bluuebunny/crossref_metadata_2025 huggingface dataset:
Regiospecific amine substitution into α‐amanitin with retention of inhibitory properties against eukaryotic class II RNA polymerase
Authors: MORRIS PAUL W. , VENTON DUANE L.
Published: August 2010

Treatment of α‐amanitin with I2‐morpholine (1:1:3 mole ratio α‐amanitin: I2: morpholine) affords 7' substitution of the α‐amanitin with the N‐atom of the morpholine rather than the expected 7'‐iodination. The 7'‐(N‐morpholino)‐α‐amanitin is isolable in yields up to 95% and shows electronic spectra similar to the parent amatoxin but with distinct Λmax and Λmin at 243 and 237 nm, respectively. Spectrophotometric titration of the phenolic hydroxyl shows an increase in pKa from 9.72 (α‐amanitin) to 10.24 for this product; an increase in pKa is expected on chemical grounds following ortho substitution with an amine. Proton magnetic resonance shows two aromatic protons (δ4H= 7.56 p.p.m.; δ5H= 6.88 p.p.m.; J = 9 Hz), thus establishing the extent and position of substitution at the 7' carbon in the tryptophan residue. The 7'‐(N‐morpholino)‐α‐amanitin inhibits RNA polymerase activity with half‐maximal inhibition at 1 times 10‐8M and 3 times 10‐4M for class II and class III sea urchin RNA polymerase, respectively. Extension of this procedure with additional dialkylamines affords the preparation of a family of 7'‐(N‐dialkylamino)‐α‐amanitins analogous to the prototypical morpholine derivative. With tertiary and aryl amines no amatoxin derivative results, while with primary alkyl amines this procedure yields an unresolved product mixture containing an as yet undefined inhibitor of the class I as well as class II and III RNA polymerases. The title reaction thus provides a facile, efficient route to modified amatoxins that do not disrupt the amatoxin: RNA polymerase binary complex formation. A means toward reporter molecule analysis of class II RNA polymerase structure and function is implied by these results.
Thermal denaturation of tryptophan synthase α‐subunit
Authors: OGASAHARA KYOKO , YUTANI KATSUHIDE , SUZUKI MINORU , SUGINO YOSHINOBU
Published: August 2010

Thermal denaturation for the wild‐type of tryptophan synthase α‐subunit from E. coli and one of its mutant proteins was followed by CD measurements at various pHs in the alkaline region and the results from van't Hoff analyses of the thermal denaturation curves were compared with those from calorimetry. Although the far‐u.v. CD spectra of the thermally denatured proteins differed from those of the completely denatured states in 3.2 M guanidine hydrochloride, the titration curves by denaturants at higher temperatures were not sigmoidal but straight lines, indicating that the cooperative structure of the proteins has been completely destroyed by heating. The ratio of calorimetric enthalpy change to van't Hoff enthalpy change obtained from calorimetric study was unity, indicating that the thermal denaturation of the proteins was a two‐state system. The unfolding heat capacity change (ΔCp) of the wild‐type protein from van't Hoff analysis of the thermal denaturation curves by CD measurement was estimated to be 2.45 kcal/mol ± deg, which was similar to that from calorimetry. The values of unfolding enthalpy change at denaturation temperatures were lower by about 15 kcal/mol compared to those from calorimetry.
Conformational study of the basic proline‐rich polypeptides from human parotid saliva
Authors: Shibata S. , Asakura J. , Isemura T. , Isemura S. , Saitoh E. , Sanada K.
Published: August 2010

The conformational study of two basic proline‐rich polypeptides from human parotid saliva, P—D and P—E of known primary structures, was performed by CD and 1H—n.m.r. spectra measurements. These polypeptides contain consecutive sequences of five prolyl residues in their amino acid sequences. The troughs in CD spectra of P—D and P—E were found at 202 and 201 nm, respectively. These wavelengths were different from the value of 206 nm of poly‐l‐proline form II conformation. In spite of this, the existence of poly‐l‐proline form II conformation was suggested in the structure of P—D, because the trough for a fragmental peptide of P—D containing five consecutive prolyl residues was found at 204 nm.

No remarkable change was detected in CD and 1H—n.m.r. spectra of P—D and P—E in the range of pH 3.0–11.0. The result suggests that no folding of polypeptide which might be affected by ionic interaction exists in its structure.
Peptide synthesis catalyzed by papain at alkaline pH values
Authors: Mitin YU.V. , Zapevalova N.P. , Gorbunova E.Yu.
Published: August 2010

The synthesis of peptides in the presence of papain at pH 8–9.5 is described. Starting substances are acylamino acid alkyl esters (the carboxyl component) and amides or tert.‐butylesters of amino acids, as well as peptide (the amino component). Under such conditions secondary hydrolysis is not essential, making the synthesis of peptides soluble in aqueous medium. The yield of peptides is 50–94%. The effect of different factors (temperature, solvents, reagent concentrations) on the result of the reaction has been studied. It has been found that an excess of the carboxyl component is expedient to increase the yield of peptides.
Vitamin‐K‐dependent carboxylation
Authors: BORY SONIA , DUBOIS JOËLLE , GAUDRY MICHEL , MARQUET ANDRÉE
Published: August 2010

Pentapeptides Phe‐Leu‐X‐Glu‐Val where X is successively L‐threo‐γ‐fluoro‐glutamyl, L‐erythro‐γ‐fluoro‐glutamyl, L‐threo‐γ‐methyl‐glutamyl or L‐erythro‐γ‐methyl‐glutamyl have been synthesized and tested as substrates for the vitamin K‐dependent carboxylation. L‐threo‐ or L‐erythro‐γ‐methyl‐glutamyl are not carboxylated but both corresponding peptides are inhibitors of the reaction. The L‐threo‐γ‐methyl‐glutamyl containing peptide has the highest affinity described so far for the active site of the carboxylase (80 M). In the γ‐fluoro‐glutamyl series, only the L‐erythro‐γ‐fluoro‐glutamyl residue is carboxylated, showing that the enzymatic hydrogen abstraction is stereospecific and corresponds to the elimination of the pro S hydrogen of glutamic acid. The lack ofin vitrodicarboxylation of model peptides in contrast with thein vivopolycarboxylation of endogenous precursors is discussed along with the regiospecificity of the reaction with the different substrates.
Cyclic analogues of bradykinin
Authors: MUTULIS F.K. , CHIPENS G.I. , MUTULE I.E. , MAUROPS G.H.
Published: August 2010

