Conformations of cyclic pentapeptide endothelin receptor antagonists

The solution conformations in methanol and chloroform of the endothelin A receptor antagonists cyclo(dV-L-dW-dD-P), 1, and cyclo(dV-^Nα-MeL-dW-dD-P), 2, have been studied by NMR spectroscopy at room temperature and below. In these solvents, both peptides were found to have a well defined peptide backbone conformation composed of a type II β turn at the Leu-D-Trp and a γ′ turn at Pro. This conformation is in agreement with results reported for 1 in other solvents and consistent with the expected location of the N-methyl substituent in that backbone. In methanol, both peptides show NOE and chemical shift evidence of close contact between the Leu and D-Trp side chains. This interaction is greatly reduced or absent in chloroform, and is stronger in methanol at 203 K than at 298 K.     

Crystal and molecular structure of Boc-Phe-Val-OMe; comparison of the peptide conformation with its dehydro analogue

The crystal structure of the peptide Boc-Phe-Val-OMe determined by X-ray diffraction methods is reported in this paper. The crystals grown from aqueous methanol are orthorhombic, space group P 2₁2₁ 2₁, a= 11.843(2), b= 21.493(4), c= 26.676(4) A and V= 6790 ų. Data were collected on a CAD4 diffractometer using MoK_α radiation (λ= 0.7107 Å) up to Bragg angle θ=26°. The structure was solved by direct methods and refined by a least-squares procedure to an R value of 6.8% for 3288 observed reflections. There are three crystal-lographically independent peptide molecules in the asymmetric unit. All the three molecules exhibit extended conformation. The sidechain of the Val² residue shows two different conformations. The conformation of the peptide Boc-Phe-Val-OMe is compared with the conformation of Ac-ΔPhe-Val-OH. It is observed that while Boc-Phe-Val-OMe exhibits an extended conformation, Ac-ΔPhe-Val-OH shows a folded conformation. The results of this comparison highlight the conformation constraining property of the ΔPhe residue. Interestingly, even though Boc-Phe-Val-OMe and Ac-ΔPhe-Val-OH are conformation ally different, they exhibit similar packing patterns in the solid state. © Munksgaard 1995.     

Structure of cyclic peptides: the crystal and solution conformation of cyclo(Phe-Phe-Aib-Leu-Pro)

A solid-state and solution conformation analyses of the cyclopentapeptide cyclo(Phe-Phe-Aib-Leu-Pro) has been carried out by X-ray diffraction and nuclear magnetic resonance techniques. The structure of the hexagonal crystals, grown from a methanol solution [a=b= 16.530(4) Å, c= 21.356(9) Å, space group P6₅, Z = 6], shows the presence of one intramolecular N-H?O=C hydrogen bond with the formation of a γ-turn (C₇). The Aib³ residue, at the center of the γ-turn, presents unexpected values of the torsion angles [φ= 70.5° and ψ= -73.8°], which have been observed only once before for this helicogenic residue. A cis peptide bond occurs between Leu⁴ and Pro⁵; all other peptide bonds are trans. The overall conformation for the cyclopentapeptide with one cis-peptide bond on one side and an intramolecular γ-turn on the opposite side results in an equatorial topology of the side-chains of the Phe¹, Phe² and Leu⁴ residues. Indeed, the C^α-C^βand C^β-C^γ bonds of these residues lie approximately in the mean plane of the cyclic ring system. The structure is compared with data in the literature on cyclic pentapeptides. In addition the Pro-Phe-Phe moiety shows a conformation similar to that observed in other larger cyclic bioactive peptides, which indicates a reduced number of conformations for this sequence. The solution study was carried out in three different solvent systems: chloroform, acetonitrile and methanol in the temperature interval 220–300 K. In all three solvents the room temperature spectra show that the peptide is conformationally nonhomogeneous. In acetonitrile at low temperatures it is possible to reduce the conformational equilibrium to two predominant conformers which differ for the cis-trans isomerism of the Leu⁴-Pro⁵ peptide bond.     

