bluuebunny/crossref_metadata_2025 huggingface dataset:
Stepwise synthesis of a GalNAc‐containing cluster glycoside ligand of the asialoglycoprotein receptor
Authors: FINDEIS MARK A.
Published: August 2010

A series of peptidylglycoside derivatives, including the tris(GalNAc‐aminohexyl) glycoside of tyrosyl(glutamyl)‐glutamate (1) which is known to have sub‐nanomolar affinity for the asialoglycoprotein receptor (ASGPr), have been synthesized using the protected tetra‐O‐acetyl aminohexyl glycoside (9) of N‐acetylgalactosamine. The N‐succinyl derivative of 1 was also prepared, providing a derivative for bioconjugate formation via carboxyl activation of the glycopeptide. Use of the protected glycoside 9 affords synthetic intermediates with increased solubility in organic solvents that are easier to purify and use in subsequent synthetic manipulations in comparison with compounds containing unprotected glycosides. These synthetic procedures will be generally applicable to the preparation of related compounds to probe binding to the ASGPr and the use of these cluster glycosides as ligands for targeted delivery to the liver.
Susceptibility of glycans to β‐elimination in Fmoc‐based O‐glycopeptide synthesis
Authors: MELDAL MORTEN , BIELFELDT TIM , PETERS STEFAN , JENSEN KNUD J. , PAULSEN HANS , BOCK KLAUS
Published: August 2010

order to investigate the possible extent of β‐elimination occuring in Fmoc‐based continuous‐flow solid‐phase glycopeptide synthesis, the influence of the pKb of the base used for Nα‐deprotection has been studied. A glycosylated pentapeptide as synthesized using 50%morpholine, 10%, piperidine or 2% DBU, respectively, in DMF for deprotection. The dehydropentapeptide Nα‐.Ac‐Thr‐Thr‐δAba‐Val‐Thr‐NH2, which would be formed in the case of β‐elimination, was prepared independently and used as a control in HPLC analysis; however, this product was not formed under any of the deprotection conditions applied. Furthermore, a 23 amino acid long glycopeptide from human intestinal mucin was prepared using 2% DBU as a base for Fmoc cleavage, and similarly no β‐elimination was observed. The glycopeptide products were subjected to a prolonged treatment with sodium hydroxide in methanol/water without significant formation of byproducts, and the pure glycopeptides were isolated and characterized by 1H‐NMR spectroscopy.
Echistatin: the refined structure of a disintegrin in solution by 1 H NMR and restrained molecular dynamics
Authors: ATKINSON R.A. , SAUDEK V. , PELTON J.T.
Published: August 2010

The structure of the disintegrin echistatin has been determined by 1H NMR, distance geometry calculations and restrained molecular dynamics simulations. The structure has been refined from the preliminary distance geometry calculations with the inclusion of additional 1H NMR data and hydrogen bonds identified in early stages of the molecular dynamics calculations. The calculations reported here allow a distinction to be made between the two possible disulfide bridging patterns ‐ echistatin is crosslinked as follows: Cys2‐Cysll, Cys7‐Cys32, Cys8‐Cys37, Cys20‐Cys39. The final set of structures gives an average pairwise root mean square distance of 0.100 nm (calculated over the backbone atoms of residues Ser4‐Cys20 and Asp30‐Pro40).

The core of echistatin is a well defined though irregular structure, composed of a series of non‐classical turns crosslinked by the disulfide bridges and stabilised by hydrogen bonds. The RGD sequence is located in a protruding loop whose stem is formed by two rigid, hydrogen‐bonded strands (Thr18‐Cys20, Asp30‐Cys32). The RGD sequence is connected to this structure by short, flexible segments. High (but not unlimited) mobility is probably necessary for fast recognition and fitting to the integrin receptors. Sequence variability among the disintegrins is found in the segments flanking the RGD sequence, suggesting that these may be important in conferring specificity for the receptors.
Structure and solubility of interleukin‐2 in sodium dodecyl sulfate
Authors: ARAKAWA TSUTOMU , PHILO JOHN , KENNEY WILLIAM C.
Published: August 2010

The solubility of an interleukin‐2 (C125A) in an aqueous sodium dodecyl sulfate (SDS) solution was examined as a function of SDS concentration. A stock interleukin‐2 solution at 1.1 or 0.31 mg/mL was mixed with one‐tenth volume of a stock SDS solution at various concentrations. At intermediate SDS concentrations, the interleukin‐2 showed nearly complete precipitation, while lower or higher SDS concentrations resulted in an increase in protein solubility. Circular dichroic analysis indicated that the protein remaining in the supernatant at low or high SDS concentrations has an altered conformation.

Sedimentation velocity analysis of interleukin‐2 in 0.1%/SDS showed that about half the protein was monomeric, with the remainder as large aggregates with a broad size distribution from 20 to 100 S (Mr≊ 105‐107). In contrast, interleukin‐2 in 0.01% SDS has a much smaller distribution of aggregates, sedimenting at ca. 2–10 S.
Characterisation of the solution conformation of a cyclic RGD peptide analogue by NMR spectroscopy allied with a genetic algorithm approach and constrained molecular dynamics
Authors: SANDERSON P.N , GLEN R.C , PAYNE A.W.R , HUDSON B.D. , HEIDE C , TRANTER GE , DOYLE PM , HARRIS CJ
Published: August 2010

The solution conformation of a cyclic RGD peptide analogue, cyclo‐(S,S)‐2‐merrcaptobenzoate‐arginine‐glycine‐aspartate ‐2‐mercaptoanilide, has been determined via two independent approaches for the searching of conformational space and identification of conformations consistent with NMR and CD spectroscopic data: (i) the use of a binary genetic algorithm and (ii) a molecular dynamics simulation. Inter‐proton distances were obtained via analysis of cross‐peak volumes from a two‐dimensional ROESY NMR spectroscopy experiment at 600 MHz and were used as constraints for the computational calculations. The mercaptoanilide amide proton resonance chemical shift had a very small temperature coefficient, indicating that this proton was hydrogen‐bonded. Circular diehroism data showed that, in solution, the torsion angle about the disulfide bond was negative, consistent with one of the distinct conformations around this bond in the 200 ps molecular dynamics simulation. The backbone conformations of the structures resulting from the two different approaches were very similar.
Crystal structure of deltakephalin: a γ‐selective opioid peptide with a novel β‐bend‐like conformation
Authors: FLIPPEN‐ANDERSON JUDITH L. , DESCHAMPS JEFFREY R. , WARD KEITH B. , GEORGE CLIFFORD , HOUGHTEN RICHARD
Published: August 2010

