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.     

Comparative conformational analysis and in vitro pharmacological evaluation of three cyclic hexapeptide NK-2 antagonists

The conformational analysis of three cyclic hexapeptides is presented. Cyclo-(-Gln⁶-Trp⁷-Phe⁸-Gly⁹-Leu¹⁰-d -Met¹¹-) (1) and cyclo-(-Gln⁶-Trp⁷-Phe⁸-Gly⁹-Leu¹⁰-Met¹¹-) (2) are NK-2 antagonists in the hamster trachea assay, whereas cyclo-(-Gln⁶-Trp⁷-Phe⁸-(R)-Gly⁹-[ANC-2]Leu¹⁰-Met¹¹-) (3), where Gly⁹[ANC-2]Leu¹⁰ represents (2S)-2-((3R)-3-amino-2-oxo-1-pyrrolidinyl)-4-methylpentanoyl, is inactive as agonist and antagonist in this assay. In DMSO, the NMR results cannot be interpreted as being consistent with a single conformation. However, the combined interpretation of results from NMR spectroscopy, restrained molecular dynamics simulations with application of proton–proton distance information from ROESY spectra, and pharmacological results leads to a reduced number of conformational domains for each peptide, which can be compared with each other and may be classified as responsible for their biological activity. Trying to match the conformational domains approximately with regular β- and γ-turns, we find a γⁿ-turn at the position of the methionine occuring in all peptides. For the active peptides 1 and 2 we arrive at an inverse γⁱ-turn at Phe⁸, and βI′- or βII-turns with Gly⁹ and Leu¹⁰ at the corner positions, these β-turns having a similar topology with respect to the linking peptide unit. Other conformational domains common to only 1 and 2 support their classification as responsible for the biological activity.     

The effect of conformation on the solution stability of linear vs. cyclic RGD peptides

Abstract: The objective of this study was to evaluate the relationship between conformational flexibility and solution stability of a linear RGD peptide (Arg-Gly-Asp-Phe-OH; 1 ) and a cyclic RGD peptide (cyclo-(1, 6)-Ac-Cys-Arg-Gly-Asp-Phe-Pen-NH₂; 2 ); as a function of pH. Previously, it was found that cyclic peptide 2 was 30-fold more stable than linear peptide 1 . Therefore, this study was performed to explain the increase in chemical stability based on the preferred conformation of the peptides. Molecular dynamics simulations and energy minimizations were conducted to evaluate the backbone flexibility of both peptides under simulated pH conditions of 3, 7 and 10 in the presence of water. The reactive sites for degradation for both molecules were also followed during the simulations. The backbone of linear peptide 1 exhibited more flexibility than that of cyclic peptide 2 , which was reflected in the rotation about the phi and psi dihedral angles. This was further supported by the low r.m.s. deviations of the backbone atoms for peptide 2 compared with those of peptide 1 that were observed among structures sampled during the molecular dynamics simulations. The presence of a salt bridge between the side chain groups of the Arg and Asp residues was also indicated for the cyclic peptide under simulated conditions of neutral pH. The increase in stability of the cyclic peptide 2 compared with the linear peptide 1 , especially at neutral pH, is due to decreased structural flexibility imposed by the ring, as well as salt bridge formation between the side chains of the Arg and Asp residues in cyclic peptide 2 . This rigidity would prevent the Asp side chain carboxylic acid from orienting itself in the appropriate position for attack on the peptide backbone.     

Structure-activity relationship of the ring portion in backbone-cyclic C-terminal hexapeptide analogs of substance P. NMR and molecular dynamics

The conformations of two backbone-cyclized substance P analogs were derived from homo- and heteronuclear NMR measurements and molecular dynamics simulations carried out in DMSO. The analogs contain subtle variations in the ring chemistry and are compared with biologically active analogs previously examined. The correlation between conformation and activity is used to gain insight into the conformational requirements from the pharmacophore.     

