bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparison of methods for the Fmoc solid‐phase synthesis and cleavage of a peptide containing both tryptophan and arginine
Authors: CHOI H. , ALDRICH J.V.
Published: August 2010

A major side reaction which can occur during the synthesis of Trp‐containing peptides is modification of the Trp indole by reactive carbonium ion species released during acidolytic cleavage. [Asn2,Trp4]Dynorphin A‐(1–13), a sequence which is very susceptible to Trp modification, was chosen as a model peptide to compare the effectiveness of various methods proposed to minimize Trp modification during Fmoc solid‐phase synthesis. The peptide was synthesized with the side chain of Trp unprotected and cleaved by Reagent K [82.5% trifluoroacetic acid (TFA)/5% phenol/5% water/5% thioanisole/2.5 % ethanedithiol (EDT)] [King, D.S. et al. (1990) Int. J. Peptide Protein Res.36, 255–2661, Reagent R [90% TFA/5 % thioanisole/3% EDT/2% anisole] [Albericio, F. et al. (1990) J. Org. Chem.55, 3730–3743], TFA containing 20% EDT and 4% water [Riniker, B. & Hartmann, A. (1990) in Peptides: Chemistry, Structure, and Biology (Rivier, J.E. & Marshall, G.R., eds.), pp. 950–952, Escom, Leiden], and TFA containing trialkylsilane, MeOH, and ethylmethyl sulfide [Chan, W.C. & Bycroft, B.W. (1992) in Peptides: Chemistry, Structure, and Biology, Op. cit., pp. 613–614]. Cleavage with Reagent K, Reagent R and TFA containing 20% EDT and 4% water yielded similar results; in addition to the desired peptide, the crude product contained 22–30% of a side product which appeared to result from Trp modification by a Pmc group. Cleavage with the triakylsilane‐containing mixture gave the lowest recovery of the desired peptide and the highest levels of Pmc‐containing peptides. In contrast, synthesis of the peptide by Fmoc solid‐phase synthesis utilizing Fmoc‐Trp(Boc) and subsequent cleavage with TFA containing 20% EDT and 5% water yielded the desired peptide in essentially pure form with < 5% of the Pmc‐containing side product. Thus, in the Fmoc solid‐phase synthesis of [Asn2,Trp4]dynorphin A‐(1–13) protection of the indole nitrogen by Boc was the most effective method for suppressing the modification of Trp by Pmc. This demonstrates the potential for improving the yield and purity of peptides containing both Trp and Arg by utilizing Fmoc‐Trp(Boc) during the Fmoc solid‐phase synthesis of these peptides.
Conformational study on trans‐ and cis‐N‐acetyl‐N′‐methylamides of Pro‐Xaa dipeptides
Authors: HAN SEONG JUN , KUNG YOUNG KEE
Published: August 2010

Conformational free energy calculations using an empirical potential (ECEPP/2) and the hydration shell model were carried out on the N‐acetyl‐N′‐methylamides of Pro‐Xaa dipeptides (Xaa = Ala, Leu, Val, Gly, Cys, Met, Phe, Tyr, Asn, Asp, and Ser) with trans and cis peptide bonds preceding proline residue in the unhydrated and hydrated states. As compared with the results obtained by using the earlier version of ECEPP, the values of β‐bend probabilities are about doubled. The average calculated population of cis‐dipeptide is about 4%, which is close to the abundance obtained from the analysis of X‐ray crystal structures of proteins. The β‐bends are the most dominant structures of cis‐dipeptides. Type I, usually having intramolecular hydrogen bonds, contributes greatly to the β‐bend conformations of trans‐ and cis‐dipeptides. However, type I β‐bends of cis‐dipeptides do not have any hydrogen bonds. By including the hydration, the β‐bend probabilities for trans‐ and cis‐dipeptides decreased, indicating that the interactions of water molecules with a backbone or side‐chain may force the dipeptides to be more distorted or extended. In particular, type II is found to be a dominant β‐bend conformation of trans‐ and cis‐Pro‐Gly dipeptides in both the unhydrated and hydrated states. In general, the calculated propensities for Pro‐Xaa dipeptides to adopt β‐bend conformations are reasonably consistent with available experimental data. From comparing conformations of Pro and Xaa residues in the dipeptides and single residues, we found that inter‐residue interactions and hydration are of importance in determining the conformational properties of the Pro‐Xaa dipeptide.
Molecular structures of two crystalline forms of the cyclic heptapeptide antibiotic ternatin, cyclo[‐β‐OH‐d‐Leu‐d–Ile–(NMe)Ala (NMe)Leu‐Leu‐(NMe)Ala‐d–(NMe)Ala‐]
Authors: MILLER R. , GALITSKY N.M. , DUAX W.L. , LANGS D.A. , PLETNEV V.Z. , IVANOV V.T.
Published: August 2010

The crystal structures of two solvated forms of ternatin, cyclo[‐β‐OH‐d‐Leu‐d‐Ile‐(NMe)Ala‐(NMe)Leu‐Leu‐(NMe)Ala‐d‐(NMe)Ala‐] are reported. The first crystallizes with two molecules of peptide and one of dioxane in the asymmetric unit: P212121, a= 11.563(1), b= 21.863(2), c= 36.330(4) Å. The second crystallizes with two molecules of peptide and one of water in the asymmetric unit: P212121, a= 14.067(2), b= 16.695(1), c= 36.824(6) Å. N‐Methylation of four of the seven residues of ternatin appears to reduce the number of low‐energy conformations the molecule can assume. The same H‐bonded macrocyclic ring conformation is adopted by the backbone of each of the four molecules observed here. All the amino‐acid side chains, with the exception of d‐Ile2, have similar orientations in each of the four conformers. The heptapeptide macrocycle is characterized by: (i) a cis peptide between (NMe)Ala3 and (NMe)Leu4, (ii) a type II β‐bend, involving residues Leu5‐(NMe)Ala6‐d‐(NMe)Ala7‐β‐OH‐d‐Leu2, stabilized by two H‐bonds, N1′05 and N5′01, between Leu5 and β‐OH‐d‐Leu1 residues, (iii) a third intramolecular H‐bond, observed in each of the four molecules, between the hydroxyl group of β‐OH‐d‐Leu1 and the carbonyl oxygen of d‐Ile2.
Molecular design of peptides
Authors: SINGH T.P. , NARULA P.
Published: August 2010

