Structural and conformational properties of (Z)-beta-(1-naphthyl)- dehydroalanine residue

To understand how chemical structure of beta-substituted alpha, beta-dehydroalanine (particularly size and pi conjugation of beta substituent) affects conformational property, x-ray crystallographic analysis was performed on Boc-Ala-Delta(Z) Nap-Val-OMe [Boc: t-butoxycarbonyl; Delta(Z) Nap: (Z)-beta-(1-naphthyl)dehydroalanine; OMe: methoxy] having the naphthyl group as a bulky beta substituent. Single crystals were grown by slow evaporation from an ethanol solution in the triclinic space group P1 with a = 9.528 (3) A, b = 12.410(4) A, c = 5.975(2) A, alpha = 96.77(3) degrees, beta = 102. 81(2) degrees, gamma = 88.74(3) degrees, V = 684.1(4) A3, and Z = 1. Phase determination was carried out by a direct method (SHELEXS), and the final structure was refined to R = 8.1% and R(w) = 9.0% for 1964 observed reflections. The bond lengths and bond angles of the Delta(Z)Nap residue, characterized by a sp(2) hybridized C(alpha) atom, did not differ from those of other dehydroresidues such as Delta(Z) Phe, Delta(Z) Leu, and DeltaVal essentially. The peptide backbone took a type II beta-turn conformation involving an intramolecular hydrogen bond between CO(Boc) and NH(Val), similar to di- or tripeptides containing a Delta(Z) Phe or Delta(Z) Leu residue in the second positions. Here the naphthyl group was found to be nonplanar [chi(2) = 55(1) degrees ] relative to the C(alpha)==C(beta)==C(gamma) plane. The nonplanarity was supported by conformational energy calculation. The molecular packing was stabilized by two kinds of intermolecular hydrogen bonds and van der Waals interactions. Naphthyl groups were arranged in a partially overlapped face-to-face orientation with a center-to-center distance of 5.97 A. For additional information, peptide Boc-(Ala-Delta(Z) Nap-Leu)(2)-OMe was synthesized and its solution conformation was investigated by (1)H-NMR spectroscopy. The hexapeptide showed the tendency to form a 3(10)-helical conformation in solution essentially. Conformational properties of Delta(Z) Nap residue, characterized by a type II beta-turn and 3(10)-helix, were supported by a conformational energy contour map of the Delta(Z)Nap residue.     

Crystal structure and solution conformation of S,S'-bis(Boc-Cys-Ala-OMe): intramolecular antiparallel beta-sheet conformation of an acyclic cystine peptide

The conformation of the acyclic biscystine peptide S,S'-bis(Boc-Cys-Ala-OMe) has been studied in the solid state by x-ray diffraction, and in solution by 1H- and 13C-nmr, ir, and CD methods. The peptide molecule has a twofold rotation symmetry and adopts an intramolecular antiparallel beta-sheet structure in the solid state. The two antiparallel extended strands are stabilized by two hydrogen bonds between the Boc CO and Ala NH groups [N...O 2.964 (3) A, O...HN 2.11 (3) A, and NH...O angle 162 (3) degrees]. The disulfide bridge has a right-handed conformation with the torsion angle C beta SSC beta = 95.8 (2) degrees. In solution the presence of a twofold rotation symmetry in the molecule is evident from the 1H- and 13C-nmr spectra. 1H-nmr studies, using solvent and temperature dependencies of NH chemical shifts, paramagnetic radical induced line broadening, and rate of deuterium-hydrogen exchange effects on NH resonances, suggest that Ala NH is solvent shielded and intramolecularly hydrogen bonded in CDCl3 and in (CD3)2SO. Nuclear Overhauser effects observed between Cys C alpha H and Ala NH protons and ir studies provide evidence of the occurrence of antiparallel beta-sheet structure in these solvents. The CD spectra of the peptide in organic solvents are characteristic of those observed for cystine peptides that have been shown to adopt antiparallel beta-sheet structures.     

