International journal of peptide and protein research最新文献

As a part of a research program aimed at studying synthesis and conformation of small ring peptides, the cyclization of diastereoisomeric N-phenylacetyl-seryl-propyl-proline tripeptides has been examined. Two 10-membered peptide lactones, PhCH2CO-DSer-Pro-Pro- 5a and PhCH2CO-DSer-Pro-DPro- 5b, have been isolated by treating the corresponding linear p-nitrophenyl esters with DBU in dry benzene. In these two compounds the serine lactone fragment (a common structural feature of several bioactive cyclodepsipeptides) is inserted into a highly strained small ring system. The conformation in the crystal of 5a and 5b has been studied by X-ray analysis. Both the 10-membered rings of 5a and 5b adopt an overall cis-cis-trans conformation in which the lactone junction is trans. The deviations from planarity of the peptide units vary from delta omega = 30 degrees for the DSer-Pro bond in 5b to delta omega = 5-6 degrees for the DSer-Pro bond in 5a and Pro-DPro bond in 5b. The skeletal atoms of 5b, containing the Pro-DPro sequence, are related by a pseudo-symmetry mirror plane passing through the Pro carbonyl and the opposite DSer C beta H2 group. In both the molecules the exocyclic amide bond adopts an extended conformation with respect to the DSer-Pro ring junction; this arrangement gives rise to a C5-type ring structure which is well evidenced in the case of 5a.

The cyclic pseudopentapeptide cyclo[Gly-Pro psi [CH2NH]Gly-D-Phe-Pro] and its TFA salt were synthesized by solution methods, and their conformations were studied by NMR spectroscopy in both DMSO-d6 and CDCl3. While intramolecular hydrogen bonding is observed with some conformers, the nature of the solvent and the presence of TFA affects the relative structural rigidities of the compounds. No evidence was found for the psi [CH2NH] or psi [CH2NH2+] units acting as H donors in this series.

The structures of two tripeptides, Cbz-glycylglycyltyrosine methyl ester (ZGGYOMe) and Cbz-glycyl(D,L)tyrosylglycine ethyl ester (ZGYGOEt) have been determined from single-crystal X-ray diffraction data. Crystals of ZGGYOMe are monoclinic, space group P2(1), with a = 12.427(3), b = 4.999(3), c = 17.401(6) A, beta = 99.98(2) degree and Z = 2. The final R-index is 0.049 for 1698 reflections with I > or = to 2 sigma (I). Crystals of ZGYGOEt are monoclinic, space group P2(1)/n with a = 12.134(8), b = 14.614(3), c = 26.154(9) A, beta = 98.78(4) degrees, Z = 8. The final R-index is 0.067 for 4457 reflections with I > or = to 2 sigma (I). Both peptides adopt highly extended structures; principal torsion angles are omega 0 = 175.0(4) degrees, phi 1 = 69.2(5) degrees, psi 1 = -154.9(4) degrees, omega 1 = -175.8(4) degrees, phi 2 = 165.4(4) degrees, psi 2 = 154.2(3) degrees, omega 2 = 169.6(3) degrees, phi 3 = -94.8(5) degrees, psi 3 = -47.6(5) degrees for ZGGYOMe and, for the two independent molecules of ZGYGOEt, omega 0 = 177.9(4) degrees, 178.9(4) degrees, phi 1 = -172.0(4) degrees, 169.7(4) degrees; psi 1 = 174.4(4) degrees, -162.5(4) degrees; omega 1 = -170.1(4) degrees, 176.7(4) degrees; phi 2 = -130.8(4) degrees, 130.3(5) degrees; psi 2 = 162.8(4) degrees, -163.3(4) degrees; omega 2 = -177.6(4) degrees, 176.2(4) degrees; phi 3 = -169.9(4) degrees, 172.9(4) degrees; psi 3 = -168.2(4) degrees, 160.9(4) degrees. The structures are of interest since the first one adopts a conformation unlike those of related GGX sequences and the latter shows an antiparallel hydrogen-bonding pattern.

The crystal and molecular structure of the fully protected dipeptide Boc-Val-(S)-alpha-MeSer-OMe has been determined by X-ray diffraction techniques. Crystals grown from ethyl acetate/n-pentane mixtures are tetragonal, space group I4(1), with cell parameters at 295 K of a = 15.307(2), c = 18.937(10)A, V = 4437.1A3, M.W. = 332.40, Z = 8, Dm = 0.99 g/cm3 and Dx = 0.995 g/cm3. The structure was solved by application of direct methods and refined to an R value of 0.028 for 1773 reflections with I > or = 3 sigma (I) collected on a CAD-4 diffractometer. Both chiral centers have the (S) configuration. The dipeptide assumes in the solid state an S shape. The urethane moiety is in the cis conformation, while the amide bond is in the common trans conformation. The conformational angles phi 1, psi 1 of the Val and phi 2, and psi 2 of the (S)-alpha MeSer fall in the F region of the phi-psi map. The isopropyl side chain of the Val residue has the (t, g-) conformation, while the Ser side chain has a g+ conformation. The hydrogen bond donor groups are all involved in intermolecular H-bond interactions. Along the quaternary axis the dipeptide molecules are linked to each other with the formation of infinite rows.

A peptide corresponding to the native (1-66) sequence of horse heart cytochrome c has been synthesized by stepwise automated solid-phase methods on PAM resin. The course of the synthesis has been monitored by several analytical methods including quantitative ninhydrin and Edman degradation. After HF cleavage, the peptide has been purified by a combination of semipreparative ion-exchange and RP-HPLC. The homogeneity of the purified synthetic peptide has been determined by different criteria including HPLC, amino-acid composition, electrophoresis, antibody binding, tryptic and chymotryptic peptide mapping. After deprotection of the Acm-Cys residues and CNBr cleavage of the Met65-Glu66 peptide bond with simultaneous transformation of the Met65 residue into the activated C-terminal [Hse65]lactone, this purified synthetic peptide has been utilized for conformation-assisted joining experiments in combination with synthetic (66-104) to produce fully synthetic [Hse65]apocytochrome c. This latter, after mitochondria-mediated stereospecific heme insertion, has given a functional molecule corresponding to native horse heart holocytochrome c.

