi)3] [V. Z. Pletnev et al. (1980) Biopolymers, Vol. 19, pp. 1517–1534]. The structure has an asymmetric conformation stabilized by six intramolecular H bonds, five bonds being of the 4 1 type and one bond being of the 5 1 type. One water molecule is caged in the internal cavity of each cyclodepsipeptide. This conformation could represent an intermediate state between free and complexed forms of valinomycin. © 1997 John Wiley & Sons, Inc. Biopoly 42: 651–658, 1997

The conformation and intermolecular association of [D-Hyi2, L-Hyi4] meso-valinomycin [cyclo[-D-Val-D-Hyi-L-Val-L-Hyi-(D-Val-L-Hyi-L-Val-D-+ ++Hyi)2-], C60H102N6O18] in a crystal form obtained from ethanol solution has been determined by x-ray crystallography. Two depsipeptides and one ethanol molecule per asymmetric unit crystallize in space group P2(1) (Z = 4); a = 14.579, b = 39.795, c = 13.928 A, beta = 116.90, Rl = 0.0757. The molecular conformation is very similar to that observed in the trigonal P3(2) crystal form obtained from acetone solution [V. Z. Pletnev et al. (1991) Biopolymers, Vol. 31, pp. 409-415]. Both independent molecules in the crystal adopt a similar distorted bracelet structure with a sterically inaccessible, partially formed, ion-binding center that is stabilized by six 4-->1 type H bonds. The observed conformation accounts for the inability of the molecule to complex ions. Close examination of the three crystallographically independent molecules reveals that differences in the backbone conformation associated with solvent interaction are significantly larger than those associated with hydrophobic van der Waals interactions of crystal packing.

Ionophores are antibiotics that induce ion transport across natural and artificial membranes. The specific function of a given ionophore depends upon its selectivity and the kinetics of ion capture, transport, and release. Systematic studies of complexed and uncomplexed forms of linear and cyclic ionophores provide insight into molecular mechanisms of ion capture and release and the basis for ion selectivity. The cyclic dodecadepsipeptide valinomycin, cyclo[(L-Val-D-Hyi-D-Val-L-Lac)]. transports potassium ions across cellular membrane bilayers selectively. The x-ray crystallographic and nmr spectroscopic data concerning the structures of Na+, K+, and Ba+2 complexes are consistent and provide a rationale for the K+ selectivity of valinomycin. Three significantly different conformations of valinomycin are observed in anhydrous crystals, in hydrated crystals grown from dimethylsulfoxide, and in crystals grown from dioxane. Each of these conformations suggests a different mechanism of ion capture. One of the observed conformations has an elliptical structure stabilized by four 4 ← 1 intramolecular hydrogen bonds and two 5 ← 1 hydrogen bonds. Ion capture could be readily achieved by disruption of the 5 ← 1 hydrogen bonds to permit coordination to a potassium ion entering the cavity. The conformation found in crystals obtained from dimethyl sulfoxide is an open flower shape having three petals and three 4 ← 1 hydrogen bonds. Complexation could proceed by a closing up of the three petals of the flower around the desolvating ion. In the third form, water molecules reside in the central cavity of a bracelet structure having six 4 ← 1 hydrogen bonds. Two of these bracelets stack over one another with their valine-rich faces surrounding a dioxane molecule. The stacked molecules form a channel approximately 20 A in length, suggesting that under certain circumstances valinomycin might function as a channel. A series of analogues of valinomycin differing in ring composition and size have been synthesized and their transport properties tested. Peptide substitution and chiral variation in the dodecadepsipeptide can result in stabilization or modification of the different conformers. While contraction of the ring size results in loss of ion transport properties, expansion of the ring size permits complexation of larger ions and small positively charged molecules. Gramicidin A is a pentadecapeptide that functions as a transmembrane channel for transporting monovalent cations. Crystal structures of the cesium chloride complex and two uncomplexed forms of gramicidin A have been reported. In all three structures the gramicidin A molecule is a left-handed, antiparallel, double-stranded helical dimer. In the cesium complex the β7.2-helix has 6.4 residues per turn with an internal cavity large enough to accommodate cesium ions. In the uncomplexed structures the channel is 31 A long and has 5.6 amino acids per turn. Because the helix is too tightly wound to permit ion transport, ion transport would require breaking and reforming of hydrogen bonds. © 1996 John Wiley & Sons, Inc.

