bluuebunny/crossref_metadata_2025 huggingface dataset:
Cyclic hexapeptides related to somatostatin Synthesis and biological testing
Authors: PATTARONI CHRISTIAN , LUCIETTO PIERLUIGI , GOODMAN MURRAY , YAMAMOTO GAIL , VALE WYLIE , MORODER LUIS , GAZERRO LUCIA , GÖHRING WALTER , SCHMIED BERNHARD , WÜNSCH ERICH
Published: August 2010

As a continuation of our program to study the structure‐function relationship of the peptide hormone somatostatin, we report the synthesis and biological potencies of a series of cyclic hexapeptide analogs related to somatostatin. The parent peptide of this series was designed by Veber and coworkers, c[‐Pro6‐Phe7‐d‐Trp8‐Lys9‐Thr10‐Phe11‐], and has been reported to be superactive in the inhibition of the release of growth hormone. (The superscript numbers refer to the positions of residues in native somatostatin.) The series of analogs has been designed to examine the role of the so‐called bridging region, Phe11‐Pro6, which has been postulated to be important in maintaining the proper conformation of the biologically active tetrapeptide, Phe‐d‐Trp‐Lys‐Thr. We have incorporated peptidomimetics and the retro‐inverso modification into the bridging region of the molecule, with the aim of affecting the conformational preferences found in the parent peptide. Results from the biological assay—in vitro inhibition of growth hormone—and the conformational analysis (adjoining paper) of our analogs will provide insight into the relationship between structure and biological activity of somatostatin.
Cyclic hexapeptides related to somatostatin Conformational analysis employing 1H‐NMR and molecular dynamics
Authors: MIERKE DALE F. , PATTARONI CHRISTIAN , DELAET NANCY , TOY ANNA , GOODMAN MURRAY , TANCREDI TEODORICO , MOTTA ANDREA , TEMUSSI PIERO A. , MORODER LUIS , BOVERMANN GUNTER , WÜNSCH ERICH
Published: August 2010

We report the conformational analysis of a series of cyclic hexapeptides related to the hormone somatostatin utilizing 1H NMR spectroscopy and NOE restrained molecular dynamics. The conformational preferences and results from biological analysis of these analogs (previous paper) allow for refinement of the current understanding of the structure‐activity relationship of somatostatin. For most of the molecules examined, a βII′ turn about the d‐tryptophan‐lysine residues, postulated to be required for biological activity, was present. From the NOE restrained molecular dynamics, it can be seen that the turn structure is important for the maintenance of the proper orientation of the side chains of the adjacent phenylalanine, tryptophan and lysine. The biologically active analogs have the side chains of lysine and d‐tryptophan extended away from the 18‐membered ring in close proximity to each other for a significant portion of the dynamic simulations. Although other conformations are accessible and monitored during the simulations, we believe this is important for biological recognition. The absence of the βII′ turn at the d‐tryptophan‐lysine disrupts this side chain array producing inactive molecules. The role of the bridging region, the Phe‐Pro dipeptide, is to stabilize the βII′ turn and help maintain the proper orientation of the biologically important side chains.
Further studies on proctolin analogues modified in position 2 of the peptide chain and their influence on heart‐beat frequency of insects
Authors: BARTOSZ‐BECHOWSKI HUBERT , ROSIńSKI GRZEGORZ , KONOPIńSKA DANUTA , SUJAK PREZEMYSŁAW , SOBÓTKA WIESLAW
Published: August 2010

Seven proctolin analogues (I‐VII) modified in position 2 of the peptide chain by Phe (p‐guanidino) (I), Phe (p‐OEt) (II), Tyr (3′‐NH2) (III), Tyr (3′‐NO2) (IV), Afb (p‐OH) (V) (Afb = 3‐amino‐4‐phenyl‐l‐butyric acid), Afb (p‐NH2) (VI), Afb (p‐NO2) (VII), and the tetrapeptide Tyr (3′‐NH2)‐Leu‐Pro‐Thr (VIII) were synthesized by the classic liquid‐phase method. The biological effects of the peptides were investigated in cardioexcitatory tests on two insect species, the cockroach Periplaneta americana L., and the yellow mealworm, Tenebrio molitor L. Within physiological concentrations (10−9 ‐ 10−7 M) peptides II, III, and IV stimulated the heart action of P. americana like proctolin itself. Under identical conditions, in the case of T. molitor, only peptide III showed cardiostimulatory properties, whereas other compounds (including II and IV) were inactive at concentrations up to 10−7m. Results reported here reflect, with reference to the analogues I‐VII, selective recognition of receptors on myocardium of both insect species. The tetrapeptide VIII revealed a weak deacceleratory effect on P. americana and T. molitor heart action.
Effects of hydrophobic substitutions at position 18 on the potency of parathyroid hormone antagonists
Authors: CHOREV MICHAEL , ROUBINI ELIAHU , GOLDMAN MARK E. , McKEE ROBERTA L. , GIBBONS SUSAN W. , REAGAN JANE E. , CAULFIELD MICHAEL P. , ROSENBLATT MICHAEL
Published: August 2010

Position 18 in a parathyroid hormone (PTH) antagonist, [Nle8,18,Tyr34]bPTH(7‐34)NH2 (ii), was shown to tolerate substitutions by a range of amino acids with retention of inhibitory activity. The effects of hydrophobic substitutions at this position as a means of enhancing binding interactions with the receptor were evaluated. Substitution of Nle at position 18 with either d‐Ala, d‐Trp, or l‐Trp in analog ii or with Trp (d or l) in the recently reported, highly potent antagonist, [Nle8,18,d‐Trp12,Tyr34]bPTH(7‐34)NH2 (in vitro activities; Kb= 15 nM and Ki= 125 nM), was performed. In terms of activity on renal receptors, one antagonist, [Nle8,d‐Trp12,18,Tyr34]bPTH(7‐34)NH2, is the most active in vitro PTH antagonist yet reported (Kb= 4nm; Ki= 30 nM). The rationale for design of this antagonist and the conclusions regarding PTH‐receptor interactions are discussed.
Differential effects of secretin‐fragments imply a dual mechanism of action for secretin
Authors: KOFOD HANS , THAMS PETER , HOLST JENS J.
Published: August 2010

