Solvent accessibility studies of β-bends and application to cyclic hexapeptides

The solvent-accessible surface areas (ASAs), of the atoms in tripeptides around the minimum-energy conformations of the β-bend types I, I′, II, and II′ have been computed as a first step in the systematic solvent accessibiity study of secondary structures. The side chains chosen at the two middle positions of the bend are L -Ala, D -Ala, and Gly. The ASAs of the hydrogen atoms are reported here and are found useful in determining the type of β-bends in six examples of cyclic hexapeptides whose crystal structures are known. Comparison with observation showed that all the β-bends in these cyclic hexapeptides were correctly identified by the present method. This points to a possible use of the method in identifying β-bend types in solution.     

Conformation and complexation with metal ions of cyclic hexapeptides: cyclo (L-Leu-L-Phe-L-Pro)2 and cyclo [L-Cys(Acm)-L-Phe-L-Pro]2

Cyclic hexapeptides, cyclo (L-Leu-L-Phe-L-Pro)2 and cyclo[L-Cys(Acm)-L-Phe-L-Pro]2, in which Acm represents an acetoamide-methyl group, were synthesized, and the conformation and complexation with metal ions were investigated. Cooperation of the carbonyl groups of the Cys(Acm) side chains with those of the cyclic skeleton in complexation was especially examined. Cyclo(L-Leu-L-Phe-L-Pro)2, which possesses no functional groups on side chains, was taken as the reference compound. 13C- and two-dimensional n.m.r. measurements revealed that cyclo(L-Leu-L-Phe-L-Pro)2 and cyclo[L-Cys(Acm)-L-Phe-L-Pro]2 took a C2-symmetric conformation containing cis L-Phe-L-Pro bonds in chloroform and acetonitrile. Both cyclic hexapeptides were found to complex selectively with Ba2+ and Ca2+ in acetonitrile. On complexation the conformation of either cyclic hexapeptide changed into a similar one. However, the binding constant of cyclo[L-Cys(Acm)-L-Phe-L-Pro]2 was higher than that of cyclo(L-Leu-L-Phe-L-Pro)2. The n.m.r. measurements showed that the amide carbonyl groups of Cys(Acm) side chains as well as those of cyclic skeleton in cyclo[L-Cys(Acm)-L-Phe-L-Pro]2 cooperatively bound the cations.     

Synthesis and conformation of the cyclic octapeptides cyclo(Phe-Pro)4, cyclo(Leu-Pro)4, and cyclo[Lys(Z)-Pro]4

Cyclic octapeptides, cyclo(X-Pro)₄, where X represents Phe, Leu, or Lys(Z), were synthesized and their conformations investigated. A C₂-symmetric conformer containing two cis peptide bonds was found in all of these cyclic octapeptides. The numbers of available conformations due to the cis–trans isomerization of Pro peptide bonds depended on the nature of the solvent and X residue: they decreased in the following order: cyclo[Lys(Z)-Pro]₄ > cyclo(Leu-Pro)₄ > cyclo(Phe-Pro)₄ in CDCl₃. ¹³C spin-lattice relaxation times (T₁) of these cyclic octapeptides were measured, and the contribution of segmental mobility to T₁ was found to vary with the nature of the X residue.     

Complex formation with alkali and alkaline earth metal ions of cyclic octapeptides,cyclo(Phe-Pro)4, cyclo(Leu-Pro)4, and cyclo[Lys(Z)-Pro]4

Complex formation with alkali and alkaline earth metal ions of cyclic octapeptides, cyclo(Phe-Pro)₄, cyclo(Leu-Pro)₄, and cyclo[Lys(Z)-Pro]₄ was investigated in relation to conformation. In an alcohol solution, cyclo(Phe-Pro)₄ did not form complexes. However, cyclo(Leu-Pro)₄ and cyclo[Lys(Z)-Pro]₄ formed complexes selectively with Ba²⁺ and Ca²⁺ ions. Changing the solvent from alcohol to acetonitrile, the complexation behavior was very different. In acetonitrile, cyclo(Phe-Pro)₄ was found to form a complex with Ba²⁺, and CD spectra of cyclo(Leu-Pro)₄ and cyclo[Lys(Z)-Pro]₄ changed sharply on complexation with K⁺. Rate constants of the complex formation between the cyclic octapeptides and metal salts were in the range of 0.7–12 L mol^?1 min^?1 in an alcohol solution. One of the two types of complex formation in acetonitrile was much faster than that in an alcohol solution.     

