bluuebunny/crossref_metadata_2025 huggingface dataset:
Complete primary structure of statherin, a potent inhibitor of calcium phosphate precipitation, from the saliva of the monkey, Macaca arctoides
Authors: SCHLESINGER DAVID H. , HAY DONALD I. , LEVINE MICHAEL J.
Published: August 2010

Human saliva, which is supersaturated with respect to basic calcium phosphate salts, is stabilized primarily by the presence of two classes of phosphoproteins, statherin and the acidic proline‐rich proteins (PRP). These molecules act by inhibiting both primary (spontaneous) precipitation of calcium phosphates in saliva and secondary (surface induced) precipitation of these salts onto dental enamel. The complete amino‐acid sequences of several human PRP and the N‐terminal sequence of PRP from saliva of M. arctoides have been determined. Similarly, the complete sequence of statherin from human and M. fascicularis saliva is known. We now report the complete structure of statherin from the saliva of the stump‐tailed monkey, M. arctoides. The structure was determined by gas‐phase sequencing of intact statherin, elucidating positions 1‐26, and sequencing an unpurified mixture of tryptic peptides which elucidated the remaining positions through the C‐terminus (residue 42) of the molecule. This latter degradation produced an eight amino‐acid overlap with that of intact statherin and was confirmed by C‐terminal analysis and amino‐acid composition of native statherin. The complete amino‐acid sequence of M. arctoides statherin is:

NH2‐Asp‐PSer‐PSer‐Glu‐Glu5‐Lys‐Phe‐Leu‐Arg‐Arg10‐Leu‐Arg‐Arg‐Phe‐Asp15‐Glu‐Gly‐Arg‐Tyr‐Gly20‐Pro‐Tyr‐Gln‐Pro‐Phe25‐Val‐Pro‐Pro‐Pro29Leu30‐Tyr‐Pro‐Gln‐Pro‐Tyr35‐Gln‐Pro‐Tyr‐Gln‐Pro40 ‐Gln‐Tyr‐COOH

This sequence differs from human statherin at positions 11, 12, 15, 16, 18, 25‐27, 38‐40 and from M. fascicularis statherin at positions 26 and 28.
Effect of divalent cations on structure‐function relationships of the antitumor protein α‐sarcin
Authors: MARTÍNEZ DEL POZO ALVARO , GASSET MARIA , OÑADERRA MERCEDES , GAVILANES JOSÉ G.
Published: August 2010

α‐Sarcin binds one Zn(II) cation per protein molecule, with a Kd value of 0.9 mM, determined by equilibrium dialysis experiments. Ca(II), Mg(II), and Mn(II) do not bind to α‐sarcin. Cd(II) and Co(II) also behave as Zn(II). The binding produces local modifications on the protein conformation affecting the microenvironment of tryptophan residues. The three cations modify the fluorescence emission of the protein. The near‐u. v. circular dichroism spectrum of the protein is also altered. The binding of Zn(II) and related cations does not modify the secondary structure of the protein. The ribonucleolytic activity of a‐sarcin is inhibited upon Zn(II) binding, but no alteration of the ability of the protein to aggregate phospholipid vesicles has been observed.
Crystal structure and conformational analysis of angiotensinogen fragments
Authors: BENKOULOUCHE M. , COTRAIT M. , GEOFFRE S. , PRECIGOUX G.
Published: August 2010

The tripeptide acetyl‐L‐prolyl‐L‐phenylalanyl‐L‐histidine crystallizes in the orthorhombic space group P212121 with eight molecules in a unit cell of dimensions a = 9.028(2), b = 140.54(6) and c = 42.41(1) Å. The structure has been solved by direct methods and refined to an R value of 0.056 for 2904 observed reflections. The molecule exists as a zwitterion with terminal (His)CO−2 and (imidazole)H+ as charged groups. The two peptide molecules in the structure adopt a type I β‐turn with Pro and Phe as the corner residues. The main conformational difference between the two crystallographically independent molecules is seen to be in the histidine side‐chain orientations. The molecules arrange themselves in sheets perpendicular to the c axis. All hydrophobic side chains lie on one side of the sheets thus generated, whereas the hydrophilic groups are located on the other side. An interesting feature of the crystal structure is the existence of a water layer between adjacent peptide sheets. The conformational study of the isolated Ac‐His‐Pro‐Phe‐His‐MA using energy calculations gives a rather limited number of stable conformers. The most stable corresponds to a type I β‐turn stabilized through two hydrogen bonds, followed by a less stable type II β‐turn (ΔE = 2.0kcal) and a partly helical structure (ΔE = 2.6 kcal).
Conformation and complexation with metal ions of cyclic hexapeptides: cyclo (l‐Leu‐l‐Phe‐l‐Pro)2and cyclo (l‐Cys(Acm)‐l‐Phe‐l‐Pro]2
Authors: OZEKI EIICHI , KIMURA SHUNSAKU , IMANISHI YUKIO
Published: August 2010

Cyclic hexapeptides, cyclo (l‐Leu‐l‐Phe‐l‐Pro)2and cyclo[l‐Cys(Acm)‐l‐Phe‐l‐Pro]2, in which Acm represents an acetoamide‐methyl group, were synthesized, and the conformation and complexation with metal ions were investigated. Cooperation of the carbonyl groups of the Cys(Acm) side chains with those of the cyclic skeleton in complexation was especially examined. Cyclo(l‐Leu‐l‐Phe‐l‐Pro)2, which possesses no functional groups on side chains, was taken as the reference compound.13C‐ and two‐dimensional n.m.r. measurements revealed that cyclo(l‐Leu‐l‐Phe‐l‐Pro)2and cyclo[l‐Cys(Acm)‐l‐Phe‐l‐Pro]2took a C2‐symmetric conformation contaIntngcisl‐Phe‐l‐Pro bonds in chloroform and acetonitrile. Both cyclic hexapeptides were found to complex selectively with Ba2+and Ca2+in acetonitrile. On complexation the conformation of either cyclic hexapeptide changed into a similar one. However, the binding constant of cyclo[l‐Cys(Acm)‐l‐Phe‐l‐Pro]2was higher than that of cyclo(l‐Leu‐l‐Phe‐l‐Pro)2. The n.m.r. measurements showed that the amide carbonyl groups of Cys(Acm) side chains as well as those of cyclic skeleton in cyclo[l‐Cys(Acm)‐l‐Phe‐l‐Pro], cooperatively bound the cations.
Influence of ions on cyclization of the amino terminal glutamine residues of tryptic peptides of streptococcal PepM49 protein
Authors: KHANDKE KIRAN M. , FAIRWELL THOMAS , CHAIT BRIAN T. , MANJULA BELUR N.
Published: August 2010

