bluuebunny/crossref_metadata_2025 huggingface dataset:
Heat stable proteinase from Thermomonospora fusca
Authors: KRISTJANSSON MAGNUS M. , KINSELLA JOHN E.
Published: August 2010

An extracellular proteinase secreted by the thermophilic bacteria Thermomonospora fusca YX (YX‐proteinase) is a serine proteinase as shown by its inactivation by the site specific reagents, phenylmethanesulfonyl fluoride, dansyl fluoride, and carbobenzoxy‐l‐phenylalanine chloromethyl ketone. This conclusion is further supported by the effect of various proteinase inhibitors on its activity. The activity of the proteinase toward small synthetic ester substrates shows that the enzyme has a primary specificity for the aromatic and hydrophobic amino acids. The amino acid composition and NH2 ‐terminal sequence, as well as its size, suggest that the enzyme is related to the chymotrypsin‐like microbial proteinase, α‐lytic protease from Myxobacter 495 and protease A and B from Streptomyces griseus.
A cleavage method which minimizes side reactions following Fmoc solid phase peptide synthesis
Authors: KING DAVID S. , FIELDS CYNTHIA G. , FIELDS GREGG B.
Published: August 2010

The success of solid phase peptide synthesis utilizing 9‐fluorenylmethoxycarbonyl (Fmoc) amino acids is often limited by deleterious side reactions which occur during TFA peptide‐resin cleavage and side‐chain deprotection. The majority of these side reactions modify susceptible residues, such as Trp, Tyr, Met, and Cys, with TFA‐liberated side‐chain protecting groups and linkers. The purpose of this study was to assess the relative effectiveness of various scavengers in suppressing these side reactions. We found that the cleavage mixture 82.5% TFA: 5% phenol: 5% H2O : 5% thioanisole : 2.5% EDT (Reagent K) was maximally efficient in inhibiting a great variety of side reactions. Synthesis and cleavage of 10 peptides, each containing 20‐50 residues, demonstrated the complementarity of Fmoc chemistry with Reagent K for efficient synthesis of complex peptides.
Solid‐phase synthesis of peptides containing phosphoserine using phosphate tert.‐butyl protecting group*
Authors: LACOMBE J.M. , ANDRIAMANAMPISOA F. , PAVIA A.A.
Published: August 2010

In this paper, we report the solid‐phase synthesis of peptides containing O‐phosphonoserine using BOP as coupling reagent. Commercially available Fmoc amino‐acids linked to p‐alkoxybenzyl resin were used in the first step and Alloc amino acids in the following. Alloc group was removed by catalytic hydrostannolytic cleavage. Acid‐labile side‐chain protecting groups (including phosphate residue) were used. Thus, both removal of side‐chain protecting groups and cleavage of the phosphopeptide from the resin were achieved in one step by treatment with TFA. Alloc serine was phosphorylated by the phosphoramidite method. This strategy enables the preparation of peptides with selectively phosphorylated residue and overcomes problems due to repetitive treatments with TFA and final cleavage with HF.
Synthetic, immunological and structural studies on repeat unit peptides of Plasmodium falciparum antigens
Authors: KAUR PARAMJEET , SHARMA PAWAN , KUMAR A. , CHAUHAN V.S.
Published: August 2010

Using solid phase methodology, we have synthesized five peptides (16‐18 residues long) corresponding to repeat sequences of four antigens of a human malarial parasite, Plasmodium falciparum. Three of these antigens (RESA, FIRA, and ABRA) are found in the asexual blood‐stages of the parasite, while the remaining one (CSP) is found in the sporozoites. The synthetic peptides, conjugated to bovine serum albumin, elicited high levels of antibodies in rabbits, and these antibodies were found to cross‐react with the heterologous peptides. The degree of cross‐reactivity, as estimated in an ELISA, was quite remarkable among all the peptides. The peptide corresponding to the RESA tetrapeptide repeat was found to be the most immunogenic and highly cross‐reactive. For this reason this tetrapeptide repeat unit, peptide 1, may be a suitable candidate for inclusion in a multiple epitope polypeptide vaccine design. Conformational studies using circular dichroism spectroscopy show that these peptides have similar conformational characteristics with a common feature OF ∼30% and ∼50% helical content in water and TFE respectively. Theoretical predictions regarding conformation using the Chou‐Fasman method have also been presented.
A simple, continuous fluorometric assay for HIV protease
Authors: TOTH MIHALY V. , MARSHALL GARLAND R.
Published: August 2010

Novel fluorogenic substrates for human immunodeficiency viral protease have been developed based on the principle of fluorescence energy transfer. Starting from a p24/p15 cleavage site‐derived hexapeptide substrate, Ac‐Thr‐Ile‐Nle‐Nle‐Gln‐Arg‐NH2, incorporation of 2‐aminobenzoic acid in place of the acetyl group as the donor and p‐NO2‐Phe at the P1′ position as acceptor gave the intramolecularly quenched fluorogenic substrate. Cleavage of the substrate by HIV protease released the fluorescent N‐terminal tripeptide from its close apposition to the quenching nitrobenzyl group, resulting in enhanced fluorescence. An automated assay based on 96=well microtiter plates and a fluorometric plate reader have been developed, which allow high throughput of compounds in the search for HIV protease inhibitors.
Conformationally restricted analogs of oxytocin; stabilization of inhibitory conformation†
Authors: LEBL MICHAL , HILL PATRICIA , KAZMIERSKI WIESLAW , KÁRÁSZOVÁ LENKA , SLANINOVÁ JIŘINA , FRIČ IVO , HRUBY VICTOR J.
Published: August 2010

Analogs of oxytocin containing tetrahydroisoquinoline carboxylic acid (Tic) of L or D configuration in position 2 were synthesized and their biological activities were tested. Both analogs showed negligible agonist activity in uterotonic, galactogogic, and pressor assays, but they are in vitro uterotonic inhibitors. In comparison with oxytocin analogs containing l‐ or D‐phenylalanine in position 2, the analog with the D‐configuration of the conformationally fixed aromatic residue has significantly increased inhibitory activity which suggests that the proper conformation for the interaction with the receptor, but not for its activation, was stabilized. 1H NMR and CD studies, supported by theoretical calculations, suggest that the conformational properties of the analog containing D‐tetrahydroisoquinoline carboxylic acid are similar to those of [2D‐phenylalaninejoxytocin.
Problems associated with use of the benzyloxymethyl protecting group for histidines Formaldehyde adducts formed during cleavage by hydrogen fluoride 1
Authors: MITCHELL MARK A. , RUNGE THOMAS A. , MATHEWS W. RODNEY , ICHHPURANI AVNEET K. , HARN NANCY K. , DOBROWOLSKI PAUL J. , ECKENRODE FRANCES M.
Published: August 2010

