bluuebunny/crossref_metadata_2025 huggingface dataset:
Enzymatic semisynthesis of porcine despentapeptide (B26–30) insulin using unprotected desoctapeptide (B23–30) insulin as a substrate
Authors: KUBIAK TERESA , COWBURN DAVID
Published: August 2010

Unprotected porcine desoctapeptide(B23–30) insulin (DOPI) and the synthetic Gly‐Phe‐Phe were used as substrates for the trypsin‐catalyzed synthesis of despentapeptide(B26–30) insulin (DPPI). The DPPI synthesis was accompanied by a moderate oligomerization and by the formation of a side produce which was identified as a DOPI derivative having an extra peptide bond between the Gly(A1) and Arg(B22) and which was named des(23–63) proinsulin (1). Despite side reactions, the conditions were found where the overall DPPI yields were comparable to those obtained via di‐Boc DOPI, and these procedures were faster and simpler since the Boc protection and deprotection steps were omitted. The reaction progress was directly monitored by HPLC.
Side reactions in peptide synthesis
Authors: Perseo Giuseppe , Forino Romualdo , Galantino Mauro , Gioia Bruno , Malatesta Vincenzo , De Castiglione Roberto
Published: August 2010

During the hydrogenolytic removal of the benzyl ester from the pentapeptide Boc‐Asp(OBzl)‐Ala‐Phe‐Ile‐Gly‐OEt, either with hydrogen gas or ammonium formate in the presence of palladium‐on‐charcoal as catalyst, variable amounts (5–30%) of an unexpected by‐product were always obtained. The compound was eventually identified as Boc‐Asc‐Ala‐Phe‐Ile‐Gly‐OEt (Asc = aminosuccinyl). The application of field desorption mass spectrometry as a diagnostic tool is reported. The study of this side reaction, carried out on the pentapeptide and two other dipeptide models, showed that: 1) palladium‐on‐charcoal may induce Asc formation; 2) the contemporary presence of the catalyst and a base (even in trace amounts) greatly increases the by‐product formation; 3) the side reaction is sequence and solvent dependent; 4) the Asc formation is completely prevented by adding a few equivalents of acetic or formic acid. Some mechanistic considerations are also reported.
Vasopressin antagonist analogs modified at position 7
Authors: MOORE MICHAEL L. , GREENE HEIDEMARIE , HUFFMAN WILLIAM F. , STASSEN FRANS , STEFANKIEWICZ JAMES , SULAT LYNN , HECKMAN GRACE , SCHMIDT DULCIE , KINTER LEWIS , McDONALD JEANNE , ASHTON‐SHUE DARYL
Published: August 2010

Vasopressin antagonist analogs having alanine or glycine at position 7 were essentially equipotent with analogs with proline,N‐methylalanine or sarcosine at position 7. This demonstrates that the conformational constraint imposed by anN‐alkyl residue at position 7 is not necessary for binding of antagonist to the receptor, the exact opposite of what is seen in agonists. This suggests that antagonists bind to the receptor in a manner which is very different from that of agonists.
Phenylethylamide derivatives of the C‐terminal tetrapeptide of gastrin
Authors: MARTINEZ JEAN , RODRIGUEZ MARC , BALI JEAN‐PIERRE , LAUR JEANINE
Published: August 2010

Peptide analogues of the C‐terminal tetrapeptide of gastrin in which the phenyl‐alanine had been replaced were synthesized and their biological activity on acid secretion evaluated. Compounds Boc‐Trp‐Leu‐Asp phenylethylamide 6, Boc‐β‐Ala‐Trp‐Leu‐Asp phenylethylamide 9, Boc‐Trp‐Leu‐Asp p‐fluorophenylethylamide 19, Boc‐Trp‐ø(CH2NH)‐Leu‐Asp phenylethylamide 23, Boc‐Trp‐Leu‐Asp 2,2‐diphenylethylamide 15, and Boc‐D Trp‐Leu‐Asp 2,2‐diphenylethylamide 21, in which the phenylalanine had been replaced by phenylethylamine, p‐fluorophenylethylamine or 2,2‐diphenylethylamine were synthesized. None of these derivatives showed activity on acid secretion in the anaesthetized rat at doses as high as 5 mg/kg. However, they were potent inhibitors of gastrin‐induced acid secretion, with ED50 varying from 0.1 to 0.6 mg/kg.
Synthesis and hormonal activity of [Tyr 22] glucagon and [desHis 1, Tyr 22] glucagon
Authors: LU GUI‐SHEN , MOJSOV SVETLANA , MERRIFIELD R.B.
Published: August 2010

[Tyr22] glucagon and [desHis1, Tyr22] glucagon were synthesized by an improved solid phase procedure on a Pam‐resin. The course of the synthesis was monitored by quantitative ninhydrin analysis and preview sequencing. Following cleavage by the low/high HF method the peptides were purified by ion exchange chromatography and reverse phase HPLC. The overall yield of homogeneous isolated peptide from the first amino acid was 41%. Circular dichroism measurements on dilute solutions in mixed aqueous organic solvents at pH 2, 6.9 and 9.2 showed increased β‐sheet structure relative to glucagon. [Tyr22]glucagon was a full agonist with 20–30% activity in the rabbit blood glucose assay and 10% activity in the rat liver membrane adenyl cyclase assay. [desHis1, Tyr22] glucagon had only a trace of activity in the adenyl cyclase assay (>0.002%) but bound to membranes in a competitive [125I] glucagon assay 1.0% as well as glucagon. The analog completely inhibited formation of cAMP by natural glucagon, with 50% inhibition at a ratio of 83:1 and pA2 = 6.7. The data are discussed in terms of models of glucagon structure in dilute solution.
Conformational analysis of pepstatin and related renin inhibitors by 400 MHz1H n.m.r. spectroscopy
Authors: ROY P. , DELEPIERRE M. , WAGNON J. , NISATO D. , ROQUES B.P.
Published: August 2010