The solid‐state structure of deltakephalin (Tyr‐D Thr‐Gly‐Phc‐Leu‐Thr) has been determined by single‐crystal X‐ray diffraction. Deltakephalin (DTLET) is a synthetic opioid peptide which differs from enkephalin in that ad‐Thr has been substituted for Gly2and a sixth residue,l‐Thr, has been added. Clear colorless plates obtained using vapor diffusion and macro‐seeding crystallization techniques were monoclinic; space group C2 with u = 27.389(5), b = 9.205(2), c = 16.788(2) Å, β= 98.87(2) ˚ and V= 4181.4(14) Å3. The asymmetric unit contained one molecule of DTLET and six molecules of water, giving a calculated density of 1.28 g cm−3. The crystal structure revealed that DTLET has a pseudo type I'β‐bend which is stabilized by an intramolecular side‐chain to backbone hydrogen bond. This is the first reported observation of a pseudo β‐bend conformation in a solid‐sate structure of an enkephalin analog.
Cyclic hexapeptide NK‐2 antagonists
Authors: HÖLZEMANN G. , Löw A. , HARTING J. , GREINER H.E.
Published: August 2010

The synthesis of 11 cyclic hexapeptides, some of which contain a carbohydrate side chain moiety, is described in this paper. A glycosylamine was coupled without hydroxyl protecting groups either directly or via a butyric acid spacer to the side chain of glutamic acid, leading to β‐N‐glycosylated peptides. All peptides described are selective NK‐2 antagonists. The binding affinity to the NK‐2 receptor ranges from 7 × 10−7 to 1 × 10−8 M, whereas at the NK‐I receptor the IC50 was > 10−5 M with the exception of cyclo(‐Lys(Boc)‐Trp‐Phe‐Gly‐Leu‐D‐Leu‐) (I), which shows low affinity to the NK‐1 receptor (IC50= 9 × 10−6 M). The antagonist activity is determined in the hamster trachea assay. pA2‐Values range from 7.1 to 7.8. The results demonstrate the broad range of side chains which can be accommodated at the glutarnine position without a major drop in activity. The different charges of the lysine and the glutamic acid peptides indicate that the interaction with the receptor at this position is not determined by ionic forces. Rather, we expect that conformational flexibility allows differently charged amino acid residues to be accommodated by the receptor.
2,4‐Dimethyl‐5(4H)‐oxazolone as reagent for activation and coupling of N‐substituted aspartic acid
Authors: BENOITON N. LEO , CHEN FRANCIS M.F.
Published: August 2010

Reaction of the title oxazolone with N‐benzyloxycarbonyl‐L‐aspartic acid in dichloromethane followed by addition of phenylalanine methyl ester. HCI and N‐methylmorpholine gave a 90% yield of a mixture of α‐and β‐isomers of Z‐aspartylphenylalanine methyl esters in a 7:3 ratio. Reaction of the oxazolone with N‐acetyl‐L‐aspartic acid anhydride gave a 75 % yield of crystalline N‐acetyl‐L‐aspartic acid anhydride. tert‐Butoxycarbonylaspartic and Z‐glutamic acids also underwent activation to give the anhydrides.
Conformational study of cyclosporin A in acetone at low temperature
Authors: VERHEYDEN PATRICIA , JASPERS HENDRIKA , WOLF ELKE DE , BINST GEORGES VAN
Published: August 2010

The conformation of cyclosporin A (CsA), an undecapeptide with seven N‐methylated amino acids, was studied in acetone at 193 K. Previous studies of the conformation of CsA in different solvents, in the cyclosporin‐cyclophilin complex and in complexes with LiCl showed that the conformation of the free and the bound CsA are different. Differences were observed at the conformation of the MeLeu9‐MeLeu10 peptide bond, which is cis in solution and trans in the complex, and in the orientation of the amide protons and the N‐Me groups. By using acetone, which is a proton acceptor, we wanted to influence the orientation of the amide protons. In the conditions used in this study a new conformation is found, which differs as well from the one previously observed in solution as from the Conformation observed in the complex. This conformation has a cis peptide bond between MeLeu9 and MeLeu10. The trans conformation of the peptide bond MeLeu9‐MeLeu10, which is necessary for biological activity, was not induced. One of the amide protons is involved in an intramolecular H‐bridge stabilising a β‐turn around Sar3MeLeu4, and three of the seven NMe groups are oriented to the centre of the molecule. © Munksgaard 1994.
Conformational preferences of oligopeptides rich in α‐aminoisobutyric acid. III. Design, synthesis and hydrogen bonding in 310‐helices
Authors: BINDRA VANDANA A. , KUKI ATSUO
Published: August 2010