Synthetic protein design: construction of a four‐stranded β‐sheet structure and evaluation of its integrity in methanol–water systems

Abstract: The characterization of a four‐stranded β‐sheet structure in a designed 26‐residue peptide Beta‐4 is described. The sequence of Beta‐4 (Arg‐Gly‐Thr‐Ile‐Lys‐^Dpro‐Gly‐Ile‐Thr‐Phe‐Ala‐^DPro‐Ala‐Thr‐Val‐Leu‐Phe‐Ala‐Val‐^DPro‐Gly‐Lys‐Thr‐Leu‐Tyr‐Arg) was chosen such that three strategically positioned ^DPro‐Xxx segments nucleate type II′β‐turns, which facilitate hairpin extension. A four‐stranded β‐sheet structure is determined in methanol from 500 MHz ¹H NMR data using a total of 100 observed NOEs, 11 dihedral restraints obtained from vicinal J_CαH‐NH values and 10 hydrogen bonding constraints obtained from H/D exchange data. The observed NOEs provide strong evidence for a stable four‐stranded sheet and a nonpolar cluster involving Ile⁸, Phe¹⁰, Val¹⁵ and Phe¹⁷. Circular dichroism studies in water–methanol mixtures provide evidence for melting of the β‐sheet structure at high water concentrations. NMR analysis establishes that the four‐stranded sheet in Beta‐4 is appreciably populated in 50% (v/v) aqueous methanol. In water, the peptide structure is disorganized, although the three β‐turn nuclei appear to be maintained.     

A crystal-state,solution and theoretical study of the preferred conformation of linear Cα,α-diphenylglycine derivatives and dipeptides with potential anticonvulsant activity

Several linear molecules containing the C^α,α-diphenylglycine residue were prepared as potential anticonvulsants. The conformational preferences of the C^α,α-diphenylglycine residue were assessed in these synthetic derivatives and dipeptides by X-ray diffraction, FTIR absorption and ¹H NMR techniques, and by conformational energy computations. Five (out of six) derivatives adopt the fully extended C₅ conformation in the crystal state. This intramolecularly H-bonded form is largely populated in chloroform solution in all the derivatives investigated. Conformational energy computations in vacuo support the view that the intramolecularly H-bonded C₇-ring form is the most stable structure for these compounds. Only one linear derivative exhibits a (modest) anticonvulsant activity.     

A solution NMR study of the selectively 13C, 15N-labeled peptaibol chrysospermin C in methanol

Abstract: The conformation of the 19-residue peptaibol chrysospermin C in methanol has been investigated by NMR spectroscopy using selective ¹⁵N and ¹³C labeling of the α-aminoisobutyric acid (Aib) residues. Complete ¹H and ¹³C sequential assignments, including stereospecific assignments for the heavily overlapped resonances from the two C^β methyl groups of the eight Aib residues, are reported for a peptaibol for the first time. An Aib residue followed by a Pro is an exception to previous suggestions regarding stereospecific assignment of the two C^β methyl groups of Aib residues. Local nuclear Overhauser effects and ³J_HNC and ³J_HNCβ scalar couplings indicate that the φ angles of the Aib residues are restricted sterically to local conformations consistent with right-handed helices. Despite these constraints on the eight Aib residues, the NMR data for chrysospermin C in methanol are generally most consistent with an ensemble of transient conformations, including backbone conformations inconsistent with helical structures. Initial NMR measurements for chrysospermin C bound to micelles suggest structural and dynamic differences relative to alamethicin bound to micelles which may be related to differences in gating voltages for formation of ion channels.     

Conformational study of two substance P hexapeptides by two-dimensional NMR

The conformation of two substance P (SP) related hexapeptides. Glp-Phe-Phe-(L-Pro)-Leu-Met.NH₂(I) and Glp-Phe-Phe-(D-Pro)-Leu-Met.NH₂ (II), in two solvents, chloroform-d and trifluoroethanol(TFE)-d₃/H₂O, was studied by two-dimensional NMR methods, including COSY, TOCSY, ROESY and HMQC. The study shows that these two peptides exist predominantly in the extended form in TFE/H₂O, but in general exhibit a reverse-turn structure in chloroform. I is clearly less ordered than II in both solvents. Furthermore, extensive Phe³-Pro⁴cis?trans isomerization was found in I but not in II. The differences in the conformational behavior of these two peptides, which are selective agonists for neurokinin NK1 and NK2 receptors, respectively, are discussed.     