Synthesis of a new dipeptide template,its X-ray structure,and modeling studies on its conformational features

The diastereoselective synthesis is reported of a dipeptide template which is closely related to H-Gly-Trp-OH. Intramolecular bond formation between α-C of Gly and ring position 2 of the Trp unit has been achieved by a Pictet-Spengler-type electrophilic aromatic substitution. The absolute configuration of the N-Moc protected dipeptide template 9·2H₂O was determined by single-crystal X-ray crystallography and found to be (2S,5S). The cis orientation of the amino and carboxy termini prompted us to investigate the potential of 9 as a β-turn mimic. MD calculations on the model pseudopeptide Ac-Ala-Gly-Trp-Ala-NHMe 11 suggest that an unusually tight turn should be favoured rather than a β-turn. The proper protective situation as a prerequisite for the incorporation of the template into a peptide has been established, and comments about its chemical properties are given. © Munksgaard 1995.     

环状氨基酸的合成及其应用

环状氨基酸是一种构型限制型氨基酸,因为它们的构象限制性特点有利于肽的生物活性构象的形成和稳定,在修饰多肽时引起的构象效应和生物学效应有着重要的理论和应用价值,常用于修饰生物活性肽,以改善其药学性质。研究表明,环状氨基酸的引入是一种改善多肽性质的有效方法之一。本文就环状氨基酸的结构特点、合成,尤其是多肽修饰方面进行了综述。     

Synthesis,biological activity and conformational analysis of cyclic GRF analogs

A novel cyclic GRF analog, cyclo(Asp⁸-Lys¹²)-[Asp⁸,Ala¹⁵]-GRF(1-29)-NH₂, i.e. cyclo^8.12[Asp⁸,Ala¹⁵]-GRF(1-29)-NH₂, was synthesized by the solid phase procedure and found to retain significant biological activity. Solid phase cyclization of Asp⁸ to Lys¹² proceeded rapidly (～2h) using the BOP reagent. Substitution of Ala¹² with d -Ala² and/or NH₂-terminal replacement (desNH₂-Tyr¹ or N-MeTyr¹) in the cyclo^8.12[Asp⁸,Ala¹⁵]-GRF(1-29)-NH₂ system resulted in highly potent analogs that were also active in vivo. Conformational analysis (circular dichroism and molecular dynamics calculations based on NOE-derived distance constraints) demonstrated that cyclo^8.12[Asp⁸,Ala¹⁵]-GRF(1-29)-NH₂ contains a long α-helical segment even in aqueous solution. A series of cyclo^8.12 stereoisomers containing d -Asp⁸ and/or d -Lys¹² were prepared and also found to be highly potent and to retain significant α-helical conformation. The high biological activity of cyclo^8.12[N-MeTyr¹,d -Ala²,Asp⁸,Ala¹⁵]-GRF(1-29)-NH₂ may be explained on the basis of retention of a preferred bioactive conformation.     

Synthesis and conformational study in solution and in solid state of oligopeptides containing l-leucine and glycine

The synthesis and conformational studies of the oligopeptide N-tert.-butyloxycarbonyl-l -Leu-(l -Leu-Gly)_n-OBzl (n = 1, 3, 5) and N-tert.-butyloxycarbonyl-(l -Leu-Gly)₂-OBzl are described. The peptides were synthesized by stepwise and fragment condensation techniques using dicyclohexylcarbodiimide as the condensing agent in solution. The conformational study of the oligopeptides was carried out using CD, u.v. and i.r. spectra. The conformation in solution was examined in trifluoroethanol, hexafluoroisopropanol. hexafluoroacetone trihydrate, and methanol. CD spectra in trifluoroethanol exhibited a gradual variation with increasing peptide chain length. This can be interpreted as a formation of an ordered structure which is already present in the heptapeptide and, to a greater extent, in the undecapeptide. The results obtained from the CD profiles and i.r. spectra showed the presence of β structure with antiparallel chains in the heptapeptide and undecapeptide. Finally, CD spectra revealed in trifluoroethanol-water solution the binding of Ca²⁺ to heptapeptide and undecapeptide together with a contemporaneous conformational change. This change is probably due to the formation of β-turns. No change in the CD profiles was obtained by using Mg²⁺, K⁺, Na⁺, and Li⁺ ions instead of Ca²⁺.     