Studies on cyclic peptides. IV. Conformation of cyclo(Sar-Sar-Gly)2, cyclo(Sar)6 and cyclo(Sar-Gly-Gly)2 and their conformational change induced by alkali thiocyanates

Conformation of cyclo (Sar-Sar-Gly)₂, cyclo(Sar)₆, and cyclo(Sar-Gly-Gly)₂ was investigated by nmr spectroscopy. cyclo(Sar-Sar-Gly)₂, were shown to assume various conformations in dimethysulfoxide. It was attributed to the distribution of cis as well as trans Gly-Sar or Sar-Sar amide links along the peptide backbone. In particular, cyclo(Sar-Sar-Gly)₂ took five or six different conformations: one or three C₂-symmetric conformations and four or three asymmetric conformations, respectively. Three of nine NH resonance signals were ascribed to the internally hydrogen-bonded glycine residues. cyclo(Sar-Sar-Gly)₂ and cyclo(Sar)₆ showed a spectral change on the addition of alkali thiocyanates, indicating a conformational change induced by a complex formation with the alkali cations. The complex nmr spectrum due to a hybridization of different conformations changed with the salt addition into a simple nmr spectrum, suggesting a preponderence of a new, single conformation. On the basis of the spectral change, the strength for the cations binding the cyclic peptides was found to be in the order of K⁺ > Na⁺ > Rb⁺ > Cs⁺ for cyclo(Sar-Gly-Gly)₂ and K⁺ > Rb⁺ > Cs⁺ for cyclo(Sar)₆. On the other hand, cyclo(Sar-Gly-Gly)₂ in dimethylsulfoxide assumed a single C₂ conformation having two glycyl peptide protons shielded from solvent and the other two exposed to solvent. This conformation did not change with the salt addition. Finally, the conformations of several cyclic peptides containing the sarcosine residue such as cyclo(Sar)₆ cyclo(Sar-Sar-Gly)₂ cyclo(Pro-Sar-Gly)₂, and cyclo (Sar-Gly-Gly)₂ were compared. It appeared that proline and glycine residues reduced the conformational multiplicity of the cyclic peptide backbone, and the ability to bind alkali metal cations decreased in the above order.     

Lactobacillus plantarum MiLAB 393 produces the antifungal cyclic dipeptides cyclo(L-Phe-L-Pro) and cyclo(L-Phe-trans-4-OH-L-Pro) and 3-phenyllactic acid

We have isolated a Lactobacillus plantarum strain (MiLAB 393) from grass silage that produces broad-spectrum antifungal compounds, active against food- and feed-borne filamentous fungi and yeasts in a dual-culture agar plate assay. Fusarium sporotrichioides and Aspergillus fumigatus were the most sensitive among the molds, and Kluyveromyces marxianus was the most sensitive yeast species. No inhibitory activity could be detected against the mold Penicillium roqueforti or the yeast Zygosaccharomyces bailii. An isolation procedure, employing a microtiter well spore germination bioassay, was devised to isolate active compounds from culture filtrate. Cell-free supernatant was fractionated on a C(18) SPE column, and the 95% aqueous acetonitrile fraction was further separated on a preparative HPLC C(18) column. Fractions active in the bioassay were then fractionated on a porous graphitic carbon column. The structures of the antifungal compounds cyclo(L-Phe-L-Pro), cyclo(L-Phe-trans-4-OH-L-Pro) and 3-phenyllactic acid (L/D isomer ratio, 9:1), were determined by nuclear magnetic resonance spectroscopy, mass spectrometry, and gas chromatography. MIC values against A. fumigatus and P. roqueforti were 20 mg ml(-1) for cyclo(L-Phe-L-Pro) and 7.5 mg ml(-1) for phenyllactic acid. Combinations of the antifungal compounds revealed weak synergistic effects. The production of the antifungal cyclic dipeptides cyclo(L-Phe-L-Pro) and cyclo(L-Phe-trans-4-OH-L-Pro) by lactic acid bacteria is reported here for the first time.     