The conformational behaviour of pepstatin (Iva‐Val‐Val‐Sta‐Ala‐Sta) and of two derived renin inhibitors, Boc‐Phe‐Nle‐Sta‐Ala‐Sta‐OMe, 1, and Boc‐Phe‐Nle‐X‐Ala‐Sta‐OMe, 2 (X = ‐NH‐CH(iPr)‐CHOH‐CH2‐CO‐) was assessed in DMSO‐d6at various temperatures and in deuteriopyridine at ‐35°. Complete assignment of almost all proton signals was achieved by 2D COSY, 2D NOESY and selective NOE experiments. The three compounds show similar extended conformations in both solvents, with the hydrophobic lateral chainsextending awayfrom the peptide backbone. In the case of pepstatin the solvated conformation is closely related to the structure found in the crystal of the pepstatin‐Rhizopus chinensiscomplex. Strong NOE effects and precise determination of vicinal coupling constants show the lack of large structural differences between1and2at the level of the internal Sta or X residues, which are assumed to interact with the aspartyl residues of the renin active‐site. This suggests that the 100‐fold lower inhibitory potency of2is mainly due to unfavorable close contacts of the β‐branched residue X with constituent amino acids of the enzyme.
Sequence dependence in the formation of pyroglutamyl peptides in solid phase peptide synthesis
Authors: ORLOWSKA ALICJA , WITKOWSKA EWA , IZDEBSKI JAN
Published: August 2010

A study was conducted to determine the effect of amino acid sequence on the rate of formation of pyroglutamate (pyrrolidone carboxylate) residues from amino‐terminal glutaminyl residues in peptides. The extent of cyclization from H‐Gln‐X‐OCH2‐resin was determined by a quantitative reaction, and found to be quite pronounced with X being Asn, Leu and Gly. The peptide‐resin with Leu was used to examine the influence of various reagents on the rate of pyroglutamate formation.
Easy cleavage of C′‐terminal iminoacids from peptide acids through acidic hydrolysis
Authors: VAN DER AUWERA C. , ANTEUNIS M.J.O.
Published: August 2010

Evidence is presented for the facile acid‐hydrolytic cleavage of C‐terminal N‐alkylated amino acid residues in peptide acids. A mechanism (defined as “back‐biting”) is proposed in which the process is initiated by a nucleophilic attack of the conjugated base of the terminal acid on the (protonated) preceding cis‐peptide bond.
Proton n.m.r. investigation of conformational influence of penicillamine residues on the disulfide ring system of opioid receptor selective Somatostatin derivatives
Authors: SUGG ELIZABETH E. , TOURWE DIRK , KAZMIERSKI WIESLAW , HRUBY VICTOR J. , VAN BINST GEORGES
Published: August 2010

Three cyclic disulfide analogs related to somatostatin, d‐Phe1 ‐Cys2‐Tyr3‐d‐Trp4‐Lys5‐Thr6‐Xxx7‐Thr8NH2 (where Xxx =l‐Pen 1; l‐Cys 3; or d‐Pen 4) were examined in DMSO‐d6 by one‐ and two‐dimensional proton n.m.r. spectroscopy in order to analyze the conformational influence of the position‐7 residue on the 20‐membered disulfide ring. From these studies it was concluded that all three analogs maintain a β II turn solution conformation for the core tetrapeptide‐Tyr3‐d‐Trp4‐Lys5‐Thr6‐. However, the disulfide conformation differs in the analogs, with 1 and 3 having a left‐handed and 4 a right‐handed disulfide chirality.
Ten‐membered cyclotripeptides
Authors: CERRINI SILVIO , GAVUZZO ENRICO , LUCENTE GINO , PINNEN FRANCESCO
Published: August 2010

The crystal structure of a conformationally restricted cyclotripeptide containing N‐methylanthranilic acid, l‐phenylalanine and l‐proline has been determined. The 10‐membered ring of cyelo(‐MeAnt‐Phe‐Pro‐) is characterized by a pseudosymmetry plane and by three cis amide bonds. The MeAnt aromatic ring makes an angle of 63.3° with the best plane of the backbone and is nearly perpendicular to the plane of the two adjacent amide bonds. The proline ring adopts βαT twist conformation (Ps = 144°) and the benzylic side chain is extended towards the nitrogen. An analysis of i.r. and n.m.r. spectral data indicates that the solution conformation retains the features found in the crystals. Crystal data: orthorhombic, P212121 with a= 10.153(3), b= 11.700(3), c= 16.402(3) Å and Z = 4. The final R and Rw are 0.047 and 0.067, respectively.
Role of steric interactions in the delta opioid receptor selectivity of [d‐Pen2,d‐Pen5]enkephalin
Authors: MOSBERG HENRY I. , HAASETH RONALD C. , RAMALINGAM KONDAREDDIAR , MANSOUR ALFRED , AKIL HUDA , WOODARD RONALD W.
Published: August 2010

In order to assess the individual effects of each of the 3‐methyl groups in residue 2 of [d‐Pen2,d‐Pen5]enkephalin on binding affinity to mu and delta opioid receptors, (2S,3S)methylcysteine ((3S)Me‐d‐Cys) and (2S,3R)methylcysteine ((3R)Me‐d‐Cys) were synthesized and incorporated into the analogs, [(3S)Me‐d‐Cys2,d‐Pen5]enkephalin and [(3R)Me‐d‐Cys2,d‐Pen5]enkephalin. Of these analogs, [(3S)Me‐d‐Cys2,d‐Pen5]enkephalin appears from1H n.m.r. spectra to assume a conformation similar to those of [d‐Pen2,d‐Pen5]enkephalin and the less delta receptor‐selective, but more potent, [d‐Cys2,d‐Pen5]enkephalin. Assessment of binding affinity to mu and delta receptors revealed that [(3S)Me‐d‐Cys2,d‐Pen5]enkephalin exhibits delta receptor affinity intermediate between [d‐Pen2,d‐Pen5]enkephalin and [d‐Cys2,d‐Pen5]enkephalin, while its mu receptor affinity is similar to that of [d‐Cys2,d‐Pen5]enkephalin. These results suggest that, for [d‐Pen2,d‐Pen5]enkephalin, adverse steric interactions between thed‐Pen2pro‐Rmethyl group and the mu receptor binding site lead to the low mu receptor binding affinity observed for this analog. By contrast, boththe pro‐Randpro‐Sd‐Pen2methyl groups lead to minor steric interactions which contribute to the somewhat lower delta receptor affinity of this compound.
X‐ray studies on crystalline complexes involving amino acids and peptides
Authors: SOMAN JAYASHREE , SURESH C.G. , VIJAYAN M.
Published: August 2010