The effects of synthetic peptides, representing different parts of the secretin molecule in isolated mouse pancreatic islets have been investigated in perifusion studies. In the presence of 10 mM D‐glucose the C‐terminal nonapeptide Leu‐Gln‐Arg‐Leu‐Leu‐Gln‐Gly‐Leu‐Val‐NH2 (S19–27) showed a 2‐fold higher activity than that earlier shown for S22–27 and had the same effect on the dynamic pattern of insulin release as secretin, while the elongating sequence Leu‐Gln‐Arg (S19–21) had no effect on the insulin release. The nonapeptide Leu‐Ser‐Arg‐Leu‐Arg‐Asp‐Ser‐Ala‐Arg (S10–18) had no influence on the insulin release. Glu‐cagon release seen after intact secretin could not be shown for any of the smaller fragments. Accumulation of CAMP in the islets as seen with secretin, could at 10mmol/L D‐glucose only be demonstrated with S22–27 or S19–27 but not with S10–18 or S1–6. Our results indicate that full size secretin has to be present to stimulate glucagon release while insulin‐releasing activity can be confined to the C‐terminal part of the hormone.
A new synthetic functionalized antigen carrier
Authors: DRIJFHOUT J. W. , BLOEMHOFF W.
Published: August 2010

A new synthetic functionalized antigen carrier is described. It consists of a core of seven branched lysine residues, of which each of the four N‐terminal lysine residues contains two N‐(S‐acetylmercaptoacetyl)‐glutamyl residues. After removal of the protecting S‐acetyl groups affording eight thiol functions, the carrier can easily be conjugated to a properly functionalized antigen, e.g. an S‐(Npys)‐cysteinyl peptide, thus affording a high molecular weight conjugate with an unusually high antigen content.
Synthesis, crystal structure and molecular conformation of the tBuCO‐D,L‐Ala‐Δz‐Phe‐NhiPr α,β‐unsaturated dipeptide
Authors: AUBRY ANDRE , PIETRZYNSKI GRZEGORZ , RZESZOTARSKA BARBARA , BOUSSARD GUY , MARRAUD MICHEL
Published: August 2010

The crystal structure of the tBuCO‐d,l‐Ala‐Δz‐Phe‐NHiPr dipeptide has been solved by X‐ray diffraction. The peptide crystallizes in monoclinic space group P2JC with a = 13.445 (3) Å, b = 35.088 (4) Å, c = 14.755(3) Å, β= 116.73(1)°, Z = 12 and dc= 1.151 g.cm−3. The three independent molecules per asymmetric unit accommodate a βII‐folded conformation, but only one of them contains the typical i + 3 → i interaction characterizing a β‐turn. In the other two molecules, the N…O distance exceeds 3.2 Å, a value generally considered the upper limit for hydrogen bonds in peptides. In solution, the βII‐turn conformation is largely predominant.
Convergent solid‐phase peptide synthesis
Authors: LLOYD‐WILLIAMS PAUL , ALBERICIO FERNANDO , GIRALT ERNEST
Published: August 2010

The solid‐phase synthesis, photolytic detachment from the solid support and purification in solution, of a fully‐protected octapeptide containing a methionine residue (protected as the sulphoxide) is described. Protection of methionine in this manner avoids problems associated with the oxidation of this residue during the photolysis. The peptide has been purified by medium pressure liquid chromatography using solvent mixtures containing a high proportion of dimethylformamide in order to avoid precipitation of the peptide on the column.
Studies on in vitro proteolytic sensitivity of peptides inhibiting herpes simplex virus ribonucleotide reductases lead to discovery of a stable and potent inhibitor
Authors: PARADIS HÉLÈNE , LANGELIER YVES , MICHAUD JACQUES , BRAZEAU PAUL , GAUDREAU PIERRETTE
Published: August 2010

The nonapeptide, HSV R2‐(329‐337). corresponding to the subunit 2 (R2) carboxyl terminus of herpes simplex virus (HSV) ribonucleotide reductases, specifically inhibits this enzyme activity. We report here that under standard reductase assay conditions, this peptide was rapidly degraded by proteases present in the partially purified enzyme extract. The main process of proteolysis involves the successive removal of Tyr329 and Ala330, which corresponds to an aminopeptidase activity. Determination of the proteolytic susceptibility of HSV R2‐(329‐337) analogs showed that natural modifications which are present in the homologous varicella zoster virus (VZV) nonapeptide decreased its susceptibility to protease action 1.5‐fold. Nα‐acetylation, a modification known to protect peptides against aminopeptidase attacks, greatly improved the proteolytic resistance of HSV and VZV nonapeptides. Moreover, Ac‐VZV R2‐(298‐306) exhibited a 15‐fold higher potency on reductase inhibition than HSV R2‐(329‐337). The degradation process of HSV R2‐(329‐337) was partially inhibited by amastatin. bestatin, and leupeptin whereas it was completely abolished by bacitracin, suggesting a combined action of more than one aminopeptidase activity. Moreover, bacitracin protected most of these nonapeptide analogs from proteolysis, although it was less effective in preventing HSV R2‐(332‐337) degradation. Our results indicate that it is possible to determine, in the presence of bacitracin, the relative inhibitory potencies of HSV R2‐(329‐337) analogs with minimal error due to proteolytic susceptibility. Moreover, HSV R2‐(329‐337) modifications that were found to protect the peptide against degradation might be useful to increase its efficacy in vivo.
Conformational features responsible for the binding of cyclic analogues of enkephalin to opioid receptorsIII. Probable binding conformations of μ‐agonists with phenylalanine in position 3
Authors: SHENDEROVICH MARK D. , NIKIFOROVICH GREGORY V. , GOLBRAIKH ALEXANDER A.
Published: August 2010

Theoretical conformational analysis was carried out for several tetrapeptide analogues of β‐casomorphin and dermorphin containing a Phe residue in position 3. Sets of low‐energy backbone structures of the μ‐selective peptides [N‐Me‐Phe3,d‐Pro4]‐morphiceptin and Tyr‐d‐Orn‐Phe‐Asp‐NH2were obtained. These sets of structures were compared for geometrical similarity between themselves and with the low‐energy conformations found for the δ‐selective peptide Tyr‐d‐Cys‐Phe‐d‐Pen‐OH and nonactive peptide Tyr‐Orn‐Phe‐Asp‐NH2. Two pairs of geometrically similar conformations of μ‐selective peptides, sharing no similarity with the conformations of peptides showing low affinity to the μ‐receptor, were selected as two alternative models of probable μ‐receptor‐bound backbone conformations. Both models share geometrical similarity with the low‐energy structures of the linear μ‐selective peptide Tyr‐d‐Ala‐Phe‐Phe‐NH2. Putative binding conformations of Tyr1and Phe3side chains are also discussed.
Determination of the structure of [Nle7]‐endothelin by1H NMR
Authors: AUMELAS ANDRÉ , CHICHE LAURENT , MAHE EVE , LE‐NGUYEN DUNG , SIZUN PHILIPPE , BERTHAULT PATRICK , PERLY BRUNO
Published: August 2010