Cyclopeptides containing the complete bradykinin sequence have been synthesized using the pentafluorophenyl esters procedure. Cyclization was performed either by directly connecting the terminal amino acids or via one or two ω‐aminododecanoic acid residues. Preparative liquid chromatography with an axial adsorbent compression system was employed for purification of intermediates. Cyclo‐bradykinin and cyclo‐(ω‐aminododecanoyl‐bradykinin) exhibit prolonged hypotensive activity, whereas cyclo‐(ω‐aminododecanoyl‐ω‐aminododecanoyl‐bradykinin) appeared inactive in our experiments.
Spectroscopic investigations on dermorphin and its [L‐Ala2] analog
Authors: ARLANDINI EMANUELE , BALLABIO MARZIA , DE CASTIGLIONE ROBERTO , GIOIA BRUNO , MALNATI MARIA LUISA , PERSEO GIUSEPPE , RIZZO VINCENZO
Published: August 2010

Dermorphin, DM, (H‐Tyr‐d‐Ala‐Phe‐Gly‐Tyr‐Pro‐Ser‐NH2) and its [l‐Ala2]‐analog, LDM, have been investigated in aqueous and DMSO solutions by means of 1H‐ and 13C‐n.m.r., CD and u.v. with the aim of establishing possible differences in the conformational preferences of the two peptides. A study of the pH dependence of 13C chemical shifts resulted in the determination of the pKa of the α‐amino group (6.97 0.02 for DM and 7.13 0.02 for LDM). A significantly higher value (7.75) is obtained for LDM from the pH dependence of CD spectra. This difference is attributed to the presence of associates at the high concentrations required for the 13C‐n.m.r. measurements. The average pKa's of the two phenol groups have also been obtained and the values determined from u.v. absorption spectra (9.79 for DM, 9.85 for LDM) are in fair agreement with those determined from the CD spectra (9.94 for DM and 10.11 for LDM). An analysis of titration shifts in 13C‐n.m.r. spectra indicates that the α‐amino group is not involved in interactions stabilizing folded conformers in aqueous solution. Temperature coefficients and exchange times of the NH protons suggest that both peptides exist in DMSO‐d6 as a mixture of rapidly interconverting conformers. A model is proposed in terms of an extended β‐sheet conformation stabilized by intermolecular association at the high concentrations required for the n.m.r. measurements. CD spectra are dominated by contributions of the aromatic chromophores and cannot be used as a source of information about the main chain conformation. However, differences between the spectra of the two peptides indicate that the conformational freedom of one tyrosine side chain (probably Tyr1) is slightly more restricted in DM than in LDM.
Oxygen‐17 n.m.r. of peptides
Authors: VALENTINE B. , STEINSCHNEIDER A. , DHAWAN D. , BURGAR M.I. , AMOUR T. ST. , FIAT D.
Published: August 2010

Peptide‐17O chemical shifts of linear dipeptides with and without protecting groups in H2O, CH3OH, CH2Cl2, CHCl3, CCl4, CH3CN and DMSO were between 256–350 ppm downfield from external water. Increasing solvent H‐bond donating ability correlated with shifts to higher field. The 17O resonance of several cyclic dipeptides appeared at higher field relative to comparable linear dipeptides (303–317 p.p.m. vs. 327–337 p.p.m.). Separate signals were simultaneously observed by 13C and 17O n.m.r. for cis and trans N‐tert.‐butyl‐formamide in binary mixtures with H2O, (CH3)2CO, and CCl4. The differences in the 17O nuclear screening of the amide isomers and most probably for cis and trans peptides were independent of contributions from H‐bonding at the amide or peptide linkage, apparently reflecting differences between geometric isomers in electron distribution and through space effects. Peptide‐17O of Gly‐Ala, Gly‐Leu and Gly‐Glu in aqueous solution experienced upfield shifts of 6–12 p.p.m. and 12–16 p.p.m. upon deprotonation of the C‐terminal COOH and of the N‐terminal NH+3 groups respectively. These observations were rationalized in terms of the attendant changes in substituent effects, especially on the ± electron donating ability of the N atom at the peptide linkage and increased partial negative charge on the peptide oxygen. Temperature studies of peptide‐17O of Gly‐Ala between pH 1.5–9.0 revealed a chemical shift coefficient of 0.08 p.p.m./K and similar behavior of T1 and T2 relaxation times. Ea for molecular rotation was 5 kcal/mol between 301–331·K. Rotational correlation times, c, were within the range expected from the Stokes‐Einstein relation.
Synthesis and properties of an all‐D model ribonuclease S‐peptide
Authors: CORIGLIANO‐MURPHY M. ANGELA , LIANG XUN , PONNAMPERUMA CYRIL , DALZOPPO DANIELE , FONTANA ANGELO , KANMERA TATSUHIKO , CHAIKEN IRWIN M.
Published: August 2010

In order to examine the effect of a defined enantiomeric sequence on protein structure, the all‐d model ribonuclease S‐peptide, H‐Ala‐Glu‐Ala4‐Lys‐Phe‐Ala‐Arg‐Ala‐His‐Met‐Ala2‐OH, has been synthesized by the solid phase method. The all‐L peptide has been synthesized previously and shown to possess 36% of ribonuclease S activity when added to ribonuclease S‐protein (Komoriya, A. & Chaiken, I.M. (1982) J. Biol. Chem. 257, 2599–2604). The synthetic d‐peptide was purified by gel filtration and semipreparative reverse phase HPLC. Amino acid composition of the synthetic peptide was in agreement with theory and gas chromatographic analysis showed that no significant racemization had occurred during synthesis. Circular dichroism (CD) studies of the d‐peptide showed a peak of positive ellipticity in the 220–230 nm region, whereas a negative ellipticity peak for the l‐peptide was observed. The effects of temperature and trifluoroethanol on the far‐ultraviolet CD spectra of d‐ and l‐peptides were similar but of opposite sign, confirming the expectation that the d‐peptide has the propensity to form an α‐helical structure which is enantiomeric with respect to that formed by the l‐peptide. In the presence of S‐protein, the l‐peptide showed hydrolytic activity against the substrate cytidine‐2′:3′‐monophosphate, whereas the d‐peptide was inactive. Addition of the d‐peptide to mixtures of l‐peptide and S‐protein did not lead to inhibition of enzymatic activity. These results indicate lack of binding of d‐peptide to S‐protein to produce either an active or inactive species.
Tryptophyllins from extracts of Phyllomedusa rhodei skin: new tetra‐, penta‐ and heptapeptides. Further studies
Authors: GOZZINI LUIGIA , MONTECUCCHI PIER CARLO , ERSPAMER VITTORIO , MELCHIORRI PIETRO
Published: August 2010

