bluuebunny/crossref_metadata_2025 huggingface dataset:
Assignment of the 1H 500 MHz n.m.r. spectra of a synthetic eight‐membered ring analogue of somatostatin in aqueous solution
Authors: WYNANTS C. , VAN BINST G. , MICHEL A. , ZANEN J.
Published: August 2010

A somatostatin octapeptide analogue has been synthesized and its conformational properties have been studied by high field n.m.r. in water. All the proton signals of the individual amino acids have been assigned except for the aromatic protons, by use of 2D homonuclear correlation spectroscopy. Neither temperature effects on the amide protons nor the values of the coupling constants provided any significant arguments for predominant conformations. As in the case of somatostatin itself, analogues with this ring size remain very flexible.
Circular dichroism and fluorescence spectroscopic analyses of a proline‐rich glycoprotein from human parotid saliva
Authors: LOOMIS RONALD E. , BERGEY E. JAMES , LEVINE MICHAEL J. , TABAK LAWRENCE A.
Published: August 2010

A proline‐rich glycoprotein (PRG) was isolated from human parotid saliva and examined by circular dichroism and fluorescence spectroscopy. Addition of guanidine hydrochloride to PRG labeled with an extrinsic dansyl probe had no effect on the fluorescence spectra's 511 nm lambda‐max location. Thermodynamic calculations supported the contention that PRG has no significant tertiary structure. Circular dichroism results for PRG were simulated by computer and a secondary structure composed of 70% random coil and 30%β‐form conformation was predicted. Circular dichroism of PRG failed to detect either poly‐L‐proline type I or II structures. Deglycosylation of PRG had no measurable effect on the circular dichroism spectrum, indicating that the carbohydrate side chains had little influence on PRG secondary structure. Based upon mathematical calculations, β‐turns were predicted around three glycosylated Asn residues of PRG. These collective data suggest that PRG is composed of a disordered polypeptide chain with at least three of theN‐linked Asn residues participating in some type of β‐turn.
Synthesis of tritium labeled Boc‐[Nle11] ‐substance P5‐11
Authors: SASAKI N. ANDRÉ , MICHELOT ROBERT , MORGAT JEAN‐LOUIS , GENET ROGER , POTIER PIERRE
Published: August 2010

An optically active, alkynyl analogue of norleucine (Nle), L‐2‐amino‐4‐hexynoic acid (Aha), was substituted for Met in the solution synthesis of the Boc‐protectedC‐terminal heptapeptide analogue of substance P. Treatment of the resulting alkynyl peptide with tritium gas in the presence of 10% Pd/C afforded a Boc‐(Nle11)‐substance P5‐11 with a specific radioactivity of 96 Ci/mmol.
Interaction of allylisothiocyanate with bovine serum albumin
Authors: MURTHY N.V. KISHORE KUMAR , RAO M.S. NARASINGA
Published: August 2010

The interaction of allylisothiocyanate with bovine serum albumin was monitored by “available” lysine estimation, electrometric and spectrophotometric titration, u.v. absorption and difference spectroscopy and electrophoresis. “Available” lysine estimation and electrometric titration indicated that the interaction increased with the reaction pH. Spectrophotometric titration indicated that at least two of the tyrosine groups became masked, probably via H‐bonding, upon interaction of the protein with allylisothiocyanate. The pKint values of the unreacted ∍‐amino groups of lysine and the titrable phenolic groups of tyrosine did not alter upon interaction. The electrophoretic mobility of the protein increased with the interaction. Difference spectral studies suggested thiourea formation.
A new solution synthesis of locust adipokinetic hormone
Authors: KISFALUDY LAJOS , KORENCZKI FERENC , MOHÁCSI TIVADAR , SAJGÓ MIHÁLY , FERMANDJIAN SERGE
Published: August 2010

An alternative synthesis of locust adipokinetic hormone (AKH‐I) is described using the pentafluorophenyl ester method. During purification a by‐product (ca. 20%) was isolated and characterized as [Thr(Bzl)]5‐AKH‐I. Hydrogenolytic cleavage of this benzyl ether bond could not be pursued to completion because partial saturation of the indole ring of Trp started. Thus, this protecting group combination should preferably be avoided. AKH‐I had no effect on the central nervous system of vertebrates.
A “helix‐scissors” mechanism for the hinge‐bending conformational change in phosphoglycerate kinase
Authors: BLAKE C.C.F. , RICE D.W. , COHEN F.E.
Published: August 2010

X‐ray studies of phosphoglycerate kinase (EC 2.7.2.3, PGK) have shown that the enzyme's single polypeptide chain is organized into two separate domains that correspond to the N‐ and C‐terminal halves of the chain. Substrate binding studies and the incorporation of the complete amino acid sequence of horse‐muscle PGK into its X‐ray model suggest that the C‐domain is an ADP/ATP binding unit and that the N‐terminal domain contains the phosphoglycerate binding site and the active site located in a prominent cluster of positively charged residues. Because the distance between these two sites is 12–15 Å, a hinge‐bending of 10°–20° has been proposed to bring the two sites together for catalysis. Independent solution studies of yeast PGK have shown that the radius of gyration decreases significantly on the formation of the ternary complex. This change has been interpreted in terms of a 9°–12° rotation about a hinge in the interdomain region that brings the two domains together. We suggest here a structural basis for the proposed hinge‐bending that involves the rotation of the two helices that form the domain interface about their contact normal carrying their respective domains with them.
Synthesis of sequences 1–16 and 63–95 of Cerebratulus lacteus toxin AIII
Authors: BALASUBRAMANIAM A. , MURPHY R.F. , BLUMENTHAL K.M.
Published: August 2010

Sequences 1–16 and 63–95 of C. lacteus toxin AIII were synthesized on benz‐hydrylamine and PAM resins respectively; the crude peptide products released by HF contained 90 and 33% of the target peptides. Fragment 63–95 was nearly a full agonist, producing > 90% of the hemolytic activity of the intact toxin molecule at 103‐fold higher concentrations. Fragment 1–16 had no activity.
Conformational properties of trans Ac‐Asn‐Pro‐Tyr‐NHMe and trans Ac‐Tyr‐Pro‐Asn‐NHMe in dimethylsulfoxide and in water determined by multinuclear n.m.r. spectroscopy
Authors: STIMSON E.R. , MEINWALD Y.C. , MONTELIONE G.T. , SCHERAGA H.A.
Published: August 2010

