bluuebunny/crossref_metadata_2025 huggingface dataset:
Crystal and molecular structure of L‐lysyl‐L‐valine hydrochloride – a new lysine conformation
Authors: BOQUÉ LL. , VERDAGUER N. , URPÍ L. , FITA I. , SUBIRANA J.A.
Published: August 2010

Thin plates of L‐lysyl‐L‐valine hydrochloride (C11H24N3O3Cl) were obtained using the vapour diffusion technique and analysed by X‐ray diffraction. The unit cell is orthorhombic, space group P212121, a = 5.465(6)Å, b = 19.657(4) Å, c = 13.522(2) Å, V = 1452.6(2.1) Å3 and Z = 4. The structure was solved by direct methods and refined to an agreement factor of 6.7% for 939 reflections with I > 3 σ(I). The lysine side chain conformation (g‐ g‐ tt) has never been found in peptide crystal structures, although it has been reported to occur in proteins. A network of hydrogen bonds between peptide molecules spreads along the a and c directions while no direct bonds are observed to occur between peptides along the b axis direction. This asymmetric pattern of interactions correlates with the crystal morphology.
Conformational analysis of NKB and potent and selective NK‐3 agonists
Authors: LOEUTLLET D. , CONVERT O. , LAVIELLE S. , CHASSAING G.
Published: August 2010

The three‐dimensional structures of NKB, [Arg0] NKB, [Cys2.5] NKB and a potent, water‐soluble, selective NK‐3 agonist, [Pro7] NKB have been studied by1H‐n.m.r. spectroscopy in dimethylsulfoxide and methanol. From this study, the postulated common conformational features of potent NK‐3 agonists are: 1) either an α‐helical structure from residues 2 to 6 or a β‐turn orientation for residues 3 to 6 and 2) a C7orientation for the common phenylalanine, Phe6. TheC‐terminal tetrapeptide should adopt an extended structure.
Design, synthesis and antibacterial activity of cecropin‐like model peptides
Authors: FINK JÜRGEN , BOMAN ANITA , BOMAN HANS G. , MERRIFIELD R.B.
Published: August 2010

In order to investigate structure‐activity relationships of cecropins, model peptides that mimic certain structural features of the cecropin molecules were designed and synthesized. The conformational analysis of cecropins and the design of the model peptides were based on Chou‐Fasman calculations. The peptides were synthesized by solid‐phase methods and purified by reverse‐phase liquid‐chromatography on C18‐silica columns. Their secondary structures were studied by circular dichroism measurements. Antibacterial activities against seven test organisms were determined and compared to the activities of the natural cecropins A and B. These results were discussed on the basis of structural features of the model peptides and on model mechanisms. It was concluded that high antibacterial activity for this class of compounds requires a basic helical amphipathic N‐terminal segment that is connected to a hydrophobic helical C‐terminal segment by a flexible non‐helical hinge region.
Synthesis of O‐phosphonotyrosyl peptides
Authors: VALERIO ROBERT M. , ALEWOOD PAUL F. , JOHNS R.B. , KEMP BRUCE E.
Published: August 2010

A general method for the synthesis of O‐phosphonotyrosyl peptides using solid phase methodology is described. Protected O‐phosphonotyrosine derivatives with the general structure Boc‐Tyr(R2PO3)‐OH (R = methyl, ethyl or benzyl) were prepared as potential synthons for the introduction of O‐phosphonotyrosine residues into peptide sequences. Using 31P n.m.r. spectroscopy, the alkyl phosphate protecting groups (R = methyl or ethyl) were shown to be stable to the coupling, deprotection and neutralization cycles of the Merrifield method of solid phase peptide synthesis. Facile removal of the methyl phosphate protecting groups from the O‐phosphonotyrosyl peptide analogue Ac‐Tyr(Me2PO3)‐NHMe was demonstrated using 45% HBr/acetic acid. The O‐phosphonotyrosyl heptapeptide H‐Leu‐Arg‐Arg‐Ala‐PTyr‐Leu‐Gly‐OH was subsequently prepared using solid phase methodology via incorporation of N2‐tert‐butyloxycarbonyl‐O‐dimethylphosphonotyrosine.
Synthesis of peptide amides by Fmoc‐solid‐phase peptide synthesis and acid labile anchor groups
Authors: STÜBER WERNER , KNOLLE JOCHEN , BREIPOHL GERHARD
Published: August 2010

The preparation and use of new anchor groups for the synthesis of peptide amides by solid‐phase peptide synthesis employing the Fmoc‐method is described. Based on the structure of the 4,4′‐dimethoxybenzhydryl group (Mbh) handles were developed, which could be cleaved by mild acid treatment to give carboxamides. The syntheses and application of Fmoc‐amino‐acid‐(4‐carboxylatomethyloxyphenyl‐4′‐methoxyphenyl) methyl amide and Fmoc‐(4‐carboxylatopropyloxyphenyl‐4′‐methoxyphenyl) methyl amide are described in detail. These handles were coupled to resins and a stepwise elongation of peptide chains proceeded smoothly with Nx‐9‐fluorenylmethoxycarbonyl (Fmoc) amino acid derivatives using a carbodiimide/HOBt mediated reaction. The final cleavage of side‐chain protecting groups and the release of the C‐terminal amide moiety was achieved by the treatment with trifluoroacetic acid, dichloromethane in the presence of scavengers. Various peptides, such as the Leu‐enkephalin amide and Leu‐Gly‐Gly‐Gly‐Gln‐Gly‐Lys‐Val‐Leu‐Gly‐NH2, which is a good substrate for F XIII, were prepared in high yields and purities.
Conformational energy analysis of the chemotactic tripeptide formyl‐Met‐Leu‐Phe and three analogs
Authors: FELLER DAVID C. , ZIMMERMAN S. SCOTT
Published: August 2010