Influence of chirality,amino acid sequence,and protecting groups on the intramolecular interaction behavior of dipeptide and tripeptide derivatives

The intramolecular interaction of protected dipeptides and tripeptides containing the amino acid units Ala, Phe, and Val was studied by means of ir spectroscopy. The NH and CO regions of the compounds dissolved in carbon tetrachloride clearly show the existence of different intramolecular hydrogen bonds. Using solvents with higher polarity such as chloroform and methylene chloride, the association bands disappear. Investigating the substances with the same amino acid sequence but opposite chirality of the central C atom in the peptide chain, we observed different band shapes in the CO and NH regions. Large effects were found when the chirality of the Phe unit in the second position was changed. This is probably due to the steric hindrance originated by the rotation of the aromatic ring in the side chain. The protecting groups, Z (benzyloxycarbonyl) or Boc (tert-butyloxycarbonyl) residues at the N-terminal group and methyl- or tert-butyl esters at the C-terminal group, influence the solubility of the substances in nonpolar solvent, as well as the NH and CO association band profiles in the methylene chloride solutions. The consequences of changing the sequence of the amino acids are discussed for the tripeptide derivatives. Besides a qualitative discussion, some quantitative considerations concerning the intramolecular interaction are also given to illustrate the different stabilities of the associates. © 1996 John Wiley & Sons, Inc.     

De novo design,synthesis and solution conformational study of two didehydroundecapeptides: effect of nature and number of amino acids interspersed between Phe residues

De novo design of peptides and proteins has recently surfaced as an approach for investigating protein structure and function. This approach vitally tests our knowledge of protein folding and function, while also laying the groundwork for the fabrication of proteins with properties not precedented in nature. The success relies heavily on the ability to design relatively short peptides that can espouse stable secondary structures. To this end, substitution with α,β‐didehydroamino acids, especially α,β‐didehydrophenylalanine (Δ^zPhe), comes in use for spawning well‐defined structural motifs. Introduction of ΔPhe induces β‐bends in small and 3₁₀‐helices in longer peptide sequences. The present work aims to investigate the effect of nature and the number of amino acids interspersed between two ΔPhe residues in two model undecapeptides, Ac‐Gly‐Ala‐ΔPhe‐Ile‐Val‐ΔPhe‐Ile‐Val‐ΔPhe‐Ala‐Gly‐NH₂ (I) and Boc‐Val‐ΔPhe‐Phe‐Ala‐Phe‐ΔPhe‐Phe‐Leu‐Ala‐ΔPhe‐Gly‐OMe (II). Peptide I was synthesized using solid‐phase chemistry and characterized using circular dichroism spectroscopy. Peptide II was synthesized using solution‐phase chemistry and characterized using circular dichroism and nuclear magnetic resonance spectroscopy. Peptide I was designed to examine the effect of incorporating β‐strand‐favoring residues like valine and isoleucine as spacers between two ΔPhe residues on the final conformation of the resulting peptide. Circular dichroism studies on this peptide have shown the existence of a 3₁₀‐helical conformation. Peptide II possesses three amino acids as spacers between ΔPhe residues and has been reported to adopt a mixed 3₁₀/α‐helical conformation using circular dichroism and nuclear magnetic resonance spectroscopy studies. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis, crystal structure and molecular conformation of Nalpha-Boc-L-leucyl-(Z)-beta-(3-pyridyl)-alpha,beta- dehydroalanyl-L-leucine methyl ester.

A protected tridehydropeptide containing (Z)-beta-(3-pyridyl)-alpha,beta-dehydroalanine (Delta(Z)3Pal) residue, Boc-Leu-Delta(Z)3Pal-Leu-OMe (1), was synthesized via Erlenmeyer azlactone method. X-ray crystallographic analysis revealed that the peptide 1 adopts an extended conformation, which is similar to that of a Delta(Z)Phe analog, Boc-Leu-Delta(Z)Phe-Leu-OMe (2).     