The crystal structure of cyclo (D-Val-D-Hyi-D-Val-L-Hyi-L-Val-D-Hyi-L-Val-L-Hyi -L-Val-D-Hyi-D-Val-L-Hyi).2H2O has been solved by x-ray direct methods. The crystals (grown from a mixture of octane/CH2Cl2) are an orthorhombic, centrosymmetric space group Pbca, cell parameters a = 11.458 (2), b = 25.613 (3), c = 23.691 (3) A, Z = 4; therefore the molecule lies on a center of inversion in the cell. The atomic coordinates for the C, N, and O atoms were refined in the anisotropic thermal motion approximation (allowing for H-atom contribution to Fcal) to a standard R-factor value of 0.081. In contrast to meso-valinomycin, the analogue under study does not adopt an octahedral cage bracelet conformation. It has an unusual centrosymmetric elongated form with two type II terminal beta-bends formed by N-H ... C=O 4-->1 type intramolecular H bonds. Two symmetry-related water molecules reside in the elongated molecular cavity of the centrosymmetric depsipeptide ring.

The crystal structure of a synthetic depsipeptide ionophore hexadecaisoleucinomycin, cyclo [-(D-Ile-L-Lac-L-Ile-D-Hyi)4-] (C80H136N8O24), has been determined by single crystal x-ray diffraction techniques. The crystals are orthorhombic, space group P2(1)2(1)2(1), number of molecules per unit cell z = 4, and cell parameters a = 11,195, b = 17.853, c = 54.835 A. The values of the standard (R) and weighted (Rw) discrepancy factors after refinement are 0.122 and 0.135, respectively. The structure is characterized by an elongated bracelet form with a twofold axis of pseudosymmetry. It is stabilized by eight intramolecular 4----1 hydrogen bonds between the amide C = O and N - H groups. The ester carbonyls are directed toward the inside of the molecule, their oxygen atoms forming an ellipsoidal internal cavity. The side chains are located on the molecular periphery. The conformational states of hexadecaisoleucinomycin in solution are discussed in the light of the data obtained.

The crystal structure of a synthetic analogue of valinomycin, cyclo[-(L-Val-L-Hyi-L-Val-D-Hyi)2-(D-Val-L-Hyi-L-Val-D -Hyi)-] ([L-Val1, L-Val5]meso-valinomycin), C60H102N6O18, has been determined. Crystals grown from petroleum ether are orthorhombic, space group P2(1)2(1)2(1), with cell parameters a = 16.41(1), b = 18.76(1), c = 25.86(1) A, and Z = 4. The atomic coordinates for nonhydrogen atoms, except those of terminal carbons on one side chain, were refined in the anisotropic thermal motion approximation. The coordinate parameters of the H atoms were incorporated into the structure factor calculations at geometrically expected positions. Values of the standard and weighted R factors after refinement are 0.074 and 0.083, respectively. The crystal structure of the molecule is asymmetric and adopts a conformation with four 4----1 type and one 6----1 type intramolecular hydrogen bonds between amide nitrogens and carbonyl oxygens. Valinomycin binds potassium more than 100 times strongly than the D,L stereoisomeric analogue, as a result of a different spatial orientation of potentially interacting carbonyl groups.

