CD-n.m.r. study of the solution conformation of bradykinin analogs containing α-aminoisobutyric acid

The conformation in aqueous solution of several α-aminoisobutyric acid (AIB)-containing analogs of bradykinin (BK) has been probed by complementary CD and ¹H n.m.r. measurements. The conclusion reached is that substitution of AIB for Pro² and/or Pro³ in BK stabilizes a degree of β-turn conformation in the N-terminal tetrapeptide moiety of the resulting analogs. Changing the solvent from water to DMSO or TFE further enhances the contribution of particular hydrogen bonded structures to the time-averaged conformation of these peptides. Bradykinin and [AIB⁷]-BK adopt similar hydrogen bonded conformations in TFE, apparently with a contribution from a β-turn involving their common Arg¹-Pro²-Pro³-Gly⁴ moiety. The contrasting biological activities of BK and its AIB-analogs are considered in terms of the conformational analogy between the AIB-residue and cis¹ Pro and the propensity for a β-turn at the N-terminus of the peptide.     

Sulfonated analogues of cyclolinopeptide A Synthesis,immunosuppressive activity and CD studies

Linear and cyclic analogues of cyclolinopeptide A (CLA) in which one or both phenylalanine residues in fragment Pro⁶-Pro⁷-Phe⁸-Phe⁹ were substituted by their sulfonated derivatives have been synthesized by SPPS method and cyclization with the BOP reagent. The peptides were examined for their immunosuppressive activity in the humoral and cellular immune response by PFC and DTH tests. All of the analogues retain some immunosuppressive activity of native CLA. Their CD spectra confirm that the optical activity of aromatic residues in CLA depends on their position in the peptide chain. Only the residue in position 8 seems to be optically active. CD spectrum of the cyclic analogue modified in position 9 is very similar to that of native CLA which correlates with its high biological activity. The chiroptical properties of the p-sulfonated Phe-residue are established. © Munksgaard 1997.     

Peptide conformations — 49(1): Synthesis and structure-activity relationships of side chain modified peptides of cyclo(-d-Pro-Phe-Thr-Lys-Trp-Phe-)

Cyclic hexapeptide analogues representing the modified retro sequence of the amino acid residues 7-11 of natural somatostatin are known to protect liver cells from phalloidin poisoning. To determine the influence of steric, lipophilic, and charge effects on (a) the conformation of the backbone and the aromatic side chains and (b) the biological response, the side chains of Phe², Lys⁴, and Phe⁶ of cyclo(-d -Pro¹-Phe²-Thr³-Lys(Z)⁴-Trp⁵-Phe⁶-), 1a, one of the most active peptides found so far, were modified by various residues. The discussion of conformationally relevant parameters proves that neither backbone conformations nor populations of aromatic side chain rotamers were altered by these substitutions. The potency of these derivatives in a cytoprotection assay varies by at most one order of magnitude (more or less active than the parent peptide 1a). A qualitative evaluation of lipophilic, steric, and charge effects reveals the dominance of lipophilic effects of aromatic residues; the most potent compounds contain aromatic substructures in the side chain of Lys⁴.     

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.     

内啡肽-morphiceptin 的构象和量子力学研究

本文用分子动力学方法对内啡肽morphiceptin(Tyr¹-Pro²-Phe³-Pro⁴-NH₂)进行了构象研究。所得结果与¹H,¹³CNMR和其它实验方法的结果一致,证明我们的构象搜索方法有效。量子力学计算表明此多肽和吗啡在空间和电性上非常相近。因此,它们应作用于同一受体。     

CONFORMATIONAL CHARACTERIZATION OF CYCLOPENTAPEPTIDE,LVAL-LPRO-GLY-LVAL-GLY: A REPEATING ANALOGUE OF ELASTIN

The cyclopentapeptide, ·L·Val¹-L· Pro²-Gly³-L· Val⁴-Gly⁵, was synthesized and its conformational characterization was carried out using n.m.r. and theoretical energy calculations. The n.m.r. studies indicated the existence of a cis Val¹-Pro² peptide bond in water and a very strong intramolecular H-bond between the Val¹ NH and Gly³ C=O groups. This H-bond forms a β-turn (type II) placing Val⁴ Gly⁵ residues within the turn. Two minimum energy conformations were derived, one of which agrees very well with the solution conformation.     

