Evidence for the bioactive conformation in a cyclic hexapeptide analogue of somatostatin containing a cis-peptide bond mimic

N-methyl- alpha -benzyl-o-aminomethylphenylacetic acid was incorporated into a cyclic somatostatin analogue in order to mimic a cis-peptide bond configuration. The high biological potency of one of the isomers of the cyclic peptide strongly argues in favour of the proposed cis-configuration of the peptide bond at that position in the parent peptide. This represents the first cis-peptide bond mimic which has high biological activity.     

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.     

Conformational mimicry. II. An obligatory cis amide bond in a small linear peptide

The structure of Z-Pro psi [CN4]-Ala-OBzl has been determined by X-ray crystallographic techniques. The structure crystallizes in space group P2(1) with cell constants a = 22.176(3) A, b = 6.141(1)A, c = 8.275(1) A, beta = 98.31(1), and Z = 2. The structure has been refined to a residual of 0.038 for 2538 independent data. The amide bond between the prolyl and alanyl residues is cis, a result of the presence of the tetrazole ring system, as is the urethane bond linking the benzyloxycarbonyl and the prolyl groups. A comparison of the structures in this study to other structures containing cis amide bonds shows that the tetrazole ring system, when incorporated into peptides, mimics a cis amide bond. Changes in the distance between the alpha-carbons adjacent to the tetrazole rings in the linear peptide as compared with the bicyclic diketopiperazine required a reassessment of the conformational mimicry with the cis amide bond.     

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

A cyclic hexapeptide analog of somatostatin, cyclo-(Pro-delta 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 analyses of cyclic hexapeptide analogs of somatostatin containing arylalkyl peptoid and naphthylalanine residues

We report the conformational analysis by ¹H‐NMR in DMSO and computer simulations involving distance geometry and molecular dynamics simulations of peptoid analogs of the cyclic hexapeptide c‐[Phe¹¹‐Pro⁶‐Phe⁷‐d ‐Trp⁸‐Lys⁹‐Thr¹⁰] L‐363,301 (the numbering refers to the positions in native somatostatin). The compounds c‐[Phe¹¹‐Nphe⁶‐Nal⁷‐d ‐Trp⁸‐Lys⁹‐Thr¹⁰] ( Nphe ⁶ ‐ Nal ⁷ analog 1 ), c‐[Nal¹¹‐Nphe⁶‐Phe⁷‐d ‐Trp⁸‐Lys⁹‐Thr¹⁰] ( Nal ¹¹ ‐ Nphe ⁶ analog 2 ) and c‐[Phe¹¹‐Nnal⁶‐Phe⁷‐d ‐Trp⁸‐Lys⁹‐Thr¹⁰] ( Nnal ⁶ analog 3 ), where Nphe denotes N‐benzylglycine and Nnal denotes N‐(1‐naphthylmethyl)glycine, are subjected to SAR studies in order to investigate the influence of the bulky naphthyl aromatic ring on the conformation. The Nal ¹¹ ‐ Nphe ⁶ and Nphe ⁶ ‐Nal ⁷ analogs exhibit potent binding to the hsst2, hsst3 and hsst5 receptors, whereas the Nnal ⁶ analog has decreased binding affinity to all receptors but is more selective towards the hsst2 than the other two analogs and L‐363,301. The conformational search employing distance geometry, energy minimization and molecular dynamic simulations gives insight into the conformational flexibility of these analogs. The molecules adopt both cis and trans orientations of the peptide bond between residues 11 and 6. The cis isomers of these analogs adopt type II′ β‐turns with d ‐Trp in the i+1 position and type VIa β‐turns with the cis peptide bond between residues 6 and 11. The results of free and distance restrained molecular dynamics simulations at 300 K indicate that the Nphe ⁶ ‐Nal ⁷ and Nal ¹¹ ‐Nphe ⁶ compounds adopt a preferred backbone conformation which can be described as ‘folded’ about residues 7 and 10. The Nnal ⁶ analog, which binds less effectively to the hsst receptors, has a more flexible backbone structure than the Nal ¹¹ ‐Nphe ⁶ and Nphe ⁶ ‐Nal ⁷ analogs and prefers a ‘flat’ structure with regard to the orientations about Phe⁷ and Thr¹⁰ during molecular dynamics simulations. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

Backbone modifications in cyclic peptides. Conformational analysis of a cyclic pseudopentapeptide containing a thiomethylene ether amide bond replacement