[Nle7]‐endothelin was synthesized and studied by1H NMR and distance geometry calculations. The NMR study was performed first in DMSO‐d6and then in 50% acetonitrile/water since this peptide aggregates in pure water. In both cases, all spin systems were identified and assigned with the aid of two‐dimensional spectroscopy (2D): COSY (for scalar couplings) and NOESY (for dipolar couplings). On the basis of the acetonitrile/water NMR parameters, and using the DISGEO program, a three‐dimensional structure of [Nle7]‐endothelin is proposed and discussed.
Modification of a zinc proteinase from Bacillus mesentericus strain 76 by diethylpyrocarbonate
Authors: STOEVA STANKA
Published: August 2010

Diethylpyrocarbonate (DEPC) inactivated the neutral zinc proteinase from Bacillus mesentericus strain 76/Bacillus subtilis (MCP 76) by ethoxycarbonylation completely. Exposure of the enzyme to DEPC together with the competitive inhibitor Z‐l‐phenylalanine prevented the loss of activity toward both peptide and protein substrates. Treatment with hydroxylamine restored the catalytic properties of the modified MCP 76 to that of the native enzyme. After chymotryptic digestion of ethoxycarbonylated MCP 76 in the presence and absence of Z‐l‐phenylalanine a single histidyl residue essential for the enzyme activity was isolated and identified as histidine 231.
Diastereoselective hydrolysis of peptide esters by alkaline protease Preparation of racemization‐free peptides
Authors: CHEN SHUI‐TEIN , WU SHIH‐HSIUNG , WANG KUNG‐TSUNG
Published: August 2010

A new and practiscal enzymatic procedure for preparative diastereoselective hydrolysis of peptide esters using the alkaline protease alcalase as a catalyst was developed. This procedure has been successfully applied to the resolution of peptide diastereomers and synthesis of racemization free peptides.
Conductance measurements in solid phase peptide synthesis
Authors: McFERRAN N. V. , WALKER B. , McGURK C.D. , SCOTT F. C.
Published: August 2010

Potentiated conductance shows promise for monitoring the progress of both coupling and deprotection reactions in solid phase peptide synthesis using Fmoc chemistry. Sterically hindered base may be added to the aprotic polar solvents used for synthesis, this induces ionisation of acids formed in the coupling and deprotection reactions, thus giving rise to a conductance signal in the solvent. Changes in this signal allow reactions occurring at the resin to be followed with no degradation of coupling efficiency.
Hydration of clupeine in solution
Authors: ISHIMURA MIYUKI , NISHI NORIO , UEDAIRA HISASHI
Published: August 2010

Spin‐lattice relaxation times, T1, of H217O at 25° were measured for aqueous solutions of clupeine and its constituent amino acids, which are serine, threonine, proline and arginine. The dynamic hydration numbers, nh, of clupeine and amino acids were determined from a concentration dependence of T1. The coordination numbers nh, and the rotational correlation times, τCh of water molecules around clupeine and amino acids were estimated and compared with that of pure water. The τch,τ° of clupeine was 1.85 and close to that of arginine. The experimental value of nDHN of clupeine was in good agreement with that calculated from the nDHN values of the constituent amino acids. This means that the clupeine molecule has a random conformation in solution.
Transesterification of peptide esters and peptidyl resins in methanol‐containing calcium acetate
Authors: MIRANDA MARIA TERÊSA MACHINI , THEOBALDO FRANCESCA CARDILLO , TOMINAGA MINEKO
Published: August 2010

The following crude peptide derivatives obtained by thermolysin catalysis in the presence of calcium acetate, were dissolved in methanol and in methanol‐containing calcium acetate to determine the possible occurrence of transesterification: Z‐Asn‐Leu‐Gly‐OEt, Boc‐Asn‐Leu‐Gly‐OEt, Moz‐Asn‐Leu‐Gly‐OBzl, Moz‐Asn‐Leu‐Gly‐OtBu, Moz‐Gln‐Leu‐Gly‐OEt, Moz‐Asn‐lle‐Gly‐OEt, and Moz‐Asn‐Leu‐Ala‐OEt. Only Z‐Asn‐Leu‐Gly‐OEt and Moz‐Gln‐Leu‐Gly‐OEt were transesterified in methanol, indicating the existence of a peptide derivative‐Ca2+ catalytic complex that may favor the reaction. In the presence of calcium acetate, all protected peptide esters except the t‐butyl esters were transesterified. The transesterification of several other di‐ and tripeptide derivatives of different structures in methanol‐containing calcium acetate was detected by HPLC and confirmed by the isolation and characterization of some of the protected peptide methyl esters obtained. Boc‐Leu‐Gly‐ and Moz‐Asn‐Leu‐Gly‐Merrifield resin were also transesterified in these solutions.
A novel reagent, dialkylphosphite, for peptide synthesis
Authors: ZHAO YU‐FEN , ZHANG DE‐QING , XUE CHU‐BIAO
Published: August 2010

The same dialkylphosphite reagent can be used for both N protection and C activation of amino acids. Two N‐diisopropyloxyphosphinyl (Dipp) tripeptide esters were prepared, and nine N‐Dipp‐dipeptide acids were synthesized through the activated amide intermediate. The positive ion FAB‐MS of N‐Dipp‐tripeptide showed novel cleavage patterns in that only the N‐phosphoryl fragment ions gave intense peaks while the C‐terminal series ions did not appear. This novel character might be useful for peptide sequence analysis. In addition, dialkyloxyphosphinyl group can be examined by 31P‐NMR for peptide conformational analysis and inspection for the degree of racemization during the coupling reaction.
Preparations, solution conformations and molecular structures of N,N′‐ethylene‐bridged dipeptides and their derivatives
Authors: KOJIMA YOSHITANE , IKEDA YOUKO , KUMATA ETSUKO , MARUO JOJI , OKAMOTO AKIHIRO , HIROTSU KEN , SHIBATA KOZO , OHSUKA AKIO
Published: August 2010