The three-dimensional structure of the Fab fragment of a monoclonal antibody (LNKB-2) to human interleukin-2 (IL-2) complexed with a synthetic antigenic nonapeptide, Ac-Lys-Pro-Leu-Glu-Glu-Val-Leu-Asn-Leu-OMe, has been determined at 3.0 A resolution. In the structure, four out of the six hypervariable loops of the Fab (complementarity determining regions [CDRs] L1, H1, H2, and H3) are involved in peptide association through hydrogen bonding, salt bridge formation, and hydrophobic interactions. The Tyr residues in the Fab antigen binding site play a major role in antigen-antibody recognition. The structures of the complexed and uncomplexed Fab were compared. In the antigen binding site the CDR-L1 loop of the antibody shows the largest structural changes upon peptide binding. The peptide adopts a mostly alpha-helical conformation similar to that in the epitope fragment 64-72 of the IL-2 antigen. The side chains of residues Leu 66, Val 69, and Leu 70, which are shielded internally in the IL-2 structure, are involved in interactions with the Fab in the complex studied. This indicates that antibody-antigen complexation involves a significant rearrangement of the epitope-containing region of the IL-2 with retention of the alpha-helical character of the epitope fragment.

The detailed analysis of conformational space of alpha-conotoxin GI in aqueous solution has been performed on the basis of two-dimensional NMR spectroscopy data using multiconformational approach. As the result, two topologically distinct interconvertible sets of GI conformations (populations of 78% and 22%) have been found. A common feature of the two sets is the Asn4-Cys7 beta-turn. The Gly8 to Tyrll region has a structure of right-handed helical turn in the major set and two sequential bends in the minor one. N-terminus and C-terminus also have different orientations, anti-parallel in the major conformational set and parallel in the minor one. An average pairwise rmsd for backbone heavy atoms is 0.56 A in the major set, 0.23 A in the minor, and 1.85 A between the structures of the two sets. The X-ray structure of GI [Guddat, L. W., Martin, J. A., Shan, L., Edmundson, A. B. & Gray, W. R. (1996) Biochemistry 35, 11329 - 11335] has the same folding pattern as the major NMR set, the average backbone rmsd between the two structures being 0.77 A.

A number of hemoglobin-derived homogenous peptides were isolated from erythrocyte lysate. The amino acid sequences of nine peptides were determined. Seven out of nine peptides were relatively long, 30–32 membered peptides covering the N- or C-terminal sequences of globin chains. The remaining two were the pentapeptide neo-kyotorphin and its tetrapeptide derivative.

The structure of the channel-forming polypeptide gramicidin A (GA) incorporated into phosphatidylcholine (PC) liposomes has been studied as a function of the degree of unsaturation of the acyl chains of PC. The initial conformational state of GA in reconstituted bilayers is determined by the solvent in which the peptide and the lipid are initially co-dissolved, whereas the equilibrium conformational state (after heat incubation) is affected by the lipid structure rather than by the nature of the solvent. The conformational equilibrium of GA has been studied in liposomes prepared from PC having a variable number of double bonds in the fatty acid moiety, by circular dichroism and Fourier transform infrared. Liposomes were prepared from trifluoroethanol or ethanol solutions and incubated at 68°C. GA was shown to retain the conformation of the right-handed π $$\overleftarrow 6$$ .3 π $$\overrightarrow 6$$ .3 helix in PC with saturated acyl chains and with one double bond, whereas in dilinoleoyl-PC, having two double bond in each chain, the thermodynamically preferred structures are left-handed antiparallel and parallel double ππ5.6 helices. Natural soybean PC also favours left-handed ππ5.6 helical structures of GA (≈75%). This finding is discussed in terms of the role of PC unsaturation in the dynamic properties of the lipid matrix. Differences between observed FTIR spectra of the ↑↓ππ=5.6 helix in solution (and to a larger extent in the membrane) and the calculated IR spectra can be interpreted as resulting from deviation of the real structure from the theoretically derived ideal helix. The data obtained provide grounds for better understanding of a GA channel functioning in lipids of variable degrees of unsaturation.