RPHPLC of the tryptic digest of lysine blocked group A streptococcal PepM49 protein (DHP‐PepM49) consistently yielded, among others, two pairs of peptides which were well resolved, eluted in tandem, and had identical amino acid compositions. In each pair, the earlier eluting peptide was readily amenable to sequencing and yielded an amino‐terminal glutamine whereas the later eluting peptide could not be sequenced. Mass spectral analysis revealed that each of these pairs of peptides differed in mass corresponding to the loss of ammonia. These data suggested that the later eluting peptide in each pair is a result of cyclization of the amino‐terminal glutamine residue to pyroglutamic acid, which apparently leads to an increase in the hydrophobicity of the peptide. A kinetic analysis of the tryptic digestion of the DHP‐PepM49 protein revealed that the cyclized form of the peptides were essentially absent during the Initial time and increased with time of incubation, with a concomitant decrease in the uncyclized form. In 0.2 M ammonium bicarbonate at 37°, nearly 44% conversion of the glutaminyl peptides to the pyroglutamyl peptides was observed in 24 h. This conversion was accelerated in sodium phosphate buffer relative to that in ammonium bicarbonate whereas it had a significantly lower rate in ammonium acetate buffer. The conversion was also temperature dependent, with essentially no cyclization at 0°, in all the three buffers. Thus, an extended digestion at 0° or a brief digestion at 37° in ammonium acetate was found to be a suitable condition for limiting the cyclization of amino‐terminal glutamine residues of PepM49 peptides. The results of the present study suggest that use of ammonium acetate should be preferred over the ammonium bicarbonate and phosphate buffers in the handling of peptides and proteins known to contain amino‐terminal glutamine in order to minimize the possibility of cyclization of these residues.
A common cytolytic region in myotoxins, hemolysins, cardiotoxins and antibacterial peptides*
Authors: KINI R. MANJUNATHA , EVANS HERBERT J.
Published: August 2010

Several proteins and polypeptides of reptilian, amphibian, insect, and microbial origin share a common cytolytic property. However, these cytolysins fulfill different objectives. They provide offensive armament in the case of toxins, but defensive systems in the case of antibacterial peptides. The sequences of several nonenzymatic cytolysins and their analogues were compared to identify the structural requirements for cytolytic activity. These cytolysins, although isolated from phylogenetically unrelated organisms, possess the common sequence features of a cationic site flanked by a hydrophobic surface. The presence of such a region apparently confers the cytolytic activity of various cytolysins. The concept of a cytolytic region is strongly supported by the existence of several natural and synthetic analogues of cytolysins and by chemical modification studies of these cytolysins. This prediction provides a new focus for cytolysin research. The understanding of this structure‐function relationship should facilitate the design, synthesis, and development of better antibacterial and anticancer peptides.
Synthesis and acid ionization constants of cyclic cystine peptides
Authors: HORVAT Š. , GRGAS B. , RAOS N. , SIMEON VI.
Published: August 2010

Cyclic peptide disulfides of the general formula were synthesized from the corresponding peptide derivatives [Boc‐Cys(Trt)(Gly)‐n‐Cys(Trt)‐OBut] by oxidation with iodine in methanol and by subsequent removal of the terminal groups with trifluoroacetic acid. Acid ionization constants of the obtained peptides were determined by potentiometric titration in aqueous KCl (0.1 mol/L) medium. All compounds have two dissociable hydrogens, corresponding to carboxyl (pK1= 2.35–2.84) and to terminal amino group (pK2= 5.61–6.93); pK1, values show first an upward and then a downward trend with the increase in ring size; the opposite is true for pK2, values. These trends could be tentatively attributed to the intramolecular salt bridge (‐COO——‐NH+3‐) formation.
Novel C‐terminal gastrin antagonists Synthesis and biological activity
Authors: YASUI AKIHIRO , DOUGLAS ALASTAIR J. , WALKER BRIAN , MAGEE DONAL F. , MURPHY RICHARD F.
Published: August 2010

The C‐terminal tetrapeptide, Trp‐Met‐Asp‐Phe‐NH2, is a full agonist of gastrin, but des‐Phe analogues, including Boc‐Trp‐Met‐Asp‐NH2, are antagonists. To ascertain the minimum structural requirement for an antagonist, we used conventional solution phase methodology to synthesize analogues with further modifications including removal of the α‐amino group of Trp, conversion of the indole to a phenyl ring, and methylation of amide bonds. These analogues were tested for their effect on pentagastrin‐stimulated acid release in dogs surgically prepared with a gastric fistula. When infused intravenously at a dose of 20pmolkg−1h−1, the peptides significantly inhibited acid secretion. The extent of inhibition ranged from 12% to 60%. Thus, tripeptide analogues based on the C‐terminal sequence of gastrin act as potent and specific antagonists of gastrin‐stimulated acid secretion.
Flexibility of tripeptides in solution: free energy molecular mechanics
Authors: RAMÉ GRACIELA L. , LAU WAN F. , PETTITT B. MONTGOMERY
Published: August 2010

A full theory of the conformations of biopolymers requires a method for treating the effects of solvent on the induced structures. This is especially critical in aqueous solvent where hydrogen‐bonding and dielectric shielding play major roles in determining the relative stability of conformers. Calculations of peptide conformations on a free energy surface are contrasted with the traditional sort of calculations which employs a simple potential energy function (in vacuo). The method employs a pairwise decomposable free energy surface determined by approximate analytical statistical mechanical theory. Applications are presented for tripeptides of alanine and glycine in water. This method, with precomputed free energy functions, takes the same amount of time and effort as traditional molecular mechanics in vacuo.
In vitvointeractions of opioid peptides with phospholipids
Authors: D'ALAGNI MARIA , GULLÁ PATRIZIA , RODA L. GIORGIO , ROSCETTI GIANNA
Published: August 2010