Vicinal coupling constants between various nuclei provide backbone and side‐chain conformational information for a series of asparagine‐ and tyrosine‐containing peptides in DMSO and in H2O. By enriching Tyr of Ac‐Asn‐Pro‐Tyr‐NHMe with 15N, it has been possible to distinguish between the resonances of the two side‐chain β protons of Tyr. Analysis of the coupling constants in terms of the distributions of side‐chain conformations in these peptides indicates that the addition of Asn to the Pro‐Tyr sequence leads to a less random conformational distribution. When compared to the side‐chain rotamer distribution of Ac‐Asn‐NHMe and Ac‐Tyr‐NHMe, particular Asn and Tyr side‐chain conformations of Ac‐Asn‐Pro‐Tyr‐NHMe are stabilized in dimethylsulfoxide solution. The interaction(s) which stabilize a unique Tyr side‐chain conformation of Ac‐Asn‐Pro‐Tyr‐NHMe in dimethylsulfoxide are not present in Ac‐Ala‐Pro‐Tyr‐NHMe and are unaffected by the addition of Val‐Pro to the C‐terminus of Asn‐Pro‐Tyr. In water, a preferential stabilization of one Asn side‐chain conformation of Ac‐Asn‐Pro‐Tyr‐NHMe is also observed, while the Tyr side‐chain rotamer distribution is similar to that of Ac‐Tyr‐NHMe. An interaction between the Asn side chain and the Pro‐Tyr‐NHMe backbone was previously shown to stabilize a β‐bend conformation at Pro‐Tyr in water. Data are also presented for Ac‐Tyr‐Pro‐Asn‐NHMe, for which local interactions do not stabilize particular backbone conformations in dimethylsulfoxide or in water. The conformations of the peptides studied here are relatively insensitive to temperatures between 27° and 62°, both in dimethylsulfoxide and in water. The sequences Asn‐Pro‐Tyr and Tyr‐Pro‐Asn occur in ribonuclease A, and these tripeptides serve as models for the interactions involved in the folding of this protein.
Isotopic labeling of tyrosine, followed by 17O n.m.r.
Authors: ECKERT H. , FIAT D.
Published: August 2010

A simple chemical procedure has been developed in order to introduce oxygen‐17 labels into the carboxylic and phenolic sites of L‐tyrosine. Detailed studies of the 17O n.m.r. chemical shift as a function of pH reveal an unusually large titration shift upon the deprotonation of the phenol group. This result suggests that 17O n.m.r. may contribute useful information about side chain properties in peptides and proteins.
N‐Methyl peptides
Authors: VITOUX BERNARD , AUBRY ANDRE , CUNG MANH THONG , MARRAUD MICHEL
Published: August 2010

The natural occurrence of N‐methyl peptides in various plant metabolites has made N‐methylation a subtle and attractive possible modification for structure‐activity relationship studies of endogeneous peptides. However, little is known about the conformational specificity induced by the N‐methylation of a given peptide, and particularly concerning the β‐turn conformation. A spectroscopic investigation (i.r., n.m.r., CD) and X‐ray diffraction experiments have been carried out on tBuCO‐X‐Me‐Y‐NHMe blocked dipeptides (X = Gly, L‐Ala, L‐Pro, and Y = Gly, and L‐ or D‐Ala, Leu, Phe) with reference to the homologous desmethylated species. The influence of the N‐methylation on conformation depends to a large extent on the chirality of the X and Y residues. Homochiral sequences are the most affected, with a strong preference for the βVI‐folded conformation containing a middle cis amide bond. Heterochiral sequences are essentially unaffected and retain the βII‐folded conformation with a trans middle amide bond. Glycine‐containing sequences undergo a more complex perturbation according to the X or Y position of the Gly residue. The available data for larger N‐methyl peptides are consistent with our observations, suggesting that these simple dipeptides well reflect the conformational perturbations induced by N‐methylation on the β‐turn conformation.
Partial specific volume and interaction of glycinin with solvent components in urea and guanidine hydrochloride
Authors: CHANDRA B.R. SURESH , PRAKASH V. , RAO M.S. NARASINGA
Published: August 2010

The interaction of glycinin with urea/guanidine hydrochloride was investigated by determining the apparent partial specific volume of the protein in these solvents. The apparent partial specific volume was determined under both isomolal and isopotential conditions. The preferential interaction parameter with solvent components had values of 0.10 and 0.11 g urea and guanidine hydrochloride respectively per g of protein. In both the cases the interaction was not preferential with water. The total binding of urea/guanidine hydrochloride to glycinin was calculated. Excellent correlation between the calculated value and the total number of peptide bonds and aromatic amino acids residues of protein was observed. The changes in volume upon transferring glycinin from dilute salt solution to urea and guanidine hydrochloride solution were also calculated.
Primary structure determination of a tryptophan‐containing tridecapeptide fromPhyllomedusa rohdei
Authors: MONTECUCCHI PIER CARLO , GOZZINI LUIGIA , ERSPAMER VITTORIO
Published: August 2010

A new tryptophan‐containing peptide has been identified in the methanol extracts of the skin ofPhyllomedusa rohdei.The primary structure proposed is the following: <Glu‐Glu‐Lys‐Pro‐Tyr‐Trp‐Pro‐Pro‐Pro‐Ile‐Tyr‐Pro‐Met‐OH.
Use of microbore high‐performance liquid chromatography for purifying subnanomole levels of polypeptides for microsequencing
Authors: GREGO BORIS , DRIEL IAN R. VAN , GODING JAMES W. , NICE EDOUARD C. , SIMPSON RICHARD J.
Published: August 2010