Binding of neurotoxin II Naja naja oxiana derivatives containing one spin label at various positions (Leu 1, Glu 2, Lys 15, Lys 25, Lys 26, His 31, Lys 44 and Lys 46) to purified solubilized acetylcholine receptor protein (AchR) from Torpedo marmorata was studied by EPR techniques. AchR interaction with several dansylated neurotoxin II derivatives was followed by difference fluorescence spectroscopy. A series of neurotoxin II p-azidobenzoyl derivatives were prepared and in three of them modified lysine residues were identified. In combination, spectroscopic data and photolabeling implicate a considerable area of the neurotoxin in association with AchR. Rigidity of the neurotoxin II conformation allowed to regard its binding surface as a mould of the AchR corresponding site and to estimate the minimal size of the latter. Conformation of the long-chain neurotoxins and their binding to AchR are briefly discussed basing on the 1H and 19F NMR studies of neurotoxin I Naja naja oxiana, toxin 3 Naja naja siamensis and its acetylated or trifluoroacetylated derivatives, as well as on Achr interaction with the derivatives spin labeled at Lys 27 and His 71.

The synthesis of ten cyclic hexapeptides containing residues of N-methyl (amino acid)s and of α-hydroxyisovaleric acid has been performed.

1. The synthesis of six functional derivatives of valinomycin and enniatine B containing amine or carboxy groups in the side chain has been effected.

1. The CD, IR, and NMR spectra of the cyclohexapeptides (1)–(7) have been studied. 2. It has been shown that in trifluoroethanol and ethanol the compounds investigated show a tendency to association which is particularly strong in the case of[(L-Val-Gly)3] (5). 3. In aqueous media, cyclohexapeptides of the type of[(X-Gly-Gly)2] adopt the “pleated sheet” II conformation, and the other cyclopeptides the “pleated sheet” I conformation. 4. In nonpolar media, conformations with three or four intramolecular hydrogen bonds predominate.

Conformation of the synthetic 32-residue polypeptide, an analog of membrane spanning segment B (residues 34–65) of the Halobacterium halobium bacteriorhodopsin in the membrane mimetic system, methanol/chloroform (1:1), was investigated by 1H-NMR spectroscopy. Previously it was shown by 19F-NMR spectroscopy that this medium retains the native conformation of membrane bound BR and its fragments. The spectrum resonance was assigned by means of the sequential signal assignment porcedure using phase-sensitive DQF-COSY, MLEV17 HOHAHA and NOESY techniques. Interproton nuclear Overhauser effects, spin-spin coupling constant of vicinal H-NCα-H protons and deuterium exchange rates of individual NH groups were derived from two-dimensional NMR spectra. The data unequivocally define the peptide conformation as the right-handed α-helix, extremely rigid in the central region from Phe 42 to Nle 60 and flexible in the N- and C-terminal parts.

A proton nuclear magnetic resonance (NMR) study at 100 and 300 MHz of neurotoxin II from the venom of Middle-Asian cobra Naja naja oxiana has been performed in 2H2O and H2O solutions. By means of chemical modification and double resonance all the aromatic residue resonances have been assigned. From the NMR titration curves, pK values of histidine 4 and histidine 31 residues have been determined. For one of the two neighbouring tryptophan residues pH dependence (in the 2–8-pH range) of the chemical shifts of indole protons has been revealed. According to the different sensitivity of the linewidth of indole NH resonances to pH in H2O solution, the accessibility of each of the tryptophan residues has been estimated. Temperature dependence has been observed for the linewidth of the aromatic resonances of the tyrosine 24 residue. Deuterium exchange rates have been measured for amide protons as well as for C(2)H histidine resonances. The NMR data obtained have allowed the conclusions to be made that the two histidine residues and one of the tryptophan residues should be localized on the surface of the protein globule, that arginine residues should be present in the environment of histidine 4, that histidine 31 and the buried tryptophan are possibly localized in close spatial proximity and that the side chain of tyrosine 24 is buried within the protein globule.

1. The successive introduction of N-methyl groups is accompanied by a decrease in the stability of the folded sheet structure in the series of cyclic hexapeptides: cyclo(-L-Val-Sar-L-Val-Gly-L-Val-Gly-) (I) — cyclo(-L-Val-Sar-L-Val-Sar-L-Val-Gly-) (II) — cyclo(-L-Val-Sar-L-Val-Sar-L-Val-Sar-) (III). 2. In polar media the cyclopeptides (I)-(III) display a tendency to form unsymmetrical conformations with oneΒ-bend and a cis-configuration of either one or two of the tertiary amide linkages. 3. In nonpolar media, cyclo[-(L-Val-Sar)3-] assumes a symmetrical conformation with three intramolecular hydrogen bonds of type 3 → 1. 4. Cyclo[-(L-MeVal-Sar)3-3 assumes a rigid unsymmetrical conformation, the parameters of which are independent of the properties of the medium.

