bluuebunny/crossref_metadata_2025 huggingface dataset:
Side reaction in peptide synthesis
Authors: CHEN SHUI‐TEIN , LO LEE‐CHIANG , WU SHIH‐HSIUNG , WANG KUNG‐TSUNG
Published: August 2010

2‐Oxazolidone derivatives formed through an intramolecular reaction in the process of alkaline treatment of urethane‐type N‐protected peptides of which the N‐terminal residues were Ser or Thr having unprotected hydroxyl groups. In order to avoid this side reaction, the esters of these peptides could be cleaved by enzymatic hydrolyses instead of saponification.
Efficient solution phase synthesis of [l‐(β‐mercapto‐β, β‐cyclopentamethylenepropionic acid)‐ 2‐(O‐ethyl‐d‐tyrosine)‐4‐valine‐9‐desglycine]arginine vasopressin
Authors: MENDELSON WILFORD L. , TICKNER ANN M. , HOLMES MONICA M. , LANTOS IVAN
Published: August 2010

A convergent synthesis of the peptide [1‐(β‐mercapto‐β,β‐cyclopentamethylenepropionic acid)‐2‐(O‐ethyl‐d‐tyrosine)‐4‐valine‐9‐desglycine]arginine vasopressin (1), based on the classical solution phase method, was developed. The molecule is assembled by a 3 + 4 coupling via the amide method; then the disulfide bridge is installed by iodine treatment of the bis‐acetamidomethyl protected thiols, and the terminal arginine amide added by a 7 + 1 coupling. The method (see Scheme 1) has been used to prepare gram quantities of 1 in more than 98% purity and in 13% yield (based on tetrapeptide intermediate 13) after a single stage purification. The method appears to be particularly suitable for the large scale preparation of 1 and other vasopressin congeners. A novel, albeit low level, transfer of acetamidomethyl group from the sulfur of cysteine to the asparagine amide side‐chain was detected following hydrogen chloride treatment of Boc‐containing intermediates.
Separation of a complete set of basic, diastereomeric pentapeptides by reversed‐phase ion‐pair chromatography
Authors: NYASSE BARTHÉLÉMY , FRANSSON BENGT , RAGNARSSON ULF
Published: August 2010

The resolving power of liquid chromatography systems containing 1‐pentane sulfonate, previously used to analyze basic hydrophilic peptides related to substrates of cAMP‐dependent protein kinase, has been studied with respect to diastereomeric peptides. A set of peptides comprising RRASV and its five diastereomers containing one D‐amino acid were used as model compounds. Complete resolution of all the peptides could be accomplished both with ethanol and acetonitrile as organic modifiers. The separation of the various peptides within the set turned out to be only modestly changed under the conditions investigated. These new results clearly demonstrate the potential of the chromatography systems studied.
Solution phase synthesis of Saccharomyces cerevisiae a‐mating factor and its analogs
Authors: XUE CHU‐BIAO , EWENSON ARIEL , BECKER JEFFREY M. , NAIDER FRED
Published: August 2010

The solution phase synthesis of the Saccharomyces cerevisiae a‐mating factor and nonfarnesylated and nonmethylated a‐factor analogs are reported. The a‐factor, a lipopeptide with the sequence Tyr‐Ile‐Ile‐Lys‐Gly‐Val‐Phe‐Trp‐Asp‐Pro‐Ala‐Cys(S‐Farnesyl)OCH3 was synthesized by the condensation of the amine terminal protected decapeptide with the carboxyl terminal farnesylated dipeptide using benzotriazol‐l‐yl‐oxy‐tris‐(dimethylamino)‐phosphonium hexafluorophosphate (BOP reagent) as the coupling agent. The synthesis of the decapeptide involved 5 + 5 fragment coupling with the BOP reagent and the successful application of 9‐fluorenylmethyl ester(OFm) and 9‐fluorenylmethoxycarbonyl(Fmoc) groups for the protection of Asp and Lys side chains and Tyr α‐amine and of phenacyl esters (OPa) for α‐carboxyl protection. The OFm and Fmoc groups tolerated repeated couplings and were completely stable to zinc powder in acetic acid, a condition under which the OPa group was removed. The synthesis of the nonfarnesylated a‐factor was accomplished by the coupling of the decapeptide with tetrapeptide (Ala‐CysOCH3)2 followed by the deprotection of the OFm and Fmoc groups with piperidine and the cleavage of the disulfide bond with zinc powder in acetic acid. The nonmethylated a‐factor was prepared by 10 + 2 fragment coupling using OFm protection of the dipeptide carboxyl group followed by removal of all protecting groups with piperidine. Attempts to saponify a‐factor were not successful. The synthetic nonfarnesylated and nonmethylated a‐mating pheromones were 100‐1000 times less active than the a‐factor, indicating that although the methyl ester and the farnesyl group are not essential for biological activity, they are necessary for high potency.
Synthesis and properties of cholecystokinin‐releasing peptide (monitor peptide), a 61‐residue trypsin inhibitor
Authors: LIN YAO‐ZHONG , ISAAC DANIEL D. , TAM JAMES P.
Published: August 2010