Ring conformation of cyclic octapeptide cyclo (L-Pro-Sar)4 in the crystalline state

A single-crystal X-ray diffraction analysis has been made of the structure of the cyclic octapeptide cyclo(L-Pro-Sar)4. The material [C32H48O8N8 X (21/4) H2O X (1/2) CH3OH, Mr = 799.43] crystallizes in the monoclinic space group C2 with cell dimensions a = 14.544 (3), b = 11.902 (2), c = 14.064 (3), and beta = 122.26 (2) degrees (lambda = 1.54178 A, T = 293 K). The final R value for the 1980 observed reflections is 0.079. The ring conformation has the peptide bond sequences of cis-cis-trans-trans-cis-cis-trans-trans (Pro-Sar-Pro peptide bond linkages are cis-cis- or trans-trans). The pyrrolidine rings in the four proline residues take an envelope form in which the gamma-carbon atom deviates from the plane of the remaining four atoms in the ring.     

Conformations of cyclic octapeptides. 4. Diastereomers of cyclo(Gly-Pro-Phe-Ala-Asn-Ala-Val-Ser)

Stereoisomers of cyclo(Gly-Pro-Phe-Ala-Asn-Ala-Val-Ser) were synthesized. NMR studies of their solution conformations, focusing on peptide N-H solvent exposure, were made. These indicated that a single proline residue in the cyclic octapeptide ring is insufficient constraint to stabilize the backbone conformations that were previously established for cyclo(Gly-Pro-Phe-Ala)2.     

Conformational and ion binding properties of a cyclic octapeptide, cyclo (Ala-Leu-Pro-Gly)2.

The conformation and ion-binding characteristics of a cyclic octapeptide, cyclo (Ala-Leu-Pro-Gly)2, in a liphophilic solvent, acetonitrile, have been studied using CD and nmr spectroscopy. The peptide binds preferentially to divalent cations such as calcium, magnesium, and barium. The conformations of the free cyclic peptide and its calcium complex are very similar with well-defined beta- and gamma-turns. The cyclic peptide readily forms equimolar and possibly 2:1 (peptide:cation) complexes with divalent cations.     

Solution conformations of two flexible cyclic pentapeptides: cyclo(Gly-Pro-D-Phe-Gly-Ala) and cyclo(Gly-Pro-D-Phe-Gly-Val).

In an effort to explore the residue preferences in three-residue reverse turns (so-called gamma-turns), two cyclic pentapeptides--cyclo(Gly1-Pro2-D-Phe3-Gly4-Ala5) (I) and cyclo(Gly1-Pro2-D-Phe3-Gly4-Val5) (II)--have been synthesized and analyzed by nmr. It was anticipated that the Gly-Pro-D-Phe-Gly portions of these molecules would favor a beta-turn conformation, leaving the remainder of the molecule to adopt a gamma turn, as seen in several previously studied model cyclic pentapeptides. The nmr data for both peptides in CDCl3 (5% DMSO-d6) and in neat DMSO-d6 indicate that the most populated conformation contains a distorted beta turn around Pro2-D-Phe3, which includes a gamma turn around D-Phe3. The distortion in the beta turn does not impede the formation of an inverse gamma turn around residue 5, and indeed, this conformation is observed in both peptides. Both the alanine and the bulkier valine residues are therefore found to be compatible with an inverse gamma turn. Molecular dynamics simulations on the title peptides are reported in the following paper. These simulations indicate that there is conformational flexibility around the D-Phe3-Gly4 peptide bond, which enables the formation of the gamma turn around D-Phe3. The third paper in this series explores the impact of a micellar environment on conformational equilibria in II.     

