bluuebunny/crossref_metadata_2025 huggingface dataset:
Acid‐base properties for each protonation site of six corticotropin fragments
Authors: NOSZÁL BÉLA , OSZTÁS ERZSÉBET
Published: August 2010

The individual basicity of each protonation site for six different fragments of aH‐ACTH (corticotropin) have been characterized in terms of group constants. Microconstants have also been estimated for the N‐ and C‐terminal moieties of ACTH (15–32) and ACTH (1–28), respectively. In these cases the bifunctional termini contain adjacent protonation sites of similar basicity in close interaction. For fragments consisting of 14 or fewer amino acids the acid‐base properties of all the protonation sites can be well interpreted by taking into account only the primary peptide structure. The group constant values for ACTH (15‐32), ACTH (1‐28) and ACTH (1‐32) provide evidence that the functional groups of these fragments are influenced by several intramolecular interactions such as H‐bonding and macromolecular hydrophobic effects. By means of the above constants the concentration of any arbitrarily chosen microspecies can be calculated.
Crystal‐state structural analysis of two γ‐lactam‐restricted analogs of Pro‐Leu‐Gly‐NH2
Authors: VALLE G. , CRISMA M. , TONIOLO C. , YU K.‐L. , JOHNSON R.L.
Published: August 2010

The crystal structures of two analogs of Pro‐Leu‐Gly‐NH, (1), containing a γ‐lactam conformational constraint in place of the ‐Leu‐Gly‐ sequences, are described. The highly biologically active (S,R)‐diastere‐omer 2a is semi‐extended at the C‐terminus, with the N‐terminal Pro residue in the unusual “C5” conformation [ψ1=– 0.8(15)°] stabilized by a (peptide)N‐H…N(amino) intramolecular H‐bond [the N(3)…N(4) separation is 2.687(11)Å]. Conversely, the N,N′‐isopropylidene aminal trihydrate of the (S,S)‐diastereomer 2b, compound 3, adopts a β‐bend conformation at the C‐terminus, as already reported for 1. However, the backbone torsion angles [φ= 57.4(4), ψ2=– 129.9(3)°; ψ3= ‐ 92.3(4), ψ3= 6.4(5)°] lie close to the values expected for the corner residues of an ideal type‐II β‐bend. A weak intramolecular 4 → 1 H‐bond is seen between the Gly carboxyamide anti‐NH and Pro C=O groups. In the newly formed 2,2,3,4‐tetraalkyl‐5‐oxo‐imidazolidin‐1‐yl moiety the ψ1 torsion angle is 12.9(4)° and the intramolecular N(3)…N(4) separation is 2.321(4)Å.
Structure and conformation of peptides involving prolyl residue III. L‐Prolyl‐L‐isoleucine monohydrate
Authors: PANNEERSELVAM K. , CHACKO K.K. , VEENA R.
Published: August 2010

The dipeptide, L‐prolyl‐L‐isoleucine monohydrate (C11 H20N2O3· H2O, molecular weight 246.3) crystallizes in the monoclinic space group P21, with a = 6.601(3)Å, b = 5.413(3) Å, c = 19.128(6) Å, β= 98.1(1)°, Z = 2, Do = 1.20g·cm‐3 and Dc = 1.208g·cm‐3. The structure was solved by MULTAN–80 and refined to a final R‐factor of 0.081 for 594 reflections measured on a Enraf Nonius CAD‐4 diffractometer. The peptide linkage exists in the trans conformation. The pyrrolidine ring is disordered with two alternate envelope conformations for the Cγ atom. The values of the sidechain torsion angles are: χ11=– 63.6(17)°, χ12= 171.1(16)° and χ2=– 59.6(21)° for isoleucine (C‐terminal). The crystal structure is stabilized by a three‐dimensional network of N—H ⋯ O, O—H ⋯ O and C—H ⋯ O hydrogen bonds. The dipeptide exists in the extended Conformation.
Conformational stability of antamanide and analogs Crystal structure of perhydrosymmetric antamanide, cyclic (‐Val‐Pro‐Pro‐Cha‐Cha‐Val‐Pro‐Pro‐Cha‐cha‐)
Authors: KARLE ISABELLA L. , FLIPPEN‐ANDERSON JUDITH L. , WIELAND THEODOR
Published: August 2010

The synthetic perhydrogenated symmetric analog of the cyclic decapeptide antamanide is biologically inactive, although the conformation of the molecule and the crystal packing are very similar to that of the active symmetric analog of antamanide. In fact, the same conformation for the molecule has now been found in six polymorphs of uncomplexed antamanide and its analogs. The differences between the active and inactive antamanide analogs are displayed dramatically in the conformations of their metal ion (Na+ or Li+) complexes, thus suggesting strongly that for physiological activity antamanide is not in the conformation assumed by the uncomplexed molecule, but rather in the conformation assumed by the complexed state of natural antamanide. The present structure crystallizes in space group P212121 with a = 20.515(14) Å, b = 21.316(16) Å, c = 17.128(16) Å and four peptide molecules in the unit cell. There are three cocrystallized water molecules at full occupancy intrinsic to the peptide, and several more water molecules or other solvent molecules at partial occupancy. The formula of the peptide is C66 H106 N10O10· 4‐H2O·2X.
Synthesis of phosphopeptides containing O‐phosphoserine or O‐phosphothreonine
Authors: ARENDT ANATOL , PALCZEWSKI KRZYSZTOF , MOORE WILLIAM T. , CAPRIOLI RICHARD M. , McDOWELL J. HUGH , HARGRAVE PAUL A.
Published: August 2010

Peptides containing phosphoserine or phosphothreonine were synthesized by solid phase methods. Phosphoserine and phosphothreonine were incorporated into peptides using Boc‐diphenylphosphono esters of serine and threonine and standard DCC/HOBt coupling. The phenylphosphoesters were not removed when the peptides were cleaved from the resin by HF or by trifluoromethane sulfonic acid, but were subsequently removed by catalytic hydrogenation. Phosphopeptides were purified by HPLC and by Fe+3‐Chelex chromatography and their identity verified by mass spectrometry. Two peptides, Leu‐Arg‐Arg‐Ala‐Ser(P)‐Leu‐Gly and Leu‐Arg‐Arg‐Ala‐Thr(P)‐Leu‐Gly, were prepared by both enzymatic and chemical methods and had identical properties.
Isolation, sequence, and conformation of seven trichorzianines from Trichoderma harzianum
Authors: REBUFFAT SYLVIE , EL HAJJI MOHAMED , HENNIG PHILIPPE , DAVOUST DANIEL , BODO BERNARD
Published: August 2010

