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.     

Solvent effects on the conformation of cyclo(-D-Trp-D-Asp-Pro-D-val-Leu-) An NMR spectroscopy and molecular modeling study

The conformations of cyclo(-D-Trp-D-Asp-Pro-D-val-Leu-) in dimethyl sulfoxide-d₆ (DMSO-d₆) and water were determined using two-dimensional nuclear magnetic resonance spectroscopy and restrained molecular dynamics. Comparisons were made between conformations of the cyclic pentapeptide in both solvents. The NMR study revealed that, while the backbone remained relatively unchanged in both solvents, the side-chains adopted distinctly different orientations in DMSO-d₆ vs. H₂O. A modeling study, minus NOE constraints, produced a set of low-energy conformers possessing agreement in backbone conformation with the NMR-derived structures; however, lowest-energy conformers did not have this agreement. These results show that different solvents can significantly affect the preferred side-chain conformation of small cyclic peptides in solution. This finding will impact the selection of solvent when determining structures for use as templates in rational drug design.     

Conformation and other biophysical properties of cyclic antimicrobial peptides in aqueous solutions

Abstract: As a step towards understanding the mechanism of the biological activity of cyclic antimicrobial peptides, the biophysical properties and conformations of four membrane‐active cyclic peptide antibiotics, based on gramicidin S (GS), were examined in aqueous environments. These cyclic peptides, GS10 [cyclo(VKLdY P)₂], GS12 [cyclo(VKLKdY PKVKLdY P)], GS14 [cyclo(VKLKVdY PLKVKLdY P)] and [d ‐Lys]⁴GS14 [cyclo(VKLdK VdY PLKVKLdY P)] (d ‐amino acid residues are denoted by d and are underlined) had different ring sizes of 10, 12 and 14 residues, were different in structure and amphipathicity, and covered a broad spectrum of hemolytic and antimicrobial activities. GS10, GS12 and [d ‐Lys]⁴GS14 were shown to be monomeric in buffer systems with ionic strength biological environments. GS14 was also monomeric at low concentrations, but aggregated at concentrations > 50 µm . The affinity of peptides for self‐assembly and interaction with hydrophobic surfaces was related to their free energy of intermolecular interaction. The effects of variations in salt and organic solvent (trifluoroethanol) concentration and temperature on peptide conformation were also examined. Similar to GS, GS10 proved to have a stable and rather rigid conformation in different environments and over a broad range of temperatures, whereas GS12, GS14 and [d ‐Lys]⁴GS14 had more flexible conformations. Despite its conformational similarity to GS10, GS14 had unique physicochemical properties due to its tendency to aggregate at relatively low concentrations. The biophysical data explain the direct relation between structure, amphipathicity and hydrophobicity of the cyclic peptides and their hemolytic activity. However, this relation with the antimicrobial activity of the peptides is of a more complex nature due to the diversity in membrane structures of microorganisms.     

Conformation of cyclosporin A in polar solvents

A major conformation of cyclosporin A in methanol and in aqueous methanol was revealed by some simple NMR experiments. Thus, a stepwise transition of cyclosporin A conformation from 100% CDCl₃ to 100% CD₃OD was followed by ¹H NMR, which showed that the chloroform conformation of cyclosporin A was still the major one in methanol. Employing the same technique, it was also shown that the chloroform conformation of cyclosporin A was one of the major conformations in 50% aqueous methanol. This may be the first experimental determination of a major conformation of cyclosporin A in polar solvents.     

Comparing conformations at low temperature and at high viscosity

The influence of low temperature and high viscosity on the conformation of somatostatin and two of its analogues was investigated by ¹H NMR in solution. The conformation of native somatostatin, a cyclic octapeptide agonist DC13-116 and a linear octapeptide agonist were compared in ethylene glycol at 303 K and in methanol at low temperature. The first goal of this study was to investigate if either low temperature or high viscosity is the more important for the reduction of the conformational freedom. Secondly we wanted to compare the amount of information concerning the conformation present in both solvents. A larger amount of NOESY cross-peaks is observed in ethylene glycol at room temperature compared to methanol at low temperature. This indicates that the raising of the viscosity is a more important factor in reducing the flexibility of peptides than the lowering of the temperature.     