Conformation of cyclo-(D-phenylalanyl-trans-4-fluoro-D-prolyl)

cyclo(D-Phenylalanyl-trans-4-fluoro-D-prolyl), c(D-Phe-D-FPro), was synthesized and its conformation determined both in solution and in the solid state by ¹H NMR and X-ray analysis, respectively. In the crystals the 2,5-diketopiperazine (DKP) ring assumes the uncommon conformation, for cyclodipeptides containing Pro residue, of a flattened chair, which seemingly results from a compromise between, on the one hand, the DKP-aromatic intramolecular ring-ring attraction (folding), requiring the C^α-C^β bond of the Phe to be axial, and, on the other hand, the intrinsic tendency of the Pro residue to have its C^α-C^β bond equatorial. Unlike the solid state, the ¹H NMR data in CDCl₃ and C₆D₆ demonstrate that in both solutions the DKP ring assumes a boat-like shape, typical for the Pro-containing cyclodipeptides, with the equatorial C^α-C^β bonds in both amino acid residues, which preclude ring-ring folding. A similar conformation was encountered in the closest analog of c(D-Phe-D-FPro), viz. in c(Phe-Pro), both in solution (21, 22, 26) and in the solid state (12). A subtle interplay of intramolecular interactions introduced into a cyclodipeptide by a Pro-type and a Phe-type residue is emphasized.     

NMR structure of a minimum activity domain of human parathyroid peptide hormone: structural origin of receptor activation

Abstract: Parathyroid hormone (PTH) which increases osteoblast numbers and bone formation by activating bone‐lining cells to osteoblasts plays an important role in calcium and phosphate homeostasis and bone remodeling by activating PTH receptors. To determine the structural origin of a minimum activity domain of hPTH, we initiated a detailed structural determination of the hPTH^H14 in aqueous solution using NMR spectroscopy. Circular dichroism and NMR data demonstrated that hPTH^H14 maintains a typical helical conformation in both membrane‐mimicking environments and 30% TFE solution. The solution structure clearly showed that the residues from Ser³ to Leu¹¹ of hPTH^H14 formed a stable helical structure, especially having charged side‐chains oriented in opposite directions relative to one another for optimum interaction with the receptor molecule.     

Synthesis and solution conformation of c(D-Trp-D-Cys(SO3-Na+)-Pro-D-Val-Leu), a potent endothelin-A receptor antagonist

The endothelin family of polypeptides are known to exert potent physiological effects which include cardiovascular regulation. The solution conformation and dynamics of c(D-Trp-D-Cys(SO₃-Na⁺)-Pro-D-Val-Leu), a potent endothelin-A receptor-selective antagonist, were characterized in aqueous solution by NMR spectroscopy and molecular modeling. NMR-derived conformational constraints were combined with computer-assisted molecular modeling using distance geometry calculations and energy minimization. The pentapeptide backbone is shown to adopt a single conformation in solution comprising a type II β-turn and an inverse γ-turn, with each residue in the trans conformation. Molecular dynamics were explored using relaxation measurements and low-temperature studies, and indicate that the peptide backbone is highly constrained with little conformational mobility present.     

丹参中水溶性酚酸类成分的薄层扫描测定法

应用高效薄层扫描法对丹参根中的原儿茶醛、咖啡酸、迷迭香酸及其甲酯、丹酚酸A,B和C7种有效成分进行了含量测定。选择了最佳提取分离方法,即生药用热水提取并酸化后用乙酸乙酯萃取,萃取液浓缩后进行薄层色谱分离。展开剂A:氯仿乙酸乙酯——苯—甲酸(2.4:2:1:0.6)分离原儿茶醛,展开剂B:氯仿-乙酸乙酯-苯-甲酸甲醇(1.5:2:1;1:0.1)分离其余6种成分。并用所建立的分析方法分析比较了4种丹参根中7种有效成分的含量。     

Evaluation of Wound Healing Potential of Bauhinia purpurea Leaf Extracts in Rats

The present study was carried out to evaluate the effect of methanol and chloroform extracts of Bauhinia purpurea on experimentally induced excision, incision, burn and dead space wound models in Sprague Dawley rats. Formulations of methanol and chloroform extracts of Bauhinia purpurea were prepared in carbopol and simple ointment base at concentrations of 2.5% and 5% and applied to the wounds. In the excision and burn wound models, animals treated with high doses of methanol and chloroform showed significant reduction in time taken for epithelization and wound contraction (50%) compared to control. A significant increase in breaking strength was found in incision wound model with methanol and chloroform extracts compared to their respective bases. In the dead space wound model, methanol and chloroform extract treatment (100 and 500 mg/kg) orally produced a significant increase in the breaking strength, dry tissue weight and hydroxyproline content of the granulation tissue when compared to control. Among the extracts, methanol extract exhibited more activity followed by the chloroform extract. In conclusion, the present study indicated that Bauhinia purpurea leaves exhibited wound healing activity.     