The interaction of methionine and leucine enkephalin with phosphatidylserine and phosphatidylcholine was studied by optical spectroscopy techniques. The data reported indicate that with both peptides the binding is controlled by ionic parameters. They also indicate that the differences in the binding behavior of the two peptides induced by changing these parameters are minor. Non‐ionic interactions are also important in the binding phenomena, but the above observations hold in this case as well. Finally, the tridimensional structure of both enkephalins appears to be modified in the presence of phospholipids. Moreover, the changes induced by these lipids appear to differ from one peptide to the other.
Solid‐phase synthesis of the native sequence of horse heart cytochrome c‐(66–104)‐nonatriacontapeptide and of a C‐terminal carboxyamide analog selectively modified at Met‐80
Authors: DI BELLO CARLO , TONELLATO MAURO , LUCCHIARI ADRIANA , BUSO ORFEO , GOZZINI LUIGIA , VITA CLAUDIO
Published: August 2010

The peptide corresponding to the (66–104) sequence of horse heart cytochrome c and its carboxyamide analog, selectively modified at the critical Met80 residue, have been synthesized by stepwise solid‐phase methods on PAM and BHA resins respectively. The correctness of the growing peptide chain as well as the homogeneity of the final products have been monitored by several analytical methods including quantitative Edman degradation. After HF cleavage both peptides were purified by semipreparative HPLC. The overall yields were 24% for the native (66–104) and 10% for the carboxyamide analog. The homogeneity of the purified synthetic peptides have been determined by different criteria including HPLC, amino acid composition, Edman degradation, electrophoresis, and tryptic peptide mapping. The synthetic fragments have been utilized for preliminary semisynthesis experiments with the native [Hse〉65](1–65)H heme‐sequence.
Ψ[CH2O] pseudodipeptide synthesis An improved approach which allows absolute configuration determination
Authors: BRETON PASCAL , MONSIGNY MICHEL , MAYER ROGER
Published: August 2010

An improved way to obtain Ψ[CH2O] pseudodipeptide units is proposed, involving an intramolecular Williamson's reaction, with displacement of bromine by an alkoxide, instead of the classical intermolecular one. Until now, Ψ[CH2O] pseudodipeptide synthesis done by this new method, has used a protected form of the amino alcohol hydroxyl group to prepare the acyclic precursor. In the present paper, the use of an active ester of the brominated carboxylic acid avoids this protection step. The pseudodipeptides Ac‐GlyΨ[CH2O]‐d,l‐Ala‐OH and Ac‐Ser(Bzl)Ψ[CH2O]‐d,l‐Ala‐OH were obtained in high yields, through a delta‐lactam intermediate, which furthermore allows the determination of the absolute configuration of compounds using HPLC and appropriate nuclear magnetic resonance (NMR) techniques.
Three‐dimensional structure of bradykinin in SDS micelles
Authors: LEE SUSANNIE C. , RUSSELL ANNE F. , LAIDIG WILLIAM D.
Published: August 2010

The conformational properties of bradykinin in five molar excess sodium dodecyl sulfate (SDS) micelles have been examined by two‐dimensional nuclear magnetic resonance (NMR) techniques at 500 MHz. Detailed structural information for bradykinin in SDS was obtained from quantitative 2‐D nuclear Over‐hauser enhancement (n.O.e.) analyses, distance geometry, and restrained molecular mechanics and dynamics calculations. The conformation of bradykinin in SDS micelles, as determined by these methods, is characterized by a β‐turn‐like structure at residues 6–9. A detailed comparison of the structures derived from distance geometry and restrained molecular mechanics and dynamics is also presented.
Repetitive BOP coupling (REBOP) in solid phase peptide synthesis
Authors: SEYER RENE , AUMELAS ANDRE , CARATY ALAIN , RIVAILLE PIERRE , CASTRO BERTRAND
Published: August 2010

The coupling reagent (benzotriazol‐1‐yloxy)tris‐(dimethylamino)phosphonium (BOP) hexafluorophosphate was tested in the synthesis of luliberin (LH‐RH) with inexpensive classically protected Boc‐amino acids, in slight excess, and benzhydryl amino resin, without any other additive. The good solubility of this reagent and its by‐products is of particular interest for automated peptide synthesis. [D‐H‐S1]LH‐RH was also synthesized and compared with LH‐RH by proton nuclear magnetic resonance spectroscopy. As shown by the biological tests and the high performance liquid chromatography study, unprotected pyroClu and Boc‐His can be used without any significant racemization but Boc‐His(Boc) was found to be preferable since it gave no detectable racemization and no by‐products. The difficult isolation of the minority D‐derivative from the crude preparation of LH‐RH was resolved by a recycling procedure in reversed phase HPLC.
Synthesis of O‐glycosylated tuftsins by utilizing threonine derivatives containing an unprotected monosaccharide moiety
Authors: FILIRA F. , BIONDI L. , CAVAGGION F. , SCOLARO B. , ROCCHI R.
Published: August 2010

Synthesis is described of four tuftsin derivatives containing a D‐ghcopyranosyl or a D‐galactopyranosyl unit covalently linked to the hydroxy side chain function of the threonine residue through either an α or βO‐glycosidic linkage. Fmoc‐threonine derivatives containing the suitable unprotected sugar were used for incorporating the O‐glycosylated amino acid residue. Z‐Thr[α‐Glc(OBzl)4]‐OBzl and Z‐Thr[α‐Gal(OBzl)4]‐OBzl were prepared from the tetra‐O‐benzylated sugar and Z‐Thr‐OBzl by the trichloroacetimidate method in the presence of trimethylsilyl trifluoromethane sulfonate. The α glycosylated threonine derivatives were converted into Fmoc‐Thr(α‐G1c)‐OH and Fmoc‐Thr(α‐Gal)‐OH by catalytic hydrogenation followed by acylation with Fmoc‐OSu. β‐Glucosylation and β‐galactosylation of threonine were carried out by reacting the proper per‐O‐acetylated sugar with Z‐Thr‐OBzl and boron trifluoride ethyl etherate in dichloromethane. Catalytic hydrogenation of the β‐O‐glycosylated threonine derivatives followed by acylation with Fmoc‐OSu and deacetylation with methanolic hydrazine yielded Fmoc‐Thr(β‐Glc)‐OH and Fmoc‐Thr(β‐Gal)‐OH, respectively. The O‐glycosylated threonine derivatives were then reacted with H‐Lys(Z)‐Pro‐Arg(NO2)‐OBzl in the presence of DCC and HOBt and the resulting glycosylated tuftsin derivatives were fully deblocked by catalytic hydrogenation, purified by HPLC, and characterized by optical rotation, amino acid analysis, and 1H NMR. The β‐galactosylated tuftsin was also prepared by the continuous flow solid phase procedure.
Synthesis, metabolic stability and chemotactic activity of peptide T and its analogues
Authors: MARASTONI MAURO , SALVADORI SEVERO , BALBONI GIANFRANCO , SPISANI SUSANNA , GAVIOLI RICCARDO , TRANIELLO SERENA , TOMATIS ROBERTO
Published: August 2010