The synthesis of seven cyclic hexapeptides containing the residues of L-valine, L-norvaline, L-leucine, and glycine has been effected.

selected groupings gives crucial information on the conformation of proteins in solution and characterizes the topography of the sites responsible for biological activity, It also enables a comparison of the protein’s spatial structure in solution and in the crystal to be made on a quantitative basis, which is essential for understanding the dynamic features and conformational potentialities of the molecule. Evaluation of the intramolecular distances in proteins in solution can be done by optical spectroscopy, EPR and NMR techniques. For instance, detailed information can be obtained through the analysis of shift and relaxation effects of paramagnetic metal ions [l] or covalent spin labels (stable nitroxyl radicals) [2] in the NMR spectra of proteins. However, there arises a serious problem of signal assignment in overlapping proton spectra of proteins. In this respect it appears promising to use line-broadening evoked by spin labels in 19F NMR spectra of fluorine-containing protein derivatives. Such derivatives are obtained via chemical modification or biosynthetically, whereas their spectra usually consist of a limited number of signals [3,4 1. Feasibility of such an approach is demonstrated here with neurotoxin II (NT-II) isolated from the venom of Central Asian cobra (Naja naja oxiuna). A spin label was incorporated at the e-amino group of the Lys 27* residue. The amino groups of Leu 1, Lys 15, Lys 26, Lys 45 and Lys 47 residues were trifluoroacetylated. The distances between the spin label and the trifluoroacetyl groups were evaluated by r9F NMR. Comparison of the results on the spatial structure of neurotoxins in solution [2,6] and in the crystal [7] points to adequacy of the utilized approach.

The conformational states in dioxane and ethanol of gramicidin A and of analogs varying in chain length and amino acid sequence have been studied. Infrared, CD, and polarization of fluorescence spectra of the peptides were measured, from which dimerization constants were determined and spectral characteristics of the monomeric and dimeric states obtained. Resonance splitting of the amide I ir band has been calculated for all gramicidin A models proposed earlier. Detailed comparison of the experimental and computed spectra showed that the four dimeric gramicidin species present in solution are predominantly antiparallel double ⇅ππ ld helices in equilibrium with smaller amounts of head-to-head associated π LD helices. The gramicidin A monomer was found to be a π LD 4.4 helix in dioxane. For each conformational form the number of residues per turn and the helical sense were determined. The relationship between the amino acid sequence and the structure and stability of the dimer in the series of gramicidin A and its analogs is discussed. The above findings are rationalized in terms of the membrane channel properties of gramicidin A, in particular the conformational rearrangements occurring during the passage of metal ions through the channel and also the differences in conformation of the antibiotic in nonpolar solutions and in the membrane.

After treatment of neurotoxin II, a component part of the venom of the Middle Asian cobra Naja naja oxiana, with acetoxysuccinimide all five possible epsilon-acetylated-lysyl derivatives were obtained and the position of the label was established. Trifluoroacetylation of both the derivatives and the parent toxin yielded, respectively, the five acetyl-penta(trifluoroacetyl)-neurotoxins II and the hexa(trifluoroacetyl)-neurotoxin II, which were studied by circular dichroism (CD), 1H and 19F nuclear magnetic resonance (NMR) spectroscopy. The availability of this series of compounds made possible assignment of all six fluorine signals (from the N-terminal and the five epsilon-amino groups) in the hexa(trifluoroacetyl)-neurotoxin II NMR spectra and disclosure of the proximity of the Lys-26 and Lys-46 trifluoroacetyl groups. The pH dependence of the 19F NMR signals was determined and the pK values of the groups affecting the signal chemical shifts were calculated by a computer iterative program. In order to ascertain the relative accessibility of the lysyl side chains, the change in halfwidths of the hexatrifluoroacetylated neurotoxin II 19F signals, with addition of varying amounts of an iminoxyl spin probe, was determined. The data obtained are compared with the X-ray data on sea snake neurotoxins and the significance of the side chain interactions observed in solution is discussed.