l‐Lysine d‐glutamate crystallizes in the monoclinic space group P21 with a= 4.902, b= 30.719. c= 9.679 Å. β= 90° and Z = 4. The crystals of l‐lysine d‐aspartate monohydrate belong to the orthorhombic space group P212121 with a= 5.458, b= 7.152, c= 36.022 Å and Z = 4. The structures were solved by the direct methods and refined to R values of 0.125 and 0.040 respectively for 1412 and 1503 observed reflections. The glutamate complex is highly pseudosymmetric. The lysine molecules in it assume a conformation with the side chain staggered between the α‐amino and the α‐carboxylate groups. The interactions of the side chain amino groups of lysine in the two complexes are such that they form infinite sequences containing alternating amino and carboxylate groups. The molecular aggregation in the glutamate complex is very similar to that observed in l‐arginine d‐aspartate and l‐arginine d‐glutamate trihydrate, with the formation of double layers consisting of both types of molecules. In contrast to the situation in the other three ld complexes, the unlike molecules in l‐lysine d‐aspartate monohydrate aggregate into alternating layers as in the case of most ll complexes. The arrangement of molecules in the lysine layer is nearly the same as in l‐lysine l‐aspartate, with head‐to‐tail sequences as the central feature. The arrangement of aspartate ions in the layers containing them is, however, somewhat unusual. Thus the comparison between the ll and the ld complexes analyzed so far indicates that the reversal of chirality of one of the components in a complex leads to profound changes in molecular aggregation, but these changes could be of more than one type.
Enkephalin‐degrading enzyme inhibitors Crucial role of theC‐terminal residue on the inhibitory potencies of retro‐hydroxamate dipeptides
Authors: FOURNIÉ‐ZALUSKI MARIE‐CLAUDE , HERNANDEZ JEAN‐FRANÇOIS , SOLEILHAC JEAN‐MARC , RENWART NATHALIE , PEYROUX JACQUES , XIE JUAN , ROQUES'SOLE B.P.
Published: August 2010

The retro‐inversion of the amide bond in kelatorphan and analogs, the first series of complete inhibitors of enkephalin metabolism, led to compounds highly efficient only against the neutral endopeptidase 24‐11 (NEP). In order to increase the recognition of the aminopeptidase N (APN) and dipeptidylaminopeptidase (DAP), without loss of affinity for NEP, the malonyl group of these retro‐inhibitors was replaced by diversely substituted succinyl moieties. All the molecules synthesized are highly efficient NEP inhibitors with Ki's in the 0.2–1 nM range, indicating that NEP possesses a relatively large and not very selective S2’subsite. In contrast, inhibition of DAP activity is crucially dependent on the size and the position of the substituent in the succinyl moiety. Inhibitory potencies in the nanomolar range are obtained with compounds containing a benzyl group in the α‐position related to thc rctro amide bond. Finally, a relatively modest inhibition of APN was observed with Ki's in the 0.5‐1 μM range for compounds with benzyl or cyclohexyl group in P2'position. However, these data demonstrate that efficient and complete inhibition of enkephalin degradation can be obtained with hydroxamate dipeptides containing a retro amide bond. The analgesic potency of the most active inhibitors was measured using the hot plate test in mice. Significant antinociceptive responses were obtained but these effects were rather weaker than those expected from thein vitroinhibitory potencies of these compounds on the three enkephalin‐degrading enzymes.
Enzymatic methyl esterification of a deamidated form of mouse epidermal growth factor
Authors: GALLETTI PATRIZIA , IARDINO PATRIZIA , INGROSSO DIEGO , MANNA CATERINA , ZAPPIA VINCENZO
Published: August 2010

The enzyme S‐adenosylmethionine:protein carboxyl‐O‐methyl‐transferase, type II (EC2.1.1.77; PCMT) from eukaryotes methyl esterifies peptides containing isoAsp residues, which can arise from spontaneous deamidation of labile Asn residues. We report here a study on in vitro methyl esterification of mouse EGF by bovine brain PCMT. This peptide contains two Asn in the sequences Asn1‐Ser2 and Asn16‐Gly17. It is known from the literature that the presence of a small residue on the carboxyl side of asparaginyl makes this residue susceptible to deamidation through the spontaneous formation of a succinimide intermediate. Therefore EGF was incubated under deamidating conditions (pH9.0, 37° for 48 h) and the extent of deamidation monitored by enzymatically measuring the NH3 produced during the alkali treatment: a release of 0.80 mol NH3/mol EGF was calculated. The alkali‐treated EGF, analyzed by anion‐exchange chromatography, shows two major components identified as native EGF (nEGF) and its deamidated form (dEGF). When incubated in the presence of purified PCMT neither nEGF nor dEGF showed any methyl accepting capability. Since it is known that the three‐dimensional structure of a protein may hinder the methyl esterification of a potential ethyl accepting site, dEGF was unfolded by reducing and alkylating the intrachain disulfide bridges. Only a slight increase in the methyl accepting capability could be observed. Conversely, when EGF was deamidated after its unfolding, the resulting protein was stoichiometrically methylated by PCMT, presumably at level of isoAsp16. Our findings strongly suggest that the three‐dimensional structure of a protein is a major specificity determinant for both deamidation and methyl esterification processes.
Conformational analysis of enkephalin analogs contaIntng a disulfide bond Models for delta‐ and mu‐receptor opioid agonists
Authors: FROIMOWITZ MARK , HRUBY VICTOR J.
Published: August 2010

Conformational analyses of the cyclic opioids H‐Tyr‐d‐Pen‐Gly‐Phe‐d‐Pen‐OH (DPDPE) and H‐Tyr‐d‐Cys‐Gly‐Phe‐d‐Cys‐OH (DCDCE) have been performed using the AMBER program. DPDPE is considerably more selective for delta‐receptors than DCDCE. Using the RNGCFM program, a large number of ways were found to close the 14‐membered disulfide‐containing ring structure. However, intramolecular hydrogen bonds were only possible in gamma‐turn and inverse gamma‐turn conformations centered on the glycine residue which were associated with opposite chiralities of the disulfide bond. With the cyclic part of the molecules in either a gamma‐turn or inverse gamma‐turn, a systematic conformational analysis was performed on the tyrosine and phenylalanine sidechains. This showed that conformers with the tyrosine and phenylalanine phenyl rings in the vicIntty of the disulfide bond were preferred due to attractive van der Waals forces. For DPDPE, however, this was only possible with a positive dihedral angle for the disulfide bond due to the presence of the beta‐carbon methyls of Pen2. In contrast, these preferred conformers were possible with both chiralities of the disulfide bond in DCDCE. Conformational entropies and free energies were computed from the translational, rotational, and vibrational energy levels available to each conformer. The conformational entropies were found to vary significantly and to result in a re‐ordering of the lowest energy mIntma. Based on these conformational differences in DPDPE and DCDCE and their differing pharmacological selectivities, tentative conformational preferences for delta‐ and mu‐receptor opioid peptides are proposed.
Structure and conformation of linear peptides XII. Structure of tryptophanyl‐glycyl‐glycine dihydrate
Authors: SUBRAMANIAN E. , SAHAYAMARY J. JAMES
Published: August 2010

