Cyclic enkephalin and dermorphin analogues containing a carbonyl bridge.

Four cyclic enkephalin analogues and four cyclic dermorphin analogues have been synthesized. Cyclization of linear peptides containing basic amino acid residues of various side chain length in position 2 and 5 (enkephalin analogues) or 2 and 4 (dermorphin analogues) was achieved by treatment with bis-(4-nitrophenyl) carbonate to form a urea unit. The peptides were tested in the guinea-pig ileum (GPI) and mouse vas deferens (MVD) assays. Diverse activity was observed, depending on the size of the ring and the location of the urea unit. The conformation of two dermorphin analogues has been studied: one of high activity (IC(50) = 4.15 nM in the GPI assay) and a second of low activity (IC(50) = 6700 nM in the GPI assay). The conformational space of these peptides was examined using the EDMC method. Using data from the NMR spectra, each peptide was described as an ensemble of conformers. Biological activity was discussed in light of the structural data.     

Highly potent side-chain to side-chain cyclized enkephalin analogues containing a carbonyl bridge: synthesis, biology and conformation.

Six novel cyclic enkephalin analogues have been synthesized. Cyclization of the linear peptides containing basic amino acid residues in position 2 and 5 was achieved by treatment with bis(4-nitrophenyl)carbonate. It was found that some of the compounds exibit unusually high mu-opioid activity in the guinea pig ileum (GPI) assay. The 18-membered analogue cyclo(N(epsilon),N(beta)-carbonyl-D-Lys2,Dap5)-enkephalinamide turned out to be one of the most potent mu-agonists reported so far. NMR spectra of the peptides were recorded and structural parameters were determined. The conformational space was exhaustively examined for each of them using the electrostatically driven Monte Carlo method. Each peptide was finally described as an ensemble of conformations. A model of the bioactive conformation of this class of opioid peptides was proposed.     

Synthesis, conformational and pharmacological studies on dermorphin N-terminal tetrapeptide analogues

Summary Dermorphin structure-activity relationships toward μ and δ opioid receptors were investigated using a series of synthetic peptides, in which the aromatic residues at positions 1 or/and 3 of the N-terminal tetrapeptide analogue H-Tyr-d-Arg-Phe-β-Ala-NH₂ were replaced by unnatural or constrained amino acids.     

Synthesis, conformational and pharmacological studies on dermorphin N-terminal tetrapeptide analogues

Dermorphin structure–activity relationships toward and opioid receptors were investigated using a series of synthetic peptides, in which the aromatic residues at positions 1 or/and 3 of the N-terminal tetrapeptide analogue H-Tyr-d-Arg-Phe--Ala-NH₂ were replaced by unnatural or constrained amino acids.     

Synthesis, conformation and biological activity of dermorphin and deltorphin I analogues containing N-alkylglycine in place of residues in position 1, 3, 5 and 6.

Syntheses are described of new dermorphin and [D-Ala2]deltorphin I analogues in which the phenylalanine, the tyrosine or the valine residues have been substituted by the corresponding N-alkylglycine residues. Structural investigations by CD measurements in different solvents and preliminary pharmacological experiments were carried out on the resulting peptide-peptoid hybrids. The contribution from aromatic side chain residues is prominent in the CD spectra of dermorphin analogues and the assignment of a prevailing secondary structure could be questionable. In the CD spectra of deltorphin analogues the aromatic contribution is lower and the dichroic curves indicate the predominance of random conformer populations. The disappearance of the aromatic contribution in the [Ntyr1,D-Ala2]-deltorphin spectrum could be explained in terms of high conformational freedom of the N-terminal residue. The kinetics of degradation of the synthetic peptoids digestion by rat and human plasma enzymes were compared with that of [Leu5]-enkephalin. The binding to opioid receptors was tested on crude membrane preparations from CHO cells stably transfected with the mu- and delta-opioid receptors. The biological potency of peptoids was compared with that of dermorphin in GPI preparations and with that of deltorphin I in MVD preparations. All the substitutions produced a dramatic decrease in the affinity of the peptide-peptoid hybrids for both the mu- and delta-opioid receptors. Nval5 and/or Nval6 containing hybrids behaved as mu-opioid receptor agonists and elicit a dose-dependent analgesia (tail-flick test) when injected i.c.v. in rats.     