The use of Nα‐tert.‐butyloxycarbonyl‐Nπ benzyloxymethylhistidine in peptide synthesis resulted in significant levels of several different side products attributable to the generation of formaldehyde during the hydrogen fluoride cleavage reaction. Methylated impurities in a decapeptide were isolated and identified. These methylated impurities were attributed to the use of the benzyloxymethyl protecting group for the histidines, since the impurities did not form when the dinitrophenyl protecting group was used. Also, peptides containing benzyloxymethyl‐protected histidines in addition to N‐terminal cysteines quantitatively yielded their respective N‐terminal thiazolidine derivatives upon isolation from standard hydrogen fluoride cleavage mixtures. Thiazolidine ring formation was circumvented by including in the cleavage reaction a formaldehyde scavenger such as cysteine hydrochloride or resorcinol.
Synthesis of insulin‐like growth factor I using N‐methyl pyrrolidinone as the coupling solvent and trifluoromethane sulphonic acid cleavage from the resin
Authors: BAGLEY CHRISTOPHER J. , OTTESON KENNETH M. , MAY BRUCE L. , MCCURDY SARAH N. , PIERCE LAURA , BALLARD F. JOHN , WALLACE JOHN C.
Published: August 2010

Insulin‐like growth factor I (IGF‐I), a protein of 70 amino acid residues and 3 cystine bridges, has been synthesized by two solid phase Boc methods. The first method used N‐methylpyrrolidinone as the solvent with single coupling cycles while the second synthesis used dimethylformamide and dichloromethane as the solvents with a double‐coupling protocol. In both cases, trifluoroacetic acid/trifluoromethanesulphonic acid cleavage of the peptide from the resin was employed. Purification of the cleavage products followed by removal of the S‐acetamidomethyl protecting groups gave reduced peptides which were then oxidized under conditions favouring the formation of the correct disulphide bonds. The purified synthetic IGF‐I peptides were full agonists of natural IGF‐I in a radioimmunoassay, in an IGF‐I radioreceptor assay, in a bioassay which measures the stimulation of protein synthesis in rat L6 myoblasts and in an IGF‐binding protein competitive binding assay. Moreover, in each of these assays, the synthetic IGF peptides were found to be at least 70% as potent as natural IGF‐I
Comparative study on proteinase R, T, and K from Tritirachiam album limber
Authors: KOLVENBACH CARL G. , NARHI LINDA O. , LAZENBY KELLY , SAMAL BABRU , ARAKAWA TSUTOMU
Published: August 2010

Proteinase R and T purified from Tritirachiam album limber were characterized in comparison with proteinase K using circular dichroism (CD), enzyme activity, thermal melting, and sodium dodecylsulfatc polyacrylamide gel electrophoresis (SDS‐PAGE). CD analysis suggested that these three proteins possess some β‐sheet structure, with little α‐helix except for proteinase R which showed about 14%α‐helix. SDS‐PAGE and gel filtration in 0.1% SDS indicated that proteinase T and K are resistant to SDS‐induced unfolding similar to subtilisin. Thermal denaturation experiments showed the melting temperature for proteinase T to be 67° and that for proteinase K to be 65° in the absence of Ca2+, with higher melting temperatures in the presence of Ca2+. However, the enzyme activities of proteinase T and R were significantly lower than those of proteinase K.
Backbone modifications in cyclic pep tides Conformational analysis of a cyclic pseudopentapeptide containing a thiomethylene ether amide bond replacement
Authors: ANWER MOHMED K. , SHERMAN DOUGLAS B. , SPATOLA ARNO F.
Published: August 2010

NMR and X‐ray crystallographic studies have shown that cyclic pentapeptides of the general structure cyclo(D‐Xxx‐Pro‐Gly‐Pro‐Gly) possess β‐ and γ‐turn intramolecular hydrogen bonds. As part of our continuing series surveying the compatibility of various amide bond replacements on peptide structure, we have synthesized cyclo(D‐Phe‐Proψ [CH2S]Gly‐Pro‐Gly). The pseudopeptide was prepared by solid phase methods and cleaved from the resin by a new procedure involving phase transfer catalysis using K2CO3 and tetrabutylammonium hydrogen sulfate. Cyclization was carried out with the use of DPPA, HOBt, and DMAP to afford the product in 69% yield. The conformational behavior of the pseudopeptide was analyzed by 1H and 13C (1D and 2D) NMR techniques. The backbone modification replaced the amide bond that is involved in a γ‐turn intramolecular hydrogen bond in the all‐amide structure. In CDCl3, the pseudopeptide adopted the same all‐trans conformation as its parent, although the remaining β‐turn hydrogen bond was weaker according to Δδ/ΔTNH measurements. In DMSO‐d6, the all‐trans conformer and a second conformer were observed in a ratio of 55:45. These conformers, which slowly inter converted on the NMR time scale, could be separately assigned; peaks due to chemical exchange were readily distinguishable by the ROESY technique as reported earlier by others. 13C and ROESY experiments suggested the minor conformer contained one cis amide bond at the Gly1‐Pro2 position. Thus, both the location and type of amide surrogate are important determinants affecting the compatibility of the replacement with a particular conformational feature.
Side reaction of pyrenylalanine‐peptides containing NG‐tosylarginine during detosylation with hydrogen fluoride
Authors: NAKAMURA HIROSHI , LEE SANNAMU , ONO SHIN , KATO TAMAKI , AOYAGI HARUHIKO
Published: August 2010