Molecular dynamics study of disulfide bond influence on properties of an RGD peptide

Three 1 ns length molecular dynamics simulations of an RGD peptide (Ac-Pen-Arg-Gly-Asp-Cys-NH₂, with Pen denoting penicillamine) have been performed in aqueous solution, one for the disulfide bridged, and two for the unbridged form. The trajectories were analyzed to identify conformations explored by the two forms and to calculate several properties: NMR vicinal coupling constants, order parameters, dipole moments and diffusion coefficients, in an effort to describe the physical role of the disulfide bond. The cyclic peptide was able to explore several distinct backbone conformations centered around a turn–extended-turn structure. However, its flexibility was limited and it appeared to be ‘locked in’ into a a family of structures characterized by a high dipole moment and a well-defined conformation of the pharmacophore, which has been previously identified as biologically active. Excellent agreement between the simulated and observed NMR vicinal coupling constants indicates that realistic structures were sampled in the cyclic peptide simulation. The linear form of the peptide was much more flexible than the cyclic one. In the two independent 1 ns simulations of the linear form the explored conformations could be roughly grouped into two classes, of cyclic-like and extended type. Within each simulation the peptide switched between the two classes of structures several times. Exact matches between conformations in the two linear peptide simulations were not found; several conformational regions with backbone rms deviations below 1 Å were identified, suggesting that representative structures of the linear form have also been identified. In the linear peptide simulations the RGD pharmacophore is able to adopt a wide range of conformations, including the one preferred by the cyclic form. The lower biological activity of the linear peptide compared to the cyclic one may be correlated with the lower population of this structure in the absence of the disulfide bond.     

Synthesis and conformational analysis of cyclic pentapeptide endothelin antagonists

Two endothelin antagonists cyclo(d -Leu-d -Val-Pro-d -Asp-Trp) (IPI-147), and cyclo(d -Trp-d -Asp-Ac₃c-d -Val-Leu) (IPI-725) have been synthesized. Their solution conformations have been studied in aqueous solution by NMR spectroscopy and dynamics simulation. Activity studies show that IPI-725 is a strong ET_A antagonist, while IPI-147 is a weak ET_A antagonist. Comparison of the solution conformations of these two ET_A antagonists suggests that the difference in their activities results from their structural differences. IPI-147 contains a type II β-turn with a hydrogen bond between NH of d -Val and the C = O of d -Asp. IPI-725, on the other hand, contains two turns, a type II β-turn with a hydrogen bond between NH of d -Asp and C=O of d -Val, as well as a γ-turn with a hydrogen bond formed between d -Val NH and d -Asp carbonyl group. Therefore IPI-147 appears to be more flexible than IPI-725. Although both γ-turns contain the same residues, their orders in the turn are reversed. The β-turn in IPI-725 is formed with d -Val:Leu:d -Trp:d -Asp, while in IPI-147, the β-turn is formed with d -Asp:Trp:d -Leu:d -val. The activities and solution conformations of IPI-147 and IPI-725 were also compared with BQ-123 [Cyclo(d -Trp-d -Asp-Pro-d -Val-Leu)], a well characterized, highly potent endothelin antagonist. © Munksgaard 1996.     

Molecular dynamics study of disulfide bond influence on properties of an RGD peptide. J. Peptide Res., 1999, 53, 188–200.

We regret that equations 1–5 on pages 191 and 192 of the above article were printed incorrectly. The correct equations and accompanying text are displayed below. We apologize for any confusion caused.     