A 61‐residue cholecystokinin‐releasing peptide (monitor peptide), which was obtained from rat pancreatic juice and found to stimulate pancreatic enzyme secretion, was recently reported to inhibit bovine trypsin and to possess epidermal growth factor (EGF)‐like activities, at a concentration of about 10nM. However, monitor peptide is structurally different from the EGF family of growth factors. To investigate whether monitor peptide contains the supposed EGF‐like activities, it has been synthesized together with its [Ala23, Ala47] analog. The purified peptides, which were fully characterized by a range of methods including Cf‐252 ionization mass spectrometry and enzymatic digestion to establish the locations of disulfide linkages, were shown to belong to the pancreatic secretory trypsin inhibitor family and not to the EGF family. Neither synthetic monitor peptide nor its analog were able to compete with 125I‐EGF in A‐431 cells or to stimulate growth of Swiss 3T3 and NRK 49F cells, up to 1 μM concentration. However, synthetic monitor peptide was as effective as the native product in the inhibition of trypsin. Replacement of the essential Arg23 in the [Ala23, Ala47]‐analog led to loss of trypsin inhibition activity.
Human recombinant CuZn‐superoxide dismutase
Authors: PERETZ MOSHE , WERBER MOSHE U. , BURSTEIN YIGAL
Published: August 2010

The complete amino acid sequence of recombinant human Cu‐Zn superoxide dismutase (CuZnSOD) is presented. The S‐carboxymethylated protein was cleaved at lysine residues (with Achromobacter protease I) to provide a set of nine non‐overlapping fragments accounting for 90% of the sequence. These fragments were then overlapped and aligned, and the sequence was completed by using peptides generated by cleavage at glutamic acid residues (with S. aureus V8 protease) and at arginine (with clostripain). The recombinant protein contains a single disulfide bond between cysteine residues 57 and 146. The primary sequence of recombinant human CuZnSOD is identical to that predicted by its cDNA sequence.
Synthetic peptides for Plasmodium vivax malaria sero‐epidemiology
Authors: BIANCHI ELISABETTA , GIUDICE GIUSEPPE DEL , VERDINI ANTONIO S. , PESSI ANTONELLO
Published: August 2010