The pseudo-βI-turn

Synthesis and conformational analysis of three cyclic hexapeptides cyclo(-Gly¹-Pro²-Phe³-Val⁴-Xra⁵-Phe⁶), Xaa= Phe (I), D-Phe (II) and D-Pro (III), were carried out to examine the influence of proline on the formation of reverse turns and the dynamics of hydrophobic peptide regions. Assignment of all ¹H and ¹³C resonances was achieved by homo- and heteronuclear 2D-NMR techniques (TOCSY, ROESY, HMQC, HMQC-TOCSY and HMBCS-270). The conformational analysis is based on interproton distances derived from ROESY spectra and homo- and heteronuclear coupling constants (E.COSY, HETLOC and HMBCS-270). For structural refinements restrained molecular dynamics (MD) simulations in vacuo and in DMSO were performed. Each peptide exhibits two conformations in DMSO solution due to cis-trans isomerism about the Gly-Pro peptide bond. Surprisingly the cis-Gly-Pro segment in the minor isomers is not involved in a βVI-turn, but forms a turn structure with cis-Gly-Pro in the i and i+ 1 positions. Although no stabilizing hydrogen bond is found in this turn, the φ and ψ-angles closely correspond to a βI-turn [Pro²:φ(i+ 1) -60°, ψ(i+ 1) -30° Phe³: φ(i+ 2) -100°, ψ(i+ 2) -50°]. Hence we call this structural element a pseudo-βI-turn. As expected, in the dominating all-trans isomers proline occupies the i+ 1 position of a standard βI-turn. Therefore, cis-trans isomerization of the Gly¹-Pro² amide bond only induces a local conformational rearrangement, with minor structural changes in other parts of the molecule. However, the geometry of the other regions is affected by the chirality of the i+ 1 amino acid for both isomers (βI for Phe⁵, βII′ for D-Phe⁵ or D-Prp⁵).     

Peptide inhibitors of E. collagenolyticum bacterial collagenase -effect of N-methylation

Peptide inhibitors of E. collagenolyticum bacterial collagenase, HS-CH₂-CH₂-CO-Pro-Yaa (Yaa = Ala, Leu, Nle), have been N-methylated at the Yaa position. The N-methylation slightly increases the inhibitory potency of the modified peptides as compared to the parent compounds. The conformational effects of the N-methylation have been investigated by both ¹H 2D-NMR and molecular mechanics energy minimization. Three low-energy conformers have been predicted for the unmethylated parent compounds (Yaa = Ala, Leu, Nle). They are characterized by the Ψ value of the central proline residue: Ψ_Pro= 150^c(trans' conformation), Ψ_pro= 70° (C₇ conformation) and Ψ_Pro= -50°(cis' conformation). The N-methylation has been found to strongly increase the energy of the C₇ conformer and to a less extent the energy of the cis' conformer. This leaves the trans' conformation as the only low-energy conformer. The ROESY experiments have established that both the N-methyl peptides and the parent compounds adopt the same preferred backbone conformation in water solution, i.e. the trans conformation. Based on these results, the activities of the N-methyl peptides are discussed and a possible conformation of the inhibitor in the bound state is proposed.     

Stereochemistry of peptides containing 1-aminocycloheptane-1-carboxylic acid (Ac7c)

The crystal structures of four peptides incorporating l-aminocycloheptane-l-carboxylic acid (Ac₇c) are described. Boc-Aib-Ac₇c-NHMe and Boc-Pro-Ac₇c-Ala-OMe adopt β-turn conformations stabilized by an intramolecular 4 × 1 hydrogen bond, the former folding into a type-I/III β-turn and the latter into a type-II β-turn. In the dipeptide esters, Boc-Aib-Ac₇c-OMe and Boc-Pro-Ac₇c-OMe, the Ac₇c and Aib residues adopt helical conformations, while the Pro residue remains semi-extended in both the molecules of Boc-Pro-Ac₇c-OMe found in the asymmetric unit. The cycloheptane ring of Ac₇c residues adopts a twist-chair conformation in all the peptides studied. ¹H-NMR studies in CDCl₃ and (CD₃)₂SO and IR studies in CDCl₃, suggest that Boc-Aib-Ac₇c-NHMe and Boc-Pro-Ac₇c-Ala-OMe maintain the β-turn conformations in solution.     