Potent side-chain to side-chain cyclized dermorphin analogues containing a carbonyl bridge.

A new family of cyclic opioid peptide analogues related to the 1-4 sequence of dermorphin/deltorphin (Tyr-D-Aaa2-Phe-Aaa4-NH2) has been synthesized. The synthesis of the linear precursor peptides was accomplished by the solid-phase method and ring formation was achieved via a ureido group incorporating the side chain amino functions of D-Aaa2 (D-Lys, D-Orn) and Aaa4 (Lys, Orn, Dab, Dap). The peptides were tested in the guinea-pig ileum (GPI) and mouse vas deferens (MVD) assays. Most showed very high agonist potency in the GPI assay. The peptide containing D-Lys in position 2 and Dab in position 4 was 210 times more active than enkephalin, and that containing Orn and Dab, respectively, was 150 times more active than enkephalin. The latter peptide was also very active in the MVD assay, and showed an IC50 MVD/GPI ratio of 0.816. NMR spectra of selected peptides were recorded, and structural parameters were determined. The conformational space of the peptides was examined using the electrostatically driven Monte Carlo method. With the help of the NMR spectra each peptide was described as an ensemble of conformations. The conformations have been interpreted with regard to the opioid activities, and comparisons have been made with a model proposed earlier for enkephalin analogues.     

N‐(ureidoethyl)amides of cyclic enkephalin analogs

Novel N‐(ureidoethyl)amides of cyclic enkephalin analogs have been synthesized. The p‐nitrophenyl carbamate of 1‐Boc‐1,2‐diaminoethane was coupled with 4‐methylbenzhydrylamine (MBHA) resin. The Boc group was removed by treatment with HCl/dioxane, and the peptide chain was assembled using Boc strategy. For deprotection of amino function, HCl/dioxane was used. D ‐Lys or D ‐Orn were incorporated in position 2, and the side chains of Lys, Orn, Dab, or Dap in position 5 were protected with Fmoc group. Side chain protection was removed by treatment with 55% piperidine in DMF, and cyclization was achieved by treatment with bis‐(4‐nitrophenyl)carbonate to form a urea bridge. The peptide was cleaved from the resin by treatment with 45% TFA in DCM. The peptides were tested in the guinea‐pig ileum (GPI) and mouse vas deferens (MVD) assays. Divers opioid activities were observed, depending on the size of the ring. In comparison with [Leu⁵]enkephalin, all peptides were more active in the GPI assay (between 125 and 12 times), and some of them were also more potent in the MVD assay. The conformational propensities of each peptide were determined using the EDMC method in conjunction with NMR experiments. This approach allows treating the dynamical behavior of small peptides properly. The results were compared with those obtained previously for corresponding nonsubstituted amides and are in agreement with the biologically active conformation proposed by us earlier. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Conformation-activity relationships of cyclo-constrained micro/delta opioid agonists derived from the N-terminal tetrapeptide segment of dermorphin/deltorphin.

