bluuebunny/crossref_metadata_2025 huggingface dataset:
S‐deoxo‐Abu1, Ile3‐amaninamide, an inactive amatoxin analogue
Authors: ZANOTTI GIANCARLO , WIELAND THEODOR , DAURIA GABRIELLA , PAOLILLO LIVIO , TRIVELLONE ENRICO
Published: August 2010

The title compound 3, an amatoxin analogue containing l‐α‐aminobutyric acid instead of l‐asparagine in position 1, as in natural toad stool peptides, has been synthesized. It does not inhibit the eukaryotic DNA‐dependent RNA polymerase form II (or B) in concentrations up to 10‐4M, whereas 50% inhibition is exerted in M solution by the corresponding Asn‐analogue S‐deoxo‐Ile3‐amaninamide 2. The striking difference seems to be due to a relatively small variation of the conformation recognized by sensitive NMR spectroscopic methods.
Synthesis of [β‐(4‐pyridyl‐1‐oxide)‐L‐alanine4]‐angiotensin I as a potential suicide substrate for protein‐tyrosine kinases
Authors: CUSHMAN MARK , CHINNASAMY PENNAMUTHIRIAR , CHAKRABORTI ASIT K. , JURAYJ JURJUS , GEAHLEN ROBERT L. , HAUGWITZ RUDIGER D.
Published: August 2010

An asymmetric synthesis of [β‐(4‐pyridyl‐1‐oxide)‐L‐alanine4]‐angiotensin I (la), which is a potential suicide substrate (mechanism‐based inhibitor) for protein‐tyrosine kinases, has been performed. Deprotonation of 6 with n‐butyllithium in THF gave the anion 7, which was alkylated with 4‐(chloromethyl)pyridine‐1‐oxide to afford intermediate 9 as a crystalline solid. Hydrolysis of 9 afforded a mixture of 11 and 12 in a ratio of 96:4 as estimated by conversion to the diastereomeric dipeptides 13 and 14 followed by HPLC analysis. The 96:4 mixture of 11 and 12 was used in the solid phase synthesis of the target angiotensin analog la and its diastereomer 1b, which were separated and tested for inhibitory activity against two thymocyte protein‐tyrosine kinases: p40 and p56lck. Neither peptide displayed significant inhibitory activity toward p40 and both served as weak competitive inhibitors of p56lck.
Peptides containing 2‐aminopimelic acid
Authors: LE ROUX PIERRE , BLANOT DIDIER , MENGIN‐LECREULX DOMINIQUE , VAN HEIJENOORT JEAN
Published: August 2010

Taking advantage of the peptide transport strategy, we have designed and synthesized several new peptides containing 2‐aminopimelic acid (Apm), an inhibitor of the diaminopimelate pathway in bacteria: L‐LYS‐ambo‐Apm, ambo‐Apm‐L‐Lys, L‐Lys‐L‐Ala‐ambo‐Apm, ambo‐Apm‐L‐Ala‐L‐Lys, L‐Ala(CI)‐ambo‐Apm and ambo‐Apm‐L‐Ala(CI). In the two latter cases, Apm was associated with antibacterial amino acid β‐chloro‐L‐alanine [L‐Ala(CI)], an inhibitor of alanine racemase and transaminase B. The peptides displayed weak or no antibacterial activities; nevertheless, those containing L‐Ala(CI) had low MIC values in the presence of amino acids restoring protein synthesis. When tested on exponential phase Escherichia coli cells grown in minimal medium, the peptides were without effect or bacteriostatic, but important bacteriolytic effects could be observed, especially for the L‐Ala(CI)‐containing peptides, when the growth medium was supplemented with specific amino acids. It was demonstrated that the weak or nil effect of the L‐lysine‐containing peptides was due to a poor uptake.
2‐Chlorotrityl chloride resin
Authors: CHATZI KLEOMENIS BARLOS. OLGA , GATOS DIMITRIOS , STAVROPOULOS GEORGE
Published: August 2010

The esterification of 2‐chlorotrityl chloride resin with Fmoc‐amino acids in the presence of DIEA is studied under various conditions. High esterification yields are obtained using 0.6 equiv. Fmoc‐amino acid/mmol resin in DCM or DCE, in 25 min, at room temperature. The reaction proceeds without by product formation even in the case of Fmoc‐Asn and Fmoc‐Gln. The quantitative and easy cleavage of amino acids and peptides from 2‐chlorotrityl resin, by using AcOH/TFE/DCM mixtures, is accomplished within 15‐60 min at room temperature, while t‐butyl type protecting groups remain unaffected. Under these exceptionally mild conditions 2‐chlorotrityl cations generated during the cleavage of amino acids and peptides from resin do not attack the nucleophilic side chains of Trp. Met, and Tyr.
Importance of the N‐terminal domain of the type I angiotension II antagonist [Sar1,Ile8]ANG II for receptor blockade
Authors: MOORE GRAHAM J. , GANTER RENEE C. , GOGHARI MAHESH H. , FRANKLIN KEVIN J.
Published: August 2010

Analogues of the Type I angiotensin (ANG) antagonist, [Sar1,Ile8]ANG II, in which the N‐terminal dipeptide was modified were synthesized by the solid phase method and purified by reversed‐phase HPLC. Antagonist potencies (pA2) of the peptides were determined on the rat isolated uterus using ANG II as the agonist. Substitution of the Arg residue occupying position 2 of [Sar1,Ile8]ANG II (pA2 8.1) by Gly, Ala, Nle, Phe, Pro or Sar reduced the antagonist potency to pA2= 7.0, 6.8, 6.7, 6.8, 5.8 and 5.3, respectively. Deletion of the N‐terminal Sar residue in these same peptides gave pA2= 6.8, 5.7, 5.5, 5.9, 6.1 and 7.5, respectively. The characteristically long duration of action of [Sar1,Ile8] was absent for all of these analogues including (des1, Sar2, Ile8]ANG II. These findings demonstrate that the antagonist potencies of Type I angiotensin antagonists for smooth muscle receptors, and also the long duration of action, are dependent on the location of positive charges within the peptide and on the conformation of the molecule in determining favorable electrostatic interactions with the receptor. A model is proposed in which the two positively charged loci on the angiotensin molecule (N‐terminus and Arg) interact with two corresponding anionic binding sites on the smooth muscle receptor. The possibility that the prolonged duration of action of [Sar1, Ile8]ANG II results from binding to a different site on the angiotensin receoptor from that occupied by ANG II is discussed in relation to the present findings.
Structure and conformation of a novel inhibitor of angiotensin I converting enzyme – a tripeptide containing phosphonic acid
Authors: HIRAYAMA N. , KASAI M. , SHIRAHATA K.
Published: August 2010