From the antagonistic fungus Triclroderma harzianum, a group of acidic new peptides, trichorzianines B (TB), was isolated in addition to neutral trichorzianines A (TA) previously studied. TA and TB exhibit various biological activities related to their membrane properties and a different behaviour of the two groups was noticed. As observed for other peptaibols, TB consist in a microheterogeneous mixture which was resolved into pure peptides by reversed‐phase C18 HPLC. The sequence of the seven main isolated TB, namely TB IIa, TB IIIc, TB IVb. TB Vb. TB VIa. TB VIb, TB VII, was determined by the combined use of positive ion FAB mass spectrometry and 2D 1H n.m.r. spectroscopy, including COSY and NOESY experiments. TB differ from the corresponding TA only by the replacement of Gln 18 in the TA sequence by a glutamic acid. The 1H n.m.r. data suggested that the TB are mainly organized in an α helix.
Neuropeptide Y and neuropeptide Y18‐36 Structural and biological characterization
Authors: BOUBLIK J. , SCOTT N. , TAULANE J. , GOODMAN M. , BROWN M. , RIVIER J.
Published: August 2010

Neuropeptide Y (NPY), a 36‐residue peptide amide, has been shown by numerous studies to be a potent vasoconstrictor. In order to gain an appreciation of the structural requirements for this action, we have previously synthesized a number of fragments of NPY. It had been shown that sequential deletions from the N‐terminus resulted in peptides with decreasing hypertensive activity. In the present study we present data supporting the unexpected finding of two fragments, NPY17‐36 and NPT18‐36 with substantial hypotensive action in vivo. This action was dose dependent (data not shown) and was also observed to a lesser extent with NPY19‐36 but not NPY16‐36 or NPY20‐36. It was, however, slower in onset and of longer duration than the hypertensive action of NPY. These differing kinetics of action may suggest that NPY and NPY18‐36 act through different mechanisms. Structural studies using circular dichroism were performed. While NPY was found to assume an ordered helical structure in both aqueous buffer and trifluoroethanol (TFE), 30% TFE in aqueous buffer was required to induce substantial helicity for NPY18‐36. This structural investigation suggests that both NPY and NPY18‐36 assume an ordered conformation upon reaching the lipid rich receptor environment.
Hormonotoxins I. Strategy for synthesis of ovine luteinizing hormone ‐ gelonin conjugate bearing the toxin in the β‐subunit
Authors: SINGH VINOD , SAIRAM M.R.
Published: August 2010

The amino groups in the β‐subunit of ovine luteinizing hormone (oLH) were modified by thiolation using N‐succinimidyl‐3‐(2‐pyridyldithio) propionate so that it may be coupled in a disulfide linkage to similarly modified ribosome inactivating protein, gelonin. The modified β‐subunit was able to hybridize with free LHα‐subunit and the complex retained full biological activity. However, when gelonin was coupled to the β‐subunit, the resulting conformational changes masked or eliminated the sites necessary for intersubunit recognition of the free α‐subunit. This has important implications for the design in the synthesis of gonadotropin‐toxin/drug conjugates.
Ostrich MSEL‐neurophysin belongs to the class of two‐domain “big” neurophysin as indicated by complete amino acid sequence of the neurophysin/copeptin
Authors: LAZURE C. , SAAYMAN H.S. , NAUDÉ R.J. , OELOFSEN W. , CHRÉTIEN M.
Published: August 2010

Mammalian neurohypophyseal hormones, oxytocin and vasopressin, are known to be synthesized as part of two larger precursors containing, respectively, a VLDV‐neurophysin and a MSEL‐neurophysin together with its associated glycopeptide. Starting from ostrich neurohypophyses, a “big” neurophysin was isolated and chemically characterized. Following sequence determination of the CNBr‐derived fragments and of peptides obtained from trypsin and V8‐protease digestion of the oxidized protein, this “big” neurophysin was found to contain an MSEL‐neurophysin moiety (94 residues) still covalently associated with the COOH‐terminal glycopeptide (38 residues, copeptin). This study demonstrates that the ostrich MSEL‐neurophysin sequence closely resembles all known MSEL‐neurophysin sequences and that, furthermore, it does not contain the single amino acid insertion shown previously in the ostrich VLDV‐neurophysin. It is also shown that the stretch of amino acids, linking the MSEL‐neurophysin and the copeptin, is clearly different from its mammalian homologues and lacks the Arg residue normally recognized by the cleaving enzyme. This study also demonstrates that the ostrich copeptin is more closely related to the amphibian copeptin sequence than to its mammalian homologue, leading to the hypothesis that two families of copeptin molecules might exist. Thus, the ostrich MSEL‐neurophysin‐copeptin molecule is the first “big” neurophysin reported in birds and, together with the guinea pig and amphibian homologues, represents the third example of partial or no neurophysin‐copeptin cleavage.
Isolation and characterization of a gonadotropin receptor binding inhibitor from porcine follicular fluid
Authors: BHAT P.J. , MOUDGAL N.R.
Published: August 2010

The presence of a gonadotropin receptor binding inhibitor in pooled porcine follicular fluid has been demonstrated. Porcine follicular fluid fractionation on DE‐32 at near neutral pH, followed by a cation exchange chromatography on SPC‐50 and Cibacron blue affinity chromatography, yielded a partially purified gonadotropin receptor binding inhibitor (GI‐4). The partially purified GI binding inhibitor inhibited the binding of both 125I labelled hFSH and hCG to rat ovarian receptor preparation. SDS electrophoresis of radioiodinated partially purified GI followed by autoradiography made it possible to identify the binding component as a protein of molecular weight of 80000. Subjecting 125I labelled GI‐4 to chromatography on Sephadex G‐100 helped obtain a homogeneous material, Gl‐5. The 125I labelled GI‐5 exhibited in its binding to ovarian membrane preparations characteristics typical of a ligand‐receptor interaction such as saturability, sensitivity to reaction conditions as time, ligand and receptor concentrations and finally displaceability by unlabelled inhibitor as well as FSH and hCG in a dose dependent manner. This material could bind ovarian receptors for both FSH and LH, its binding being inhibited by added FSH or hCG in a dose dependent manner.
Weak dependence of octapeptide solubility in organic solvents on the amino acid sequence*
Authors: NARITA MITSUAKI , UMEYAMA HIROSHI , SASAKI CHIE
Published: August 2010