The peptide N‐Boc‐l‐Phe‐dehydro‐Abu‐NH‐CH3 was synthesized by the usual workup procedure. The crystals grown from methanol at 4°C belong to the space group P212121 with a= 7.589(2), b= 13.690(4), c= 21.897(6) Å, Z= 4 and dc= 1.149(5) g cm−3 for C19H29N3O5·CH3OH. The peptide crystals were highly sensitive to radiation. The final agreement factor R was 0.055 for 1109 observed reflections (I > 2σ) with data extending to a 2θ value of 103°. The methanol oxygen atom is split into two occupancies. Both sites are involved in identical hydrogen bonding. As a result of substitution of a dehydro‐Abu residue at the (i+ 2) position the peptide adopts an ideal β‐turn II′ conformation with torsion angles of corner residues as φ1=63(1)°, ψ1= ‐ 127(1)°, φ2= ‐66(1)° and ψ2= ‐ 10(1)°, and an intramolecular hydrogen bond N—H ⋯ O of length 3.01(1) Å. This shows that the conformational constraints produced by dehydro‐Abu are similar in nature to but different in magnitude than those produced by dehydro‐Phe and dehydro‐Leu. The methanol–peptide interactions show characteristic features of multiple hydrogen‐bond formations involving polar sites of participating peptide and methanol molecules. The packing of the molecules in the unit cell is stabilized by interactions through methanol molecules with the help of several hydrogen bonds.
Synthesis and bioassay of LHRH‐antagonists with N‐Ac‐d‐O‐phenyltyrosine and N‐Ac‐d‐3‐(2‐dibenzofuranyl)alanine in position 1
Authors: LJUNGQVIST ANDERS , BOWERS CYRIL Y. , FOLKERS KARL
Published: August 2010

N‐Ac‐d‐O‐phenyltyrosine was synthesized via the corresponding azlactone. Resolution of the dl methyl esters was achieved by Subtilisin Carlsberg. Treatment with palladium(II) acetate in trifluoroacetic acid converted N‐Ac‐d‐O‐phenyltyrosine into N‐Ac‐d‐3‐(2‐dibenzofuranyl)alanine. These two amino acids were incorporated instead of N‐Ac‐d‐2‐Nal into position 1 of the LHRH‐antagonist (N‐Ac‐d‐2‐Nal1, d‐pClPhe2, d‐3‐Pal3, c‐PzACAla5, d‐PiCLyS6, ILys8,d‐Ala10)‐LHRH. The more rigid N‐Ac‐d‐3‐(2‐dibenzofuranyl)alanine was structurally more effective than N‐Ac‐d‐O‐phenyltyrosine; the AOAs for the corresponding analogs were 82 and 38%, respectively, at 0.5 μg. Replacement of c‐PzACAla in position 5 by O‐phenyltyrosine significantly decreased potency.
Three‐dimensional structure of human insulin‐like growth factor‐I (IGF‐I) determined by 1H‐NMR and distance geometry
Authors: SATO AKIHIRO , NISHIMURA SHIGENORI , OHKUBO TADAYASU , KYOGOKU YOSHIMASA , KOYAMA SATOSHI , KOBAYASHI MASAKAZU , YASUDA TSUTOMU , KOBAYASHI YUJI
Published: August 2010

The three‐dimensional structure of human insulin‐like growth factor‐I has been determined through a combination of NMR measurements and distance geometry calculations. A total of 320 interatomic distance constraints, including 12 related to the disulfide bridges, were used in these calculations. The resulting structure is characterized by the presence of three helical rods corresponding to the sequence regions, Ala8‐Cys18, Gly42‐Cys48 and Leu54‐Cys61. Furthermore, a turn structure and an extended structure exist in the Gly19‐Gly22 and Phe23‐Asn26 regions, respectively. Neglecting the N‐ and C‐termini, with their expectedly high degree of mobility as well as a fluctuating C‐domain, the r.m.s.d. value is 1.9 Å for backbone atoms. Those of the three α‐helical regions are 1.0, 0.9 and 0.8 Å, respectively, 1.8 Å being that for the total backbone atoms participating in the formation of these three helices, showing the good convergence of their spatial arrangements. The overall structure obtained here shows that the human IGF‐I molecule folds into a spatial structure very similar to that of insulin in an aqueous solution.
Structure–activity relationships of cyclic and linear peptide T analogues
Authors: MARASTONI M. , SALVADORI S. , BALBONI G. , SCARANARI V. , SPISANI S. , REALI E. , TRANIELLO S. , TOMATIS R.
Published: August 2010

Using the potent cyclic peptide T analog‐Thr‐Thr‐Asn‐Tyr‐Thr‐Asp‐ as parent compound, a series of analogues were synthesized and their potencies in a monocyte chemotaxis assay were compared with those of correspondingly modified linear peptides. Structure‐activity relationships observed with cyclic compounds did not always parallel those determined with linear analogues. ‐Thr‐Hse‐Asn‐Tyr‐Thr‐Asp‐ showed the highest affinity to CD4 receptor of monocytes of any peptide thus far studied. It also proved to be highly resistant to degradation by plasma or brain enzymes.
Structure and conformation of the calcium complex of cyclo(Ala‐Leu‐Pro‐Gly)2 in two crystal forms
Authors: JOIS D.S. SEETHARAMA , PRASAD G. SRIDHAR , BEDNAREKT MARIA , EASWARAN K.R.K. , VIJAYAN M.
Published: August 2010