Formation of by‐products of pyrenylalanine‐peptides was observed during the cleavage of a tosyl group in pyrenylalanine‐peptides containing an Arg(Tos) residue with HF, NMR and fluorescence experiments showed that by‐products were compounds in which a pyrenyl group was modified with a tosyl group(s). The side reaction was little suppressed by the addition of usual scavengers such as anisole, 1,2‐ethanedithiol, thioanisole and p‐cresol. Under the conditions used for the cleavage of Nα‐Boc group, i.e., trifluoroacetic acid or 4m HCl in dioxane, the pyrenylalanine residue was stable. No side reaction was, furthermore, observed in the reductive cleavage with sodium in liquid ammonia. These results suggest that the side reaction may be due to an electrophilic attack of tosyl cations to the electron‐rich pyrenyl group in the pyrenylalanine residue under the HF‐mediated acidic conditions.
Thermolysin as a catalyst in enzymatic synthesis of asparagine‐containing peptides II
Authors: MIRANDA MARIA TERÊSA MACHINI , TOMINAGA MINEKO
Published: August 2010

Thermolysin was used as a catalyst to obtain the following protected di‐ and tripeptide esters: Z‐Asn‐Leu‐OEt, Z‐Asn‐Phe‐OEt, Moz‐Asn‐Leu‐Gly‐OEt, Boc‐Asn‐Leu‐Gly‐OEt, Z‐Asn‐Leu‐Gly‐OEt, Moz‐Asn‐Leu‐Gly‐OBzl, Moz‐Asn‐Leu‐Gly‐OtBu, Moz‐Gln‐Leu‐Gly‐OEt, Moz‐Asn‐Ile‐Gly‐OEt, and Moz‐Asn‐Leu‐Ala‐OEt. These compounds were obtained in pure form and the yields exceeded 50 %, except for Moz‐Asn‐Leu‐Gly‐OtBu and Boc‐Asn‐Leu‐Gly‐OEt. H‐Cys(Bz1)‐OtBu and H‐Cys(Bz1)‐Pro‐Leu‐Gly‐NH2 were both inadequate as amino components for obtaining Moz‐Asn‐Cys(Bz1)‐OtBu, Z‐Asn‐Cys(Bz1)‐OtBu and Moz‐Asn‐Cys(Bzl)‐Pro‐Leu‐Gly‐NH2 in the thermolysin‐catalyzed reactions. In the attempted synthesis of the protected pentapeptide amide, this protease cleaved the Pro‐Leu bond of the amino component H‐Cys(Bz1)‐Pro‐Leu‐Gly‐NH2 and catalyzed the coupling between the resulting dipeptide amide and Moz‐Asn‐OH, thus yielding Moz‐Asn‐Leu‐Gly‐NH2 as the main product.
Solid‐phase synthesis of neurokinin A antagonists
Authors: ROVERO PAOLO , QUARTARA LAURA , FABBRI GAETANO
Published: August 2010

During the preparation of the NK‐2 selective tachykinin antagonist MEN 10208 (Thr‐Asp‐Tyr‐D‐Trp‐Val‐D‐Trp‐D‐Trp‐Arg‐NH2) and its analogs by the solid‐phase method employing the Boc strategy routinely used in our laboratory, we encountered difficulties in the coupling of hydrophobic amino acids D‐Trp and Val. To study the coupling problems several syntheses of MEN 10208 and analogs were carried out with different activation strategies. These syntheses yielded considerable amounts of deletion sequences even though a negative Kaiser test was obtained after each coupling. Inaccessibility of the free amino group of the growing peptide due to steric hindrance of the hydrophobic residues during coupling, and for the ninhydrin complex during the Kaiser test, may account, at least in part, for the unsatisfactory synthetics results and for the false‐negative ninhydrin tests. Repetition of each synthesis with the Fmoc strategy on a newly developed DOD resin for peptide amides using the DCC/HOBt chemistry gave superior results in terms of the yield and purity of the crude peptides. Therefore, the Fmoc strategy appears to offer advantages over the Boc method for the preparation of these peptides containing hydrophobic amino acids.
Synthesis of biologically active porcine secretin and [ITyr10] porcine secretin
Authors: KOFOD HANS
Published: August 2010

Porcine secretin, [Tyr10] secretin, and [Tyr13] secretin were synthesized by solid phase methodology and purified by stepwise gradient elution from a short reversed‐phase column with ethanol and acetic acid as organic modifiers. [Tyr10] secretin and [Tyr13] secretin were iodinated by the chloramine‐T method and non‐, mono‐, and di‐iodinated products separated and isolated by reversed‐phase HPLC. Batch incubation analysis in isolated mouse pancreatic islets revealed that secretin and the [Tyr10] analogue were indistinguishable in their effect on the glucose‐induced insulin release and cAMP accumulation. [Tyr13] secretin in contrast was significantly less potent in its effect on the glucose‐induced insulin release.
Synthesis of novel immunologically active tripalmitoyl‐S‐glycerylcysteinyl lipopeptides as useful intermediates for immunogen preparations
Authors: METZGER JÖRG , WIESMÜLLER KARL‐HEINZ , SCHAUDE RENATE , BESSLER WOLFGANG G. , JUNG GÜNTHER
Published: August 2010

The synthesis and characterization of lipopeptides consisting of the lipoamino acid N‐palmitoyl‐S‐[2,3‐bis(palmitoyloxy)‐(2RS)‐propyl]‐[R]‐cysteine (Pam,Cys‐OH) and different peptide segments and/or spacer molecules is described. Pam,Cys‐peptides, which are derived from the immunologically active TV‐terminus of bacterial lipoprotein, were obtained either by solution or solid phase peptide synthesis. In particular, the amphiphilic and water‐soluble lipohexapeptides Pam3Cys‐Ser‐(Lys)4 and Pam3Cys‐Ser‐(Glu)4 proved to be potent macrophage and B‐cell activators and non‐toxic, non‐pyrogenic immune adjuvants in combination with or covalently linked to antigens and haptens.
Effect of tertiary amine on the carbodiimide‐mediated peptide synthesis
Authors: BEYERMANN MICHAEL , HENKLEIN PETER , KLOSE ANNEROSE , SOHR REINHARD , BIENERT MICHAEL
Published: August 2010