NMR and X-ray crystallographic studies have shown that cyclic pentapeptides of the general structure cyclo(D-Xxx-Pro-Gly-Pro-Gly) possess beta- and gamma-turn intramolecular hydrogen bonds. As part of our continuing series surveying the compatibility of various amide bond replacements on peptide structure, we have synthesized cyclo(D-Phe-Pro psi[CH2S]Gly-Pro-Gly). The pseudopeptide was prepared by solid phase methods and cleaved from the resin by a new procedure involving phase transfer catalysis using K2CO3 and tetrabutylammonium hydrogen sulfate. Cyclization was carried out with the use of DPPA, HOBt, and DMAP to afford the product in 69% yield. The conformational behavior of the pseudopeptide was analyzed by 1H and 13C (1D and 2D) NMR techniques. The backbone modification replaced the amide bond that is involved in a gamma-turn intramolecular hydrogen bond in the all-amide structure. In CDCl3, the pseudopeptide adopted the same all-trans conformation as its parent, although the remaining beta-turn hydrogen bond was weaker according to delta delta/delta TNH measurements. In DMSO-d6, the all-trans conformer and a second conformer were observed in a ratio of 55:45. These conformers, which slowly interconverted on the NMR time scale, could be separately assigned; peaks due to chemical exchange were readily distinguishable by the ROESY technique as reported earlier by others. 13C and ROESY experiments suggested the minor conformer contained one cis amide bond at the Gly1-Pro2 position. Thus, both the location and type of amide surrogate are important determinants affecting the compatibility of the replacement with a particular conformational feature.     

Synthesis and conformational study of a cyclic hexapeptide analogue of somatostatin: cyclo(Phe-D-Trp-Lys-Thr-o-AMPA)

The active sequence Phe7-D-Trp8-Lys9-Thr10 of somatostatin has been cyclized through o-(aminomethyl)phenylacetic acid, a spacer molecule, designed to mimic a Gly-Gly dipeptide containing a cis-constrained peptide bond. The resulting analogue shows no GH-inhibition. A 2D n.m.r. study reveals conformations different from the proposed bio-active one and still sensitive to the medium (solvent).     

Crystal and molecular structure of cyclo(L-prolyl-glycyl)3. A cyclic hexapeptide with a cis peptide bond

The crystal structure of cyclo(L-Pro-Gly)3 was solved using X-ray crystallographic techniques. The backbone of the peptide is asymmetric and is made up of five trans peptide units and one cis peptide. There is a hydrogen bonded water bridge that links the carbonyl oxygens, O1 and O4. The molecules exist as dimers in the crystal lattice. The two molecules of the dimer are related by crystallographic twofold symmetry and are linked by two N-H ... O hydrogen bonds. The crystals are trigonal, space group P3(2)12 with a = 11.379(3), c = 32.93(1) and z = 6. The structure was solved by multisolution methods and refined by least squares technique to an R of 0.083.     

Inhibition of neurotensin release by a cyclic hexapeptide analog of somatostatin

Studies were performed to determine whether the cyclic hexapeptide analog of somatostatin, cyclo(N-Me-Ala-Tyr-D-Trp-Lys-Val-Phe) II, could alter circulating levels of neurotensin (NT) and inhibit the release of NT from small intestine following the intraluminal perfusion of lipid and ETOH. The small intestine of anesthetized rats was perfused with 0.9% NaCl, 1mM ETOH, 100 mM ETOH or 1 mM oleic acid with and without the intravenous infusion of the somatostatin analog. Plasma samples collected from the superior mesenteric vein were extracted, chromatographed on HPLC and assayed with both C-terminal and N-terminal antisera to NT. The basal circulating levels of chromatographically and immunochemically identified NT observed during the perfusion of the small intestine with 0.9% NaCl were significantly lower (p less than 0.01) during the IV infusion of the somatostatin analog as compared to animals infused IV with saline. The 2-3 fold increase in plasma levels of NT observed with the intestinal perfusion of oleic acid and ETOH did not occur in animals simultaneously infused IV with the somatostatin analog. The somatostatin analog was also effective in decreasing the basal levels of NT metabolite NT(1-8) as well as inhibiting the increase in this metabolite that accompanies the stimulated release of NT.     

Solution conformation of a model hexapeptide containing RGD sequence.