The crystal structure of a tripeptide, tryptophanyl‐glycyl‐glycine dihydrate (C15H18N4O4·2H2O, molecular weight = 354) has been determined. The crystals are orthorhombic, space group P212121 with a= 7.875 (1) A,b= 9.009(1), c= 24.307(1) and Z = 4. The final R‐index is 0.058 for 1488 reflections ((sin θ/λ≤ 0.6 A−1) with I < 2σ(I). The molecule exists as a zwitterion, with terminal NH+3 and COO− groups. The peptide units are trans and nearly perpendicular to the plane of the carboxyl group. The backbone torsion angles are: ψ1= 132.7°, ω1= 174.2°, φ2 88.2°, ψ= 8.6°, ω2 ‐ 179.8°, φ= ‐ 85.2°, ψ31, = ‐ 178.1°, ψ32 5.0°. For the sidechain of tryptophan, χ1= ‐ 171.6°, χ2 101.0°.
Sequence characterization of venom toxins from Thailand cobra
Authors: CHIOU S‐H. , LIN W‐W. , CHANG W‐P.
Published: August 2010

Several toxins with distinct pharmacological properties were isolated from the venom of Thailand cobra(Naja naja siamensis)by cation‐exchange chromatography. Two neurotoxins and one basic toxin with cardiotoxic activity were further purified and sequenced. The neurotoxins characterized were closely similar to the previously reported long‐ and short‐chain neutrotoxins. The complete sequences of one minor neurotoxin and one cardiotoxin analogue were determined with the automatic protein sequencer in non‐stop single runs of Edman degradation coupled withC‐terminal sequence determination with carboxypeptidase digestion. The minor neurotoxin consists of 62 amino‐acid residues with 8 cysteine residues and is found to be almost identical to cobrotoxin, a major toxic component of Formosa cobra(Naja naja atra).The sequence comparison of the 60‐residue cardiotoxin with other reported cytotoxins of snake venoms indicates that 8 cysteine residues at the positions 3, 14, 21, 38, 42, 53, 54, and 59 are invariant among all sequences, with only two conservative changes at other positions along the sequence. The upshot of this report exemplified the facile sequence analysis of venom toxins by the application of pulsed‐liquid phase protein sequencer and also revealed new analogues of a minor neurotoxin and one major cardiotoxin reported previously on the same species of Thailand cobra.
Chemical synthesis of a neurotoxic polypeptide from the sea anemoneStichodactyla helianthus+
Authors: PENNINGTON MICHAEL W. , KEM WILLIAM R. , NORTON RAYMOND S. , DUNN BEN M.
Published: August 2010

The sea anemoneStichodactyla helianthusneurotoxin I, a 48‐residue polypeptide, was synthesized by automated solid phase methodology. The fully reduced polypeptide was subsequently refolded in the presence of a glutathione oxidoreduction buffer to the biologically active species containing three disulfide bonds. The overall yield after rigorous purification was 12.5%. The circular dichroism (CD), and proton nuclear magnetic resonance (1H NMR) spectra of the HPLC‐purified synthetic toxin were indistinguishable from those obtained concurrently with the natural toxin. A subtilisin digest of the synthetic neurotoxin generated peptide fragments identical to that of a sample of the natural toxin subjected to the same treatment. The toxicity of the synthetic polypeptide was identical to that of the natural toxin (crab LD50, 3.1μg/kg). The equilibrium dissociation constant (28 nM) for interaction of the synthetic toxin with crab axolemma vesicles was nearly identical to that of the natural toxin (25 nM).
Transesterification of Moz‐Asn‐Leu‐Gly‐OEt in methanol Confirmation of Ca2+ mediated catalysis
Authors: MIRANDA MARIA TERÊSA MACHINI , MORITA HIROSHI , TOMINAGA MINEKO
Published: August 2010

During the recrystallization of the crude protected tripeptide ester Moz‐Asn‐Leu‐Gly‐OEt (obtained by an enzymatic coupling reaction) in methanol/water, the transesterification of this compound to methyl ester was observed. The involvement of Ca2+ in this process was indicated by the results obtained in the following experiments: 1) incubation of crude Moz‐Asn‐Leu‐Gly‐OEt in methanol solutions of o‐phenanthroline and EDTA; 2) recrystallization of the crude Moz‐Asn‐Leu‐Gly‐OEt in ethanol/water followed by incubation in methanol; 3) determination of the Ca2+ content of the crude Moz‐Asn‐Leu‐Gly‐OEt. After recrystallization Moz‐Asn‐Leu‐Gly‐OEt lost the ability to be transesterified in methanol. However, in the presence of crude Moz‐Asn‐Leu‐Gly‐OEt, or calcium acetate, or a mixture of calcium chloride/sodium acetate, the compound was transesterified, suggesting that the transesterification of crude and recrystallized compounds occurs through different mechanisms. On the basis of these results, and of the conformational data obtained for these peptides by 1H‐NMR, we suggest models for the transesterification reactions.
Peptides from chiral Cα,α‐disubstituted glycines Synthesis and characterization, conformational energy computations and solution conformational analysis of Cα‐methyl, Cα‐isopropylglycine [(αMe)Val] derivatives and model peptides*
Authors: TONIOLO C. , CRISMA M. , BONORA G.M. , KLAJC B. , LELJ F. , GRIMALDI P. , ROSA A. , POLINELLI S. , BOESTEN W.H.J. , MEIJER E.M. , SCHOEMAKER H.E. , KAMPHUIS J.
Published: August 2010