The effect of tertiary amine (DIEA) on reaction rate and product purity of a carbodiimide/HOBt‐mediated peptide synthesis was studied. It was found that very rapid activation can be achieved using carbodiimide/HOBt in non‐polar solvents, such as DCM. Although the HOBt is poorly soluble in DCM, the activation proceeds within 2 min, probably forming the HOBt‐ester. By such a preactivation followed by a coupling in the presence of DIEA the rate of coupling is comparable with other rapid methods using BOP or TBTU, and no racemization was found in a model coupling (< 0.1%). For comparison, syntheses of neurotensin by means of different coupling reagents (BOP, TBTU, OPfp‐esters) and the DIEA‐catalyzed coupling after carbodiimide/HOBt‐activation under comparable conditions have shown that these procedures are of the same value in view of coupling efficiency and product purity.
Synthesis, conformational studies, and molecular dynamics calculations of two cyclic tetrapeptides with 17‐ and 18‐membered rings
Authors: HÖLZEMANN G. , PACHLER K.G.R. , EBERHART B. , HÖLZEL H. , KRAFT M. , BARNICKEL G.
Published: August 2010

Two cyclic tetrapeptides [Boc‐cyclo(‐Xxx‐Pro‐Asn‐Lys‐)OMe (Xxx = Asp or Glu)] were synthesized and investigated by NMR spectroscopy. They were designed in order to mimic the salt bridge found in physalaemin. Isomers of the urethane bond were observed in DMSO solution. The ROESY spectrum allowed the assignment of many signals of the minor isomer of both compounds. Conformational studies based on the temperature gradients of the NH chemical shifts, coupling constants, and ROEs revealed a similar conformation for the Asp analogue as proposed for physalaemin. A β1 turn with Pro and Asn in the corner positions was found for the major isomer. No hydrogen bonds were detected for the major isomer of the cyclic Glu analogue. Molecular dynamics calculations, using the NMR based initial structures, yielded sets of conformations in agreement with the experimental data. It is concluded that the salt bridge in physalaemin is best approximated by a lactam formed from the original amino acids.
Conformation of MeAla6‐cyclosporin A by NMR
Authors: GOOLEY P. R. , DURETTE P. L. , BOGER J. , ARMITAGE I. M.
Published: August 2010

MeAla6‐cyclosporin A (MeAla6‐CsA) is a unique CsA analog that shows weak immunosuppressive activity and yet binds strongly to the proposed cytosolic protein receptor, cyclophilin (CyP). Preliminary 1H NMR data showed significant chemical shift differences between spectra of MeAla6‐CsA and CsA, suggesting different preferred conformations. A more detailed study, however, revealed that the backbone conformations of the two molecules are essentially identical, and that the differences can be accounted for, principally, by the sidechain motions of the MeBmt‐1, MeLeu‐9, and ‐10 residues. ROE and coupling constant data show that in MeAla6‐CsA, the preferred χ1 rotamers for MeLeu‐9 and ‐10 are + 180° (T), whereas in CsA there is a more even distribution of rotamer populations for MeLeu‐10, and a preferred – 60° (G−) χ1 rotamer for MeLeu‐9. Similar data argue that the sidechain of MeBmt‐1 is more restricted in its motion in MeAla‐CsA than in CsA. Temperature studies suggest that these preferred rotamers for MeAla6‐CsA may increase the stability of the hydrogen bond between NH(7) and CO(II), but prevent particular residues, especially the essential MeBmt‐I sidechain, from adopting orientations required to elicit immunosuppressive activity. The significant changes observed in the preferred orientations for the side‐chains of the MeBmt‐1, MeLeu‐9, and MeLeu‐10 residues in MeAla6‐CsA argue that the particular orientations which they assume in CsA are not essential for cyclophilin binding.
Minimum analogue peptide sets (MAPS) for quantitative structure‐activity relationships
Authors: HELLBERG SVEN , ERIKSSON LENNART , JONSSON JÖRGEN , LINDGREN FREDRIK , SJÖSTRÖM MICHAEL , SKAGERBERG BERT , WOLD SVANTE , ANDREWS PETER
Published: August 2010

The information contents in previously published peptide sets was compared with smaller sets of peptides selected according to statistical designs. It was found that minimum analogue peptide sets (MAPS) constructed by factorial or fractional factorial designs in physicochemical properties contained substantial structure‐activity information. Although five to six times smaller than the originally published peptide sets the MAPS resulted in QSAR models able to predict biological activity. The QSARs derived from a MAPS of nine dipeptides, and from a set of 58 dipeptides inhibiting angiotensin converting enzyme were compared and found to be of equal strength. Furthermore, for a set of bitter tasting dipeptides it was found that an incomplete MAPS of 10 dipeptides gave just as good a model as the model based on a set of 48 dipeptides. By comparison other non‐designed sets of peptides gave QSARs with poor predictive power. It was also demonstrated how MAPS centered on a lead peptide can be constructed as to specifically explore the physicochemical and biological properties in the vicinity of the lead. It was concluded that small information‐rich peptide sets MAPS can be constructed on the basis of statistical designs with principal properties of amino acids as design variables.
General method for rapid synthesis of multicomponent peptide mixtures
Authors: FURKA ÁRPÁD , SEBESTYÉN FERENC , ASGEDOM MAMO , DIBÓ GÁBOR
Published: August 2010

A method is suggested for the synthesis of multicomponent peptide mixtures. The method is a solid phase synthesis modified in order to give a closely equimolar mixture of peptides with predetermined sequences. The main point of modification is that before every coupling cycle the resin is divided into equal parts and each portion is coupled with a different amino acid. Then the portions are mixed and before the next coupling cycle the resin is again distributed into equal portions. The method is illustrated by the synthesis of a mixture of 27 tetrapeptides and that of 180 pentapeptides.
Peptides from chiral Cα,α‐disubstituted glycines
Authors: VALLE G. , CRISMA M. , TONIOLO. C. , POLINELLI S. , BOESTEN W. H. J. , SCHOEMAKER H. E. , MEIJER E. M. , KAMPHUIS J.
Published: August 2010

