bluuebunny/crossref_metadata_2025 huggingface dataset:
Structure, solubility and reactivity of peptides
Authors: TONIOLO CLAUDIO , BONORA GIAN MARIA , HEIMER EDGAR P. , FELIX ARTHUR M.
Published: August 2010

A conformational study of two protected peptide segments, (1–10 and 11–28), spanning the entire sequence of thymosin α1, in solvents of different polarity and capability of forming hydrogen bonds, is reported. By using infrared absorption and circular dichroism techniques the occurrence of the random coil conformation, the self‐associated β‐structure, and the α‐helix (the latter adopted only by the longer peptide) was established. The self‐associated species of the two peptide segments were disrupted either by adding increasing amounts of hexamethylphosphoramide or by dilution. This structural transition was monitored by the disappearance of the amide‐I C=O stretching band of strongly intermolecularly hydrogen‐bonded molecules (near 1630 cm‐1) in the infrared absorption spectra. The tendency of these peptides to aggregate is paralleled by a decrease in their solubility. The conformational findings are discussed in terms of the solvent‐dependent product yields obtained in the reaction of segment (1–10) with the Nα‐deprotected (11–28) segment to give the fully protected thymosin α1.
Synthesis and binding properties to DNA and to opioid receptors of enkephalin‐ellipticinium conjugates
Authors: RIGAUDY P. , GARBAY‐JAUREGUIBERRY Ch. , JACQUEMIN‐SABLON A. , PECQ J‐B. LE , ROQUES B.P.
Published: August 2010

In order to specifically direct cytotoxic agents against tumor cells bearing δ opioid receptors, the DNA intercalating agents ellipticine and 9‐OH‐ellipticine were coupled by quaternarization of the pyridine nitrogen to an enkephalin modified pentapeptide through a short chemical linker. The ellipticine ring of these conjugates was shown to intercalate into DNA, with DNA affinity constants close to those of the non‐conjugated ellipticines. Despite the addition of a polycyclic ring to theC‐terminal amino acid, the D‐Ala2‐D‐Leu5‐enkephalin‐ellipticine conjugates bind to the opioid receptor from rat brain and NG 108–15 cells with an affinity constant close to 108M‐1. Other derivatives were synthesized as a control using a tripeptide which does not bind to the opioid receptor.
Comparison of conformational properties of linear and cyclic δ selective opioid ligands DTLET (Tyr‐D‐Thr‐Gly‐Phe‐Leu‐Thr) and DPLPE (Tyr‐c[D‐Pen‐Gly‐Phe‐Pen]) by1H n.m.r. spectroscopy
Authors: BELLENEY JOËL , ROQUES BERNARD P. , FOURNIE‐ZALUSKI MARIE‐CLAUDE
Published: August 2010

The preferential conformations of the δ selective opioid peptides DPLPE (Tyr‐c[D‐Pen‐Gly‐Phe‐Pen]) and DTLET (Tyr‐D‐Thr‐Gly‐Phe‐Leu‐Thr) were studied by 400 MHz1H n.m.r. spectroscopy in DMSO‐d6solution. In neutral conditions, the weak NH temperature coefficients of theC‐terminal residue (Pen5or Thr6), associated with interproton NH‐NH and α‐NH NOE's (ROESY experiments), indicated large analogies between the backbone folding tendency of both the linear and cyclic peptides. Various γ and/or β turns may account for these experimental data. A similar orientation of theN‐terminal tyrosine related to the folded backbones is observed for the two agonists, with a probable γ turn around the amino acid in position 2. Finally, a short distance, about 10 Å, between Tyr and Phe side chains and identical structural roles for threonyl and penicillamino residues are proposed for both peptides. These results suggest the occurrence of similar conformers in solution for the constrained peptide DPLPE and the flexible hexapeptide DTLET. Therefore, it may be hypothesized that the enhanced δ selectivity of DPLPE is related to a very large conformational expense of energy needed to interact with the μ opioid receptor, a feature not encountered in the case of DTLET. These findings might allow peptides to be designed retaining a high affinity for δ opioid receptors associated with a very low cross‐reactivity with μ binding sites.
Microheterogeneity in H1 histones and its consequences
Authors: COLE R. DAVID
Published: August 2010

The extent of microheterogeneity of H1 histones in individual higher organisms, without considering post‐translational modifications, is such that five to eight molecular species can be recognized. The H1 variants differ among themselves in their ability to condense DNA and chromatin fragments, and they are non‐uniformly distributed in chromatin. This review assembles data that support the notion that the differences in chromatin condensation (heterochromatization) observed through the microscope are maintained by the non‐uniform distribution of H1 variants, and that this pattern of chromatin condensation may determine the dynamics of chromatin during replication and may represent the commitment aspect of differentiation. The differential response of the multiple H1 variants with regard to their synthesis and turnover is consistent with this notion.
Synthesis of analogues of amaninamide, an amatoxin from the white Amanita virosamushroom
Authors: ZANOTTI GIANCARLO , MÖHRINGER CLAUDIUS , WIELAND THEODOR
Published: August 2010

Analogs of amaninamide, due to the absence of a 6‐hydroxy group in the tryptophan moiety, are more easily accessible by synthesis than derivatives of α‐amanitin. Syntheses of bicyclic octapeptide thioethers 1f‐1m have been carried out starting from linear Hpi‐S‐trityl‐octapeptides (3), cyclization by intramolecular 2′‐indolylthioether formation yielding monocyclic peptides (2) and final cyclization by DCCI. One of the bicyclic thioethers was oxidized to yield the corresponding chromatographically separated (R)‐ and (S)‐sulfoxides, respectively. The products were characterized by RF‐values, u.v. and CD spectra as well as by mass (FAB) spectroscopy. The widely differing inhibitory activities on RNA polymerase II (or B) from calf thymus are listed.
Effect of N‐methylation of selected peptide bonds on the biological activity of insulin. [2‐N‐Methylisoleucine‐A]insulin
Authors: OGAWA HIROSHI , BURKE G. THOMPSON , CHANLEY JACOB D. , KATSOYANNIS PANAYOTIS G.
Published: August 2010