Two sterically constrained peptides {iBoc‐Aib‐Aib‐Aib‐DkNap‐Leu‐Qx‐Ala‐Aib‐Aib‐F1, (Dk4Qx6[7/9]) and iBoc‐Aib‐Aib‐Aib‐DkNap‐Leu‐Aib‐Ala‐Aib‐Aib‐Fl, (Dk47/9)} containing α‐aminoisobutyric acid (Aib) and Aib‐class amino acids in conjunction with selected mono‐α‐alkyl amino acids were synthesized by an optimized TBTU/HOBt procedure. The use of Aib‐class amino acids (e.g. DkNap and Qx), defined and discussed here, gives rise to the same overwhelmingly 310‐helical backbone conformation as that provided by simpler Aib‐rich peptides and homopeptides. The synthetic α,α‐dialkylamino acids (DkNap, Qx) are aromatic homologues of the known alicyclic variants of Aib, the Ac5c and Ac6c amino acids. Two new organic solubilizing groups for peptides, iBoc and 2‐methoxyethylamine, are introduced. The 1H nuclear magnetic resonance analyses of the Dk4s/p[7/9] and Dk4Qx6[7/9] peptides demonstrate the unambiguous 310s/b‐helical hydrogen bonding pattern of these peptides, confirming the design objective of these sequence patterns containing greater than 50% Aib and Aib‐class composition. © Munksgaard 1994.
Synthesis and characterization of site‐specific biotinylated probes for the motilin receptor*
Authors: MACIELAG MARK J. , PEETERS THEO , DEPOORTERE INGE
Published: August 2010

The solid‐phase synthesis of two porcine motilin derivatives, specifically biotinylated on the side chain of Lys20), was accomplished by preactivation of the protected amino acids Nx‐(9‐fluorenylmethoxycarbonyl)‐Nε‐biotinyl‐L‐lysine and Nx‐(9‐fluorenylmethoxycarbonyl)‐Nε‐[N‐(biotinyl)‐6‐aminohexanoyl]‐L‐lysine with BOP/ HOBt/DIEA (1:1:2.5) followed by coupling to the support‐bound peptide substrate. The biotin moiety was stable to TFA cleavage and repetitive cycles of acylation, as evidenced by the high level of purity (>80%) of the crude peptides. This direct synthetic approach complements existing orthogonal protection strategies for the site‐specific biotinylation of peptides. The derivatized peptides were purified by RP‐HPLC and characterized by mass spectral and amino acid analysis. In binding studies using a rabbit antral smooth muscle homogenate, both [Leu13, Lys20 (Nε‐biotinyl)]porcine motilin (3) and [Leu13, Lys20 (Nε‐[N‐(biotinyl)‐6‐aminohexanoyl])]porcine motilin (4) possessed nearly equal affinities for the motilin receptor (IC50= 0.89 and 1.2 nM, respectively) as native porcine motilin (1) (IC50= 0.76 nM). The biotinylated peptides were also highly potent in tissue bath assays employing rabbit duodenal smooth muscle segments. In contrast, commercially available [Nx‐biotinylPhe1]porcine motilin (5) had markedly lower affinity in the binding assay (IC50= 30 nM). The relative bioactivities of these receptor probes are in accord with previous synthetic studies on motilin which demonstrated the importance of the amino‐terminal segment in the high affinity interaction between the peptide and its receptor. Analog 3 retained high affinity for the motilin receptor in the presence of avidin. Therefore, this peptide is expected to be a valuable tool for the isolation and identification of motilin receptors. © Munksgaard 1994.
Interaction of 3′ ‐O‐caffeoyld‐quinic acid with human serum albumin
Authors: MURALIDHARA B.K. , PRAKASH V.
Published: August 2010

The interaction of chlorogenic acid (CGA) with human serum albumin (HSA) was studied from the viewpoint of thermodynamics and mechanism of binding at pH 6.0. The association constants (Ka) for the HSA‐CGA interaction at 10, 25 and 40° C were 6.0 × 104, 9.0 × 103and 2 × 104M−1, resulting in AG of ‐6.21, ‐5.80, ‐6.32 kcal/mol, respectively. These high Ka‐values showed that the interaction between CGA and HSA is strong, endothermic and entropically driven. Binding of chlorogenic acid induces conformational change in HSA as indicated by quenching of fluorescence emission intensity along with a red shift in the emission maxima from 338 to 350 mm. This suggested the involvement of the lone tryptophan residue in the region of binding. Far‐ultraviolet circular dichroic data showed a decrease in the α‐helical content of HSA from 56 to 50% upon binding of CGA. These data are also supported by the decrease in the apparent Tm of HSA by 4°C upon binding of CGA causing destabilization of the HSA molecule. The kinetics of the interaction involves a single step in the binding, and the kinetic curve attains equilibrium within 180 ± 5 s. Data on caffeic acid (CA) and quinic acid (QA), which are the hydrolysis products of the bidentate CGA molecule, indicate that CA interacts more strongly than CGA. CA binds with an association constant of 8 × 104M−1and with a maximum number of binding sites of four. Microcalorimetric investigation of the interaction of these ligands with HSA suggests that the strength of binding follows the order CA≪CGA≪>QA with a single class of binding sites. The effect of temperature on the binding of CGA to HSA showed that the interaction is dominated by hydrophobic forces and hydrogen bonding.
Structure‐activity relationships of dermorphin analogues containing N‐substituted amino acids in the 2‐position of the peptide sequence
Authors: SCHMIDT RALF , KÁLMÁN ANDRÁS , CHUNG NGA N. , LEMIEUX CAROLE , HORVÁTH CSABA , SCHILLER PETER W.
Published: August 2010