The molecular and crystal structures of one derivative and three model peptides (to the pentapeptide level) of the chiral Cα,α‐disubstituted glycine Cα‐methyl, Cα‐isopropylglycine [(αMe)Val] have been determined by X‐ray diffraction. The derivative is mClAc‐l‐(αMe)Val‐OH, and the peptides are Z‐l‐(αMe)Val‐(l‐Ala)2‐OMe monohydrate, Z‐Aib‐L‐(αMe)Val‐(Aib)2‐OtBu, and Ac‐(Aib)2‐l‐(αMe)Val‐(Aib)2OtBu acetonitrile solvate. The tripeptide adopts a type‐I β‐turn conformation stabilized by a 1 ← 4N‐H . O=C intramolecular H‐bond. The tetra‐ and pentapeptides are folded in regular right‐handed 310‐helices. All four L‐(αMe)Val residues prefer φ, Ψ angles in the right‐handed helical region of the conformational map. The results indicate that: (i) the (αMe)Val residue is a strong type‐I/III β‐turn and helix former, and (ii) the relationship between (αMe)Val chirality and helix screw sense is the same as that of Cα‐monosubstituted protein amino‐acids. The implications for the use of the (αMe)Val residue in designing conformationally constrained analogues of bioactive peptides are briefly discussed.
Conformation and inhibitory properties of peptides based on the tissue kallikrein‐aprotinin complex
Authors: DESHPANDE MILIND S. , BOYLAN JOHN , HAMILTON JAMES A. , BURTON JAMES
Published: August 2010

A series of peptides encompassing the primary binding segment (residues 12–19) of aprotinin has been synthesized and tested for their ability to inhibit procine pancreatic kallikrein. A minimum sequence of five amino acids spanning residues 12‐16 of aprotinin is necessary for inhibition of porcine pancreatic kallikrein. An octapeptide homologous with the binding segment of aprotinin has a Kivalue of 1.2 × 10−4M. The solution structure of the octapeptide was studied by one‐ and two‐dimensional NMR methods for comparison with the known structure of the segment of aprotinin that contacts tissue kallikrein. NMR experiments suggest that the peptide is either a random coil or that it samples several conformations on the NMR time scale. Analysis of the molecular dynamics trajectory of the octapeptide also suggests that the peptide is highly flexible. Thus, inhibition by the octapeptide occurs because of its homology with residues 12‐19 of aprotinin. Moreover, the absence of a stable solution conformation similar to that of the binding segment of aprotinin is consistent with the 150 000‐fold increase in Ki of the octapeptide compared to intact aprotinin.
Fmoc/solid‐phase synthesis of Tyr(P)‐containing peptides through t‐butyl phosphate protection
Authors: PERICH JOHN W. , REYNOLDS ERIC C.
Published: August 2010

The synthesis is of Tyr(P)‐containing peptides by the use of Fmoc‐Tyr(PO3Me2)‐OH in Fmoc/solid phase synthesis is complicated since, firstly, piperidine causes cleavage of the methyl group from the ‐Tyr(PO3 Me2)‐residue during peptide synthesis and, secondly, harsh conditions are needed for its final cleavage. A very simple method for the synthesis of Tyr(P)‐containing peptides using t‐butyl phosphate protection is described. The protected phosphotyrosine derivative, Fmoc‐Tyr(PO3tBu2)‐OH was prepared in high yield from Fmoc‐Tyr‐OH by a one‐step procedure which employed di‐t‐butyl N,N‐diethyl‐phosphoramidite as the phosphorylation reagent. The use of this derivative in Fmoc/solid phase peptide synthesis is demonstrated by the preparation of the Tyr(P)‐containing peptides, Ala‐Glu‐Tyr(P)‐Ser‐Ala and Ser‐Ser‐Ser‐Tyr(P)‐Tyr(P).
Sulfonation of arginine residues as side reaction in Fmoc‐peptide synthesis
Authors: BECK‐SICKINGER ANNETTE G. , SCHNORRENBERG GERD , METZGER JÖRG , JUNG GÜNTHER
Published: August 2010

Several arginine‐rich peptides containing the C‐terminus of neuropeptide Y (NPY) were prepared by solid phase peptide synthesis using Fmoc chemistry and cleaved from the resin with trifluoroacetic acid (TFA). The products were characterized by fast atom bombardment‐MS, LC‐thermospray‐MS, ion spray‐MS/MS, and Edman degradation. The side products could be identified as peptides with sulfonated arginine residues resulting from an unexpected cleavage of Mtr or Pmc protecting groups. The degree of sulfonation depended on the choice and composition of the cleavage solution. Several scavenger mixtures were used and a mixture of thioanisole/thiocresol was found to be the most efficient for suppressing sulfonation. Furthermore treatment with the enzyme arylsulfate‐sulfohydrolase desulfonated the peptides yielding the correct sequence.
Chemical synthesis of human β‐endorphin(1–27) analogs by peptide segment coupling
Authors: CHENG HEUNG‐CHIN , YAMASHIRO DONALD
Published: August 2010

[Gly8]βhEP(1–27)NH2 and [l‐Leu8]βhEP(1–27)NH2, two analogs of human β‐endorphin, were synthesized by both all‐stepwise solid phase synthesis and peptide segment coupling. For the peptide segment coupling method, two thiocarboxyl peptides, Msc‐[Gly8]βhEP(1–8)SH and Msc‐[l‐Leu8]βhEP(1–8)SH, were synthesized by standard solid phase method on 4‐[α‐(Boc‐Gly‐S)benzyl]phenoxyacetamidomethy‐resin and 4‐[α‐(Boc‐l‐Leu‐S)benzyl]phenoxyacetamidomethy‐resin. These two thiocarboxyl peptides were coupled to H‐[Lys(Cit)9, 19, 24]‐βhEP(9–27)NH2 [Gly8]βhEP(1–27)NH, and [l‐Leu8]βhEP(1–27)NH2 were obtained after removal of Msc groups and citraconyl groups from products of the segment coupling reaction. The yields of both [Gly8]βhEP(1–27)NH2 and [l‐Leu8]βhEP(1–27)NH2 in the segment coupling reaction were approximately 18%. Less than 1 % of racemization of Leu‐8 occurred during coupling of Msc‐[l‐Leu8]βhEP(1–8)SH to H‐[Lys(Cit)9, 19, 24]‐βh EP(9–27)NH2. Results of amino acid composition analysis, analysis by reverse phase high pressure liquid chromatography and receptor binding activity assays of the analogs showed that peptide analogs prepared by segment coupling method and those prepared by all‐stepwise solid phase synthesis were identical. Results of receptor binding activity assays suggested that the molecular charge properties of β‐endorphin(1–27) and its analogs influenced the receptor binding activity.
Aminopeptidase resistant Arg‐Gly‐Asp analogs are stable in plasma and inhibit platelet aggregation
Authors: FOK K.AM F. , PANZER‐KNODLE SUSAN G. , NICHOLSON NANCY S. , TJOENG FOE S. , FEIGEN LARRY P. , ADAMS STEVEN P.
Published: August 2010