The solution conformations of novel dipeptides, methyl (2S, 3′S)‐3‐methyl‐2‐(2′‐oxo‐3′‐isopropyl‐1′‐piperazinyl)‐butanoate (EVV‐OCH3), methyl (2S, 3′S)‐3‐phenyl‐2‐(2′‐oxo‐3′‐benzyl‐1′‐piperazinyl) propionate (EFF‐OCH3), and their derivatives (Boc‐Gly‐EW‐OH, Boc‐Gly‐EVV‐Gly‐OH, and Boc‐Gly‐EFF‐OH), were studied by ‘H NMR measurements and molecular mechanics calculations (1). The molecular structures of Boc‐Gly‐EVV‐OH, Boc‐Gly‐EFF‐OH, and the hydrochloride of EVV‐OCH3 were determined by X‐ray analyses. The conformations of the piperazinone rings and the side chains of these oligopeptides were clarified.
Hydrolysis of β‐casein by gastric proteases
Authors: GUILLOU H. , MIRANDA G. , PELISSIER J‐P.
Published: August 2010

Hydrolysis of βA2‐casein by bovine chymosin and pepsin A was performed in order to compare the hydrolysis of the two enzymes on this protein. Different conditions have been tested: pH 5.5 for 116 h and pH 3.5 for 7h[E/S = 1/100 (w/w)] for chymosin. pH 3.0 for 24 h [E/S = 1/1000 (w/w)] for pepsin A. Under these conditions 17 peptides were obtained after the action of chymosin and 23 after the action of pepsin A. They corresponded respectively to the cleavage of 14 and 15 peptide bonds for chymosin and pepsin A. However, six of the peptide bonds were only hydrolyzed by chymosin and seven other bonds only by pepsin A. Our results showed a preferential splitting at the Leu‐X, Ser‐X, and Trp‐X bonds for chymosin and Leu‐X, Met‐X, and Thr‐X, for pepsin A. Some of the identified peptides contained sequences with possible physiological roles.
Infrared spectroscopic discrimination between α‐ and 310‐helices in globular proteins
Authors: PRESTRELSKI STEVEN J. , BYLER D. MICHAEL , THOMPSON MARVIN P.
Published: August 2010

We have undertaken a new and more detailed Fourier‐transform infrared (FTIR) spectroscopic study of α‐lactalbumin (in D2O solution) aimed at correlating its secondary structures to observed Amide I’ infrared bands. The spectra reported here were interpreted in light of the recently determined crystal structure of α‐lactalbumin and by comparison with the spectra and structure of the homologous protein lysozyme. Of particular importance is the new evidence supporting the assignment of the band at 1639 cm−1 to 310‐helices. This assignment is in excellent agreement with one based on theoretical and experimental studies of 310‐helical polypeptides. The frequency observed for 310‐helices is distinctly different from that at which α‐helices are typically found (viz., around 1655 cm−1). In the present study, two bands are clearly resolved in the latter region at 1651 and 1659 cm−1. Both are apparently associated with α‐helices. These results suggest that for D2O solutions of globular proteins, FTIR spectroscopy can be a facile method for detecting the presence of these two different types of helical conformation and distinguishing between them. This provides a distinct advantage over ultraviolet circular dichroism spectroscopy (UV‐CD). This work also provides a basis for future studies of α‐lactalbumin which examine the effects of environment (e.g., pH, temperature) and ligands (e.g., Ca2+, Mn2+) on its conformation.
Synthesis and biological evaluation of mouse growth hormone‐releasing factor
Authors: HEIMER EDGAR P. , AHMAD MUSHTAQ , LAMBROS THEODORE J. , FELIX ARTHUR M. , DOWNS THOMAS R. , FROHMAN LAWRENCE A.
Published: August 2010

The recently described mouse growth hormone‐releasing factor (mGRF) was synthesized by the solid phase procedure, purified by 2 stages of preparative high performance liquid chromatography and fully characterized. The biologic activity of the 42‐amino acid peptide (H‐His‐Val‐Asp‐Ala‐Ile‐Phe‐Thr‐Thr‐Asn‐Tyr‐Arg‐Lys‐Leu‐Leu‐Ser‐Gln‐Leu‐Tyr‐Ala‐Arg‐Lys‐Val‐Ile‐Gln‐Asp‐Ile‐Met‐Asn‐Lys‐Gln‐Gly‐Glu‐Arg‐Ile‐Gln‐Glu‐Gln‐Arg‐Ala‐Arg‐Leu‐Ser‐OH) was assessed in primary cultures of both mouse and rat anterior pituitary cells and compared to synthetic rat (rGRF) and human (hGRF) growth hormone‐releasing factors. mGRF was equipotent to rGRF in mouse somatotrophs but slightly less potent in rat somatotrophs, while hGRF was 3‐5 times less potent in both rodent species.
Enhancement of solubility by temporary dimethoxybenzyl‐substitution of peptide bonds
Authors: BLAAKMEER J. , TIJSSE‐KLASEN T. , TESSER G. I.
Published: August 2010

The synthesis of the model compound Aloe‐Ala‐Ala‐Dma‐Ala‐Ala‐OMe has been described as an illustration of the fact that a large group reversibly alkylating the amido group of an oligomer can disturb the regularity of a peptide backbone, oppose its aggregation and thus enhance its solubility greatly, affording synthons for further oligomerization. Application of such a group not only affects the solubility, but alters also the properties of the intermediates. The concomitant change in reactivity may run to such an extent that N‐alkylation of oligomers has to be abandoned (this was encountered in the attempted synthesis of Lys‐Glu‐Dmg). Consequently, the solubility of the growing protected peptide chain will become progressively less and in the mentioned example the oligomerization had to be terminated at the dodecapeptide level, indicating the severe need for reversible „structure‐breaking” functions.
Preparation of N‐9‐fluorenylmethoxycarbonylamino acid chlorides from mixed anhydrides by the action of hydrogen chloride1
Authors: CHEN FRANCIS M.F. , LEE YOUNG C. , BENOITON N. LEO
Published: August 2010