Synthetic and conformational studies on dehydroalanine-containing model peptides

Three model dipeptides containing a dehydroalanine residue (delta Ala) at the C-terminal, Boc-X-delta Ala-NHCH3 [X = Ala, Val, and Phe,] have been synthesized and their solution conformations investigated by 1H-NMR, IR, and CD spectroscopy. NMR studies on these peptides in CDCl3 clearly indicate that the NH group of dehydroalanine is involved in an intramolecular hydrogen bond. This conclusion is supported by IR studies also. Nuclear Overhauser effect (NOE) studies are also accommodative of an inverse gamma-turn-type of conformation that is characterized by conformational angles of phi approximately -70 degrees and psi approximately +70 degrees around the X residue, and a C alpha i + 1 H-Ni + 2H interproton distance of 2.5 A. It appears that unlike dehydrophenylalanine or dehydroleucine, which tend to stabilize beta-turn type of structures occupying the i + 2 position of the turn, dehydroalanine favors the formation of an inverse gamma-turn, centered at the preceding L-residue in such solvents as CDCl3 and (CD3)2SO. A comparison of solution conformation of Boc Val-delta Ala-NHCH3 with the corresponding saturated analogue, Boc-Val-Ala-NHCH3, is also presented and shows that dehydroalanine is responsible for inducing the turn structure. It may be possible to design peptides with different preferred conformations using the suitable dehydroamino acid.     

Investigation of α/γ hybrid peptide self‐assembled structures with antimicrobial and antibiofilm properties

The present work describes the synthesis and characterization of α/γ hybrid peptides, Boc‐Phe‐γ⁴‐Phe‐Val‐OMe, P1 ; Boc‐Ala‐γ⁴‐Phe‐Val‐OMe, P2 ; and Boc‐Leu‐γ⁴‐Phe‐Val‐OMe, P3 together with the formation of self‐assembled structures formed by these hybrid peptides in dimethyl sulfoxide (DMSO)/water (1:1). The self‐assembled structures were characterized by infrared (IR) spectroscopy, circular dichroism (CD), and scanning electron microscopy (SEM). Further, α/γ hybrid peptide self‐assembled structures were evaluated for antibacterial properties. Among all, the self‐assembled peptide P1 exhibited the antimicrobial activity against Escherichia coli and Klebsiella pneumoniae, while self‐assembled peptide P3 inhibited the biofilms of Salmonella typhimurium and Pseudomonas aeruginosa. In this study, we have shown the significance of self‐assembled structures formed from completely hydrophobic α/γ hybrid peptides in exploring the antibacterial properties together with biofilm inhibition.     

Conformational analysis of cyclolinopeptide A, a cyclic nonapeptide: nuclear Overhauser effect and energy minimization studies

The conformation of cyclolinopeptide A [cyclo(Pro-Pro-Phe-Phe-Leu-Ile-Ile-Leu-Val)], a naturally occurring cyclic nonapeptide has been investigated in dimethylsulfoxide solution by 270 MHz 1H-nmr. A complete assignment of all C alpha H and NH resonances has been accomplished using two-dimensional correlated spectroscopy and nuclear Overhauser effects (NOEs). Analysis of interresidue NOEs and JHNC alpha H values permit construction of a molecular model for the cyclic peptide backbone. The crude model derived from nmr has been used as a starting point for energy minimization, which yields a refined structure largely compatible with nmr observations. The major features of the conformation of cyclolinopeptide A are a Type VI beta-turn centered at Pro(1)-Pro(2), with a cis peptide bond between these residues and a gamma-turn (C7 structure) centered at Ile(6). Two intramolecular hydrogen bonds Val(9) CO--Phe(3)NH (4----1) and Leu(5) CO--Ile(7)NH (3----1) are observed in the low-energy conformation. The limited solvent accessibility observed for the Val(9) and Leu(5) NH groups in the nmr studies are rationalized in terms of steric shielding.     

Controlling the helical screw sense of peptides with C‐terminal L‐valine

One chiral L ‐valine (L ‐Val) was inserted into the C‐terminal position of achiral peptide segments constructed from α‐aminoisobutyric acid (Aib) and α,β‐dehydrophenylalanine (Δ^ZPhe) residues. The IR, ¹H NMR and CD spectra indicated that the dominant conformations of the pentapeptide Boc‐Aib‐ΔPhe‐(Aib)₂‐L ‐Val‐NH‐Bn (3) and the hexapeptide Boc‐Aib‐ΔPhe‐(Aib)₃‐L ‐Val‐NH‐Bn (4) in solution were both right‐handed (P) 3₁₀‐helical structures. X‐ray crystallographic analyses of 3 and 4 revealed that only a right‐handed (P) 3₁₀‐helical structure was present in their crystalline states. The conformation of 4 was also studied by molecular‐mechanics calculations. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Crystal and molecular structure of the doubly unsaturated dehydropeptide Ac-delta Phe-Ala-delta Phe-NH-Me.