The solution conformation of a model hexapeptide Asp-Arg-Gly-Asp-Ser-Gly (DRGDSG) containing the RGD sequence has been studied in DMSO-d6 as well as in aqueous solution (H2O:D2O/90:10%) by 1H NMR spectroscopy. The unambiguous identification of spin systems of various amino acid residues and sequence specific assignment of all proton resonances was achieved by a combination of two dimensional COSY and NOESY experiments. The temperature coefficient data of the amide proton chemical shifts in conjunction with the vicinal coupling constants, i.e. 3JNH-C alpha H, NOESY and ROESY results indicate that the peptide in both the solvents exists in a blend of conformers with beta-sheet like extended backbone structure and folded conformations. The folded conformers do not appear to be stabilised by intramolecular hydrogen bonding. Our results are consistent with the flexibility of RGD segment observed in the NMR studies on the protein echistatin containing the RGD motif (references 23-25).     

Unusual thionation of a cyclic hexapeptide. Conformational changes and dynamics.

One carbonyl oxygen of the cyclic hexapeptide cyclo(-Gly1-Pro2-Phe3-Val4-Phe5-Phe6-) (A) can be selectively exchanged with sulphur using Yokoyama's reagent. Surprisingly it was not the C=] of Gly1 but that of Phe5 which was substituted and cyclo(-Gly1-Pro2-Phe3-Val4-Phe5 psi [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 beta VIa-turn (minor) and a trans Gly-Pro bond in a beta II'-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 beta I- and beta II-turn about the Val4-Phe5 amide bond in both the cis and trans isomers. Additionally proton relaxation rates in the rotating frame (R1 rho) were measured to verify the assumption of this fast beta I/beta II equilibrium within each isomer. Significant contributions to R1 rho-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.     

Biological activities of cyclic enkephalin pseudopeptides containing thioamides as amide bond replacements

Analogs of H-Tyr-cyclo(N epsilon-D-Lys-Gly-Phe-Leu) have been prepared which contain thioamides at the 3-4 position (monothio), 3-4 and 5-2 positions (dithio), and 2-3, 3-4, and 5-2 positions (trithio). These compounds have been tested for opioid activity in mu- and delta-receptor selective bio- and binding assays. As the number of sulfurs increased, the biological activities dropped on the guinea pig ileum and fluctuated modestly on the mouse vas deferens assay. Surprisingly, the compounds displayed increasing delta selectivity as the number of sulfurs increased. In the binding assay, the thioamide analogs tended to retain affinity toward the mu receptor. The mono- and dithio-analogs were more mu selective than the parent, while the trithio-analog was more delta selective. These results suggest that the subtle exchange of sulfur for oxygen can have a significant impact on receptor selectivity and affinity, and probably reflect the different conformation/structural requirements for binding vs. the biological transduction event.     

Synthesis and hydrolysis by pepsin and trypsin of a cyclic hexapeptide containing lysine and phenylalanine.

1. A cyclic hexapeptide, cyclo(-Gly2-Phe2-Gly-Lys-), and the corresponding open-chain hexapeptides, Gly2-Phe2-Gly-Lys and Phe-Gly-Lys-Gly2-Phe, have been synthesized and their susceptibilities to the hydrolytic action of pepsin and trypsin were determined. 2. The cyclic peptide was hydrolyzed slowly by trypsin to a hexapeptide Gly2-Phe2-Gly-Lys, the value of the Michaelis constant for this reaction being Km equals 0.00022 M. 3. The cyclic peptide was not cleaved by pepsin at all, but Gly2-Phe2-Gly-Lys was hydrolyzed rapidly at a Phe-Phe bond; Km equals 0.0091 M. 4. The cyclic peptide inhibits the hydrolysis of Gly2-Phe2-Gly-Lys by pepsin in a linear non-competitive manner, the value of the inhibition constant being Ki equals 0.004 M.     

Synthesis and conformation of cyclic hexapeptide cyclo(Pro-Sar-Sar)2

A cyclic hexapeptide, cyclo(Pro-Sar-Sar)₂, which consists of N-substituted amino acids only was synthesized, and its solution conformation was investigated by n.m.r. spectroscopy. Seven different C₂-symmetric conformations were detected, which were distinguishable from each other on the n.m.r. time scale. This is due to the cis/trans isomerization of N-substituted peptide bonds. Allowed C₂-symmetric conformations were computed on the basis of a hard-sphere model. Some conformations detected in n.m.r. spectra were not allowed in the calculation. This disagreement suggests that some asymmetric conformations with regard to the single bond rotation are averaged out due to a rapid rotation on the n.m.r. time scale. These points indicate that the molecule of cyclo (Pro-Sar-Sar)₂ is very flexible     