Crystal structures of two different forms of the calcium perchlorate complex of cyclo(Ala‐Leu‐Pro‐Gly)2 have been determined and refined using X‐ray crystallographic techniques. Orthorhombic form: C32H52N8O8·Ca(ClO4)2·7H2O·2CH3OH, space group C2221, a= 14.366, b= 18.653, c= 19.824 Ä, Z=4, R= 0.068 for 2208 observed reflections. Monoclinic form: C32H52N8O8·Ca(ClO4)2·4H2O, space group C2, a= 21.096, b= 10.182, c= 11.256 Å, β= 103.33°, Z= 2, R= 0.075 for 2165 observed reflections. The cyclic peptide molecule in both the structures has the form of a twofold symmetric, slightly elongated bowl. Type II′β‐turns, involving Gly and Ala at the corners, exist at the two ends of the molecule. The interior of the molecule is substantially hydrophilic, and the external surface of the bowl is largely hydrophobic. The calcium ion is located at the centre of the mouth of the bowl‐like molecule. In both crystal forms, four peptide carbonyl oxygens from the cyclic peptide and two solvent oxygens coordinate to the metal ion. The mode of complexation may be described as incomplete encapsulation as, for example, in the case of metal complexes of antamanide. In the crystal structures the complex ions are held together by hydrogen bonds involving perchlorate ions and water molecules. The molecular structure observed in the crystals is entirely consistent with the results of solution studies, which also indicate the conformation of the cyclic peptide in the complex to be similar to that of the uncomplexed molecule.
Investigation of the structural parameters involved in the δ‐opioid selectivity of several families of opioid peptides
Authors: GUIS CHRISTINE , BRUETSCHY LUCE , MEUDAL HERVÉ , ROQUES BERNARD P. , GACEL GILLES A.
Published: August 2010

Three series of highly δ‐opioid selective peptides are now available, and each family is used as template to investigate the structural parameters involved in δ‐receptor recognition and in the modulation of the selectivity of the parent peptide. The first series includes cyclic derivatives such as Tyr‐D‐Pen‐Gly‐Phe‐D‐Pen(DPDPE) and Tyr‐D‐Pen‐Gly‐Phe‐Pen(DPLPE); the second are the synthetic linear constrained peptides [Tyr‐D‐Ser(OtBu)‐Gly‐Phe‐Leu‐Thr(DSTBULET), Tyr‐D‐Ser(OtBu)‐Gly‐Phe‐Leu‐Thr(OtBu) (BUBU) and especially Tyr‐D‐Cys(StBu)‐Gly‐Phe‐Leu‐Thr(OtBu) (BUBUC)] and the last one the natural peptides [Tyr‐D‐Met‐Phe‐His‐Leu‐Met‐Asp‐NH2(deltorphin or dermenkephalin) and Tyr‐D‐Ala‐Phe‐Asp‐Val‐Val‐GlyNH2([D‐Ala2] deltorphin I)].

In the present study, the possibly of transposing some of the decisive factors of δ‐selectivity evidenced in the two other families, to the linear constrained peptides series was examinated. With this aim in view, residues such as Phe3, pClPhe4or Asp were introduced in the sequence of DSTBULET, BUBU or BUBUC.

Direct comparison between the biochemical profiles of the [pClPhe4] analogs of the linear constrained peptides and their parent compounds shows that the addition of an electronegative atom on the Phe4residue of enkephalin sequences is not an absolute parameter for δ‐selectivity improvement. The hydrophobic δ‐receptor subsite seems able to receive a range of molecular volumes and electronegativities. By contrast, this subsite cannot interact with a Phe3aromatic ring introduced in this series of peptides. Moreover, the results obtained with linear peptides including additional negatively charged residues demonstrate that the proposed location of the δ‐receptors in a cationic membrane environment is not adequate to explain the selectivity profile of a number of compounds.
Synthesis, biological activity, conformational analysis by NMR and molecular modeling of N‐formyl‐L‐Met‐L‐Pro‐L‐Phe‐OMe, a praline analogue of the chemotactic peptide N‐formyl‐L‐Met‐L‐Leu‐L‐Phe‐OH
Authors: DUGAS H. , LAROCHE M. , PTAK M. , LABBÉ H.
Published: August 2010

The tripeptide N‐formyl‐Met‐Pro‐Phe‐OMe (f‐MPF‐OMe), an analogue of the signal peptide N‐formyl‐Met‐Leu‐Phe‐OH (f‐MLF‐OH), was synthesized and its chemotactic activity evaluated; it showed no activity in either superoxide production or calcium mobility with human neutrophils. However, the corresponding acid f‐MPF‐OH retained about 25% activity in the production of superoxide. The conformation of the f‐MPF‐OMe analogue was evaluated by NMR spectroscopy and molecular simulation and shown to predominate in a γ‐turn with a hydrogen bond between Met CO and Phe NH. Since this analogue is not chemotactic, it is suggested that for recognition the receptor prefers a peptide with a flexible backbone, favoring an extended conformation in the binding site.
Conformation‐function relationships in LHRH analogs
Authors: NIKIFOROVICH GREGORY V. , MARSHALL GARLAND R.
Published: August 2010

Systematic energy calculations were performed for a series of LHRH analogs including five agonists with substitutions of d‐ or N‐Me‐amino acid residues in positions 4, 6 and 7, and five antagonists with substitutions of d‐, N‐Me‐ or α‐Me‐amino acid residues in positions 1, 2, 3, 6, 7 and 10, as well as a bicyclic LHRH antagonist. The geometrical shapes of the calculated low‐energy backbone structures for each compound were compared to those of LHRH itself. It appeared that the β‐II′ turn at the Tyr5‐Gly6‐Leu7‐Arg8 central tetrapeptide is the common structure for all LHRH agonists considered. LHRH antagonists also possess a common chain reversal in the central tetrapeptide, but it is different from that for LHRH agonists. The LHRH agonists share a similar low‐energy conformer at the level of the entire peptide backbone. A characteristic feature of this conformer is a ‘surface’ formed by a ‘polygon’ with hydrophobic moieties of pGlu1, Trp3, Tyr5, Leu7 and Pro9 in the corners and with the side chain of the His2 residue in the middle, the latter being crucial for a manifestation of LHRH agonistic activity. Since the N‐terminal tripeptide of LHRH presumably participates in a direct interaction with specific receptors, it is legitimate to suggest that the β‐II′ turn in the central tetrapeptide maintains the proper spatial arrangement of the N‐terminal tripeptide. On the other hand, LHRH antagonists considered in this study were shown to possess low‐energy structures, with the spatial arrangement of the residues in the N‐terminal tripeptide similar to that of agonists. This would suggest a new approach to the design of LHRH antagonists, namely by stabilizing this specific arrangement, rather than the β‐II′ turn in the central tetrapeptide.
Synthesis and solution conformation of c(D‐Trp‐D‐Cys(SO3‐Na+)‐Pro‐D‐Val‐Leu), a potent endothelin‐A receptor antagonist
Authors: BOGUSKY MICHAEL J. , BRADY STEPHEN F. , SISKO JOHN T. , NUTT RUTH F. , SMITH GRAHAM M.
Published: August 2010