Conformations and conformational interconversions of diastereomeric cyclic tetraprolines

Cyclic tetrapeptides exclusively composed of L- and D-Pro have been studied by theoretical means (conformational searches and molecular mechanics calculations using the CHARMM program) supported by ¹H-NMR spectroscopy, X-ray analysis and chiroptical measurements. We explored the entire conformational space of the diastereomers cyclo(LLLL-Pro₄) (I), cyc1O(LDLD-Pro₄) (II) and CYClo(LLDD-Pro₄) (III) including the low-energy conformations and the related interconversion paths. The conformational interconversions were found to be restricted to cis/trans isomerisations of the amide bonds. Owing to the polycyclic nature of cyclo(Pro₄) most of the cis/trans transitions are hindered by energy barriers higher than 30 kcal/mol (up to 150–200 kcal/mol). A few transitions are characterized by computed energy barriers comparable to those found in linear -Xxx-Pro- sequences (～ 18 kcal/mol), and are therefore experimentally significant. Experimental evidence has been obtained in the case Of CyClo(LDLD-Pro₄), where two enantiomers are interconverted by a series of 4 cis/trans isomerisations ctct→cttt→tttt→tctt→tctc. The Eyring activation parameters of this reaction were determined in H₂O and in DMF by chiroptical measurements (ΔH^#= 44 and 28 kcal/mol; ΔS^#=59 and 22 cal K ^?1 mol^?1, respectively), and correlated with the calculated barriers. In I and III comparable series of four cis/trans isomerisations relate two main conformations with the peptide bond sequences ctct and tctc. In compound I pseudorotational images are interconverted via ctct→ccct→cctt→cctc→tctc. The pathway ctct→ccct→cctt→cctc→tctc. that relates diastereomeric main conformations of III involves exclusively low-energy intermediates; however, the transitions leading to the all-cis conformation are energetically unfavourable, and the conformational space is divided in three insulated domains.     

Reversible scaling of dihedral angle barriers during molecular dynamics to improve structure prediction of cyclic peptides

Abstract: Peptide cyclization or chemical cross‐linking has frequently been used to restrict the conformational freedom of a peptide, for example, to enhance its capacity for selective binding to a target receptor molecule. Structure prediction of cyclic peptides is important to evaluate possible conformations prior to synthesis. Because of the conformational constraints imposed by cyclization low energy conformations of cyclic peptides can be separated by large energy barriers. In order to improve the conformational search properties of molecular dynamics (MD) simulations a potential scaling method has been designed. The approach consists of several consecutive MD simulations with a specific lowering of dihedral energy barriers and reduced nonbonded interactions between atoms separated by three atoms followed by gradually scaling the potential until the original barriers are reached. Application to four cyclic penta‐ and hexa‐peptide test cases and a protein loop of known structure indicates that the potential scaling method is more efficient and faster in locating low energy conformations than standard MD simulations. Combined with a generalized Born implicit solvation model the low energy cyclic peptide conformations and the loop structure are in good agreement with experiment. Applications in the presence of explicit water molecules during the simulations showed also improved convergence to structures close to experiment compared with regular MD.     

Synthesis and biological activity of a cyclic hexapeptide related to peptide T

The cyclo [Thr-Thr-Thr-Tyr-Asn-Thr] hexapeptide related to peptide T, H-Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr-OH, competitor of the Human Immunodeficiency Virus in the binding to human T cells, was synthesized and tested for its ability to stimulate monocyte migration (chemotaxis). The new cyclic derivative showed negligible biological activity.     