A procedure for the purification of subnanomole levels of polypeptides has been developed. Reversed‐phase high performance liquid chromatography on short (10 cm or less) microbore (1–2 mm internal diameter) columns has been used to fractionate and purify a number of tryptic peptides generated from approximately 600 pmol of purified murine plasma cell antigen PC‐1, a major membrane glycoprotein on all cells secreting immunoglobulins. The use of reversed‐phase microbore columns permits the recovery of subnanomole amounts of polypeptides from large volumes in high yield (> 90%) and in small eluent volumes (40–60μL) which can be loaded directly onto the gas‐phase sequencer without further concentration. This procedure avoids the severe sample loss which frequently occurs with other concentration procedures such as lyophilization and evaporation. The use of a photodiode‐array detector for identifying tryptophan‐containing peptides from on‐the‐fly, ultraviolet spectra is described. This procedure permits the selection of tryptophan‐containing peptides from complex tryptic digests for use as candidate peptides for oligonucleotide probe construction. Automated Edman degradation was performed on seven tryptic peptides, yielding 110 unique assignments; this corresponds to approximately 11% of the molecule.
Synthesis and biological activity of dynorphin‐(1 ‐ 13) and analogs substituted in positions 8 and 10
Authors: LEMAIRE SIMON , LAFRANCE LUCIE , DUMONT MICHEL
Published: August 2010

Dynorphin‐(1–13) (Dyn‐(1–13)) and various analogs substituted in positions 8 and 10 were synthesized by the solid‐phase technique and analyzed for their ability to inhibit the electrically evoked contraction of the guinea pig ileum (GPI) and to compete with the binding of [3H]‐ethylketocyclazocine (EKC, k ligand), [3H]‐[D‐Ala2, MePhe4‐Gly‐ol5]‐enkephalin (DAGO, μ ligand) and [3H]‐[D‐Ser2, Thr6]‐Leu‐enkephalin (DSLET, δ ligand) to membrane preparations of the guinea pig cerebellum or rat brain. Introduction of Ala in position 8 decreased the activity of the peptide on the GPI by 50% but induced a 2.22‐fold increase in its affinity for the k receptor ([3H]‐EKC binding displacement from guinea pig cerebellum; Ki of 0.05 nM as compared with 0.11 nM for Dyn‐(1–13)). On the other hand, the ability of [Ala8]‐Dyn‐(1–13) to displace the binding of [3H]‐DSLET from rat brain membranes was decreased by a factor of 1.7 while its affinity for the μ receptor was not greatly affected ([3H]‐DAGO displacement; Ki of 0.44nM as compared with 0.50nM for Dyn‐(1–13)). Replacement of position 8 by D‐Ala caused similar changes in the activity of the peptide but the increase in its affinity for the k site was somewhat smaller (Ki of 0.08 nM as compared with 0.11 nM). [D‐Pro10]‐Dyn‐(1–13) was equipotent to [Ala8]‐Dyn‐(1–13) in the GPI but its affinity for the μ binding site was decreased by a factor of 2.7 as compared with Dyn‐(1–13). The affinity of [D‐Pro10]‐Dyn‐(1–13) for the other binding sites (k and δ) was not greatly affected. Replacement of positions 8 or 10 by Trp or D‐Trp decreased the activity on the GPI by more than 50% as well as the affinity for most receptor types. These data indicate that the selectivity of Dyn(1–13) for the k opioid receptor can be increased either by increasing its affinity for the K binding site ([Ala8]‐Dyn‐(1–13)) or lowering its potency for other binding sites, [Ala8]‐Dyn‐(1–13) being less potent on δ sites and [D‐Pro10]‐Dyn‐(1–13) less potent on μ sites.
Ontogeny of bovine neurohypophysial hormone precursors
Authors: LEVY BRIGITTE , CHAUVET MARIE‐THÉERÈESE , CHAUVET JACQUELINE , ACHER ROGER
Published: August 2010

Vasopressin, MSEL‐neurophysin and a glycopeptide, here referred to as copeptin, are three fragments of a common protein precursor processed during axonal transport from hypothalamus to neurohypophysis. Neurohormones and neurophysins purified from 7–9‐month‐old bovine foetuses have previously been shown to be identical with those found in the adult. Copeptin has now been isolated from 7–9‐month and 3‐month‐old bovine foetuses and chemically characterized. It can be concluded from the nature of the three precursors that the same vasopressin gene is expressed in the adult and the 7–9‐month‐old foetus.
General procedure for preparing a reference mixture and determining the amount of dipeptide contaminant in N‐alkoxycarbonylamino acids by high‐performance liquid chromatography
Authors: Benoiton N. Leo , Chen Francis M.F. , Steinauer Rene , Chouinard Michele
Published: August 2010

The amount of N‐alkoxycarbonyl‐dipeptide contaminant in a sample of N‐alkoxycarbonylamino acid is determined by reversed phase high‐performance liquid chromatography on a μBondapak‐C18 column using aqueous methanol or acetonitrile as solvent and u.v.‐absorbance monitoring. Reference mixtures of the N‐substituted monomer and dipeptide are prepared by reaction of the symmetrical anhydride of the N‐alkoxycarbonylamino acid with the sodium salt of the amino acid in aqueous dimethylformamide. The proportions of the two components in the reference mixture are established by 1H‐n.m.r. spectroscopy. N‐Protected‐dipeptide was detected (0.1–0.7%) in seven out of eight commercial benzyloxycarbonylamino acids examined.
Theoretical π‐π* absorption and circular dichroic spectra of β‐turn model peptides
Authors: Sathyanarayana B.K. , Applequist Jon
Published: August 2010