Conformational energy analyses were carried out on the chemotactic tripeptide fMLF (CHO‐Met‐Leu‐Phe) and three analogs fALF (CHO‐Ala‐Leu‐Phe), fMF (CHO‐Met‐Phe), and MLF (H‐Met‐Leu‐Phe). A near‐folded or puckered conformation predominates in all four peptides. The calculated average end‐to‐end distance R of each of the peptides is 7.4 Å 7.6 Å, 7.0 Å, and 7.3 Å, respectively (where bends have R ≤ 7 Å and extended structures have R ≅ 10.5 Å). The puckered conformation calculated for fMLF is similar to that determined experimentally for fMLF in nonpolar solvents and in the protein receptor. The results suggest that maximum chemotactic activity of the peptides depends on a combination of the chemical structure (the presence of N‐formyl‐methionine) and backbone conformation (C, conformation of the first amino acid residue).
Inhibitors of the enkephalin degrading enzymes Modulation of activity of hydroxamate containing compounds by modifications of theC‐terminal residue
Authors: XIE J. , SOLEILHAC J.M. , RENWART N. , PEYROUX J. , ROQUES B.P. , FOURNIÉ‐ZALUSKI M.C.
Published: August 2010

To further characterize the S′2subsite of both the neutral endopeptidase (EC 3.4.24.11, NEP) and aminopeptidase N (EC 3.4.11.2, APN), two enzymes physiologically involved in enkephalin metabolism, a new series of hydroxamate inhibitors containing a cyclic amino acid as the Pi component were synthesized. These amino acids differ by the size of the cycle, the relative position of the functional groups, and their absolute configuration. Highly efficient inhibitors of NEP were obtained whatever the modification on the Pi component, while for APN inhibition, a cyclic β‐amino acid was preferred. The most active inhibitors contained atranscyclopentyl β‐amino acid and aCisor atranscyclohexyl β‐amino acid. When injected intracerebroventricularly in mice, these two latter compounds elicited potent antinociceptive responses on both the jump latency and the fore paw lick times.
Application of the trimethylsilyl trifluoromethanesulfonate deprotecting procedure for the synthesis of porcine peptide YY (PYY)
Authors: ONO SEISHI , KIYAMA SHINYA , KITAGAWA KOUKI , FUTAKI SHIROH , NAKAMURA TOMOHIKO , TAIKE TAKASHI , AKITA TADASHI , SUMI SHOICHIRO , INOUE KAZUTOMO , FUJIMURA MASAKI , TOBE TAKAYOSHI , YAJIMA HARUAKI
Published: August 2010

A 36‐residue peptide amide corresponding to the entire amino acid sequence of porcine peptide YY (PYY) was synthesized by assembling eight peptide fragments of established purity, followed by hard acid deprotection with 1m trimethylsilyl trifluoromethanesulfonate in trifluoroacetic acid. β‐Cycloheptylaspartate, Asp(OChp), was employed to minimize the base‐catalyzed succinimide formation. When administered to dogs, synthetic PYY was active as natural peptide in its effects on exocrine pancreatic secretion and pancreatic tissue blood flow.
Solid phase synthesis of bioadhesive analogue peptides with trifluoromethanesulfonic acid cleavage from PAM resin
Authors: SWERDLOFF MICHAEL D. , ANDERSON SUSAN B. , SEDGWICK R. DONALD , GABRIEL MINA K. , BRAMBILLA RAYMOND J. , HINDENLANG DAVID M. , WILLIAMS JON I.
Published: August 2010

Several decapeptides related to consensus peptide sequences found in the natural polyphenolic proteins from the sea mussels M. edulis or M. californianus have been synthesized in high yields and purities. The peptides were prepared by solid phase peptide synthesis on PAM support resins utilizing BOC protection strategies. The product peptides were then deprotected and cleaved from the PAM resins with TFMSA in a two‐step procedure. The course of selective peptide deprotection with TFMSA was followed by high resolution 13C n.m.r. on aliquots of the peptide resin swollen in DMF and optimal deprotection times for batch processing of the peptide resins were chosen based on the n.m.r. results. Our two‐step deprotection and cleavage procedure using TFMSA was shown to be at least as good as the usual HF procedure for the class of synthetic peptides described in this paper. N to O rearrangements of threonine and/or serine residues in the synthesized decapeptides were observed in both procedures but were reversible following treatment with ammonium bicarbonate. Coupled HPLC‐u.v./vis spectroscopy, HPLC‐MS and FAB MS analytical methods were used to characterize the product peptides and to provide substantial amino acid sequence information.
Physico‐chemical properties of the lectin from winged bean (Psophocarpus tetragonolobus (L.) DC) tubers
Authors: SHET MANJUNATH S. , MADAIAH M.
Published: August 2010

A galactose specific lectin from the tubers of winged bean, Psophocarpus tetragonolobus (L.) DC, has been isolated and purified to homogeneity by chromatography on DEAE‐cellulose, Biogel P‐60, and CM‐Sephadex C‐50 columns. It is a glycoprotein containing 6% total sugar, comprising mannose, fucose, and xylose in a ratio of 7:2:1, and 0.85% amino sugars. The Mr of the lectin by SDS‐PAGE and analytical ultracentrifugation is 29000. In contrast, gel filtration gave an Mr of ca. 48 000. The lectin has a pI value of ∼ 9.5 and a sedimentation coefficient value of 3.0. The purified lectin agglutinates both trypsinized and untrypsinized erythrocytes of human (types A, B, and AB), rabbit, and rat erythrocytes but not human (type O) and sheep erythrocytes. The hemagglutinating activity of the lectin is inhibited by D(+) galactose and other galactose containing oligosaccharides, and its derivatives. The sugar specificity of the lectin appears to be directed to glycosides containing non‐reducing α‐d‐galactopyranosyl residues; v‐d‐galactosides are poor inhibitors. The amino acid composition of the lectin has revealed that it is rich in aspartic acid and poor in sulphur‐containing amino acids. Lysine alone appears to be the NH2‐terminal residue of the lectin. The purified lectin is fairly stable over a wide range of pH and temperature. The lectin is found to be mitogenic by transforming normal human lymphocytes at a concentration of 0.1 mg/mL.
N.m.r. study on conformation of Ac‐Thr(α‐GalNAc)‐Ala‐Ala‐OMe as a model for mucin type glycoprotein
Authors: MIMURA YOSHIMASA , INOUE YOSHIO , MAEJI N. JOE , CHÛJÔ RIICHRIÔ
Published: August 2010