Use of Intrauterine Microdialysis to Investigate Methanol-Induced Alterations in Uteroplacental Blood Flow

Methanol is teratogenic in rodents; it has been postulated that this teratogenicity may be mediated in part by conceptal hypoxia. To construct a model to predict conceptal risk following maternal methanol exposure, conceptal disposition of methanol must be determined and any effects of such exposure on blood flow must be quantitated. In the present study, these toxicokinetic and toxicodynamic parameters were evaluated byin vivointrauterine microdialysis. Microdialysis probes were inserted into the uteri of Gestational Day (gd) 20 rats; methanol was administered as either an iv bolus (100 or 500 mg/kg) or infusion (100 or 1000 mg/kg/hr). In separate experiments, methanol (100 or 500 mg/kg) and³H₂O (20 μCi/kg) were administered iv to gd 20 and 14 rats and gd 18 mice. In both experiments, maternal blood and uterine microdialysate were collected and analyzed for methanol or³H₂O content. The methanol concentration–time data were consistent with saturable maternal elimination and apparent first-order transfer between maternal and conceptal compartments; at distribution equilibrium, conceptal methanol concentrations exceeded those in the dam by approximately 25%. The initial rate of conceptal permeation of methanol was proportional to the reciprocal of maternal blood methanol concentration (r²= 0.910). Methanol also reduced significantly the rate of³H₂O uptake into the conceptus in a concentration-dependent fashion in gd 14 and 20 rats and gd 18 mice. These data indicate that methanol may decrease uteroplacental blood flow, decreasing methanol presentation to the conceptus and possibly producing conceptal hypoxia.     

Chiral recognition in dipeptides containing 1-aminocyclopropane carboxylic acid or α-aminoisobutyric acid: NMR studies in solution

Protected dipeptides containing 1-aminocyclopropane carboxylic acid (Ac₃c) or α-aminoisobutyric acid (Aib) residues at the C-terminus and Phe, Val or Ala residues at the N-terminus displayed different proton NMR spectra for the pure enantiomers and the racemic mixtures in deuterochloroform (CDCl₃) solution. An unequal mixture of enantiomers showed two sets of resonances (NMR nonequivalence), one corresponding to major and the other to minor enantiomer. The NMR nonequivalence was originated by the presence of the C-terminal Ac₃c or Aib residues, which have been known for their unique spatial preferences in avoiding an extended (C₅) conformation. When a C₅ conformation favoring residue such as glycine was incorporated in place of Ac₃c or Aib, negligible NMR nonequivalence was observed. The magnitude of the NMR nonequivalence depended on the side chain as well as on the protecting groups at N-terminus α-amino acid. For the same peptide, the magnitude of nonequivalence increased with increasing solution concentration and/or with decreasing the solution temperature. The NMR nonequivalence disappeared in polar solvent-like deuterated dimethylsulfoxide (DMSO-d₆). A preference for hetero-chiral recognition leading to dimeric association under fast exchange conditions had been invoked to explain the observed phenomenon. The dipeptides thus prepared could well serve as ‘model peptides’ for the evaluation of any preparative methods.     

Conformational studies of N-Tyr-MIF-1 in aqueous solution by 1H nuclear magnetic resonance spectroscopy

N-Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH₂) is an endogenous brain peptide with multiple effects on animal behavior. However, there have been no studies on the conformation of this tetrapeptide. In this report, we studied the conformation of N-Tyr-MIF-1 in aqueous solution by conventional one-dimensional and two-dimensional (COSY and NOESY) ¹H nuclear magnetic resonance spectroscopy at 300 MHz. A complete set of assignments for the resolved resonances and approximate assignments for the overlapping resonances were made. The results demonstrate that N-Tyr-MIF-1 is in slow exchange between two conformers, most likely determined by the cis and trans states of the proline residue. The minor conformation represents 30 ± 3% of the population over the temperature range from 3° to 73°. In the major conformation, the tyrosine aromatic ring appears to be close enough to interact directly with the proline pyrrolidine ring, as indicated by a strong temperature dependence of the proline CβH, CδH and CδH′ chemical shifts. In contrast, this interaction of the tyrosine and proline rings is not present in the minor conformation.     