Hydrogen bonding involving peptide bonds of the backbone of the insulin molecule may play an important role in insulin‐receptor interaction. Our previous work suggested that the A2‐A8 helical segment of the hormone molecule participates in this interaction. To investigate the possible involvement of peptide bonds of this segment in insulin‐receptor interaction the [2‐N‐methylisoleucine‐A]insulin and [3‐N‐methylvaline‐A]insulin ([MeIle2‐A]‐ and [MeVal3‐A]insulins) were synthesized. The circular dichroic spectra of the analogues were obtained and their properties were examined in several biological assays. The circular dichroic spectra suggested that the analogues remained monomeric at concentrations at which insulin is predominantly dimeric, and that their A2‐A8 helical segments are distorted. The in vitro biological activity and the receptor binding affinity of these analogues were compared with that of natural insulin. Both analogues are weak full agonists. [MeIle2‐A]insulin displayed a potency of 5.4 ± 0.3% in stimulating lipogenesis and 4.6 ± 2.3% in receptor binding affinity in rat fat cells and rat liver plasma membranes respectively. [MeVal3‐A]insulin displayed a potency of 2.1 ± 0.2% in lipogenesis and 1.0 ± 0.3% in receptor binding assays. In radioimmunoassays [MeIle2‐A]‐ and [MeVal3‐A]insulins exhibited potencies of 13% and 11% respectively relative to the natural hormone. The substantially decreased biological activity and receptor binding affinity of these analogues may be attributed partly to the change of conformation and partly to the loss of hydrogen bonding capacity of the A2‐A8 segment brought about by N‐methylation of the A1‐A2 or A2‐A3 peptide bonds.
Solid‐phase peptide synthesis using tert.‐butyloxycarbonylamino acid pentafluorophenyl esters
Authors: OTVOS LASZLO , DIETZSCHOLD BERNHARD , KISFALUDY LAJOS
Published: August 2010

Boc‐amino acid pentafluorophenyl esters have been successfully used in solid‐phase peptide synthesis. The coupling rates and the purity of the products are comparable to those with the symmetrical anhydrides. These active esters require modified Merrifield resin, polar medium for coupling and in some cases, base catalysis.
Conformational study of a somatostatin analogue by high‐field n.m.r. spectroscopy, in aqueous solution
Authors: WYNANTS C. , SUGG E. , HRUBY V.J. , VAN BINST G.
Published: August 2010

The cyclic analogue of somatostatin (SRIF), D‐Phe‐Cys‐Tyr‐D‐Trp‐Lys‐Thr‐Cys‐Thr‐NH2 (CTC), exhibits good affinity for both opioid and SRIF receptor systems. Its conformational properties were examined in water by high‐field proton n.m.r. spectroscopy and compared with results previously obtained with structurally related analogues SMS 201–995 and Sandoz 204–090 in the same solvent. The assignments were made using 2 D‐n.m.r. methods, especially long‐range connectivities between neighbouring α protons, and between β and aromatic protons. The 3JNH‐cαH and Δδ/ΔT values are compatible with an equilibrium between two γ turns involving residues 2, 3 and 4 and residues 3, 4, and 5, respectively.
Synthesis of side chain‐modified iodothyronines
Authors: HAN SO‐YEOP , GORDON JANICE T. , BHAT KRISHNA , DRATMAN MARY B. , JOULLIÉ MADELEINE M.
Published: August 2010

New side chain‐modified iodothyronines have been synthesized. They include: 1‐[4‐(4‐hydroxyphenoxy)‐3,5‐diiodophenyl]‐1,2‐ethanediol (T2EG); α‐hydroxy‐4‐(4‐hydroxyphenoxy)‐3,5‐diiodobenzeneacetic acid (T2HAA) and their 4‐methyl ether derivatives (MT2EG, MT2HAA); 1‐[4‐(4‐hydroxyphenoxy)‐3,5‐diiodophenyl]‐2‐aminoethanol (T2EA); 1‐[4‐(4‐hydroxy‐3‐iodophenoxy)‐3,5‐diiodophenyl]‐1,2‐ethanediol (T3EG); 1‐[4‐(4‐hydroxy‐3‐iodophenoxy)‐3,5‐diiodophenyl]‐2‐amino‐ethanol (T3EA); and α‐hydroxy‐4‐(3‐iodo‐4‐hydroxyphenoxy)‐3,5‐diiodobenzeneacetic acid (T3HAA). These model compounds are being used to study thyroid hormone metabolism and to determine structure‐activity relationships of iododiphenylether derivatives.
Purification and comparative studies of several mitochondrial aspartate aminotransferases from avian liver
Authors: CASCANTE M. , CORTÉS A. , BOZAL J.
Published: August 2010

A new purification method has been developed which only exploits the chromatographic behaviour of avian liver mitochondrial aspartate aminotransferase enzymes (m‐AAT), and permits a rapid isolation of the protein (4 days) in large quantities with high yield and low cost. m‐AAT from turkey, chicken and quail livers have been isolated by chromatography on CM‐Sepharose, Sephadex G‐100 and 5′ AMP‐Sepharose using TEA‐acetate buffer (pH 7.4), and specific activities (A.E.) of 311.6, 318.9, 320.1 I.U./mg respectively were obtained. Preparations were homogeneous as judged by various electrophoretic techniques and by size exclusion HPLC. The amino acid composition, Stokes Radius, subunit molecular weight and pI values have been determined and compared, finding no appreciable differences among them. In contrast, the absorption spectrum of the turkey enzyme differed from those of chicken and quail at both pH 7.4 and pH 5.0.
Guinea pig MSEL‐neurophysin. Sequence comparison of eight mammalian MSEL‐neurophysins
Authors: CHAUVET MARIE‐THÉRÈSE , CHAUVET JACQUELINE , ACHER ROGER
Published: August 2010

