International journal of peptide and protein research最新文献

The beta-casomorphin-5 analog H-Tyr-c[-D-Orn-2-Nal-D-Pro-Gly-] (2-Nal = 2-naphthylalanine) was the first reported cyclic opioid peptide with mixed mu agonist/delta antagonist properties [R. Schmidt et al. (1994) J. Med. Chem. 37, 1136-1144]. The 2-Nal3 residue in this peptide was replaced with benzothienylalanine (Bta) (3), His(Bzl) (4), Tyr(Bzl) (5), 4'-benzoylphenylalanine (Bpa) (6), 4'-benzylphenylalanine (Bzp) (7), thyronine (Thy) (8), thyroxine (Thx) (9), 4'-biphenylalanine (Bip) (10), 4'-biphenylglycine (Bpg) (12) and 3,3-diphenylalanine (Dip) (14), and the in vitro opioid activity profiles of the resulting compounds were determined in mu and delta receptor-representative binding assays and bioassays. Analogues 3, 12 and 14 were full agonists in the mu receptor-representative guinea-pig ileum (GPI) assay and also were agonists in the delta receptor-representative mouse vas deferens (MVD) assay. The agonist effects of the latter compounds in the MVD assay were antagonized by the highly selective delta antagonist H-Tyr-Tic-Phe-Phe-OH (TIPP), indicating that they were triggered by delta receptor activation. The Bzp3- and Bip3- containing peptides 7 and 10 turned out to be mu antagonists against the mu selective agonist H-Tyr-D-Ala-Phe-Phe-NH2 in the GPI assay. The other analogues were weak partial mu agonists which displayed remarkably decreased mu receptor affinity as compared to parent peptide 1. Compounds 4-10 were found to be delta antagonists in the MVD assay. Analogues 4 and 9 exhibited delta antagonist potency similar to that of parent peptide 1, while compounds 5-8 and 10 showed 3-12-fold higher delta antagonist potency against DPDPE and deltorphin I and, in most cases, increased delta receptor affinity. These results indicate that the delta receptor tolerates bulky aromatic side chains in the 3-position of cyclic beta-casomorphin analogs with either delta agonist or delta antagonist properties. However, these compounds displayed drastically reduced mu receptor affinity in nearly all cases.

The crystal structure and solution conformation of Ac-Pro-deltaAla-NHCH3 and the solution conformation of Ac-Pro-(E)-deltaAbu-NHCH3 were investigated by X-ray diffraction method and NMR, FTIR and CD spectroscopies. Ac-Pro-deltaAla-NHCH3 adopts an extended-coil conformation in the crystalline state, with all-trans peptide bonds and the deltaAla residue being in a C5 form, phi(1)=-71.4(4), psi(1)=-16.8(4), phi(2)= -178.4(3) and psi(2)= 172.4(3) degrees. In inert solvents the peptide also assumes the C5 conformation, but a gamma-turn on the Pro residue cannot be ruled out. In these solvents Ac-Pro-(E)-deltaAbu-NHCH3 accommodates a beta(II)-turn, but a minor conformer with a nearly planar disposition of the CO-NH and C=C bonds (phi(2) approximately 0 degrees) is also present. Previous spectroscopic studies of the (Z)-substituted dehydropeptides Ac-Pro-(Z)-deltaAbu-NHCH3 and Ac-Pro-deltaVal-NHCH3 reveal that both peptides prefer a beta(II)-turn in solution. Comparison of conformations in the family of four Ac-Pro-deltaXaa-NHCH3 peptides let us formulate the following order of their tendency to adopt a beta-turn in solution: (Z)-deltaAbu > (E)-deltaAbu > deltaVal; deltaAla does not. None of the folded structures formed by the four compounds is stable in strongly solvating media.

The total enzymatic synthesis of a model peptide Leu-enkephalin on a preparative scale was accomplished in the so-called solvent-free system. The syntheses were carried out in a rotary glass homogenizer by admixing solid reactants with native proteases and Na2CO3.10H2O. The most feasible way leading to biologically active Leu-enkephalin, was based on the strategy of 2 + (1 + 2) condensation catalyzed by alpha-chymotrypsin, thermolysin and papain for the final segment coupling. Subtilisin was used for the ester hydrolysis of peptide intermediates. Alternative strategies as well as the influence of several reaction conditions on the yield of the protease-catalyzed synthesis of Leu-enkephalin or Leu-enkephalin amide were also investigated.