This study report on the results of high resolution 1H n.m.r. investigations on Ac‐Thr(α‐GalNAc)‐Ala‐Ala‐OMe 1 as a mucin type model glycopeptide of antifreeze glycoprotein (AFGP) in both dimethyl sulfoxide (DMSO) and H2O. The temperature dependence of amide proton chemical shifts strongly suggested the presence of the intramolecular hydrogen bond between the amide proton of GalNAc and the carbonyl oxygen of the Thr residues. Due to this bond, the orientation of the sugar residue of 1 appears to be fairly restricted relative to its peptide backbone. Despite the lack of the clear evidence for such intramolecular hydrogen bond in H2O, 1H coupling constant data suggested the structural similarity of 1 in DMSO and H2O, indicating the presence of the intramolecular hydrogen bond even in H2O, which may play an important role in determining the orientation of the sugar moiety with respect to the peptide backbone in glycoprotein.
Effects of thioamide substitution for the enkephalin conformation
Authors: DOI MITSUNOBU , TAKEHARA SHIGERU , ISHIDA TOSHIMASA , INOUE MASATOSHI
Published: August 2010

The crystals of Boc‐Tyr‐Gly‐Gly‐PheΨ[CSNH]Leu‐OBzl monohydrate (C40H51N5O8S H2O), a monothionated Leu‐enkephalin analogue, were obtained with space group P21 a = 12.616(3), b= 9.347(2), c= 18.548(5) Å, β= 96.31(4)°. The structure was elucidated by X‐ray diffraction analysis, and refined to the R value of 0.091 for the observed 3294 reflections. Two antiparallel molecules related by a pseudo twofold symmetry were stabilized to each other by four intermolecular hydrogen bonds. The molecular conformation was bent at the Phe residue, and the extended moiety of the Tyr‐Gly‐Gly fragment was almost perpendicular to that of the Phe‐Leu residues. Consequently the molecule, as a whole, formed an L‐shape conformation with a slightly left‐handed helicity.
Role of hydrophobicity in protein structure is overestimated
Authors: BLOEMENDAL MICHAEL , MARCUS YIZHAK , SIJPKES ANDRE H. , SOMSEN GUS
Published: August 2010

Some microcalorimetrically measured and theoretically calculated thermodynamic interaction coefficients of amino acid compounds in aqueous and peptidic systems have been collected, and are reported here. They indicate that although hydrophobicity contributes to the stability of the native structure of proteins, its influence on their exact configuration is limited.
Characterization of β‐endorphin in human pituitary by fast atom bombardment mass spectrometry of trypsin‐generated fragments*
Authors: DASS CHHABIL , FRIDLAND GENEVIEVE H. , TINSLEY PETER W. , KILLMAR JOHN T. , DESIDERIO DOMINTC M.
Published: August 2010

A novel mass spectrometric method possessing a high level of structural specificity is described for characterization in biological fluids and tissues of endogenous β‐endorphin of the human amino acid sequence (βh‐EP). The method is based upon purification of tissue extracts by an RP‐HPLC gradient, followed by trypsinolysis of that particular HPLC fraction corresponding to the elution time of synthetic βh‐EP. The tryptic digest of that endogenous βh‐EP fraction was purified further by a second RP‐HPLC gradient. A unique tryptic fragment selected from the second gradient was analyzed by fast atom bombardment mass spectrometry and B/E linked‐field scan MS/MS techniques to provide molecular weight and amino acid sequence‐determIntng fragment ion information, respectively, of that fragment. Collectively, these independent analytical methodologies provided unequivocal structure evidence for the presence of endogenous βh‐EP in human pituitary. The method was established first by utilizing synthetic Ph‐EP to optimize experimental parameters, and then applied to the analysis of βh‐EP in post‐mortem human pituitary extracts. The suitability of the present method for semiquantitation of tissue extracts is also demonstrated. The corresponding detection limit of the synthetic βh‐EP was 90fmol, and human pituitary contained 1.5pmol of βh‐EPmg−1 protein. The method can be extended readily to the analysis of β‐endorphin derived from other species and tissues.
Solid‐phase synthesis of phosphopeptides
Authors: OTVOS LASZLO , ELEKES ILONA , LEE VIRGINTA M‐Y.
Published: August 2010

We report the solid‐phase synthesis of peptides containing O‐phosphoserine. Coupling was with commercially available Fmoc‐amino acid pentafluorophenyl esters, with base used at each cycle to cleave Fmoc. Phosphorylation of those serine residues left unprotected on the peptide‐resin was achieved with dibenzylphosphochloridate, and finally trifluoroacetic acid was used to remove side‐chain protecting groups (including the benzyl groups used for the phosphate), and to cleave the peptide from the resin in the same step. This synthetic strategy enables the preparation of peptides with individual, selectively phosphorylated residues. Alternative approaches to introduce protected phosphate and continue with coupling of further amino acids were less advantageous due to the lability of the phosphate group to base and to steric hindrance.
Amino acid sequence of a neutral phospholipase A2 (III) in the venom of Bungarus fasciatus
Authors: LIU C.‐S. , LEU H.‐L. , CHANG C.‐S. , CHEN S.‐W. , LO T.‐B.
Published: August 2010

Two phospholipases were found in the venom of Bungarus fasciatus, one in fraction III, the other in fraction X of the chromatographic separation. A neutral PLA2(III) purified from fraction III was subjected to amino acid sequencing by means of an automated sequenator applied to the intact RCM‐PLA2 (III) and the individual peptides obtained from HPLC separation of the three types of enzymatic peptides. PLA(III) was shown to consist of 118 amino acid residues with 14 half‐cystines. It is 65% homologous to the basic PLA2 obtained from fraction X.
1H n.m.r. conformational studies on the C‐terminal octapeptide of oxyntomodulin, a β‐turn locked by a salt bridge
Authors: AUMELAS ANDRÉ , AUDOUSSET‐PUECH MARIE‐PASCALE , HEITZ ANNIE , BATAILLE DOMINIQUE , MARTINEZ JEAN
Published: August 2010