The endothelin family of polypeptides are known to exert potent physiological effects which include cardiovascular regulation. The solution conformation and dynamics of c(D‐Trp‐D‐Cys(SO3‐Na+)‐Pro‐D‐Val‐Leu), a potent endothelin‐A receptor‐selective antagonist, were characterized in aqueous solution by NMR spectroscopy and molecular modeling. NMR‐derived conformational constraints were combined with computer‐assisted molecular modeling using distance geometry calculations and energy minimization. The pentapeptide backbone is shown to adopt a single conformation in solution comprising a type II β‐turn and an inverse γ‐turn, with each residue in the trans conformation. Molecular dynamics were explored using relaxation measurements and low‐temperature studies, and indicate that the peptide backbone is highly constrained with little conformational mobility present.
Transglutaminase covalently incorporates amines into human immunodeficiency virus envelope glycoprotein GP120 in vitro
Authors: MARINIELLO LOREDANA , ESPOSITO CARLA , GENTILE VITTORIO , PORTA RAFFAELE
Published: August 2010

Human immunodeficiency virus envelope glycoprotein gp120, but not its precursor gp160, covalently incorporates both spermidine and glycine ethyl ester in the presence of Ca2+ and transglutaminase purified from guinea pig liver. The examined ability to act as enzyme substrate of various glutamine‐containing gp120 fragments, including the principal neutralizing determinant, the CD4 binding domain, and the sequence 254–274, suggested to be involved in post‐binding events and in virus entry in the host cell, indicated the glutamine‐265 as possible reactive acyl donor site of the protein.
Solution structure of the toxic octapeptide, lophyrotomin
Authors: DALY NORELLE L. , ATKINS ANNETTE R. , SMITH ROSS
Published: August 2010

Lophyrotomin is a toxic octapeptide, first isolated from larvae of the sawfly Lophyrotoma interrupta, which causes the death of cattle and sheep. It appears to act principally‐ on the liver, however very little is known about the cellular site and mechanism of action. In the present study lophyrotomin was synthesized using solid‐phase peptide synthesis and the structure examined with two‐dimensional nuclear magnetic resonance (NMR) spectroscopy.

Two‐dimensional correlation experiments (COSY and TOCSY) enabled the assignment of many of the resonances. Conventional NOESY experiments did not produce inter‐residue information, however the alternative rotating frame NOE experiment (ROESY) resulted in intra‐residue αN, and sequential αN and NN NOES, permitting the sequence‐specific assignment of all resonances. The presence of few additional short‐range NOES and the absence of any long‐range NOES in the ROESY spectra indicated a lack of persistent secondary structure. The results from circular dichroism (CD) spectroscopy experiments were consistent with the NOE data, as addition of high concentrations of the denaturant urea produced no changes in the lophyrotomin CD spectrum. This conclusion was further supported by 13C spin‐lattice relaxation studies, which indicated that the peptide is a flexible molecule by examination of the α‐carbon chemical shifts, and by amide proton exchange rate measurements. Consequently it appears that if this peptide has to adopt a well defined structure to exert its biological activity it must do so on interaction with other molecules, such as a receptor.
1H and 31P NMR spectroscopy of phosphorylated model peptides
Authors: HOFFMANN RALF , REICHERT INGMAR , WACHS WOLFGANG O. , ZEPPEZAUER MICHAEL , KALBITZER HANS ROBERT
Published: August 2010

The model peptides glycylglycyltyrosylalanine (Gly‐Gly‐Tyr‐Ala), glycylglycylthreonylalanine (Gly‐Gly‐Thr‐Ala) and glycylglycylserylalanine (Gly‐Gly‐Ser‐Ala) were phosphorylated at the hydroxyl groups of their tyrosyl, threonyl and seryl residues, respectively, and characterized by 31P and 1H NMR spectroscopy. The pKa‐value of the phosphoryl group in the tyrosine‐containing peptide determined from the pH dependence of chemical shifts is 5.9, the 31P chemical shifts at low pH (4.0) and high pH (8.0) are ‐3.8 and 0.2 ppm, respectively. Phosphorylation also leads to significant shifts of the 1H NMR resonances of the tyrosine residue; the amide resonance is shifted ‐0.02 ppm, the Hα resonance 0.06 ppm, the Hβ resonances 0.10 and ‐0.04 ppm, the H§ resonances 0.02 ppm and the Hω resonances 0.26 ppm. The pKa‐value of the phosphoryl group in the threonine peptide determined from the pH dependence of chemical shifts is 6.1; the 31P chemical shifts at low pH (4.0) and high pH (8.0) are ‐0.1 and 4.8 ppm, respectively. The corresponding values for the serine peptide are 6.1 (pKa), 0.6 ppm and 4.9 ppm. Phosphorylation also leads to significant shifts of the 1H NMR resonances of the threonine and serine residues. In the threonine residue the amide resonance is shifted 0.25 ppm, the Hα‐resonance ‐0.43 ppm, the Hβ‐resonance 0.03 ppm and the Hγ‐resonance 0.09 ppm. In the serine residue the amide resonance is shifted 0.21 ppm, the Hα‐resonance ‐0.17 ppm, and the Hβ‐resonances 0.17 ppm.
Protected peptide disulfides by oxidative detachment from a support
Authors: RIETMAN B.H. , SMULDERS R.H.P.H. , EGGEN I.F. , VAN VLIET A. , VAN DE WERKEN G. , TESSER G.I.
Published: August 2010

A new and efficient procedure for the preparation of protected cyclized and protected symmetrical dimeric peptide disulfides is described. A thiol is immobilized onto a solid phase through coupling of the thiol function with a resin‐linked trityl group. Following conventional peptide assembly using the Fmoc‐strategy, detachment is performed by oxidation with iodine in a suitable organic solvent. When N,N‐dimethylformamide is used as the solvent, and the peptide chain contains an acetamidomethylthio function, located N‐terminally in a Nx‐(9‐fluorenylmethyloxycarbonyl), or Nx‐tert‐butyloxycarbonyl cysteinyl residue, or occurring in the chain, then the corresponding fully protected cyclic peptide disulfide will be obtained in high yield and purity. In other solvents (e.g. dioxane or chloroform‐methanol 1:1, v/v), the iodine‐mediated oxidation gave not only the cyclic product, but also substantial amounts of the parallel symmetrical dimeric peptide retaining Cys(Acm) at the two identical N‐termini.
Structural characteristics of diproline: a new crystal form of tBoc‐Pro‐Pro‐OH
Authors: THOMAS L.M. , RAMASUBBU N. , BHANDARY K.K.
Published: August 2010