The amatoxins, highly toxic components of death cap Amanita mushrooms, bind strongly to RNA polymerase II (or B) in cell nuclei thus preventing the transcription of DNAs to hn-RNAs (Pre-mRNAs), the precursors of messenger RNAs. Three of the binding sites of the bicyclic octapeptides have been identified: an isoleucine side chain in position 6, a trans-4-hydroxyl group at proline in position 2 and a hydroxylated L-isoleucine side chain in position 3. No information exists about the stereochemical conditions at the beta-C-atom (C-atom 3) of this side chain. We have now synthesized the diastereomeric S-deoxo-amaninamides (Fig. 1) containing, in position 3, L-allo-isoleucine (analog 1), (2S, 3R)-2-amino-4-hydroxy-3-methyl butyric acid (analog 2), the diastereomer (2S, 3S)-2-amino-4-hydroxy-3-methylbutyric acid (analog 3) and D-isoleucine (analog 4). In the last synthesis, besides the "normal" bicyclic octapeptide 4, an isomeric Iso-4 was formed. The affinities for Drosophila RNA polymerase II were 100 times weaker as compared to gamma-amanitin for 1, 10 times weaker for 2, 200 times weaker for 3, 100 times weaker for 4, and more than 1000 times weaker for Iso-4. The results point to the importance of a methyl group in (R)-configuration at the beta-C atom of side chain 3.

Beaded cellulose (Perloza) was modified with acrylonitrile followed by reduction with diborane to give a functionalised support containing aminopropyl groups. This spacer arm was then further extended with a glycolamido or an Fmoc-amino acid-4-oxymethylphenoxyacetyl moiety. A number of peptides, including the Merrifield test peptide leucyl-alanyl-glycyl-valine, leucine-enkephalin, Acyl Carrier Protein (65-74), angiotensin I and II, ACTH(4-11) and LHRH were synthesised on the aminopropyl beaded cellulose support using modified t-butyloxycarbonyl (Boc) or fluorenylmethoxycarbonyl (Fmoc) synthesis protocols. The peptides were cleaved from the support and further purified.

We have developed an expeditious method for the incorporation of the biotinylaminocaproyl moiety on the epsilon-amino group of a lysine residue within a peptide chain in a site-specific manner. Using t-Boc chemistry for the solid phase synthesis approach and a base labile, acid stable protecting group (Fmoc-) for the epsilon-amino group of the target lysine, we prepared fully protected resin bound peptides which are site-specifically biotinylated. Following HF cleavage, the uniquely biotinylated peptides were obtained in a high degree of purity. Using this approach, a number of biotinylaminocaproyllysyl derivatives of a monocyclic Endothelin-1 analog were prepared. Synthesis of selected bicyclic analogs of high affinity monocycles led to the preparation of the bicyclic [Nle7]ET-1 analog containing epsilon-biotinylaminocaproyllysine at position-9. This peptide, with Kd = 0.08 nM, has 1000-fold higher affinity for the ETA receptor than the commercially available N alpha-biotinylated Endothelin-1. The general utility of this biotinylation methodology was demonstrated by the synthesis of a site-specifically biotinylated PTH analog which contained several side chain functionalized amino acid residues in its sequence. The synthetic method reported here is convergent in that it allows the facile variation of the length of the spacer and also offers the potential to introduce in a site specific manner other groups such as affinity labels and fluorescent tags.

The value of the mass balance strategy is illustrated in the sequence determination of S. aureus V8 protease. Capillary electrophoresis, electrospray mass spectrometry, and high performance tandem mass spectrometry are used as well as proteolysis and Edman degradation. The carboxy terminus is found to contain 17 irregularly repeating units of the triptych motifs NNP and DNP, which provide a challenge to any strategy involving mapping, sequencing, and overlapping of hydrolytic peptides.

Solution conformations of three series of model peptides, homochiral Ac-Pro-L-Xaa-NHCH3 and heterochiral Ac-Pro-D-Xaa-NHCH3 (Xaa = Val, Phe, Leu, Abu, Ala) as well as alpha,beta-unsaturated Ac-Pro-delta Xaa-NHCH3 [delta Xaa = delta Val, (Z)-delta Phe, (Z)-delta Leu, (Z)-delta Abu] were investigated in CDCl3 and CH2Cl2 by 1H-, 13C-NMR, and FTIR spectroscopy. NH stretching absorption spectra, solvent shifts delta delta for NH (Xaa) and NHCH3 on going from CDCl3 to (CD3)2SO, diagnostic interresidue proton NOEs, and trans-cis isomer ratios were examined. These studies performed showed the essential difference in conformational propensities between homochiral peptides (L-Xaa) on the one hand and heterochiral (D-Xaa) and alpha,beta-dehydropeptides (delta Xaa) on the other. Former compounds are conformationally flexible with an inverse gamma-bend, a beta-turn, and open forms in an equilibrium depending on the nature of the Xaa side chain. Conformational preferences of heterochiral and alpha,beta-dehydropeptides are very similar, with the type-II beta-turn as the dominating structure. There is no apparent correlation between conformational properties and the nature of the Xaa side chain within the two groups. The beta-turn formation propensity seems to be somewhat greater in alpha,beta-unsaturated than in heterochiral peptides, but an estimation of beta-folded conformers is risky.