A novel phosphotripeptide, (IR)‐1‐(N‐(N‐acetyl‐l‐isoleucyl)‐l‐tyrosyl)amino‐2‐(4‐hydroxyphenyl)ethyl‐phosphonic acid, is a potent inhibitor of angiotensin I converting enzyme (ACE). ACE inhibitory activity in vitro of the peptide is comparable to that of captopril. Its diethylester (C29H42N3O8P, molecular weight, 591.6) crystallizes in the monoclinic space group C2, with cell constants: a = 25.666(9), b = 9.590(8), c = 13.557(2)Å, β= 91.65(2)°, Z = 4, Dc = 1.17g/cm3. The structure was solved by MULTAN 11/82 and refined by full matrix least‐squares methods to a final R‐factor of 0.063 for 2123 unique reflections (F > 3σ(F)) measured on an Enraf‐Nonius CAD‐4 diffractometer (CuKα, λ= 1.541 8 Å, T = 295 K). The absolute configuration of the α‐carbon where the phosphonic acid is attached was determined unequivocally by referring to the l‐isoleucyl moiety whose absolute configuration is known. The conformation of the molecule is relatively rigid owing to the intramolecular requisites and the resultant relative disposition of hetero atoms, which are necessary to its biological activities, are confined to the corresponding disposition in captopril.
Immunosuppressive analogues of hexapeptide Tyr‐Val‐Pro‐Leu‐Phe‐Pro, an immune system stimulant
Authors: SIEMION IGNACY Z. , KUBIK ALEKSANDRA , LISOWSKI MAREK , SZEWCZUK ZBIGNIEW , ZIMECKI MICHAL , WIECZOREK ZBIGNIEW
Published: August 2010

It was found that substitution of Val2 and/or Leu4 residue in a hexapeptide Tyr‐Val‐Pro‐Leu‐Phe‐Pro (I) transforms this peptide immunostimulant into analogues possessing the immunosuppressor activity – Tyr‐Gly‐Pro‐Leu‐Phe‐Pro (II), Tyr‐Val‐Pro‐Gly‐Phe‐Pro (III), and Tyr‐Gly‐Pro‐Gly‐Phe‐Pro (IV). Biological effects of peptides I‐IV were studied using PFC (plaque forming cell) test, GvH (graft vs host) reaction in mice, and ARFC (autologous rosette forming cell) test. The strongest immunosuppressor activity was observed for III – in this case the immunosuppressor effect was observed even in PFC test in vitro in which II and IV showed no such activity. These results suggest that simultaneous presence of both Val2 and Leu4 residues is necessary for the generation of immunostimulation. The CD study of I‐IV in methanol solution suggests that the conformational preferences of II‐IV change towards stabilization of the β‐turn structure, whereas in the case of I the γ‐turn on Leu4 and cis orientation of the Pro3 carbonyl (distorted β‐turn of type I) was found (1) as the preferred conformation. Competition in biological effects observed for I and III in PFC in vitro test suggests that these analogues may interact with the same cellular receptor. Drastic changes in the activity which accompany changes in the sequence are discussed in the terms of our stereochemical hypothesis (1).
Solid phase synthesis of human growth hormone‐releasing factor analogs containing a bicyclic β‐turn dipeptide
Authors: SATO KAZUKI , HOTTA MARI , DONG MING‐HUI , HU HSIAO‐YU , TAULENE JOSEPH P. , GOODMAN MURRAY , NAGAI UKON , LING NICHOLAS
Published: August 2010

Three analogs derived from the N‐terminal 29‐residue fragment of human growth hormone‐releasing factor (hGRF) which contained a bicyclic β‐turn dipeptide (BTD) at 7‐8,8‐9, and 9‐10 positions were synthesized by solid phase methodology to ascertain if the β‐turns are important for the biological activity of hGRF and also to show the applicability of the BTD unit to solid phase synthesis. All three analogs were obtained in good yield and purity indicating that the BTD unit can be used in the usual condition of solid phase synthesis. The capacity of these analogs to release growth hormone (GH) was tested in an in vitro bioassay using rat anterior pituitary cells. All three BTD‐containing analogs showed the same maximal GH secretion with parallel dose‐response curves to that of hGRF(1‐29)NH2, except their relative potencies were very low.
Synthesis of histidine‐containing dipeptide affinity‐labelling agents
Authors: ANGLIKER H. , ZUMBRUNN A. , SHAW E.
Published: August 2010

Peptidyl diazomethyl ketones and fluoromethyl ketones containing histidine in the C‐terminal position were synthesized to determine their properties as proteinase inactivators. These were examined chiefly with derivatives of Z‐Ala‐His. The protection of histidine during conversion of the C‐terminal residue to the diazomethyl ketone required unblocking conditions which avoid acid due to the lability of this function. This was achievable with a Cbz‐imidazole derivative since aminolysis provided deblocking without disturbance of the diazomethyl ketone function. In the case of the fluoromethyl ketone synthesis using fluoroacetic anhydride (Dakin‐West procedure), the desired product could be isolated without ring blocking. The Z‐Ala‐His products showed enhanced selectivity for inactivation of cathepsin B over L when compared to analogous dipeptide inhibitors.
Periodate oxidation products derived from methylated α‐amanitin: evidence for distinct aldehydic and non‐aldehydic forms
Authors: MULLERSMAN JERALD E. , BONETTI SANDRA J. , PRESTON JAMES F.
Published: August 2010