Acquired immunodeficiency syndrome (AIDS) is initiated by the attachment of the human immunodeficiency virus (HIV) to a surface glycoprotein CD4 present on T4 helper/inducer lymphocytes, monocytes/macrophages and other cells. A simple octapeptide (H‐Ala‐Ser‐Thr‐Thr‐Thr‐Asn‐Tyr‐Thr‐OH, peptide T) seems to inhibit HIV infectivity and to activate human monocyte chemotaxis. In order to study in vitro metabolic stability and structure‐activity relationships, peptide T and a number of analogues were prepared and tested on human monocytes by chemotactic assay. Peptide T and the shorter fragments T(3‐8)‐OH and T(4–8)‐OH displayed potent bioactivity (maximal chemotactic activity in the range 10‐11‐10‐10M). The C‐terminal heptapeptide showed a reduction of potency, while further truncations at N‐terminus of T(4–8)‐OH abolished the biological action. In the octapeptide series, whereas the α‐amino butyric acid (Abu) substitution for Thr4 was well tolerated, the same “slight” structural change at Thr5 or Thr8 was very detrimental. Finally, [d‐Asn6]T(1‐8)‐OH analogue has low chemotactic activity. All these results indicate that i) the C‐terminal pentapeptide is the minimum sequence required for bioactivity, ii) residues 5 to 8 appear to play a crucial biological role, iii) peptide T chemotaxis is mediated, at least in part, through the polar properties of Thr side chains at the critical positions 5 and 8, while the Thr4 does not interfere with biological characteristics of peptides. With regard to the enzymic degradation, the in vitro experiments showed the susceptibility of peptide T to rapid metabolism by human or rat plasma (T1/2 = 5.2 min), rat brain (T1/2 = 2.1 min) and kidney (T1/2 = 0.4 min) enzymes. The main metabolic product appears to be the C‐terminal heptapeptide, which retains, in turn, a low enzymatic stability.
Divalent metal ion mediated interaction of proteins with negatively charged membranes A model study employing molecular mechanics
Authors: CAIN J.D. , DEERFIELD D.W. , HISKEY R.G. , PEDERSEN L.G.
Published: August 2010

Previous molecular mechanics calculations on the effect of Ca(II) and Mg(II) ions on the conformation of the 18‐23 loop of bovine prothrombin [Maynard et al. 1988, Int. J. Peptide Protein Res. 31, 137–1491 are extended to include the effect of a model phospholipid head group methyl[l‐seryl] phosphate. Whereas the conformation of the Gla‐21 Pro‐22 amide bond remains decidedly trans in the absence of the model head group, in its presence, the cis Ca(II) ion induced (but not Mg(II)) form is significantly lowered in relative energy. The low energy Ca(II) structures establish a coordination sphere with more ligands than do the low energy Mg(II) ion structures.
Enzymatic synthesis of dipeptide units of the d‐d‐configuration in aqueous media
Authors: KEMPE MARIA , EKBERG BJÖRN , MOSBACH KLAUS
Published: August 2010

The enzymatic synthesis of dipeptide units of the d‐d‐configuration in aqueous media, catalysed by muramoyl‐pentapeptide carboxypeptidase (E.C.3.4.17.8), is described. Ac‐l‐Lys(Ac)‐d‐Ala‐d‐Lac‐OH and Ac‐d‐Ala‐OMe were used as acyl‐components. Neutral, basic, and hydrophobic amino acids acting as nucleophiles were incorporated. The enzyme is stereospecific in that only the d‐enantiomers of amino acids or amino acid derivatives were incorporated. As nucleophiles, the unmodified amino acids resulted in higher product yields compared with using the corresponding amino acid derivatives. Product yields ranged from 40 to 87%
5,5‐Dimethylthiazolidine‐4‐carboxylic acid (DTC) as a proline analog with restricted conformation
Authors: SAMANEN J. , ZUBER G. , BEAN J. , EGGLESTON D. , ROMOFF T. , KOPPLE K. , SAUNDERS M. , REGOLI D.
Published: August 2010