Conformational energy computations on Ac‐l‐(αMe)Val‐NHMe indicate that turns and right‐handed helical structures are particularly stable conformations for this chiral Cα‐methyl, Cα‐alkylglycyl residue. We have synthesized and characterized a variety of l‐(αMe)Val derivatives and peptides (to the pentamer level). The results of the solution conformational analysis, performed using infrared absorption, 1H nuclear magnetic resonance, and circular dichroism, are in general agreement with those obtained from the theoretical investigation, in the sense that the l‐(αMe)Val residue turns out to be a strong β‐turn and right‐handed helix former. A comparison is also made with the conclusions extracted from published work on peptides rich in other Cα‐methyl, Cα‐alkylglycyl residues.
Isolation of a heptapeptide Val‐Val‐Tyr‐Pro‐Trp‐Thr‐Gln (valorphin) with some opiate activity
Authors: ERCHEGYI JUDIT , KASTIN ABBA J. , ZADINA JAMES E. , QIU XIU‐DI
Published: August 2010

Bovine hypothalamic tissue was extracted and purified by solid phase extraction and several reversed‐phase HPLC steps. The amino acid sequence of the purified peptide was determined by Edman degradation to be Val‐Val‐Tyr‐Pro‐Trp‐Thr‐Gln. This was confirmed by comparison of its chromatographic behavior with that of the synthetic peptide, and mass spectrometric analysis resulted in a mass identical to the calculated mass for this peptide. This heptapeptide shows homology with residues 32‐38 of the beta‐chain of bovine hemoglobin. The peptide inhibited the electrically induced contractions of the guinea pig ileum muscle preparation; this inhibition was reversible by naloxone. It also inhibited the binding of 125I‐DAMGO (selective for μ receptors) to rat brain with an IC50 of 10 μm and the binding of 3H‐DPDPE (selective for σ receptors) with an IC50 of 185 μm. With two valines at the N‐terminus and some opiate activity, valorphin seems a suitable name for this newly isolated peptide.
Peptide inhibitors of E. collagenolyticum bacterial collagenase ‐effect of N‐methylation
Authors: DIVE VINCENT , YIOTAKIS ATHANASIOS , ROUMESTAND CHRISTIAN , GILQUIN BERNARD , LABADIE JEAN , TOMA FLAVIO
Published: August 2010

Peptide inhibitors of E. collagenolyticum bacterial collagenase, HS‐CH2‐CH2‐CO‐Pro‐Yaa (Yaa = Ala, Leu, Nle), have been N‐methylated at the Yaa position. The N‐methylation slightly increases the inhibitory potency of the modified peptides as compared to the parent compounds. The conformational effects of the N‐methylation have been investigated by both 1H 2D‐NMR and molecular mechanics energy minimization. Three low‐energy conformers have been predicted for the unmethylated parent compounds (Yaa = Ala, Leu, Nle). They are characterized by the Ψ value of the central proline residue: ΨPro= 150c(trans' conformation), Ψpro= 70° (C7 conformation) and ΨPro= ‐50°(cis' conformation). The N‐methylation has been found to strongly increase the energy of the C7 conformer and to a less extent the energy of the cis' conformer. This leaves the trans' conformation as the only low‐energy conformer. The ROESY experiments have established that both the N‐methyl peptides and the parent compounds adopt the same preferred backbone conformation in water solution, i.e. the trans conformation. Based on these results, the activities of the N‐methyl peptides are discussed and a possible conformation of the inhibitor in the bound state is proposed.
Synthesis and pharmacological activity of the N‐terminal dermorphin tetrapeptide analogs with CH2‐NH peptide bond isosteres
Authors: SALVADORI SEVERO , GUERRINI REMO , BOREA PIERO ANDREA , TOMATIS ROBERTO
Published: August 2010

The synthesis of pseudotetrapeptides H‐Tyr‐D‐Ala‐Phe‐NH‐(CH2)2‐NH2 (1a), H‐Tyr‐D‐Ala‐Phe‐ψ(CH2‐NH)‐Gly‐NH2 (2a), H‐Tyr‐D‐Ala‐ψ(CH2‐NH)‐Phe‐Gly‐NH2 (3a), and H‐Tyr‐ψ(CH2‐NH)‐D‐Ala‐Phe‐Gly‐NH2 (4a), representing the N‐terminal tetrapeptide sequence of dermorphin, in which amide bonds are replaced by CH2‐NH bond, is described. N‐acetyl‐Tyr and desamino‐Tyr pseudopeptide analogs (1‐4b), (1‐3c) are also described. The analogs were assayed in binding studies based on displacement of μ and δ‐receptor selective radiolabels from rat brain membrane and in a bioassay using guinea pig ileum (GPI). Pseudopeptides in which the C‐terminal (1a) or D‐Ala‐Phe (3a) amide bond are substituted, exhibit higher μ‐affinities and μ‐receptor selectivity than the corresponding Phe‐Gly or Tyr‐D‐Ala analogs (2a, 4a). Acetyl‐and desamino‐Tyr pseudopeptide analogs (1‐4b) and (1‐3c) did not exhibit μ and δ‐opioid receptor affinity at nM concentration. The relevance of the single peptide replacement and of its association to acetylation or amino group elimination of Tyr, is discussed on the basis of a receptor model for μ and δ opioids.
Modification of the Phe3aromatic moiety in delta receptor‐selective dermorphin/deltorphin‐related tetrapeptides Effects on opioid receptor binding
Authors: HEYL DEBORAH L. , MOSBERG HENRY I.
Published: August 2010