The immunodominant epitope of Plasmodium vivax, one of the major causative agents of malaria in man, consists of the tandem repetitions of a nonapeptide sequence, AspArgAlaAsp/AlaGlyGlnProAlaGly, with Asp (variant d) or Ala (variant a), in the fourth position. Synthetic peptides corresponding to the P. vivax epitope, containing a different number of nonapeptide sequences, were prepared by solid‐phase synthesis according to the Fmoc‐polyamide method. Three peptides, containing 1, 2, and 4 copies of the d variant, were assembled on the gel polymer; none of these peptides, however, was suitable for P. vivax sero‐epidemiology. A 45‐peptide containing both the d and a variants, ddaad, was prepared by continuous‐flow Fmoc‐polyamide (flow‐polyamide). Among the cleavage procedures evaluated for the removal of the five Mtr groups only TFMSA/TFA/ 1,2‐ethanedithiol (1:89:10 by vol.) brought deblocking to completion; a substantial level of impurities originated, however, from these procedures. The product was purified by reversed‐phase displacement chromatography, a technique only recently applied to peptides, which shows distinct advantages over conventional, linear elution chromatography. In a single experiment, 107 mg of the crude mixture were loaded onto an analytical column (250 × 4 mm), obtaining in purified form 85% of the desired material present in the sample. An ELISA test base on the ddaad peptide was developed and is being applied to the sero‐epidemiology of P. vivax malaria.
Synthesis and biological activities of angiotensin II and Sarmesin analogues containing cyclohexylalanine
Authors: HONDRELIS JOHN , MATSOUKAS JOHN , CORDOPATIS PAUL , GANTER RENEE C. , FRANKLIN KEVIN J. , MOORE GRAHAM J.
Published: August 2010

Analogues of angiotensin II with cyclohexylalanine (Cha) at position 4 or 8, and analogues of the competitive (type II) angiotensin antagonist [Sar1,Tyr(Me)4]ANG II (Sarmesin) with Cha at position 8, have been prepared by the solid phase method and purified by reversed‐phase HPLC. Analogues of ANG II with Cha at position 8 in which the position 1 residue was substituted with sarcosine (Sar) or amino‐isobutyric acid (Aib) or was deleted (Des), were slowly reversing (Type I) antagonists with „pA2” values in the rat isolated uterus assay of ∼ 8.5. The additional substitution of Tyr(Me) for Tyr at position 4 of these peptides gave reversible competitive (Type I/II) antagonists with pA2 values of 6.7, 5.8, and < 5, while substitution of Phe for Tyr gave pA2 values of 7.4, 6.7, and < 5, respectively. All 19 peptides synthesized in this study had low intrinsic agonist activity in the rat isolated uterus assay except for the type I antagonists [Sar1,Cha8]ANG II (7%), [Aib1, Cha8]ANG II (12%) and [Des1,Cha8]ANG II (20%). These data illustrate that the substitution of Cha at position 8 of ANG II analogues produces potent antagonists; however, Type I antagonists retain significant agonist activity whereas Type I/II antagonists do not. In contrast, substitution of Cha at position 4 in a variety of ANG II analogues resulted in severely diminished biological activity, illustrating that the presence of an aromatic ring quadrupole at position 4 is obligatory for receptor binding and activity.
Spectroscopic analysis of [Trp3]‐β‐casomorphin analogs Comparative structure conformation‐activity studies
Authors: EPPS DE. , HAVEL HA. , SAWYER T.K. , STAPLES D.J. , CHUNG N.N. , SCHILLER P.W. , HARTRODT B. , BARTH A.
Published: August 2010