Crystal structure and conformation of the cyclic tetramer of a repeat tripeptide of elastin, cyclo(L-valyl-L-prolylglycyl)4

X-ray diffraction data were used to determine the crystal structure of cyclo-(L-Val-L-Pro-Gly)4, the cyclic tetramer of a repeat tripeptide of elastin. The crystals are monoclinic, space group C2, with a = 29.639(3), b = 7.099(1), c = 20.325 (2) A, and beta = 130.4(4) degrees. The structure was solved by direct methods and refined by least squares to R = 0.082 for 2603 observed reflections. The cyclic dodecapeptide contains two beta (II) turns. Hydrophilic and hydrophobic channels that run parallel to the b axis are formed by the stacking of cyclic peptides on twofold axes.     

Synthesis and conformation of cyclic hexapeptide cyclo(Pro-Sar-Sar)2

A cyclic hexapeptide, cyclo(Pro-Sar-Sar)₂, which consists of N-substituted amino acids only was synthesized, and its solution conformation was investigated by n.m.r. spectroscopy. Seven different C₂-symmetric conformations were detected, which were distinguishable from each other on the n.m.r. time scale. This is due to the cis/trans isomerization of N-substituted peptide bonds. Allowed C₂-symmetric conformations were computed on the basis of a hard-sphere model. Some conformations detected in n.m.r. spectra were not allowed in the calculation. This disagreement suggests that some asymmetric conformations with regard to the single bond rotation are averaged out due to a rapid rotation on the n.m.r. time scale. These points indicate that the molecule of cyclo (Pro-Sar-Sar)₂ is very flexible     

Characterization of β-turns in cyclic hexapeptides in solution by fourier transform IR spectroscopy

The β-turn represents a structural element frequently encountered in globular proteins. However, in spite of various theoretical and experimental studies the ir signature bands of pure β-turns are still not established beyond doubt. Although considerable information exists now on the ir spectra of β-helical and β-sheet structures, the lack of knowledge concerning turn structures in general, and that of β-turns in particular, presents a major uncertainty in the estimation of global protein secondary structures from ir spectroscopic data. To obtain more specific information about the characteristic amide bands in β-turns, we report herein an ir spectroscopic analysis of a series of five cyclic pseudo-hexapeptides known to form β-turns from previous CD and nmr studies [A. Perczel, M. Hollósi, B. M. Foxman, and G. D. Fasman (1991) Journal of the American Chemical Society, Volume 113, pp. 9772-9784 ]. We show here that in these cyclic peptides the amide groups involved in β-turns that comprise a ten-membered hydrogen-bonded ring (and represent the first H-bond pair in a β-sheet), give rise to characteristic amide I bands in the range 1638–1646 cm^?1, with the exact position depending on the solvent and the nature of the side-chain substituents. © 1993 John Wiley & Sons, Inc.     

An improved synthesis of (2S, 4S)‐ and (2S, 4R)‐2‐amino‐4‐methyldecanoic acids: assignment of the stereochemistry of culicinins

An improved synthesis of (2S, 4S)‐ and (2S, 4R)‐2‐amino‐4‐methyldecanoic acids was accomplished using a glutamate derivative as starting material and Evans' asymmetric alkylation as the decisive step. The NMR data of the two diastereomers were measured and compared with those of the natural product. As a result, the stereochemistry of this novel amino acid unit in culicinins was assigned as (2S, 4R). Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Studies on the aggregation and possible channel formation in membranes of a cyclic hexapeptide, cyclo (D-Ala-L-Pro-L-Ala)2.