The N-terminal tetrapeptide segments of dermorphin (Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH(2)) and deltorphin (Tyr-D-Ala-Phe-Asp/Glu-Val-Val-Gly-NH(2)) are agonists at the opioid receptors micro and delta, respectively. [D-Arg(2), Lys(4)]-dermorphin-(1-4) amide (Tyr-D-Arg-Phe-Lys-NH(2), DALDA) and [Dmt(1)]DALDA (where Dmt is 2',6'-dimethyltyrosine) are among the most potent and selective micro-agonists reported to date, both in vitro (having picomolar micro receptor affinity) and in vivo. In this communication, conformation-activity studies of the following four cyclic analogs of DALDA are presented and discussed: the lead peptide S(2),S(4)-cyclo (Tyr-D-Cys-Phe-Cys-NH(2)), constrained by means of an S(4.2)--S(4.4) disulfide between Cys(2) and Cys(4); its two cis and trans C(4.2)--C(4.4)-olefinic dicarba analogs, and the product of saturation of them both. They are potent nonselective or moderately micro-selective opioid agonists in vitro.They have been synthesized and tested earlier [Berezowska I, Chung NN, Lemieux C, Wilkes BC, and Schiller PW, Acta Biochim Polon 53, 2006, 73-76]. We have studied their conformations using NMR and molecular dynamics. With major conformational constraints imposed by the 11-membered ring spanning residues 2-4, they show well defined conformations of this ring, while the exocylic Tyr(1) and Phe(3) side chains still have significant conformational freedom. The more active and selective micro versus delta disulfide and saturated dicarba agonists seem to have in common: (i) their ring structures more flexilble than those of the other two and (ii) their ring structures similar to each other and more diverse than those in the other two. Given this and the small size of the peptides having confirmed bioactivity profiles, there is a chance that their conformations determined in solution approach receptor-bound conformations. Copyright (c) 2008 European Peptide Society and John Wiley & Sons, Ltd.     

N‐terminal guanidinylation of the cyclic 1,4‐ureido‐deltorphin analogues: the synthesis,receptor binding studies,and resistance to proteolytic digestion

The synthesis of a series of N‐guanidinylated cyclic ureidopeptides, analogues of 1,4‐ureido‐deltorphin/dermorphine tetrapeptide is described. The δ‐ and μ‐opioid receptor affinity of new guanidinylated analogues and their non‐guanidinylated precursors was determined by the displacement radioligand binding experiments. Our results indicate that the guanidinylation of cyclic 1,4‐ureidodeltorphin peptide analogues does not exhibit a uniform influence on the opioid receptor binding properties, similarly as reported earlier for some linear peptides. All analogues were also tested for their in vitro resistance to proteolysis during incubation with large excess of chymotrypsin, pepsin, and papain by means of mass spectroscopy. Guanidinylated ureidopeptides 1G–4G showed mixed μ agonist/δ agonist properties and high enzymatic stability indicating their potential as therapeutic agents for treatment of pain. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Novel glycosylated [Lys(7)]-dermorphin analogues: synthesis, biological activity and conformational investigations.

Syntheses of the [Lys(7)]- and [Hyp(6),Lys(7)]-dermorphin analogues in which either Tyr(5) or Hyp(6) are O-glucosylated are described. For comparison, the carbohydrate-free peptides have also been prepared. Structural investigations by FT-IR and CD measurements were carried out on the synthetic analogues and some preliminary pharmacological experiments were also performed.The biological potency of the glucosylated analogues was compared with that of the micro-opioid receptor agonist dermorphin in GPI preparations. Glucosylation of either Tyr(5) or Hyp(6) reduces the potency of both [Lys(7)]-dermorphin and [Hyp(6),Lys(7)]-dermorphin. The effect induced by the Tyr(5) glucosylation is quite strong and the potency of both peptides is reduced by about 150 times. A similar but less dramatic effect is induced by the glucosylation of the Hyp(6) residue, and the potency of the parent peptide is reduced by about 15 times. The presence of acetyl groups on the sugar hydroxyl functions further reduces the agonistic potency of the glucosylated analogues. The analgesic potency of [Hyp(6),Lys(7)]-, [Hyp(betaGlc)(6),Lys(7)]- and [Tyr(betaGlc)(5),Lys(7)]-dermorphin were also tested in vivo by the tail-flick test. The glucosylated hydroxyproline-containing analogue is 8-10 times less active than the parent peptide, but its analgesic effect lasts significantly longer.     