The dipole interaction model, treated by the partially dispersive normal mode method, is used to calculate π‐π* absorption and circular dichroic spectra of β‐turn model peptides in certain conformations. These include Ac‐Gly‐Gly‐NHMe, Ac‐L‐Ala‐L‐Ala‐NHMe, and Ac‐L‐Ala‐Gly‐NHMe in the standard β‐turn conformations I, II, and III of Venkatachalam and cyclo(L‐Ala‐Gly‐ε‐aminocaproyl), cyclo(L‐Ala‐L‐Ala—aminocaproyl), and cyclo(L‐Ala‐D‐Ala‐ε‐aminocaproyl) in the minimum‐energy conformations of Nemethy et al. Boltzmann average circular dichroic spectra of the cyclic compounds agree with experimental spectra in most respects. The results are compared with previous theoretical CD spectra for these molecules and with conformational assignments based on other evidence. Absorption spectra in the π‐π* band are predicted to be moderately sensitive to conformation.
Cyclic peptides
Authors: MIHARA HISAKAZU , IKESUE KOICHI , LEE SANNAMU , AOYAGI HARUHIKO , KATO TETSUO , UENO TAMIO , IZUMIYA NOBUO
Published: August 2010

In order to investigate the role of the L‐2‐hydroxy‐3‐methylbutanoic acid residue at position 2 of AM‐toxin I (cyclic tetradepsipeptide) on necrotic activity for apple leaves, two analogs, [L‐lactic acid2] AM‐toxin I and [L‐2‐hydroxy‐4‐methylpentanoic acid2] AM‐toxin I, were synthesized by the conventional method for peptide synthesis. The toxic activity of the former analog was much weaker than that of the natural AM‐toxin I and the latter analog. CD spectra of AM‐toxin I and these analogs were correlated to those toxic activities, indicating that bulkiness of the side chain at position 2 is important for the maintenance of active conformation and the induction of necrotic activity.
Dipeptide synthesis catalyzed by aminoacyl‐tRNA synthetases from Bacillus stearothermophilus
Authors: NAKAJIMA HIROSHI , KITABATAKE SENJI , TSURUTANI RYOICHI , YAMAMOTO KEIICHI , TOMIOKA ISAO , IMAHORI KAZUTOMO
Published: August 2010

A new approach to enzymatic peptide synthesis by using aminoacyl‐tRNA synthetase (ARS) as a catalyst has been investigated. Four ARSs (AspRS, HisRS, LeuRS and TyrRS) have been purified from a thermophilic bacterium, Bacillus stearothermophilus. By using TyrRS as a catalyst, tyrosine and leucinamide were shown to be condensed in the presence of ATP to give tyrosylleucinamide. In this manner, all of the ARSs investigated catalyzed the peptide synthesis reactions. TyrRS did not have strict specificity for the amino acid derivatives used as substrates and even D‐amino acids were incorporated into peptides fairly easily in this enzymatic reaction. Preparative scale synthesis of L‐Tyr‐L‐LeuNH2 was carried out and from this the scope and limitation of this new enzymatic reaction as a tool to the peptide synthesis has been described.
Interaction of urea with the high molecular weight protein fraction, carmin, from safflower seed (Carthamus tinctorius)
Authors: PRAKASH V.
Published: August 2010

The effect of urea on the high molecular weight protein fraction, carmin, from safflower seedCarthamus tinctorius L, was investigated by the techniques of sedimentation velocity, viscosity, fluorescence and difference spectral measurements, partial specific volume and circular dichroism. The protein underwent dissociation and denaturation simultaneously in the presence of increasing concentration of urea. The protein dissociated to a 2S component and in the process, the buried and exposed residues of tyrosines and tryptophans were perturbed. At 8 M urea concentration, the protein existed completely as a random coil; nearly 535 mol of urea were bound per mol of protein. The results indicated that the subunits of carmin appears to be held principally by noncovalent interactions and are dissociated/denaturated by increasing concentration of urea, leading to the destabilization of the native oligomer.
In vitrointeractions of opioid peptides with phospholipids
Authors: D'ALAGNI MARIA , RODA L. GIORGIO , VITA FABRIZIO
Published: August 2010

The interaction of leu‐enkephalin with phosphatidylserine has been studied with ultraviolet and circular dichroism spectroscopy methods as well as with fluorescence anisotropy techniques. The data reported hereunder confirm the existence of binding between the two species, and also support the hypothesis that not only the tyrosine, but also the phenylalanine residue in the leu‐enkephalin molecule is involved in peptide‐lipid interaction. In addition, ultraviolet and CD evidence, taken together, tend to suggest that both aromatic residues are bound, with a different degree of involvement, to the same region of the lipid molecule. The data reported are discussed in terms of the interaction model previously proposed by us.
Peptides containing dialkylglycines
Authors: COTTON RON , HARDY PAUL M. , LANGRAN‐GOLDSMITH ANDREW E.
Published: August 2010

Three novel peptide analogues containing a residue of the hindered amino acid dibenzylglycine (Dbg) were prepared. Although [Glp6, Dbg7]‐substance P‐(6–11) possessed no substance P agonist or antagonist activity, [Dbg4, Leu5]‐enkephalinamide was 8.4 times as potent as [Leu5]‐enkephalinamide in isolated tissue assays for opioid activity, and proved highly δ‐selective (300‐fold). [N,N‐Di‐allyl Tyr1, Dbg4, Leu5]‐enkephalinamide was a moderately potent opioid antagonist, but showed little μ or δ selectivity. Dbg residues were incorporated using 2‐trifluoromethyl‐4,4‐dibenzyl‐oxazolin‐5‐one. Problems were experienced in coupling even dipeptides to peptides containing amino‐terminal Dbg, and the ‘encapsulation’ of any dialkylglycine residue at the centre of a tripeptide before further coupling offered the best synthetic strategy for future work in such analogues.
Enzymatic semisynthesis of [Leu B30] insulin
Authors: SAKINA KIYOSHI , UENO YOSHIHIKO , OKA TATSUSHI , MORIHARA KAZUYUKI
Published: August 2010