Amatoxins are cyclic octapeptides which can be purified from various mushroom species. They have found widespread use due to their potent inhibition of eukaryotic RNA polymerase II. In the course of our efforts to prepare additional semisynthetic derivatives of the amatoxin, α‐amanitin, we examined the products formed through the periodate oxidation of 6′‐0‐methyl‐α‐amanitin. Periodate oxidation under conditions of near‐neutral pH yielded two chemically similar, yet chromatographically separable, products. On the basis of their proton NMR spectra and their lack of reactivity with sodium chlorite these products did not contain a free aldehydic group. However, both forms were interconvertible in aqueous neutral solution and could be converted to the same product, 6′‐O‐methyldemethyl‐γ‐amanitin, through reduction with sodium borohydride. Periodate oxidation under mildly acidic conditions generated a single product which has properties of a free aldehyde. It exhibited a proton NMR spectrum with signals characteristic of an aliphatic aldehyde and was readily oxidized to the carboxylic acid with sodium chlorite. Conditions have been defined to synthesize the free aldehyde derivative of 6′‐O‐methyl‐α‐amanitin in generous yield to provide a precursor for oxidation and further derivatization.
Formation of a dehydroalanyl residue from S‐benzylcysteine upon HF cleavage of a [Sar1, Cys8]‐angiotensin II peptide resin
Authors: HALLINAN E. ANN
Published: August 2010

Attempted removal with hydrogen fluoride (HF) of an S‐benzyl group from a cysteine‐containing peptide resulted in the formation of a dehydroalanine residue by elimination of benzyl mercaptan.
Efficient solid phase peptide synthesis
Authors: KISO YOSHIAKI , FUJIWARA YOICHI , KIMURA TOORU , NISHITANI AKIKO , AKAJI KENICHI
Published: August 2010

An efficient method for solid phase peptide synthesis was developed, which consists of Nα‐selective deprotection by dilute methanesulfonic acid, in situ neutralization and rapid coupling reaction using benzotriazol‐1‐yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP) or 2‐(benzotriazol‐1‐yl)oxy‐1,3‐dimethylimidazolidinium hexafluorophosphate (BOI) reagent. Selective removal of the Nα‐Boc group by dilute methanesulfonic acid was of more advantage than removal by TFA in terms of stability of semipermanent protecting groups and suppression of undesired side reactions. The use of in situ neutralization and rapid coupling method reduced intramolecular aminolytic cyclization by shortening exposure of the deprotected nucleophilic amino group. A successful synthesis of porcine brain natriuretic peptide (pBNP) has been achieved using this efficient solid phase peptide synthesis scheme.
Phase transfer catalysis in solid phase peptide synthesis
Authors: SPATOLA ARNO F. , ANWER MOHMED K. , RAO MEENA N.
Published: August 2010

Relatively small cyclic peptides that contain functionalized side chains provide interesting model compounds for studying side chain‐side chain interactions, peptide backbone flexibility (especially if X‐Pro bonds are included), and as potential enzyme mimetics. In order to develop more efficient synthetic routes to compounds such as cyclo(Xxx‐Pro‐Gly‐Yyy‐Pro‐Gly), using the Merrifield method, we have investigated several orthogonal solid phase synthesis strategies and contrasted the use of two solid phase peptide‐resin cleavage techniques for preparing partially protected linear sequences. Phase transfer catalysis using tetrabutyl ammonium hydrogen sulfate in THF with saturated aqueous K2CO3 provides peptide acid salts in which most of the common protecting groups (Arg(NO2), Tyr(Bzl), Z‐Lys, Lys(Boc), and Glu(tBu)) are not affected. Using 500 MHz proton NMR, peptides having a cyclo (l‐l‐Gly‐l‐l‐Gly) sequence generally display two conformers in DMSO‐d6 with the major isomer being the bis‐cis conformer, while the minor form contains two δ turns. For peptides with a cyclo(d‐l‐Gly‐l‐l‐Gly) sequence, the major conformer contains one cis and one trans X‐Pro bond and one Type II δ turn, as previously predicted for related structure by Kopple and others.
Conformational regions of Boc‐Ala‐Aib‐Ala‐OMe
Authors: BRUNNE ROGER M. , LEIBFRITZ DIETER
Published: August 2010

The method of time averaging of distance restraints in molecular dynamics simulations is applied to Boc‐Ala‐Aib‐Ala‐OMe in order to demonstrate the improved sampling properties of this method compared to conventional distance restraining. Two conformational regions, β‐turn type II and γ‐turn, are seen during MD runs at a simulation temperature of 500 K, while in simulations with conventional distance restraining, no conformational transitions could be observed for temperatures up to 1000 K.
A bifunctional heterodimeric insect neuropeptide analog
Authors: NACHMAN RONALD J. , COAST GEOFF M. , HOLMAN G. MARK , HADDON WILLIAM F.
Published: August 2010

The synthesis and biological activity of a bifunctional, heterodimeric insect neuropeptide analog are described. The heterodimer is composed of the C‐terminal pentapeptide active core regions of the leucokinin/achetakinin and pyrokinin neuropeptide families linked via their N‐terminal amino groups with a succinyl diacid moiety. Members of the leucokinin/achetakinin family can induce fluid secretion in malpighian tubules of the house cricket, Acheta domesticus, whereas the pyrokinins demonstrate activity in a cricket oviduct myotropic bioassay. No cross‐activity is observed for the two neuropeptide families in these bioassays. However, the heterodimer elicits responses in both Acheta bioassays. Such a bifunctional analog may in future serve as a template for the design of stable, bifunctional pest insect control agents of greater efficiency.
Synthesis of α‐, β‐ and cyclic spaglumic acids
Authors: REDDY A. VEERA , RAVINDRANATH B.
Published: August 2010

A short, one‐pot synthesis of α‐ and β‐spaglumic acids (N‐acetyl‐L‐aspartyl‐L‐glutamic acids, NAAGA) has been developed based on ultrasound‐promoted acetylation of aspartic acid, followed by dehydration, condensation with glutamic acid dibenzyl ester and hydrogenolysis. The α‐ and β‐peptides were separated by anion‐exchange chromatography. The α‐peptide shows a remarkable tendency to cyclize during methylation with diazomethane and yields cyclic N‐acetylaspartylglutamic acid dimethyl ester, which could be hydrolysed to the hitherto unreported diketopiperazine dicarboxylic acid, cyclic spaglumic acid (cyclic NAAGA).
Continuous flow solid (gel) phase peptide synthesis using unsupported ultra‐high load polymers *
Authors: COFFEY A.F. , JOHNSON T.
Published: August 2010