A series of dermorphin analogues containing an N‐alkylated amino‐acid residue Xaa in the 2‐position of the peptide sequence was synthesized (Xaa =N‐methylalanine, proline, pipecolic acid, N‐methylphenylalanine, 1,2,3,4‐tetrahydroisoquinoline‐3‐carboxylic acid [Tic]). These peptides have the potential of assuming a cis Tyrl‐Xaa2 peptide bond. Their in vitro opioid activity profiles were determined in μ and δ‐receptor‐representative binding assays and bioassays. Aside from [D‐Pro2]dermorphin, all analogues showed high affinity for μ and/or δ‐opioid receptors. Whereas most compounds were found to be full μ‐agonists in the guinea pig ileum (GPI) assay, [Tic2]dermorphin (compound 7) was a partial μ‐agonist. Replacement of Gly4in 7 with Phe resulted in an analogue (8) with weak μ‐antagonist activity. Furthermore, analogues 7 and 8 both were potent § antagonists (Kc= 3–40 nM) against the §‐agonists Leuenkephalin, DPDPE and deltorphin I in the mouse vas deferens (MVD) assay. Compound 3, containing l‐Pro in the 2‐position, turned out to be one of the most μ receptor‐selective linear dermorphin analogues reported to date. Low‐temperature HPLC experiments using micropellicular octadecyl silica as stationary phase revealed conformational heterogeneity of the dermorphin analogues which was ascribed to cis‐trans isomerization around the Tyrl‐Xaa2‐and Tyr5‐Pro6 peptide bonds. In the case of analogue 7 four separate peaks corresponding to the four possible isomers were apparent at ‐5°C. Since opioid peptide analogues with a non‐N‐akylated l‐amino acid residue in the 2‐position are nearly inactive and cannot assume a cis peptide bond at the 1–2 position, these results support the hypothesis that the bioactive conformation of opioid peptides containing an N‐alkylated l‐amino acid residue in position 2 is characterized by a cis Tyrl‐Xaa2 peptide bond.
Conformational studies of a synthetic cyclic decapeptide fragment of rat transforming growth factor‐α
Authors: JAYARAMAN GURUNATHAN , BHASKARAN RAJAGOPALAN , KUMAR THALLAMPURANAM KRISHNASWAMY SURESH , YU HUI‐MING , CHEN SHUI‐TEIN , YU CHIN
Published: August 2010

The solution conformation of a synthetic cyclic decapeptide [with sequence mimicking the third disulfide loop of rat transforming growth factor‐α (rTGF‐α)] in deuterated dimethyl sulfoxide was studied by 2D NMR. The determination of solution structures was based on NOE interproton distances, using a combination of distance geometry and simulated annealing protocols. The convergence of the selected structures was evident from the small atomic pairwise root‐mean‐square deviation values among them. Good agreement was noted between the experimental and simulated NOESY spectra, thereby reflecting the accuracy of the calculated solution structures. Analysis of the structures indicates that the residues Tyr5 and Arg9 exhibit similar side chain orientation as that in the corresponding disulfide loop of human transforming growth factor‐α.
Novel microbial inhibitors of ACE*
Authors: BAUER K. , MÜLLER H. , SCHEDEL M. , TRUSCHEIT E. , SCHNABEL E.
Published: August 2010

Two novel microbial ACE‐ inhibitors BAY o 6997 and BAY q 1313 were detected in the fermentation broths of streptomyces spec. WS 464 and spec. WS 1065, respectively. Both were isolated and purified by ion exchange chromatography as initial steps, and final purification was achieved by HPLC or additional chromatography of the Cu‐chelate (BAY q 1313). Both inhibitors are reversibly inactivated on chelation with Cu2+ or Zn2+. Irreversible inactivation occurs on standing in aqueous and acidic solution or in ammonium hydroxide at room temperature and more rapidly on heating. In 4 M sodium hydroxide solutions BAY o 6997 is completely stable, and BAY q 1313 still remarkably stable even on longer heating to 80°C. Thus, BAY o 6997 was alternatively and advantageously isolated after heating of its solution in 4 M sodium hydroxide to 37°C for 2 days and subsequent fractional precipitation with ethanol in a relatively pure state. Total hydrolysis yielded His, 2‐methylamino‐4‐amino‐butyric acid and α‐keto butyric acid (BAY o 6997) and pyruvic acid (BAY q 1313) respectively. The unusual stability of both inhibitors in sodium hydroxide solution on the one hand and their instability on heating and storage in aqueous or acidic solutions on the other hand clearly prove that the constituents are not linked by amide bonds.
Studies on the structure and function of the carp gonadotropin a subunit by site‐directed mutagenesis
Authors: HUANG CHANG‐JEN , HUANG FORE‐LIEN , CHANG GEEN‐DONG , CHANG YEA‐SHA , LO TUNG‐BIN
Published: August 2010

There are two genes encoding the α subunit of carp gonadotropin (α1 and α2 subunits). Our previous data have demonstrated that both α1 and α2 subunits expressed in insect cells are able to associate with the β subunit, but only the α1β heterodimer displays biological activity. In the mature protein, there are only four amino‐acid residues different between the α1 and α2 subunits. In this study we used site‐directed mutagenesis and expressed different mutant α subunits in insect cells to identify which residue might be important for biological activity of the α subunit. Our results suggested that the change of Arg‐71 to Gln‐71 affected the activity of the carp gonadotropin α subunit.
d‐Enantiomers of 15‐residue cecropin A‐melittin hybrids
Authors: MERRIFIELD E.L. , MITCHELL SCOTT A. , UBACH JOSEP , BOMAN HANS G. , ANDREU DAVID , MERRIFIELD R.B.
Published: August 2010

The all‐D enantiomers of six 15‐residue hybrids of cecropin A and melittin were synthesized. They contained the seven N‐terminal residues of cecropin A, followed by eight residues from the N‐terminal region of melittin. They were pure and of the correct composition and structure. The peptides were compared with their all‐L enantiomers. The b and D isomer pairs were each exact mirror images by circular dichroism at several concentrations of hexafluoroisopropanol, and at 12 or 20°, were highly helical. The L analogs were rapidly hydrolyzed by trypsin but the D analogs were very resistant, making them suitable candidates for orally active drugs. These 15‐mers did not form ion channels in normal lipid bilayers made in decane, but those bilayers made in squalene were thinner and the peptides did form ion‐conducting channels. The D/L pairs of peptides were very active antibiotics against five representative Gram‐negative and Gram‐positive bacteria. In each case the D and L isomers were essentially equally active within experimental error. This is interpreted to mean that the peptides do not act by tight interactions with chiral receptors, enzymes or lipids. The action of these peptides against these organisms is best explained by self‐aggregation and the formation of ion‐conducting pores across bacterial membranes.
Synthesis, crystal structure and conformation in solution of four stereoisomeric cyclo‐lanthionine derivatives
Authors: ÖSAPAY GEORGE , ZHU QIN , SHAO HUI , CHADHA RAJ K. , GOODMAN MURRAY
Published: August 2010