Spectroscopic evidence is presented for the lack of intramolecular hydrogen bonding in a simple peptide derivative of 5,5‐dimethylthiazolidine‐4‐carboxylic acid (Dtc). The infrared spectrum of Boc‐Pro‐Ile‐OMe 1 in nonpolar solvents displays two N‐H stretching bands at 3419 and 3330 cm‐1 in CCl4 and one at 3417 and 3328cin‐1 in CHCl3. The low frequency band at 3328–3330cm‐1 may be assigned to conformations with an intramolecular hydrogen bond between the Ile N‐H and Boc C=O. The band at 3417‐3419 cm‐1 is the normal Ile N‐H stretch. In the polar solvent CH3 CN only one NH stretching band at 3365 cm‐1 is observed. The IR spectrum of Boc‐Dtc‐Ile‐OMe 2, on the other hand, displays one N‐H stretching band at 3423cm‐1 in CCI, and one at 3418cm‐1 in CHCI3. The IR spectrum of 2 does not display the N‐H stretching band that would arise from intramolecular hydrogen bonding between the Boc C=O and Ile N‐H. The lack of intramolecular hydrogen bonding for Boc‐Dtc‐Ile‐OMe 2 was evident also in the NMR spectra in nonpolar solvents. The 1H‐NMR spectrum of the Pro dipeptide 1 in 50% CDCl3/C6D6 at 20° displayed two Ile‐NH signals at 6.58 and 7.74 ppm. The latter signal corresponds to the intramolecularly hydrogen bonded Ile‐NH in the trans‐Boc isomer of 1 (60% of the total population), while the former signal corresponds to the nonhydrogen bonded Ile‐NH in the cis‐Boc isomer. The 1H‐NMR spectrum of the Dtc dipeptide 2 displayed two slowly exchanging cis‐ and trans‐Boc amide isomers as well, but both amide proton resonances were observed upfield at 6.67 and 6.74 ppm, which correspond only to a nonhydrogen bonded Ile N‐H. The X‐ray crystal structure of Boc‐Dtc displays only a cis‐Boc‐Dtc urethane amide group and two conformations for the Dtc ring, one in which the beta carbon atom is anti to the carboxyl group and the other in which the gamma sulfur atom is anti to the carboxyl group. Conformational analysis of Ac‐Dtc‐NHMe suggests that in the hydrogen bonded C7 conformation steric interaction between the syn‐beta methyl group and carbonyl group of Dtc adds nonbonded and angle strain energies to counteract the stabilizing coulombic interaction between the Boc C=O and terminal amide N‐H. Whereas the C7 conformation is a prominent conformation for peptide derivatives of proline, other conformations are favored in peptide derivatives of Dtc (ψ ‐ñ 110‐150° or ñ 320‐360°). These results suggest that, in peptides where substitution of Pro appears to maintain or enhance biological activity, the substitution of Dtc for Pro may test the functional importance of the C7 conformation in that position of the peptide sequence.
Release of peptides from sustained delivery systems (microcapsules and microparticles) in vivo
Authors: CSERNUS VALER J. , SZENDE BELA , SCHALLY ANDREW V.
Published: August 2010

Sustained delivery systems (microcapsules, microparticles, or implants) developed for once a month administration of peptides are efficacious and convenient. Long acting formulations of several bioactive peptides are based on microcapsules of a biodegradable polymer poly(dl‐lactide‐co‐glycolide) (PLG), but a better understanding is required of the mechanism of the peptide release from the microcapsules, which is assumed to be primarily by diffusion through pores. In order to clarify this mechanism, microcapsules and microparticles of the agonist [d‐Trp6]‐LHRH and microcapsules of the LHRH antagonist SB‐75 were given i.m. to rats 2 h and 1, 2, 4, 7, 14 and 21 days before histological and immunohistochemical investigation. Signs of biodegradation of the PLG matrix could be seen the first day after the injection, in a form of vacuole development in the interior of the particles and connected with the presence of macrophages within the matrix. The microcapsules showed excellent tissue‐compatibility, and no significant foreign body reaction was detected. Immunohistochemical study on the microcapsules revealed no visible decrease in peptide concentration in the remnants of the matrix even 2 weeks after the injection. Evaluation of serum [d‐TrP6]‐LHRH showed that after an initial burst, both microcapsules and microparticles maintained elevated serum [d‐Trp6]‐LHRH levels for more than 3 weeks. Our results suggest that the previously proposed mechanisms do not reflect the experimental findings, particularly for the insoluble peptides. The peptide release from the PLG microcapsules or microparticles appears to be controlled mostly by the speed of the biodegradation of the polymer matrix and the diffusion of the peptides from the PGL is negligible.
Large fragments of nef‐protein and gp110 envelope glycoprotein from HIV‐1
Authors: SABATIER J.‐M. , FONTAN G. , LORET E. , MABROUK K. , ROCHAT H. , GLUCKMAN J.‐C. , MONTAGNIER L. , GRANIER C. , BAHRAOUI E. , VAN RIETSCHOTEN J.
Published: August 2010

Chemical synthesis of large peptide fragments (from 18 to 66 amino acid residues long) of the gp110 envelope glycoprotein and of nef‐protein from HIV‐1 was achieved by the solid phase method. Stepwise assembling of the peptide chains was carried out automatically on 4‐(oxymethyl)‐phenylacetamidomethyl resin using the N‐α‐butyloxycarbonyl amino acids with benzyl‐based side chain protecting groups. Two elongation protocols were used depending on the peptide chain length: a standard cycle, mainly characterized by a single coupling step (Boc‐amino acid symmetrical anhydride in dimethylformamide), and an optimized one for large peptides, based on a double coupling strategy (Boc‐amino acid symmetrical anhydride first in dimethylformamide, then in dichloromethane). Final cleavage of the peptide from the solid support was carried out by anhydrous hydrogen fluoride and crude peptides were purified by C18 reverse phase medium pressure liquid chromatography after molecular filtration. Characterization of the purified peptides was done by analytical HPLC, amino acid content determination, and circular dichroism analysis both in polar (H2O) and in non‐polar (TFE) environments. Immunoreactivity of anti‐nef positive sera from HIV‐1 infected patients by ELISA with the synthetic peptides was investigated. The results showed four major antigenic regions of nef‐protein and mainly the immunodominance of the N‐ and C‐termini of the molecule. Several of these peptides should prove to be useful for both diagnosis and vaccination purposes.
Synthesis of peptide‐immunogens corresponding to amino acid sequences from human histocompatibility class II membrane glycoproteins
Authors: CHILLEMI FRANCESCO , CAPPELLETTI SILVANA , FRANCESCATO PIERANGELO , CHERSI ALBERTO
Published: August 2010

Six peptides with amino acid sequences of human histocompatibility Class II membrane glycoproteins were synthesized by conventional solution methods. Five peptides were prepared by stepwise procedures from the carboxyterminus. The sixth was synthesized by fragment condensation (5 + 10 coupling). Antibodies to synthetic peptides were then used to locate exposed and buried regions in the membrane glycoproteins.
Synthesis of a cyclic hexapeptide with sequence corresponding to murine tumor necrosis factor‐(127–132) as a novel potential antitumor agent
Authors: SHEH LEUNG , CHENG JAI‐YI , KUAN YEN‐HUI , CHEN CHIA‐FU
Published: August 2010