Tryptophyllins (TPH's) are members of a new family of amphibian skin peptides containing in their structures a tryptophyl residue. In this paper we describe the identification of tetra‐, penta‐, and heptapeptides occurring in the 70% ethanol eluates from alkaline alumina columns of skin extracts of Phyllomedusa rhodei.
Interaction of dipalmitoyl‐phosphatidylcholine with calf thymus histone H1
Authors: GAVILANES JOSE G. , LIZARBE MARIA A. , MUNICO ANGEL M. , OÑNADERRA MERCEDES
Published: August 2010

The interaction between dipalmitoyl‐phosphatidylcholine and calf thymus histone H1 has been studied. A protein‐phospholipid complex, resulting from this interaction, has been isolated by centrifugation in a sucrose gradient. The phospholipid‐histone interaction causes an increase in the α‐helix content of the protein; the corresponding conformational transition is observed by CD studies in the far‐u.v. region. The only tyrosine residue of the protein can be advantageously used as an intrinsic fluorescent probe; thus, fluorescence spectra indicate that protein folding induced by phospholipids is concomitant with the tyrosine transfer into a more hydrophobic environment. The trypsin‐resistant core of the histone is also folded in the presence of the phospholipid but the conformational transition occurs at lower lipid concentration than for the intact protein. Fluorescence polarization of 1,6‐diphenyl‐1,3,5‐hexatriene indicates that the protein shifts the transition temperature of the phospholipid from 41.5 to 44.0°. Secondary structure prediction of the trypsin‐resistant core of the histone indicates the existence of an amphipathic helix that could be responsible for the lipid‐protein interaction.
Synthesis of analogues of the Des‐Phe‐NH2C‐terminal hexapeptide of cholecystokinin showing gastrin antagonist activity
Authors: LAUR JEANINE , RODRIGUEZ MARC , AUMELAS ANDRE , BALI JEAN‐PIERRE , MARTINEZ JEAN
Published: August 2010

Four analogues of Z‐CCK‐27–32‐NH2, Z‐Tyr(SO3‐)‐Met‐Gly‐Trp‐Met‐Asp‐NH2, a cholecystokinin receptor antagonist have been synthesized by solution methodology. In these analogues, Z‐Tyr(SO3‐)‐Nle‐Gly‐Trp‐Met‐Asp‐NH2 16, Z‐Tyr(SO3‐)‐Nle‐Gly‐Trp‐Nle‐Asp‐NH2 17, BOC‐Tyr(SO3‐)‐Met‐Gly‐Trp‐Met‐Asp‐NH2 24 and Boc‐Tyr(SO3‐)‐Met‐Gly‐Trp‐Nle‐Asp‐NH2 25 methionyl residues were replaced by norleucyl residues. Preliminary biological activity on gastrin‐induced acid secretion, in rat, are reported. These derivatives proved to antagonize the action of gastrin, with ED 50 of between 0.5 and 3 mg/kg.
Theoretical conformational analysis of brain peptides
Authors: AKHMEDOV N.A. , AKHVERDIEVA G.A. , GOD'JAEV N.M. , POPOV E.M.
Published: August 2010

Using a semi‐empirical method, an a priori conformational analysis of the octadecapeptide β‐melanocyte‐stimulating hormone (β‐MSH) was carried out. The spatial structure of β‐MSH can be described by eight low‐energy conformations, yielded by combinations of the most stable states of the respective free fragments. Calculations produced the values of all dihedral angles of the backbones and side chains of these forms as well as intra‐ and inter‐residue interaction energies.
Solid state and solution conformation of Boc‐L‐Met‐Aib‐L‐Phe‐OMe
Authors: BARDI R. , PIAZZESI A.M. , TONIOLO C. , RAJ P. ANTONY , RAGHOTHAMA S. , BALARAM P.
Published: August 2010

The conformation of the peptide Boc‐L‐Met‐Aib‐L‐Phe‐OMe has been studied in the solid state and solution by X‐ray diffraction and 1H n.m.r., respectively. The peptide differs only in the N‐terminal protecting group from the biologically active chemotactic peptide analog formyl‐L‐Met‐Aib‐L‐Phe‐OMe. The molecules adopt a type‐II ß‐turn in the solid state with Met and Aib as the corner residues (øMet =‐ 51.8o, øMet = 139.5o, øAib = 58.1o, øAib = 37.0o). A single, weak 4 ‐> 1 intramolecular hydrogen bond is observed between the Boc CO and Phe NH groups (N—O 3.25 Å, N‐H—O 128.4o). 1Hn.m.r. studies, using solvent and temperature dependencies of NH chemical shifts and paramagneti radical induced line broadening of NH resonances, suggest that the Phe NH is solvent shielded in CDCI3 and (CD3)2SO. Nuclear Overhauser effects observed between Met Cα H and Aib NH protons provide evidence of the occurrence of Met‐Aib type‐II ß‐turns in these solvents.
Water structure in modified bovine plasma albumin (BPA*) gel and bovine mercaptalbumin solution† 1 H‐n.m.r. studies
Authors: SOGAMI MASARU , NAGAOKA SHUNJI , ERA SEIICHI , INOUYE HIROSHI , KUWATA KAZUO , KIDA KIMIHIRO , WATARI HIROSHI , SHIGA KIYOSHI , AKASAKA KAZUYUKI
Published: August 2010