Experimental conditions for the preparation of [LeuB30] insulin by coupling of des‐AlaB30 insulin with Leu‐OBut were determined using Achromobacter protease I and trypsin as catalysts. Successful coupling required a large excess of the amine component (0.8 M), a high concentration of organic cosolvent (35–50%) and neutral pH of the reaction mixture. The coupling yield of Achromobacter protease I after 24 h at 37°C was almost the same or a little higher than that at 25°C. With trypsin, the coupling yield at 37°C after 24 h was considerably lower than at 25°C. This was partly ascribed to the difference in concentration of organic cosolvent at 37°C and 25°C; 35% and 50%, respectively, or possibly of enzyme stability at these temperatures. The maximum product yield was about 90% with both enzymes under optimal conditions. A preparative scale experiment was performed with Achromobacter protease I; the yield of [LeuB30] insulin was 51% using porcine insulin as the starting material. This semisynthetic insulin was identified by HPLC and amino acid analysis. No difference was observed in CD spectra between [LeuB30] insulin and human insulin.
Solution synthesis of antigenic and inhibiting peptides of the human renin prosegment‐2
Authors: FEHRENTZ JEAN‐ALAIN , SEYER RENÉE , HEITZ ANNIE , FULCRAND PIERRE , CASTRO BERTRAND , CORVOL PIERRE
Published: August 2010

The [Tyr40]preprorenin (40–50) peptide methyl ester, an undecapeptide related to the human renin prosegment, has been synthesized using a stepwise strategy with hydrogenolisable protections on the side chains. The final deprotection was very difficult as observed by 1 H NMR and reversed phase HPLC. 2D 1 H NMR spectroscopy of the purified peptide allowed the assignment of all protons.
β‐Endorphin. Biological activity of analogs containing dermorphin and dynorphin sequences: ileum and vas deferens assays
Authors: HO CHEWN‐LAN , LI CHOH HAO
Published: August 2010

Biological activity of synthetic β‐endorphin (β‐EP) analogs containing dermorphin or dynorphin‐A‐(1 – 13) structure has been investigated using the guinea pig ileum and the vas deferens of the mouse, rat and rabbit. Replacement of NH2‐terminal 1–7 segment of camel β‐EP [βc‐EP‐(1–7)] with dermorphin caused a great increase in opiate potency of the analog. [Dermorphin (1–7)] ‐βc‐EP was 120 times more potent than βc‐EP in the guinea pig ileum assay, 49 times more potent in the mouse vas deferens assay; and only 4 times more potent in the rat vas deferens assay. Replacement of NH2‐terminal 1–13 segment of human β‐EP [βh‐EP‐(1–13)] with dynorphin‐A‐(1–13) caused an increase in opiate potency in both the guinea pig ileum and rabbit vas deferens assays, a complete loss of potency in the rat vas deferens assay, and no change in the mouse vas deferens assay. In comparison with dynorphin‐A‐(1–13), the hybrid peptide was less potent in the guinea pig ileum assay as well as in mouse and rabbit vas deferens assay. It is suggested that βc‐EP‐(8–31) facilitates the dermorphin moiety to act on opiate μ and δ receptors but not on the ± receptor, while βh‐(14–31) reduces the action of dynorphin on μ, δ and k receptors.
Chemical modification of proteins: comments and perspectives*
Authors: FEENEY ROBERT E.
Published: August 2010

The use of chemical modification of proteins has increased exponentially during the past two decades. Today the many different uses of chemical modification include determination of relative reactivities of side chain groups, the quantitation of individual amino acids, development of affinity reagents, mechanism‐based reagents for pharmaceutical uses, cross‐linking reagents, special techniques for bioprostheses, blocking reagents for peptide synthesis, and reagents for specific cleavages of peptide bonds. Chemical modification should continue to be a primary tool in protein chemistry. It can supply information or products difficult or impossible to attain by the newer powerful technique ofin vitromutagenesis as well as serve as a supplementary procedure for the latter.
Synthesis of two biologically active fluorescent probes of thymopentin
Authors: AMOSCATO ANDREW A. , BABCOCK GEORGE F. , SRAMKOSKI R. MICHAEL , HYND BARBARA A. , ALEXANDER J. WESLEY
Published: August 2010

This study reports on the synthesis of two fluorescent analogues of thymopentin (TP‐5; Arg‐Lys‐Asp‐Val‐Tyr). A fluorescein isothiocyanate labeled analogue (FITC‐TP‐5) and a stilbene isothiocyanate labeled analogue (SITS‐TP‐5) were extensively purified by ion‐exchange and gel filtration chromatography. Characterization of the coupling site through amino acid analysis, dansylation and N‐terminal cleavage of the fluorescent amino acid yielded results which indicated that both were mono‐labeled analogues derivatized at the N‐terminal. These analogues were shown to be TP‐5‐like in nature by their ability to induce the expression of the Thy 1.2 surface marker on nude mouse prothymocytes in both in vivo and in vitro assays. In addition, these analogues were able to inhibit the specific binding of radiolabeled TP‐5 to human lymphocytes. Initial studies describing the interaction of FITC‐TP‐5 with human lymphocytes are shown.
Interaction of atriopeptin III with lipids and detergents
Authors: EPAND RICHARD M. , STAHL GLEN L.
Published: August 2010

Atriopeptin III, a potent natural hypotensive agent, contains little α‐helical structure but substantial amounts of β‐structure. The peptide can self‐associate at millimolar concentrations or can associate with the anionic phospholipid, dimyristoylphosphatidylglycerol. Both of these processes are accompanied by a conformational change suggesting the formation of an increased amount of β‐structure. The peptide can broaden the transition and lower the transition enthalpy of dimyristoylphosphatidylglycerol. The results demonstrate that a peptide hormone can associate with lipid largely in the form of a β‐structure.
Synthesis of modified tuftsins containing monosaccharides or monosaccharide derivatives
Authors: ROCCHI R. , BIONDI L. , FILIRA F. , GOBBO M. , DAGAN S. , FRIDKIN M.
Published: August 2010