The dehydropeptide Ac-delta Phe-L-Ala-delta Phe-NH-Me, containing two dehydro-phenylalanine (delta Phe) residues, crystallizes from methanol/water in space group P2(1)2(1)2(1), with a = 12.508 (2), b = 12.746 (1) and c = 15.465 (9). In the crystalline state, the peptide chain assumes a right-handed 3(10)-helical conformation stabilized by two intramolecular hydrogen bonds, between the N-terminal acetyl group and the NH of delta Phe3, and between the CO of delta Phe1 and the NH of the C-terminal methylamide group, respectively. The two consecutive 10-membered rings formed by the hydrogen bonds have torsion angles quite close to the standard values for type III beta-bends. delta Phe1 is located in the (i + 1) position of the first beta-bend, while delta Phe2 is located in the (i + 2) position of the other beta-bend. In the crystal, the molecules are linked head to tail by intermolecular hydrogen bonds to form long helical chains. The axes of the helices are parallel to the c axis, but neighboring helices run in antiparallel directions. This crystal packing is similar to the packing motifs frequently observed in Aib-containing peptides.     

Solid state and solution conformation of Boc-L-Met-Aib-L-Phe-OMe. Beta-turn conformation of a sequence related to an active chemotactic peptide analog

The conformation of the peptide Boc-L-Met-Aib-L-Phe-OMe has been studied in the solid state and solution by X-ray diffraction and 1H n.m.r., respectively. The peptide differs only in the N-terminal protecting group from the biologically active chemotactic peptide analog formyl-L-Met-Aib-L-Phe-OMe. The molecules adopt a type-II beta-turn in the solid state with Met and Aib as the corner residues (phi Met = -51.8 degrees, psi Met = 139.5 degrees, phi Aib = 58.1 degrees, psi Aib = 37.0 degrees). A single, weak 4----1 intramolecular hydrogen bond is observed between the Boc CO and Phe NH groups (N---O 3.25 A, N-H---O 128.4 degrees). 1H n.m.r. studies, using solvent and temperature dependencies of NH chemical shifts and paramagnetic radical induced line broadening of NH resonances, suggest that the Phe NH is solvent shielded in CDCl3 and (CD3)2SO. Nuclear Overhauser effects observed between Met C alpha H and Aib NH protons provide evidence of the occurrence of Met-Aib type-II beta-turns in these solvents.     

Conformational effects on peptide aggregation in organic solvents: spectroscopic studies of two chemotactic tripeptide analogs

The aggregation behavior of the chemotactic peptide analogs, Formyl-Met-Leu-Phe-OMe ( 1 ) and Formyl-Met-Aib-Phe-OMe ( 2 ), has been studied in chloroform and dimethylsulfoxide over the concentration range of 0.2–110 mM by ¹H-nmr spectroscopy. Both peptides associate in CDCl₃ at concentrations ≥ 2 mM, while there is no evidence for aggregation in (CD₃)₂SO. Analog 1 adopts an extended conformation in both solvents favoring association to form β-sheet structures. A folded, γ-turn conformation involving a 3 → 1 hydrogen bond between Met CO and Phe NH is supported by ¹H-, ¹³C-nmr, and ir studies of analog 2 . The influence of backbone conformation on the ease of peptide aggregation is demonstrated by ir studies in CHCl₃ and CD studies in dioxane.     

Conformational investigation of alpha,beta-dehydropeptides. Part. IV. Beta-turn in saturated and alpha,beta-unsaturated peptides Ac-Pro-Xaa-NHCH3: NMR and IR studies.