A series of [3‐tryptophan]‐β‐casomorphin‐5([Trp3]‐β‐CM‐5) analogs were investigated by circular dichroism (CD) and fluorescence spectroscopy to explore their structure‐conformation properties in solution. In addition, the comparative opioid activities of these compounds were evaluated using the in vitro guinea pig ileum (GPI) and mouse vas deferens (MVD) assays. Specifically, the pentapeptide sequence of [Trp3]‐β‐CM‐5, H‐Tyr‐Pro‐Trp‐Pro‐Gly‐OH (I) was modified at Pro‐2 and Pro‐4 by d‐Pro substitutions to provide two diastereometric analogs, [Trp3‐d‐Pro‐4]‐β‐CM‐5 (II) and [d‐Pro2,4,Trp3]‐β‐CM‐5 (III). In the GPI and MVD assays, β‐CM‐5 effected IC50 values of 1.3 μm and 8.9 μm, respectively, which confirmed its known μ/δ‐selectivity on these two peripheral opioid receptor subtypes. The potencies of compounds I, II, and III were 0.2, 2.0, and < 0.005 relative to β‐CM‐5 on the GPI assay. Compounds I and II exhibited pronounced μ/δ‐selectivities (> 18.9‐ and 12.4‐fold respectively), whereas compound III was essentially inactive in both the GPI and MVD assays. CD studies of β‐CM‐5 and its [Trp3]‐β‐CM‐5 analogs showed striking differences in their near‐UV and far‐UV spectra in aqueous or organic solvents. In the far UV CD spectra, weak (20%) α‐helicity (maximum at 193 nm and minima at 208 and 222 nm) for β‐CM‐5 was obtained in trifluoroethanol (TFE); however, none of the [Trp3]‐β‐CM‐5 analogs showed such CD bands. Of potential relevance to γ‐turn or C7 secondary structure was the observation of a strong negative band at 245 nm for compounds II and III which was not solvent‐dependent in H2O or TFE, whereas compound I showed this CD band exclusively in TFE. In the near‐UV CD at 275 nm (Trp electronic transition), the relative order of intensities of this band were determined for the [Trp3]‐β‐CM‐5 compounds to be II > I > III, which was identical to their relative biological potencies in both the GPI and MVD assays. Fluorescence energy transfer (FET) experiments of compounds I‐III provided the intramolecular distances (r) between their Tyr (donor) to Trp (acceptor) side‐chains, by the Förster method, and were as follows: [Trp3]‐β‐CM‐5, r = 10.6Å; [Trp3, d‐Pro4]‐β‐CM‐5, r = 9.6Å; and [d‐Pro2,4,Trp3]‐β‐CM‐5, r = 11.0Å. A rank order correlation existed between the Tyr‐Trp intramolecular distances and biological activity with shorter distance corresponding to higher biological potency. Furthermore, based on the fluorescence lifetime data analysis (Globals software) of the [Trp3]‐β‐CM‐5 analogs, which were best fitted to a double exponential decay model, the relative ranking of long (> 1.5 ns) lifetime fractions of these three compounds was II > I > III. In summary, detailed spectroscopic analysis of three [Trp3]‐β‐CM‐5 diastereomeric analogs by CD and FET have provided intriguing data indicating a possible structure conformation‐activity relationship among these μ/δ‐selective opioid‐mimetic compounds.
Contact area of bovine somatotropin dimer: involvement of tyrosine 142
Authors: OPPEZZO OSCAR J. , FERNANDEZ HORACIO N.
Published: August 2010

The presence of tyrosine residues in the contact area between protomers of bovine somatotropin dimers (Fernandez & Delfino, Biochem. J.209, 107‐115, 1983) was investigated taking advantage of the impaired self‐associating ability of molecules iodinated at such residues. Reaction of bovine somatotropin dissolved in 8 m urea with the NaI‐Chloramine T couple (2.1 × 10−4m) rendered a preparation with 3.1 iodine atoms per molecule which, by stepwise elimination of the denaturant and gel filtration through Sephadex G‐100, originated two distinguishable populations: one able (iododerivatives I), the other unable (iododerivatives II) to self‐associate. After frontal analysis, iododerivatives II were found to be unable to interact even with native molecules. Identification of the reacting tyrosine residues indicated that iodination of tyrosine 142 was responsible for the loss of the ability to form dimers in iododerivatives II. Iodohormones retained the ability to bind to somatogenic mouse hepatocyte receptors ‐ the relative potency for iododerivatives I and II being 0.60 (0.34–1.03) and 0.71 (0.41–1.22) respectively.
Computational analysis of conformational behavior of cholecystokinin fragments
Authors: PATTOU D. , MAIGRET B. , FOURNIE‐ZALUSKI M.‐C. , ROQUES B. P.
Published: August 2010

A conformational analysis has been performed on several peptide fragments (CCK4 to CCK7) of the cholecystokinin neuromodulator. The Monte‐Carlo Metropolis method was used to explore the conformational space of all these flexible units and different electric charge distributions were introduced in order to mimic pH effects. Results agree reasonably well with experimental data from NMR and fluorescence experiments. The CCK4 fragment displays a peculiar conformational behavior when compared to all other longer peptides with short range interaction between the Trp and Phe aromatic side‐chains. Several H‐bonded conformers including C7 or β‐turns are found for CCK5 to CCK7. These findings are correlated to the central and peripheral actions of these compounds and hypotheses concerning the best possible templates for each one are discussed.
Conformations of neurotensin in solution and in membrane environments studied by 2‐D NMR spectroscopy
Authors: XU GUANG‐YI , DEBER CHARLES M.
Published: August 2010