A cyclic hexapeptide cyclo(Lys‐Gly‐Asp‐Gln‐Leu‐Ser‐) 10 was synthesized stepwise in solution by acylation of peptide ester trifluoroacetates directly with preactivated Boc‐amino acids using the DCC/HOBt method; the final cyclization reaction was performed using the pentafluorophenyl ester method in solution (1–4). This peptide is a cyclic derivative of murine tumor necrosis factor‐(127–132) and is designed as a potential antitumor agent. The cyclic peptide 10 displayed weak cytotoxic activity on three of the four human tumor cell lines tested.
Conformations of disulfide bridges in proteins
Authors: SRINIVASAN N. , SOWDHAMINI R. , RAMAKRISHNAN C. , BALARAM P.
Published: August 2010

The conformational characteristics of disulfide bridges in proteins have been analyzed using a dataset of 22 protein structures, available at a resolution of 2.0 Å, containing a total of 72 disulfide crosslinks. The parameters used in the analysis include (φ, Ψ) values at Cys residues, bridge dihedral angles χss, χ1i, χ1j, χ2i and χ2j the distances Cαi‐Cαj and Cβi‐Cβj between the Cα and Cβ atoms of Cys(i) and Cys(j). Eight families of bridge conformations with three or more occurrences have been identified on the basis of these stereochemical parameters. The most populated family corresponds to the “left handed spiral” identified earlier by Richardson ((1981) Adv. Protein Chem. 34, 167–330). Disulfide bridging across antiparallel extended strands is observed in α‐lytic protease, crambin, and β‐trypsin and this structure is shown to be very similar to those obtained in small cystine peptides. Solvent accessible surface area calculations show that the overwhelming majority of disulfide bridges are inaccessible to solvent.
Synthesis and biological activity profiles of atrial natriuretic factor (ANF) analogs
Authors: GOGHARI M.H. , DELEAN A. , GARCIA R. , CANTIN M. , SCHILLER P.W.
Published: August 2010

Several analogs of the atrial natriuretic factor (ANF) were synthesized by the solid‐phase method using the acetamidomethyl (Acm) group for sulfhydryl protection. The compounds were tested in a receptor binding assay using bovine adrenal zona glomerulosa cell membranes and in the rat diuresis/natriuresis assay. Substitution of tyrosine in position 116 of ANF(101–126) and of the analog [3‐Mpr105]ANF(105–126)(3‐Mpr = 3‐mercaptopropionic acid) did not alter the biological activity profiles and, therefore, these two analogs in radioiodinated form will be useful for enzymatic degradation and clearance studies. Replacement of 3‐mercaptopropionic acid with 2‐mercaptopropionic acid in [3‐Mpr105]ANF(105–126) resulted in an analog with very low potency in both assay systems, presumably as a consequence of the steric bulk and/or local conformational restriction produced by the methyl group attached to the α‐carbon in position 105. The analog [3‐Mpr105, Nva109]ANF(105–126)(Nva = norvaline) showed very low affinity in the receptor binding assay but displayed considerable diuretic/natriuretic activity. The obtained biological activity profiles suggest that in comparison with other ANF peptides the des‐amino ANF(105–126) analogs may have a somewhat longer half‐life in vivo or, alternatively, may indicate a more complex situation of ANF receptor or binding site heterogeneity.
Synthesis and 11C‐labelling of the ACTH fragment analogue H‐Met(O2)‐Glu‐His‐Phe‐d‐Lys‐Phe‐OH (Org 2766) via its homocystine‐containing precursor
Authors: VAN NISPEN J.W. , JANSSEN W.P.A. , MELGERS P.A.T.A. , JANSSEN P.S.L. , JANSEN J.F.G.A. , VAALBURG W.
Published: August 2010

The hexapeptide dimer (H‐Hcy‐Glu‐His‐Phe‐d‐Lys‐Phe‐OH)2 was synthesized using solution methods and characterized. Its conversion into H‐Met(O2)‐Glu‐His‐Phe‐d‐Lys‐Phe‐OH, Org 2766, was studied on a small scale in as short a time as possible; reduction of the disulfide bond using Na/NH3, reaction with CH3I, oxidation with H2O2 and catalyst and purification by HPLC were carried out starting with 2 mg of the dimer in a total preparation time of approximately 22min, starting with the addition of CH3I. The preparation of the 11C‐labelled analogue was carried out by methylation with 11CH3I. Restrictions imposed by working with carbon‐11 will be discussed.
Intramolecular H‐bonds and thioamide rotational isomerism in thiopeptides
Authors: HOLLÓSI MIKLÓS , ZEWDU MEDHEN , KOLLÁT EMMA , MAJER ZSUZSA , KAJTÁR MÁRTON , BATTA GYULA , KÖVÉR KATALIN , SÁNDOR PÉTER
Published: August 2010

Mono‐ and dithionated N‐acyl amino acid and dipeptide N′‐methylamides were synthesized using Lawesson's reagent and 5‐thioacetyl thioglycolic acid. The conformation of the thionated models was characterized by IR, 13C, and 1H NMR spectroscopy, including NOE experiments. The formation of —C=S…H—N—C=X (X = O or S) intramolecular H‐bonds of the type 2 → 2. 1 → 3 and 1 → 4 was evidenced by the characteristic shifts of the IR stretching frequencies of the NH group. Act‐Pro‐NHCH3 (4) and Act‐Prot‐NHCH3 (5) were found to be present as mixtures of rotational isomers about the CS—N bond. 13C chemical shifts of the γ‐ and β‐carbons of the proline ring elucidated the conformation (Z or E) of the tertiary thioamide group. Our results suggest that the conformation of thiopeptides is determined by two factors: 1) the H‐bond donating and accepting ability of the thioamide group and 2) the repulsion between the thiocarbonyl sulfur atom and the side chain groups of the neighbouring amino acid residues.
Isolation, identification, and characterization of a palladium complex in the catalytic deprotection of a protected peptide
Authors: HEAVNER GEORGE A. , CODY WAYNE L. , CROWTHER JONATHAN , DUHL‐EMSWILER BARBARA , MAY KAREN , SALOMONS PAUL , PASCONE JOHN
Published: August 2010