A simple, manually operated, continuous flow apparatus is described for solid (gel) phase peptide synthesis. The approach uses an unsupported phenolic bead form core network at an initial matrix loading of 5 mmol g‐1, the theoretical maximum. The synthesis is performed in a flow reactor under low pressure conditions. “Layered displacement” of reagent solutions and washing solvents is an essential feature that has been developed to facilitate efficient peptide synthesis. The usefulness of the present system in conjunction with Nx Boe protected amino acids is illustrated by the syntheses of [Leu5]‐enkephalin and dermorphin. The potential for scale up synthesis has also been investigated.
Ab initio studies of aromatic‐aromatic and aromatic‐polar interactions in the binding of substrate and inhibitor to dihydrofolate reductase
Authors: SAPSE ANNE‐MARIE , SCHWEITZER BARRY S. , DICKER ADAM P. , BERTINO JOSEPH R. , FRECER VLADIMIR
Published: August 2010

Aromatic‐aromatic and aromatic‐polar interactions are investigated by performing ab initio Hartree‐Fock calculations. Binding energies and optimum distances between subsystems are obtained. It is found that the binding energy between two benzene rings is of 3.1 kcal/mol when correlation effects are included, while the serine aromatic complexes energies of binding range from 1.9 to 3.1 kcal/mol.
NMR and molecular modeling characterization of RGD containing peptides
Authors: BOGUSKY M.J. , NAYLOR A.M. , PITZENBERGER S.M. , NUTT R.F. , BRADY S.F. , COLTON C.D. , SISKO J.T. , ANDERSON P.S. , VEBER D.F.
Published: August 2010

The tripeptide sequence arginine‐glycine‐aspartic acid (RGD) has been shown to be the key recognition segment in numerous cell adhesion proteins. The solution conformation and dynamics in DMSO‐d6 of the cyclic pentapeptides, Ac‐Cys‐Arg‐Gly‐Asp‐Cys‐OH (CRGDC), a potent fibrinogen receptor antagonist, and Ac‐Cys‐Arg‐Gly‐D‐Asp‐Cys‐OH (CRGdC), a weak fibrinogen receptor antagonist, have been characterized by nuclear magnetic resonance (NMR) spectroscopy and molecular modeling. 1H‐1H distance constraints derived from two‐dimensional NOE spectroscopy and torsional angle constraints obtained from 3JNH‐hα coupling constants, combined with computer‐assisted modeling using conformational searching algorithms and energy minimization have allowed several low energy conformations of the peptides to be determined. Low temperature studies in combination with molecular dynamics simulations suggest that each peptide does not exist in a single, well‐defined conformation, but as an equilibrating mixture of conformers in fast exchange on the NMR timescale. The experimental results can be fit by considering pairs of low energy conformers. Despite this inherent flexibility, distinct conformational preferences were found which may be related to the biological activity of the peptides.
Suppression of tuftsin activity by the partial sequences of adenovirus type 2 proteins
Authors: SIEMION IGNACY Z. , SION JACEK J. , WIECZOREK ZBIGNIEW
Published: August 2010

The synthesis of tuftsin inhibitor‐like partial sequences of adenovirus type 2 proteins: TVPPR (l), TRPPR (2), TGPPT (3), PRPPT (4), FVPPR (5), ARPPA (6) and YGPPK (7) are described. The choice of peptides for synthesis was performed by using a simple computer‐aided method for predicting antigenic determinants of proteins. For all these peptides an inhibitory activity against tuftsin was found.
Reactions of C‐terminal ω‐amino acid residues in liquid hydrogen fluoride
Authors: BEDNAREK MARIA A. , SPRINGER JAMES P. , CUNNINGHAM BARRY R. , BERNICK AMY M. , BODANSZKY MIKLOS
Published: August 2010

The benzyl‐ester bond linking the C‐terminal δ‐aminovaleric acid residue of a peptide to a polymeric support was cleaved with liquid hydrogen fluoride in the presence of anisole, added as scavenger. Instead of the expected peptide with a free carboxyl group at the C‐terminus, a peptide terminating in a ketone derivative was obtained. The unusual extent of this known side‐reaction was attributed to the effect of the distance between the amino group and the carboxyl group in the C‐terminal residue. The results of model experiments corroborated this view.
N→S phosphoryl migration in phosphoryl glutathion
Authors: HOU‐JUN YANG , JIAN LIU , YU‐FEN ZHAO
Published: August 2010

It was found that in the case of N‐(diisopropylphosphoryl) glutathion (reduced form), 2, N→ S phosphoryl migration took place, but not for N,N‐bis(diisopropylphosphoryl) glutathion (oxidized form) or N‐diisopropylphosphoryl cysteine. These results were deduced by 31P‐NMR tracing experiments. It was shown that phosphoryl migration was catalyzed by an intramolecular carboxyl group, and a mechanism involving a mixed carboxyl‐phosphoric anhydride was proposed. A competitive reaction between the amino and thiol group toward diisopropyl phosphite indicated that the phospho‐thiol derived from N‐(diisopropylphosphoryl) glutathion (reduced form), 2, did not result from direct phosphorylation of the thiol group. N, S‐Bis(diisopropylphosphoryl) glutathion provides an authentic sample to confirm the migrated phosphoryl thiol product.
Infrared analysis of peptide succinimide derivatives
Authors: PISTORIUS ARTHUR M.A. , GROENEN PATRICIA J.T.A. , GRIP WILLEM J. DE
Published: August 2010

In order to establish parameters to identify imide derivatives formed during in vitro aging of aspartylalanyl‐containing proteins, a dipeptide and a tetrapeptide containing this sequence were acidified and heated in vacuo. The formation of succinimide derivatives could be confirmed by FTIR and Raman spectroscopy. Hereto, earlier assignments of succinimide vibrations had to be revised.