The octapeptide Lys‐Arg‐Asn‐Lys‐Asn‐Asn‐Ile‐Ala (Arg4 in the human sequence) is the C‐terminal part of porcine oxyntomodulin, an endogeneous peptide which is a potent inhibitor of stimulated acid secretion. This octapeptide exhibits the whole range of biological activities of the parent hormone. In the present work we report an 1H n.m.r. investigation of the conformational properties of the octapeptides of pig and human sequences in dimethylsulfoxide‐d6 (DMSO) solution. The various resonances were assigned on the basis of two‐dimensional COSY and NOESY experiments. Other experiments such as (i) temperature and concentration dependence of the amide proton chemical shifts, (ii) effects of ionic strength, (iii) comparison of the spectra with different analogues, were performed. We showed that in DMSO, the conformation of the octapeptide is directly related to the ionisation state of the C‐terminus carboxyl group of alanine. In carboxylic state, the peptide adopts an extended conformation, while in the carboxylate state the four last residues (Asn‐Asn‐Ile‐Ala) are involved in a type II β‐turn structure probably locked by a salt bridge between the carboxyl group of Ala8 and the e ammonium group of Lys4 (or the guanidinium group of Arg4). These observations provide an insight into the possible conformational tendencies of this peptide in biological media.
Asparagine coupling in Fmoc solid phase peptide synthesis
Authors: GAUSEPOHL H. , KRAFT M. , FRANK R.W.
Published: August 2010

To investigate side reactions during the activation of side chain unprotected asparagine in Fmoc‐solid phase peptide synthesis the peptide Met‐Lys‐Asn‐Val‐Pro‐Glu‐Pro‐Ser was synthesized using different coupling conditions for introduction of the asparagine residue. Asparagine was activated by DCC/HOBt, BOP (Castro's reagent) or introduced as the pentafluorophenyl ester. The resulting peptide products were analyzed by HPLC, mass spectrometry and Edman degradation. In the crude products varying amounts ofβ‐cyano alanine were found, which had been formed by dehydration of the side chain amide during carboxyl activation of Fmoc‐asparagine. A homogeneous peptide was obtained by using either side chain protected asparagine derivatives with BOP mediated activation or by coupling of Fmoc‐Asn‐OPfp. Fmoc‐Asn(Mbh)‐OH and Fmoc‐Asn(Tmob)‐OH were coupled rapidly and without side reactions with BOP. For the side chain protected derivatives the coupling was as fast as that of other Fmoc‐amino acid derivatives, whereas couplings of Fmoc‐Asn‐OH proceed more slowly. However, during acidolytic cleavage both protection groups, Mbh and Tmob, generate carbonium ions which readily alkylate tryptophan residues in a peptide. Tryptophan modification was examined using the model peptide Asn‐Trp‐Asn‐Val‐Pro‐Glu‐Pro‐Ser. Alkylation could be reduced by addition of scavengers to the TFA during cleavage and side chain deprotection. A homogeneous peptide containing both, asparagine and tryptophan, was obtained only by coupling of Fmoc‐Asn‐OPfp.
Correlation of β‐bend conformations of tetrapeptides with their activities in CD4‐receptor binding assays
Authors: SHAH DIPAK , CHEN JAMES M. , CARTY ROBERT P. , PINCUS MATTHEW R. , SCHERAGA HAROLD A.
Published: August 2010

Conformational analysis, based on ECEPP (Empirical Conformational Energy Program for Peptides) using the chain build‐up procedure, was applied to determine the low‐energy conformations for a series of tetrapeptides. The tetrapeptides are components of larger peptides which have been found to bind to the CD4 receptor of monocytes. Several previous studies have implicated the tetrapeptide units investigated here as being critical to the biological activities of the full peptides. Five such tetrapeptides were studied: Ser‐Ser‐Asn‐Tyr (from ribonuclease A), Thr‐Thr‐Asn‐Tyr (from peptide T, known to block human immunodeficiency virus from attaching to CD4+ T cells), Thr‐Ile‐Asn‐Tyr (from polio virus coat protein, which is less active than the other peptides in binding to CD4 receptors), Ser‐Ser‐Ala‐Tyr (from the gp 120 coat protein of human immunodeficiency virus, a variant of the peptide T sequence, active in blocking viral attachment to CD4+ cells), and the tetrapeptide from an active synthetic pentapeptide, Asn‐Thr‐Lys‐Tyr (from Asn‐Thr‐Lys‐Tyr‐Thr). Using a 7 kcal/mol cutoff, the low‐energy conformations for each peptide were computed. Approximately 20000 conformations were computed for each tetrapeptide. Residue probability profiles were determined for each tetrapeptide. All tetrapeptides except for the polio sequence showed flexibility in the sense that many low‐energy conformations were possible. In previous studies, it was postulated that the critical tetrapeptide units would adopt conformations similar to the one observed in a segment of ribonuclease A, residues 22–25, a β‐bend, which is part of an octapeptide segment (residues 19–26) that is homologous to the sequence of peptide T. It was found that the four most active tetrapeptides exhibited 5–10 times the probability of being superimposed on the native structure of the 22–25 segment of ribonuclease A as the less active polio tetrapeptide. The results are compatible with the hypothesis that peptide T adopts the native ribonuclease β‐bend (at residues 22–25) for its Thr‐Thr‐Asn‐Tyr sequence.
X‐ray studies on crystalline complexes involving amino acids and peptides
Authors: PRASAD G. SRIDHAR , VIJAYAN M.
Published: August 2010

Crystals of dl‐arginine hemisuccinate dihydrate (I)(monoclinic; P21/c; a = 5.292, b = 16.296, c = 15.203 Å; α= 92.89°; Z = 4) and l‐arginine hemisuccinate hemisuccinic acid monohydrate (II) (triclinic; P1; a = 5.099; b = 10.222, c = 14.626 Å; α= 77.31, β= 89.46, γ= 78.42°; Z = 2) were grown under identical conditions from aqueous solutions of the components in molar proportions. The structures were solved by direct methods and refined to R = 0.068 for 2585 observed reflections in the case of (I) and R = 0.036 for 2154 observed reflections in the case of (11). Two of the three crystallographically independent arginine molecules in the complexes have conformations different from those observed so far in the crystal structures containing arginine. The succinic acid molecules and the succinate ions in the structures are centrosymmetric and planar. The crystal structure of (II) is highly pseudosymmetric. Arginine‐succinate interactions in both the complexes involve specific guanidyl‐carboxylate interactions. The basic elements of aggregation in both the structures are ribbons made up of alternating arginine dimers and succinate ions. However, the ribbons pack in different ways in the two structures. (II) presents an interesting case in which two ionisation states of the same molecule coexist in a crystal. The two complexes provide a good example of the effect of change in chirality on stoichiometry, conformation, aggregation, and ionisation state in the solid state.
Acid‐base properties and microspeciation of six angiotensin‐type octapeptides
Authors: NYÉKI OLGA , OSZTAS ERZSÉBET , NOSZÁL BÉLA , BURGER KÁLMÁN
Published: August 2010