The final catalytic deprotection in the large scale synthesis of thymopentin (Arg‐Lys‐Asp‐Val‐Tyr) produced an impurity which had not previously been observed. Isolation by preparative HPLC and spectroscopic characterization led to a postulated structure of the impurity as the 1: 1 thymopentin‐palladium complex. A complex corresponding to the proposed structure was independently synthesized and shown to have identical chromatographic and spectroscopic properties with the isolated material. Proton and carbon (13) NMR were used to determine the coordination sites of the peptide with palladium. The susceptibility of the complex to hydrogenation indicated a possible source for its formation.
Rapid determination and NMR assignments of antiparallel sheets and helices of a scorpion and a cobra toxin
Authors: LAPLANTE STEVEN R. , MIKOU AFAF , ROBIN MICHEL , GUITTET ERIC , DELSUC MARC‐ANDRE , CHARPENTIER ISABELLE , LALLEMAND JEAN‐YVES
Published: August 2010

An NMR method is described which should provide a rapid means for determining and assigning antiparallel sheets and helices in small proteins. It begins by locating apparent NOESY crosspeaks which suggest the presence of the secondary structure; this is followed by searches for MCD patterns (Englander & Wand (1987) Biochemistry 22, 5953) which are characteristic of these structures. As a result, only spin‐systems of the amino acids within the secondary structure need to be defined. A triple‐stranded, antiparallel sheet and a helix have been found and assigned for both α‐cobratoxin and the scorpion toxin AaH III.
Effect of somatostatin analogs on gastric acid secretion in dogs and rats
Authors: SCHALLY ANDREW V. , COLALUCA JOHN , PAULSON DALE , CARTER WELDON H. , NEITZSCHMAN HAL R. , LAFAYE HADEN , CAI R.‐Z.
Published: August 2010

The effects of several superactive analogs of somatostatin on gastric acid response to various exogenous and endogenous stimulants were investigated in conscious dogs and rats with gastric fistulae (GF). The inhibition was compared to that induced by somatostatin‐14 (S‐S‐14) at two dose levels. Several octapeptide analogs of somatostatin including (RC‐160) and (RC‐121), which were superactive in tests on suppression of GH levels, were 4‐5 times more potent than S‐S‐14 in inhibiting desglugastrin‐stimulated gastric acid secretion in GF dogs. The analog RC‐160 also reduced the rise in serum gastrin levels and gastric acid secretion induced by sham feeding (SF) in dogs with gastric and esophageal fistulae (EF), but did not decrease food consumption. Gastric acid secretion induced by histamine (80μg/kg/h) in dogs was not affected by 1‐5μg/kg/h of analog RC‐121 or by 5μg/kg/h of S‐S‐14. Analogs RC‐160, RC‐121, and RC‐98‐I and others also powerfully inhibited desglugastrin‐induced gastric acid secretion in GF rats. Hexapeptide cyclo(‐Pro‐Phe‐D‐Trp‐Lys‐Thr‐Phe) was only about half as potent as S‐S‐14 in dogs but its activity was higher in rats. The results indicate that octapeptide analogs which are superactive in GH‐inhibition tests are also more potent than S‐S‐14 in suppressing gastric acid secretion. These findings may be of clinical value.
Evidence for presence of Tyr‐MIF‐1 (Tyr‐Pro‐Leu‐Gly‐NH2) in human brain cortex
Authors: HORVATH ANIKO , KASTIN ABBA J.
Published: August 2010

Tyr‐MIF‐1 (Tyr‐Pro‐Leu‐Gly‐NH2) was previously isolated from bovine hypothalamus. We have now purified it from the parietal cortex of human brain tissue by gel filtration chromatography and four subsequent high performance liquid chromatographic steps. During isolation, the peptide content was followed by radioimmunoassay and compared with the elution of synthetic Tyr‐MIF‐1 in identical chromatographic systems. This extends evidence for the presence of Tyr‐MIF‐1 from bovine to human brain tissue and from hypothalamus to cortex.
Conformation of cyclo‐(D‐phenylalanyl‐trans‐4‐fluoro‐D‐prolyl)
Authors: CIARKOWSKI J. , GDANIEC M. , KOŁODZIEJCZYK A. , LIBEREK B. , BORREMANS F.A.M. , ANTEUNIS M.J.O.
Published: August 2010

cyclo(D‐Phenylalanyl‐trans‐4‐fluoro‐D‐prolyl), c(D‐Phe‐D‐FPro), was synthesized and its conformation determined both in solution and in the solid state by 1H NMR and X‐ray analysis, respectively. In the crystals the 2,5‐diketopiperazine (DKP) ring assumes the uncommon conformation, for cyclodipeptides containing Pro residue, of a flattened chair, which seemingly results from a compromise between, on the one hand, the DKP‐aromatic intramolecular ring‐ring attraction (folding), requiring the Cα‐Cβ bond of the Phe to be axial, and, on the other hand, the intrinsic tendency of the Pro residue to have its Cα‐Cβ bond equatorial. Unlike the solid state, the 1H NMR data in CDCl3 and C6D6 demonstrate that in both solutions the DKP ring assumes a boat‐like shape, typical for the Pro‐containing cyclodipeptides, with the equatorial Cα‐Cβ bonds in both amino acid residues, which preclude ring‐ring folding. A similar conformation was encountered in the closest analog of c(D‐Phe‐D‐FPro), viz. in c(Phe‐Pro), both in solution (21, 22, 26) and in the solid state (12). A subtle interplay of intramolecular interactions introduced into a cyclodipeptide by a Pro‐type and a Phe‐type residue is emphasized.
Structure and absolute configuration of pyrophen, a novel pryrone derivative of L‐phenylalanine from Aspergillus niger
Authors: BARNES CHARLES L. , STEINER JORGE RIOS , TORRES EDITH , PACHECO ROSA , MARQUEZ HECTOR
Published: August 2010

Pyrophen, a novel 4‐methoxy‐2‐pyrone derivative of L‐phenylalanine isolated from cultures of Aspergillus niger, crystallizes in the orthorhombic space group P212121, with a = 8.918(1), b = 9.199(3), and c = 18.092(4) Å. The structure was solved by direct methods and the absolute configuration assigned by the Bijvoet method. Refinement gave R = 0.045 and S = 1.78 for 1677 reflections with I > 3σ(I). Though numerous other pyrone natural products are known, including other products of A. niger, this is the first report of an amino acid‐pyrone derivative.
Studies on somatostatin with time‐resolved spectroscopy and molecular dynamics simulations
Authors: ELOFSSON ARNE , NILSSON LENNART , RIGLER RUDOLF
Published: August 2010