The six kinds of the octapeptides 1‐6 to be conceivable from permutation of three peptide fragments, i.e. Gln‐Val‐Gly, Asn‐Ala‐Ile, and Phe‐Leu, were prepared to confirm that their solubility was not strongly dependent on their amino acid sequences. The solubility of the octapeptides 1‐6 resembled each other and they were insoluble in MeOH, EtOH, THF, and CH2Cl2, and soluble in HMPA and DMSO. I.r. absorption spectroscopic analysis of the octapeptides 1‐6 indicated that they mainly had a β‐sheet structure in the solid state. The results of the solubility and conformation of the octapeptides 1‐6 strongly suggested the adequacy of the hypothesis that their solubility and their potential for β‐sheet formation in the solid state were not strongly dependent on their amino acid sequences but on their amino acid compositions.
Crystal structure and conformation of glycyl‐glycyl‐sarcosine
Authors: SUBRAMANIAN E. , PARTHASARATHY R.
Published: August 2010

Crystals of the tripeptide, glycyl‐glycyl‐sarcosine (C7H13N3O4) from aqueous methanol are orthorhombic, space group Pbcn with cell parameters at 294 K of a = 8.279(1), b = 9.229(4), c = 24.447(5) Å, V = 1868.0 Å3, M.W. = 203.2, and Z = 8. The crystal structure was solved and refined using CAD‐4 data (1171 reflections ≥ 3σ) to a final R‐value of 0.053. The first peptide linkage is trans and planar whereas the second peptide link between Gly and sarcosine is cis and appreciably non‐planar (w = 7.4°). The peptide backbone has an extended conformation at the N‐terminal part but adopts a polyglycine‐II type of conformation at the C‐terminal part. The backbone torsion angles are: Ψ1, =− 173.9, w1=− 177.8, (φ, Ψ2) = (‐178.8, ‐170.8), w2= 7.4, (φ3, Ψ3) = (‐81.6, 165.6°).
Synthesis and characterization of retro gramicidin A‐dAla‐gramicidin A, a 31‐residue‐long gramicidin analogue
Authors: LELIEVRE D. , TRUDELLE Y. , HEITZ F. , SPACH G.
Published: August 2010

A 31‐residue peptide, retro gramicidin A‐dAla‐gramicidin A, mimicking a gramicidin A dimer was prepared by solid phase method using a 4‐(oxymethyl)‐Pam resin and BOC as α amino protecting group. To avoid any possible formation of gramicidin A, couplings of fragments were used together with stepwise growing of the peptide chain. After purification by silica gel chromatography, this peptide was shown to form long living channels in lipid bilayer. Preliminary physicochemical investigations suggest that the peptide does not adopt the expected π6.3DL helical conformation.
Anchoring of Fmoc‐amino acids to hydroxymethyl resins
Authors: GRANDAS ANNA , JORBA XAVIER , GIRALT ERNEST , PEDROSO ENRIQUE
Published: August 2010

A method to anchor Fmoc‐amino acids to p‐alkoxybenzyl ester resins without using DMAP is described. Esterification takes place with 3–4 equiv. of Fmoc‐amino acid plus DCC and a slightly lower excess of HOBt in good yields, without racemization or dipeptide formation. Addition of N‐methylmorpholine to the reaction medium enhances the reaction yield but is accompanied by a small amount of racemization and dipeptide formation. Coupling via Fmoc‐amino acid chlorides has also been evaluated.
Effect of centrally and peripherally administered β‐endorphin on fooc intake in rats
Authors: TRISCARI J. , NELSON D. , VINCENT G.P. , LI CHOH HAO
Published: August 2010

A role for β‐EP in the regulation of food intake has been suggested as a contributory factor in the obesity of some genetically obese animal models. Studies undertaken to determine whether continuous administration of β‐EP could alter food intake in normal rats are described. The present studies demonstrated that continuous subcutaneous infusion with β‐EP was ineffective in modulating food intake, but that acute intraperitoneal or intracerebroventricular administration stimulated food intake in previously food deprived or satiated animals, respectively. These results suggest that β‐EP is not involved in the long‐term regulation of food intake, but under certain conditions it may play some role in the regulation of individual meals. It is speculated that the latter activity may result from the action of other appetitive regulatory hormones.
1H‐n.m.r. studies of squash seed trypsin inhibitor
Authors: LIKOS J.J.
Published: August 2010

1H‐n.m.r. studies at 500MHz have been performed on a trypsin inhibitor (CMTI‐III) found in squash seed (Cucurbita maxima). The sequential resonance assignments have been made using two‐dimensional techniques. The chemical shifts for the assigned protons are reported at 30°, pH 2.8 and form a basis for the determination of the solution structure of CMTI‐III. Analysis of the NOE data, NH‐αCH vicinal coupling constants and pattern of slowly exchanging amide protons indicates that the predominant feature of the solution conformation is a triple stranded β sheet consisting of residues 8‐10, 21‐23, and 26‐29. Residues 12‐15 appear to form a β turn.
Solid‐phase synthesis of bombesin by continuous flow procedure using Fmoc‐amino acids*
Authors: SCOLARO B. , GOZZINI L. , ROCCHI R. , DI BELLO C.
Published: August 2010