Tetrapeptides containing the sequence Arg‐Gly‐Asp (RGD) antagonize fibrinogen binding to its platelet receptor (gp IIb/IIIa, integrin α11bβ3) and inhibit platelet aggregation in vitro. The peptides RGDS and RGDY(Me)‐NH2 were rapidly degraded when incubated in human, rat, and dog plasma. HPLC analysis indicated that amino acids were sequentially removed from the peptide N‐terminus, and this degradation was prevented by the aminopeptidase inhibitor bestatin. Analogs of RGDY(Me)‐NH2 with an acetylated or deleted α‐amino group were prepared. Both analogs were stable when incubated in plasma, blocked 125I‐fibrinogen binding to activated platelets (IC50= 10–30μm) and inhibited ADP induced platelet aggregation (IC50= 10–30μm). This study concludes that aminopeptidase rapidly degrades RGD peptides in plasma, an important issue for in vivo testing of RGD peptides and analogs. RGD analogs intrinsically stabilized against aminopeptidase are stable in plasma and are important tools for antithrombotic studies involving antagonism of gp IIb/IIIa.
Acid‐base properties of thymopoietin‐type tri‐ and tetrapeptides and their derivatives
Authors: NOSZÁL BÉLA , KASSAI‐TÁNCZOS RÓZSA , NYÍRI JUDIT , NYÉKI OLGA , SCHÕN ISTVÁN
Published: August 2010

The submolecular basicities of 21 immuno‐modulating, thymopoietin‐type di‐, tri‐, and tetrapeptides were studied and characterized in terms of group constants and partial microconstants. All compounds were derivatives of the H‐Arg‐Lys‐Asp‐OH tripeptide. Modifications within four covalent bonds of the basic site (esterification, acylation, curtailment or addition at C‐terminal end, exchange of amino acids) cause significant changes in the scheme of protonation and in the individual basicity of proton binding sites. Configurational changes of the component amino acids, however, do not cause significantly different basicities in the diastereomers.
Primary structure of alpaca growth hormone
Authors: De JIMÉNEZ BONINO MIRTHA BISCOGLIO , De NUÉ INÉS ARNAO , ORÉ RAQUEL , SÁNCHEZ DORIS , FERRARA PASCUAL , CAPDEVIELLE JOEL , CASCONE OSVALDO
Published: August 2010

Reduced and carbamidomethylated alpaca growth hormone was submitted to tryptic digestion. Peptides in the mixture were purified by reverse phase HPLC and N‐terminal determination and an amino acid analysis of each was performed. Data obtained and the already known primary structure of the equine growth hormone allowed the assembly‐by homology‐of a definite sequence of amino acids for the polypeptide chain of the protein. Present data provide further information about the relationship between growth factors.
Stereocontrolled synthesis of Ψ[CH=CH] dipeptide isosteres
Authors: KEMPF DALE J. , WANG XIU CHUN , SPANTON STEPHEN G.
Published: August 2010

A new Stereocontrolled synthesis of the Ψ[CH=CH] dipeptide isostere is described. They key step of the sequence relies on the stereospecific α‐alkylation of δ‐amino‐γ‐mesyloxy‐α,β‐unsaturated esters. The broad availability of nucleophilic α‐side chains by this method allows the preparation of a wide variety of Ψ[CH=CH] isosteres with predictable stereochemistry.
Circular dichroic and 1 H‐NMR studies on the aged form of bovine plasma albumin
Authors: ERA SEIICHI , KUWATA KAZUO , SOGAMI MASARU , KATO KAZUO , WATARI HIROSHI
Published: August 2010

Bovine plasma albumin (BPA) has 17 disulfide bonds and approximately one SH group at Cys‐34 which catalyzes the intramolecular SH, S‐S exchange reaction in the alkaline region at low ionic strength, resulting in the formation of the aged form (A‐form). 1) Fractions of α‐helix (fα) and β‐form (fβ) of iodoacetamide‐blocked non‐aged BPA (IA‐BPA) at pH 6.5 (the N‐form) and 9.0 (the B‐form) in the absence of added salt were 0.70, 0.12 and 0.62, 0.18, respectively (Era et al. (1990)). However, there were no changes in fα and fβ of the iodoacetamide‐blocked A‐form (IA‐A‐form) over the pH range from 5.5 to 9.1 in the absence of added salt or in 0.10 m KCl(fα∼ 0.60, fβ∼ 0.20), indicating that the secondary structure of the IA‐A‐form might be similar to that of non‐aged IA‐BPA at pH 9.0 (B‐form) in the absence of added salt, that is, the frozen B‐form, stabilized covalently by the repairing of disulfide bonds. 2) The rigidity of the A‐ and IA‐A‐forms, as monitored by cross‐relaxation times between irradiated and observed protein protons, was similar to or slightly higher than that of non‐aged IA‐BPA or BMA. However, spin‐echo 1H‐NMR spectra indicated that side chains of the IA‐A‐form, such as ɛ‐CH2 of Lys, ‐CH3, in the absence of added salt are more mobile than those of non‐aged IA‐BPA. 3) The ratio of the hydrodynamic volume of the A‐form to that of non‐aged BPA at pH5.5 was approximately 1.1.
Design and synthesis of nonpeptide mimetics of jaspamide
Authors: KAHN MICHAEL , NAKANISHI HIROSHI , SU TING , LEE JESSIE Y.‐H. , JOHNSON MICHAEL E.
Published: August 2010