N‐9‐Fluorenylmethoxycarbonyl‐ (Fmoc) amino‐acid chlorides have been prepared by reaction of hydrogen chloride on purified mixed Fmoc‐amino acid‐monoalkyl carbonic acid anhydrides in dichloromethane. The products partially undergo subsequent conversion to the corresponding esters due to the presence of the liberated alcohol, the extent depending on the nature of the alkyl group. Esterification occurred to 5–20% when the alkyl group was isopropyl. Anhydrides of monoisopropenyl carbonic acid which liberate acetone instead of an alcohol gave products uncontaminated with ester. The three components in a reaction mixture could be determined as the reaction progressed by normal phase high‐performance liquid chromatography of aliquots, which had been freed of excess hydrogen chloride, on a μPorasil (underivatized silica) column using tert‐butanol‐hexane (1.5:98.5) as solvent.
Synthetic glucagon antagonists and partial agonists
Authors: ZECHEL CHRISTIAN , TRIVEDI DEV , HRUBY VICTOR J.
Published: August 2010

This paper reports the synthesis and the biological activities of six new glucagon analogues. In these compounds N‐terminal modifications of the glucagon sequence were made, in most cases combined with changes in the C‐terminal region which had been shown previously to enhance receptor affinity. The design of these analogues was based on [Lys17.18,Glu21]glucagon,1 a superagonist, which binds five times better than glucagon to the glucagon receptor, and on the potent glucagon antagonist [d‐Phe4,Tyr5,Arg12]glucagon, which does not stimulate adenylate cyclase system even at very high concentrations. The N‐terminal modifications involved substitution of His1 by the unnatural conformationally constrained residue, 4,5,6,7‐tetrahydro‐1H‐imidazo[c]pyridine‐6‐carboxylic acid (Tip) and by desaminohistidine (dHis). In addition we prepared two analogues (6 and 7), in which we deleted the Phe6 residue, which was suggested to be part of a hydrophobic patch and involved in receptor binding. The following compounds were synthesized: [Tip1, Lys17.18,Glu21]glucagon (2); [Tip1,d‐Phe4,Tyr5,Arg12,Lys17.18,Glu21 glucagon (3); [dHis1,d‐Phe4,Tyr5,Arg12, Lys17.18,Glu21 glucagon (4); [dHis1,Asp3,d‐Phe4,Tyr5,Arg12,Lys17.18,Glu21]glucagon (5); des‐Phe6‐[Tip1,D‐Phe4,Tyr5Arg12,Glu21 glucagon (6); des‐Phe6‐[Asp3,d‐Phe4,Tyr5,Arg12,Glu21]glucagon (7) The binding potencies of these new analogues relative to glucagon (= 100) are 3.2 (2), 2.9 (3), 10.0 (4), 1.0 (5), 8.5 (6), and 1.7 (7). Analogue 2 is a partial agonist (maximum stimulation of adenylate cyclase (AC) approximately 15% and a potency 8.9% that of glucagon, while the remaining compounds 3‐7 are antagonists unable to activate the AC system even at concentrations as high as 10−5m. In addition, in competition experiments, analogues 3‐7 caused a right‐shift of the glucagon stimulated adenylate cyclase dose‐response curve. Hence these compounds are glucagon receptor antagonists with respect to the glucagon receptor coupled to the adenylate cyclase system.
Enzymatic peptide synthesis in organic solvent mediated by gels of copolymerized acrylic derivatives of α‐chymotrypsin and polyoxyethylene
Authors: FULCRAND VALÉRIE , JACQUIER ROBERT , LAZARO RENÉ , VIALLEFONT PHILIPPE
Published: August 2010

Copolymers of acrylated derivatives of α‐chymotrypsin and polyethylene glycol (PEG) have been prepared and used as biocatalysts for the synthesis of model peptides in organic solvent containing a low quantity of water. Other peptide couplings have been tried to point out the chemico‐ and stereoselectivity and examples of segment couplings are given.
Preparation and properties of des‐Tyr98 and des‐Arg97‐Tyr98 acylphosphatase (muscular isoenzyme)
Authors: STEFANI MASSIMO , CAPPUGI GIANNI , PAZZAGLI LUIGIA , CAMICI GUIDO , MANAO GIAMPAOLO , TADDEI NICCOLO , BUCK MATTHIAS , RAMPONI GIAMPIETRO
Published: August 2010

Previous NMR reports indicated that Tyr98, the C‐terminal residue of the muscular form of acylphosphatase, is likely to be part of the enzyme's active site. In addition, there is evidence that an arginine residue participates to the catalyzed reaction, possibly as phosphate binding site. Among all Arg residues present in the muscular forms of acylphosphatase, four, i.e. Arg23, Arg74, Arg77, and Arg97, appear to be conserved in all species checked thus far. We prepared the des‐Tyr98 and des‐Arg97‐Tyr98 derivatives of the native acylphosphatase to investigate the properties of both modified enzymes. The enzyme lacking Tyr98 was found to be catalytically less effective than the native one, whereas the des‐Arg97‐Tyr98 acylphosphatase was completely inactive. This evidence suggests that Arg97 participates directly to the active site catalytic mechanism. Fluorescence and CD spectra revealed that the latter enzyme could have been undergone some conformational change that could account for the loss of activity; on the other hand, the one‐dimensional NMR spectra of either native and des‐Arg97‐Tyr98 enzymes were strictly similar, thus demonstrating that the removal of the two C‐terminal residues does not markedly affect the fold of the enzyme. The results reported are proof of a critical contribution of Arg97 to the acylphosphatase active site; however, we cannot exclude that the function of this residue is merely to stabilize the active site conformation and dynamics.
Ten‐membered cyclotripeptides
Authors: CERRINI S. , GAVUZZO E. , LUCENTE G. , LUISI G. , PINNEN F. , RADICS L.
Published: August 2010