The interaction of zwitterionic lipid DMPC and DPPC with cyclic hexapeptide, cyclo (D-Ala-L-Pro-L-Ala)2 was studied using circular dichroism (CD) and differential scanning calorimetry (DSC). Preliminary membrane conductance results showed that the peptide has a tendency to form channels inside the lipid bilayer. CD studies indicated that as the lipid/peptide (L/P) ratio (DMPC/peptide) was increased, the magnitude of the negative CD band having a lambda(max) around 200 nm decreased. At a L/P ratio of 210:1, this band disappeared completely, indicating dramatic conformational changes in the peptide on interaction with the lipid bilayer. Reduction of the phase transition temperature and the maximum heat capacity of the lipid bilayer (DPPC) for gel-to-liquid crystalline phase transition indicates a strong interaction of the peptide with the lipid bilayer.     

Crystal structure and conformation of a cyclic tetrapeptide cyclo(L-Pro-Sar)2 containing all-cis peptide units

The crystal structure and conformation of the synthetic cyclic tetrapeptide, cyclo(L -Pro-Sar)₂, was determined by x-ray analysis. The peptide crystallizes in the orthorhombic space group P2₁2₁2₁ with cell parameters a = 9.277(1), b = 12.884(1), and c = 15.581(2) Å. The crystal structure was solved by the symbolic addition procedure for direct phase determination and least-squares refinement using 1796 reflections, which led to the final R value of 0.043. This structure provides the first example observed in a crystal of a cyclic tetrapeptide in which all four peptide units have been found in the cis conformation with ω angles deviating slightly by 2°–10° from the ideal value of 0°. It was also found that the two Pro C^α-CO single bonds assumed a trans′ (ψ = 159.6° and 158.4°) conformation. Adjoining average planes of the peptide groups fall at nearly right angles to each other. The pyrrolidine ring conformations of the two prolyl residues are in the envelope form, with C^γ carbon out of the least-squares planes for the remaining four atoms.     

Cyclic peptides. XVIII. 13C spin-lattice relaxation times of (X-pro-Y)2 cyclic hexapeptides

¹³C spin-lattice relaxation times (T₁'s) of four cyclic hexapeptides of sequence, (X-L -Pro-Y)₂, are reported. The T₁'s of the protonated carbons, which undergo dipolar relaxation, are interpreted qualitatively in terms of the overall tumbling motion of the molecule and in terms of internal motion. It is found that three of the cyclic hexapeptides, those which adopt all-trans β-conformers, tumble isotropically and appear to lack internal motion in the peptide backbone. The method of Torchia and Lyerla was applied to these compounds in order to compare the mobility of the proline rings. The results show that the sequence and particular type of β-turn present affect the internal motion of the Pro ring. Data on a fourth cyclic hexapeptide, which occurs in a conformation with two-cis X-Pro bonds, suggests that internal motion of the backbone contributes an additional frequency component to the motion of the Y residue α-carbons. A consideration of the mobility of the proline rings in the conformer with two-cis peptide bonds revealed that they are significantly more rigid in the two-cis structure than in the all-trans.     

Interactions of cyclic hexapeptide cyclo(Pro-Sar-Sar)2 with small molecules

N.m.r. and c.d. spectroscopy have been used to study the interactions of cyclic hexapeptide cyclo(Pro-Sar-Sar)₂ with metal ions and ammonium ions. Cyclo(Pro-Sar-Sar)₂ was found to form complexes with Li⁺, K^?, Ba²⁺ and Cu²⁺, accompanying the conformational change into a single conformer, and the conformation of cyclo(Pro-Sar-Sar)₂ in the Li⁺-complex was different from that in the Cu²⁺-complex. These findings indicate conformational flexibility of cyclo(Pro-Sar-Sar)₂. The equilibrium constant for the complexation with Li⁺ was 2.3 × 10²l mol^?1, and cyclo(Pro-Sar-Sar)₂ adopted an asymmetric conformation in the complex. The addition of α-amino acid ester hydrochloride also caused the conformational change of cyclo(Pro-Sar-Sar)₂), but in this case it did not converge into a single conformation. This type of interaction was strengthened with aromatic α-amino acid ester hydrochloride due to the aromatic-amide interactions. Finally, the rates of exchange between unbound α-amino acid ester hydrochlorides and those complexed with cyclo(Pro-Sar-Sar)₂ were found to be different, according to the nature of α-amino acid.