The structure of a new crystalline form of tBoc‐Pro‐Pro‐OH (C15 H24 N2 O5) has been determined. The crystals were monoclinic, P21 a = 14.667(5), b = 16.600(4), c = 15.502(3) Å, β= 117.84(2)˚, V= 3337.2 Å3 and Z= 8, Dc= 1.24g/cm3. There are four molecules in the asymmetric unit, each displaying polyproline‐type structure but differing in the proline pucker. All four molecules display a twist conformation in the first proline ring, with molecules A, B and C being βγT (P ∼ 183˚, τ 33 for A and B, t∼18 for C) and molecule D between βγT and γE (P= 10°, τ38). The second residue of all four molecules has an envelope conformation. Molecules A and B display an αE conformation (P∼126˚, t∼25) and molecules C and D display a βE conformation (P∼168˚, τ37). The molecules are hydrogen‐bonded (O…OH), forming helical channels along the a‐axis.
Circular dichroism (CD) studies on biological activity of mast cell degranulating (MCD) peptide analogs*
Authors: BUKU ANGELIKI , MIRZA UROOJ , POLEWSKI KRZYSZTOF
Published: August 2010

Analogs of MCD peptide were synthesized by solid‐phase methods. Positive charges were deleted at the N‐and/or C‐terminus, including the helical portion of the molecule. Four peptides were prepared by removing residues 16–18 (Arg‐Lys‐Ile), 1–2 (Lys), 1–2 and 16–18 and by acetylation of the amino end (Ile). Analogs were tested on mast cells for histamine‐releasing activity. Although the helicity of these derivatives, determined by circular dichroism (CD), was not significantly different from the native MCD peptide, two analogs with C‐terminal deletions showed a 5‐ to 10‐fold decrease in activity. These findings suggest that the C‐terminus is more important than the N‐terminus in determining bioactivity of MCD peptide.
Structural stability of lipase from wheat germ
Authors: RAJESHWARA A.N. , PRAKASH V.
Published: August 2010

Purified lipase from wheat germ was used for the determination of preferential interaction parameters under different stabilizing cosolvent conditions. The partial specific volume of the enzyme was measured under both isomolal and isopotential conditions in phosphate buffer at pH 7.0, 0.02 M, and the value was found to be 0.730 ± 0.001 and 0.731 & 0.002 mL/g, respectively. The partial specific volume measurements with different cosolvents indicated that the enzyme has a (δg3/δg2)T,μ1,μ3 values of −0.119 ± 0.012, −0.073 ± 0.009 and −0.141 ± 0.020 g/g, respectively, in 25% glucose, 25% sucrose and 25% DMSO. The (δg3/δg2)T,μ1μ3, values in 10 and 20% glycerol were −0.054 ± 0.012 and −0.073 ± 0.016 g/g, respectively. Based on these values it is clear that the enzyme is stabilized in the presence of these cosolvents by increasing its hydration, of which DMSO is stabilizing to the maximum extent. The stabilization of the enzyme was also confirmed by the thermal denaturation measurements in the presence of these cosolvents which indicated a shift in the apparent thermal denaturation temperature of the enzyme towards higher temperatures. The data are supported further by the ultraviolet difference spectral as well as fluorescence measurements in the presence of these cosolvents. The stabilization has been attributed to the preferential hydration of the enzyme in the presence of these cosolvents.
Tetrapeptide tyrosine kinase inhibitors
Authors: KIM MOON H. , LAI JACK H. , HANGAUER DAVID G.
Published: August 2010

A convenient enantioselective synthesis of p‐hydroxymethyl‐L‐phenylalaninc was developed which produces a 4/1 ratio of L/D enantiomers resulting from a chiral phase‐transfer‐catalyzed alkylation. This amino acid was coupled into the p561ck tyrosine kinase substrate Ac‐Leu‐Pro‐Tyr‐Ala‐NHCH3 as a replacement for Tyr and can subsequently be elaborated into a variety of potential tyrosine kinase inhibitor designs of general structure Ac‐Leu‐Pro‐AA‐Ala‐NHCH3, wherein AA is an unnatural amino acid. The contaminating D enan‐tiomer was readily removed after coupling to L‐Ala‐NHCH3 of this sequence. The utility of the p‐hydroxymethyl functionality in an efficient divergent synthetic strategy leading to various inhibitor designs is illustrated with the synthesis of Ac‐Leu‐Pro‐AA‐Ala‐NHCH3, wherein AA is p‐(R,S‐hydroxyphosphonomethyl)‐L‐phenylalanine.
Enzymatic approach to the synthesis of taurine‐containing peptides
Authors: ČEŘOVSKÝ VÁCLAV , JAKUBKE HANS‐DIETER
Published: August 2010

Subtilisins (subtilopeptidase A, nagarse) and proteinase K were able to catalyze the synthesis of taurine‐containing peptides from various N‐acylated amino acid or peptide esters and nonprotected taurine. The synthesis was optimized using a model reaction between Boc‐Tyr‐OMe and taurine. The best results were obtained under strongly alkaline conditions in acetonitrile with low water content as the reaction medium. The choice of the base added to the reaction medium had a substantial effect on the product yield. A preparative synthesis of Tyr‐Tau and Ala‐Phe‐Tau is described.
Conformation of tetragastrin in DMSO
Authors: KURODA MASATAKA , YAMAZAKI KAZUTO , TAGA TOORU
Published: August 2010

A conformation analysis of tetrapeptide in DMSO was carried out by the Monte Carlo simulation including the solvent effect in the energy function. The lowest energy conformations in the zwitterionic and cationic states are a similar compact form, in which the two aromatic rings are close to each other. The distance distributions between the specified atoms in the molecule show that most conformations in the local energy minima are similar to this folded conformation, although there is another type of conformation of interest, which has an extended form with the two separate aromatic rings. Such results are consistent with the NMR and CD experiments, and suggest that the compact form of tetragastrin is essential as a biological active conformation. The folded structure is stabilized by the solute‐solvent interaction. In particular, the non‐bonding interactions between the solute and solvent molecules cause a folding effect on the molecular conformation in total. This study is a first case taking account of the solvent effect into the conformation analysis of tetragastrin.
Incomplete Fmoc deprotection in solid‐phase synthesis of peptides
Authors: LARSEN BJARNE DUE , HOLM ARNE
Published: August 2010