The synthesis of the four stereoisomeric cyclo‐lanthionine derivatives: Cbz‐[RS]‐AlaL.‐[R S]‐AlaL‐OMe‐S

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(where AlaL, denotes one end of the lanthionine unit) was carried out on a Kaiser‐oxime resin starting from orthogonally protected lanthionine units. The peptide ring was prepared in 79–85%, yield via amide bond formation by utilizing the method of peptide cyclization on an oxime resin (PCOR). The crystal and molecular structures of the protected cyclic dipeptides have been determined by X‐ray diffraction techniques. The two cyclic lanthionine derivatives with chiralities of [R.S] and [S.R] crystallized in the orthorhonibic space group P212121 and the [R.R]‐ and [S.S]‐cyclo‐lanthionine derivatives in the monoclinic space group C2. The structures were solved by direct methods and refined to an R factor of 0.0368–0.0573. The ring amide bonds in all four compounds are cis, while the urethane group is trans and extended. The NMR spectra of the four stereoisomers in DMSO‐d6 were used to determine their conformation in solution. The analysis of the NMR data with constrained distance geometry search showed the same conformational features in solution as in the crystalline state.
Structures of BAY o 6997 and BAY q 1313 microbial ACE inhibitors
Authors: BAUER K. , KURZ J. , ROSENTRETER H. , SCHEDEL M. , WEBER B. , WÜNSCHE C. , SCHNABEL E.
Published: August 2010

BAY o 6997 and BAY q 1313 are two novel ACE inhibitors produced by Streptomyces WS 464 and Streptomyces WS 1065, respectively. Their structures were elucidated by NMR and MS analysis of the inhibitors and a substance which formed on decomposition of BAY o 6997 on heating in 4 M acetic acid. Both inhibitors are composed of the same amino acids, namely His and 2‐methylamino‐4‐aminobutyric acid. The 2‐amino group of His and the 4‐amino group of the 2‐methylamino‐4‐aminobutyric acid are bridged by differently substituted ethylene moieties. As determined by gas chromatography on a chiral phase, both amino acids isolated from the total hydrolysate after derivatisation, at least in BAY o 6997, possess the l‐configuration.
Determination of disulfide bridge pattern in ω‐conopeptides
Authors: CHUNG DAVID , GAUR SMITA , BELL JOHN R. , RAMACHANDRAN J. , NADASDI LASZLO
Published: August 2010

Synthetic versions of seven naturally occurring ω‐conopeptides were subjected to structural analyses in order to determine their disulfide bridge pattern. The method applied in this study uses a combination of amino‐acid composition and peptide sequence analysis of various peptide fragments generated by different enzymatic digestions. A temperature modification in the Edman degradation cycles of a protein sequencer allowed the unambiguous detection of the cleavage of cystine residues. The appearance of the cystine residues in particular cycles of the sequence analysis was characteristic of one or several of the theoretically possible 15 isomers. In the case of multiple choices, possible isomers were further eliminated by the amino‐acid and sequence analysis of peptide fragments generated by the enzymatic digestion. All synthetic peptides, SNX‐111, ‐157, ‐159, ‐183, ‐185, ‐230 and ‐231, were found to have the same disulfide bridge pattern as determined for the naturally occurring %omega;‐conopeptide G‐VI‐A, i.e. disulfide bridges between the half‐cystines 1–16, 8–20 and 15–25 (using the amino‐acid numbering of SNX‐111).
Oxygen effect in the radiolysis of proteins
Authors: PUCHALA M. , SCHUESSLER H.
Published: August 2010

Radiolysis of myoglobin was carried out under air and under nitrogen in phosphate buffer a1 pH 5 and 7. The radiation products were separated by SDS‐polyacrylamide gel electrophoresis and by HPL gel chromatography with guanidine HCl. Under nitrogen the main reaction is the aggregation caused by covalent crosslinks. Under air the radiolysis leads to peptide chain scission, which is not a random process, but produces specific protein fragments. The estimated molecular weights of these fragments gave further support to the assumption that the aminoacyl‐proline peptide group is the preferential breaking site. In contrast to haemoglobin, myoglobin showed nearly no radiation‐induced fragmentation under nitrogen.
Conformation and structure of linear peptides with regularly alternating l‐ and d‐residues: structure of the blocked hexapeptide tert‐butyloxycarbonyl‐(d‐alloisoleucyl‐l‐isoleucyl)3 methyl ester monohydrate
Authors: DI BLASIO BENEDETTO , DEL DUCA VALERIO , LOMBARDI ANGELA , PEDONE CARLO , LORENZI GIAN PAOLO , BENEDETTI ETTORE
Published: August 2010

Peptides with a regular sequence of enantiomeric residues (l and d) along the chain have received considerable attention because of their accessibility to unique conformations and because they are model compounds for the naturally occurring peptide gramicidin A, which shows monovalent cation selective transmembrane transport. The solid‐state structure of the linear hexapeptide t‐Boc‐(d‐aIle‐l‐Ile)3‐OMe has been determined by X‐ray diffraction techniques and refined to a final R factor of 0.068. The molecule shows a bent U‐shaped conformation stabilized by three intramolecular H‐bonds of the N—H⋯O=C type: a type II β‐bend (4 → 1 bend or C10 ring structure) with l‐Ile2 and d‐aIle3 at positions 2 and 3 of the bend, an α‐turn (5 → 1 bend or C13 ring structure) and a 1 → 5 bend or C17 ring structure. The first two 10‐membered and 13‐membered bends are enclosed in the latter 17‐membered hydrogen‐bonded ring structure. This structural motif is common to hepta‐ and octa‐peptide cyclic molecules, showing that ring closure is not required to achieve a particular topology in the molecular design of specific bended conformations. © Munksgaard 1995.
Variability analysis of the T‐cell receptors using three variability indexes
Authors: ALMAGRO JUAN C. , ZENTENO ROBERTO , VARGAS‐MADRAZO ENRIQUE , LARA‐OCHOA FRANCISCO
Published: August 2010

In the absence of a three‐dimensional structure for TCR molecules, several attempts to identify their hyper‐variable regions by variability methods have been made; this subject is still troublesome. In this paper three different variability indexes were used: (i) the Kabat index, which is the classical measure of sequence variability, (ii)the modified Kabat index, successfully used in the β‐chain of T‐cell receptors and (iii) an information‐theoretical entropy concept, recently proposed as an improved measure of the variability. In order to identify the hypervariable regions in the TCR sequences, a Fourier filtering was applied on each variability profile.