Peptide conformation

Six cyclic retro-analogues of the peptide hormone somatostatin have been synthesized using the solid phase technique. The peptides cyclo(-Xaa¹-Phe²-Thr³-Lys⁴-Ybb⁵-Phe⁶-) and cyclo(-Phe¹-Xaa²-Thr³-Lys⁴-Ybb⁵-Phe⁶-) with Xaa =d - or l -Pro and Ybb =d - or l -Trp were cyclized via the azide method. The conformations of the cyclic hexapeptides in DMSO-d₆ solution were determined by a number of homo- and heteronuclear two-dimensional n.m.r.-techniques including 2D rotating frame NOE-spectroscopy. Two-step coherence transfers, ROE and chemical exchange, are observed for the first time in ROESY spectra. The backbone conformation of the all-trans cyclopeptides consists of a β-turn containing the Pro residue in the position i + 1. These retro-analogues of somatostatin exhibit a high activity in the inhibition of cholate and phalloidin uptake by liver cells (cytoprotective effect); however, the hormonal activities of the natural hormone are completely suppressed. The constitutional and conformational requirements for the cytoprotective activity are discussed.     

Unusual thionation of a cyclic hexapeptide

One carbonyl oxygen of the cyclic hexapeptide cycle(-Gly¹-Pro²-Phe³-Val⁴-Phe⁵-Phe⁶-) (A) can be selectively exchanged with sulphur using Yokoyama's reagent. Surprisingly it was not the C=O of Gly¹ but that of Phe⁵ which was substituted and cyclo(-Gly¹-Pro62-Phe³-Va1⁴-Phe⁵ψ[CS-NH]Phe⁶-) (B)was obtained. Thionation results in a conformational change of the peptide backbone although the C=O of Phe⁵ and the corresponding C=S are not involved in internal hydrogen bonds. Two isomers in slow exchange, containing a CIS Gly¹-Pro² bond in a βVIa-turn (minor) and a trans Gly-Pro bond in a βII′-turn (major), were analyzed by restrained molecular dynamics in vacuo and in DMSO as well as using time dependent distance constraints. It is impossible to fit all experimental data to a static structure of each isomer. Interpreting the conflicting NOES, local segment flexibility is found. MD simulations lead to a dynamic model for each structure with evidence of an equilibrium between a βI- and βII-turn about the Val⁴-Phe⁵ amide bond in both the cis and trans isomers. Additionally proton relaxation rates in the rotating frame (R_1p) were measured to verify the assumption of this fast βI/βII equilibrium within each isomer. Significant contributions to R_1p-rates from intramolecular motions were found for both isomers. Therefore it is possible to distinguish between at least four conformers interconverting on different time scales based on NMR data and MD refinement. This work shows that thionation is a useful modification of peptides for conformation-activity investigations.     

Stabilization of β-turn conformations in enkephalins

Stereochemical constraints have been introduced into the enkephalin backbone by substituting α-aminoisobutyryl (Aib) residues at positions 2 and 3, instead of Gly. ¹H n.m.r. studies of Tyr-Aib-Gly-Phe-Met-NH₂, Tyr-Aib-Aib-Phe-Met-NH₂ and Tyr-Gly-Aib-Phe-Met-NH₂ demonstrate the occurrence of folded, intramolecularly hydrogen bonded structures in organic solvents. Similar conformations are also favoured in the corresponding t-butyloxycarbonyl protected tetrapeptides, which lack the Tyr residue. A β-turn centred at positions 2 and 3 is proposed for the Aib²-Gly³analog. In the Gly²-Aib³analog, the β-turn has Aib³-Phe⁴as the corner residues. The Aib²-Aib³analog adopts a consecutive β-turn or 3₁₀ helical conformation. High in vivo biological activity is observed for the Aib²and Aib²-Aib³analogs, while the Aib³peptide is significantly less active.     