The amino acid sequence of guinea pig MSEL‐neurophysin has been determined using tryptic peptides derived from the performic acid‐oxidized protein and staphylococcal proteinase peptides obtained from the reduced‐carboxamidomethylated neur‐ophysin. Guinea pig MSEL‐neurophysin consists of a 93‐residue polypeptide chain that shows 12 substitutions and 2 deletions when compared to bovine MSEL‐neurophysin. It displays the highest number of variations among known mammalian MSEL‐neurophysins. These variations are mainly found in the C‐terminal region (residues 88–93). Moreover guinea pig MSEL‐neurophysin, like rat homologous protein, exhibits substitutions in positions 2, 5, 29 and 81 and lacks an arginine in the penultimate position. Comparison between eight mammalian MSEL‐neurophysins reveals a highly conserved region (residues 1 to 88) and a hypervariable region (residues 89 to 93/95). On the other hand the eight species examined are endowed with arginine vasopressin except pig, which has a lysine vasopressin. In the vasopressin‐MSEL‐neurophysin precursor, the hormonal moiety and the MSEL region of neurophysin (residues 1–9) are encoded by a common exon in ox, rat and man; it can be concluded that this exon is evolutionarily conservative in contrast to the one encoding the C‐terminal region of MSEL‐neurophysin.
Solid‐phase peptide synthesis: a silver anniversary report*
Authors: BARANY GEORGE , KNEIB‐CORDONIER NANCY , MULLEN DANIEL G.
Published: August 2010

It has been a quarter of a century since Merrifield's initial report on solid‐phase peptide synthesis. The field has matured significantly in recent years with a better understanding of the underlying chemistry. This is reflected by new, milder orthogonal protection schemes and more efficient coupling methods, some of which have been incorporated into automated systems. Advances in purification, especially high performance liquid chromatography, have had a major impact. The efficacy of these improvements has been demonstrated by an impressive litany of applications to biological problems.
A convenient general method for synthesis of Nα‐ or Nω‐dithiasuccinoyl (Dts) amino acids and dipeptides: application of polyethylene glycol as a carrier for functional purification*
Authors: ZALIPSKY SHMUEL , ALBERICIO FERNANDO , SLOMCZYNSKA URSZULA , BARANY GEORGE
Published: August 2010

N
α‐Dithiasuccinoyl (Dts) amino acids (1) needed for solid‐phase peptide synthesis have been prepared in good yields and excellent purities by a new method that exploits the solubility properties of polyethylene glycol (PEG; bifunctional with average molecular weight 2000 was found to be optimal). Suitably side‐chain protected amino acid derivatives are first reacted with a polymeric xanthate (11), following which the free α‐carboxyl is blocked by silylation and the Dts heterocycle is elaborated in the same pot by reaction with chlorocarbonylsulfenyl chloride (4). Upon aqueous workup, the polymeric carrier removes any urethane blocked amino acids which arise during the process. Exaggerated conditions were explored to prove the power of this functional purification approach, and mechanisms of formation of polymer‐bound urethanes are proposed and supported by solution model studies. The preparation and characterization of the companion N‐(iso‐propyldithio)carbonyl derivative of proline is also presented.
Geometrical analysis of Cys‐Cys bridges in proteins and their prediction from incomplete structural information
Authors: GOLDBLUM AMIRAM , REIN ROBERT
Published: August 2010

Analysis of C‐alpha atom positions from cysteines involved in disulphide bridges in protein crystals shows that their geometric characteristics are unique with respect to other Cys‐Cys, non‐bridging pairs. They may be used for predicting disulphide connections in incompletely determined protein structures, such as low resolution crystallography or theoretical folding experiments. The basic unit for analysis and prediction is the 3 times 3 distance matrix for Cα positions of residues (i – 1), Cys(i), (i + 1) with (j – 1), Cys(j), (j + 1). In each of its columns, row and diagonal vector‐outer distances are larger than the central distance. This analysis is compared with some analytical models.
Protamines. VII. Total synthesis of [perornithine]thynnine Z1
Authors: MARCHIORI FERNANDO , BORIN GIANFRANCO , CALDERAN ANDREA , CHESSA GAVINO
Published: August 2010

The total synthesis is reported of [perornithine]thynnine Z1, one analog of thynnine Z1 in which all arginines are substituted by ornithine. The peptide has been prepared as intermediate to obtain the synthetic thynnine Z1 by the amidination procedure. The entire sequence has been carried out by coupling three new suitable protected fragments synthesized by the condensation method in solution.
Structural and functional differences between fetal and adult serum albumin
Authors: JORI G. , REDDI E. , RUBALTELLI F.F.
Published: August 2010

The binding of bilirubin with adult or fetal human serum albumin has been studied by steady‐state fluorescence emission spectroscopy. The 1:1 complex between bilirubin and the two albumin samples shows very similar fluorescence properties, as well as essentially identical accessibility of the protein‐bound bilirubin to fluorescence quenchers added to the aqueous medium. The intramolecular distance between bilirubin and the single tryptophyl residue can be estimated to be 2.4 ± 0.2 nm for both proteins by singlet‐singlet energy transfer. These findings suggest that fetal and adult human serum albumin have a very similar three‐dimensional structure; the different binding capacity for bilirubin displayed by the two proteins is likely to be the consequence of small differences in the physico‐chemical properties of some amino acid residues close to the bilirubin binding site, as indicated by pH‐titration experiments of the intrinsic albumin fluorescence.
Side reactions of monoaminodicarboxylic acids in peptide synthesis
Authors: LIBEREK BOGDAN , KASPRZYKOWSKA REGINA
Published: August 2010

N‐benzyloxycarbonyl‐l‐homoglutamine was studied to answer the question whether or not there is any tendency for the dehydration of the δ‐carboxamide group during activation and coupling. Only a minor conversion to nitrile by‐products was found in the activation of the α‐carboxyl group and formation of peptide bond with the help of dicyclohexylcarbodiimide. Addition of N‐hydroxybenzotriazole to the reacting mixture eliminates the side chain dehydration reaction. The dehydration reaction is also overcome by protection of the side chain carboxamide with the benzyloxycarbonylcarbamoyl substituent.
Conformation of d‐Phe‐Cys‐Tyr‐d‐Trp‐Lys‐Thr‐Pen‐Thr‐NH2 (CTP‐NH2), a highly selective mu‐opioid antagonist peptide, by 1H and 13C n.m.r.
Authors: PELTON JOHN T. , WHALON MICHAEL , CODY WAYNE L. , HRUBY VICTOR J.
Published: August 2010