Tetrazole analogues of cyclolinopeptide A: synthesis, conformation, and biology

Linear and cyclic analogues of cyclolinopeptide A (CLA) with two dipeptide segments (Val(5)-Pro(6) and Pro(6)-Pro(7)) replaced by their tetrazole derivatives were synthesized by the SPPS technique and cyclized using TBTU (O-(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate) reagent. The conformational properties of the c(Leu(1)-Ile(2)-Ile(3)-Leu(4)-Val(5)-Pro(6)-psi[CN(4)]-Ala(7)-Phe(8)-Phe(9)) were investigated by NMR and computational techniques. The overall solution structure of this cyclic peptide resembles that observed for the CLA in the solid state. These studies of cyclic tetrazole CLA analogue confirm that the 1,5-disubstituted tetrazole ring functions as an effective, well-tolerated cis-amide bond mimic in solution. The peptides were examined for their immunosuppressive activity in the humoral response test. For cyclic analogues the immunosuppressive activity, at low doses, is equal in magnitude to the activity presented by cyclosporin A and native CLA. The conformational and biological data seem indicate that the Pro-Pro-Phe-Phe moiety and the preservation of the CLA backbone conformation are important for immunosuppressive activity.     

Solid-phase synthesis of cyclic analogues related to the hypoglycaemic peptide hGH(6-13): comparison of two i-->i + 4 lactam cyclization procedures.

The use of 1,3-diisopropylcarbodiimide (DIC) for the synthesis of cyclic analogues of the hypoglycaemic peptide fragment derived from the N-terminus of human growth hormone (hGH), namely hGH[6-13], is described. Different strategies were examined to achieve improved yields for the on resin side-chain to side-chain cyclization of the corresponding linear peptides containing reverse beta-turn motifs. When compared with the more reactive Castro's reagent, the results confirm that DIC in the presence of HOBt can be successfully employed to minimize the formation of intermolecular oligomeric byproducts associated with the preparation of cyclic hGH[6-14] peptide analogues based on an i-->(i + 4)Lys-->Glu or Glu-->Lys cyclization strategy.     

New antitumour cyclic astin analogues: synthesis, conformation and bioactivity.

Astins, antitumour cyclic pentapeptides, were isolated from the Aster tataricus. Their chemical structures, consist of a 16-membered ring system containing a unique beta,gamma-dichlorinated proline [Pro(Cl)2], other non-coded amino acid residues and a cis conformation in one of the peptide bonds. The astin backbone conformation, along with the cis peptide bond in which the beta,gamma-dichlorinated proline residue is involved, was considered to play an important role in their antineoplastic activities on sarcoma 180A and P388 lymphocytic leukaemia in mice, but the scope and potential applications of this activity remain unclear. With the aim at improving our knowledge of the conformational properties influencing the bioactivity in this class of compounds, new astin-related cyclopeptides were synthesized differing from the natural products by the presence of some non-proteinogenic amino acid residues: Aib, Abu, -(S)beta3-hPhe and a peptide bond surrogate (-SO2-NH-). The analogues prepared c(-Pro-Thr-Aib-beta3-Phe-Abu-), c[Pro-Thr-Aib-(S)beta3-hPhe-Abu], c[Pro-Abu-Ser-(S)beta3-hPhe psi(CH2-SO2-NH)-Abu] and c[Pro-Thr-Aib-(S)beta3-hPhe psi(CH2-SO2-NH)-Abu] were synthesized by classical methods in solution and tested for their antitumour effect. These molecules were studied by crystal-state x-ray diffraction analysis and/or solution NMR and MD techniques.     

Structural characterization of cyclic kallidin analogues in DMSO by nuclear magnetic resonance and molecular dynamics.