During solid‐phase peptide synthesis of homo‐oligopeptides containing leucine or alanine using the Fmoc strategy, we have observed ineffective N‐α‐deprotection with piperidine in a sequence‐dependent manner. Incomplete deprotection was found to be associated with subsequent slow or incomplete amino acid coupling. Optimization of the deprotection step was carried out by varying the experimental conditions e.g. deprotection time, temperature, solvents and addition of chaotropes. Coupling and deprotection steps have been investigated using color monitoring, as well as FAB MS and HPLC for product analysis. The phenomena of difficult coupling and deprotection steps in the investigated systems have been demonstrated to have the same physical chemical origins, p‐sheet formation. © Munksgaard 1994.
Sequence‐dependent modification of Trp by the Pmc protecting group of Arg during TFA deprotection
Authors: STIERANDOVÁ ALENA , SEPETOV NIKOLAI F. , NIKIFOROVICH GREGORY V. , LEBL MICHAL
Published: August 2010

The extent of transfer of the Pmc protecting group from the guanidino group of arginine to the side chain of tryptophan depends on the spacial distance of these side chains. When these two amino acids are separated by one amino acid, the transfer of the Pmc protecting group is the most pronounced, and it cannot be completely prevented by the use of currently utilized scavenger mixtures. The extent of this side reaction also depends on the amino acid separating the arginine and tryptophan residues and position of tryptophan within the peptide chain as well as on the type of the solid‐phase carrier.
Synthesis of methylated phenylalanines via hydrogenolysis of corresponding 1,2,3,4‐tetrahydroisoquinoline‐3‐carboxylic acids
Authors: MAJER PAVEL , SLANINOVÁ JIŘINA , LEBL MICHAL
Published: August 2010

A new method of synthesizing ortho‐methylated phenylalanines has been developed. Phenylalanines with at least one free ortho‐position undergo a Pictet—Spengler cyclization with formaldehyde followed by hydro‐genolytic splitting of the endocyclic benzylic C—N bond of 1,2,3,4‐tetrahydroisoquinolines and afford corresponding ortho‐methyl derivatives. Repeating this reaction sequence on the ortho‐substituted phenylalanines yielded ortho, ortho‐disubstituted derivatives, and pro‐substituted phenylalanines yielded ortho,para‐disubstituted analogs. Our modified method of cyclization preserved the configuration at the chiral center: hydrogenolysis, on the other hand, led to racemization. Incorporation of the methylated phenylalanines into position 2 of oxytocin led to, in the case of the D‐isomers, potent uterotonic inhibitors.
Peptidic p‐nitroanilide substrates of interleukin‐1β‐converting enzyme
Authors: REITER LAWRENCE A.
Published: August 2010

Peptidic p‐nitroanilides are useful colorimetric substrates for enzymes. With the aim of developing a convenient, quantitative assay for inhibitors of interleukin‐1β‐converting enzyme (ICE), we have explored three approaches to the synthesis of peptidic p‐nitroanilides relevant to this enzyme. The first approach involved a late stage oxidation of a p‐aminoanilide such as CbzValAlaAsp(β‐tert‐butyl)‐p‐(t‐Boc‐amino)anilide. The second and third approaches used the preformed amino acid p‐nitroanilides HAsp‐p‐nitroanilide hydrochloride and HAsp(7beta;‐tert‐butyl)‐p‐nitroanilide which were coupled iteratively with preactivated amino acid derivatives or with an appropriate peptide, respectively. While each approach had it merits and limitations, all three produced p‐nitroanilides that were substrates for ICE.
Structural investigation of Cetrorelix, a new potent and long‐acting LH‐RH antagonist
Authors: MÜLLER A. , BUSKER E. , ENGEL J. , KUTSCHER B. , BERND M. , SCHALLY A.V.
Published: August 2010

The new decapeptide SB‐75 (INN: Cetrorelix) has been characterized as a potent antagonist of luteinizinghormone releasing hormone (LH‐RH). Such derivatives are of great medicinal interest owing to their potential application in areas such as hormone‐dependent tumors, uterine fibroids, and in diseases and conditions which result from inappropriate hormone levels or which can be treated by suppression of estrogens. SB‐75 is the subject of intensive ongoing clinical evaluation and is an accepted standard for the design of new LH‐RH antagonists. We characterized SB‐75 by means of modern MS and NMR techniques to demonstrate the significance of both sequencing methods on a complicated unnatural decapeptide. Our structural elucidations with nuclear Overhauser (NOE) experiments revealed clear evidence for a highly flexible molecule with no single predominant conformation even in sodium dodecyl sulfate (SDS) mimicking a cellular membrane.
Conformational study of a series of somatostatin analogues with antitumor and/or GH inhibitory activity
Authors: JASPERS H. , HORVÁTH A. , MEZÖ I. , KÉRI G. , BINST G. VAN
Published: August 2010

A series of somatostatin analogues with varying activities have been studied by 1H NMR in CD3OH at low temperature in order to find a possible structural explanation for the differentiation of biological activities. In somatostatin analogues with GH release inhibitory activity a β‐turn/β‐sheet backbone conformation is present, which is shown to be characteristic of somatostatin‐derived peptides exhibiting this biological activity. On the other hand, among the analogues with antitumor activity, a deviation from these typical structural features is clearly observed, but no general conformational model can be proposed.
Conformation of a biologically active C‐terminal hexapeptide analog of the pheromone biosynthesis activating neuropeptide by NMR spectroscopy
Authors: WANG YU‐SEN , KEMPE TOMAS G. , RAINA ASHOK K. , MAZZOCCHI PAUL H.
Published: August 2010