Synthesis of some modified tuftsins is described in which a monosaccharide or a monosaccharide derivative was incorporated in the molecule. Acylation of H‐Thr‐Lys(Z)‐Pro‐Arg(NO2)‐OBzl with D(+)‐gluco‐1,5‐lactone followed by catalytic hydrogenation gave Nα‐gluconyl‐tuftsin. Glycosylation of the carboxyl function of the C‐terminal arginine has been achieved by reacting, through the mixed anhydride procedure, Boc‐Thr‐Lys(Z)‐Pro‐OH with 2‐deoxy‐2‐(NG‐nitroargininamido)‐D‐glucopyranose followed by catalytic hydrogenation and trifluoroacetic acid treatment. O‐Glucosyl‐tuftsin has been prepared by reacting o‐nitrophenyl N‐benzyloxycarbonyl‐O‐[(α + β)2,3,4,6‐tetra‐O‐benzyl‐D‐grucopyranosyl]‐threoninate with H‐Lys(Z)‐Pro‐Arg(NO2)‐OBzl in the presence of 1‐hydroxybenzotriazole. Flash chromatography on silica gel allowed a partial separation of the diastereoisomers, one of which has been isolated in a reasonable yield. The single diasteroisomer and the α + β anomeric mixture were separately deblocked by catalytic hydrogenation and purified by RP‐HPLC.
1H‐n.m.r. investigation of conformational features of cyclic, penicillamine‐containing enkephalin analogs
Authors: MOSBERG HENRY I.
Published: August 2010

Conformational features of a series of cyclic, penicillamine‐containing enkephalin analogs, all of which display selectivity for the delta opioid receptor, were studied by 1H n.m.r. in aqueous solution. Comparison of chemical shifts, coupling constants, and temperature dependence of amide proton chemical shifts suggests different conformational features among the analogs, some of which can be related to the different primary sequences of these peptides. The observation that some of the analogs display disparate individual conformational features while exhibiting similar opioid potency and receptor selectivity suggests that such analogs may share a similar overall topography or at the least maintain the same relative orientations of key portions of the molecule.
Conjugates of catecholamines. VII. Synthesis and β‐adrenergic activity of peptide catecholamine conjugates†
Authors: FUKUDA MUTSUMI , BEECHAN CURTIS M. , VERLANDER MICHAEL , GOODMAN MURRAY , KHAN MANZOOR M. , MELMON KENNETH L.
Published: August 2010

A series of novel catecholamine derivatives has been prepared in which one of the N‐methyl substituents of isoproterenol has been extended by a spacer consisting of a chain of four methylenes which terminates with an amide linkage to a peptide, the point of attachment being via the aromatic amino group ofp‐aminophenylalanine. In one of the derivatives, two catecholamines are attached to the same peptide in this manner. The peptides, which range in size from three to eight amino acid residues and contain phenylalanine, glycine, and L‐α‐amino‐δ‐hydroxyvaleric acid, were synthesized via stepwise and fragment condensation techniques. The β‐adrenergic agonist activities of the derivatives were evaluatedin vitroby measuring the intracellular accumulation of cyclic AMP in S49 mouse lymphoma cells.
Synthesis and conformational study of two cyclic analogues of somatostatin containing an AMPA‐spacer unit
Authors: ELST P. VANDER , GONDOL D. , WYNANTS C. , TOURWÉ D. , van BINST G.
Published: August 2010

Two cyclic somatostatin analogues containing the active sequence Phe7‐D‐Trp8‐Lys9‐Thr10 and a meta‐ or para‐(aminomethyl) phenylacetic acid (AMPA) spacer unit, have been synthesized. A conformational study using 2D n.m.r. techniques (COSY, NOESY) reveals that the conformation of the meta‐AMPA analogue has some analogy with the bio‐active conformation proposed earlier by Veber and colleagues, while in the para‐AMPA analogue an equilibrium exists between a β‐II' turn and a γ‐turn structure. Both analogues show no GH‐inhibition or LH‐inhibition in in vitro assays.
Isosteric conversion of protein carboxyl groups into carboxamide groups. II. Application to lysozyme
Authors: KOOISTRA C. , SLUYTERMAN L.A.æ.
Published: August 2010

For isosteric conversion of carboxyl groups of proteins into amide groups, ammonolysis of protein esters under mild conditions was attempted. Ammonolysis of methyl esters of lysozyme and bovine serum albumin proved to be incomplete. Highly reactive N‐ethylsalicylamide esters of guanylated lysozyme were therefore prepared by subjecting the protein to reaction with N‐ethylbenz‐isoxazolium ion at pH 4.2, 0°. Per molecule, 5–7 ester groups were introduced, with concomitant decrease of activity of 80–90%. Only 0.3 tyrosine was modified. On hydrolysis at pH 9.2 the activity was completely restored. At pH 7.9 three classes of ester groups could be distinguished: one group of high rate of hydrolysis (k1 = 1.5 min‐1), three groups of intermediate rate (k2 = 0.13 min‐1) and two groups of low rate (k3 = 0.018 min‐1). The intermediate rate approximated the rate of hydrolysis of the model compound benzoylglycine N‐ethyl‐salicylamide ester (k = 0.15 min‐1). Ammonolysis at pH 9.2 in 2.0 M ammonia/ ammonium acetate provided complete conversion of the ester groups into amide groups without restoration of activity, confirming the essentiality of certain carboxyl groups. In particular, rearrangement of the ester groups into relatively stable imide groups by O–N acyl migration was found to be completely absent. When native lysozyme was esterified with N‐ethylbenzisoxazolium ion the activity did not completely return on hydrolysis.
Synthesis of biologically active transforming growth factor alpha
Authors: TAM JAMES P.
Published: August 2010

A 50‐amino acid residue transforming growth factor, type alpha (TGFα), secreted in culture by feline‐sarcoma‐virus‐transformed rat embryo fibroblasts, was synthesized by an improved stepwise solid‐phase method with an overall yield of 31%. A deprotection strategy based on the SN2 mechanism using either a low concentration of HF or CF3SO3H‐CF3CO2H in dimethylsulfide was employed to remove most of the benzyl‐derived protecting groups. The more acid resistant protecting groups of Cys and Arg were removed by the SN2 condition using a high concentration of HF. Synthetic TGFα was purified to homogeneity in three steps. Synthetic and natural TGFα were indistinguishable from each other in HPLC and in different assays, including the assay for anchorage‐independent growth of normal rat kidney fibroblasts in soft agar, binding, and stimulating to epidermal growth factor (EGF)‐receptor protein kinase. Furthermore, synthetic TGFα showed similar biological activities when compared with EGF in these assays. Thus, the chemical synthesis of TGFα provided convincing evidence that TGFα is functionally related to EGF and is one of the active principles required for cellular transformation.
Synthesis of conjugates between luteinizing hormone releasing hormone (LH‐RH) and N‐acetyl‐muramyl‐L‐alanyl‐D‐isoglutamine (MDP) models of totally synthetic vaccines
Authors: BERNARD J.M. , GRAS‐MASSE H. , DROBECQ H. , TARTAR A. , LEFRANCIER P. , HOSMALIN A. , CARELLI C. , CHEDID L.
Published: August 2010