Jaspamide is a novel metabolite of mixed peptide/polyketide biosynthesis that was isolated from sponges of the genus Jaspis, and that has been reported to exhibit both insecticidal and antifungal activity. We have evaluated three nonpeptide mimetic designs, and have synthesized a nonpeptide mimic of the proposed bioactive region to investigate the structure activity relationship. Structural investigations of potential mimetics, utilizing molecular modeling in conjunction with spectroscopic and crystallographic data, indicate that positioning of the critical functional groups in two mimetics corresponds closely to that observed in jaspamide, and that the flexibility of mimetic 4 approximates that of jaspamide. Initial biological evaluation suggests that lactam mimetic 4 exhibits a biological profile similar to jaspamide.
Conformational analysis of peptide surrogates
Authors: DAUBER‐OSGUTHORPE PNINA , CAMPBELL MALCOLM M. , OSGUTHORPE DAVID J.
Published: August 2010

We have investigated the conformational effects of modifying the amide link of peptides. We studied a reverse amide bond Ψ[NHCO], a reduced amide bond Ψ[CH2NH] and a retro‐reduced bond Ψ[NHCH2] as surrogates for the amide link [CONH] in native peptides. A complete search of the conformational space available to residues with these modified links was carried out. The local minima and the rotational barriers were described and compared to the minima of the native residue. The results are compatible with the available observed structural data. These modified links have been incorporated in secondary structure units such as β turns, α helices, and parallel and anti‐parallel β sheets. It was found that a reduced amide link can lead to stabilised β turns, while the retro modification can be incorporated in stable β sheets. A significant reduction in the stability of α helices is caused by the retro links, while a reduced amide link results in only a small destabilisation.
Processed forms of neuroendocrine proteins 7B2 and secretogranin II are found in porcine pituitary extracts
Authors: LAZURE C. , BENJANNET S. , SEIDAH N.G. , CHRÉTIEN M.
Published: August 2010

The complete structure of the novel polypeptide 7B2 recently deduced from cDNA clones has been reported to be highly conserved in a variety of species. The deduced amino acid sequence of the mature protein is predicted to be 185 or 186 amino acids long. While its biological role is still unknown, its occurrence in neuroendocrine secretory granules has been largely documented. This report shows: (i) that the protein, isolated from a large quantity of porcine pituitary glands, does not correspond to the full predicted cDNA structure but, on the contrary, to a truncated form; (ii) that the latter could arise from proteolytic cleavage at position ISO following pairs of basic residues; (iii) that it contains an extra residue at position 100 which is absent in the cDNA sequence; and, finally, (iv) that it displays a higher than expected molecular weight on SDS‐polyacrylamide gel electrophoresis. In addition, a copurifying peptide was identified as an NH2‐terminal related fragment of the secretogranin II molecule. Protein sequencing of the latter demonstrates (i) that the correct amino terminus of mature porcine secretogranin II is an Ala residue and not the previously proposed Gin residue and (ii) that this fragment could also arise from proteolytic cleavage at a pair of basic residues located within the secretogranin II sequence.
Solution synthesis of the glyco‐hex a peptide sequence of the human oncofetal fibronectin defined by monoclonal antibody FDC‐6
Authors: GOBBO M. , BIONDI L. , FILIRA F. , ROCCHI R.
Published: August 2010

The glyco‐hexapeptide sequence H‐Val‐(GalNAc‐α)Thr‐His‐Pro‐Gly‐Tyr‐OH, was synthesized in solution by the segment condensation procedure and the stepwise procedure. A peracetylated, O‐galactosaminyl threonine derivative was used for incorporating the glycosylated amino acid residue into the peptide chain. A consistent racemization occurred during the acylation of H‐His‐Pro‐Gly‐Tyr(Bzl)‐OBzl with Z‐Val‐[GalNAc(Ac)3‐α]Thr‐OH by the BOP‐HOBt procedure and the D‐allothreonine containing glycohexapeptide was isolated in about 20% yield. Stepwise elongation of the C‐terminal tetrapeptide with Frnoc‐[GalNAc(Ac)3‐a]Thr‐OH and Z‐Val‐OH, in the presence of the same coupling reagents, yielded the l‐threonine containing diastereoisomer without detectable racemization. A side product, the Nim‐ethoxy‐carbonylated hexapeptide derivative, formed during the EEDQ‐mediated condensation of Fmoc‐[GalNAc(Ac)3‐α]Thr‐OH with the C‐terminal tetrapeptide, was isolated and characterized. Preliminary studies showed that the synthetic glycohexapeptide is a good competitive inhibitor of the binding of the FDC‐6 monoclonal antibody to the oncofetal fibronectin, supporting the idea that it should represent the minimum essential structure required for the FDC‐6 activity.
Effect of reductive alkylation on thermal stability of ribonuclease A and chymotrypsinogen A
Authors: FUJITA YUKIHISA , NODA YUKINAO
Published: August 2010

In order to probe changes in the structural stability induced by the introduction of hydrophobic groups into proteins, the amino groups of ribonuclease A and chymotrypsinogen A were reductively alkylated by reaction with various aliphatic aldehydes, formaldehyde, acetaldehyde, n‐butylaldehyde and n‐hexylaldehyde, and their thermal stabilities were investigated by differential scanning calorimetry (DSC) at different acidic pH values. Ribonuclease A was thermally unstabilized by reductive alkylation, while chymotrypsinogen A was slightly stabilized, depending on both the size of the introduced alkyl groups and the extent of modification. These observations suggest that the effects induced by alkylation involve not only steric hindrance due to the entering bulky groups but also certain other factors such as the participation of the chemically introduced alkyl groups in hydrophobic interactions.
Crystal and molecular structure of the dehydropeptide Ac‐ΔPhe‐Val‐ΔPhe‐NH‐Me
Authors: CIAJOLO M. ROSARIA , TUZI ANGELA , PRATESI CLAUDIO R. , FISSI ADRIANO , PIERONI OSVALDO
Published: August 2010