Inhibition of homotypic adhesion of T-cells: secondary structure of an ICAM-1-derived cyclic peptide

The objective of this study was to elucidate the solution conformation of cyclo-(1, 12) Pen1-Pro2-Ser3-Lys4-Val5-Ile6-Leu7-Pro8-Arg9-Gly10-Gly11-Cys12 (1) derived from the intercellular adhesion molecule-1 (ICAM-1). Cyclic peptide 1 inhibits homotypic adhesion of T-cells (Molt-3) mediated by ICAM-1 and the leukocyte function-associated antigen-1 (LFA-1) on the surface of T-cells. Cyclic peptide 1 is more potent than is the linear peptide Pen1-Pro2-Ser3-Lys4-Val5-Ile6-Leu7-Pro8-Arg9-Gly10-Gly11-Cys12 (2) in inhibiting homotypic adhesion. The difference in biological activity of peptides 1 and 2 may be due to the more stable conformation of cyclic peptide 1 compared to linear peptide 2 or because cyclization prevents the peptide from adopting non-productive conformation. Therefore, conformational studies of cyclic peptide 1 will give a better understanding of its biological active conformation. The conformational studies of cyclic peptide 1 were done by NMR, CD and molecular dynamics simulations. NMR studies indicated that the major conformation of cyclic peptide 1 contained trans-configuration at both X-Pro peptide bonds. Type I β-turns at Lys4-Val5-Ile6-Leu7 and Leu7-Pro8-Arg9-Gly10 were found in cyclic peptide 1. The C- and N-terminal regions of this peptide were stabilized by antiparallel β-sheet-like structure with the presence of intramolecular hydrogen bonds. The overall structure of this peptide exposed the hydrophobic side chains on one face of the molecule and the hydrophilic side chains on the other.     

Synthesis of cyclic dipeptide templates,their incorporation into peptides and studies on their conformational and biological properties

This study investigated the diastereoselective synthesis of three dipeptide templates 1, 2 and 3, which may be regarded as conformationally restricted analogs of H-Gly-Xaa-OH, in which Xaa constitutes an aromatic amino acid. Bond formation between α-C of Gly and the aromatic moiety was achieved by proton-catalyzed intramolecular electrophilic aromatic substitution. The absolute configuration of the dipeptide templates was determined by single-crystal X-ray crystallography or by nuclear Overhauser enhancement measurements. A protective group strategy was elaborated to allow their incorporation into peptide sequences by liquid phase as well as by solid-phase peptide synthesis. The templates were used to generate an enkephalin analog 15, a modified peptidic neurokinin antagonist 20 and two dermorphin derivatives (24 and 33). Molecular dynamic simulations with 15 and 20 revealed the preference for a turn-like motif for 15. The biological activity, as investigated by respective receptor binding and functional assays, was strongly diminished with all four derivatives, indicating that their receptor-relevant molecular geometries lie outside the examined conformational space. © Munksgaard 1998.     

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.     

Synthesis and conformational study of a cyclic hexapeptide analog of somatostatin containing dehydrophenylalanine

A cyclic hexapeptide analog of somatostatin, cyclo-(Pro-Δ^z-Phe-d -Trp-Lys-Thr-Phe) ( II )? has been synthesized by a combination of solid phase and solution methodology. It shows a potency for inhibition of growth hormone release in vitro about one-tenth that of the corresponding saturated analog, cyclo-(Pro-Phe-d -Trp-Lys-Thr-Phe) ( I ). N.m.r. studies indicate comparable backbone conformations for analogs I and II . However, the sum of our findings from biological evaluation and solution physical data suggest that on the receptor the position-7 phenyl ring of I is adopting a conformation which differs from that of one of the major solution conformers defined previously by n.m.r. studies.     

Conformational modifications of cyclic hexapeptide somatostatin analogs

A model for the bioactive conformation of the highly active cyclic hexapeptide somatostatin analog cyclo-(Pro-Phe-d -Trp-Lys-Thr-Phe) has been proposed. As a test of this model, several compounds containing lactam and N-Me amino acid conformational modifications in the Thr-Phe-Pro-Phe beta turn were synthesized. The N-Me alanine and sarcosine substitutions for proline gave highly active analogs, while lactam dipeptides in place of Phe-Pro decreased potency. ¹H n.m.r. and CD spectra of these analogs illustrate the conformational effects in solution of these modifications. The results provide additional support for the proposed conformational model.