The previously described cyclic delta opioid receptor‐selective tetrapeptide H‐Tyr‐d‐Cys‐Phe‐d‐Pen‐OH (JOM‐13) was modified at residue 3 by incorporation of both natural and unnatural amino acids with varying steric, electronic, and lipophilic properties. Effects on mu and delta opioid receptor binding affinities were evaluated by testing the compounds for displacement of radiolabeled receptor‐selective ligands in a guinea pig brain receptor binding assay. Results obtained with the bulky aromatic 1‐Nal3and 2‐Nal3substitutions suggest that the shape of the receptor subsite with which the side chain of the internal aromatic residue interacts differs for delta and mu receptors. This subsite of either receptor can accommodate the transverse steric bulk of the 1‐Nal3side chain but only the delta receptor can readily accept the more elongated 2‐Nal3side chain. Several analogs with pi‐excessive heteroaromatic side chains in residue 3 were examined. In general, these analogs display diminished binding to mu and delta receptors, consistent with previous findings for analogs with residue 3 substitutions of modified electronic character. Several analogs with alkyl side chains in residue 3 were also examined. While delta receptor binding affinity is severely diminished with Val3, Ile3, and Leu3substitutions, Cha3substitution is very well tolerated, indicating that, contrary to the widely held belief, an aromatic side chain in this portion of the ligand is not required for delta receptor binding. Where possible, comparison of results in this delta‐selective tetrapeptide series with those reported for analogous modification in the cyclic delta‐selective pentapeptide [d‐Pen2,d‐Pen5]enkephalin (DPDPE) and linear pentapeptide enkephalins reveals similar trends.
Dermorphin tetra‐ and heptapeptide analogues containing a [3,4] amide bond replacement by a carbon‐carbon double and single bond
Authors: JASPERS H. , TOURWÉ* D. , VAN BINST G. , PEPERMANS H. , BOREA P. , UCELLI L. , SALVADORI S.
Published: August 2010

Seven dermorphin hepta‐ and tetrapeptide analogues containing [3,4] amide bond replacement by a carbon‐carbon double and single bond were prepared. 1H NMR studies of the pseudoheptapeptide in DMSO indicate the presence of extended conformations with stacking of the side chains in the N‐terminal part and an inverse γ‐turn around Ser7 in the conformational equilibrium. The binding data show that the affinity of the analogues for the μ‐receptor is only slightly diminished in the d‐Ala2 series and is more affected in the d‐Arg2 series. Since the Gly4NH is not present in these compounds we conclude that this NH is not required to stabilize the bioactive conformation nor is it directly involved in binding to the receptor.
Conformation of glutathione adduct and oxidized forms of platelet‐derived growth factor
Authors: NARHI LINDA O. , KENNEY WILLIAM C. , PRESTRELSKI STEVEN J. , ARAKAWA TSUTOMU , LYONS DAVID , LARY JEFFREY , YPHANTIS DAVID A.
Published: August 2010

The conformational properties of several platelet‐derived growth factors (PDGFs) were characterized by circular dichroism (CD), Fourier transform infrared spectroscopy (FTIR), gel filtration and sedimentation equilibrium. Three different forms of disulfide linked dimer, PDGF‐AA, PDGF‐AB, and PDGF‐BB, showed similar far UV CD spectra with evidence for slight p‐structure, but little evidence of other regular secondary structures. These spectra were, however, different from the far UV CD spectra of the glutathione adducts of PDGF‐A and B, suggesting that the latter two proteins adopt different conformations in the absence of intra‐ or inter‐molecular disulfide bonds. FTIR studies confirmed this by showing that the glutathione adducts of the PDGF‐B protein have a significantly lower amount of regular secondary structures than PDGF‐BB. Additionally, the increased bandwidths of the amide I components of the FTIR spectrum of the glutathione adduct indicates a more flexible structure relative to the dimeric form. Sedimentation equilibrium analysis showed that PDGF‐BB is primarily a dimer and that the glutathione form is primarily a monomer. Thus, it was concluded that the glutathione derivative has little affinity to form non‐covalent dimers in neutral solution.
Endothelin‐converting enzyme: the binding of metal ions
Authors: KUNDU GOPAL C. , WILSON IRWIN B.
Published: August 2010

The metal ion of endothelin‐converting enzyme (ECE) was investigated by inhibiting the enzyme with ethylenediaminetetraacetic acid (EDTA) and restoring activity by adding divalent metal salts in quantities less than the concentration of EDTA. Under these conditions, only metal ions that bind to ECE with high affinity can affect the enzyme, The ferrous enzyme had an activity of 76% relative to the native enzyme, the manganous enzyme 76%, the nickelous enzyme 77%, the cupric enzyme 17%, the zinc enzyme 98% and the cobaltous enzyme 122%. Of these first transition series elements only zinc can be the metal of the native enzyme. The zinc enzyme has the same Km and turnover number as the native enzyme.
The aqueous conformation of cyclo(1,6)Ac‐Cys‐Arg‐Gly‐Asp‐Phe‐Pen‐NH2
Authors: SIAHAAN TERUNA J. , BRUSS DAN , POWELL NOEL A. , CHAKRABARTI SOMA , CONRAD MATIHEW
Published: August 2010

RGD peptides are known as important ligands for integrin receptors in the cell adhesion process. The selectivity of RGD peptides for a certain integrin receptor is partly dependent on the RGD conformation and the residues surrounding the RGD sequence. This paper investigates the effect of the addition of a phenyl‐alanine residue on the RGD conformation in cyclo(1,6)Ac‐Cys‐Arg‐Gly‐Asp‐Phe‐Pen‐NH2 (1) as compared to the previously studied cyclo(1,5)Ac‐Pen‐Arg‐Gly‐Asp‐Cys‐NH2 (2). The conformational study of peptide I was done in aqueous solution using nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics simulations. This work will increase the understanding of the flanking residue's effect in RGD peptides.
Solid‐phase synthesis of (tyrosyl‐alanyl‐glutamyl)n, by segment condensation
Authors: OBEYESEKERE NIHAL U , CROIX JEAN‐NOEL LA , BUDDE RAYMOND JA , DYCKES DOUGLAS F , MCMURRAY JOHN S.
Published: August 2010

(Tyr‐Ala‐Glu)n, n= 1–9, were synthesized by segment condensation using the Fmoc/tert‐butyl protection strategy and solid‐phase techniques. The C‐terminal residue was coupled to the resin and the peptides were built out by adding Fmoc‐Glu(O‐r‐Bu)‐Tyr(t‐Bu)‐Ala‐OH units. When the desired lengths were reached the peptides were capped with Fmoc‐Tyr(t‐Bu)‐Ala‐OH units. Fmoc‐Tyr(t‐Bu)‐Ala‐OH and Fmoc‐Glu(O‐t‐Bu)‐Tyr(t‐Bu)‐Ala‐OH were synthesized in aqueous solution by the successive addition of N‐hydroxysuccinimide esters of Fmoc‐Tyr(t‐Bu) and Fnioc‐Glu(0‐t‐Bu) to the growing chain. Neither sequential amino acid addition or segment condensation techniques were successful on polystyrene supports. However, the segment condensations were highly successful on kieselguhr‐supported polydimethylacrylamide based resins. (Tyr‐Ala‐Glu)n, n= 1–9, were tested as inhibitors of the protein [yosine kinase, pp60Cc‐src. Inhibition, as measured by IC50 values, increased with increasing size of the peptide.
Potent pseudopeptide bombesin‐like agonists and antagonists
Authors: EDWARDS J. VINCENT , MCLEAN LARRY R. , WADE ARLENE C. , EATON SCOTT R. , CASHMAN ELIZABETH A. , HAGAMAN KAREN A. , FANGER BRADFORD O.
Published: August 2010