The acid‐base chemistry of six angiotensin II analogues (A 11) was analyzed by determining two types of terms. Macroconstants were used to characterize the molecular proton‐binding and dissociating ability of the octapeptides, and group constants to quantitate the submolecular basicity of each individual protona‐tion site of the derivatives. The group constant values indicated that some sites (Arg‐guanidino, Tyrphenolate, C‐terminal carboxylate) were of similar basicity in the different analogues, while others (Hisimidazole, N‐terminal amino) were significantly different. The group constant values are interpreted by taking into consideration the intramolecular effects of the adjoining moieties and are used for microspeciation.
Synthesis of cyclic analogues of cholecystokinin highly selective for central receptors
Authors: RODRIGUEZ MARC , AMBLARD MURIEL , GALAS MARIE‐CHRISTINE , LIGNON MARIE‐FRANÇOISE , AUMELAS ANDRÉ , MARTINEZ JEAN
Published: August 2010

Cyclic CCK analogues in which positions 28 and 31 have been replaced by lysine residues and whose side chains are bridged by a succinic moiety, were synthesized. They were tested for their ability to inhibit the binding of 125I‐BH‐CCK‐8 to isolated rat pancreatic acini and to guinea pig brain membranes. These cyclic CCK‐analogues were compared to the potent CCK analogue Boc‐[Nle28,31‐CCK‐7 and to Boc‐Trp‐Leu‐Asp‐Phe‐NH2, analogue of CCK‐4. These cyclic compounds appeared to be highly selective for central CCK receptors.
Synthesis and immunosuppressive activities of conformationally restricted cyclosporin lactam analogues
Authors: LEE JIHAE PARK , DUNLAP BRIAN , RICH DANIEL H.
Published: August 2010

Cyclosporin A (CsA, 1), an immunosuppressive cyclic undecapeptide, is known to adopt predominantly a single conformation in chloroform solution, characterized in part by a type II′β‐turn encompassing Abu‐Sar‐MeLeu‐Val (residues 2–5). In order to evaluate whether this β‐turn is bound by the receptor, we previously had prepared a conformationally restricted β‐turn analogue, (δ‐lactam)3,4CsA (3), which was found to retain only weak immunosuppressive activity, a result that could indicate that steric hindrance between receptor and the lactam atoms in 2 diminished activity or that the type II′β‐turn is not a feature in the bioactive conformation of CsA. In an attempt to distinguish between these two possibilities, we have synthesized two new CsA analogues, (γ‐lactam)3,4 CsA (3) and (des‐N‐methyl‐lactam)3,4 CsA (4), which contain less sterically demanding conformational restrictions. The immunosuppressive activity of each analogue (4–13% and 7–17%, respectively, relative to CsA), measured in an assay that determined the inhibition of concanavalin A stimulated thymocytes, is essentially equipotent with that of the δ‐lactam. The chemical shifts and temperature dependencies of the protons in analogues 3 and 4 are very similar to the corresponding protons in CsA and in 2, which suggests that the solution conformations of the small lactam analogues are very similar to that of the δ‐lactam 2. The synthesis of the lactam components and the corresponding CsA derivatives is described. Reduction in the size of the lactam ring does not lead to enhanced immunosuppressive activity.
Analgesic, receptor binding, and peripheral opioid activities of synthetic dermorphin‐dynorphin hybrid peptides
Authors: HO CHEWN‐LANG , KO JANE‐LING , HWANG LING‐LING , WANG KUNG‐TSUNG
Published: August 2010

The effects of substituting the enkephalin moiety of dynorphin with the dermorphin sequence were studied on the receptor preference, analgesic, and peripheral opioid potencies by using synthetic dermorphin‐dynorphin hybrid peptides as the probe. Replacement of the enkephalin moiety of dynorphin with the dermorphin or dermorphin1–5sequence caused a remarkable increase in analgesic potency, and a 3‐6 fold increase in potency of binding against [3H]‐dihydromorphine. The potency of receptor binding against [3H]‐EKC was also increased by incorporation of the whole dermorphin sequence into the dynorphin molecule. In the presence of NaCl (100mM), the effect of enhancing binding against [3H]‐EKC due to dermorphin substitution disappeared, suggesting the contribution of opioid μreceptor. Peripheral opioid activities assayed by various smooth muscle preparations showed that dermorphin incorporation caused a decrease in the potency of inhibition of the contractions of the guinea pig ileum and the rabbit vas deferens, no change in potency on the mouse vas deferens, and a marked increase in the inhibition of the rat vas deferens. Among the peripheral opioid activities only that assayed with the rat vas deferens appears to correlate approximately with the analgesic and the receptor binding activities. Judging from the relative potencies obtained from all assays, it is evident that theN‐terminal dermorphin moiety, but not theC‐terminal dynorphin fragment, dominates the opioid activity and receptor preference of the hybrid peptide.
Equine growth hormone Detection of immunoreactive sequences using poly‐ and monoclonal antibodies
Authors: MOLLERACH‐GOBBI BEATRIZ , RETEGUI LILIA A. , PEÑA CLARA
Published: August 2010

The immunochemical behavior of several fragments of equine growth hormone (eGH) was examined using competitive binding assays with antibodies (Abs) to eGH obtained from different sources. Antigenicity was detected within the sequences 5–72 and 73–123 by rabbit Abs to eGH and by three mouse monoclonal antibodies (MAbs) produced by using bovine growth hormone as immunogen, but showing heteroclitic properties towards eGH. The polyclonal Abs to eGH also recognized as immunoreactive two smaller peptides corresponding to the amino acid residues 52–72 and 110–123. By contrast, the heteroclitic Abs to eGH developed by hypopituitary patients therapeutically injected with human growth hormone failed to react with any eGH‐derived fragment. The rabbit polyclonal Abs and the mouse MAbs scarcely discriminated between native and S‐carbamidomethylated eGH, while the heteroclitic human Abs detected a clear difference between the native and the modified hormone.
Probes for vasopressin receptors Attachment of affinity and fluorescent groups in vasopressin
Authors: BUKU ANGELIKI , GAZIS DIANA
Published: August 2010