The conformational properties in DMSO of two head-to-tail cyclic analogues of kallidin ([Lys(0)]-bradykinin, KL) as well as those of the corresponding linear peptides were studied by NMR and molecular dynamics (MD) simulations. The modifications in the sequence were introduced at position 6, resulting in the four peptides, [Tyr(6)]-KL (YKL), [Trp(6)]-KL (WKL), cyclo-([Tyr(6)]-KL) (YCKL) and cyclo-([Trp(6)]-KL) (WCKL).The linear WKL analogue was significantly more potent than kallidin on rat duodenum preparations, whereas YKL was significantly less potent. Both cyclic peptides, YCKL and WCKL displayed similar activity, lower than that of the linear analogues and also of cyclo-KL.The two linear analogues display high conformational flexibility in DMSO. In the predominant conformer, for both peptides, all three X-Pro bonds adopt a trans configuration. Three out of four conformers present in YCKL and WCKL were completely assigned. The configurations at the X-Pro bonds are the same for the two analogues. All cyclic conformers show a cis configuration in at least one X-Pro bond and always opposite configuration for the two consecutive X-Pro bonds.The NOE-restrained MD calculations resulted in the detection of several elements of secondary structure in each of the conformers. Such elements are described and their possible relevance to biological activity is discussed.     

Conformational studies of cyclic enkephalin analogues with L- or D-proline in position 3.

The conformation of a series of cyclic enkephalin analogues of a general formula X(1)-cyclo[Y(2)-Z(3)-Nal(4)-Leu(5)] (Nal: beta-(2-naphthyl)alanine), where X = Tyr, Phe, or Phe(NO(2)), Y = D-Dab or L-Dab (Dab: 2,4-diaminobutyric acid), and Z = D-Pro or L-Pro, was studied by means of NMR spectroscopy and theoretical conformational analysis with the Empirical Conformational Energy Program for Peptides and Proteins force field plus solvation. The NMR measurements were performed in dimethyl sulfoxide solution. The nuclear Overhauser effect intensities and coupling constants were used to compute the statistical weights of the conformations of the ensemble generated in global conformational searches. The purpose of this study was to determine whether introducing the D- or L-proline residue in position 3 can produce peptides with both rigid backbone and significant separation of the pharmacophore groups in position 1 and 4 (as required for high affinity for the mu-type opioid receptors). It was found that the analogues with D-Dab in position 2 and D-Pro in position 3 possess a stable type II' beta-turn at positions 3 and 4, which rigidifies the cyclic backbone; this finding was confirmed by independent measurements of the temperature coefficients of the amide protons, which indicated very significant screening of the Leu(5) amide proton from the solvent. However, these analogues were found to possess a short interchromophore distance. The analogues containing both Dab and Pro in the L-configuration are characterized by a larger interchromophore distance; however, they do not possess a stable beta-turn and have therefore a higher conformational flexibility. The modifications proposed in this work are therefore not likely to lead to enkephalin analogues with a high affinity for the mu-receptors.     

D-Ala2]-Deltorphin I peptoid and retropeptoid analogues: synthesis, biological activity and conformational investigations.

The synthesis is described of a [D-Ala2]-deltorphin I peptoid analogue in which all amino acid residues have been substituted by the corresponding N-alkylglycine residues. The [D-Ala2]-deltorphin I retropeptoid was also prepared as well as [Ala1 ,D-Ala2]-deltorphin 1 and the corresponding peptoid. Structural investigations by FT-IR and fluorescence measurements were carried out on the synthetic analogues and on some [D-Ala2]-deltorphin 1 peptide-peptoid hybrids previously prepared. According to the fluorescence measurements the distance between the aromatic residues in the deltorphin I peptoid and retropeptoid is similar to that suggested for the delta- and micro-opioids, respectively. Measurements of CD in the presence of beta-cyclodextrin, and some preliminary pharmacological experiments were also performed. No dichroic bands are present in the spectrum of the [Ntyr1,D-Ala2]-deltorphin I, but an increasing dichroic effect appears in the spectra of both the deltorphin I peptoid and retropeptoid. Activity tests on isolated organ preparations showed that the modifications made produced a dramatic decrease in the agonistic activity of the synthetic derivatives.     

Deltorphin analogs restricted via a urea bridge: structure and opioid activity.