FTIR absorbance spectra of the succinimide, derived from H‐Asp‐Ala‐OH, in the solid state give five bands between 1700 and 1800 cm−1. In this case, owing to Fermi resonance, the antisymmetric imide carbonyl stretching vibration gives rise to an apparent doublet, centered around 1715 cm−1. The symmetric stretching mode is found at 1793 cm−1. The other bands are assigned to carboxylic acid stretching modes (1728 cm−1: COOH dimer and 1751 cm−1: COOH monomer).

Fermi resonance does not occur in succinimide derivatives occurring in larger peptides. As a consequence, the imide bands of the succinimide, generated from H‐Val‐Asp‐Ala‐Gly‐OH, are observed at 1716 and 1791 cm−1 (antisymmetric and symmetric stretching modes, respectively).
Implications of replacing peptide bonds in the COOH‐terminal B chain domain of insulin by the Ψ(CH2‐NH) linker
Authors: NAKAGAWA SATOE H. , JOHANSEN NILS L. , MADSEN KJELD , SCHWARTZ THUE W. , TAGER HOWARD S.
Published: August 2010

To evaluate more thoroughly the importance of main‐chain structure and flexibility in ligand interactions with the insulin receptor, we undertook to synthesize analogues with reduced peptide bonds in the COOH‐terminal B chain domain of the hormone (a stable, but adjustable β‐strand region). By use of solid‐phase, solution‐phase and semisynthetic methods, analogues were prepared in which ArgB22 of des‐octapeptide(B23–B30)‐insulin was extended by the sequences Gly‐Phe‐(CH2‐NH)‐Phe‐NH2, Gly‐Gly‐(CH2‐NH)‐Phe‐Phe‐NH2, Gly‐Phe‐(CH2‐NH)‐Phe‐Phe‐Thr‐Pro‐Ala‐Thr‐OH, and Gly‐Phe‐Phe‐(CH2‐NH)‐Phe‐Thr‐Pro‐Ala‐Thr‐OH, and were studied with respect to their abilities both to interact with the hepatocyte insulin receptor and to form soluble anion‐stabilized hexamers in the presence of Co2+ and phenol. Additional analogues of des‐pentapeptide(B26–B30)‐insulin were also examined. Overall, our results show that, whereas all analogues retain considerable ability to form organized metal ion‐coordinated complexes in solution, the reduction of peptide bonds both proximal and distal to the critical side chain of PheB25 results in analogues with severely diminished receptor binding potency. We conclude that the peptide carbonyls from both Phe24 and PheB25 are important for insulin–receptor interactions and that the structural organization of the region when insulin is bound to its receptor differs from that occurring during simple monomer–monomer and higher‐order interactions of the hormone.
Methionine–enkephalin related glycoconjugates
Authors: HORVAT Š. , HORVAT J. , VARGA‐DEFTERDAROVIĆ L. , PAVELIĆ K. , CHUNG N.N. , SCHILLER P.W.
Published: August 2010

A series of glycoconjugates, in which [Met5]enkephalin or [d‐Ala2, Met5]enkephalin have been linked through an ester bond to the HO‐6 of various d‐glycopyranose moieties, were synthesized by classical solution methods. The biological activities of these compounds were determined on selective pharmacological preparations: guinea pig ileum and mouse vas deferens for opioid activity, and two mouse cell lines, fibroblasts L929 and melanoma B16BL6, to study the influence on growth processes. The results reported in this study demonstrate the differential effect of the carbohydrate part in enkephalin‐related glycoconjugates on receptor recognition. In addition, synthesized neo‐glycopeptides stimulate growth of the examined mouse cell lines, whereas parent peptide demonstrated some growth inhibitory properties. Full growth curves showed a dose‐dependent effect at concentrations of 10−7 to 10−10 M.
Application of anti‐peptide antibodies to the assignment of the inter‐chain disulphide bond in tetanus toxin
Authors: FISCHER PETER M. , HOWDEN MERLIN E.H.
Published: August 2010

Cysteine‐containing peptides corresponding to the putative hinge region connecting the heavy and light polypeptide chains of tetanus toxin were synthesised utilising a solid phase with an acid hyper‐labile linkage agent. Both the single‐chain cysteine peptides, as well as a disulphide‐bonded double‐chain peptide, obtained by selective iodine‐oxidation of S‐trityl and S‐acetamidomethyl protected peptides, were conjugated to carrier proteins for the purpose of immunisation and immunoassay. Comparison of the immunochemical specificity of mouse antibodies raised against these constructs, as well as antibodies against tetanus toxoid, permitted the assignment of the location of the inter‐chain disulphide bond of tetanus toxin.
Protein and gene structure analysis of an albumin genetic variant: proalbumin Wu Yang (–2 Arg → His)
Authors: ZAN WEI‐CHANG , XU WEN‐FENG , CHI CHENG‐WU
Published: August 2010

An electrophoretically slow genetic variant of human serum albumin was isolated from a patient with bisalbuminemia in Xin Jiang who originated in the province of He Nan, county Wu Yang. Based on the FPLC‐CNBr peptide profiles of the variant and normal albumins, the mutated CNBr peptide of the variant was found. Both the mutated and corresponding normal CNBr peptides were further subjected to trypsin digestion. An additional peptide peak from the mutated CNBr peptide was found in the HPLC mapping. The amino acid sequence of this peptide was determined to be Gly‐Val‐Phe‐His‐Arg corresponding to the fragment of albumin propeptide from position –5 to –1 with an –2 Arg → His mutation. The N‐terminal sequence of the mutated CNBr peptide was also determined to be Arg‐Gly‐Val‐Phe‐His*‐Arg‐Asp‐Ala‐His‐Lys‐, namely the N‐terminal part of the mutated proalbumin. According to the known genomic structure of proalbumin, the gene fragments neighbouring the propeptide DNA sequences of the normal and variant albumins were amplified in vitro by polymerase chain reaction. The PCR products with 390 bp were then cloned into M13 vector, respectively. The nucleotide sequence analyses demonstrated that the codon CGT for –2 Arg was mutated to CAT for His. Thus, the point substitution was confirmed both at protein and gene levels. The above genetic variant of albumin was named proalbumin Wu Yang (–2 Arg → His) being the first instance of the Lille type found in China.
Racemization during aminolysis of activated esters of N‐alkoxycarbonylamino acids by amino acid anions in partially aqueous solvents and a tactic to minimize it
Authors: BENOITON N. LEO , LEE YOUNG C. , CHEN FRANCIS M.F.
Published: August 2010