Bombesin has been synthesized by the continuous flow solid‐phase procedure on the derivatized Kieselguhr‐supported polydimethylacrylamide resin. Preformed Fmoc‐amino acid symmetrical anhydrides (Met, Leu, and Arg) and Fmoc‐amino acid active esters were used for amine acylation. The Mtr and the Pmc groups have been alternatively used for masking the side chain function of Arg‐3. The progress of the synthesis was monitored by different analytical methods including quantitative solid‐phase Edman degradation. Cleavage from the resin and simultaneous formation of the C‐terminal amide function were achieved with a methanolic ammonia solution yielding indistinguishable crude peptides which have been purified by HPLC and fully characterized. Preliminary pharmacological experiments indicated that the activity of the synthetic peptides is similar to that previously measured for other synthetic bombesins. For comparison bombesin has also been prepared by solid‐phase synthesis on 4‐methyl benhydrylamine resin using the Boc chemistry. The results of the two strategies are discussed and compared.
Paralytic activity of (des‐Glu1)conotoxin GI analogs in the mouse diaphragm
Authors: ALMQUIST RONALD G. , KADAMBI SRINIVASA R. , YASUDA DENNIS M. , WEITL FREDERICK L. , POLGAR WILLMA E. , TOLL LAWRENCE R.
Published: August 2010

A series of 20 peptide analogs of (des‐Glu1) conotoxin GI were prepared by solid phase synthesis. The peptides were tested for their abilities to inhibit contractions in the mouse‐diaphragm‐with‐phrenic‐nerve assay. (Des‐Glu1) conotoxin has an IC50, of 2.7 × 10−7 M in this assay. Results from this assay show that total loss of paralytic activity occurs when Pro is replaced by Gly, Tyr by D‐Tyr, or Gly by D‐Phe. In most cases loss or change in length of one of the disulfide rings eliminates paralytic activity except with compound 17, which is weakly active, IC50= 7.0 × 10−5 M. Replacement of the Cys1‐Cys6 disulfide bond with an amide bond (compound 9) greatly lowers paralytic activity, IC5, = 3.7 × 10−5 M.
Structure and conformation of peptides containing the sulfonamide junction II. Synthesis and conformation of cyclo[‐MeTau‐Phe‐DPro‐]
Authors: CALCAGNI A. , GAVUZZO E. , LUCENTE G. , MAZZA F. , PINNEN F. , POCHEITI G. , ROSSI D.
Published: August 2010

By applying the method of amino‐acyl incorporation to sulfonamido peptides, cyclo(‐MeTau‐Phe‐DPro‐) 3 has been synthesized in high yield starting from Z‐MeTau‐Phe‐Pro‐OH. The crystal structure and the molecular conformation of 3 have been determined. Crystals are orthorhombic, s.g. P212121, with a = 5.454, b = 13.486, c = 24.025 Å. The structure has been solved by direct methods and refined to R = 0.039 for 1974 reflections with I > 1.50 σ(I). The 10‐membered cyclopeptide adopts a backbone conformation in the crystals characterized by Phe‐DPro and DPro‐MeTau peptide bonds in trans and cis conformation, respectively. Both the peptide bonds deviate significantly from planarity and the corresponding |δω| values are ca. 12°. The sulfonamide SO2NH junction adopts a cisoidal conformation with a Cα1‐ S1‐N2‐ C2α torsion angle of 70.8°. 13C n.m.r. data show that the trans geometry at the Phe‐DPro junction found in the crystals is retained in DMSO solution. The 10‐membered ring of 3 is characterized by a pseudo mirror‐plane passing through the Phe nitrogen and the DPrO carbonylic carbon. The DPrO ring adopts a half‐chair conformation. The Phe side chain conformation corresponds to the statistically most favored g− rotamer (χ1= ‐ 68.6°). The crystal packing is characterized by a weak intermolecular hydrogen bond between the NH group and the MeTau O1’ oxygen.
Conformational perturbations in retro‐analogs of the tBuCO‐Ala‐Gly‐NHiPr dipeptide Crystal structure of the retro‐dipeptide with a reversed Ala‐Gly amide bond
Authors: GOMEZ EMMANUEL J. , VITOUX BERNARD , MARRAUD MICHEL , SAKARELLOS CONSTANTINOS , MASDOURI LARBI EL , AUBRY ANDRÉ
Published: August 2010

The three retro‐analogs of the tBuCO‐Ala‐Gly‐NHiPr dipeptide, in which each amide bond had been successively reversed, were studied in solution by 1H‐n.m.r. and i.r. spectroscopy with reference to the conformational properties of their parent dipeptide. Reversal of the Ala‐Gly amide bond proved to perturb the folding tendency of the backbone less than the inversion of either of the terminal amide bonds. The crystal structure of the retro‐peptide containing a reversed Ala‐Gly amide bond was also solved by X‐ray diffraction and constitutes the first available data for this retro‐peptide series. In contrast to the βII‐folded structure of the parent dipeptide, the retro‐peptide molecule adopts an open conformation in the crystal.
Synthesis and interaction with metal ions of cyclic oligopeptides bearing carboxyl groups
Authors: FUSAOKA YOSINARI , OZEKI EIICHI , KIMURA SHUNSAKU , IMANISHI YUKIO
Published: August 2010

Cyclic di‐ and tetrapeptides bearing carboxyl or carboxylate groups, cyclo[Glu(OBzl)‐Glu(OMe)], cyclo [Glu‐Glu(OMe)], cyclo(Glu‐Glu), cyclo[Glu(OMe)‐Pro)2 and cyclo(Glu‐Pro)2 were synthesized and investigated on the intramolecular interaction of carboxyl side chains in the complexation with metal ions in relation with the conformation. The three kinds of cyclic dipeptides were found to take a flagpole boat conformation. Folded conformation of side chains was predominant for cyclo[Glu(OBzl)‐Glu(OMe)] and cyclo[Glu‐Glu(OMe)]. However, cyclo(Glu‐Glu) took an unfolded conformation. Intramolecular interaction of carboxyl groups was observed neither in free state nor in complexation with metal ions. The intramolecular interaction of carboxyl groups was observed in the case of cyclo(Glu‐Pro)2 in the absence of metal ions added. Cyclo[Glu(OMe)‐Pro], and cyclo(Glu‐Pro)2 formed a complex with Ca2+ and Ba2+ without participation of side chains.
pH dependent conformation of physalaemin
Authors: HÖLZEMANN G. , PACHLER K.G.R.
Published: August 2010