Fluorescent, photoreactive, and biotinylated analogs of vasopressin have been prepared in which one of these three groups has been attached to a reactive amino group in either position 4 or position 7. Using solid phase methodology, we have synthesized two active parent compounds, [l‐desamino,4‐lysine,7‐hydroxy‐prolinelarginine vasopressin and [l‐desamino,7‐aminoproline]arginine vasopressin, and acylated them to obtain biotinyl, azidobenzoyl, and fluoresceinyl derivatives. We have also prepared analogs in which a “spacer arm” was inserted between lysine in position 4 and the marker group. Some of these derivatives have good antidiuretic activity and could be valuable probes in studying hormone‐receptor interaction and in receptor visualization and purification.
Synthesis of cyclic peptides on solid support Application to analogs of hemagglutinin of influenza virus
Authors: PLAUÉ S.
Published: August 2010

In order to mimic a well‐known loop structure (site A) of the hemagglutinin of influenza virus, a series of cyclic peptides derived from thc region 139–147 were synthesized. The lactam analogs cyclised between the N‐terminus Cys 139 and the β‐carboxyl of aspartic acid 148 (small loop) or the ENHZ of lysine 148 via succinimidyl linker (large loop) were synthesized by the solid phase method. Cyclisation was directly performed on the solid support prior to final cleavage of the peptide. We describe two protection schemes which allow us to obtain different loop sizes derived from the same sequence. Eight of the analogs contained relatively large ring structures (up to 38 membered). For protection of the side chain of aspartic acid in combination with N‐α‐Fmoc protection, the cyclohexyl ester was more satisfactory than the benzyl ester with respect to imide formation. When the rate of cyclodimerisation, as a function of resin substitution, was compared to the rate of cyclic monomer formation, it was found that dimerisation was proportional to the charge of the resin. Furthermore, a comparison of the recently reported BOP reagent over the classical DIPC/HOBt method for the cyclisation reaction shows that in our case the reaction proceeded more rapidly by the BOP procedure although it gave a less pure crude product.
Thiolysis of the 3‐nitro‐2‐pyridinesulfenyl (Npys) protecting group An approach towards a general deprotection scheme in peptide synthesis
Authors: ROSEN OREN , RUBINRAUT SARAH , FRIDKIN MATI
Published: August 2010

The hydroxylic side‐chain functional groups of serine, threonine, hydroxproline and tyrosine, the α and ε‐amino moieties of lysine and the thiol group of cysteine were masked by the 3‐nitro‐2‐pyridinesulfenyl (Npys) protecting group. Deprotection was mildly affected by thiolysis with either 2‐mercaptopyridine and 2‐mercaptomethyl imidazole (O‐ and N‐Npys) or with 3‐mercaptoacetic acid and 2‐mercaptoethanol (S‐Npys). Thiolysis was monitored spectrophotometrically and was completed in a rather short time. Incorporation of the Npys group into a whole and single thiolyzable deprotection scheme is suggested.
Synthesis, purification, and properties of a peptide that enhances the activation of human [Glu1]plasminogen by tissue plasminogen activator and retards fibrin polymerization
Authors: CHIBBER BAKSHY A.K. , URANO SHOKO , CASTELLINO FRANCIS J.
Published: August 2010

Solid phase synthesis of the hexapeptide, GPRVVE, which represents the amino terminal six amino acids of the α‐chain of human fibrin, yielded a product that contained a modified glutamic acid. The nature of the modification was established as the Friedel‐Crafts acylation product of the peptide and anisole, the latter reagent employed in the HF deblocking step. The anisoylated peptide selectively enhanced the activation rate of native [Glu1]plasminogen by recombinant tissue plasminogen activator, accelerated clot lysis, and retarded the polymerization of nascent fibrin
Solid phase peptide synthesis utilizing 9‐fluorenylmethoxycarbonyl amino acids
Authors: FIELDS GREGG B. , NOBLE RICHARD L.
Published: August 2010

9‐Fluorenylmethoxycarbonyl (Fmoc) amino acids were first used for solid phase peptide synthesis a little more than a decade ago. Since that time, Fmoc solid phase peptide synthesis methodology has been greatly enhanced by the introduction of a variety of solid supports, linkages, and side chain protecting groups, as well as by increased understanding of solvation conditions. These advances have led to many impressive syntheses, such as those of biologically active and isotopically labeled peptides and small proteins. The great variety of conditions under which Fmoc solid phase peptide synthesis may be carried out represents a truly “orthogonal” scheme, and thus offers many unique opportunities for bioorganic chemistry.
Conformational free energy of armadillo metmyoglobin
Authors: KELLY LENORE , HOLLADAY LESLIE A.
Published: August 2010

The conformational free energy of armadillo metmyoglobin was examined over a pH range of 4.4–8.0 and a guanidinium chloride concentration of 0–2.3 M. For isothermal unfolding at 25′ essentially the same value was obtained for the conformational free energy from all the data: 27 ± 2 kJ/mol. These data suggest that the armadillo has the least stable metmyoglobin of any mammal thus far examined. The cooperativity of the unfolding with respect to denaturant is considerably less than for other mammalian myoglobins. On unfolding only three to four side chains with a pKAof 6 in the unfolded protein are protonated instead of the six found for horse and sperm whale myoglobins.
Isolation and structure elucidation of bovine pineal arginine vasopressin: arginine vasotocin not identified
Authors: BENSON BRYANT , EBELS IETSKINA , HRUBY VICTOR J.
Published: August 2010