Results show that the three variability indexes have distinct resolutions for different levels of variability. Thus, the simultaneous use of these indexes compensates for the deficiency of any one of them in estimating variability. Applying the Fourier filtering, it is found that the hypervariable regions here identified, roughly coincide with the defined CDR‐2 and CDR‐3 in TCR by analogy with Ig. However, no hypervariable in the CDR‐1 of α‐ and β‐chains was found. The study on the influence of sample size in variability analysis, indicates that results are independent of the sample size. Considering current structural models of TCR‐peptide‐MHC interaction, one can suggest that the low‐variability characteristics of these regions is inherently related to the interaction with relatively conserved region on the α‐helices of MHC. © Munksgaard 1995.
In vitro studies on a novel micelle‐forming peptide with anticoagulant activity
Authors: RAMESH C.V. , JAYAKUMAR R. , PUVANAKRISHNAN R.
Published: August 2010

A novel peptide, tert‐butyloxycarbonyl‐L‐arginine‐L‐proline lauryl ester laurate, synthesised by a solution‐phase method, formed micelles in aqueous solutions and was observed to exhibit anticoagulant activity as shown by clotting assays such as the thrombin time (TT) test, the prothrombin time (PT) test and the activated partial thromboplastin time (APTT) test. TT and PT were found to be normal up to a particular concentration of peptide, and above that level they increased with increasing concentration of peptide, while APTT did not exhibit much significance. Conductometric and potentiometric studies showed that the peptide formed a stable micelle, and the anticoagulant activity of this peptide was also compared with a non‐arginine‐containing peptide (control) known to form micelles. The anticoagulant action in the micellar form could be due to the inhibition of thrombin, as seen from the decrease in amidolytic activity. The inhibitory activity of the peptide was explained in the light of micelle formation.
Theoretical conformational analysis of three vasopressin antagonists with a modified cyclohexyl ring in the first thioacid residue
Authors: KAŹMIERKIEWICZ RAJMUND , LIWO ADAM , LAMMEK BERNARD
Published: August 2010

Analogues of arginine vasopressin (AVP) with bulky thioacid residues in position 1 of the amino acid sequence are known to be effective antagonists of the pressor response. Some of the most effective ones are those that have the first cysteine residue replaced with β,β‐cyclopentamethylene‐β′‐mercaptopropionic acid (Cpp) and its derivatives, such as 4‐mercapto‐4‐tetrahydropyraneacetic acid (OCA) and 4‐mercapto‐4‐tetrahydrothiopyraneacetic acid (SCA). The SCA analogues are more potent and the OCA ones slightly less potent antagonists than the Cpp ones. In this study we carried out conformational calculations on [Cpp1]AVP, [OCA1]AVP and [SCA1]AVP, using the ECEPP/3 force field both with and without hydration (to simulate an aqueous and non‐polar receptor environment, respectively). It was found that most of the low‐energy conformations are common in geometry and relative energy for all three compounds studied. It can therefore be concluded that the modifications of the cyclohexyl ring in position 1 influence the binding to the receptor because of changing the lipophilicity of the first residue, rather than by changing the conformational space. This is further supported by the fact that the lowest‐energy conformations in the absence of water have closely located the Phe3 side chain (which is critical for the interaction with vasopressin receptors) and the (modified) cyclohexyl ring. The lowest‐energy conformations in the presence and absence of water had β‐turns at residues Phe3‐Gln4 and Gln4‐Asn5, and Gln4‐Asn5, respectively. The conformation with the turn at Gln4‐Asn5 was most similar to the crystal structure of the pressinoic acid (the cyclic moiety of vasopressin). © Munksgaard 1995.
Preparation of the very acid‐sensitive Fmoc‐Lys(Mtt)‐OH Application in the synthesis of side‐chain to side‐chain cyclic peptides and oligolysine cores suitable for the solid‐phase assembly of MAPs and TASPs
Authors: ALETRAS ALEXIOS , BARLOS KLEOMENIS , GATOS DIMITRIOS , KOUTSOGIANNI SOPHIA , MAMOS PETROS
Published: August 2010

N
α‐9‐Fluorenylmethoxycarbonyl‐Nε‐4‐methyltrityl‐lysine, [Fmoc‐Lys(Mtt)‐OH], was prepared in two steps from lysine, in 42% overall yield. The Nε‐Mtt function can be quantitatively removed upon treatment with 1% TFA in dichloromethane or with a 1:2:7 mixture of acetic acid/trifluoroethanol/dichloromethane for 30 min and 1 h at room temperature, respectively. Under these conditions, groups of the tert‐butyl type and peptide ester bonds to TFA‐labile resins, such as the 2‐chlorodiphenylmethyl‐ and the Wang‐resin, remained intact. The utility of the new derivative in peptide synthesis has been exemplified with the synthesis of a cyclic cholecystokinin analog. As an example of further application, five types of lysine cores suitable for the solid‐phase synthesis of one, two or three epitopes containing antigenic peptides or template‐assembled synthetic proteins have been synthesized on Merrifield, Wang and 2‐chlorodiphenylmethyl resin. © Munksgaard 1995.
Chemical and enzymatic treatment of endothelin
Authors: PEREGO RITA , GOZZINI LUIGIA , ARLANDINI EMANUELE , BOLIS GIORGIO , DE CASTIGLIONE ROBERTO
Published: August 2010