The 10‐membered cyclotripeptide cyclo(‐MeβAla‐Phe‐Pro) 3 and its diastereoisomer cyclo(‐MeβAla‐Phe‐dPro‐) 4 have been synthesized under mild cyclization conditions starting from linear precursors containing C‐terminal proline. The crystal and molecular structure of the two models has been determined by X‐ray crystallography. Analysis of the NMR spectra supported by NOE data clearly indicates that the conformations found in the crystals are retained in solution. Both cyclotripeptides exhibit a cis‐cis‐trans backbone conformation. The two tertiary peptide bonds, at the proline and MeβAla nitrogen atoms, adopt a cis conformation whereas the CO—NH junctions are trans in both the models. The deviations from planarity of the peptide units vary from Δω values of ca. 18° for the Pro‐MeβAla and dPro‐MeβAla bonds to ca. 7° for Phe‐Pro and Phe‐dPro bonds. Relevant conformational details of 3 and 4, as revealed by X‐ray and NMR analysis, are reported. Crystals of 3 are monoclinic: P21, a= 5.317(2), b= 17.059(6), c= 9.514(3)Å, β= 99.18(3), Z = 2. The final R and Rw are 0.054 and 0.071 respectively. Crystals of 4 are orthorhombic: P212121, a= 8.797(2), b= 19.440(9), c= 21.605(10)Å, Z = 8. The final R and Rw are 0.069 and 0.104 respectively.
Synthesis of human angiotensinogen (1‐17) containing one of the putative glycosylation binding sites and its hydrolysis by human renin and porcine pepsin
Authors: HIRATA IZAURA Y. , BOSCHCOV PAULO , OLIVEIRA MARIA C.F. , JULIANO MARIA APARECIDA , MIRANDA ANTONIO , CHAGAS JAIR R. , TSUBOI SATOSHI , OKADA YOSHIO , JULIANO LUIZ
Published: August 2010

The N‐terminal heptadecapeptide of human angiotensinogen (Asp‐Arg‐Val‐Tyr‐Ile‐His‐Pro‐Phe‐His‐Leu‐Val‐Ile‐His‐Asn‐Glu‐Ser‐Thr‐NH2), with the C‐terminal carboxyl group amidated, was synthesized in order to study the role of Asn‐Glu‐Ser, a putative carbohydrate binding site, on the hydrolysis by human renin. The synthesis was performed by fragment condensation using the Honzl and Rudinger azide procedure. In our conditions for azide segment condensation, histidine racemization was demonstrated to be negligible for most of the condensation reactions. Human renin liberates angiotensin I from h‐angiotensinogen (1‐17)‐NH2 with a Km value of 3.4 × 10−5m, at pH 7.3 and 37° being similar to h‐angiotensinogen (1‐13), an analog without the carbohydrate binding site. However, the Vmax value of 4.1 × 10−9mol/G.U. min is one order of magnitude higher. Porcine pepsin was demonstrated to cleave preferentially Leu10‐Val11 bond and, surprisingly, His9‐Leu10 as well.
Synthesis and NMR conformational analysis of a β‐turn mimic incorporated into gramicidin S
Authors: BACH ALVIN C. , MARKWALDER JAY A. , RIPKA WILLIAM C.
Published: August 2010

The solution structure of a gramicidin S (GS) analog containing a β‐turn mimic[BTD4‐5, Lys2,2′]GS has been compared to that of native GS. The linear [BTD4‐5, Lys2,2′]GS was synthesized by solid phase methodology and the cyclized peptide was analyzed by NMR. In the peptide portion of [BTD4‐5, Lys2,2′]GS, the intramolecular hydrogen bonding pattern, inter‐residue NOEs, including a transannular Hα‐Hα NOE, and JNα coupling constants all describe a solution structure which is equivalent to that of native GS. These data confirm that the BTD group is a competent Type II' β‐turn mimic since it does not disrupt the native conformation of GS. It also supports the use of GS as a conformational model in which to test β‐turn mimics.
Solid‐phase synthesis of a range of O‐phosphorylated peptides by post‐assembly phosphitylation and oxidation
Authors: ANDREWS D. M. , KITCHIN J. , SEALE P. W.
Published: August 2010

A completely general method for the O‐phosphorylation of peptides of any given composition using solid‐phase methodology is described. Peptides were assembled using Fmoc amino acid active esters, with base used for Fmoc deprotection. Unprotected amino acid side chain hydroxyl groups were phosphitylated and oxidised at the end of the assembly using bis (benzyloxy)(diisopropylamino)phosphine and tert.‐butylhydroperoxide respectively. TFA was used for final deprotection of the amino acid side chains and for simultaneous cleavage from the resin. The synthesis of O‐phosphopeptides of up to 15 residues in length is described.
Synthesis and properties of chemotactic peptide analogs
Authors: TORRINI I. , ZECCHINI G. PAGANI , PARADISI M. PAGLIALUNGA , LUCENTE G. , GAVUZZO E. , MAZZA F. , POCHETTI G. , SPISANF S. , GIULIANI A.L.
Published: August 2010

As a part of a research program aimed at studying structure activity relationship in the field of chemotactic peptides, modified analogs of the potent chemoattractant HCO‐Met‐Leu‐Phe‐OH (fMLP) of the general formula HCO‐Xaa‐Leu‐Yaa‐OMe are examined. 4‐Aminotetrahydrothiopyran‐4‐carboxylic acid (Thp) and 2‐aminoindane‐2‐carboxylic acid (Ain) have been chosen as achiral, conformationally restricted amino acids suitable to mimick the external Met and Phe residues of fMLP‐OMe. Studies on a first model, namely [Ain3]fMLP‐OMe 1, have already been reported (12). Here the two remaining analogs (Thp1, Ain3) 2 and [Thp1] 3 have been synthesized. The conformation in the crystal of the disubstituted analog 2 has been determined and compared with those adopted by the parent fMLP‐OMe and by previously studied models. The backbone conformation of 2 is characterized by helical folding centred at each of the three residues with the central Leu presenting helical handedness opposite to those of the two adjacent achiral residues. This conformation presents strong similarities with that adopted in the crystal by fMLP‐OMe and resembles the conformation of fMLP bound to immunoglobulin (Bence‐Jones dimer). The conformationally restricted analogs 2 and 3 are more active than the parent in the stimulation of directed mobility of human neutrophils but are practically inactive in the superoxide production. Crystals of 2 are orthorhombic, s.g. P 21 21 21, with a = 21.934 (8), b = 10.856 (2), c = 10.380 (2) Å. The structure has been refined to R = 0.071 for 2301 independent reflections with I > 1.5 σ.
Amino acid sequence pattern in the regulatory peptides
Authors: BAKALKIN GEORGY Y , RAKHMANINOVA ALEXANDRA B. , AKPAROV VALERY K. , VOLODIN ALEXANDER A. , OVCHINNIKOV VALERY V. , SARKISYAN RAPHIK A.
Published: August 2010