A large number of reports have demonstrated the presence of neurohypophysial hormone‐like peptides in mammalian pineal glands and an antigonadotropic function has been ascribed to pineal arginine vasotocin (AVT). We have undertaken large scale purification of bovine pineal neurohypophysial hormone‐like substances which demonstrate mouse mammary milk‐ejection activity (ME‐activity) in vitro. Peptides with ME‐activity were extracted from more than 5 kg of bovine pineal glands. ME‐activity containing peptides were found in both high (Mr∼ 10 000–15 000) and low (Mr∼ 500–1000) Mr species from Sephadex G‐25 chromatography of 0.2 n acetic acid extracts. After ultrafiltration in 5% formic acid, the neurohypophysial hormone‐like peptides were localized to an ultrafiltration Mr 500–1000 retentate. A homogeneous peptide, which shared an identical retention time (RT) and amino acid sequence with synthetic 8‐arginine vasopressin (AVP), was isolated by serial semipreparative high performance liquid chromatography. On the other hand, the non‐mammalian nonapeptide AVT was not identified.
Proposed solution structure of endothelin
Authors: PERKINS T.D.J. , HIDER R. C. , BARLOW D.J.
Published: August 2010

A model is proposed for the 3‐dimensional structure of endothelin, a potent vasoconstrictor and pressor peptide from vascular endothelium. The model is derived through protein structure prediction and circular dichroism studies, and is based on the atomic coordinates for the bee‐venom peptide apamin. The model derived shows the same turn‐helix motif as observed for apamin and mast‐cell degranulating peptide. On the basis of this model we suggest possible strategies for endothelin antagonist design, and note that this motif may be common in a number of peptides acting on channel proteins.
A facile method for preparation of t‐butyloxycarbonylamino acid p‐nitroanilides
Authors: NODA KOSAKU , ODA MIYUKI , SATO MIYAKO , YOSHIDA NORIO
Published: August 2010

A series of p‐nitroanilides of t‐butyloxycarbonylamino acids, including Boc‐Trp, Boc‐Asn, Boc‐Gln, Boc‐Ser(Bu′), Boc‐Thr(Bu′), Boc‐Asp(OBu′), Boc‐Lys(TFA), and Boc‐His(Boc), were prepared conveniently by the mixed anhydride method using 2,2‐dimethylpropanoic chloride (pivaloyl chloride). The products were obtained in 40‐60% yields after purification by column chromatography on Sephadex LH‐20. p‐Nitroanilide of histidine was purified after deprotection of Boc‐His(Boc)‐pNA and obtained as H‐His‐pNA‐2HCl.
A synthetic protein corresponding to the entire vpr gene product from the human immunodeficiency virus HIV‐1 is recognized by antibodies from HIV‐infected patients
Authors: GRAS‐MASSE H. , AMEISEN J.C. , BOUTILLON C. , GESQUIÈRE J.C. , VIAN S. , NEYRINCK J.L. , DROBECQ H. , CAPRON A. , TARTAR A.
Published: August 2010

The 95 amino acid‐protein encoded by the non‐structural vpr gene of the human immunodeficiency virus type 1(LAV‐1BRU isolate) was chemically synthesized by solid phase methodology. The sythetic vpr protein was characterized by amino acid analysis, sequence analysis, RP‐HPLC, and urea‐SDS PAGE. Using a radioimmunoassay, antibodies to the synthetic protein were detected in sera of 25% of HIV 1‐seropositive patients tested. Western blot analysis suggested that the antibodies preferentially recognize the dimeric form of vpr
Cyclization under mild conditions of salicyloyl‐dipeptides
Authors: CALCAGNI A. , LUISI G. , LUCENTE G. , PINNEN F. , ROSSI D.
Published: August 2010

Carboxy‐activated linear peptides 6(a‐c) of general formula Sal‐Xaa‐Pro‐ONp (Xaa = Phe; Gly; Aib) were synthesized and treated at room temperature with 1,8‐diazabicyclo [5,4,0] undec‐7‐ene (DBU) in benzene solution. The tetrahedral adducts (oxa‐cyclols) 7, 11 and 12, tautomeric forms of the corresponding 10‐membered cyclodepsitripeptides, have been isolated in each of the three models examined. These adducts, which contain the hydroxylated carbon atom located at the junction between two 6‐membered rings, do not show a tendency to isomcrize into the corresponding macrocyclic lactones, regardless of the nature of the substituents on the Cα carbon atom of the central residue. Partially cyclized dimeric products 8 and 13, identified as N‐(Sal‐Xaa‐Pra)‐dioxopiperazines (Xaa = Phe: Aib), have been also isolated from the cyclization reactions.
Conformations of the central transforming region (Ile 55‐Met 67) of the p21 protein and their relationship to activation of the protein
Authors: CHEN JAMES M. , LEE GRACE , BRANDT‐RAUF PAUL W. , MURPHY RANDALL B. , GIBSON KENNETH D. , SCHERAGA HAROLD A. , RACKOVSKY SHALOM , PINCUS MATTHEW R.
Published: August 2010

The GTP‐binding p21 protein, encoded by the ras‐oncogene, becomes transforming if amino acid substitutions are made at critical positions in the polypeptide chain, e.g., at Gly 12, Gly 13, Ala 59, Gln 61 and Glu 63. Most of these substitutions occur in two phosphate‐binding loop regions, Tyr 4‐Thr 20, herein designated as segment 1, and Ile 55‐Met 67, herein designated as segment 2. These two segments are homologous to two corresponding regions in the two purine nucleotide binding proteins, bacterial elongation factor (EF‐tu) (Val 12‐Thr 28 corresponds to segment 1; His 78‐Ile 92 corresponds to segment 2) and adenylate kinase (ADK) (Lys 9‐Cys 25 corresponds to segment 1 and Tyr 95‐Arg 107 corresponds to segment 2). We find that the conformations of the segment 1 region in the p21 protein, EF‐tu and ADK are similar to one another and that the conformation of the segment 2 region of EF‐tu is superimposable on that of segment 2 of ADK. Furthermore, the relative position of the two segments in EF‐tu is strikingly similar to that of the two segments in ADK. In the originally proposed X‐ray structure for the p21 protein, the conformation of segment 2 in the p21 protein is not similar to that found for the other two proteins, and its disposition relative to segment 1 and the remainder of the protein is also different from that observed for the other two proteins. We previously computed the low‐energy conformations for segment 2 in the p21 protein using conformational energy calculations based on ECEPP (Empirical Conformational Energy Program for Peptides) with Gln, Pro, Leu, Lys, and Arg at positions 61. We also predicted that the position of segment 2 in the p21 protein adopts a position and conformation identical to the ones observed in EF‐tu and ADK in the activated protein. We now find that the computed global minimum lowest‐energy structure is superimposable on segment 2 of EF‐tu and on the 95‐107 sequence of ADK and that our predicted position for this segment in the activated protein coincides with that found in a more recent X‐ray crystallographic structure for the p21 protein.
Crystal and molecular structure of two geometrically restricted chemotactic tripeptides, analogues of formyl‐methionine‐leucine‐phenylalanine*
Authors: MICHEL ANDRÉ G. , LAJOIE GILLES , HASSANI CHAKIB AMEZIANE
Published: August 2010