Two glycopeptides associating the aminoacid sequence of LH‐RH with MDP were prepared, using a Lys residue as a linker. These conjugates, Nα MDP Ne (LH‐RH)‐Lys and Nα MDP Ne (LH‐RH)‐Lys‐NH2 obtained by condensation of fragments were synthesized by liquid as well as solid phase methods. Both compounds were able to induce anti LH‐RH antibodies and immunological castration. They retained the immuno‐adjuvant activity of MDP. Such antigen‐adjuvant constructs, devoid of carrier and obtained by chemically defined and reproducible synthetic methods could offer suitable tools for structure‐activity relationship studies aiming at defining synthetic vaccines.
Synthesis of TPH‐13, a new tridecapeptide from Phyllomedusa rohdei
Authors: PERSEO GIUSEPPE , GIGLI MAURO , DE CASTIGLIONE ROBERTO
Published: August 2010

The synthesis of TPH‐13 (Glp‐Glu‐Lys‐Pro‐Tyr‐Trp‐Pro‐Pro‐Pro‐Ile‐Tyr‐Pro‐Met‐OH), a tridecapeptide isolated from the skin of the South American frog Phyllomedusa rohdei, is described and alternative approaches are discussed.
Digestion by pancreatic juice of a beta‐casomorphin‐containing fragment of buffalo beta‐casein
Authors: PETRILLI PASQUALE , PUCCI PIERO , PELISSIER J. PIERRE , ADDEO FRANCESCO
Published: August 2010

Degradation by pig pancreatic juice of a beta‐casomorphin‐containing fragment (tryptic peptide corresponding to residues 49–68 of buffalo beta‐casein) was investigated. The FAB/MS (fast atom bombardment mass spectrometry) technique was used to identify the fragments produced by the concerted action of pancreatic proteases. Pancreatic juice, under our experimental conditions, is not able to release beta‐casomorphins or morphiceptin from the tryptic peptide sequence. Furthermore, the present report shows that the rapid hydrolysis of a peptide bond by a single protease can prevent the cleavage of peptide bonds by a different protease. Therefore the formation of some peptides in the gastrointestinal tract can depend on the protease ratio.
Analgesic dipeptide derivatives.
Authors: GARCÍA‐LÓPEZ M.T. , GONZÁLEZ‐MUNIZ R. , HERRANZ R. , MOLINERO M.T. , del RIO J.
Published: August 2010

The syntheses of dipeptide derivatives of the general formula X‐Trp(Nps)‐OR (R = H or Me; X = Lys, Gly, Ala, Leu, Ser, Glu, His, Arg, Orn, Dpr, Gpr and Har) are described. The analgesic activities of Gpr‐ and Har‐containing dipeptides have been evaluated and, in the light of these results, the influence of the side chain length of the basic amino acid on the analgesic effect is studied.
Conformation of reduced glutathione in aqueous solution by 1H and 13C n.m.r.
Authors: YORK MICHAEL J. , BEILHARZ GEORG R. , KUCHEL PHILIP W.
Published: August 2010

We report on the conformation of reduced glutathione in solutions at low and physiological pH, examined with 1H and 13C n.m.r. spectroscopy. The tripeptide in 1H2O was shown to interconvert rapidly between an array of conformers; in addition, the carbon backbone of the glutamyl was more rigid than anticipated if the residue were freely mobile. This restricted motion results from interaction of the α‐amino and α‐carboxyl groups on the glutamyl, with the γ‐Glu‐Cys peptide‐carbonyl and amino, respectively. Our results support theoretical predictions of the conformation but they are at variance with previous ultraviolet spectroscopic and lower field n.m.r. studies.
Stereochemical studies of cyclic peptides.
Authors: RAMAKRISHNAN C. , RAMNARAYAN K. , MANJULA G.
Published: August 2010

Conformational variety in cyclic tripeptides was examined using a combination of stereochemical and energy minimization methods. Four different conformations were found possible: (i) a threefold symmetrical one with all peptide units incisconfiguration, with very little nonplanarity; (ii) an asymmetrical one with allcisunits and with a larger nonplanarity (ccc); and (iii) two possible conformations with twocisand onetranspeptide unit, (cct)1and (cct)2. All these conformations can accommodate both sarcosyl and prolyl residues, which are known to occur incispeptide units. The symmetric and the (cct)2conformations can accommodate only a homo‐isomeric sequence of prolyl residues, and the other two asymmetric conformations (ccc) and (cct)1can accommodate hetero‐isomeric sequence of Pro. In order to relieve some severe steric hindrancescispeptide units show a need‐based flexibility in the peptide bond angles. All the four conformations are known to occur in solid state as well and the results obtained compare reasonably well with the available crystal structure information.
Synthesis and use of peptide substrates for the isolation and characterization of proenkephalin processing enzymes
Authors: MARET GERARD , BLANOT DIDIER , WELTI MANFRED , FAUCHERE JEAN‐LUC
Published: August 2010