Proton NMR Conformational Analysis of Cyclic β-Casomorphin Analogues of the Type Tyr-cyclo[-Nω-D-Orn-Xaa-Yaa-Gly-]

A conformational study of the cyclic β-casomorphin-5 analogues H-Tyr-cyclo[-D-Orn-2-Nal-Pro-Gly-] ( 1 ) (μ-selective agonist; 2-Nal = 2-naphthylalanine), H-Tyr-cyclo[-D-Orn-2-Nal-D-Pro-Gly-] ( 2 ) (mixed μ agonist/δ antagonist) and H-Tyr-cyclo[-D-Orn-Phe-D-Pro-Gly-] ( 3 ) (highly potent μ and δ agonist) has been carried out using ¹H NMR spectroscopy. A complete assignment of the proton resonances of the three pentapeptides has been achieved. Compound 1 was shown to exist in two conformations, a major one (90%) characterized by a cis amide bond between 2-Nal³ and Pro⁴, and a minor one (10%) showing cis amide bonds both between D-Orn² and 2-Nal³ and between 2-Nal³ and Pro⁴. Peptides 2 and 3 each showed only one conformer with all-trans peptide bonds in both cases. Temperature dependence studies of the amide proton chemical shifts indicated the existence of several intramolecular hydrogen bonds in the case of compounds 2 and 3 but not in the case of peptide 1. The backbone conformations of 2 and 3 were found to be similar, both being characterized by two consecutive γ turns around the D-Pro⁴ and D-Orn² residues, respectively, and by a D-Orn²-CO←HN^δ-D-Orn² hydrogen bond. Altogether, the overall backbone conformation and the preferred side chain conformation were found to be roughly similar for the three title peptides. For all three compounds a close proximity between the aromatic moiety of the 3-position residue (2-Nal or Phe) and the D(or L)-Pro⁴ residue was established on the basis of ROESY experiments. The examination of low energy conformations obtained in molecular modelling studies by taking into account the various experimentally found NMR parameters (NOEs, vicinal H,H coupling constants, torsion angles, H-bonds) led to proposals of the solution conformation for each peptide. These conformations are in close agreement with a pharmacophore model for μ opioid receptor binding compounds.     

Selective ion transport and ion extraction by synthetic cyclic octapeptide

Cyclic octapeptide, cyclo[Gly-L-Lys(Z)-Sar-L-Pro]₂ (CGLSP2) was synthesized as an ionophore model. Its ion-transport ability through a liquid membrane was investigated in connection with ion extractability and conformational properties. CGLSP2 transported the picrate salts of Ba²⁺ and Ca²⁺ efficiently through chloroform membrane in a U-tube (Ba²⁺> Ca²⁺ > K⁺ > Rb⁺ > Mg²⁺> Na⁺). Cyclo[Gly-L-Lys(Z)-Sar-L-Pro] or Boc-[Gly-L-Lys(Z)-Sar-L-Pro]_n-OR (n = 1 or 2; R = H or CH₃) showed a significant decrease in ion-transport ability. Extraction equilibrium constants (K_ex) of CGLSP2 with metal picrates were determined in chloroform-water system. In this case, CGLSP2/cation complexes were found to have a stoichiometry of 1:1. The K_ex sequences were Ba²+ > Ca²⁺ > Mg²⁺ and K⁺ > Rb⁺ > Na⁺, showing good agreement with the selectivity in ion transport. Conformation of CGLSP2 in chloroform (CDC1₃) was investigated by ¹H and ¹³C-n.m.r. spectroscopy. Predominant conformation takes a C₂ -symmetric structure containing two cis Lys-Sar peptide bonds. Upon complexation with Ca²⁺ in CDC1₃/CD₃CN mixture (5/3, v/v), the conformation of CGLSP2 converged into a single C₂ -symmetric one that was predominant in a free state in CDC1₃.     

Type II‘β-bend conformation of tert.-butyloxycarbonyl-L-amino-succinyl-L-alanyl-glycine methyl ester in the solid state

Boc-L-Asu-L-Ala-Gly-OMe crystallizes in the monoclinic space group P2₁ with cell dimensions a = 14.315(3) Å, b = 9.280(2) Å, c = 14.358(3) Å, β= 103.63 (1) d?, V= 1853.4(9) ų, with two molecules in the asymmetric unit. The conformation of the two molecules is characterized by a type II' β-bend, similar to that predicted earlier by potential energy calculations, stabilized by an intramolecular hydrogen bond. I.r. and ¹H-n.m.r. data show that the folded conformation is also stable in chloroform solution.