Racemization during the aminolysis of activated esters of N‐alkoxycarbonylamino acids by amino acid anions in aqueous dimethylformamide was examined by determining the epimeric products by high‐performance liquid chromatography. Partial racemization occurred for a variety of esters, particularly when sodium hydrogen carbonate was used to generate the anion of d‐valine. The racemization results from prolonged contact of unconsumed ester with the alkaline medium. Variation of the stoichiometry of reagents for reactions with N‐benzyloxycarbonylphenylalanine (Z‐Phe) 4‐nitrophenyl ester revealed that racemization could be minimized by using Na2CO3 as base and a 50% excess of amino acid anion. An efficient synthesis of optically pure Z‐l‐Phe‐D‐Val‐OH was achieved with a reaction time of 15 min.
Identification and suppression of decomposition during carbodiimide‐mediated reactions of Boc‐amino acids with phenols, hydroxylamines and amino acid ester hydrochlorides
Authors: BENOITON N. LEO , LEE YOUNG C. , CHEN FRANCIS M.F.
Published: August 2010

N‐tert‐Butoxycarbonylamino acids (Boc‐Xaa‐OH) were coupled with p‐nitrophenol (HONp) in dichloromethane using N,N′‐dicyclohexylcarbodiimide (DCC) and N‐ethyl‐N′(3‐dimethylaminopropyl)carbodiimide hydrochloride (EDC), and the products were identified and quantitated by high‐performance liquid chromatography. Boc‐Xaa‐OH with Xaa = Val was coupled also with pentafluorophenol (HOPf) and hydroxy‐containing additives (HOR), and the products were similarly determined as the methylamides. EDC‐mediated reactions of Boc‐Xaa‐OH gave 8–25% of Boc‐Xaa‐Xaa‐OR as well as Boc‐Xaa‐OR for R = Ph, Np, Pf, benzotriazole (Bt) and 5‐norbornene‐endo‐2,3‐dicarboxamide; DCC‐mediated couplings, 5–7% for R = Np and Bt. No dimer was formed in couplings with N‐hydroxysuccinimide or 3‐hydroxy‐4‐oxo‐3,4‐dihydro‐1,2,3‐benzotriazine. Dimerization was eliminated from DCC‐mediated reactions by the addition of 1 equiv. of N‐methylmorpholine, from the EDC‐mediated reactions by carrying them out in pyridine. Dimerization is attributed to formation of the intermediate 2‐alkoxy‐5(4H)‐oxazolone that undergoes fragmentation to the N‐carboxyanhydride, which reacts with the alcohol giving amino acid ester. Ester produces dimer by aminolysis of the O‐acylisourea. Decomposition (1.4%) was also detected by analysis for H‐Val‐Phe‐OMe in DCC‐mediated reactions of Boc‐valine with H‐Phe‐OMe, and was demonstrated to be caused by the hydrochloride of the ester salt that had been neutralized with N‐methylmorpholine. Decomposition was eliminated by the addition of 5%, of pyridine, which also had the beneficial effect of suppressing N‐acylurea formation.
Conformation‐function relationships in LHRH analogs
Authors: NIKIFOROVICH GREGORY V. , MARSHALL GARLAND R.
Published: August 2010

A systematic conformational build‐up procedure was performed for the LHRH molecule, pGlu1‐His2‐Trp3‐Ser4‐Tyr5‐Gly6‐Leu7‐Arg8‐Pro9‐Gly10‐NH2. The results showed a very high flexibility of the LHRH backbone, with 300 conformers being regarded as having low energy. At the same time, the conformational flexibility of LHRH differs among the fragments of the molecule. The most rigid fragments of LHRH are the Ser4‐Tyr5‐Gly6‐Leu7 and Tyr5‐Gly6‐Leu7‐Arg8 central tetrapeptides, the latter possessing only eight different types of low‐energy backbone conformers. These eight conformer types belong to different kinds of chain reversals which are stabilized by different systems of intramolecular hydrogen bonds. Some of them resemble the β‐II′ turn, which was derived as the LHRH structure from energy calculations by others. The results obtained are in good agreement with the experimental data on LHRH flexibility in solution.
Experimental studies and potential energy calculations of the blocked tetrapeptide Ac‐Lys‐Pro‐Gly‐Ile‐NMA from the third loop of short‐chain snake venom neurotoxins
Authors: ROOS H. MARITA , VAN ROOYEN PETRUS H. , WESSELS PHILIPPUS L.
Published: August 2010

The conformational space of the tetrapeptide Ac‐Lys‐Pro‐Gly‐Ile‐NMA from the β‐bend present in the third loop of short‐chain snake venom neurotoxins was investigated with the aid of energy calculations, resulting in the identification of an ensemble of β‐turn conformations. These results were compared with the experimentally determined conformations, as observed using NMR and CD spectroscopy. A random coil conformation of the peptide is indicated in polar hydrogen‐bonding solvents. In less polar solvents the peptide backbone assumed a more rigid conformation, as reflected by the existence of at least a type II β‐turn conformation.
Pepsin‐catalyzed peptide synthesis in organic media: studies with free and immobilized enzyme
Authors: BEMQUERER MARCELO PORTO , ADLERCREUTZ PATRICK , TOMINAGA MINEKO
Published: August 2010

Pepsin‐catalyzed synthesis of protected peptides was studied in two‐phase systems containing up to 50% (by volume) of aqueous phase. A methodological study was carried out to determine the optimum conditions for the synthesis of the model protected peptide Z‐Phe‐Phe‐OMe. Several parameters such as concentrations of carboxylic and amino components, pH of the aqueous phase, ratio of organic to aqueous phase volumes and nature of the organic solvent were investigated. It was observed that the most hydrophobic solvents produced the best yields, despite the low solubility of substrates in these media. The log P of the solvent could be used to predict the solvent effect over the reaction yields. Pepsin immobilized by adsorption onto the solid supports Celite and Chromosorb was employed to perform a study of secondary specificity of the enzyme in organic media through the coupling between Z‐X‐Phe‐OH (X = Ala, Asp, Glu, Gly, Phe, Ile, Val, Trp and Tyr) and Phe‐OMe. This investigation was performed in two solvent systems: (A) ethyl acetate:citrate buffer pH 4.5 (98:02, v:v) and (B) acetonitrile:citrate buffer pH 4.5 (96:04, v:v). Reaction rate data showed that pepsin had a preference for more hydrophilic substituents in the P2 position. These data are in contrast to the literature for a similar reaction performed in predominantly aqueous media. Thus, for mainly organic media, partition phenomena are very important and may cause an apparent modification of enzyme specificity.
New, highly active antagonists of LHRH with acylated lysine and p‐aminophenylalanine in positions 5 and 6
Authors: JANECKA ANNA , JANECKI TOMASZ , BOWERS CYRIL Y. , FOLKERS KARL
Published: August 2010