Somatostatin was studied by time‐resolved fluorescence spectroscopy and by molecular dynamics simulations. The results obtained indicate the existence of mainly one conformation in DMSO, and the existence of several conformations in other solvents. Molecular dynamics simulations showed that the most important region for activity (residue 7‐9) is also the most flexible region in the peptide.
Non‐mammalian “big” neurophysins — complete amino acid sequence of a two‐domain MSEL‐neurophysin from goose
Authors: MICHEL GILLES , LÉVY BRIGITTE , CHAUVET MARIE‐THÉRÈSE , CHAUVET JACQUELINE , ACHER ROGER
Published: August 2010

Vasotocin‐associated neurophysin (MSEL‐neurophysin) has been purified from goose neurohypophysis through molecular sieving and high‐pressure reverse‐phase liquid chromatography (HPLC). The protein has a molecular mass (measured by SDS‐polyacrylamide gel electrophoresis) of 17kDa in contrast to 10kDa found for the mammalian MSEL‐neurophysins. Complete amino acid sequence (131 residues) has been determined mainly through tryptic or staphylococcal proteinase peptides derived from carboxyamido‐methylated neurophysin, isolated by HPLC and microsequenced. N‐ and C‐terminal sequences have been established by Edman degradation or action of carboxypeptidase Y, respectively, applied on the native protein. Goose MSEL‐neurophysin is homologous to the two‐domain “big” MSEL‐neurophysin previously identified in the frog. It appears that in non‐mammalian tetrapods, namely birds and amphibians, the proteolytic processing of the pro‐vasotocin involves only one cleavage, releasing the hormone moiety and a “big” neurophysin with two domains homologous to mammalian MSEL‐neurophysin and copeptin, respectively. Comparison of the avian protein with its mammalian and amphibian counterparts reveals that the first half of the polypeptide chain is evolutionarily much less variable than the second and that the goose protein resembles the frog protein much more than the mammalian one.
Synthesis of α‐MSH fragments containing phenylalanine mustard for receptor studies
Authors: SÜLI‐VARGHA H. , BOTYÁNSZKI J. , MEDZIHRADSZKY‐SCHWEIGER H. , MEDZIHRADSZKY K.
Published: August 2010

For chemical affinity labeling of the melanotropin receptor several α‐MSH fragments containing phenylalanine mustard were synthesized in solution. Tested in the frog skin bioassay the derivatives roughly preserved the biological activity of the corresponding natural sequences. Alkylating peptides with prolonged biological activity containing phenylalanine mustard in place of arginine, phenylalanine or methionine are inhibitors of α melanotropin, suggesting an irreversible binding to reactive nucleophiles on the part of the MSH receptor, where the Met‐Glu‐His‐Phe‐Arg sequence is attached.
Structure and immunological properties of thymosin β9 Met, a new analog of thymosin β4 isolated from porcine thymus
Authors: LOW TERESA L.K. , LIN CHING‐YUANG , PAN TAIR‐LONG , CHIOU AIH‐JING , TSUGITA AKIRA
Published: August 2010

During the course for the studies of thymosin β4 and prothymosin α from porcine thymus, a new analog of thymosin β4 has been identified. This peptide consists of 41 amino acid residues. The amino terminus is blocked by an acetyl group as revealed by fast atom bombardment mass spectrometric analysis. Amino acid sequence studies disclosed that this peptide is identical to bovine thymosin β9 except that leucine at position 6 in β9 is substituted by methionine. Thus, this new peptide has been termed thymosin β9 Met. The recoveries of β9Met, β4, and prothymosin α in porcine tissues have been determined (in μg/g tissue) as follows: thymus (43, 85, 133); spleen (68, 203, 37); liver (10, 31, 27); heart (1.5, 10, 0); kidney (5, 51, 37); brain (0.8, 31, 5). Biologically, thymosin β9 Met was found to be more active than β4 in enhancing γ‐interferon production in cord blood lymphocytes. However, β4 appeared to stimulate higher amounts of interleukin 2 and tumor necrotic factor. The significance for the coexistence of two homologous peptides with similar functions in the thymus and a number of other organs is not clear, and deserves further investigation.
Peptides related to the active fragment of “proline rich polypeptide”, an immunoregulatory protein of the ovine colostrums
Authors: SIEMION IGNACY Z. , FOLKERS GERD , SZEWCZUK ZBIGNIEW , JANKOWSKI ANDRZEJ , KUBIK ALEKSANDRA , VOELTER WOLFGANG
Published: August 2010

The preferred solution conformation of the PRP‐hexapeptide (Tyr‐Val‐Pro‐Leu‐Phe‐Pro) and of some of its structural analogues was investigated by NMR‐ spectroscopy, spectrofluorimetry and computer simulation technic. It was found that the preferred conformation is characterized by cis′‐conformation of Pro3 and the γ‐turn on the Leu4‐residue: for Val2 and Phe5 a β‐structure seems to be privileged. In such a conformation Val2 and Leu4 residues occupy exactly the same positions in space as residues i and i+ 3 in an α‐helix. It suggests that the PRP‐hexapeptide can interact with receptor protein inducing or stabilizing its helical conformation by “knobs into holes” packing.
Neuropeptide Y: identification of the binding site
Authors: BECK‐SICKINGER ANNETTE G. , GAIDA WOLFRAM , SCHNORRENBERG GERD , LANG RUDOLPH , JUNG GÜNTHER
Published: August 2010

Based on the hypothetical 3D structure of neuropeptide Y (NPY), NPY 1‐4‐Aca‐25‐36, a 17 amino acid analogue, has been synthesized replacing the sequence NPY 5‐24 by ε‐aminocaproic acid (Aca). This low‐molecular weight deletion analogue showed nearly comparable receptor affinity to NPY. In order to elucidate the structural requirements for receptor recognition each amino acid of 1‐4‐Aca‐25‐36 was exchanged by its D‐enantiomer, glycine and L‐alanine. In addition distinct amino acids were replaced by closely related residues. Multiple peptide synthesis was applied using Fmoc‐strategy and BOP activation. Binding assay was performed on rabbit kidney membrane preparations. The results of structure affinity studies suggest that the C‐terminal tetrapeptide NPY 33‐36 is essential for receptor recognition.