The 1 H and 13C n.m.r. spectral parameters of CTP‐NH2 [d‐Phe‐Cys‐Tyr‐d‐Trp‐Lys‐Thr‐Pen‐Thr‐NH2], a potent, highly selective μ‐opiate antagonist, were measured in aqueous solution and a possible conformation has been deduced from the spectral data. The data are consistent with a type II'β‐turn for the tetrapeptide sequence ‐Tyr3‐d‐Trp4‐Lys5‐Thr6‐. Solvent shielding of the Cys2 amide proton, observed in variable temperature experiments, suggests an orientation of this amide proton toward the gem dimethyls of Pen7 with possible hydrogen bonding to the Thr6 carbonyl oxygen, and a dihedral angle of −110° for the disulfide bond. Partially relaxed Fourier transform 13C relaxation studies confirm a constrained cyclic system, with the Cα carbons in the “hinge” of the β‐turn having the shortest t1 times. Segmental motion was observed for the side chain of Lys5.
Thermolysin and α‐chymotrypsin mediated synthesis of tripeptides containing proline
Authors: CHENG ELISABETH , MIRANDA MARIA TERÊSA MACHINI , TOMINAGA MINEKO
Published: August 2010

The tripeptides Z‐Pro‐Leu‐Gly‐OEt, Z‐Pro‐Leu‐Gly‐NH2 and Z‐Pro‐Leu‐Gly‐OBzl were synthesized by thermolysin and α‐chymotrypsin catalysis. The optimum conditions for the couplings between Z‐Pro‐OH and H‐Leu‐OEt or H‐Leu‐Gly‐OEt catalysed by thermolysin were determined by a systematic study involving analysis of pH effect, ammonium sulfate concentration, reaction time, enzyme concentration, and relative proportion of the carboxyl and amine components. The best yield obtained for Z‐Pro‐Leu‐OEt was 77% and for Z‐Pro‐Leu‐Gly‐OEt, 100%. Z‐Pro‐Leu‐OEt was coupled to H‐Gly‐OEt, H‐Gly‐NH2 and H‐Gly‐OBzl. The best conditions to obtain Z‐Pro‐Leu‐Gly‐OEt and Z‐Pro‐Leu‐Gly‐NH2 were determined by the study of some factors that affect the reaction yield, such as organic solvent presence, substrate ratio and aqueous and organic solvent ratio. The yield obtained under optimum synthesis conditions was 55% for Z‐Pro‐Leu‐Gly‐OEt and 61% for Z‐Pro‐Leu‐Gly‐NH2. Z‐Pro‐Leu‐Gly‐OBzl was synthesized with 42% yield.
Applications of BOP reagent in solid phase synthesis
Authors: FELIX ARTHUR M. , WANG CHING‐TSO , HEIMER EDGAR P. , FOURNIER ALAIN
Published: August 2010

Using a variety of activating agents, a kinetic study was carried out to evaluate the rate of solid phase side‐chain to side‐chain cyclization of Asp3 to Lys12 in the model peptide‐resin, [Ala15]‐GRF(1–29)‐BHA‐resin. Asp3 and Lys12 were introduced in the peptide chain by using Nα‐Boc amino acids in conjunction with the OFm/Fmoc side‐chain protection scheme. The OFm and Fmoc side‐chain protecting groups were shown to be stable to diisopropylethylamine and selectively deprotected on treatment with 20% piperidine in DMF. Solid phase side‐chain to side‐chain cyclization (lactamization) was shown to proceed to completion within 2 h using benzotriazol‐1‐yl‐oxy‐tris(dimethylamino)‐phosphonium hexafluorophosphate (BOP reagent) while the reaction was only 55% completed in 24h using DCC/HOBt. Solid phase side‐chain to side‐chain cyclization by the BOP procedure not only proceeded more rapidly but also gave a purer cyclic product.
Enzymatic synthesis of delta sleep‐inducing peptide
Authors: SAKINA KIYOSHI , KAWAZURA KEIKO , MORIHARA KAZUYUKI
Published: August 2010

The delta sleep‐inducing peptide was assembled enzymatically from three tripeptide fragments. All the peptide bonds were prepared by either papain‐ or α‐chymotrypsin‐mediated synthesis. Secondary hydrolysis was suppressed by introducing Nα‐protected amino acid or peptide esters as carboxyl components and using an alkaline pH. The protected nonapeptide was oxidized with ferric chloride to deprotect the C‐terminal phenylhydrazide and then hydrogenated. The homogeneous peptide was obtained by reversed phase high‐performance liquid chromatography. Comparison of enzymatic and chemical preparations showed no obvious differences.
Trichosanthin, α‐momorcharin and β‐momorcharin: identity of abortifacient and ribosome‐inactivating proteins
Authors: YEUNG HIN WING , LI WAI WAN , FENG ZHANG , BARBIERI LUIGI , STIRPE FIORENZO
Published: August 2010

The abortifacient proteins trichosanthin, α‐momorcharin and β‐momorcharin at nM concentrations inhibit cell‐free protein synthesis. The momorcharins and the ribosome‐inactivating proteins isolated from Momordica charantia seeds cross‐react with the respective antisera. The ribosome‐inactivating proteins saporins, pokeweed antiviral protein (PAP) and, to a lesser extent, gelonin have abortifacient activity on pregnant mice.
Synthesis of keto‐methylene and dehydro‐keto‐methylene pseudo‐dipeptides
Authors: EWENSON ARIEL , COHEN‐SUISSA RACHEL , LEVIAN‐TEITELBAUM DINA , SELINGER ZVI , CHOREV MICHAEL , GILON CHAIM
Published: August 2010

A new synthetic pathway is proposed for the preparation of keto‐methylene and dehydro‐keto‐methylene dipeptide isosteres bearing the general formulas (RS)XxxΨ(COCH2)(RS)Yyy and (RS)XxxΨ(COCH2)ΔYyy. The method involves the condensation of an oxazolone derived from anN‐benzoyl amino acid (3a, b) via a modified Dakin‐West reaction with the appropriately α‐substituted succinoyl chloride half ester (2a, b). This last compound is obtained from a Stobbe condensation followed by reaction with oxalyl chloride. The fully protected pseudo‐dehydro dipeptides (41, b) thus obtained can then be catalytically hydrogenated to the desired pseudo‐dipeptides (5a, b). Acid hydrolysis of (4) and (5) gives the pure fully deblocked dipeptide surrogates which are thenN‐protected for coupling into the desired peptide analogs. For preparative applications, the prior isolation of intermediates is not required throughout the procedure up to the final products. In this manner, the following compounds were prepared: Boc‐(RS)PheΨ(COCH2)(E, Z)Δ Phe‐OH (9), Boc‐(RS)Pheψ(COCH2)(RS)Phe‐OH (7a), and Boc‐Glyψ(COCH2)(RS)Leu‐OH (7b). Characterization of isolated intermediates and final products was carried out.
Guanidinium‐carboxylate interactions
Authors: YOKOMORI YOSHINOBU , HODGSON DEREK J.
Published: August 2010