Bombesin‐like pseudopeptides have been synthesized, and certain physicochemical properties and biological activities have been examined. Bombesin and the related peptide litorin were modified at positions 13–14 and 8–9, respectively, with ψ[CH2S] and ψ[CH2N(CH3)]. [Phe13ψ[CH2S]Leu14]bombesin and [Phe8ψ[CH2S]‐Leu9]litorin bound to the murine pancreatic bombesin gastrin releasing peptide receptor with similar dissociation constants (Kd= 3.9 and 3.4 nM. respectively). Increased potency was achieved by oxidation of the thiomethylene ether to two diastereomeric sulfoxides (isomer I, Kd= 1.6 nM and isomer II, Kd= 0.89nM. Further oxidation to the sulfone decreased potency ([Phe8ψ[CH2SO2]Leu9]litoin, Kd= 9.9nM). All five analogs were receptor antagonists as determined by phosphatidylinositol turnover in murine pancreas. In contrast to these peptide backbone substitutions, a ψCH2N(CH3)] at the 8–9 amide bond position resulted in an agonist. The analogs were compared with those of litorin (Kd= 0.1 nM) and [Leu9]litorin (Kd= 0.17 nM) by CD and fluorescence spectroscopy. The CD spectra demonstrated ordered conformation for all the peptides in TFE. Different conformations corresponding to agonist and antagonist peptides were suggested by CD. Based on the pH‐dependence of the fluorescence spectra of the peptides in a zwitterionic detergent, two titratable groups were identified (pKa= 6.3 and 8.5). The lower pKa is found in the agonist analogs but not in the ψ[CH2S]‐containing antagonist.
Activity of two synthetic amphiphilic peptides and magainin‐2 against herpes simplex virus types 1 and 2
Authors: ABOUDY Y. , MENDELSON E. , SHALIT I. , BESSALLE R. , FRIDKIN M.
Published: August 2010

The in vitro antiviral activity of two amphiphilic synthetic peptides, modelin‐1 (mod‐1) and modelin‐5 (mod‐5), and of the natural antibacterial peptide magainin‐2 (mag‐2) against herpes simplex viruses type 1 (HSV‐1) and 2 (HSV‐2) were evaluated. The peptides were incubated with the virus, i.e. direct inactivation, and their effects examined by means of plaque reduction assay and/or reduction in virus yield. Only mod‐ displayed a strong antiviral effect against HSV‐1 and HSV‐2, with 50% effective dose (ED50) values of 4.6 and 4.1 μg/mL, respectively. Mag‐2, mod‐5 and a mixture of both had no significant inhibitory effect. Addition of mod‐1 up to a concentration of 100μg/mL to the culture medium had no significant cytotoxic effect on host vero cells, as measured by the trypan blue‐exclusion method. It showed, however, considerable hemolytic activity against human red blood cells. Experiments including acyclovir (ACV) as a reference viral inhibitor indicated that the mode of action of mod‐1 is different from that of ACV. In contrast to ACV, the peptide inactivates the virus following a very short incubation before vero cell infection, suggesting some kind of direct interaction of the peptide with the viral envelope, rather than inhibition of viral DNA replication or gene expression. Our results suggest that mod‐1 may be an effective topical antiviral agent against herpes viruses.
Chemical synthesis and characterization of peptides and oligomeric proteins designed to form transmembrane ion channels
Authors: IWAMOTO TAKEO , GROVE ANNE , MONTAL MYRTA OBLATT , MONTAL MAURICIO , TOMICH JOHN M.
Published: August 2010

A strategy for the synthesis of peptides and oligomeric proteins designed to form transmembrane ion channels is described. A folding motif that exhibits a functional ionic pore encompasses amphipathic α‐helices organized as a four‐helix bundle around a central hydrophilic pore. The channel‐forming activity of monomeric amphipathic peptides may be examined after reconstitution in lipid bilayers in which peptides self‐assemble into conductive oligomers. The covalent attachment of channel‐forming peptides to the lysine ε‐amino groups of a template molecule (KKKPGKEKG) specifies oligomeric number and facilitates the study of ionic permeation and channel blockade. Here we describe detailed protocols for the total synthesis of peptides and template‐assembled four‐helix bundle proteins, exemplified with the sequence of M2δ (EKM‐STAISVLLAQAVFLLLTSQR), considered involved in lining the pore of the nicotinic acetylcholine receptor channel. For comparison, the synthesis of a second four‐helix bundle, T4CaIVS3 with the sequence of predicted transmembrane segment S3 (DPWNVFDFLIVIGSIIDVILSE) of the fourth repeat of the l‐type voltage‐gated calcium channel, is included. Peptides and proteins are synthesized step‐wise by solid‐phase methods, purified by reversed‐phase HPLC, and homogeneity ascertained by analytical HPLC, capillary zone electrophoresis, SDS/PAGE, amino acid analysis and sequencing. Optimization of synthetic procedures for hydrophobic molecules include reducing resin substitution to avoid steric hindrance and aggregation of the final product. Protocols for the preparation of the samples prior to HPLC purification as well as the conditions and columns required for successful purification are presented. The methods developed are generally applicable for the chemical synthesis, purification and characterization of amphipathic peptides and template directed helical bundle proteins.
Effect of the environment and role of the π‐π stacking interactions in the stabilization of the 310‐helix conformation in dehydroalanine oligopeptides
Authors: ALEMÁN CARLOS
Published: August 2010