The undecapeptide physalaemin was investigated by n.m.r. spectroscopy in DMSO solution under acidic and neutral conditions. Large changes of the NH chemical shifts and the temperature gradients of the NH protons occurred on going from pH 3.5 to pH 7.0 for residues around the charged amino acids Asp and Lys. At pH 3.5 the data are in accord with a flexible conformation of the peptide. The results at neutral pH are interpreted in terms of a folded structure having two interresidue and one intraresidue hydrogen bond. They include a β turn with proline in position i + 1 and asparagine in position i + 2.
Synthesis and application of acid labile anchor groups for the synthesis of peptide amides by Fmoc‐solid‐phase peptide synthesis
Authors: BREIPOHL GERHARD , KNOLLE JOCHEN , STÜBER WERNER
Published: August 2010

The preparation and application of a new linker for the synthesis of peptide amides using a modified Fmoc‐method is described. The new anchor group was developed based on our experience with 4,4′‐dimeth‐oxybenzhydryl (Mbh)‐protecting group for amides. Lability towards acid treatment was increased dramatically and results in an easy cleavage procedure for the preparation of peptide amides. The synthesis of N‐9‐fluorenylmethoxycarbonyl‐[(5‐carboxylatoethyl‐2.4‐dimethoxyphenyl)‐4′‐methoxyphenyl]‐methyla‐mine is reported in detail. This linker was coupled to a commercially available aminomethyl polystyrene resin. Peptide synthesis proceeded smoothly using HOOBt esters of Fmoc‐amino acids. Release of the peptide amide and final cleavage of the side chain protecting groups was accomplished by treatment with trifluoroacetic acid‐dichloromethane mixtures in the presence of scavengers. The synthesis of peptide amides such as LHRH and C‐terminal hexapeptide of secretin are given as examples.
Studies on racemization associated with the use of benzotriazol‐1‐yl‐tris (dimethylamino)phosphonium hexafluorophosphate (BOP)
Authors: STEINAUER RENE , CHEN FRANCIS M.F. , LEO BENOITON N.
Published: August 2010

The influence of the amount and nature of the necessary tertiary amine on racemization attending the BOP‐mediated coupling of N‐benzyloxycarbonylglycyl‐dipeptides has been examined by determining the epimeric products by high‐performance liquid chromatography. Racemization was encountered in all cases examined that included dichloromethane as solvent. The least racemization occurred when the base was diisopropylethylamine, the most when the base was N‐methylmorpholine. Excess base promoted racemization. Racemization was significantly diminished but not eliminated when 1‐hydroxybenzotriazole was added to the reaction mixtures. The above results pertain to the coupling of peptide segments; they do not pertain to the coupling of N‐alkoxycarbonylamino acids.
3‐Amino‐2‐piperidinone‐6‐carboxylic acid
Authors: LI JORGE P. , YELLIN TOBIAS O. , DEBROSSE CHARLES W. , EGGLESTON DRAKE S.
Published: August 2010

The cis (2a) and trans (2b) isomers of methyl 3‐benzamido‐2‐piperidinone‐6‐carboxylate (Apca) were prepared and separated by fractional recrystallizations. Proton n.m.r. studies in dimethylsulfoxide solution indicate that the six‐membered lactam ring adopts a distorted chair conformation with an equatorially oriented benzamido substituent in both 2a and 2b. The carboxyl function also is equatorially oriented in the trans isomer 2b, but is disposed axially in the cis isomer 2a. In the crystal structure, the six‐membered lactam ring of 2a is clearly in a boat conformation with the benzamido and carboxyl functions attached to the two apex carbon atoms equatorially. The trans isomer, 2b, exists as two crystallographically independent, conformationally distinct molecules in one unit cell. The lactam ring in both molecules adopts a distorted chair conformation, as is the case in solution, with both the benazamido and carboxyl functions attached equatorially. The rotameric orientation for the endocyclic lactam differs between the two molecules. Both structures show evidence of C—H.O hydrogen bond formation intermolecularly in the solid state. This ability, along with the distinctive conformational features of Apca, may be exploitable in the design of unique features of polypeptides.
Stoichiometry of slow binding of palmitoyl‐CoA to liver glucokinase*
Authors: LIN SHENG‐XIANG , VOGEL WALTER K. , NEET KENNETH E.
Published: August 2010

The interaction of palmitoyl‐CoA with porcine glucokinase was studied by the gel permeation technique. The finding that glucokinase “bound” up to 60 molecules was unexpected from the specific inhibition of rat glucokinase by long chain acyl‐CoA (Tippett & Neet, J. Biol. Chem. (1982) 287, 12839–12845). Sephacryl S‐200 gel filtration in the presence of palmitoyl‐CoA demonstrated a protein peak without enzyme activity that was eluted earlier than the active enzyme peak, indicating a large molecular weight shift for the inactivated enzyme form and confirming a large number (≥ 30) of associated palmitoyl‐CoA molecules. The binding was also verified by analyzing the absorption characteristics of the inactivated enzyme peak. In the presence of glycerol, the size of the inactivated peak greatly decreased, but the separation between the two peaks remained unchanged. Therefore, the amphiphile bound predominantly to the inactive enzyme and not to the active form, suggesting that the rapid inhibitory interactions between palmitoyl‐CoA and glucokinase previously observed are specific. Parallel enzyme activity studies showed that in the time range of the column experiments (4–20 h), both the rat and pig enzyme were greatly inactivated (< 90%) in the presence of palmitoyl‐CoA (15 μM) in the absence of glycerol. This slow inactivation is different from the immediate specific inhibition previously reported and depends on both enzyme and palmitoyl‐CoA concentrations. The presence of up to 20% glycerol slowed this inactivation process. These results demonstrated that even below the critical micelle concentration, partial inactivation of glucokinase occurs in the presence of palmitoyl‐CoA over a long period of time.
Site‐directed mutagenesis of ricin A chain Trp 211 to Phe
Authors: BRADLEY JANICE L. , McGUIRE PETER M.
Published: August 2010

Oligonucleotide‐directed site‐specific mutagenesis has been used to make a conservative change in the putative active site of the catalytic subunit of the toxin ricin. The substitution of phenylalanine for tryptophan at position 211 of the A chain reduces but does not abolish catalytic activity, as assayed by inhibition of cell‐free protein synthesis.
Comparisons between the three‐dimensional structures of the chemotactic protein Che Y and the normal Gly 12‐p21 protein
Authors: CHEN JAMES M. , LEE GRACE , MURPHY RANDALL B. , BRANDT‐RAUF PAUL W. , PINCUS MATTHEW R.
Published: August 2010