Two‐dimensional HOHAHA and ROESY nuclear magnetic resonance techniques are used to obtain complete proton resonance assignments and to perform a conformational investigation of the neuropeptide neurotensin (pGlu‐Leu‐Tyr‐Glu‐Asn‐Lys‐Pro‐Arg‐Arg‐Pro‐Tyr‐Ile‐Leu) in aqueous solution, methanol, and membrane‐mimetic [deuterated sodium dodecylsulfate (SDS)] environments. Results suggest the absence of discernible elements of secondary structure in water and methanol. ROESY spectra confirm that Lys‐Pro and Arg‐Pro peptide bonds are all‐trans, but that a significant population of cis Arg‐Pro bonds arises in aqueous solution, which increases in the environment of SDS micelles. The conformational ensemble of the peptide is observed to narrow as it becomes bound through its cationic mid‐region to SDS micelles, with the accompanying advent of local extended structure. The overall results indicate the inherent conformational flexibility of neurotensin, and emphasize the environmental dependence of conformation in peptides of medium length.
Conformational analysis and aqueous hydration studies of model peptides for the adhesive protein of the mussel, Mytilus edulis L
Authors: NAGY P.I. , PATEL H.C. , DREYER W. , HOPFINGER A.J.
Published: August 2010

Conformations of model peptides of the adhesive protein of the mussel, Mytilus edulis L were investigated using molecular mechanics. The protein structure was represented as the repeat of a 10‐residue unit. This decamer, and di‐ and tri‐decamers of it, were considered in the modeling. Incorporation of the unusual dopamine residue in the decamer repeat may be explained by its hydrogen bond forming ability via its 3‐OH group to a proline carbonyl oxygen. This bond contributes to maintaining a double reverse β‐turn structure in the decamer. This conformation was found more stable than 31 and α helical conformations. Adjacent reverse β‐turn structures are connected by short segments (2 to 3 residues) having little conformational preference. Thus, the overall protein can possess a significant random nature, yet have a highly ordered embedded conformational component. Hydrophilic character is in line with the larger number of OH groups on the phenyl ring for residue 9 (the site of the Dopa residue). The dehydration free energy of the (3‐OH)‐Phe as compared to the Dopa derivative is less by 1.4kcal per decamer unit. This amounts to more than 100kcal energy gain in the dehydration process for the total protein.
Mapping the receptor‐recognition site of human transforming growth factor‐α
Authors: TAM JAMES P. , LIN YAO‐ZHONG , LIU WEN , WANG DE‐XIN , KE XIAO‐HONG , ZHANG JING‐WEN
Published: August 2010

The receptor‐recognition site human transforming growth factor‐α (TGFα), a 50‐residue tricyclic peptide with three disulfide bonds, was mapped by a set of 46 peptide analogs consisting of linear, monocyclic, bicyclic, and tricyclic structures representing individual and overlapping subdomains of human TGFα. Linear overlapping fragments ranging from 7 to 18 residues and spanning the entire length of TGFα as well as monocyclic analogs with one disulfide linkage were found to be inactive in both receptor‐binding and mitogenic assays. Bicyclic analogs with two disulfide linkage and representing either the amino or carboxyl two‐thirds of TGFα showed low activity at 0.1–0.9 mm concentrations. Tricyclic analogs containing all three disulfide linkages but lacking either the amino or carboxyl terminal heptapeptide was, respectively, 3% and 0.1 % as active as TGFα. These results show that determinants for the receptor binding cannot be represented by a short continuous fragment or a single subdomain, but are located on a discontinuous surface on a folded structure with disulfide restraints. Furthermore, these results when combined with our previous results which shows that the middle subdomain (second disulfide loop) is not involved in the receptor binding suggest that the receptor‐binding residues are constituted of three fragments located at the first and third subdomains as well as the external carboxyl peptide.
Conformation and hydrogen bonding of N‐formylpeptides: crystal and molecular structure of N‐formyl‐l‐alanyl‐l‐aspartic acid†
Authors: SRIKRISHNAN T. , PARTHASARATHY R.
Published: August 2010