Solution conformations of three series of model peptides, homochiral Ac-Pro-L-Xaa-NHCH3 and heterochiral Ac-Pro-D-Xaa-NHCH3 (Xaa = Val, Phe, Leu, Abu, Ala) as well as alpha,beta-unsaturated Ac-Pro-delta Xaa-NHCH3 [delta Xaa = delta Val, (Z)-delta Phe, (Z)-delta Leu, (Z)-delta Abu] were investigated in CDCl3 and CH2Cl2 by 1H-, 13C-NMR, and FTIR spectroscopy. NH stretching absorption spectra, solvent shifts delta delta for NH (Xaa) and NHCH3 on going from CDCl3 to (CD3)2SO, diagnostic interresidue proton NOEs, and trans-cis isomer ratios were examined. These studies performed showed the essential difference in conformational propensities between homochiral peptides (L-Xaa) on the one hand and heterochiral (D-Xaa) and alpha,beta-dehydropeptides (delta Xaa) on the other. Former compounds are conformationally flexible with an inverse gamma-bend, a beta-turn, and open forms in an equilibrium depending on the nature of the Xaa side chain. Conformational preferences of heterochiral and alpha,beta-dehydropeptides are very similar, with the type-II beta-turn as the dominating structure. There is no apparent correlation between conformational properties and the nature of the Xaa side chain within the two groups. The beta-turn formation propensity seems to be somewhat greater in alpha,beta-unsaturated than in heterochiral peptides, but an estimation of beta-folded conformers is risky.     

Designing of peptides with left handed helical structure by incorporating the unusual amino acids

The conformational behaviour of delta Ala has been investigated by quantum mechanical method PCILO in the model dipeptide Ac-delta Ala-NHMe and in the model tripeptides Ac-X-delta Ala-NHMe with X = Gly, Ala, Val, Leu, Abu and Phe and is found to be quite different. The computational results suggest that in the model tripeptides the most stable conformation corresponds to phi 1 = -30 degrees, psi 1 = 120 degrees and phi 2 = psi 2 = 30 degrees in which the > C = 0 of the acetyl group is involved in hydrogen bond formation with N-H of the amide group. Similar results were obtained for the conformational behaviour of D-Ala in Ac-D-Ala-NHMe and Ac-Ala-D-Ala-NHMe. The conformational behaviour of the amino acids delta Ala, D-Ala, Val and Aib in model tripeptides have been utilized in the designing of left handed helical peptides. It is shown that the peptide HCO-(Ala-D-Ala)3-NHMe can adopt both left and right handed helix whereas in the peptide Ac-(Ala-delta Ala)3-NHMe the lowest energy conformer is beta-bend ribbon structure. Left handed helical structure with phi = 30 degrees, psi = 60 degrees for D-Ala residues and phi = psi = 30 degrees for delta Ala is found to be more stable by 4 kcal mole-1 than the corresponding right handed helical structure for the peptide Ac-(D-Ala-delta Ala)3-NHMe. In both the peptides Ac-(Val-delta Ala)3-NHMe and Ac-(D-Val-delta Ala)3-NHMe the most stable conformer is the left handed helix. Comparisons of results for Ac-(Ala-delta Ala)3-NHMe and Ac(Val-delta Ala)3-NHMe and Ac-(D-Ala-delta Ala)3-NHMe and Ac-(D-Val-delta Ala)3-NHMe also reveal that the Val residues facilitate the population of 3(10) left handed helix over the other conformers. It is also shown that the conformational behaviour of Aib residue depends on the chirality of neighbouring amino acids, i.e. Ac-(Aib-Ala)3-NHMe adopts right handed helical structure whereas Ac-(Aib-D-Ala)3-NHMe is found to be in left handed helical structure.     

Synthesis and conformational study of peptides possessing helically arranged ΔZPhe side chains