Structural analysis of fertilinβ cyclic peptide mimics that are ligands for α6β1 integrin

Abstract: The NMR structural analysis of two fertilinβ mimics cyclo(EC²DC¹)YNH_2, 1 , and cyclo(D²EC²D¹C¹)YNH_2, 2 is described. Both of these mimics are moderate inhibitors of sperm?egg binding with IC₅₀ values of 500 µm in a mouse in vitro fertilization assay. For peptide 1 , the optimized conformations that best match the NMR data have a pseudo‐type II′β‐turn with the linker and Glu at the i+1 and i+2 positions, respectively. The EC²D¹C¹ sequence is in a nonclassical (type IV) β‐turn. For peptide 2 , the conformation that best matches the NMR data has two turns: a pseudo‐type II′β‐turn in the D²EC²D¹ sequence followed by a nonclassical β‐turn in the EC²D¹C¹ sequence. The Cβ?Cβ distance between E and D¹ in peptide 1 is 9.1 Å, in peptide 2 , it is 7.7 Å. Thus, one possibility for the high IC₅₀ values of these cyclic peptides is that the acidic residues are not constrained to a sufficiently tight turn, and thus much entropy must still be lost upon binding to the α₆β₁ integrin. This explains why the cyclic peptides are the same as linear peptides at inhibiting sperm?egg binding.     

Design,synthesis, and testing of potential antisickling agents. 9. Cyclic tetrapeptide homologs as mimics of the mutation site of hemoglobin S

As part of our continuing search for new agents which might be useful for the treatment of sickle-cell anemia, we have synthesized two cyclic tetrapeptide homologs, cyclo(-Val-Glu[-Thr-Pro-]-OH) (1a) and cyclo(-Phe-Glu[-Thr-Pro-]-OH (1b), and a tetrapeptide lactone homolog cyclo(H-T-hr-Pro-Val-Glu-OH) (2). The intent was that these peptides would mimic a tetrapeptide region around the mutation site of HbS and thus be able to bind at the acceptor site of HbS and thereby inhibit polymerization. The synthesis of the linear peptides was accomplished in solution using both the polymeric reagent (PHBT) and DCC/HOBT methods; cyclization was accomplished by an improved method. ¹³C-n.m.r. studies were performed which allowed us to assign the conformation about the Thr-Pro bond in 1a and 2 as trans. The cyclic peptides were tested for their ability to increase the solubility of HbS under deoxygenating conditions, but only 1a had any antigelling activity, albeit low.     

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.     

Conformational energy analysis of the chemotactic tripeptide formyl-Met-Leu-Phe and three analogs

Conformational energy analyses were carried out on the chemotactic tripeptide fMLF (CHO-Met-Leu-Phe) and three analogs fALF (CHO-Ala-Leu-Phe), fMF (CHO-Met-Phe), and MLF (H-Met-Leu-Phe). A near-folded or puckered conformation predominates in all four peptides. The calculated average end-to-end distance R of each of the peptides is 7.4 Å 7.6 Å, 7.0 Å, and 7.3 Å, respectively (where bends have R ≤ 7 Å and extended structures have R ? 10.5 Å). The puckered conformation calculated for fMLF is similar to that determined experimentally for fMLF in nonpolar solvents and in the protein receptor. The results suggest that maximum chemotactic activity of the peptides depends on a combination of the chemical structure (the presence of N-formyl-methionine) and backbone conformation (C, conformation of the first amino acid residue).     

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.     

Survey of conformational role of ester bonds in a cyclic depsipeptide

The effect of ester bond on the conformation of peptide molecule was studied by designing and synthesizing a model tetradepsipeptide cyclo(-l -Ala-l -Hmb-)z and by analyzing the conformation both theoretically and experimentally. Theoretical analysis showed that both ester and peptide bonds in the calculated low-energy conformations within 3 kcal/mol of the global minimum take a trans but distorted configuration. The distortion is larger in ester bonds than in peptide bonds. Further, the four carbonyls project from one side of the plane of the cyclic backbone, whereas the side chains project from the other side. These results are consistent with the experimental results obtained by NMR measurement; first, the coupling constant deduced from ¹H-NMR species in DMSO-d₆ is consistent with the dihedral angles of the calculated low-energy conformations; second, results of NOE measurement can reproduce the calculated configuration of the carbonyls and side chains. From the consistency between theoretical and experimental results, it is concluded that this model tetradepsipeptide takes an all-trans backbone conformation in solution and this backbone conformation is stabilized by large distortion in the ester bond, which compensates the strain resulted from the 12-membered cyclic backbone structure consisting only of L-residues.     