The solution conformation of a biologically active C‐terminal hexapeptide analog of the pheromone biosynthesis activating neuropeptide Tyr‐d‐Phe‐Ser‐Pro‐Arg‐Leu‐NH2 has been studied by NMR spectroscopy. A β‐turn conformation was identified from the NOE connectivities observed for the peptide in a mixed solvent of water and DMSO, indicating that this is the biologically active conformation of the peptide. This study also suggests that the use of such an aqueous‐like solvent mixture allows the observation of a preferred conformation for small linear peptides in the presence of conformational averaging.
Does the edge‐to‐face interaction between aromatic rings occur in cyclolinopeptide A analogues?
Authors: SIEMION IGNACY Z. , CEBRAT MAREK , JANKOWSKI ANDRZEJ , LISOWSKI MAREK , PFDYCZAK ARTUR , WYSLSOUCH ALEKSANDRA
Published: August 2010

We measured 1H‐NMR, fluorescence and CD spectra of cyclolinopeptide A (CLA), its tyrosine analogues with each or both phenylalanines substituted by tyrosine (c‐[LeuIleIleLeu ValProProTyrPhe], c‐[LeuIleIleLeu ValProProPheTyr] and c‐[LeuIleIleLeu ValProProTyrTyr]), and their linear counterparts with the starting sequence Leu‐Ile‐Ile‐Leu‐Val‐Pro‐Pro‐Phe‐Phe (LA). It follows from CD spectra that the conformations of all cyclic peptides are similar to that of CLA; the conformations of linear peptides are more diversified, with the conformation of [Tyr9]LA being most similar to CLA. NMR studies suggest that aromatic rings in cyclic peptides are situated perpendicular to each other, manifesting edge‐to‐face pairing. Accordingly, the residue in position 9 is shielded (‘edge’), and a residue in position 8 is the shielding one (‘face’). This effect is not present in the case of linear peptides. Fluorescence quantum yields were much lower for cyclic peptides than for linear ones, indicating the interaction of closely located aromatic chromophores. Those quantum yields depend on the relative position of Tyr in the peptide chain. Another factor influenced by the position in the peptide chain is the optical activity of aromatic side chains (optically active in position 8, inactive in position 9). This phenomenon could be explained by the differences in the side‐chain conformation of both aromatic residues.
Structural studies of two antiaggregant RGDW peptides by lH and 13C NMR
Authors: KIEFFER BRUNO , MER GEORGES , MANN ANDRÉ , LEFÈVRE JEAN‐FRANÇOIS
Published: August 2010

The structural features of Arg‐Gly‐Asp‐related sequences have been investigated by 1H and 13C NMR. Two linear peptides which inhibit platelet aggregation with a high efficiency have been studied: D‐Arg‐Gly‐Asp‐Trp and L‐Arg‐Gly‐Asp‐Trp. Analysis of pH titration effects, amide proton exchange rates and inter‐proton distances obtained from ROESY spectra suggest that these small fragments predominantly adopt a type II′β‐turn structure in solution. Folding features of a non‐active cyclic peptide based on the same sequence (cyclo‐[Arg‐Gly‐Asp‐Trp]2) have also been investigated. The biological relevance of these structures is discussed.
A crystal‐state, solution and theoretical study of the preferred conformation of linear Cα,α‐diphenylglycine derivatives and dipeptides with potential anticonvulsant activity
Authors: TONIOLO CLAUDIO , CRISMA MARCO , FABIANO NICOLETTA , MELCHIORRI PIETRO , NEGRI LUCIA , KRAUSE JEANETTE A. , EGGLESTON DRAKE S.
Published: August 2010

Several linear molecules containing the Cα,α‐diphenylglycine residue were prepared as potential anticonvulsants. The conformational preferences of the Cα,α‐diphenylglycine residue were assessed in these synthetic derivatives and dipeptides by X‐ray diffraction, FTIR absorption and 1H NMR techniques, and by conformational energy computations. Five (out of six) derivatives adopt the fully extended C5 conformation in the crystal state. This intramolecularly H‐bonded form is largely populated in chloroform solution in all the derivatives investigated. Conformational energy computations in vacuo support the view that the intramolecularly H‐bonded C7‐ring form is the most stable structure for these compounds. Only one linear derivative exhibits a (modest) anticonvulsant activity.
Chemical synthesis of o‐thiophosphotyrosyl peptides
Authors: KÜNG ERIC KITAS. ERICH , BANNWARTH WILLI
Published: August 2010

The synthon for O‐thiophosphotyrosine, Fmoc‐Tyr[PS(OBzl)2]‐OH (1c), was prepared in 63%;, yield from Fmoc‐Tyr‐OH by first transient protection as the tBuMe2Si‐ester and phosphinylation with (BzlO)2PNiPr2/ tetrazole followed by oxidation of P(III) to P(V) vith S8x in CS2. Building block 1cwas incorporated in the Fmoc solid‐phase synthesis of two O‐thiophosphotyrosine‐containing peptides H‐Thr‐Glu‐Pro‐Gln‐Tyr(PS)‐Gln‐Pro‐Gly‐Glu‐OH (2) and H‐Thr‐Arg‐Asp‐Ile‐Tyr(PS)‐Glu‐Thr‐Asp‐Phe‐Phe‐Arg‐Lys‐OH (3), corresponding to sequences of the p60src (523–531) protein and an insulin receptor (IR) (1142–1153) analogue, respectively. An alternative approach of synthesis, the global phosphorylation of a resin‐bound peptide, also proved useful. Thus, the free tyrosyl side‐chain containing‐peptide IR (1142–1153) on support was phosphinylated with the above phosphoramidite reagent followed by oxidation with either S8/CS2 or tetraethylthiuram disulfide/CH3CN solutions. Deprotection and peptide‐resin cleavage was performed with a TFA/thiophenol (H2O) mixture. Crude peptides 2 and 3 were stable to the acidolytic deprotection. Preparative RP(C18)HPLC was initially performed using 0.1% TFA(aq) EtOH solvents. However, analyses of fractions resulting from the purification step indicated significant decomposition of thiophosphopeptide in solution. Stability measurements both as a function of time and pH. further confirmed this initial finding. Purifications performed at intermediate pH using a triethylammonium acetatc (pH 7.5) CH3CN solvent system overcame this problem.
Synthesis and surface activity properties of hydrophobic/hydrophilic peptides
Authors: INFANTE M. ROSA , MOSES VIVIAN
Published: August 2010