The crystal structures of the 1:1 complexes methylguanidinium benzylhydrogenmalonate, (C2N3H8)+(C10H9O4)−, MGD.BMAL, and methylguanidinium ethylhydrogenmalonate, (C2N3H8)+(C5H7O4)−, MGD.EMAL, and of the 2:1 complex methylguanidinium sulfate, (C2N3H8)2SO4, have been determined from three‐dimensional X‐ray data. For MGD.BMAL, the complex crystallizes in the triclinic space group P with two formula units in a cell of dimensions a = 6.277(5), b = 8.470(3), c = 13.191(6)Å, α= 91.01(1), β= 99.64(9), γ= 90.83(5)°. The structure has been refined to a final value of R = 0.061 based on 1511 intensities. The MGD.EMAL complex is also triclinic, space group P with two molecules in a cell of dimensions a = 9.254(7), b = 9.625(6), c = 6.778(2) Å, α= 109.6(1), β= 100.8(1), γ= 62.7(1)Å. The crystals of this compound are of low quality, and the final value is R = 0.109 based on 706 intensities. (MGD)2SO4 is orthorhombic, space group P212121, with four molecules in a cell of dimensions a = 7.100(4), b = 12.151(3), c = 13.108(2) Å. Refinement has converged to R = 0.054 based on 907 data. All three crystals exhibit extensive interionic hydrogen bonding. The hydrogen bonding in MGD.BMAL includes a Type B interaction and a Type 1 interaction, the latter being a pairwise interaction from both amino nitrogen atoms on the cation to two carboxylate oxygen atoms from the two different carboxylate groups in an anion. In MGD.EMAL, the anion participates in both a Type A and a Type B pairwise interaction with two neighboring cations. The possible implications of the hydrogen bonding patterns in these two compounds for the role of arginyl side chains in protection of γ‐carboxyglutamate residues from decarboxylation are discussed.
Mixed carboxylic‐carbonic acid anhydrides of acylamino acids and peptides as a convenient source of 2,4‐dialkyl‐5(4H)‐oxazolones
Authors: CHEN FRANCIS M.F. , SLEBIODA MAREK , BENOITON N. LEO
Published: August 2010

Racemic 2‐R1‐4‐R2‐5(4H)‐oxazolones (R1= CH3, C6H5, C6H5 CH2 OCONHCH2; R2= aliphatic and C6H5CH2) have been prepared in good yields by reaction of the parent N‐acetyl‐, N‐benzoyl‐, and N‐benzyloxycarbonylglycyl‐amino acids with methyl chloroformate in cold dichloromethane in the presence of N‐methylmorpholine, followed by washing the mixtures with aqueous solutions and removal of the solvent. The products derived from optically active substrates contained enantiometric excesses of 83–98%. The method cannot be applied to the preparation of oxazolones from N‐formylamino acids.
Conformations of dehydrophenylalanine containing peptides Nuclear Overhauser effect study of two acyclic tetrapeptides
Authors: UMA K. , CHAUHAN V.S. , KUMAR ARUN , BALARAM P.
Published: August 2010

Two isomeric, acyclic tetrapeptides containing a Z‐dehydrophenylalanine residue (Δz‐Phe) at position 2 or 3, Boc‐Leu‐Ala‐Δz‐Phe‐Leu‐OMe (1) and Boc‐Leu‐Δz‐Phe‐Ala‐Leu‐OMe (2), have been synthesized and their solution conformations investigated by 270MHz 1H n.m.r. spectroscopy. In peptide 1 the Leu(4) NH group appears to be partially shielded from solvent, while in peptide 2 both Ala(3) and Leu(4) NH groups show limited solvent accessibility. Extensive difference nuclear Overhauser effect (n.O.e.) studies establish the occurrence of several diagnostic inter‐residue n.O.e.s (CαjH ⇆ Ni+1H and NiH ⇆ Ni+1H) between backbone protons. The simultaneous observation of “mutually exclusive” n.O.e.s suggests the presence of multiple solution conformations for both peptides. In peptide 1 the n.O.e. data are consistent with a dynamic equilibrium between an ‐Ala‐Δz‐Phe‐ Type II β‐turn structure and a second species with Δz‐Phe adopting a partially extended conformation with Ψ values of ± 100° to ± 150°. In peptide 2 the results are compatible with an equilibrium between a highly folded consecutive β‐turn structure for the ‐Leu‐Δz‐Phe‐Ala‐ segment and an almost completely extended conformation.
N.m.r. studies on the conformation of cyclo(‐l‐Trp‐l‐His) in solutions
Authors: SHEINBLATT M. , ANDORN M. , RUDI A.
Published: August 2010

N.m.r. studies on the conformations of cyclo(‐l‐Trp‐l‐His) in acidic and basic D2O and DMSO solutions are presented here. The detailed conformational analysis of the cyclodipeptide molecule is based on 1H chemical shifts, spin‐spin coupling constants and NOE measurements. The conformational analysis consists of the following steps: 1) Calculations of the allowed {χn} states of the peptide molecule, (an allowed {χn} state is characterized by defining the two torsional angles χ1 and χ2 for each amino acid residue, which does not violate the excluded volume criteria). 2) Calculations of the induced chemical shifts of the His H5 and Hβ resonances arising from the indole and imidazole ring current effects, in the various allowed {χn} states and the comparison of the calculated and measured chemical shifts of the above resonances. 3) The conformation of the DKP ring and the distribution of the relative populations among the three rotational isomers of each of the side chains were estimated from the measured coupling constants. Finally, the NOE measurements enabled us to determine the various pairs of protons which are spatially in close proximity to each other.
Immunochemistry of scorpion toxins Synthesis and antigenic properties of a model of a loop region specific to α‐toxins
Authors: FOURQUET P. , BAHRAOUI E. , FONTECILLA‐CAMPS J.C. , VAN RIETSCHOTEN J. , ROCHAT H. , GRANIER C.
Published: August 2010