The crystal structures of HCO‐Met‐Leu‐Phe‐OC(CH3)3, (CH25H39N3O5S), fMLP‐OtBu, and HCO‐Metψ[CSNH]‐Leu‐Phe‐OCH3, (C33H33N3O4S2), fMSLP‐OMe, have been determined by single crystal X‐ray diffraction, and their conformational properties investigated by molecular mechanics energy calculations. Crystals of fMLP‐OtBu are monoclinic, space group P21, a = 12.027(4), b = 9.492(3), c = 12.660(4) Å, β= 101.99(3)°, Z = 2; those of fMSLP‐OMe are orthorhombic, space group P212121, a = 7.130(1), b = 12.097(2), c = 31.060(5) Å, Z = 4. The first compounds fMLP‐OtBu is the t‐butyl ester of the tripeptide fMLP that represents one of the most potent compounds in inducing the lysozyme release from human neutrophils that reflects the chemotactic activity. From the crystal structure, it is shown that the orientation of the phenylalanine side chain is largely affected by the presence of the bulky group. fMSLP‐OMe was shown to be inactive after thionation of the methionine residue in the original tripeptide. Nevertheless, the crystal structure does not reveal any influence of the presence of the thionated peptidic bond on the backbone conformation. The X‐ray results have been used to generate parameters for empirical energy calculations. Subsequently, a strategy based on random generation of conformations followed by energy‐minimization was applied to investigate the conformational space of thiopeptides, in comparison with normal peptides. From molecular free energy calculations, it is shown that the main influence of the introduction of a thioamide bond on the molecular structure is to prevent the existence of C7eqconformations involving the thiomethionine residue. Consequently, a larger number of conformers are found to form intramolecular hydrogen bonds involving the formyl group, reducing its availability to interact with the receptor. For the first time, the theoretical prediction of the existence of C7eq conformations for fMLP is made. The resulting conformers are compared to previously active structures of these chemotactic agents.
Synthesis and crystal structures of Boc‐L‐Asn‐L‐Pro‐OBzl·CH3OH and dehydration side product, Boc‐β‐cyano‐L‐alanine‐L‐Pro‐OBzl
Authors: STROUP A.N. , COLE L.B. , DHINGRA M.M. , GIERASCH L.M.
Published: August 2010

Boc‐L‐Asn‐L‐Pro‐OBzl:C21H29O6N3·CH3OH, Mr= 419.48 + CH3OH, monoclinic, P21, a= 10.049(1), b= 10.399(2), c= 11.702(1)Åβ= 92.50(1)°, V = 1221.7(3)Å3, dx= 1.14g cm‐3, Z = 2, CuKα (λ= 1.54178 Å), F(000) = 484 (with solvent), 23°, unique reflections (I > 3σ(I)) = 1745, R = 0.043, Rw= 0.062, S = 1.66. Boc‐β‐cyano‐L‐alanine‐L‐Pro‐OBzl: C21H27O5N3, Mr= 401.46, orthorhombic, P212121, a= 15.741(3), b= 21.060(3), c= 6.496(3)ÅV= 2153(1)Å3, dx= 1.24g·cm‐3, Z = 4, CuKα (λ= 1.54178 Å), F(000) = 856, 23°, unique reflections (I > 3σ(I)) = 1573, R = 0.055, Rw= 0.078, S = 1.86. The tert.‐butyloxycarbonyl (Boc) protected dipeptide benzyl ester (OBzl), BOC‐L‐Asn‐L‐Pro‐OBzl, prepared from a mixed anhydride reaction using isobutylchloroformate, BOC‐L‐asparagine, and HCI·L‐proline‐OBzl, crystallized with one methanol per asymmetric unit in an extended conformation with the Asn‐Pro peptide bond trans. Intermolecular hydrogen bonding occurs between the methanol and the Asn side chain and between the peptide backbone and the Asn side chain. A minor impurity due to the dehydration of the Asn side chain to a β‐CNaia crystallized with a similar extended conformation and a single intermolecular hydrogen bond.
Solid phase synthesis of the retro viral nucleocapsid protein NCp10 of Moloney Murine Leukaemia virus and related “zinc‐fingers” in free SH forms
Authors: CORNILLE FABRICE , MELY YVES , FICHEUX DAMIEN , SAVIGNOL ISABELLE , GERARD DOMINIQUE , DARLIX JEAN‐LUC , FOURNIE‐ZALUSKI MARIE‐CLAUDE , ROQUES BERNARD P.
Published: August 2010

The core of retroviruses contains a highly conserved, low molecular weight, basic protein that binds nucleic acids and is essential for genomic RNA packaging. The 56 amino acid protein, NCp10, of Moloney Murine Leukaemia virus (MoMuLV) has the CysX2CysX4HisX4Cys zinc finger‐like motif shared by all retrovirus nucleocapsid proteins. The native protein and five modified peptides containing the zinc binding domain were synthesized by solid phase in order to investigate the structural and biochemical role of Zn2+ chelation in MoMuLV NCp10 activity. The purity of the synthetic molecules was verified by HPLC and their sequences were confirmed by amino acid analysis and sequencing in the case of NCp10. Thiol dosage agreed with the theoretical value of free cysteine for all these molecules. Fluorescence measurements performed on synthetic NCp10 and zinc finger fragments showed that the tryptophan quantum yield was Zn2+ ‐dependent allowing a 1:1 stoichiometry for the complex to be determined. The apparent affinity constant of NCp10 for the metal was estimated to be superior to 106 M‐1. The synthetic protein, in the presence of Zn2+ ions, possesses all the biological properties of NCp10 isolated from virions. It catalyzes both the MoMuLV RNA dimerization and the annealing of the replication primer tRNAPro onto MoMuLV RNA.