The three‐dimensional structure of a chemotactic protein Che Y from Salmonella typhimurium has recently been determined by X‐ray crystallography. The structure of this small protein, containing 129 amino acid residues, shows a domain consisting of a central beta‐pleated sheet surrounded on both sides by alpha‐helices. We have examined the sequence and the arrangement of the structural domains of the Che Y protein and have compared them with other nucleotide binding protein sequences and structures. We find that the Che Y protein has significant sequence homology to the ras‐gene encoded p21 protein. In addition, the structural domains of the two proteins are arranged in a fundamentally similar manner, including the phosphate‐binding site (both proteins bind phosphate‐containing ligands). The striking similarity in the arrangement of the structural domains of the two proteins suggests that both may serve similar functions as signal transducers.
Syntheses of argininal semicarbazone containing peptides and their applications in the affinity chromatography of serine proteinases†
Authors: BASAK AJOY , GONG YUEH T. , CROMLISH JIM A. , PAQUIN JOHANNE A. , JEAN FRANÇOIS , SEIDAH NABIL G. , LAZURE CLAUDE , CHRÉTIEN MICHEL
Published: August 2010

Eight argininal semicarbazone containing peptides prepared by liquid phase synthesis were all found to be reversible inhibitors of model serine proteinases including trypsin and plasma kallikrein (PK). Among the peptides tested, those having a Lys residue at position P2 displayed the maximum binding potency towards PK. One of the peptides, Leu‐enkephalin‐argininal semicarbazone, a comparatively weak inhibitor, was chosen in order to develop an affinity‐based purification protocol for PK. The affinity column was prepared by covalent attachment of the NH2 ‐terminal moiety of the peptidyl semicarbazone to a solid‐phase matrix bearing a spacer group. For efficient binding of PK, it was found necessary to optimize parameters like the concentration of inhibitor linked to the solid matrix, the ionic strength of the buffer used, the temperature and the pH. The majority of the bound enzyme could be recovered following elution with guanidine hydrochloride or benzamidine hydrochloride in a high salt buffer at pH 6.0. The usefulness of the affinity procedure towards the purification of other serine proteinases is also discussed.
Practical approach to solid‐phase synthesis of C‐terminal peptide amides under mild conditions based on a photolysable anchoring linkage1
Authors: HAMMER ROBERT P. , ALBERICIO FERNANDO , GERA LAJOS , BARANY GEORGE
Published: August 2010

Several 3‐nitro‐4‐(N‐protected aminomethyl)benzoic acids, with protection provided by tert.‐butyloxycar‐bonyl (Boc), 9‐fluorenylmethyloxycarbonyl (Fmoc), trifluoroacetyl (Tfa), dithiasuccinoyl (Dts), or phthaloyl (Phth), have been prepared by reproducible routes. Synthesis of Dts‐handle 6 illustrates some particularly novel and efficient chemistry, and is preferred over more intricate routes to Boc‐handle 3 and Fmoc‐handle 4. The five handles were each evaluated for their application to the synthesis of peptide amides. Coupling onto amino‐functionalized supports provided a general starting point for peptide chain assembly. The handle amino function was deblocked (Boc, Fmoc, Dts), the C‐terminal residue was coupled as its Nx‐protected free acid, and ultimately the ortho‐nitrobenzylamide anchoring linkage was cleaved photolytic‐ally to give the corresponding amide. Starting with handles 3, 4, and 6, several free and protected peptide amides were synthesized.
Effect of inhibitors on conformational changes in hen lysozyme around thermal transition point in solution and solid state
Authors: BERNARD M. , CANIONI P. , COZZONE P. , BERTHOU J. , JOLLÉS P.
Published: August 2010

Carbon‐13 NMR spectroscopy has been used to further document the interaction, at low and high temperatures, of N‐acetylglucosamine and its short polymrs with hen egg‐white lysozyme. The results have been compared with the corresponding X‐ray crystallographic data. Two domains, the active site and the hydrophobic box, have been found by NMR to undergo conformational rearrangement while X‐ray crystallography only detected changes located in the active site. The extent of the modifications induced by inhibitor binding was proportional to the inhibitor size. The two techniques concurred to show that even in the presence of monosaccharide (N‐acetylglucosamine), more than one subsite of the enzyme was occupied at high temperature, the binding at the C‐site being the best defined. The thermal transition of lysozyme still occurred in solution when inhibitors were bound. However, in the solid state, crystallographic data showed that the transition was hindered.
Enthalpic and entropic determinants for the specificity of alkylation of the histidine‐12 residue of ribonuclease A by four bromoacetamido nucleoside affinity labels and bromoacetamide
Authors: PINCUS MATTHEW R. , HUMMEL CHARLES F. , BRANDT‐RAUF PAUL W. , CARTY ROBERT P.
Published: August 2010

The binding and alkylation rate constants for the reaction of four bromoacetamido pyrimidine nucleosides and bromoacetamide with bovine pancreatic ribonuclease A (RNase A) have been determined as a function of temperature. The four nucleoside derivatives react exclusively or preferentially with the NE2 atom of histidine‐12 and include 2′‐bromoacetamido‐2′‐deoxyuridine, 2′‐bromoacetamido‐2′‐deoxyxylofuranosyl‐uracil, 3′‐bromoacetamido‐3′‐deoxythymidine and 3′‐bromoacetamido‐3′‐deoxyarabinofuranosyluracil. Transition‐state parameters, δH‡ and AS‡, reveal that nucleosides with “up” OH groups experience relative rate enhancements which have been attributed to contacts between these groups and the enzyme in the transition state (1). Variations in alkylation rates are explained in terms of different degrees of entropic destabilization (2) of the nucleosides in the enzyme affinity label complex.
A novel Fmoc‐based anchorage for the synthesis of protected peptides on solid phase
Authors: LIU YIN‐ZENG , DING SHAO‐HUA , CHU JI‐YU , FELIX ARTHUR M.
Published: August 2010