Stereochemical analysis of the antigenic tip of the V3 loop peptide of HIV-1 gp120

A novel multiple turn conformation has been observed for a segment GPGRAFY in the crystal structure of a complex of HIV-1 gp120 V3 loop peptide with the Fab fragment of a neutralizing antibody [Ghiara et al. (1994) Science 264 , 82–85]. A structural motif has been defined for the peptide segment, employing idealized backbone conformations characterized by ranges of virtual C^α torsion angles and bond angles. A search of 122 high-resolution protein crystal structures has permitted identification of 24 examples of similar structural motifs. Two major conformational families have been identified, which differ primarily in the conformation at residue 3. The observed conformation at residue 3 in family 1 is left-handed helical (α_L) and that in family 2 is right-handed helical (α_R). Of the 10 examples in family 1, 9 examples have Gly residues at position 3. Of the 12 examples in family 2, 7 examples have Asn/Asp at position 3. Computer modeling of the V3 loop tip sequence using the two backbone conformational families as starting points leads to minimum-energy conformations in which antigenically important side-chains occupy similar spatial arrangements. This stereo-chemical analysis of the V3 loop tip sequence suggests a rational basis for the design of synthetic analog peptides for use as viral antagonists or synthetic antigens. © Munksgaard 1995.     

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.     

Solution conformations of deltorphin-I obtained from combined use of quantitative 2D-NMR and energy calculations: A comparison with dermenkephalin

Deltorphin-I, Tyr-d -Ala-Phe-Asp-Val-Val-Gly-NH₂ and dermenkephalin, Tyr-d -Met-Phe-His-Leu-Met-Asp-NH₂, two highly related opioid peptides from frog skin, display very similar N-termini but strikingly different C-terminal tails. Nevertheless, both peptides are highly potent at, and exquisitely selective for the δ-opioid receptor. To identify common determinants concuring to the remarkably efficient targeting of deltorphin-I and dermenkephalin, combined use of quantitative two-dimensional nuclear magnetic resonance (53 dipolar interactions studied at four temperatures) and energy calculations using simulated annealing generated five groups of deltorphin-I conformers. These groups were pooled into two families whose overall conformation could be described either by a left-handed helix (Family I) or by a big loop (Family II), both stabilized by H-bonds. Proximity of D-Ala²-Phe³-Asp⁴ and Val⁵-Val⁶-Gly⁷ triads is an obvious structural similarity between almost all groups in both families of structures. Whereas differences between the two families originated mostly from a transition at Ψ Asp⁴ backbone dihedral angle, the backbone structures at segment 1–4 are similar and spatial arrangements of Tyr¹ (t) and Phe³ (g^–) are identical in one group of each family. Moreover, these two groups have a N-terminal tetrapeptide whose conformation most closely resembles that of a well-defined group of structures for dermenkephalin. Altogether, these results suggest that conformational attributes that are common to dermenkephalin and deltorphin-I, i.e., the backbone conformation of the N-terminal tetrapeptide and preferential orientations in the side-chain of Tyr¹ (t) and Phe3 (g^–) underlie their ability to bind with high selectivity to the δ-opioid receptor.     

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 contaIntng cisl -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], cooperatively bound the cations.     

Cyclo(l-propyl-l-N-methylphenylalanyl) Conformation in solution and in the crystal

Detailed analysis of the proton and carbon-13 NMR spectra of cyclo(l -prolyl-l -N-methylphenylalanyl) in chloroform and methanol in relation to its nonmethylated analog provided information on the conformation of the title compound in solution as well as on the effect of N-methylation and solvation. The N-ray structure of the title compound in the crystalline state showed the same conformational features as the solution structure. The phenyl group folds over the diketopiperazine ring which resembles a flattened half-chair. Both amide bonds are considerably nonplanar. The pyrrolidine ring of proline shows a strong pucker at the ring junction with the largest χ5 value hitherto observed.     

N-acyl-diketopiperazines.