Bovine plasma albumin Fr. V (BPA) has been known to contain small amounts of proteolytic enzyme. Wilson & Foster (1971) found a very limited and specific cleavage of BPA catalyzed by the enzyme with BPA in the F‐form near pH 3.8, resulting in the formation of partially hydrolyzed BPA (BPA*). BPA* had a tendency to form a transparent gel at pD 4.0 (pD range of the F‐form) above 8%, though proteolytic enzyme‐free bovine mercaptalbumin (BMA) was in a transparent solution at pD 4.0 even at 12%. Water structures of the F‐form of BMA in the solution state and of BPA* in the gel state were studied by measuring 1 H‐n.m.r. spectra, spin‐lattice relaxation time (T1) and cross relaxation time (TIS) between irradiated and observed protons. Protein concentration‐dependent changes of T1 of water protons indicated that the amount of hydrated water of BPA* in the gel state is far greater than that of the F‐form of BMA in the solution state. TIS values from protein protons to water protons also indicated a large amount of hydration of BPA*, strong interaction between water and BPA* and rapid exchange between bound and bulk water in the gel state.
Assignment and conformation of neurotensin in aqueous solution by 1H NMR
Authors: NIETO J.L. , RICO M. , SANTORO J. , HERRANZ J. , BERMEJO F.J.
Published: August 2010

A complete assignment of exchangeable and unexchangeable proton resonances of neurotensin 1–13 in aqueous solution has been carried out with the help of its 1–8 and 8–13 fragments. To detect formation of a secondary structure, the effects of peptide fragmentation, temperature decrease, pH changes and addition of denaturing agents on the neurotensin 1H NMR spectrum were investigated. The small changes observed in all cases support the conclusion that neurotensin exists mainly as a flexible random coiled polypeptidic chain in aqueous solution in agreement with previous CD studies.
Synthesis of neuropeptide Y*
Authors: BALASUBRAMANIAM A. , GRUPP I. , SRIVASTAVA L. , TATEMOTO K. , MURPHY R.F. , JOFFE S.N. , FISCHER J.E.
Published: August 2010

Neuropeptide Y (NPY), a hexatriacontapeptide amide, was synthesized on benzhydrylamine resin. The peptide product obtained by HF treatment contained 63% of the target peptide, NPY. A comparison of the chemical, immuno‐chemical and biological properties of the synthetic peptide with natural NPY indicated that they were identical.
Synthesis of the trypsin fragment 10–25/75–88 of mouse nerve growth factor.
Authors: ROMANI S. , MORODER L. , GÖHRING W. , SCHARF R. , WÜNSCH E. , BARDE Y.A. , THOENEN H.
Published: August 2010

The synthesis of the unsymmetrical double chain cystine peptide corresponding to sequence 10–25/75–88 of mouse nerve growth factor is described. The synthetic product was inactive in all bioassays examined; consequently, this portion of the NGF molecule did not represent or contain a lower molecular weight form of the neurotrophic factor.
Photoreactivity of histidyl residues in subtilisins Novo and DY. Photooxidation of subtilisins
Authors: GENOV NICOLAY , IDAKIEVA KRASIMIRA
Published: August 2010

Subtilisins Novo and DY were photoinactivated in the presence of methylene blue according to first order kinetics. The competitive inhibitor Nα‐benzoyl‐L‐arginine protected significantly against inactivation. Under the conditions employed in this study a selective photooxidation of the active site histidine 64 was achieved. Rate constants of 0.32 times 10‐2, s‐1 and 0.35 times 10‐2, s‐1, were calculated for the Novo enzyme and subtilisin DY, respectively. Apparent pKa values of the catalytically important imidazole group of 7.0 ± 0.1 (s. Novo) and 7.1 ± 0.1 (s. DY) were directly determined. The histidyl residues in the two proteases, except the active site histidine, which is the first target of photooxidation, are “buried” in the interior of the protein globule. Conformational studies suggested that the photoreactive histidine is not involved in the stabilization of the protein conformation.
17O and 14 N n.m.r. studies of the Co (II) interaction with cyclo(Ala*‐Ala) in aqueous solution
Authors: TRITT‐GOC JADWIGA , GOC ROMAN , PONNUSAMY ETTIGOUNDER , FIAT DANIEL
Published: August 2010

The complexation of cyclo(Ala*‐Ala) with the cobaltous ions in aqueous solution was investigated by 17O and 14N n.m.r. spectroscopy. The 17O and 14N transverse relaxation time (T2p) and chemical shift (Δωa) of cyclo(Ala*‐Ala) were measured as a function of the temperature at pH = 7.03 ± 0.02, and pH = 6.45 ± 0.02, and as a function of pH at room temperature. No effects of pH on the transverse relaxation time and chemical shift were observed. Complementary 17O studies of the solvent water molecules were also carried out. The hyperfine coupling constant and the entropy and enthalpy of activation for the exchange of cyclo(Ala*‐Ala) and water molecules between the coordinated and noncoordinated states were determined by least‐square fit of theoretical equation for the chemical shift Δωa to experimental data. The hyperfine coupling constant of the peptide bound oxygen was determined to be (– 1.6 ± 0.1) ± 105 Hz and the entropy and enthalpy (32.0 ± 3.0) kJ/mol and (– 12.0 ± 1.0)e.u, respectively. Information obtained from 17O n.m.r. study allows some inferences concerning the probable coordination sphere of the cobaltous ion. There are three types of complexes: Co(H2O)2+6, CoL ± 5H2O and CoL2 ± 4H2O, with relative concentrations 19.9%, 2.9%, and 77.2%, respectively. The 17O n.m.r. line broadening is due to the presence of a minor species, CoL ± 5H2O, in rapid exchange and not due to the major species, CoL2 ± 4H2O, in solution, which has a large ±tm. 14N n.m.r. transverse relaxation rates are unaffected by the cobaltous ion, indicating either that the cobaltous ions do not bind to the nitrogen peptide bond or that the rate of exchange is very slow and does not affect the free peptide.
N,N'‐bis(2‐oxo‐3‐oxazolidinyl) phosphinic chloride (BOP‐Cl); a superb reagent for coupling at and with iminoacid residues
Authors: VAN DER AUWERA C. , ANTEUNIS M.J.O.
Published: August 2010