In order to explore the ability of amphiphilic peptides to behave as surface‐active agents with emulsifying properties, several short peptides of leucine and glutamine were synthesized with different periodicity, length and hydrophobic characteristics. The stepwise liquid‐phase procedure using the N‐hydroxysuccinimide ester was deployed in all chain‐lengthening steps, and the same procedure was also used subsequently to modify some of the products by introducing a lipophilic moiety such as a palmitoyl residue. The surface‐active properties of these products were evaluated by measuring the variation of surface (ys) and interfacial (yi) tensions and the formation of micelles as a function of concentration in aqueous solution. The alternating sequence (Leu‐Gln)n showed good surface activity behaviour, similar to those of recognized surfactants, albeit with no emulsifying function. More hydrophobic compounds, such as lipopeptides, lowered the surface tension of water at concentrations markedly below those usually observed for classical surfactants, and emulsifying properties were observed in all the peptides substituted with lipophilic moieties.
Analogues of theC‐terminal fragments of neurokinins with modifications at theirC‐terminal methionyl residue
Authors: ANTONIOU MARIA , POULOS CONSTANTINE
Published: August 2010

Analogues of SP4–11have been synthesized in which the methionyl residue is replaced successively by the Glu(OCH2CH3), Glu(OBzl), Hse(CH3) and Glu(CONHCH3) residues, and analogues of NKA4–10and NKB4–10have been prepared in which the methionyl residue is replaced by the Hse(Bz1) and Hse(CH3) residues, respectively. The SP4–11analogues were tested in threein vitropreparations representative of NK‐1, NK–2 and NK‐3 receptor types. Substitution of the SCH3group of the Met11side chain by the groups COOCH2CH3and COOBzl has little affect on the agonist activity in NK‐1 preparations, while in NK‐2 the corresponding analogues are more potent than the parent octapeptide; that substituted with COOBzl being 8.2 times more potent than SP4–11. In NK‐3 preparations all analogues are weak agonists. The selectivity of all the analogues is reduced compared with the corresponding hexapeptide analogues. The SP4–11analogues, along with those of NKA4–10, and NKB4–11, were tested for their binding ability in the three receptor subtypes above. The SP4–11analogues show reduced affinity for NK‐1 receptors, while the NKA4–10and NKB4–10analogues have almost the same affinities as NKA and NKB for NK‐2 and NK‐3 receptors, respectively. The effect of the lipophilicity of the Met11side chain, especially when a phenyl group is present in the side chain, at the NK‐2 receptor is discussed.
A free‐energy simulation study of a bacterial collagenase inhibitor
Authors: GILQUIN B. , PERAHIA D. , DIVE V.
Published: August 2010

The cis‐trans conformational isomerisation of the N‐methylated peptide bond of a bacterial collagenases peptide inhibitor (HS‐CH2‐CH2‐CO‐Pro‐NMe‐Ala) has been investigated by molecular dynamics simulations, energy minimisations and free‐energy simulations in presence of solvent molecules. The free‐energy difference between the cis and trans forms obtained by the thermodynamics integration method is equal to 0.95 kcal/mol in favour of the trans form, in accord with the experimental result. The main contribution to this free‐energy difference comes from solute‐water electrostatic interactions. Interestingly, we show that the number of interactions between water molecules and the oxygen atoms of the inhibitor is larger in the trans form than in the cis. Thus the organisation of water molecules around the inhibitor appears crucial in determining the population of the cis and trans conformers.
Primary structure, spectroscopic and inhibitory properties of a two‐chain trypsin inhibitor from the seeds of charlock (Sinapis arvensis L), a member of the napin protein family
Authors: SVENDSEN IB , NICOLOVA DIANA , GOSHEV IVAN , GENOV NICOLAY
Published: August 2010

A protein with inhibitory activity toward trypsin has been isolated from Sinapis arvensis L (charlock). It has a molecular weight of 15500 and consists of two chains connected by disulfide bonds. The amino acid sequence was determined and showed that it belongs to the napin family of storage proteins. CD studies showed an α‐helix content of 12% and a β‐structure of about 50%.
Human parathyroid hormone: efficient synthesis in Escherichia coli using a synthetic gene, purification and characterization
Authors: OSHIKA YURI , YAMADA TAKAO , NAKAGAWA SHIZUE , FUJISHIMA AKIRA , KAWASE MASAHIRO , ISHIBASHI YOSHIHIRO , FUKUDA TSUNEHIKO
Published: August 2010

Human parathyroid hormone is a peptide hormone consisting of 84 amino acid residues. Production of small proteins by direct expression in Escherichia coli is often unsuccessful owing to susceptibility of the mRNA and/or the product to endogenous enzymes. In this study, direct expression of the hormone at an excellent level (over 100 mg/L) has been achieved by using a suitably designed synthetic gene under the control of the T7 promoter.

The protein produced in bacteria was extracted and easily purified in a good yield of 27 mg/L. The purified product was physico‐chemically identified as intact human parathyroid hormone from the results of amino acid analysis, N‐terminal sequencing, and peptide mapping using fast atom bombardment mass spectrometry. In biological assays the purified product stimulated adenylate cyclase in vitro, promoted bone growth and increased the serum osteocalcin in rats to the same extent as the authentic hormone.
Effects of Cα‐methyl substitution on the conformation of linear GnRH antagonist analogs
Authors: LIFF MARK I. , KOPPLE KENNETH D. , TIAN ZHENPING , ROESKE ROGER W.
Published: August 2010

We have examined the effect of Cα‐methyl groups on the conformational ensemble of GnRH analog peptides by comparing 1H 2D NMR data from two analogs, Ac‐D‐Nal1‐D‐4‐Cl‐Cα‐Me‐Phe2‐D‐Pal3‐Ser4‐Tyr5‐D‐Arg6‐Leu7‐Arg8‐Pro9‐D‐Ala10‐NH2(1)andAc‐D‐Nal1‐D‐4‐CI‐Cα‐Me‐Phe2‐D‐Pal3‐Ser4‐Cα‐Me‐Tyr5‐D‐Arg6‐Leu7‐Cα‐Me‐Arg8‐Pro9‐D‐Ala10‐NH2 (2). The two additional Cα‐methyl groups in residues 5 and 8 of 2 do not influence significantly the pattern of the observable main chain NOE intensities, or of the backbone HN proton chemical shifts, which indicates that they do not produce global changes in the conformational ensemble of the peptide. A local change induced by the substitution was observed in the conformation at d‐Arg8‐Pro9.