Z-Dehydrophenylalanine (Δ^zPhe) possessing four oligopeptides, Boc-(L -Ala-Δ^zPhe-Aib)_n-OCH₃ (n = 1–4: Boc, t-butoxycarbonyl; Aib, α-aminoisobutyric acid), were synthesized, and their solution conformations were investigated by ¹H-nmr, ir, uv, and CD spectroscopy and theoretical CD calculation. ¹H-nmr (the solvent accessibility of NH groups) and ir studies indicated that all the NH groups except for those belonging to the N-terminal L -Ala-Δ^zPhe moiety participate in intramolecular hydrogen bonding in chloroform. This suggests that the peptides n = 2–4 have a 4 → 1 hydrogen-bonding pattern characteristic of 3₁₀-helical structures. The uv spectra of all these peptides recorded in chloroform and in trimethyl phosphate showed an intense maximum around 276 nm assigned to the Δ^zPhe chromophores. The corresponding CD spectra of the peptides n = 2–4 showed exciton couplets with a negative peak at longer wavelengths, whereas that of the peptide n = 1 showed only weak signals. Theoretical CD spectra were calculated for the peptides n = 2–4 of several helical conformations, on the basis of exciton chirality method. This calculation indicated that the three peptides form a helical conformation deviating from the perfect 3₁₀-helix that contains three residues per turn, and that their side chains of Δ^z Phe residues are arranged regularly along the helix. The center-to-center distance between the nearest phenyl pair(s) was estimated to be ～ 5.5 Å. The chemical shifts of the Δ^zPhe side-chain protons (H^β and aromatic H) for the peptides n = 2–4 indicated anisotropic shielding effect of neighboring phenyl group(s); the effect also supports a regular arrangement of the Δ^z Phe side chains along the helical axis. © 1993 John Wiley & Sons, Inc.     

Role of N-t-Boc group in helix initiation in a novel tetrapeptide.

Protecting groups in N- and C-terminal positions play a decisive role in the conformational preference of smaller peptides. Conformational analysis of tetrapeptide derivatives containing Ala, Ile and Gly residues was performed. Peptide 1, Boc-Ala-Ile-Ile-Gly-OMe (Boc: tert-butyloxycarbonyl) has a predominantly helical turn conformation in all the alcoholic solvents studied, whereas in the solid state it has a beta-sheet conformation. In contrast, peptide 2, Ac-Ala-Ile-Ile-Gly-OMe (Ac: acetyl) has a random coil conformation in solution. The FTIR spectrum of peptide 1 shows a lower frequency of urethane carbonyl, indicating involvement of the carbonyl group in hydrogen bonding in the helical turn.     

Stereochemistry of peptides containing 1-aminocyclopentanecarboxylic acid (Acc5): Solution and solid-state conformations of Boc-Acc5-Acc5-NHMe

The conformational analysis of a protected homodipeptide of 1-aminocyclopentanecarboxylic acid (Acc⁵) has been carried out. ¹H-nmr studies establish a β-turn conformation for Boc-Acc⁵-Acc⁵-NHMe in chloroform and dimethylsulfoxide solutions involving the methylamide NH in an intramolecular hydrogen bond. Supportive evidence for the formation of an intramolecular hydrogen bond is obtained from ir studies. X-ray diffraction studies reveal a type III β-turn conformation in the solid state stabilized by a 4 → 1 hydrogen bond between the Boc CO and methylamide NH groups. The ?,ψ values for both Acc⁵ residues are close to those expected for an ideal 3₁₀-helical conformation (?? ± 60°, ψ～ ±30°).     

310-Helices,Helix Screw Sense and Screw Sense Reversal in the Dehydro-peptide Boc-Val-ΔPhe-Gly-ΔPhe-Val-OMe

The pentapeptide Boc-Val-ΔPhe-Gly-ΔPhe-Val-OMe, containing two dehydro-phenylalanine (ΔPhe) residues, has been synthesized and its structure investigated. In the crystalline state, the molecule adopts a right-handed 3₁₀-helical conformation stabilized by two intramolecular hydrogen bonds between CO of Val¹ and NH of ΔPhe⁴, and between CO of ΔPhe² and NH of Val⁵, respectively. NMR measurements are consistent with the presence of 3₁₀-helical structures also in acetonitrile and dimethylsulphoxide solution: the distances between backbone protons estimated from NOE connectivities are in overall agreement with those observed in the solid state; the chemical shifts of the amide protons show the smaller temperature coefficients for the NHs that in solid state are involved in intramolecular hydrogen bonds. The CD spectra in acetonitrile, chloroform, methanol and dimethylsulphoxide display exciton couplets of bands corresponding to the ΔPhe electronic transition at 280nm; the sign of the bands is consistent with the presence of helical structures having a prevalent left-handed screw sense. Addition of 1,1,1,3,3,3-hexafluoro- propan-2-ol gives rise to the gradual appearance of a couplet of opposite sign, suggesting the helix reversal from left-handed sense to right-handed sense. The conformational behaviour is discussed on the basis of the specific sequence of the peptide.     