Eight cyclic heptapeptides related to the full sequence of deltorphin have been synthesized. The synthesis of linear peptides containing diamino acid residues in positions 2 and 4 was carried out on a 4-methylbenzhydrylamine resin. Depending on protection procedures, the N-protected peptide-resins or N-protected peptide amides with free amino groups in the side chains were obtained, which were subsequently treated with bis-(4-nitrophenyl)carbonate to form a urea unit. Opioid activities of the peptides were determined in the guinea pig ileum (GPI) and mouse vas deferens (MVD) assays. Several compounds showed high delta opioid agonist potency and high selectivity for delta receptors. The results were compared with those obtained earlier for respective 1-4 deltorphin analogs. The conformations of these peptides have been studied using 2D-NMR in H2O/D2O and molecular dynamics. We observed that the backbone rings had well defined conformations, while the Tyr and Phe side chains and the C-terminal tail had significant conformational freedom. The bioassay data and conformational parameters of these peptides were compared with those of previously described, corresponding 1-4 deltorphin analogs. This comparison permitted an assessment of the role of the C-terminal peptide segment in defining the conformation and receptor interaction of the N-terminal portion and provided insight into the relationship between the putative bioactive conformations and bioactivity.     

The impact of β‐azido(or 1‐piperidinyl)methylamino acids in position 2 or 3 on biological activity and conformation of dermorphin analogues

The synthesis of new dermorphin analogues is described. The (R)‐alanine or phenylalanine residues of natural dermorphin were substituted by the corresponding α‐methyl‐β‐azidoalanine or α‐benzyl‐β‐azido(1‐piperidinyl)alanine residues. The potency and selectivity of the new analogues were evaluated by a competitive receptor binding assay in rat brain using [³H]DAMGO (a μ ligand) and [³H]DELT (a δ ligand). The most active analogue in this series, Tyr‐(R)‐Ala‐(R)‐α‐benzyl‐β‐azidoAla‐Gly‐Tyr‐Pro‐Ser‐NH₂ and its epimer were analysed by ¹H and ¹³C NMR spectroscopy and restrained molecular dynamics simulations. The dominant conformation of the investigated peptides depended on the absolute configuration around C^α in the α‐benzyl‐β‐azidoAla residue in position 3. The (R) configuration led to the formation of a type I β‐turn, whilst switching to the (S) configuration gave rise to an inverse β‐turn of type I′, followed by the formation of a very short β‐sheet. The selectivity of Tyr‐(R)‐Ala‐(R) and (S)‐α‐benzyl‐β‐azidoAla‐Gly‐Tyr‐Pro‐Ser‐NH₂ was shown to be very similar; nevertheless, the two analogues exhibited different conformational preferences. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Conformational and biological characterization of human parathyroid hormone hPTH(1-34) analogues containing beta-amino acid residues in positions 17-19

The N-terminal 1-34 fragment of parathyroid hormone (PTH) elicits the full spectrum of bone-related biological activities of the intact native sequences. It has been suggested that the structural elements essential for bioactivity are two helical segments located at the N-terminal and C-terminal sequences, connected by hinges or flexible points around positions 12 and 19. In order to assess the relevance of the local conformation around Gly(18) upon biological function, we synthesized and characterized the following human (h) PTH(1-34) analogues containing beta-amino acid residues: [analogues: see text]. Biological activity and binding affinity of analogue I are one order of magnitude lower than those of the parent compound. In analogue II, both binding affinity and biological activity are partially recovered. Analogues III and V have no binding affinity and very low biological activity. Both bioactivity and binding affinity are partially recovered in analogue IV. The conformational properties of the analogues in aqueous solution containing dodecylphosphocholine micelles were studied by CD, 2D-nuclear magnetic resonance and molecular dynamics calculations. The results confirmed the presence in all analogues of two helical segments located at the N-terminal and C-terminal sequences. The insertion of beta-amino acid residues around position 18 does not cause appreciable conformational differences in the five analogues. The differences in biological activity and binding affinity among the five analogues cannot be related to structural differences in the membrane mimetic environment reported in this study. Our results stress the importance of the side-chain functionalities in the sequence 17-19 for biological function.