N-(N-phenylacetyl-L-alanyl)-cyclo-(L-phenylalanyl-D-prolyl) (I) was synthesized in one step starting from the linear precursor phenylacetyl-L-alanyl-L-phenyl-alanyl-L-proline. X-ray crystallographic analysis of (I) shows that the diketopiperazine ring adopts a boat conformation appreciably more puckered than that found in the unacylated cyclo(L-Pro-D-Phe). The side chain of Phe residue is in quasi-axial flagpole orientation with the aromatic ring folded over the diketopiperazine ring. ¹H-n.m.r. data indicate that the same conformation is preferred in chloroform solution. The proline ring assumes a β-envelope conformation. No intramolecular interactions between the diketopiperazine system and the aromatic ring of the N-phenylacetyl-alanyl side chain have been evidenced. Crystals: space group P2₁ with a= 9.956(3), b= 8.809(2), c= 13.615(2) Å, β= 111.0(1)° and Z = 2. The final R and R_w are 0.037 and 0.052, respectively.     

Crystal structure and conformation of glycyl-glycyl-sarcosine

Crystals of the tripeptide, glycyl-glycyl-sarcosine (C₇H₁₃N₃O₄) from aqueous methanol are orthorhombic, space group Pbcn with cell parameters at 294 K of a = 8.279(1), b = 9.229(4), c = 24.447(5) Å, V = 1868.0 ų, M.W. = 203.2, and Z = 8. The crystal structure was solved and refined using CAD-4 data (1171 reflections ≥ 3σ) to a final R-value of 0.053. The first peptide linkage is trans and planar whereas the second peptide link between Gly and sarcosine is cis and appreciably non-planar (w = 7.4°). The peptide backbone has an extended conformation at the N-terminal part but adopts a polyglycine-II type of conformation at the C-terminal part. The backbone torsion angles are: Ψ₁, =? 173.9, w₁=? 177.8, (φ, Ψ₂) = (-178.8, -170.8), w₂= 7.4, (φ₃, Ψ₃) = (-81.6, 165.6°).     

Conformational studies of cyclic tetrapeptides

The conformations of chlamydocin and cyclo (Ala-Aib-Phe-D-Pro)(Ala⁴-chlamy-docin) in chloroform have been investigated by nuclear magnetic resonance, infrared and circular dichroism spectroscopy. The data obtained from these experiments establish an all transoid, bis γ-turn conformation for both compounds in chloroform with the following torsional angles (± 20d?): Ala⁴4-chlamydocin: Aib, Ø+ 60d?, Ψ– 50d?; ω+ 160d?; Phe Ø– 120d?, Ψ+ 120d?, ω– 160d?; D-Pro Ø+ 60d?, Ø– 55d?, ω+ 160d?; Ala Ø– 110d?, Ψ+ 110d?, ω– 160d?. Chlamydocin adopts a closely related conformation in neat chloroform. Nuclear Overhauser Effect (NOE) data are utilized to assign amide bond geometries in the cyclic tetrapeptide ring system.     

Role of azaamino acid residue in β‐turn formation and stability in designed peptide

Abstract: The structural perturbation induced by C^αH→N^α exchange in azaamino acid‐containing peptides was predicted by ab initio calculation of the 6‐31G* and 3‐21G* levels. The global energy‐minimum conformations for model compounds, For‐azaXaa‐NH₂ (Xaa = Gly, Ala, Leu) appeared to be the β‐turn motif with a dihedral angle of φ = ± 90°, ψ = 0°. This suggests that incorporation of the azaXaa residue into the i + 2 position of designed peptides could stabilize the β‐turn structure. The model azaLeu‐containing peptide, Boc‐Phe‐azaLeu‐Ala‐OMe, which is predicted to adopt a β‐turn conformation was designed and synthesized in order to experimentally elucidate the role of the azaamino acid residue. Its structural preference in organic solvents was investigated using ¹H NMR, molecular modelling and IR spectroscopy. The temperature coefficients of amide protons, the characteristic NOE patterns, the restrained molecular dynamics simulation and IR spectroscopy defined the dihedral angles [ (φ_i+1, ψ_i+1) (φ_i+2, ψ_i+2)] of the Phe‐azaLeu fragment in the model peptide, Boc‐Phe‐azaLeu‐Ala‐OMe, as [(?59^°, 127^°) (107^°, ?4^°)]. This solution conformation supports a βII‐turn structural preference in azaLeu‐containing peptides as predicted by the quantum chemical calculation. Therefore, intercalation of the azaamino acid residue into the i + 2 position in synthetic peptides is expected to provide a stable β‐turn formation, and this could be utilized in the design of new peptidomimetics adopting a β‐turn scaffold.