Classical methods of peptide synthesis in solution were used for the preparation of the two tetrapeptides alanyl‐lysyl‐arginyl‐tyrosine and acetyl‐alanyl‐lysyl‐arginyl‐tyrosine‐(N‐methylamide). The two compounds were able to be recognized as substrates by proenkephalin processing enzymes and were used for the development of a quantitative assay for these enzymes. The first substrate proved to be convenient, although it was also partially degraded by amino‐ and carboxypeptidases under the conditions of the assay. The second was found to be hydrolyzed by the endopeptidases at too slow a rate to allow its routine use in the assay.
Inhibition of angiotensin‐converting enzyme by angiotensin I analogue peptide inhibitors. A kinetic study
Authors: PÁLDI ANDRÁS , MÓRA MELINDA , BAJUSZ SÁNDOR , GRÁF LÁSZLÓ
Published: August 2010

Angiotensin I analogues with a phosphonic acid group replacing the C‐terminal carboxyl group were shown to be competitive inhibitors of angiotensin‐converting enzyme. This new class of inhibitors was used to study the binding requirements of the angiotensin I‐like ligands to the enzyme's active site. These studies indicate that angiotensin‐converting enzyme recognizes at least five amino acid residues at the C‐terminus of the peptide. The effect of pH on the binding of the most potent inhibitor peptide was compared to Captopril. The two inhibitors showed similar Ki‐pH profiles despite their structural differences. Chloride enhanced the binding of the peptide inhibitor at both pH 9.0 and pH 6.5. At pH 9.0 the inhibitor peptide and the anion bind randomly to the enzyme, while at pH 6.5 the mechanism is ordered. In the latter case, the anion binds first to the enzyme.
Synthesis of HC toxin and related cyclopeptides containing the (L‐Ala‐D‐Ala‐L‐Ada‐D‐Pro) sequence
Authors: JACQUIER ROBERT , LAZARO RENÉE , RANIRISEHENO HANTA , VIALLEFONT PH.
Published: August 2010

In studies leading to HC toxin synthesis, a phytotoxic cyclic tetrapeptide with the sequence cyclo (L‐Ala‐D‐Ala‐L‐Aoe‐D‐Pro), we have determined optimal conditions for the cyclization which constitutes one of the most important steps in the synthesis of the toxin. All four possible sequences containing an optically active precursor, i.e. L‐Ada = (2 S)‐2‐amino‐9‐decenoic acid instead of Aoe, have been prepared and subjected to cyclization. Owing to the differences in racemization risk during activation of the terminal carboxyl aminoacid different cyclization procedures have been applied. Cyclopeptide yields and selectivity between cyclomonomer and dimer both containing the title sequence are mainly controlled by the linear precursor sequence. The cyclic tetrapeptide is only obtained with D‐proline in the C‐terminal position, the best yield reached by the ‐ONSu activation method. Starting from the peptide, the (9S, 9R) HC toxin epimer on the epoxidic carbon atom has been further synthesized in two steps.
Conformation of dioxopiperazines
Authors: JANKOWSKA RENATA , CIARKOWSKI JERZY
Published: August 2010

2,5‐Dioxopiperazines (DOPs), due to their conformational variability, provide a unique type of six‐membered ring, for which the general ring‐puckering formalism may be fully explored. First, it is shown, through the analysis of 65 X‐ray DOP structures, that any experimental conformation of the DOP ring (e.g. one described in terms of its six endocyclic torsion angles) is perfectly described by the formalism, with an average precision being not worse than 0.2° for any torsion angle type. Next, some applications of the treatment are visualized. They include: (i) A very simple test for an immediate check if originally reported values of endocyclic torsion angles are mutually consistent; (ii) Some generalizations concerning conformational dynamics of the DOP ring; (iii) Implications for solution studies and for the investigations of other types of six‐membered rings.
Biochemical characterization of crystallins from frog lenses
Authors: CHIOU SHYH‐HORNG
Published: August 2010

Lens crystallins were isolated from the homogenate of frog (Rana catesbeiana) eye lenses by gel permeation chromatography and characterized by gel electrophoresis, amino acid analysis and circular dichroism. Four well‐defined fractions corresponding to α/β‐, β‐, frog 39.5 kDa and γ‐crystallins comprising the relative weight percentages in the total soluble cytoplasmic proteins of 18%, 15%, 14% and 48% respectively were obtained. The native molecular masses for each purified fraction were determined to be 432, 207, 40 and 23 kDa, respectively. The polypeptide compositions as determined by SDS‐gel electrophoresis revealed the typical subunit structures of mammalian crystallins with the exception of 39.5 kDa monomeric crystallin, which has not been shown in other classes of vertebrate lenses. The spectra of circular dichroism indicate a predominant β‐sheet structure in all four fractions, which also bears a resemblance to the secondary structure of mammalian crystallins. Comparison of the amino acid compositions of frog crystallins with those of mammalian and fish crystallins suggests that γ‐crystallin from the frog is more closely related to that of porcine than fish crystallins, and the frog 39.5 kDa, frog β‐ and lamprey 48 kDa crystallins are probably mutually interrelated.
Reactions of N‐hydroxysulfosuccinimide active esters*
Authors: ANJANEYULU P.S.R. , STAROS JAMES V.
Published: August 2010

N‐Hydroxysulfosuccinimide esters are reactive functional groups employed in a variety of protein modification reagents, especially cross‐linking reagents. For these compounds, hydrolysis is the most important reaction competing for reaction of the esters with nucleophilic groups in proteins. We have employed model compounds to investigate the rates of hydrolysis of N‐hydroxysulfosuccinimide esters and their reactions with the α‐amino group and the side chains of naturally occurring amino acids, under conditions comparable to those used for protein modification studies. The rates of hydrolysis observed were found to be very low, as compared with their rates of reaction with nitrogen nucleophiles found in proteins. Further, within the ranges investigated, the rate of aminolysis was observed to increase more rapidly than the rate of hydrolysis with increasing pH or with increasing temperature. Four amino acid side chains and the α‐amino group were found to react measurably with N‐hydroxysulfosuccinimide esters. At pH 7.4 and room temperature, the order of reactivity was found to be Nα‐Cbz‐histidine < Nα‐Cbz‐lysine ≤ phenylalanine (α‐amino group) ˜N‐acetylcysteine ˜N‐acetyltyrosine; however, the acylimidazole adduct formed with the side chain of histidine was found to be a transient product, subject to hydrolysis or reaction with another nucleophile.