The essential properties of the primary structure of regulatory peptides, i.e. amino acid residues and their combinations, which are characteristic of the whole population of regulatory peptides, have been revealed using statistical methodology. These properties are as follows: increased content of certain residues (Gly, Pro, Phe, Arg, Tyr, Met and Trp) as well as an increased rate of occurrence of certain pairs of residue as compared with proteins, a random sequence of residues and “nonregulatory” peptides. By representing regulatory peptides as a sequence of hydrophobic (2 types) and hydrophilic (3 types) segments, the pattern for alternation of these segments in regulatory peptides has been determined. The segments were classified into 5 types according to the peculiarities of mutual localization of hydrophobic and hydrophilic residues within the primary structure of regulatory peptides. As compared with proteins, “nonregulatory” peptides and a random sequence of segments, regulatory peptides were characterized by an increased frequency of 4 particular pairs of segments among 12 theoretically possible pairs. These 4 pairs are fragments of the periodic segment sequence with periods of 4 segments. The revealed pattern indicates that there exists a general principle of the regulatory peptide primary structure organization and possibly a common type of the regulatory peptides flexible peptide chain folding at the ligand‐receptor complex formation.
Conformational analysis of [Cpp1, Sar7, Arg8] vasopressin by 1H‐NMR spectroscopy and molecular mechanics calculations
Authors: SHENDEROVICH MARK D. , KASPRZYKOWSKI FRANCISZEK , LIWO ADAM , SEKACIS ILMARS , SAULITIS JURIS , NIKIFOROVICH GREGORY V.
Published: August 2010

A combined 1 H‐NMR and molecular mechanics study of [Cpp1, Sar7]AVP was performed in order to select the most probable conformations in DMSO solutions. The NMR constraints obtained were employed in the selection of starting conformations of the cyclic moiety of the analog. In particular, the diminished accessibility of the Asn5 NH proton to solvent and the close contact between Cpp1 and Cys6 C2H protons suggests a β‐turn conformation at the Phe3‐Gln4 residues. Energy minimization was carried out both in the ECEPP/2 (rigid‐valence geometry) and in the AMBER (flexible‐valence geometry) force fields. Comparison of the experimental and calculated values of NMR characteristics has revealed that conformations containing type I, II, and III β‐turns at the Phe3‐Gln4 residues are in reasonable agreement with the experimental data, with a dynamic equilibrium between the βI (βIII) and βII type structures of the cyclic part being the most probable. All of these conformations prefer the negative chirality of the disulfide bridge (X3∼ 90°). Five representative conformations were chosen for the acyclic tail: one with a βI, one with a βII′‐turn at the Sar7‐Arg8 residues, two extended‐type conformations, and a conformation with a γ‐turn at Sar7. Because only high‐energy extended conformations were in agreement with NMR data, it was concluded that the acyclic tail has considerable conformational flexibility in solution. The conformations obtained are discussed in terms of the structure‐function relationship of the neurohypophyseal hormone analogs.
Solid phase peptide synthesis of human endothelin precursor peptide using two‐step hard acid deprotection/cleavage methods
Authors: NOMIZU MOTOYOSHI , INAGAKI YOSHIMASA , IWAMATSU AKIHIRO , KASHIWABARA TOMOKO , OHTA HIDEO , MORITA AKIHITO , NISHIKORI KOJI , OTAKA AKIRA , FUJII NOBUTAKA , ROLLER PETER P.
Published: August 2010

Syntheses are described for the putative human and porcine biosynthetic precursors (hET‐38 and pET‐39) of endothelin, with the sequence previously deduced from human‐ and porcine‐cDNA coding for prepro‐endothelin. The Boc based solid phase synthetic method was applied, followed by weak hard acid, trimethylsilyl bromide, cleavage. The peptide removal from the resin was optimally accomplished with hydrogen fluoride. Disulfide bridges were formed by air‐oxidation, and the linkage modes determined by enzymic (Endoproteinase Asp‐N) digestion and HPLC. Five additional C‐terminally elongated endothelin homologs were also synthesized. For alternative synthesis of pET‐39, the use of trimethylsilyl tri‐fluoromethanesulfonate for the removal of peptide from the resin generated a major side product, which was characterized. hET‐38 was found to be less effective in vitro, when compared to endothelin. The vasoconstrictor activity in vitro of other related peptides was comparable to that of hET‐38.
Peptide crystal growth via vapor diffusion
Authors: EGGLESTON DRAKE S. , BAURES PAUL W.
Published: August 2010

The structure of a dihydrated form of glycyl‐L‐tyrosyl‐L‐alanine (GYA) has been determined as part of a series of peptide structural investigations and development of microscale vapor diffusion experiments for peptide crystal growth. Crystals were grown by the hanging‐drop method against sodium acetate. The tripeptide is a zwitterion in the crystal, adopting an extended conformation through glycine, a nearly perpendicular bend attyrosine and a reverse turn for the C‐terminal carboxylate. Principal backbone torsion angles are ψ1 175(1)°, ω2 173(1)°, φ2 ‐119(1)°, ψ2 120(1)°, ω33 172(1)°, φ3 ‐73(1)°,ψ31 ‐9(1)°, ψ32 171(1)°. The tyrosyl side chain adopts an unusual orientation (χ1/2= ‐86(1)°). The relationship of the GYA.2H2O structure to GYA sequences in proteins is examined, particularly as regards its helix‐forming potential. Crystal data: C14H19N3O4.2H2O, Mr = 345.36, orthorhombic, P212121, a = 4.810 (4), b= 11.400(7), c = 30.162(23)Å, V=1653.8(24)Å−3, Z = 4, Dx= 1.387 Mgm−3, λ(CuKα−)= 1.540 Åμ= 9.053 mm−1, F(000) = 736, T = 199K, R = 0.041 for 1458 observations with I ≥ 3σ(I).
Synthesis and biological activity of the mono‐ and di‐galactosyl‐vespulakinin 1 analogues
Authors: GOBBO M. , BIONDI L. , FILIRA F. , SCOLARO B. , ROCCHI R. , PIEK T.
Published: August 2010