NMR studies on the structure of some cyclic and linear antagonists of luteinizing hormone-releasing hormone (LHRH)

The structure of cyclic antagonists of luteinizing hormone-releasing hormone (LHRH), Ac-D-Phe(p-Cl)¹-D-Phe(p-C1)²-Trp³-Ser⁴-c(Asp⁵-D-Arg⁶-Leu⁷-Lys⁸)-Pro⁹-D-Ala¹⁰-NH₂ ( I ), Ac-D-Phe(p-Cl⁾l-D-Phe(p-Cl)²-D-Trp³-Ser⁴-c(Glu⁵-Arg⁶-Leu⁷-Lys⁸)-Pro⁹-D-Ala¹⁰-NH₂ ( II ) and their linear analogues, Ac-D-Phe(p-Cl)¹-Phe(p-C1)²-Trp³-Ser⁴-Asp⁵-D-Arg⁶-Leu⁷-Lys⁸-Pro⁹-D-Ala¹⁰-NH₂ ( III ) and Ac-D-Phe(p-Cl)¹-D-Phe(p-C1)²-Trp³-Ser⁴-Glu⁵-D-Arg⁶-Leu⁷-Lys⁸-Pro⁹-D-Ala¹⁰-NH₂ ( IV ), have been studied by NMR spectroscopy. The cyclic peptides I and II are more potent antagonists than the corresponding linear peptides in an in vivo assay. All the peptides show propensity of an unusual type II′β-turn involving residues 3–6. Cyclic analogues also show some additional structure around residues 7 and 8 which is absent in the linear peptides. This additional structure in the cyclic peptides may be due to a minor conformation with a β-turn between residues 5 and 8. © Munksgaurd 1995.     

EFFECT OF TEMPERATURE UPON THE CIRCULAR DICHROISM OF BRADYKININ

Analysis of the effect of temperature on the circular dichroism spectrum of bradykinin has led to a more precise understanding of the solution conformation of the peptide. Circular dichroism and ¹³C n.m.r. have been used in a complementary fashion to support the picture that bradykinin spends a maximum of about 20% of its time in a partially ordered conformation featuring a γ-turn with Pro⁷ as the second residue. Since the γ-turn probability is insensitive to temperature, some other conformational effect dominated by the structure of water presumably produces the pronounced change in the circular dichroism spectrum with increasing temperature.     

SPECTROSCOPIC STUDY OF THE CONFORMATIONS OF PROLINE-CONTAINING OLIGOPEPTIDES IN THE CRYSTALLINE STATE AND IN SOLUTION

A conformational study has been carried out on a series of linear proline-containing oligopeptides (ZGP, ZGPL, ZGPLG and ZGPLGP) in both the crystalline state and in DMSO-d₆, solution, using Raman and n.m.r. spectroscopy. The amide I and HI bands in the Raman spectra of the crystalline forms indicate the presence of a type I β-bend conformation in ZGPLG and ZGPLGP, but not in ZGP and ZGPL. This result is in agreement with X-ray data on these mole cules. The Raman spectra of these peptides in solution indicate that more than one conformation is present, i.e. that the β-bend structure of the solid form of ZGPLG and ZGPLGP is destabilized by DMSO-d₆. ¹³C and ¹H n.m.r. data also demonstrate the presence of more than one conformation in ZGP, ZGPL, ZGPLG and ZGPLGP in DMSO-d₆ solution. These isomers differ in their con formation (cis and trans) about their Gly-Pro peptide bonds and possibly about the Cα-C' bond of the C-terminal proline in ZGPLGP. For ZGP, ZGPL, ZGPLG and ZGPLGP, the ensemble of conformations in DMSO-d₆ includes C₅ and C₇ hydrogen-bonded rings; in addition, ZGPLGP may contain a small percentage of a β-bend conformation (at Pro₂-Leuj₃) with trans peptides in both Gly-Pro moieties.     