The dehydropeptide Ac‐ΔPhe‐l‐Val‐ΔPhe‐NH‐Me, containing two dehydrophenylalanine (ΔPhe) residues, crystallizes from methanol/water in space group P212121 with a= 12.622 (1), b= 12.979 (1), and c= 15.733 (1) Å. In the solid state, the molecular structure is characterized by the presence of two intramolecular hydrogen bonds which form two consecutive β‐bends. The (φ,Ψ) torsion angles of the three residues are very similar and close to the standard values of type III β‐bends, so the molecular conformation corresponds to an incipient right‐handed 310 ‐helix, only slightly distorted. In the crystal, the molecules are linked by head‐to‐tail hydrogen bonds, thus forming continuous helical columns packed in antiparallel mode. There are no lateral hydrogen bonds; the only interactions are hydrophobic contacts between the apolar side chains of neighboring helical columns.
Studies on asymmetric induction associated with the coupling of N‐acylamino acids and N‐benzyloxycarbonyldipeptides
Authors: BENOITON N. LEO , LEE YOUNG C. , CHEN FRANCIS M.F.
Published: August 2010

The asymmetric induction occurring during aminolysis by an amino acid benzyl ester of the 5(4H)‐oxazolones obtained from N‐acyl‐dl‐valine for acyl = formyl, acetyl, benzoyl, trifluoroacetyl and A′‐benzyloxycarbonyl‐Xaa where Xaa = Gly, Ala and Leu in dichloromethane and dimethylformamide at + 5° and – 5° was determined by analysis of the epimeric products by high‐performance liquid chromatography after removal of protecting groups by hydrogenolysis. The influence of the side‐chain of the activated residue on induction was assessed by examining aminolysis of the 5(4H)‐oxazolones from N‐benzyloxycar‐bonyl giycyl‐Xaa‐OH for Xaa = Ala, Leu, Val, and Phe. The contribution of induction to the epimeric content of products obtained from couplings mediated by N,N′‐dicyclohexylcarbodiimide in the presence and absence of 1‐hydroxybenzotriazole, and by the mixed‐anhydride method, were calculated. The induction was affected at varying levels by the nature of the N‐acyl group, the side‐chain of Xaa, the nature of the aminolyzing nucleophile, the nature of the solvent, and the temperature, with diastereomeric excesses reaching – 32 and +53. The influence of the side‐chain of Xaa on the induction was different in the two solvents. For the N‐acyl series, the epimeric content of products did not always correctly reflect the relative tendencies of the derivatives to racemize. The order for epimeric content of the products also depended on the method of coupling.
Conformation of a neurokinin antagonist in solution
Authors: MALIKAYIL J. A. , HARBESON SCOTT L.
Published: August 2010

The hexapeptide [cyclo(Leu1Ψ(CH2NH2)Leu2‐Gln3‐Trp4‐Phe5‐Gly6)] +1 is a potent antagonist of neurokinin A activity in tissues of hamster urinary bladder. The solution conformation of this cyclic hexapeptide has been characterized by the combined use of two dimensional nuclear magnetic resonance spectroscopy and restrained molecular dynamics. The proton spectrum of the peptide was fully assigned by the sequential assignment procedure. Interproton distances were derived from crosspeak volumes in two dimensional Nuclear Overhauser Effect spectra, and dihedral angles were calculated from appropriate coupling constants. Temperature coefficients of the amide protons were determined. Restrained molecular dynamics simulations were carried out using the backbone interproton distances as constraints. During 210 ps of restrained molecular dynamics the peptide interconverted among three closely related families of conformations. These interconversions occurred at picosecond timescales under the simulation conditions.
Racemization free coupling of peptide segments
Authors: XAUS N. , ALBERICIO F. , JORBA X. , CALVET S. , CLAPÉS P. , TORRES J.L. , VALENCIA G.
Published: August 2010

The total synthesis of the insect neuropeptide derivative Z‐Gly‐Gly‐Ser‐Leu‐Tyr‐Ser‐Phe‐Gly‐Leu‐NH2 has been carried out by a convergent solid phase strategy. For the coupling of the N‐terminal pentapeptide to the C‐terminal tetrapeptide, three different methods were assayed. Racemization of the acyl activated amino acid during the fragment condensation reaction was monitored by HPLC. Best results were obtained by enzymatic coupling in a low water containing media using adsorbed α‐chymotrypsin. An optically pure product was obtained in 82% yield after 1 h of reaction. Chemical methods such as DIC/HOBt and BOP/HOBt NMM always rendered highly optically impure products containing 10‐20% of the d‐epimer.
Design, synthesis and structural characterization of model heterodimeric coiled‐coil proteins
Authors: ZHU BING‐YAN , ZHOU NIAN E. , SEMCHUK PAUL D. , KAY CYRIL M. , HODGES ROBERT S.
Published: August 2010

We report the design and synthesis of model heterodimeric coiled‐coil proteins and the packing contribution of interchain hetero‐hydrophobic side‐chains to coiled‐coil stability. The heterodimeric coiled‐coils are obtained by oxidizing two 35‐residue polypeptide chains, each containing a cysteine residue at position 2 and differing in amino acid sequences in the hydrophobic positions (“a” and “d”) responsible for the formation and stabilization of the coiled‐coil. In each peptide, a single Ala residue was substituted for Leu at position “a” or “d”. The formation and stability of heterodimeric coiled‐coils were investigated by circular dichroism studies in the presence and absence of guanidine hydrochloride and compared to the corresponding homodimeric coiled‐coils. The coiled‐coil proteins with an Ala substitution at position “a” were less stable than those with an Ala substitution at position “d” in both the homodimeric (Ala‐Ala interchain interactions) and heterodimeric (Leu‐Ala interchain interactions) coiled‐coils. The 70‐residue disulfide bridged peptides (homoand heterodimeric coiled‐coils) can be readily separated by reversed‐phase chromatography (RPC) even though they have identical amino acid compositions as well as in the hydrophobic “a” and “d” positions. The elution of the 70‐residue peptides prior to their corresponding 35‐residue monomers suggests that these proteins are retaining a large portion of their coiled‐coil structure during RPC at pH 2 and their retention behavior correlates with protein stability.
Effects of resin swelling and substitution on solid phase synthesis
Authors: PUGH KATHLEEN C. , YORK EUNICE J. , STEWART JOHN M.
Published: August 2010

Resins sold by several companies were examined for swelling, uniformity of beads, and substitution in the case of resins sold with the first amino acid attached. Effects of resin swelling, uniformity, and substitution on the solid phase synthesis of long, structured and/or branched peptides were evaluated.