Analytically pure dipeptides containing two iminoacids [Pro, MeAla, MePhe, MeVal and SPip (γ‐thiapipecolinic acid)] were prepared with BOP‐Cl [N,N]‐bis(2‐oxo‐3‐oxazolidinyl) phosphinic chloride] at normal temperature (0° — r.t.) in high yields (80–100%) and with excellent optical purity (below noise limit in 1H n.m.r.). Maximum yields were obtained with Zervas' preactivation procedure, using, for example, an excess of BOP‐Cl followed by the addition of (an equal excess of) the aminocomponent. Racemization suppressors (HOBt, imidazole, etc.) are of no use in these difficult couplings. Z‐Me Val‐Me Val‐OBut (yield: 89%, without racemization (!)) and Z‐SPip‐SPip‐OMe (yield: 96%, less than 5% stereomutation) were prepared for the first time, whereas all other coupling reagents have failed. Peptide acids with N‐methylaminoacids as the COOH‐terminal residues were coupled with excellent yields (70–90%), but unfortunately chiral integrity could not be preserved. Pronounced chiral induction was observed during the preparation of Z‐Pro‐MePhe‐MeAla‐OBut, and an influence of the penultimate protecting group was detected. In contrast, Z‐MeVal‐MeVal‐MeVal‐MeVal‐OBut was formed with probably only 5% stereomutation (yield: 52%). Some side reactions with BOP‐Cl are observed: e.g. symmetrical anhydride and DKP formation.
Structure‐activity relationships of melanin‐concentrating hormone
Authors: KAWAZOE ICHIRO , KAWAUCHI HIROSHI , HIRANO TETSUYA , NAITO NOBUKO
Published: August 2010

Melanin‐concentrating hormone (MCH) is a cyclic heptadecapeptide (H‐Asp‐Thr‐Met‐Arg‐Cys‐Met‐Val‐Gly‐Arg‐Val‐Tyr‐Arg‐Pro‐Cys‐Trp‐Glu‐Val‐OH) that induces aggregation of melanin granules within the melanophores of teleost fishes. Chemical and enzymatic modifications of MCH were conducted in order to deduce the structure‐activity relationship using an in vitro bioassay with fish scales, and a radioimmunoassay using a specific antiserum to synthetic MCH. Micro‐modification of MCH was employed with the natural peptide, and the modified form was purified by reverse‐phase HPLC. MCH1–14 and NPS‐Trp15‐MCH were equipotent to MCH. Reduction and carboxamidomethylation of MCH caused complete loss of biological activity. Modification of the Tyr residue with tetranitromethane and Arg residues with 1,2‐cyclohexadione significantly reduced activity, while oxidation with hydrogen peroxide caused only partial loss (10%) of activity. These results suggest that the configuration of the S‐S loop is essential for activity, and Arg and Tyr may play an important role in the biological activity. In the radioimmunoassay, MCH1–14, MCH5–14 and CAM‐Cys5,14‐MCH showed no cross‐reactivity, whereas MCH5–14 and other derivatives gave inhibition slopes parallel to the MCH standard, suggesting that the antigenic determinant of the antiserum is located in the carboxy‐terminal.
Complete amino acid sequence of a VLDV‐type neurophysin from ostrich differs markedly from known mammalian neurophysins
Authors: LAZURE C. , SAAYMAN H.S. , NAUDÉ R.J. , OELOFSEN W. , CHRÉTIEN M.
Published: August 2010

The neurohypophyseal hormones vasopressin and oxytocin are known to be synthesized in eutherian mammals as part of larger precursors containing either MSEL‐or VLDV‐neurophysins. A neurophysin has been isolated from ostrich neurohypophyses and shown by partial amino acid sequence determination to be related to mammalian VLDV‐neurophysin. The present report describes the complete amino acid sequence of this ostrich neurophysin containing 93 residues. This amino acid sequence, the first reported in birds, differs in a remarkable manner from its mammalian homolog. Indeed, it contains a large number of substitutions, including one insertion, distributed throughout the polypeptide chain when compared to known VLDV‐neurophysins. Whereas many of these substitutions are localized inside the so‐called constant region of the neurophysin, the highest variation can be found in the COOH‐terminal region.
Cyclic tetrapeptides with sequences related to HC toxin. Conformations and cation binding
Authors: HEITZ F. , KADDARI F. , HEITZ A. , RANIRISEHENO H. , LAZARO R.
Published: August 2010

Peptides with sequences related to HC toxin (cyclo(lAla‐dAla‐l‐Aoe‐dPro)) can adopt a conformation locked by three γ turns. A “structure — spectroscopy characteristics” relationship is proposed. These peptides can complex Mg++ cations and the binding is accompanied by a transconformation of the peptide backbone. The relevance with the biological activity of the toxin is discussed.
Purification and characterization of a lectin from Euphorbia nivulia Buch. Ham. latex
Authors: INAMDAR SHASHIKALA R. , MURUGISWAMY B. , MADAIAH M
Published: August 2010

A lectin from Euphorbia nivulia Buch. Ham. latex was purified by affinity chromatography on galactose‐Sepharose‐6B. Euphorbia nivulia latex (ENL‐L) was resolved into four lectin fractions on CM‐Sephadex C‐50 column. All the four fractionated lectins were found to be homogeneous. One of the major fractionated lectins (ENL‐L‐IV) was further characterised. ENL‐L‐IV shows a single band on PAGE at pH 4.5 and SDS‐PAGE. Both ENL‐L and ENL‐L‐IV are glycoproleins containing 13.6% and 9% of carbohydrates respectively. ENL‐L has a molecular weight of 45 700 and has two subunits with Mr of 19 000 and 22 800 and a sedimentation coefficient of 4S. ENL‐L‐IV has a molecular weight of 44 000 and appears to be a dimer with subunits of Mr of 22 000. Ultracentrifugation studies showed that it has a Mr of 29 000 and a sedimentation coefficient of 4.8S. Both ENL‐L and ENL‐L‐IV are galactose specific and are also inhibited by galactose derivatives and its oligosaccharides. with increased β‐anomeric specificity. They are found to be mitogenic against human lymphocytes and blood group non‐specific. ENL‐L‐IV was found to be immunologically identical with ENL‐L and other fractionated ENL lectins.
17O n.m.r. studies of amino acids in the solid state, in single‐ and polycrystalline forms
Authors: GOC ROMAN , PONNUSAMY ETTIGOUNDER , TRITT‐GOC JADWIGA , FIAT DANIEL
Published: August 2010