Synthesis and solution structure of an S-glycosylated cyclic hexapeptide

Synthesis and conformational analysis of the S-glycosylated cyclic hexapeptide cyclo(-d -Pro¹-Phe²-Cys³(tetra-O-acetyl-β-d -galactopyranosyl)-Trp⁴-Lys(Z)⁵-Phe⁶-) I was carried out to examine the influence of a saccharide residue in position i of a standard β-turn on the formation of reverse turns and on the biological activity. Synthesis was carried out in the liquid phase employing a galactosylated cysteine building block. The cyclization reagents DPPA/NaHCO₃ avoided high dilution conditions. Spectroscopic data were extracted from homo- and heteronuclear 2D-NMR techniques (TOCSY, NOESY, HMQC, HMQC-TOCSY, HMBCS-270). For structural refinement restrained molecular dynamics (MD) simulations in vacuo and with explicit DMSO as solvent were performed. Finally, simulations in DMSO without experimental restraints provided insight in stability and dynamics of the structural model. A comparison of the S-glycosylated Cys³ peptide with the analogous Thr³ peptide exhibits a similar overall conformation of the hexapeptide [βII’d -Pro-Phe and another β-turn about Trp⁴-Lys⁵(Z)]. However, the latter shows a distinct dynamic flip βI, βII in the glycopeptide, whereas the Thr-analogue only populates βI. This influence is attributed to a βI stabilizing effect of a hydrogen bridge of Thr-O, in position i to the NH of the amino acid in position i+ 2, which is lacking in the glycosylated compound.     

Conformational features responsible for the binding of cyclic analogues of enkephalin to opioid receptors III. Probable binding conformations of μ-agonists with phenylalanine in position 3

Theoretical conformational analysis was carried out for several tetrapeptide analogues of β-casomorphin and dermorphin containing a Phe residue in position 3. Sets of low-energy backbone structures of the μ-selective peptides [N-Me-Phe³, d -Pro⁴]-morphiceptin and Tyr-d -Orn-Phe-Asp-NH₂ were obtained. These sets of structures were compared for geometrical similarity between themselves and with the low-energy conformations found for the δ-selective peptide Tyr-d -Cys-Phe-d -Pen-OH and nonactive peptide Tyr-Orn-Phe-Asp-NH₂. Two pairs of geometrically similar conformations of μ-selective peptides, sharing no similarity with the conformations of peptides showing low affinity to the μ-receptor, were selected as two alternative models of probable μ-receptor-bound backbone conformations. Both models share geometrical similarity with the low-energy structures of the linear μ-selective peptide Tyr-d -Ala-Phe-Phe-NH₂. Putative binding conformations of Tyr¹ and Phe³ side chains are also discussed.     

Conformation of sequential peptides containing proline residues

The conformational properties of Boc-Leu₃-(Pro₂GlyLeu₃)_n-Pro₂Gly-OH(n = 0, 1, 3, and 5) and Boc-Leu₃-(Pro₂GlyLeu₃)_n-Pro₂Gly-OBzl (n = 7, 9, and 11) were investigated in various organic solvents of high polarity using molar rotation and CD measurements. The peptides examined are assembled by the N-terminal, internal, and C-terminal segments having the sequences of Boc-Leu₃, Pro₂Gly-Leu₃, and Pro₂Gly-OX (X = H or Bzl), respectively. The conformational studies using the molar rotation of the oligopeptides and polypeptides in MeOH, DMSO, DMF, DMA, and NMP have made it clear that all the peptides have a randomly coiled structure. CD spectral patterns of the peptides in MeOH were essentially the same as each other and indicative of the randomly coiled structure. The additive nature of the total molar rotation and total molar ellipticity in Boc-Leu₃-(Pro₂GlyLeu₃)_n-Pro₂Gly-OX (X = H or Bzl) indicates that the “peptide segment separation” is indeed in operation in organic solvents of high polarity and that the peptide molecule can be considered to be assembled with the small peptide segments having a randomly coiled structure.