Mass spectrometry was used to determine the molecular mass of rat pituitary beta-endorphin1-31 (BErat, 1-31). The measured molecular mass (3435 +/- 1 Da, n = 5) of endogenous BErat, 1-31 differed from the molecular mass of commercially available synthetic BErat, 1-31 (3465 +/- 1 Da, n = 9), but corresponded to the molecular mass of synthetic BEbovine, 1-31 (3436 +/- 3 Da, n = 3). Based on the combination of these ESIMS molecular mass measurements, HPLC retention time data, LSIMS measurement of the molecular mass of selected tryptic fragments, and consideration of codon sequences, we suggest that the amino-acid sequence of endogenous BErat, 1-31 differs from the DNA-deduced sequence of BErat, 1-31, and that endogenous BErat, 1-31 contains Ala instead of Val in position 26.

A quantum-mechanical study of the chain-length dependent stability of the extended, 2(7)-ribbon and 3(10)-helix conformations in dehydroalanine (delta Ala) oligopeptides has been performed. To address the study, the oligopeptides delta Ala(n), where n varies from 1 to 6, were computed by using the semiempirical AMI methodology. Cooperative free-energy effects permit one to predict the stabilization of the 3(10)-helix with respect to the extended and 2(7)-ribbon conformations when the number of residues in the polypeptide chain increases. The interactions associated with the pi-electron density of the side chains can easily explain this finding. The effects of the solvent and the crystalline packing on the different conformations were modeled using a self-consistent reaction field (SCRF) method and a molecular mechanics approach to the packing, respectively. Both the aqueous and crystal environments seem to be a key factor in the stabilization of the helical conformation. Finally, the variations of electrostatic parameters such as atomic point charges and dipole moments in delta Ala-containing peptides with internal (conformation) and external (solvent) effects are discussed.

Two novel microbial ACE-inhibitors BAY o 6997 and BAY q 1313 were detected in the fermentation broths of streptomyces spec. WS 464 and spec. WS 1065, respectively. Both were isolated and purified by ion exchange chromatography as initial steps, and final purification was achieved by HPLC or additional chromatography of the Cu-chelate (BAY q 1313). Both inhibitors are reversibly inactivated on chelation with Cu2+ or Zn2+. Irreversible inactivation occurs on standing in aqueous and acidic solution or in ammonium hydroxide at room temperature and more rapidly on heating. In 4 M sodium hydroxide solutions BAY o 6997 is completely stable, and BAY q 1313 still remarkably stable even on longer heating to 80 degrees C. Thus, BAY o 6997 was alternatively and advantageously isolated after heating of its solution in 4 M sodium hydroxide to 37 degrees C for 2 days and subsequent fractional precipitation with ethanol in a relatively pure state. Total hydrolysis yielded His, 2-methylamino-4-amino-butyric acid and alpha-keto butyric acid (BAY o 6997) and pyruvic acid (BAY q 1313) respectively. The unusual stability of both inhibitors in sodium hydroxide solution on the one hand and their instability on heating and storage in aqueous or acidic solutions on the other hand clearly prove that the constituents are not linked by amide bonds.

Conditions have been developed for the site-specific pegylation (NH2-terminus, side-chain and carboxy-terminus) of a potent analog of growth hormone-releasing factor, [Ala15]-hGRF(1-29)-NH2. These pegylated peptides were prepared by solid-phase peptide synthesis using the Fmoc/tBu strategy, and were fully characterized by analytical HPLC, amino-acid analysis, 1H-NMR spectroscopy and laser desorption mass spectrometry. Biological activities of hGRF analogs were determined in vitro utilizing stimulation of growth hormone release by cultured rat pituitary cells as an index. GH-releasing potencies of the pegylated hGRF analogs were compared to a series of model analogs of [Ala15]-hGRF(1-29)-NH2 that were acetylated or protected as the ethylamides at the pegylation sites. It was found that acetylation at the NH2-terminus resulted in reduced potency, which was not further affected when the NH2-terminus was pegylated, regardless of the size of poly(ethyleneglycol) (PEG) employed (e.g. PEG2000 or PEG5000). Pegylation at Asp8 or Lys12 decreased biological potency, a situation which was exacerbated by increasing the molecular weight of PEG. Pegylation at Lys21 or Asp25 did not significantly affect biological activity. The C-terminal model peptide, [Ala15,Orn(Ac)30]-hGRF(1-29)-NH2, was the most potent analog identified in this series (ca. 4-5-fold that of hGRF(1-44)-NH2. The COOH-terminal pegylated analogs retained this increased level of biological activity independent of PEG molecular weight. These studies demonstrate that a biologically active peptide can be pegylated and retain the full in vitro potency of the peptide. However, the biological activity is highly dependent on the site of pegylation and, in some cases, the molecular weight of PEG (degree of pegylation) moiety used.