A single crystal of 17O enriched α‐glycine was grown by slow evaporation of aqueous solution. 17O n.m.r. studies of the α‐glycine molecule in single crystalline form revealed five 17O transitions each consisting of two lines due to inequivalency of the oxygen atoms in the unit cell, with each of these lines revealing a dipolar interaction between the 17O and the nearest hydrogen atom. The spectral width was found to be of the order of magnitude of MHz and the linewidths of the order of magnitude of 2 kHz.
Protected amino acid tetrafluorophenyl esters for peptide synthesis
Authors: HUI KWAN Y. , HOLLERAN EUGENE M. , KOVACS JOSEPH
Published: August 2010

New 2,3,5,6‐tetrafluorophenyl active esters of protected amino acids useful for peptide synthesis were prepared in high yield. For certain amino acid derivatives such as Boc‐Pro‐OH, Boc‐Ile‐OH, and Boc‐Val‐OH, their tetrafluorophenyl esters have significantly higher melting points than the corresponding pentafluorophenyl esters. Kinetic studies were carried out to compare the racemization rate constants (with triethylamine) and coupling rate constants (with valine methyl ester) of the tetrafluorophenyl and pentafluorophenyl esters of protected histidine and tyrosine. Results of the second‐order kinetics showed similarly large kcoupling/kracemization ratios for both tetra‐ and pentafluorophenyl esters. In particular, the use of 2,3,5,6‐tetrafluorophenyl or pentafluorophenyl ester prevents extensive racemization of the N‐tert.‐butyloxycarbonyl‐Nim‐benzyl‐histidine.
Synthesis of inhibitors of the meso‐diaminopimelate‐adding enzyme from Escherichia coli
Authors: ABO‐GHALIA MOHAMED , FLEGEL MARTIN , BLANOT DIDIER , VAN HEIJENOORT JEAN
Published: August 2010

In order to obtain inhibitors of the meso‐diaminopimelate‐adding enzyme, which participates in the biosynthesis of bacterial peptidoglycan, several Nα ‐propionyl‐dipeptides of the general formula Pr‐l‐Ala‐ambo‐Xaa‐OH were synthesized. Xaa represented methionine S,S‐dioxide, methionine S‐oxide, methionine sulfoximine, and 2‐amino‐4‐phosphonobutyric acid, i.e. transition state analogs of glutamine synthetase and γ‐glutamyl‐cysteine synthetase, which catalyze the same type of reaction as our target enzyme. After synthesis, the diastereoisomers were separated by preparative HPLC or t.l.c.; those containing methionine derivatives could be identified thanks to previously synthesized reference compounds. After preincubation with the meso‐diaminopimelate‐adding activity from Escherichia coli. the ld diastereoisomers displayed moderate inhibitory effects, whereas the ll ones were inefficient. The best inhibition was obtained with one diastereoisomer of Pr‐l‐Ala‐ζ‐2‐amino‐4‐phosphonobutyrate, presumably the ld one. A chloromethylketone derivative Pr‐l‐Ala‐d‐Glu(CH2Cl)‐OH, potential affinity labeler of the meso‐diaminopimelate‐adding enzyme, was also synthesized. In the assay with preincubation, this compound behaved as the best inhibitor.
Potentiation of glucose‐induced insulin release in islets by desHis1[Glu9]glucagon amide
Authors: KOFOD† HANS , UNSON CECILIA G. , MERRIFIELD R.B.
Published: August 2010

Glucagon and secretin and some of their hybrid analogs potentiate glucose‐induced release of insulin from isolated mouse pancreatic islets. It was recently shown that the synthetic glucagon analog, desHis1[Glu9]glucagon amide, does not stimulate the formation of cyclic adenosine monophosphate in the rat hepatocyte membrane, but binds well to the glucagon receptor and is a good competitive antagonist of glucagon. In the present study the effect of this analog on isolated islets was examined. desHis1‐[Glu9]glucagon amide at 3 x 10−7m, in the presence of 0.01 md‐glucose, increased the release of insulin by 30% and maintained that level for the full 30‐min test period. The rate of insulin release returned to the glucose‐induced base line after removal of the peptide. The same insulin level was produced by 3 x 10−9m glucagon, and at 3 x 10−7m glucagon insulin release was enhanced 290% above the glucose base line.
Synthesis of a substrate of protein kinase C and its corresponding phosphopeptide
Authors: DE BONT H.B.A. , LISKAMP R.M.J. , O'BRIAN C.A. , ERKELENS C. , VEENEMAN G.H. , VAN BOOM J.H.
Published: August 2010

The syntheses of a protein kinase C (PKC) peptide substrate, H‐Lys‐Arg‐Thr‐Leu‐Arg‐OH, and a phosphopeptide analog of the synthetic substrate, H‐Lys‐Arg‐Thr(P)‐Leu‐Arg‐OH, are reported. PKC phosphorylates the peptide with an apparent KMof 0.30 ± 0.04 mM and an apparent Vmaxequal to one‐tenth that of histone III‐S. The synthesis of the phosphopeptide features a recently developed convenient phosphorylation procedure for serine and threonine usingN,N‐diethylamino‐dibenzylphosphoramidite. A complete characterization of the PKC substrate and its corresponding phosphopeptide by C‐H COSY 2D n.m.r. is included.
Circular dichroism studies on sequential polypeptides (Arg‐X‐Gly) when X = L‐Ala, Val, Leu, Ile, Nva, Nle in amphiphilic environments
Authors: TSIKARIS VASSILIOS , SAKARELLOS‐DAITSIOTIS MARIA , PANOU‐POMONIS EUGENIA , SAKARELLOS CONSTANTINOS , MARRAUD MICHEL
Published: August 2010

A series of (L‐Arg‐X‐Gly)n sequential polypeptides, where X represents the amino acid residues L‐Ala, Val, Leu, Ile, Nva, and Nle, have been prepared and their conformational properties as well as their association with nucleic acids in various water fluoroalcohol mixtures have already been reported. In order to gain further insight into the conformational behavior of these polypeptides in amphiphilic environments, we now report their secondary structure, in the presence of increasing amounts of sodium dodecylsulfate and dodecylphosphorylcholine using CD spectroscopy. The CD spectral pattern of all the polypeptides studied is indicative of an equilibrium between α‐helix, β‐turn, and random coil. They are analyzed in relation to the nature of the X side‐chain (length, branching, linearity). The results are explained on the basis of Lundahl's model for polypeptide‐micelle interactions.
Protease inhibitors from Ecballium elaterium seeds
Authors: FAVEL A. , MATTRAS H. , COLETTI‐PREVIERO M.A. , ZWILLING R. , ROBINSON E.A. , CASTRO B.
Published: August 2010