Syntheses are described of some mono‐ and di‐glycosylated analogues of vespulakinin 1. The solid phase procedure, based on the Fmoc chemistry, was used to prepare (Gal α)Thr3‐vespulakinin 1, (Galβ)Thr3‐vespulakinin 1 and the di‐glycosylated analogue ((Gal α)Thr3. (Gal α)Thr4‐vespulakinin 1. The β‐glycosylated derivative was also prepared by the continuous flow variant of the Fmoc polyamide method. The synthesized glycopeptides were purified and characterized by amino acid analysis, optical rotation, analytical HPLC, 1H‐and 13C‐NMR and FAB‐MS. Preliminary pharmacological experiments showed that the carbohydrate‐free vespulakinin 1 is less active than bradykinin (about 0.3 times on a molar basis) when tested by guinea pig rectum contraction, and the two monoglycosylated analogues are equiactive (about 0.9 times the bradykinin activity). The most active derivative, the (Gal α)Thr3, (Gal α)Thr4‐vespulakinin 1 analogue, was about 2.5 times as active as bradykinin.
FMOC solid phase synthesis of an endothelin converting enzyme substrate
Authors: HANDA BALRAJ K. , KEECH ELIZABETH
Published: August 2010

The synthesis of a substrate for the endothelin converting enzyme using the continuous flow FMOC solid phase method has been accomplished. The allyl group, employed to protect Glu side chain, was cleanly removed during the synthesis in the presence of tert.‐butyl based protecting groups to enable the introduction of the Edans moiety. The substrate as obtained in 31% yield after reverse phase hplc purification.
Synthesis and conformation of the trimeric coiled‐coil segment of laminin*
Authors: NOMIZU MOTOYOSHI , UTANI ATSUSHI , SHIRAISHI NORIO , YAMADA YOSHIHIKO , ROLLER PETER P.
Published: August 2010

A disulfide linked 95‐mer parallel hetero‐trimeric active site segment of laminin was designed and synthesised. The three subunits, A (32‐mer), B1 (30‐mer) and B2 (33‐mer), were prepared by Boc‐based solid‐phase peptide synthesis involving a two‐step trimethylsilyl bromide‐thioanisole and HF deprotection procedure. The interlinking of the three subunits was accomplished by the stepwise selective formation of two disulfide bridges using air‐oxidation and thallium (III) trifluoroacetate oxidation. The conformations of the synthetic peptides were studied by circular dichroism (CD) spectroscopy, showing that the hetero‐dimer, B1‐B2, one of the homo‐dimers, B1‐B1, and the trinier are 30 to 40% in the α‐helical conformation in aqueous buffer. Variable temperature CD studies demonstrated that the trimer is considerably more stable (melting temperature (Tm) = 61°) than the hetero‐dimer, B1‐B2 (Tm = 36°).
Bradykinin and angiotensin II analogs containing a conformationally constrained proline analog
Authors: JUVVADI PADMAHA , DOOLEY DAVID J. , HUMBLET CHRISTINE C. , LU GINA H. , LUNNEY ELIZABETH A. , PANEK ROBERT L. , SKEEAN RICHARD , MARSHALL GARLAND R.
Published: August 2010

Three analogs of bradykinin and one of angiotensin II have been prepared in which the naturally occurring proline residues have been replaced by the bicyclic amino acid, 2,4‐methanoproline (2,4‐MePro). The relative binding affinities for these analogs were determined to be significantly reduced in the cases of the three bradykinin analogs; [2,4‐McPro3]‐BK retains 1.3%, [2,4‐McPro7]‐BK retains 0.3% and [2,4‐MePro3]‐BK retains 0.021% of the binding affinity of bradykinin. Results from other modification at positions three and seven indicate preference for thetrans‐amide bond preceding these residues implying that other factors, either steric or conformational, are responsible for the decreased affinity for the receptor seen with 2,4‐MePro substitution. The retention of significant binding affinity (26%) in the case of [Ile5,2,4‐McPro7]‐angiotensin II gives direct evidence that thetrans‐conformation of the proline amide bond is the one recognized by the All receptor. Only significant retention of activity can be interpreted unambiguously with the use of this proline analog because of its known conformational differences from Pro as well as its increased steric requirements at the receptor.
HIV protease (HIV PR) inhibitor structure‐activity‐selectivity, and active site molecular modeling of high affinity Leu [CH(OH)CH2]Val modified viral and nonviral substrate analogs
Authors: SAWYER TOMI K. , STAPLES DOUGLAS J. , LIU LI , TOMASSELLI ALFREDO G. , HUI JOHN O. , O'CONNELL KATHY , SCHOSTAREZ HEINRICH , HESTER JACKSON B. , MOON JOSEPH , HOWE W. JEFFREY , SMITH CLARCK W. , DECAMP DIANNE L. , CRAIK CHARLES S. , DUNN BEN M. , LOWTHER W. TODD , HARRIS JESSICA , POORMAN ROGER A. , WLODAWER ALEXANDER , JASKOLSKI MARIUSZ , HEINRIKSON ROBERT L.
Published: August 2010

This report details the structure‐activity relationships of the HIV gag substrate analog Val‐Ser‐Gln‐Asn‐LeuΨ[CH(OH)CH2]Val‐Ile‐Val (U‐85548E), an inhibitor exhibiting subnanomolar affinity towards HIV type‐1 aspartic proteinase (HIV‐1 PR). Our data show that the P1‐P′2 tripeptidyl sequence provides the minimal chemical determinant for HIV‐1 PR binding. We describe the structure‐activity properties of LeuiΨ[CH(OH)CH2]Val substitution in other peptidyl ligands of nonviral substrate origin (e.g., angiotensinogen, insulin and pepstatin). Furthermore, the aspartic proteinase selectivities of a few key compounds are summarized relative to evaluation against human renin, human pepsin, and the fungal enzyme, rhizopuspepsin. These studies have led to the rational design of nanomolar potent inhibitors of both HIV‐1 and HIV‐2 PR. Finally, a 2.5 Å resolution X‐ray crystallographic structure of U–85548E complexed to synthetic HIV‐1 PR dimer (Jaskolski etal, Biochemistry 30, 1600 [1991]) provided a 3‐D picture of the inhibitor bound to the enzyme active site, and we performed computer‐assisted molecular modeling studies to explore the possible binding modes of the above series of LeuΨ[CH(OH)CH2]Val substituted HIV‐1 PR inhibitors.