A region of the toxin II of the scorpion Androctonus australis Hector, possessing a loop structure, is shown to be antigenic. Some clear hints for the probable antigenic character of this region were obtained by the protruding properties of the loop region, as assessed by accessibility computations using atomic coordinates of the toxin and Lee‐Richards algorithm. A synthetic replica of the loop region was obtained in a linear and cyclised form. Within the total anti‐toxin antibody population, we have found and isolated those that recognize the model peptides. A high affinity binding of these specific antibodies to the parent toxin was demonstrated, affording experimental evidence for the antigenic properties of the loop region.
Racemization during aminolysis of mixed and symmetrical anhydrides of N‐alkoxycarbonylamino acids by amino acid anions in aqueous dimethylformamide
Authors: BENOITON N. LEO , LEE YOUNG , CHEN FRANCIS M.F.
Published: August 2010

Racemization attending the aminolysis of symmetrical (Sy) and mixed (Mx) anhydrides (An) of N‐alkoxycarbonylamino acids by amino acid anions in aqueous dimethylformamide has been examined by reversed phase high‐performance liquid chromatography analysis of the N‐protected dipeptides produced. Racemization occurred in most cases when the anion was generated using 1 equiv. sodium hydrogen carbonate, in most cases for MxAn reactions when the base was sodium carbonate, and only in a few cases for SyAn reactions when the base was sodium carbonate. Less racemization was associated with MxAn reactions when the chloroformate of a secondary alcohol had been used for their generation. The change in chirality is explained on the basis of the formation and racemization of the 2‐alkoxy‐5(4H)‐oxazolone, which is greater in the presence of the weaker base because of incomplete deprotonation of the amino acid zwitter‐ion.
Secondary structure of interleukin‐2(Alal25) in unfolded state*
Authors: ARAKAWA TSUTOMU , KENNEY WILLIAM C.
Published: August 2010

Secondary structure of interleukin‐2(Alal25) in unfolded state was examined by circular dichroism (CD). Unfolding of tertiary structure of the protein, as determined by CD, was observed when the solvent pH was decreased below 3.0 or the disulfide bond was reduced. Consistent with the CD results, a stronger fluorescence enhancement of 1‐anilinonaphthalene‐8‐sulfonic acid was observed on acidification or reduction of interleukin‐2(Alal25) relative to that of the native protein, indicating a larger hydrophobic surface exposed to solvent. However, the secondary structure was fully retained in 5% acetic acid or aqueous HCl, pH 3.0. It seemed that α‐helical content of the protein is even greater at pH 2.0. Reduced protein showed a far u.v. CD spectrum indistinguishable from the oxidized one at pH 4.0. These results suggest that the secondary structure of interleukin‐2(Alal25) does not require tertiary structure.
Peptide conformation
Authors: KESSLER HORST , KLEIN MECHTILD , WAGNER KLAUS
Published: August 2010

Six cyclic retro‐analogues of the peptide hormone somatostatin have been synthesized using the solid phase technique. The peptides cyclo(‐Xaa1‐Phe2‐Thr3‐Lys4‐Ybb5‐Phe6‐) and cyclo(‐Phe1‐Xaa2‐Thr3‐Lys4‐Ybb5‐Phe6‐) with Xaa =d‐ or l‐Pro and Ybb =d‐ or l‐Trp were cyclized via the azide method. The conformations of the cyclic hexapeptides in DMSO‐d6 solution were determined by a number of homo‐ and heteronuclear two‐dimensional n.m.r.‐techniques including 2D rotating frame NOE‐spectroscopy. Two‐step coherence transfers, ROE and chemical exchange, are observed for the first time in ROESY spectra. The backbone conformation of the all‐trans cyclopeptides consists of a β‐turn containing the Pro residue in the position i + 1. These retro‐analogues of somatostatin exhibit a high activity in the inhibition of cholate and phalloidin uptake by liver cells (cytoprotective effect); however, the hormonal activities of the natural hormone are completely suppressed. The constitutional and conformational requirements for the cytoprotective activity are discussed.
Ovine prolactin and human growth hormone derivatives
Authors: CARIDAD J.J. , NOWICKI C. , SANTOMÉ J.A. , WOLFENSTEIN‐TODEL C.
Published: August 2010

The α‐amino group of ovine prolactin (oPRL) and human growth hormone (hGH) was selectively modified by transamination with glyoxylic acid. No difference was found in the binding capacity of transaminated oPRL to rat liver lactogenic receptors with respect to its control, although both samples showed a decrease in its binding capacity with reference to the native hormone. This decrease was due to conformational changes caused by the reaction conditions and not by the transamination itself, as shown by the circular dichroism spectra. Transaminated hGH retained the full binding capacity of the hormone. These results suggest that the α‐amino group is not relevant for the binding to lactogenic liver receptors in both lactogenic hormones.
Cholecystokinic activity of Nα‐hydroxysulfonyl‐[Nle28,31]CCK26‐33 analogues modified at the C‐terminal residue
Authors: SUGG ELIZABETH E. , SERRA MARIANGELA , SHOOK JENNIFER E. , YAMAMURA HENRY I. , BURKS THOMAS F. , KORC MURRAY , HRUBY VICTOR J.
Published: August 2010

Three new analogues of Nα‐hydroxysulfonyl‐[Nle28,31]CCK26‐33 are reported in which the C‐terminal l‐Phe33 residue has been replaced by l‐Leu, d‐Phe or N‐methyl‐l‐Phe. Biological evaluation in a series of binding and bioassays demonstrates that both l‐stereochemistry and an aromatic side chain at position‐33 are essential for full agonist activity. While the l‐Leu33 and d‐Phe33 analogues had reduced potencies in stimulating contraction of the guinea pig ileum or gall bladder, the d‐Phe33 analogue was fourfold selective for the ileum. This latter analogue also exhibited apparent partial agonism in the rat pancreatic amylase release assay. The N‐methyl‐l‐Phe33 analogue was almost equipotent to the parent analogue in all bioassays, suggesting that this modification might be useful for introducing enzymatic stability in CCK analogues.
Chemical synthesis of five tubulin antigenic sequences
Authors: ANDREU DAVID , DE LA VIÑA SONSOLES , ANDREU JOSÉ MANUEL
Published: August 2010