A novel bifunctional compound, 9‐(hydroxymethyl)‐2‐fluoreneacetic acid, was synthesized, coupled to benzhydrylamine‐resin, and evaluated for its application to the solid phase synthesis of protected peptide fragments. Anchor‐bond cleavage was achieved with 15% pipetidine/DMF. A protected heptapeptide, Boc‐Val‐Val‐Ser(Bzl)‐His(Tos)‐Phe‐Asn‐Lys‐(Z)‐OH, corresponding to the sequence (1–7) of rat‐transforming growth factor‐α, was synthesized using this new support with an overall yield of 46%.
Multiple peptide synthesis using a single support (MPS3)
Authors: TJOENG FOE S. , TOWERY DEREK S. , BULOCK JOSEPH W. , WHIPPLE DEBORAH E. , FOK KAM F. , WILLIAMS MARK H. , ZUPEC MARK E. , ADAMS STEVEN P.
Published: August 2010

An automated multiple peptide synthesis method to synthesize, cleave, and purify several peptides simultaneously in a single batch has been developed. The technique is based on the synthesis of multiple peptides on a single solid phase support and is easily adapted to manual or to automated methods. The approach relies on coupling of amino acid mixtures to the resin and it has been found that DCC/HOBt gives the best coupling performance. Fast Atom Bombardment Mass Spectrometry (FAB‐MS) was used to rapidly and efficiently identify the peptides in each synthetic mixture which significantly assisted the purification process by HPLC. The method has been successfully applied to the synthesis of magainin 2 and angiotensinogen peptides.
Antagonists of luteinizing hormone releasing hormone with novel unnatural amino acids at position six
Authors: LIU KELIANG , HE BINGLIN , XIAO SHAOBO , XIA QIMING , FANG XI , WANG ZHONGYI
Published: August 2010

Five new antagonists of luteinizing hormone releasing hormone (LHRH) containing novel unnatural amino acids at position six are reported. They are very effective in the rat antiovulatory assay. Using saline as vehicle, antagonist‐[N‐ Ac‐d‐2‐Nal1, d‐4‐Cl‐Ph2, d‐3‐Pal3, Arg5, d‐A26,d‐Ala10‐LHRH inhibited ovulation completely at 1 μg/rat and three of the other antagonists showed some antiovulatory activity at μpg/rat.
Formation of optically pure N‐acyl‐N,N′‐dicyclohexylurea in N,N′‐dicyclohexylcarbodiimide‐mediated peptide synthesis
Authors: ŚLEBIODA MAREK , WODECKI ZBIGNIEW , KOŁODZIEJCZYK ALEKSANDER M.
Published: August 2010

N‐acylurea, a side product in peptide synthesis from DCC, preserves its chiral integrity although peptides formed simultaneously in the same reaction are racemized to a large extent. This observation is inconsistent with the generally accepted opinion that racemization‐prone O‐acylisourea is a common intermediate for both peptides and N‐acylurea. Chiral purity of N‐acylureas and peptides was determined by HPLC using chiral stationary phases. An efficient method of synthesis of chirally pure N‐acylureas is also presented.
Receptor selectivity of enkephalin analogs carrying artificial address peptides
Authors: KIMURA SHUNSAKU , SASAKI‐YAGI YOSHIKO , IMANISHI YUKIO
Published: August 2010

The synthetic [Leu]enkephalin analogs YGGFLGP(KS′S′S′)2‐OMe (S′ represents Sar residue, ENK‐S′8), YGGFLGP(KPPP)2‐OMe (ENK‐P8), and YGGFLGP(KA′LA′)2‐OMe (A′ represents 2‐aminoisobutyric acid (Aib), ENK‐A′8) were designed to investigate the role of membrane affinity in opioid receptor binding. CD measurement in ethanol revealed that ENK‐S′8 took a random coil conformation, while ENK‐S′8 and ENK‐A′8 partly adopted a 31 ‐ and α‐helical conformation, respectively. ENK‐A′8 was shown to be distributed to lipid bilayer membrane due to the amphiphilic α‐helical structure, while ENK‐S′8 and ENK‐P8 stayed in aqueous phase. Opioid receptor affinities of ENK‐S′8 and ENK‐P8 were similar to [Leu]enkephalinamide, indicating that the connection of hydrophilic peptide segments to the C‐terminal of [Leu]enkephalinamide did not affect the receptor affinity. On the other hand, δ and μ‐receptor affinities of ENK‐A′8 were about 1/20 and 1/6 times, respectively, those of [Leu]enkephalinamide. Therefore, ENK‐A′8 has higher selectivity for μreceptor than [Leu]enkephalinamide. These results are explained in terms of the membrane compartment concept proposed by Schwyzer.
Role of guanidine group at the N‐terminal proctolin chain in cardioexcitatory effects in insects
Authors: KONOPIÑSKA DANUTA , ROSIÑSKI GRZEGORZ , BARTOSZ‐BECHOWSKI HUBERT , LESICKI ANDRZEJ , SUJAK PRZEMYSLAW , SOBÓTKA WIESLAW
Published: August 2010

Six proctolin analogues (I‐VI) modified in position 1 of the peptide chain by the following amino acids: homo‐Arg, Gac, Gav, Gap, Phe (p‐guanidino) and Orn, were synthesized by conventional liquid phase method. The myotropic activity of the obtained peptides was investigated in cardioexcitatory test on two insect species, cockroach, Periplaneta americana L., and yellow mealworm, Tenebrio molitor.
Conformational characteristics of legume 7S globulins as revealed by circular dichroic., derivative u. v. absorption and fluorescence techniques
Authors: DESHPANDE S.S. , DAMODARAN SRINIVASAN
Published: August 2010