A series of antagonists of the luteinizing hormone releasing hormone (LHRH) with substitutions in position 5 and/or 6 that included acylated lysine or p‐aminophenylalanine were synthesized, characterized and tested for antiovulatory activity (AOA) in rats, and histamine releasing activity. Some of these antagonists were considerably more soluble at neutral pH than antagonists like Antide.

Of 37 new antagonists, the best physico‐chemical and biological properties were found for the following two analogs: NAcDNal‐DCpa‐DPal‐Ser‐PicLys‐D(PicSar)Lys‐Leu‐ILys‐Pro‐DAlaNH2 (named Sartide) and NAcDNal‐DCpa‐DPal‐Ser‐Tyr‐D(PicSar)Lys‐Leu‐ILys‐Pro‐DAlaNH2. They are both soluble in water, inhibit ovulation completely at 0.5 μg per rat, and have ED50 values for histamine release of about 30 μg/mL.
X‐Ray studies on crystalline complexes involving a mino acids and peptides
Authors: SURESH STEPHEN , PRASAD G. SRIDHAR , VIJAYAN M.
Published: August 2010

L‐Histidine acetate crystallizes in two forms: (I) orthorhombic; P212121; a= 5.027, b= 11.126, c= 17.473 Å; Z = 4; (II) monoclinic; C2; a= 15.649, b= 9.276, c= 8.566 Å; β= 94.65°; Z = 4. The structures were solved by direct methods and refined to R‐values of 0.056 and 0.089 for 1131 and 1330 observed reflections, respectively. The conformations of the histidine molecule in the two forms are different. However, both are such that they facilitate the occurrence of a specific interaction of the histidine molecule with a carboxylate group. The basic elements of aggregation are hydrogen‐bonded histidine ribbons, but they are of different types in the two structures. The ribbons are interconnected by acetate ions to form the crystals. The structures contain two characteristic interaction patterns involving amino and carboxylate groups, one of which is observed for the first time. The two water molecules in form II and their symmetry equivalents form an uninterrupted hydrogen‐bonded chain running through the crystal. They also present an interesting case of disorder in hydrogen bonds. A comparative study involving amino acid complexes of acetic acid shows that the presence of acetate ion could lead to new aggregation patterns, specific interactions and characteristic interaction patterns with varying degrees of similarity with those observed in other structures containing amino acids.
Conformation of the oxalamide group in retro‐bispeptides
Authors: KARLE ISABELLA L. , RANGANATHAN DARSHAN , SHAH KAVITA , VAISH NARENDRA K.
Published: August 2010

We have determined the structures of a range of peptides having the oxalamide (‐NH‐CO‐CO‐NH‐) unit located at their center. The oxalamide group has the trans conformation in two retropeptides and an approximately orthogonal conformation in the peptide with Pro residues. Torsional angles about the CO‐CO bond are 180° in MeO‐Aib‐CO‐CO‐Aib‐OMe (1). 175 in MeO‐L‐Leu‐CO‐CO‐L‐Leu‐OMe (2), and ‐108 and ‐106° for the two independent molecules in the crystal of MeO‐L‐Pro‐CO‐CO‐L‐Pro‐OMe (3), owing to steric hindrance between CO and the pyrolidine ring. Crystal data are: (1) C12H20N2O6, triclinic, space group 1, a= 6.190(1), b ‐ 10.044(2), c= 11.989(2) A, α= 86.38(2), β= 83.13(2), γ= 80.16(2)°, R= 0.057 for 1646 observed reflections [F0., >3σ(F0)]; (2) C16H28N2O6, tetragonal, space group P41, a = b= 11.121(3), c= 15.775(6) Å, R= 0.058 for 1216 observed reflections [F0, > 3σ(F0)]; (3) C14H20N2O6, monoclinic space group P21, a= 9.556(2), b= 17.861(3), c= 9.618(2) Å, β= 104.35(1)°; R= 0.051 for 2100 observed reflections [F0 > 3σ(F0)].
N‐9‐Fluorenylmethoxycarbonylpyroglutamate
Authors: BENOITON N. LEO , CHHN FRANCIS M.F.
Published: August 2010

Fmoc‐glutamic acid is converted by thionyl chloride into the dichloride, which spontaneously cyclizes to Fmoc‐pyroglutamyl chloride. The latter is stable to water. Pure Fmoc‐pyroglutamyl chloride is obtained by washing the reaction mixture with water, which destroys uncyclized dichloride by converting it into the 2‐alkoxy‐5(4H)‐oxazolone that is readily hydrolyzed. Fmoc‐pyroglutamic acid and succinimidyl ester are obtained from the chloride by acid hydrolysis and reaction with N‐hydroxysuccinimide, respectively.
Interaction of mammalian sperm nuclear protamines and peptides derived thereof with immobilized zinc
Authors: BIANCHI FABIEN , ROUSSEAUX‐PREVOST ROSELYNE , HUBLAU PATRICK , ROUSSEAUX JEAN
Published: August 2010