Design of peptides with alpha,beta-dehydro-residues: synthesis, and crystal and molecular structure of a 3(10)-helical tetrapeptide Boc-L-Val-deltaPhe-deltaPhe-L-Ile-OCH3.

The peptide Boc-L-Val-deltaPhe-deltaPhe-L-Ile-OCH3 was synthesized using the azlactone method in the solution phase, and its crystal and molecular structures were determined by X-ray diffraction. Single crystals were grown by slow evaporation from solution in methanol at 25 degrees C. The crystals belong to an orthorhombic space group P2(1)2(1)2(1) with a = 12.882(7) A, b = 15.430(5) A, c = 18.330(5) A and Z = 4. The structure was determined by direct methods and refined by a least-squares procedure to an R-value of 0.073. The peptide adopts a right-handed 3(10)-helical conformation with backbone torsion angles: phi1 = 56.0(6)degrees, psi1 = -38.0(6)degrees, phi2 = -53.8(6)degrees, psi2 = 23.6(6)degrees, phi3 = -82.9(6)degrees, psi3 = -10.6(7)degrees, phi4 = 124.9(5)degrees. All the peptide bonds are trans. The conformation is stabilized by intramolecular 4-->1 hydrogen bonds involving Boc carbonyl oxygen and NH of deltaPhe3 and CO of Val1 and NH of Ile4. It is noteworthy that the two other chemically very similar peptides: Boc-Val-deltaPhe-deltaPhe-Ala-OCH3 (i) and Boc-Val-deltaPhe-deltaPhe-Val-OCH3 (ii) with differences only at the fourth position have been found to adopt folded conformations with two overlapping beta-turns of types II and III', respectively, whereas the present peptide adopts two overlapping beta-turns of type III. Thus the introduction of Ile at fourth position in a sequence Val-deltaPhe-deltaPhe-X results in the formation of a 3(10)-helix. The crystal structure is stabilized by intermolecular hydrogen bonds involving NH of Val1 and carbonyl oxygen of a symmetry related (-x, y - 1/2, 1/2 + z) deltaPhe2 and NH of deltaPhe2 with carbonyl oxygen of a symmetry related (x, y1/2, 1/2 + z) Ile4. This gives rise to long columns of helical molecules linked head to tail running along [010] direction.     

Effect of asymmetric modification on the conformation of ascidiacyclamide analogs.

Ascidiacyclamide (ASC), cyclo(-Ile1-Oxz2-d-Val3-Thz4-)2 (Oxz=oxazoline and Thz=thiazole) has a C2-symmetric sequence, and the relationships between its conformation and symmetry have been studied. In a previous study, we performed asymmetric modifications in which an Ile residue was replaced by Gly, Leu or Phe to disturb the symmetry [Doi et al. (1999) Biopolymers49, 459-469]. In this study, the modifications were extended. The Ile1 residue was replaced by Gly, Ala, aminoisobutyric acid (Aib), Val, Leu, Phe or d-Ile, and the d-Val3 residue was replaced by Val. The structures of these analogs were analyzed by X-ray diffraction, 1H NMR and CD techniques. X-Ray diffraction analyses revealed that the [Ala1], [Aib1] and [Phe1]ASC analogs are folded, whereas [Val1]ASC has a square form. These structures are the first examples of folded structures for ASC analogs in the crystal state and are similar to the previously reported structures of [Gly1] and [Phe1]ASC in solution. The resonances of amide NH and Thz CH protons linearly shift with temperature changes; in particular, those of [Aib1], [d-Ile1] and [Val3]ASCs exhibited a large temperature dependence. DMSO titration caused nonlinear shifts of proton resonances for all analogs and largely affected [d-Ile1] and [Val3]ASCs. A similar tendency was observed upon the addition of acetone to peptide solutions. Regarding peptide concentration changes, amide NH and Thz CH protons of [Gly1]ASC showed a relatively large dependence. CD spectra of these analogs indicated approximately two patterns in MeCN solution, which were related to the crystal structures. However, all spectra showed a similar positive Cotton effect in TFE solution, except that of [Val3]ASC. In the cytotoxicity test using P388 cells, [Val1]ASC exhibited the strongest activity, whereas the epimers of ASC ([d-Ile1] and [Val3]ASCs), showed fairly moderate activities.