Several protease inhibitors were found in the seeds of a Cucurbitacea, Ecballium elaterium, and were separated from one another by affinity and molecular sieve chromatography. Three main trypsin iso‐inhibitors were purified by ion‐exchange chromatography and the sequence of the major one, EETI II, was elucidated and compared with other inhibitors of the squash family. It is a peptide of M.W. 3020 of strong inhibitory activity (Ka = 8 × 1011 M‐1) against trypsin, showing high Gly content, six half‐cystine residues, but devoid of histidine, threonine, tryptophan, and tyrosine residues.
Structure‐activity relationships of CCK26‐33‐related analogues modified in position 33
Authors: MARSEIGNE I. , DOR A. , PELAPRAT D. , REIBAUD M. , ZUNDEL J.L. , BLANCHARD J.C. , ROQUES B.P.
Published: August 2010

In the sequence of the C‐terminal octapeptide of cholecystokinin, the phenylalanine amide residue in position 33 is of primary importance for the biological activity. Indeed, removal of Phe33‐NH2 is a modification known to induce antagonist properties. The influence of the chemical nature of the Phe33‐NH2 side chain on the biological activity of CCK8 was investigated through replacement of this residue by several amino acids with different lipophilic properties in the sequence of Boc(Nle28, Nle31)CCK27‐33, an equipotent analogue of CCK8. The binding properties of these new CCK‐related analogues: Boc(Nle28,Nle31,X33)CCK27‐33 were measured on both mouse brain and guinea pig pancreatic membranes, and their peripheral activities on amylase secretion and contractions of guinea pig ileum. Among the various peptides modified in position 33, Boc(Nle28,Nle31,Naa33‐NH2)CCK27‐33(Naa = naphthylalanine) and Boc(Nle28,Nle31,Cha33‐NH2)CCK27‐33(Cha = cyclohexylalanine) displayed high affinities for central and peripheral CCK‐receptors and proved to be full agonists of CCK8 in the peripheral tests while Boc(Nle28,Nle31,Ala33‐NH2)CCK27‐33 was completely inactive. This suggests that, at the level of the Phe33‐NH2 subsite, the critical factor for optimal interaction with CCK‐receptors is not the aromatic nature of the side chain but its size and hydrophobicity.
Immuno‐affinity purification of specific antibodies against human gastrin releasing peptide (h‐GRP) by the h‐GRP(1–8)‐linked poly dimethylacrylamide resin
Authors: TAKEYAMA MASAHARU , KINO TOSHIYA , GUO LILI , OTAKA AKIRA , FUJII NOBUTAKA , YAJIMA HARUAKI
Published: August 2010

A new method for immuno‐affinity purification of specific antibodies against human gastrin releasing peptide(h‐GRP) was developed. The antiserum GP(No. 6201) elicited by h‐GRP‐BSA conjugate was hetereogeneous and reacted not only with h‐GRP and its fragments but also partially with other structurally related peptides, such as other GRPs (porcine, canine, and chicken), bombesin, and neuromedin‐C. To obtain specific antibodies against human GRP, antiserum GP was purified by column chromatography on the amino‐terminal octapeptide h‐GRP(1–8)‐linked polydirnethylacrylamide resin. The antibody thus obtained was highly specific to amino‐terminal sequence of h‐GRP and hardly reacted with other GRPs (porcine, canine and chicken), bombesin, and even carboxy‐terminal h‐GRP fragments in ELISA.
Regularities in the primary structure of proteins
Authors: CSERZÖ M. , SIMON I.
Published: August 2010

In this paper the latest protein database consisting of more than a million amino acids is analyzed to characterize the short range regularities in the primary structure. The amino acid distributions along the polypeptide chain and among the proteins have been studied first. Their influence on the amino acid pair statistics was taken into account. We are primarily interested in the distances of the covalent structure, where the amino acid pair frequencies show non‐random characters. The amino acid pairs separated by at least 20 residues in the covalent structure exhibit an exact Gaussian distribution. We found that there is a range of non‐random pairing in the covalent structure. We conclude that the pair preference characters are different for each of the 20 × 20 amino acid pairs. The range of the non‐random pairing varies from pair to pair, and in most cases it does not extend beyond the 9th neighbour. The preferences of a certain pair in a certain position can not be derived from the character of that pair in another position. The preference values of 400 amino acid pairs are listed for up to the pairs in 9th neighbour position. Some fields of potential application of these data have also been discussed.
Preparation of oligomer‐free Nα‐Fmoc and Nα‐urethane amino acids
Authors: BOLIN DAVID R. , SYTWU IOU‐IOU , HUMIEC FRANK , MEIENHOFER JOHANNES
Published: August 2010

A variety of Nα‐urethane blocked amino acids, in particular 9‐fluorenylmethyloxycarbonyl (Fmoc) derivatives, have been synthesized by utilizing intermediate O,N‐bis‐trimethylsilyl‐amino acids, formed in situ by treating an amino acid with trimethylsilylchloride and a base in an aprotic solvent. The intermediate is then reacted with an acylating agent. A general procedure is given which eliminates the oligomerization side reactions normally observed in Schotten‐Baumann type methods. Protected amino acids obtained from this procedure are of high purity as judged by t.l.c, HPLC, ion exchange chromatography, and other physical parameters.
Crystal structure, conformation, and potential energy calculations the chemotactic peptide N‐ formyl‐l‐Met‐l‐Leu‐l‐Phe‐OMe
Authors: GAVUZZO ENRICO , MAZZA FERNANDO , POCHETTI GIORGIO , SCATTURIN ANGELO
Published: August 2010