Endothelin‐1 (ET), the most potent vasoconstrictor yet discovered, is a peptide containirig 21 amino acids with two intrachain disulfide bridges. With the aim of obtaining two‐chain derivatives, Et was submitted to chemical and enzymatic treatments. Reaction of ET with CNBr in 70% HCOOH gave, in addition to the expected [Hse7 lactone]‐7,8‐seco‐ET and unreacted material, a by‐product whose molecular weight was 25 m.u. greater than that of ET. When the reaction mixture, after lyophilisation, was immediately quenched with NH3‐saturated dry MeOH, two products could be recovered in a 5:1 ratio, both obtained by nucleophilic attack of the homoserine lactone: the expected [Hse7‐NH2]‐7,8‐seco‐ET and [Hse7]ET, resulting from competitive intramolecular reaction of the deprotonated α‐amino group of the Asp8 residue. The Lys9‐Glu10 bond turned out to be very resistant to enzymatic attack both by Lys‐C‐endopeptidase and trypsin. The 9,10‐seco‐ET derivative could be obtained by treatment with Lys‐C‐endopeptidase only by using a high enzyme/ET ratio and after a prolonged incubation time. Cleavage of the Lys9‐Glu10 bond could not be achieved by treatment with trypsin, even with a high enzyme/substrate ratio. The main product was 13, 14‐seco‐ET, deriving from the action of chymotripsin (present as an impurity in the trypsin preparation) on Tyr13. The structure of these peptides was confirmed by amino‐acid sequence analysis and fast atom bombardment mass spectrometry (FAB‐MS). Nicking of the ET structure at different positions had different impact on the biological properties of the resulting derivatives. © Munksgaard 1995.
Chemical synthesis and biological activities of the EGF‐like domain of mouse schwannoma‐derived growth factor (SDGF)
Authors: TAKENOUCHI TAKATO , KADOSAKA MISA , SHIN SONG YUB , MUNEKATA EISUKE
Published: August 2010

Schwannoma‐derived growth factor (SDGF), an epidermal growth factor (EGF) family peptide recently discovered, has an EGF‐like domain in the carboxyl terminal portion. In this study, we synthesized mSDGF(38‐80) corresponding to the EGF‐like domain of mouse SDGF by means of stepwise solid‐phase method using Fmoc chemistry in order to evaluate the biological function of the EGF‐like domain in SDGF. The linear peptide of mSDGF(38‐80) was folded by direct oxidation with reduced and oxidized glutathione to form intramolecular disulfide bridges in synthetic peptide. On the biological activity, we examined mitogenic activity induced by mSDGF(38‐80) in NIH/3T3 fibroblast cells and interaction with EGF receptor in A431 cells. In the results, mSDGF(38‐80) was confirmed to form three disulfide linkages that were similar in pattern to EGF by amino acid and sequence analysis of fragments obtained after thermolytic digestion. However, mSDGF(38‐80) possessed weak mitogenic activity in NIH/3T3 cells and weak binding affinity for the EGF receptor in A431 cells compared with those of human EGF. These results suggest that the EGF‐like domain of SDGF may have little effect upon mitogenic activity and the EGF receptor binding of SDGF. © Munksgaard 1995.
The role of the C‐terminus of the insulin B‐chain in modulating structural and functional properties of the hormone
Authors: LEYER SIGMAR , GATTNER HANS‐GREGOR , LEITHÄUSER MARGOT , BRANDENBURG DIETRICH , WOLLMER AXEL , HÖCKER HARTWIG
Published: August 2010

In memoriam Howard S. Tager

Within the scope of structure‐function studies on the proteohormone insulin, the role of the C‐terminal segment B26‐B30 for self‐association and receptor interaction was analyzed. Insulin derivatives with modifications in the region B26‐B30 were synthesized by trypsin‐catalyzed coupling reactions of des‐(B23‐B30)‐insulin with synthetic peptides. The peptides were obtained by Fmoc solid‐phase peptide synthesis. Insulins with multiple amino acid → glycine substitutions were examined to distinguish between the influence of the side chains and the influence of the main chain in positions B27‐B30 on the self‐association of the hormone. The analogues [GlyB27,B28,B29,B30]insulin and [GlyB27,B28,B30]insulin exhibit relative receptor affinities of 80% and self‐associate. The successive extension of [AlaB26]des‐(B27‐B30)‐insulin‐B26‐amide (relative receptor binding 273%) with amino xids corresponding to the native sequence B27‐B30 showed the influence of the length of the B‐chain on receptor affinity: the extension by B27‐threonine amide reduces receptor binding to 71%, all further prolongations have only small effects on the binding. The effect of the B28‐side chain on main‐chain conformation, self‐association and receptor binding was examined with [XB28]des‐(B29‐B30)‐insulin‐B28‐amides (X = Phe, Gly, D‐Pro). While the glycine and D‐proline analogues (relative binding 104 and 143%, respectively) retain the self‐association properties typical of insulin, [PheB28]des‐(B29‐B30)‐insulin‐B28‐amide (relative binding 50%) shows diminished self‐association. The backbone‐modified insulin derivative [SarB26]des‐(B27‐B30)‐insulin‐B26‐amide (sarcosine =N‐methylglycine) exhibits an unexpectedly high receptor affinity of 1100% which demonstrates that the B26‐amide hydrogen of the native hormone is not important for receptor binding. © Munksgaard 1995.
Amino‐acid sequence of bone Gla protein from the African clawed toad Xenopus laevis and the fish Sparus aurata1
Authors: CANCELA M. LEONOR , WILLIAMSON MATTHEW K. , PRICE PAUL A.
Published: August 2010

As an initial step in the analysis of bone Gla protein (BGP; osteocalcin) function in lower vertebrates, we have developed a simple and rapid method for the isolation of BGP from bone and have applied this to the isolation of BGP from the African clawed toad Xenopus laevis and the fish Sparus aurutu. We have also determined the complete amino‐acid sequence of Sparus and Xenopus BGP, including the identification of the sites of γ‐carboxylation.