19 F NMR and CD spectra reveal that bacteriorhodopsin as well as its 5-fluorotryptophan-labeled analog solubilized in a CH 3 OH-CHCl 3 mixture (i) retains a secondary structure of the fully active chromoprotein in the purple membrane and (ii) possesses a folded structure in which modifications at the Lys-216 bound retinal are sensed by sequentially remote tryptophan residues. Individual fragments isolated after limited proteolysis and NaBH 4 -cleavage of bacteriorhodopsin keep the spatial structure of the intact polypeptide chain in the organic solvent.

1. Employing the optical rotation dispersion, IR, and NMR spectroscopy methods, a study was made of the conformation states of some valinomycin analogs with modified side chains, and also of their K+ complexes. 2. A change in the side chains of valinomycin affects the relative stability of its conformation forms, but does not lead to the appearance of new forms. 3. In the “propeller” conformation the L-valine isopropyl groups shield the intramolecular H bonds from the action of the solvent. 4. Replacements of the valine residues by alanine moieties in the K+ complex of valinomycin are accompanied by an enhancement of the reaction of the solvent with the central ion. Such “unilateral” solvation leads to additional distortion of the symmetry of the system of chromophore groups in the complexes. The complexes of the analogs, obtained by the replacements L-Val→ L-Ala and D-Val→-D-Ala, have opposite chiralities of the depsipeptide chains.

1. The UV spectra and CD and ORD curves of methylamides and dimethylamides of acetylamino acids have been studied in polar and nonpolar media. 2. On the basis of an analysis of the spectropolarimetric results and those of other methods, the predominating types of spatial forms in media of different polarities have been determined. 3. It has been shown that the conformational state of the main peptide chain is affected both by the volume of the substituent on the Cα atom and by the presence of tertiary amide groups.

1. The dipole moments of methylamides of N-acetyl α-amino acids and their N-methyl derivatives in CCl4 and CHCl3 solutions have been measured.

The IR and NMR-H 1 spectra of alanine dipeptides and their N-Me derivatives have been investigated. Measurement of the integral intensities of the NH stretching vibrations in dilute solutions of CCl 4 and of a (1:9) CHCl 3 + CCl 4 mixture showed that in these solvent approximately 70% of the alanine dipeptide molecules are in an intramolecular hydrogen-bonded folded form. It was found from analysis of the vicinal proton spin-spin coupling constant of the CONHCH fragment that the stable conformer with respect to this bond is that with cis -arrangement of the NH and CH hyrogens. The conformation of the 7-membered hydrogen-bonded ring of the dipeptides has been elucidated. An empirically found stereochemical dependence of the constant 3 J NHCH upon the dihedral angle θ of the fragment has served as basis for discussing the possible conformations of the extended form of the dipeptides in polar (including aqueous) solvents.

Conformational study of gramicidin S and N,N′-diacetyl gramicidin S has been carried out by a number of techniques. Optical rotatory dispersion measurements showed both cyclodecapeptides to possess similar conformations in different solvents. Quantitative infra red studies in dilute chloroform solutions lead to the inference that there are ca. six intramolecular hydrogen bonds in the N,N′-diacetyl derivative. Of these deuterium exchange rates by proton magnetic resonance measurements indicate four strong H-bonds due to the Val and Leu NH groups and two weaker bonds due to the Orn α-NH groups. The NH-CαH proton coupling constants show the protons of this fragment to be trans in Val, Orn and Leu residues and gauche in D-Phe residues. The data obtained provide unequivocal proof of the Hodchkin-Oughton-Schwyzer model of gramicidin S, for which φ and ψ coordinates are given.

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.

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.

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