The tripeptide N‐formyl‐L‐Met‐l‐Leu‐l‐Phe‐OMe (FMLP‐OMe) crystallizes in the orthorhombic system, space group P 212l21, with the following unit‐cell parameters: a = 21.727, b = 21.836, c = 5.133Å, Z = 4. The structure has been solved and refined to a final R of 0.068 for 1838 independent reflexions with I > 2σ(I). The peptide backbone is folded at the Leu residue (φL=−67.7,ΨL=−49.1°) without intramolecular hydrogen bonds. Considering each peptide plane, the Leu side‐chain is oriented on the same side of that of the Phe residue and on the opposite side of that of the Met residue, respectively. The crystal conformation differs from all the other conformations proposed for FMLP‐OMe and the anionic form of N‐formyl‐l‐Met‐l‐Leu‐l‐Phe‐OH (FMLP) in solution accounts for the amphiphilic character of the peptide, giving rise, through intermolecular hydrogen bonds, to a stacking of molecules which could be maintained in the aggregation states experimentally observed in solvents of low polarity. Intramolecular potential energy calculations have ben carried out in order to compare the energies of the various backbone conformers.
Didemnin B Conformation and dynamics of an antitumour and antiviral depsipeptide studied in solution by 1H and 13C. n.m.r. spectroscopy
Authors: SEARLE MARK S. , HALL JON G. , KYRATZIS ILIAS , WAKELIN LAURENCE P.G.
Published: August 2010

The solution conformation of didemnin B, the most potent member of a family of depsipeptides that shows antitumour, antiviral, and immunosuppressive activity, has been studied in chloroform solution using n.m.r. spectroscopy. 1H and 13C spectra have been assigned from analysis of a number of two‐dimensional homonuclear and heteronuclear chemical shift correlation experiments which confirm the recently corrected primary structure of the molecule. The conformation of the peptide has been deduced from measurements of the temperature dependence of the NH chemical shifts, analysis of coupling constant data and primarily through NOE effects observed in the rotating frame. Interproton distance bounds determined from a quantitative analysis of the ROE data provide 41 constraints from which a family of closely related structures were calculated using distance geometry algorithms. A type II β‐turn involving residues Thr6, Leu7, and Pro8 is well represented in the computed conformers as is a hydrogen bonding interaction between the NH of Leu3 and the carbonyl oxygen of Thr6. This latter interaction causes the linear portion of the structure to fold back over the depsipeptide ring, imparting to it a degree of structural stability as well as giving the molecule a somewhat globular character. Only one transannular hydrogen bond, between 1st1 NH and Leu3 carbonyl, stabilizes the conformation of the depsipeptide, which has an irregular non‐planar configuration. The small temperature coefficients (< 2.0 × 10−3ppm/°C) for the NHs of 1st1 and Leu3 are consistent with their involvement in these hydrogen bonding interactions. We find that many of the structural features observed in the crystalline form of didemnin B are conserved in solution. Analysis of the 13C spin‐lattice relaxation rates of the protonated carbons reveals small variations in effective correlation times at specific sites in the molecule. The data suggests that the peptide segment encompassing residues Leu3through to Thr6 is in a more motionally restricted part of the structure.
Crystal structure of l‐2‐oxothiazolidine‐4‐carboxylic acid
Authors: RAMASUBBU N. , PARTHASARATHY R.
Published: August 2010

Crystals of the title compound, l‐2‐oxothiazolidine‐4‐carboxylic acid, OTC (C4 H5 NO3 S), grown from an aqueous solution are orthorhombic, space group P21 21 21 with the following cell parameters at 22 ± 3°; a = 5.381(1), b = 5.961(1), c = 17.929(3)Å V = 575.lÅ3 Mr= 146.2, Dc= 1.688 g.cm−3, μ 43.9cm−1 and Z = 4. The crystal structure was solved by the application of direct methods and refined to an R value of 0.032 for 596 reflections with I > 3σ(I). The thiazolidine ring adopts a “twist” conformation. This structure contains a short (2.619(3)Å) intermolecular hydrogen bond between the carboxyl OH and the oxygen of the 2‐oxo moiety, a feature common to most acyl amino acids and acyl peptides.
Interaction of gossypol with amino acids and peptides as a model of enzyme inhibition*
Authors: STRØM‐HANSEN THORBJØRN , CORNETT CLAUS , JAROSZEWSKI JERZY W.
Published: August 2010

In order to clarify the interaction of gossypol with proteins, the pure diastereoisomeric Schiff bases froml‐tryptophan methyl ester and both gossypol enantiomers were prepared. Their c.d. and n.m.r. spectra demonstrate that the interaction between gossypol and tryptophan, previously reported to involve a weakly associated complex, consists in Schiff base formation. Recent studies on enzyme inhibition by gossypol are discussed; it is suggested that nonspecific covalent binding of gossypol to proteins may be responsible for a significant proportion of thein vitroeffects of gossypol.
Complete amino acid sequence of arachin subunit of molecular weight 21000
Authors: BHUSHAN R. , REDDY K.R.N.
Published: August 2010

A subunit of molecular weight 21 000 from arachin, the major peanut protein, was isolated in pure form and primary structure was determined. The subunit was fragmented with CNBr, trypsin, and NBS; the fragments were separated and isolated by PAGE, gel filtration, Dowex treatment, and paper electrophoresis, and Edman degradation on each fragment, including the intact subunit, was performed. The PTH‐amino acids thus obtained were identified by UV spectroscopy and TLC. The complete sequence of 176 residues was established by overlapping technique.
Local interactions in peptides
Authors: FERMANDJIAN SERGE , SAKARELLOS CONSTANTIN , AUMELAS ANDRÉ , TOMA FLAVIO , GRESH NOHAD
Published: August 2010

We report the results of a joint NMR and theoretical investigation devoted to the conformational properties of N‐acetyl‐N′‐methylamides of aliphatic amino acids with side chains of increasing bulkiness: Gly, Ala, Leu, Ile, and tert. Leu. In this series, determination of the coupling constants 3JHNCαH together with the coupling constants 3JC′NCαH (thanks to specific carbon‐13 labeling of the N‐acetyl carbonyl group) led to the derivation of alternative A, B, and C parameters in a Karplus‐type relation expressing the dependence of 3JC′NCαH upon the φ dihedral angle. The value of the latter is found to increase regularly following the increase of the side‐chain bulkiness. The theoretical conformational analysis is performed by applying the SIBFA procedure, which uses empirical formulas based on ab initio SCF computations. The conformational energy maps illustrate the progressive distortion of the backbone conformation incurred in the series Gly to tert.Leu. Theoretical values computed for 3JHNCαH and 3JC′NCαH are found to be in a good quantitative agreement with the experimental ones.