Five peptides comprising several potential epitopes of α and β‐tubulin were synthesized by solid phase methods and purified to homogeneity by HPLC. These are RRNLDIERPTYTN (corresponding to positions 214–226 of the α chain of porcine brain tubulin), KDYEEVGVDSVEGE (α, 430–443), EGEFSEAREDMAALEKDYEEVGVDSVEGE (α, 415–443), RYPGQLNADLRKLAVN (β, 241–256) and ESNMNDLVSEYQQYQDATAD (β, 412–431). Appropriate conjugates with carrier proteins rendered all peptides immunogenic, raising antibodies that were cross‐reactive with plate‐adsorbed tubulin in ELISA. This reaction was completely inhibited by the corresponding free peptides, which indicates that these antibodies react specifically with the regions of α and β‐tubulin encompassed by the peptides. The reaction was also fully inhibited by soluble tubulin in a stabilising buffer. In immunoblotting experiments, the anti‐peptide antibodies were useful as specific markers for either α‐ or β‐tubulin in brain extracts. The specificity of the anti‐peptide antibodies for cellular microtubules was also examined by indirect immunofluorescence experiments.
Synthetic amatoxin analogues
Authors: ZANOTTI GIANCARLO , D'AURIA GABRIELLA , PAOLILLO LIVIO , TRIVELLONE ENRICO
Published: August 2010

Amatoxin analogues with d and l‐Ala substitutions in position 5 have been studied by means of 1‐ and 2‐dimensional n.m.r. spectroscopy at 500 MHz. The assignment of all resonances for both analogues has been carried out mostly with the use of COSY and NOESY type experiments. Temperature coefficients for the amide NH protons have been measured and the data compared to known amatoxin structures. The results obtained demonstrate that the rigidity of the bicyclic amatoxin framework is preserved in the d and l‐Ala5 analogues, although the temperature coefficients point to intramolecular hydrogen bonds stronger in the case of the l‐Ala analogue. The 10‐fold decrease of biological activity is discussed in terms of structural features involving also the Trp4 indole accessibility.
Chymotrypsin‐catalyzed peptide synthesis
Authors: BIZZOZERO S.A. , DUTLER H. , RÜCKERT P.
Published: August 2010

The kinetics of peptide‐bond formation catalyzed by δ‐chymotrypsin has been studied for a number of peptide products of different length using fixed concentrations of the acyl component (Ac‐Phe‐OMe. Ac‐Ala‐Ala‐Phe‐OMe, or Ac‐Ala‐Ala‐Tyr‐OMe) and varying concentration of the amino component (H‐Ala‐NH2 or H‐Ala‐Ala‐NH2). The time course of the reactions was followed by monitoring ester consumption and peptide product formation by analytical HPLC. On the basis of a plausible four‐centre mechanistic model, the theoretical time course of these reactions was calculated using rate and equilibrium constants determined by separate kinetic experiments. The excellent agreement observed between the theoretical and the experimental time courses supports the proposed mechanism and provides evidence for the validity of the present kinetic approach. By focusing attention on the rate constants which are critical for efficient synthesis, this mechanistic information constitutes a valuable basis for the use of the enzymatic peptide synthesis in preparative applications.
Peptides as potential virus inhibitors
Authors: LOBL T.J. , RENIS H.E. , EPAND R.M. , MAGGIORA L.L. , WATHEN M.W.
Published: August 2010

Five carbobenzoxylated and d‐amino acid containing‐peptide analogs of the respiratory syncytial virus (RSV) Fl glycoprotein amino terminus were chemically synthesized by solution and FMOC‐solid phase peptide synthesis methods. Several of these peptides, ranging from 3 to 6 residues in length, raised the bilayer to hexagonal phase transition temperature of dielaidoylphosphatidylethanolamine. None of these peptides were specific inhibitors of RSV or herpes simplex virus infection. Two of the series, CBZ‐d‐Phe‐l‐Leu‐Gly‐d‐Phe‐d‐Leu‐d‐Leu and CBZ‐d‐Phe‐l‐Leu‐Gly‐d‐Phe‐d‐Leu‐d‐Leu‐Gly, were active in reducing measles virus‐induced cytopathic effect at 62μg/mL. Others in the series showed some activity at higher doses or activity simultaneously with some cell toxicity. These results support the view that membrane‐stabilizing agents may have non‐specific effects on membranes which are responsible for their antimeasles activity.
Characterization of the receptor binding profile of (3H)‐dermorphin in the rat brain
Authors: AMICHE MOHAMED , SAGAN SANDRINE , MOR AMRAM , DELFOUR ANTOINE , NICOLAS PIERRE
Published: August 2010

Amphibian skin synthesizes a variety of biologically active peptides. Of these, dermorphin (Tyr‐d‐Ala‐Phe‐Gly‐Tyr‐Pro‐Ser‐NH2) is an extraordinarily potent opioid peptide up to 1000 times more active than morphine in inducing analgesia after intracerebroventricular administration. Dermorphin has little in common with the sequence of all hitherto known mammalian opioid peptides and is unique in having ad‐amino acid residue in position 2. Specific binding properties of tritium labeled dermorphin were characterized in the rat brain. Scatchard or Hill analysis of equilibrium measurements performed over a large range of concentrations revealed a single population of dermorphin binding sites with a Kd value of 0.46 nM. Dermorphin and the selective μ‐receptor ligand (d‐Ala2, MePhe4, Gly5‐ol)‐ enkephalin (DAGO) had similar high potencies in competing with (3H)‐dermorphin binding, whereas the inverse holds for the prototypical delta receptor ligand (d‐Pen2,d‐Pen5)‐enkephalin (DPDPE), which exhibited a potency three orders of magnitude lower. Dermorphin was tested for its relative affinity to μ and delta binding sites by determining its potency in displacing (3H)‐DAGO and (3H)‐DPDPE from rat brain membrane preparations. Based on these comparisons, dermorphin exhibited a selectivity ratio Ki(DPDPE)/Ki(DA‐GO) = 100, a value almost identical to that of DAGO, this ligand being considered as the protypical μ‐receptor probe. The high affinity and selectivity of (3H)‐dermorphin together with its very low nonspecific binding make this peptide a useful tool for dissecting the role(s) of the μ‐receptor(s).
Primary structures of carp gonadotropin subunits deduced from cDNA nucleotide sequences
Authors: CHANG YEA‐SHA , HUANG CHANG‐JEN , HUANG FORE‐LIEN , LO TUNG‐BIN
Published: August 2010