The interaction of mammalian and human protamines with zinc was studied by immobilized metal ion affinity chromatography (IMAC). The affinity of protamines containing blocked cysteine residues was found to correlate in part with the presence and number of histidine residues in the protamine structure: absence or low affinity of P1 protamines containing 0 or 1 histidine residue; high affinity of human P2 protamine containing 9 histidines. Nevertheless a fraction strongly retained on an IDA‐Zn(II) column was observed for P1 protamines with one histidine in the N‐terminal sequence (ram and boar protamines). The strong binding was found to be related to the presence of tyrosine, serine and threonine closely spaced to the histidyl side chain. In the case of human protamine P2, the strong retention on the IDA‐Zn(II) column seems to result from the additive contribution of all the histidine residues of the molecule. Thus, strong retention of protamines in IMAC seems to depend on an additive contribution of amino‐acid side chains: histidine, tyrosine, serine, threonine and perhaps arginine. The high affinity of protamines, more especially P2 protamines, for zinc suggests that this metal ion could play a role for their correct folding and binding to DNA.
Synthesis and biological activity of locust AKH‐I and its analogues with modifications at the threonine residues
Authors: POULOS CONSTANTINE , KARAGIANNIS KOSTAS , LEE MICHAEL , GOLDSWORTHY GRAHAM
Published: August 2010

A convenient method of synthesis, using a combination of solid and liquid phase methodology, for locust Adipokinetic Hormone‐I (AKH‐I) and its analogues with modifications at the threonine residues are reported. TheN‐terminal nonapeptide acid of AKH‐I is synthesized in the solid phase using the 2‐chlorotrityl chloride resin and the Fmoc/t‐Bu strategy. Quantitative cleavage of the nonapeptide acid from the resin, with thetert‐butyl type side‐chain protection intact, is achieved with a mixture of acetic acid/trifluoroethanol/ dichloromethane. The nonapeptide acid is then coupled in solution to the threonine derivatives, H‐Thr‐NH2or H‐Thr(Bz1)‐NH2, with the DCC/HOBt method. The efficiency of this approach in the synthesis of AKH‐I is demonstrated by the high yields and purity of the synthesized peptides. All the synthesized peptides were tested in two ways: first, in a lipid mobilization assay in locustsin vivo; and second, in a novel assayin vitroconcerned with the uptake of radiolabelled acetate into locust tissue. Replacement of the hydroxyl hydrogen in Thr5of locust AKH‐I by the bulky and highly lipophilictert‐butyl group reduced the potency markedly, whereas efficacy is unaffected, but when the hydroxyl hydrogen of Thr10in AKH‐I is replaced by a benzyl group, the activity of the resulting analogue is identical to that of the natural peptide. Structure‐activity relationships are discussed. © Munksgaard 1994.
Crystal structures of a D‐residue containing tetrapeptides 1. tert‐Boc‐D‐Valyl‐alanyl‐leucyl‐alanyl methoxide, butanol solvate
Authors: BOBDE V. , DURANI S. , PATTABHI VASANTHA , SADASWAN C.
Published: August 2010

The crystal structure of a heterochiral peptide, viz. Boc‐D‐Val‐Ala‐Leu‐Ala‐OMe, with a D‐residue in the beginning of the sequence has been determined (a= 9.464(5), b= 35.615(5), c= 9.703(2) Å, space group P21212, Z= 4, R= 0.09). The peptide is in the extended β‐conformation and the packing is stabilised by four N—H. O hydrogen bonds in an antiparallel β‐sheet arrangement. The solvent molecule is disordered and does not have any specific interactions with the peptide.
Trypsin‐mediated semisynthesis of salmon calcitonin
Authors: DAVEY M.W. , ROMMELAERE H. , BOECK S. DE , GOETHALS M. , DAMME J. VAN , VANDEKERCKHOVE J.
Published: August 2010

Salmon calcitonin, CT(1‐32)·NH2, was synthesised by the trypsin‐mediated coupling of the peptide fragments CT(1‐24) and CT(25‐32)·NH2, prepared by conventional Fmoc solid‐phase chemistry. Optimal conditions regarding reaction time course, pH, proportion of catalyst, substrate concentration and composition of the reaction medium were determined from initial studies on the coupling of CT(1‐11) to CT(12‐24) and of CT(12‐24) to CT(25‐32)·NH2. For the final successful semisynthesis, we found that it was unnecessary to protect lysine residues not involved in the coupling, and that secondary hydrolysis at these sites could be prevented by increasing the pH of the reaction medium. The reaction achieved equilibrium after 30‐45 min, with overall conversion of around 30% of the initial amount of CT(1‐24) substrate into product. Yields were depressed due to cyclisation of the CT(1‐24) substrate via air‐oxidation of the Cys1 and Cys7 residues.
Convergent solid‐phase peptide synthesis 12. * Chromatographic techniques for the purification of protected peptide segments
Authors: GAIRÍ MARGARIDA , LLOYD‐WILLIAMS PAUL , ALBERICIO FERNANDO , GIRALT ERNEST
Published: August 2010

The purification of a range of protected peptide segments has been carried out using modified reversed‐phase chromatographic techniques in which DMF was added to the water and acetonitrile mixtures used as eluents. The purity of the recovered peptides was excellent and recoveries were high in all cases, even for longer hydrophobic segments. In several cases purifications were carried out on the hundreds of milligrams scale. For protected peptide segments containing Met, protection as the sulfoxide avoids its unwanted alkylation and oxidation, and the increased overall polarity can be useful in the purification of protected peptides incorporating this residue. © Munksgaard 1995.
Amino‐acid conjugates of the hapten 2‐phenyl‐4‐ethoxymethylene‐5(4H)‐oxazolone Synthesis and confirmation of structure
Authors: BENOITON N. LEO , HUDECZ FERENC , CHEN FRANCIS M.F.
Published: August 2010

2‐Phenyl‐4‐ethoxymethylene‐5(4H)‐oxazolone (PhOx=CHOEt) was reacted with methylamine, and 2‐phenyloxazole‐4‐carboxylic acid was coupled with methylamine. The spectroscopic properties of the two products were compared in order to confirm that aminolysis of PhOx=CHOEt occurs by displacement of the ethoxy group to give 2‐phenyl‐4‐(substituted‐methylene)‐5(4H)‐oxazolones and not by attack at the oxazolone‐carbonyl followed by rearrangement to give 2‐phenyloxazole‐4‐carboxamides. Ten crystalline conjugates were prepared and characterized by reacting PhOx=CHOEt with an excess of unprotected di‐ and trifunctional amino acid anions followed by purification by washing them with hydrochloric acid.