Since the addition of Xenopus and Sparus BGP sequences significantly extends the range of species whose BGP structures are known, we have compared the 18 presently known BGP sequences. Twelve amino acids are invariant in these 18 RGP sequences and are therefore presumably critical to BGP conformation or function. Eight of these 12 invariant amino acids are also invariant in all presently known matrix Gla protein sequences (shark, mouse, rat, cow, human), an observation which strongly supports the evolutionary relationship between these two vitamin K‐dependent bone proteins and suggests that the proteins may adapt similar tertiary structures. © Munksgaard 1995.
A new clustering system for protein sequences and its application to constraints discovery in protein evolution
Authors: NAKAGAWA HIROYUKI , KOYAMA YASUSHI , KAWAI TOSHIKAZU , OKADA TAKASHI
Published: August 2010

A conceptual clustering system, CLUSMOL/S, has been developed to classify protein sequences from a user‐defined point of view. Given a grouping of amino acids as a viewpoint, the system constructs taxonomic trees of sequences based on minimum information criterion. Every tree node expresses itself as a generic consensus sequence that consists of specific consensus amino acids, insertionideletion points, and generic amino acids with a specified character. The resulting tree and generic sequences show the similarity‐based relationships among sequences and their characteristics. Application to vertebrate cytochromes c yields an acceptable cladrogram only when amino acids are grouped by volume and length of sidechains. The result indicates that the steric factor is the most important constraint in the process of protein evolution. © Munksgaard 1995.
NMR conformational study of a model tetradecapeptide mimicking the RXVRG consensus cleavage site of a Xenopus luevis skin endoprotease
Authors: CHALAOUX FRANÇOIS‐RÉGIS , RIAND JACQUES , LESENEY ANNE‐MARIE , BARON DANIEL
Published: August 2010

A model tetradecapeptide, used for the purification of the RXVRG‐endoprotease from Xenopus laevis skin exudate, has been studied by two‐dimensional NMR, correlation (COSY) and NOE (NOESY) spectroscopy. This peptide has the 5–9 consensus sequence (RXVRG), along with an acidic moiety (1–4) and a hydrophobic domain (10–14). Variations with temperature of NH chemical shifts in a dimethyl sulfoxide solution (low thermal coefficients at residues 6, 7 and 8) and quantified NOE values from four spectra at different mixing times clearly showed a structural organization in the consensus domain with φ‐angles around [–40, –10°] for residues 7 and 8, and two NOE correlations of αHiNHi+2 type (5–7 and 6–8). Moreover, a privileged rotamer in the side chain is established for three residues (Val2, Asp3 and Val7) and limited possibilities are discussed for seven others. Most of the folding trends were not observed in the [Ser7] derivative, underlying the relationship between the conformations and a full consensus sequence. In the model tetradecapeptide an equilibrium between two β‐turns of type I, fragments 4–7 and 5–8, seems the most probable. Comparison between this tetradecapeptide and its 4–14 fragment, also a substrate for RXVRG‐endoprotease, shows that the 1–3 moiety (DVD) influences the consensus domain structure(s) and clearly stabilizes the folded one(s). Finally, two analytical methods are developed in order to determine: (1) the trifluoroacetic acid content of the peptide samples, on the basis of 19F NMR spectroscopy; (2) the mean φ‐ and φ‐angles of each residue, from the whole set of NH/αH coupling constants (3JNα) and NOE data at a local level. © Munksgaard 1995.
1H and 13C NMR assignments and molecular modelling of a minor groove DNA‐binding peptide from the HMG‐I protein
Authors: EVANS JEREMY N.S. , ZAJICEK JAROSLAV , NISSEN MARK S. , MUNSKE GERHARD , SMITH VIRGINIA , REEVES RAYMOND
Published: August 2010

The HMG‐I subfamily of high mobility group (HMG) chromatin proteins consists of DNA‐binding proteins that preferentially bind to stretches of A·T‐rich sequence both in vitro and in vivo. Recently, members of the HMG‐I family have been suggested to bind in vitro to the narrow minor groove of A·T‐DNA by means of an 11 amino acid peptide binding domain (BD) which, because of its predicted structure, is called the‘A·T‐hook motif [Reeves, R. & Nissen, M. (1990) J. Biol. Chem.265, 8573–8582], and would appear to be crescent‐shaped. A BD peptide with 13 amino‐acid residues was synthesized and examined by proton and carbon‐13 nuclear magnetic resonance (NMR) spectroscopy. The peptide contains four proline residues, and on the basis of NOES and 13C chemical shifts was found to exist in an all‐trans conformation. Molecular modelling based on this result provides evidence for a dynamic equilibrium between turn‐like conformations in solution, the most populated of which is likely to be an S‐shaped conformer, on the basis of amide exchange data. © Munksgaard 1995.
Acid catalysis in the formation of dioxopiperazines from peptides containing tetrahydroisoquinoline‐3‐carboxylic acid at position 2
Authors: CAPASSO SANTE , SICA FILOMENA , MAZZARELLA LELIO , BALBONI GIANFRANCO , GUERRINI REMO , SALVADORI SEVERO
Published: August 2010

The kinetics of the spontaneous formation of 2,5‐dioxopiperazines from peptides containing the Tic (1,2,3,4‐tetrahydroisoquinoline‐3‐carboxylic acid) residue in the 2‐position of the sequence has been studied in DMSO and water solution.

The reaction is first order in Tic‐peptide and subject to general‐acid catalysis. Moreover, only the fraction of peptide having the amino terminal group in the deprotonated state reacts with appreciable rate. In pure organic solvent, and in aqueous solution with low buffer concentration, the degradation reaction of Tic‐peptides is very low; at 20°C for the peptide H‐Tyr‐Tic‐Phe‐Phe‐NH2, in DMSO and in neutral water in the absence of buffer, the half‐lives (t1/2) are 3 × 104 and 1.2 × 104 h, respectively. The addition of carboxylic acids or buffers to the reaction solutions markedly increases the reaction rate; in 0.01 M HAc in DMSO and in 0.1 M phosphate buffer in water, pH 7.1, t1/2 values for the tetrapeptide are 61 and 121 h, respectively. © Munksgaard 1995.