A quantum‐mechanical study of the chain‐length dependent stability of the extended, 27ribbon and 310‐helix conformations in dehydroalanine (ΔAla) oligopeptides has been performed. To address the study, the oligopeptides ΔAlan, where n varies from 1 to 6, were computed by using the semiempirical AM1 methodology. Cooperative free‐energy effects permit one to predict the stabilization of the 310‐helix with respect to the extended and 27‐ribbon conformations when the number of residues in the polypeptide chain increases. The interactions associated with the π‐electron density of the side chains can easily explain this finding. The effects of the solvent and the crystalline packing on the different conformations were modeled using a self‐consistent reaction field (SCRF) method and a molecular mechanics approach to the packing, respectively. Both the aqueous and crystal environments seem to be a key factor in the stabilization of the helical conformation. Finally, the variations of electrostatic parameters such as atomic point charges and dipole moments in ΔAla‐containing peptides with internal (conformation) and external (solvent) effects are discussed. © Munksgaard 1995.
Solid‐phase synthesis of peptide haptens containing a cysteine—sulfur mustard adduct
Authors: NOORT D. , JACOBS E.H. , FIDDER A. , DE JONG L.P.A. , DRIJFHOUT J.W. , BENSCHOP H.P.
Published: August 2010

Solid‐phase synthesis of peptide haptens containing 2‐[2‐(S‐cysteinyl)ethylthio]ethanol has been achieved by solution‐phase synthesis of a properly protected S‐alkylated cysteine derivative and subsequent solid‐phase incorporation. © Munksgaard 1995.
Synthesis of a selenomethionine peptide and a preliminary study of transport into Escherichia coli monitored by high‐performance liquid chromatography*
Authors: KOCHANSKY JAN P. , TSCHURSIN ELIZABETH , LUSBY WILLIAM R. , LACROIX DENIS , WOLF WAYNE R.
Published: August 2010

The tripeptide Gly‐SeMet‐Gly has been synthesized by a combination of solution and solid‐phase methods. Increase in weight of the resin was very nearly theoretical, and purification was straightforward. Its absorption was compared to that of the corresponding peptide, Gly‐Met‐Gly, in E. coli using HPLC ion‐exchange separation and fluorometric determination of the disappearance of peptides in the culture medium and the appearance of methionine and selenomethionine in the same culture medium. As E. coli are not known to possess extracellular peptidases, and in fact have been shown to possess transport systems for peptides, this absorption is interpreted as transport of the peptide through the cell wall and membrane into the cytoplasm, endohydrolysis of the peptide, and efflux of the peptides' amino acids. Uptake of both peptides was approximately equal, but was slowed when both peptides were present simultaneously. © Munksgaard 1995.
The preferred solid‐state conformation of (αMe)Trp peptides
Authors: FORMAGGIO F. , TONIOLO C. , CRISMA M. , VALLE G. , KAPTEIN B. , SCHOEMAKER H.E. , KAMPHUIS J. , DI BLASIO B. , MAGLIO O. , FATTORUSSO R. , BENEDETTI E. , SANTINI A.
Published: August 2010

The two Z‐l‐Ala‐dl‐(xMe)Trp‐NH2 diastereomeric dipeptides were synthesized from (Z‐l‐Ala)2O and H‐dl‐(xMe)Trp‐NH2. The latter racemate, prepared by phase‐transfer catalyzed alkylation of the Nα‐benzylidene derivative of alanine amide followed by acidic hydrolysis of the resulting Schiff base, was characterized by X‐ray diffraction. The molecular and crystal structure of Z‐l‐Ala‐l‐(αMe)Trp‐NH2, separated from its diastereomer by silica‐gel column chromatography, was determined by X‐ray diffraction analysis. Both independent molecules in the asymmetric unit of the dipeptide adopt a type‐II β‐bend conformation. However, only the more regularly folded conformation of molecule B is stabilized by a 1←4 C=O…H—N intramolecular H bond. The present results indicate that: (i) the Cα‐methylated (αMe)Trp residue is a strong β‐bend and helix former, and (ii) the relationship between (αMe)Trp chirality and helix screw sense tends to be opposite to that of protein amino acids. The implications for the use of the (αMe)Trp residue in designing conformationally restricted analogs of bioactive peptides are briefly discussed. ©Munksgaard 1995.
Solution conformation of bovine lens α‐and βB2‐crystallin terminal extensions
Authors: BRETON EUGENE R. LE , CARVER JOHN A.
Published: August 2010

α‐and βB2Crystallin are the major proteins in the mammalian lens. Each of these crystallins has short, flexible terminal extensions from its domain core; the two α‐crystallin subunits have C‐terminal extensions of eight and ten amino acids whilst βB2‐crystallin has N‐ and C‐terminal extensions of 15 and 11 amino acids, respectively. The solution conformations of these chemically synthesised extensions have been examined by two‐dimensional 1H NMR spectroscopy, The N‐terminal extension of βB2‐crystallin and the C‐terminal extensions of α‐crystallin adopt little ordered structure. In the membrane‐mimicking solvent trifluoroethanol, the α‐crystallin extensions are also unstructured. In contrast, the C‐terminal extension of βB2‐crystallin in water has a structural preference towards turn‐like structures, creating a hydrophobic region involving G198, F200 and P202. In the lens, the C‐terminal extension of βB2‐crystallin is the only one of these extensions that interacts to any large extent with other crystallins. The structural preference of the C‐terminal extension of βB2‐crystallin may therefore have implications for the role of this extension in crystallin‐crystallin interactions. ©Munksgaard 1996.
Solution conformation of a peptide corresponding to residues 151–172 of HIV‐1 integrase using NMR and CD spectroscopy
Authors: CHENG JYA‐WEI , CHENG CHING‐CHOU , LYU PING‐CHIANG , CHEN SHUI‐TEIN , LIN THY‐HOU
Published: August 2010

The solution structure of a synthetic peptide corresponding to residues 151–172 of HIV‐1 integrase has been determined by NMR and CD spectroscopy. Residues 151–172 of HIV‐1 integrase were predicted to be an α‐helix and to be responsible for the oligomerization of HIV‐1 integrase. Two‐dimensional 1H NMR and CD studies indicate that this synthetic peptide adopts an amphipathic α‐helical conformation in TFE‐containing solution. However, concentration‐dependent CD studies reveal that this peptide motif does not form dimers or oligomers in solution as predicted. These results are in agreement with the crystal structure of the catalytic domain of HIV‐1 integrase reported recently. ©Munksgaard 1996.