The α and β subunits of carp gonadotropin (cGTH) were isolated by high performance liquid chromatography. They were identified to be the subunits of cGTH by bioassay and by partial N‐terminal amino acid sequence analysis. To elucidate the complete primary structures of the α and β subunits of cGTH, cDNA cloning technique was employed. The α and β subunits consist of 95 and 115 amino acid residues, respectively. Homology of the α subunit of cGTH to those of mammalian GTH is around 70%. In comparison, the extent of homology of the β subunit between carp and salmon GTH (75%) is higher than that between fish and mammalian GTH (39–47%). Such comparative data suggest that the α subunit is highly conserved while the β subunit is diversified during the molecular evolution of vertebrate GTH.
Isolation and characterization of growth hormone from a marine fish, bonito (Katsuwonus pelamis)
Authors: NOSO T. , YASUDA A. , KAWAZOE I. , TAKEHARA H. , TAKAHASI A. , SAKAI K. , KAWAUCHI H.
Published: August 2010

Growth hormone (GH) was extracted under alkaline conditions (pH 10) from pituitary glands (6.3 g) of bonito (Katsuwonus pelamis), and subsequently purified by gel filtration, ion exchange chromatography, and reversed‐phase HPLC. The GH was monitored by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) and by immunoblotting with yellowtail GH antiserum at each step of purification. GH activity was determined by an in vivo bioassay. The yield of this hormone was 4.8mg/g wet tissue. Intraperitoneal injection of bonito GH at doses of 0.1 and 1 μg/g body wt at 7‐day intervals resulted in a significant increase in body weight and length of juvenile rainbow trout. Bonito GH antiserum exhibited both species and hormone specificity in radioimmunoassay. However, the bonito GH antiserum as well as yellowtail GH antiserum exhibited hormone specificity but not species specificity in immunoblotting. A molecular weight of 21 000 and an isoelectric point of 7.0 for bonito GH were estimated by SDS‐PAGE and gel electrofocusing, respectively. The complete amino acid sequence of 185 residues was determined by sequencing fragment peptides prepared by chemical and enzymatic cleavages. Sequence comparison of bonito GH with other GHs revealed that there is a significant deletion in the middle of the molecule.
Purification and characterization of Locusta migratoria chymotrypsin
Authors: SAKAL EDNA , APPLEBAUM SHALOM W. , BIRK YEHUDITH
Published: August 2010

A chymotrypsin‐like enzyme (CTLE) was isolated from the digestive tract of the African migratory locust Locusta migratoria migratorioides by ion‐exchange chromatography on diethylaminoethyl (DEAE) cellulose followed by affinity chromatography on phenylbutylamine (PBA) Sepharose. The purity and homogeneity of CTLE have been shown by SDS‐PAGE and on cellulose acetate strips. The enzyme has a molecular weight of 24 000, determined by SDS‐PAGE and on a Sephadex G‐75 calibrated column. It has an isoelectric point of 10.1 and contains 0‐1 half cystine residues. Sequence analysis of the first 20 N‐terminal amino acids has shown 25% homology with bovine chymotrypsin and 40% homology with Vespa crabo and Vespa orientalis chymotrypsins and with Hypoderma lineatum trypsin. The optimal pH for enzyme activity and stability was in the range of 8.5–9.0. The Km and kcat values, determined on substrates for proteolytic, esterolytic and amidolytic activity, were similar to those for bovine chymotrypsin. CTLE was inactivated by PMSF and TPCK, indicating the involvement of serine and histidine in its active site. The enzyme was fully inhibited by the proteinaceous, double‐headed, chymotrypsin‐trypsin inhibitors BBI from soybeans and CI from chickpeas, by chicken ovomucoid (COM) and turkey ovomucoid (TOM), as well as by the Kunitz soybean trypsin inhibitor (STI), which hardly inhibits bovine chymotrypsin. Inhibition studies of CTLE with amino acid and peptide‐chloromethylketones point towards the existence of an extended binding site.
N.m.r. studies of internal mobility in a cyclic tetrapeptide
Authors: KOPPLE KENNETH D. , WANG YU‐SEN
Published: August 2010

The conformation of cyclo(D‐Phe‐D‐Pro‐Ala‐Pro) is reported. Measurements of spin‐lattice relaxation in the rotating frame indicate that this peptide is conformationally less mobile on the microsecond time scale than larger cyclic peptides previously studied. Libration of the Pro‐Ala and Pro‐Phe peptide bond planes is suggested as the source of the small exchange contributions to 1/T1ρ.
Structure of N‐Boc‐L‐Pro‐dehydro‐Leu‐NHCH3
Authors: SINGH T.P. , NARULA PUNIT , CHAUHAN V.S. , SHARMA ASHWANI K. , HINRICHS WINIFRIED
Published: August 2010

The peptide N‐Boc‐L‐Pro‐dehydro‐Leu‐NHCH3 was synthesized to examine the nature of β‐bend as a result of dehydro‐Leu in the sequence. The peptide crystallizes from methanol‐water mixture at 4° in orthorhombic space group P22121 with a = 5.726(1)Å, b = 14.989(4) Å, c = 24.131(9) Å, V = 2071(1) Å3, Z = 4, dm = 1.064(5)gcm‐3 and dc = 1.0886(5)gcm‐3. The structure was solved by direct methods using SHELXS 86 and it was refined by full‐matrix least‐squares procedure to an R value of 0.059 for 957 observed reflections. The peptide is found to adopt a β‐bend type II conformation with φ1=– 51(1)°, ψ1= 133(1)°, φ2= 74(2)° and ψ2= 8(2)°. The β‐bend is stabilized by an intra‐chain hydrogen bond between the carbonyl oxygen of ith residue and the NH of (i + 3)th residue. The five‐membered pyrrolidine ring of Pro‐residue adopts an ideal Cγ‐exo conformation with torsion angles of χ11=– 25(1)°, χ12= 38(1)°, χ2=– 34(1)°, χ14= 20(1)° and χ10= 2(1)°. The side chain conformation angles of dehydro‐Leu residue are χ2= 12(2)°, χ22.1=– 112(2)° and χ22.2= 136(2)°. The crystal structure is stabilized by a network of hydrogen bonds and van der Waals interactions.