The 7S globulin storage proteins, phaseolin, vicilin, and β‐conglycinin of, respectively, dry bean, field pea, and soybean, are highly homologous, have similar predicted protein structures, and yet exhibit considerable differences in their susceptibility to various proteinases [Nielsen, S.S., Deshpande, S.S., Hermodson, M.A. & Scott, M.P. (1988) J. Agric. Food Chem.36, 896‐902]. These differences in their proteolytic behavior were studied in relation to their solution conformational states. The secondary structures of these three proteins determined by far u. v. circular dichroism were characterized by predominantly β‐sheet and β‐turn parameters. However, characterization of tertiary and quaternary structures using second derivative u. v. absorption spectroscopy, surface hydrophobicity using cis‐parinaric acid as hydrophobic probe, and fluorescence quenching studies of intrinsic Trp fluorescence using an ionic (iodide) and a neutral (acrylamide) quencher indicated sharp differences in the conformation of these proteins. About 9.6 and 10.2 out of 13 and 15 tyrosyls/subunit of phaseolin and β‐conglycinin, respectively, were exposed to polar solvent, while the surface hydrophobicity varied β‐conglycinin > vicilin > phaseolin. The Trp residues in phaseolin were not accessible to iodide, while half those of vicilin and β‐conglycinin were quenched. The order of Trp accessibility to acrylamide was vicilin > β‐conglycinin > phaseolin. The relative compactness of these three proteins based on these studies was related to the observed differences in their susceptibility to various proteinases.
Synthesis of cyclic peptide homologs of glutathione as potential antitumor agents
Authors: SHEH LEUNG , CHEN BO‐LING , CHEN CHIA‐FU
Published: August 2010

Two cyclic tripeptide homologs, cyclo(Glu[Cys‐β‐Ala‐]‐OH) 8a, and cyclo(Glu[Cys‐Gaba‐]‐OH) 8b, were synthesized by the pentafluorophenyl ester method in solution (1‐3). These cyclic peptides are cyclo homologs of glutathione and are designed as potential antitumor agents. The 1H‐ and 13C‐n.m.r. spectral parameters of cyclo(Glu[Cys(Bzl)‐β‐Ala‐]‐OH) 7a were measured in DMSO‐d6 and a possible conformation has been proposed. The cyclic peptide 8a showed low cytotoxic activities against three human tumor cell lines: KB, HeLa, and Colo 205.
Solid‐phase synthesis of peptides with C‐terminal asparagine or glutamine
Authors: ALBERICIO FERNANDO , VAN ABEL ROBERT , BARANY GEORGE
Published: August 2010

Attempts to anchor Fmoc‐asparagine or glutamine as p‐alkoxybenzyl esters for solid‐phase peptide synthesis are fraught with difficulties. A convenient and effective method to prepare peptides with C‐terminal asparagine or glutamine involves quantitative attachment of Nx ‐Fmoc‐Cx ‐tert.‐butyl aspartate or glutamate via the free ω‐carboxyl groups to a tris(alkoxy)benzylamino (PAL) support. Chain elongation proceeds normally by standard Fmoc chemistry, and treatment with acid, e.g., CF3COOH—CH2Cl2, 90 min at 25°, releases the desired peptides in >95% yields without side reactions at the C‐terminus. Feasibility of the approach has been demonstrated by the syntheses of the C‐terminal octapeptide from human proinsulin, H‐Leu‐Ala‐Leu‐Glu‐Gly‐Ser‐Leu‐Gin‐OH, and the serum thymic factor pGlu‐Ala‐Lys‐Ser‐Gln‐Gly‐Gly‐Ser‐Asn‐OH.
Synthesis of glutathione analogues with modified ionizable groups for the study of enzyme: ligand interactions
Authors: JANES WOLFGANG , SCHULZ GEORG E.
Published: August 2010

The syntheses and analyses of GSSG analogues with modified ionizable groups are described. Using the mixed anhydride method, three symmetrical and three asymmetrical peptide analogues were obtained in moderate overall yields. The products were analyzed by HPLC, 1H‐NMR, thin‐layer electrophoresis, and amino acid analysis. They were used to study enzyme: ligand interactions in glutathione reductase.
Single residue modifications of the delta opioid receptor selective peptide, [d‐Pen2,d‐Pen5]‐enkephalin (DPDPE)
Authors: HAASETH RONALD C. , SOBCZYK‐KOJIRO KATARZYNA , MEDZIHRADSKY FEDOR , SMITH CHARLES B. , MOSBERG HENRY I.
Published: August 2010

Six analogs of the highly delta opioid receptor selective, conformationally restricted, cyclic peptide [d‐Pen2,d‐Pen5]enkephalin, Tyr‐d‐Pen‐Gly‐Phe‐d‐PenOH (DPDPE), were synthesized and evaluated for opioid activity in rat brain receptor binding and mouse vas deferens (MVD) smooth muscle assays. All analogs were single amino acid modifications of DPDPE and employed amino acid substitutions of known effects in linear enkephalin analogs. The effect on binding affinity and MVD potency of each modification within the DPDPE structural framework was consistent with the previous reports on similarly substituted linear analogs. Conformational features of four of the modified DPDPE analogs were examined by1H NMR spectroscopy and compared with DPDPE. From these studies it was concluded that the observed pharmacological differences with DPDPE displayed by diallyltyrosine1‐DPDPE ([DAT1]DPDPE) and phenylglycine4‐DPDPE ([Pgl4]DPDPE) are due to structural and/or conformational differences localized near the substituted amino acid. The observed enhanced μ receptor binding affinity of the carboxamide terminal DPDPE‐NH2appears to be founded solely upon electronic differences, the NMR data suggesting indistinguishable conformations. The observation that the α‐aminoisobutyric acid substituted analog [Aib3]DPDPE displays similarin vitroopioid behavior as DPDPE while apparently assuming a significantly different solution conformation suggests that further detailed conformational analysis of this analog will aid the elucidation of the key structural and conformational features required for action at the δ opioid receptor.
Acid‐labile anchoring linkages for solid phase synthesis of C‐terminal asparagine peptides using the Fmoc strategy
Authors: SHAO JUN , LI YOU‐HE , VOELTER WOLFGANG
Published: August 2010

Two acid‐labile substituted benzylamine type anchoring linkages, 4‐benzoxy‐2,6‐dimethoxybenzylamine and 2‐benzoxy‐4,6‐dimethoxybenzylamine, for solid phase synthesis of peptide amides were prepared. The Na‐9‐fluorenylmethyloxycarbonyl (Fmoc) amino acids could be easily attached to the resins with DCC/HOBt (loading 0.5–0.6 mmol/g resin). After final removal of the Na‐protecting groups, treatment with TFA (50–95%) yielded amino acid and peptide amides in high purity. As we could show for the synthesis of thymulin (FTS, pGlu‐Ala‐Lys‐Ser‐Gln‐Gly‐Gly‐Ser‐Asn), these two resins with anchoring linkages are well suited for the synthesis of C‐terminal Asn peptides using protected aspartic acid derivative as starting material.