Synthesis of oligonucleotide 2'-conjugates via amide bond formation in solution

An efficient method for synthesis of 2'-O-carboxymethyl oligonucleotides is described. Fully deprotected oligonucleotides containing a carboxymethyl group at the 2'-position of sugar residue were obtained by a two-step procedure by periodate cleavage of an oligonucleotide containing 1,2-diol group followed by oxidation of the 2'-aldehyde resulted with sodium chlorite. 2'-O-Carboxymethyl oligonucleotides prepared were efficiently coupled in aqueous solution in the presence of a water-soluble carbodiimide to a number of amino acid derivatives or short peptides to afford novel 2'-conjugates of high purity in good yield. The method is thus shown to be suitable in principle for preparation of oligonucleotide-peptide conjugates containing an amide linkage between the 2'-carboxy group of a modified oligonucleotide and the amino terminus of a peptide.     

Synthesis and binding potencies of cyclic hexapeptide analogs of somatostatin incorporating acidic and basic peptoid residues.

The synthesis, binding affinity, and structure-activity relationships of compounds related to the cyclic hexapeptide, c[Pro6-Phe7-D-Trp8-Lys9-Thr10-Phe11], L-363,301 (the numbering in the sequence refers to the position of the residue in native somatostatin) is reported. The Pro residue in this compound is replaced with the peptoid residues Nasp [N-(2-carboxyethyl) glycine], Ndab [N-(2-aminoethyl) glycine] and Nlys [N-(4-aminobutyl) glycine]. This series of compounds enables us to draw conclusions about the influence of positively or negatively charged residues in the bridging region on the binding affinity towards the isolated human somatostatin receptors. A loss of binding to the recombinant human somatostatin (hsst) receptors in the Nasp analog compared with L-363,301 and compared with the Ndab and Nlys analogs clearly demonstrates that the presence of an acidic residue in the bridging region is unfavorable for binding to the hsst receptors. Comparison between the Ndab analog and the Nlys analog suggests that the presence of a basic residue in the bridging region might be advantageous for binding to the hsst5 receptor provided that the residue bearing the basic group extends far enough to allow for interaction with the receptor, while the length of the basic peptoid residue does not influence binding to the hsst2 receptor. These results are useful for the design of hsst5 selective somatostatin analogs.     

Stereochemical studies on cyclic peptides. IX. Conformational studies on cyclic tetrapeptides containing alternating cis and trans peptide units

Conformational analyses of cyclic tetrapeptides consisting of alternating cis and trans peptide units have been made using contact criteria and energy calculations. This study has been restricted to those structures having a symmetry element in the backbone ring, such as a twofold axis (d) or a center of inversion (i). There are five main results. (1) There are two distinct types of conformations, which are stereochemically favorable corresponding to each of twofold and inversion-symmetrical structures, designated as d₁, d₂ (for twofold symmetrical) and i₁, i₂ (for inversion-symmetrical). Among these, the i₁ type has the lowest energy when glycyl residues occur at all four α-carbon atoms. (2) With the glycyl residue at all four α-carbon atoms, methyl substitution at the cis peptide nitrogen atoms is possible in all the four types, whereas the substitution at trans peptide nitrogen atoms is possible only for the i₁ type. Thus only in the i₁ type can all the nitrogen atoms be methylated simultaneously. The conformation of the molecule in the crystal structure of cyclotetrasarcosyl belongs to the i₁ type. (3) When alanyl residues occur at all four α-carbon atoms, the possible symmetrical type is dependent on the enantiomorphic form and the actual sequence of the alanyl residues. (4) The methyl substitution at peptide nitrogen atoms for cyclic tetrapeptides having alanyl residues causes more stereochemical restriction in the allowed conformations than with glycyl residues. (5) The prolyl residue can be incorporated favorably at the cis-trans junction of both d and i types of structures. The results of the present study are compared with the data on cyclic tetrapeptides available from the crystal structure and nmr studies. The results show an overall agreement both regarding the type of symmetry and the conformational parameters.     

Peptide conformation. 48. Conformation and biological activity of proline containing cyclic retro-analogues of somatostatin

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 beta-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.     

Synthesis of a cyclic peptide library based on the somatostatin sequence using the backbone amide linker approach

Herein, we report on the synthesis of a library of cyclic peptides targeted at the somatostatin receptor using the backbone amide linker strategy. After optimising head-to-tail cyclisation and cleavage conditions, a library of discrete cyclic peptides was assembled in high purity and good overall yield.