Crystals of N‐formyl‐l‐alanyl‐l‐aspartic acid (C8H11N2O6) grown from aqueous methanol solution are orthorhombic, space group, P212121 with cell parameters at 294 K of a = 13.619(2), b = 8.567(2), c = 9.583(3)Å, V = 1118.1Å3, M.W. = 232.2, Z = 4, Dm= 1.38g/cm3 and Dx= 1.378g/cm3. The crystal structure was solved by the application of direct methods and refined to an R value of 0.075 for 1244 reflections with I ≥ 3σ collected on a CAD‐4 diffractometer. The structure contains two short inter‐molecular hydrogen bonds: (i) between the C‐terminal carboxyl OH and the N‐acyl oxygen (2.624(3)Å), a characteristic feature found in many N‐acyl peptides and (ii) between the aspartic carboxyl OH and the peptide oxygen OP1 (2.623(3)Å). The peptide is nonplanar (ω= 165.5(6)°). The molecule takes up a folded conformation in contrast to N‐formyl peptides which form extended β‐sheets; the values of ø1, Ψ1, ø2, Ψ12, and Ψ22 are, respectively –65.7(6), 152.0(5), –107.2(5), 30.9(5), and –150.3(6). The aspartic acid side chain conformation is g− with χ1= 73.1(5). The formyl group, as expected, is transplanar [OF‐CF‐N1‐CA1 = ‐4.0(8)°]. The presence of the short O–H … O hydrogen bond emerges as a structural feature common to this peptide and several other N‐formyl peptides. There are no C‐H … O hydrogen bonds in this structure.
Characterization of growth hormone releasing factor analog expression in Saccharomyces cerevisiae
Authors: CRAIG WILLIAM S. , WONDRACK LILLIAN , SIEGEL ROBERT , PATTHI SARASWATHI , DAVIS GENEVA R. , VELICELEBI GONUL , MOWLES THOMAS F. , THILL GREGORY P.
Published: August 2010

An analog of growth hormone releasing factor (GRF), [Leu27]GRF(1‐40)‐OH, has been expressed and secreted in Saccharomyces cerevisiae under the control of the α‐factor gene promoter and prepro sequence. A single pair of consecutive basic residues served as a processing site between the a‐factor sequences and the GRF sequences. [Leu27]GRF(1‐40)‐OH from fermentor broth containing 20‐30 mg/L of immuno‐reactive peptides was shown to be correctly processed and to possess biological activity as measured in vitro and in vivo. Additional peptides purified from broth appear to result from proteolytic degradation of the original translation product. Analysis of the amino acid compositions and sequences of these peptides suggests that processing enzymes may be responsible for some of the degradation.
Antibacterial peptides designed as analogs or hybrids of cecropins and melittin
Authors: WADE D. , ANDREU D. , MITCHELL S.A. , SILVEIRA A.M.V. , BOMAN A. , BOMAN H.G. , MERRIFIELD R.B.
Published: August 2010

Eight new analogs of cecropin A, two new analogs of melittin and 30 hybrid peptides containing sequences from cecropins and melittin have been synthesized. The lengths of the peptides have varied from 37 residues (the length of cecropin A) to 18 residues. The peptides have been assayed for lysis of sheep red blood cells and for antibacterial activity against two Gram negative and three Gram positive bacteria. The best analogs of cecropin A maintained the anti‐Escherichia coli activity of the parental peptide, and were not lytic for red blood cells. Melittin and its replacement analogs were all lytic for red blood cells, but an analog with transposed segments was not. Several of the hybrid peptides were found to be both non‐hemolytic and highly active against all test bacteria. The data were used to define the structural requirements for antibacterial activity.
Spectroscopic studies on proteinase K and subtilisin DY
Authors: DOLASHKA PAVLINA , FILIPPI BRUNO , WILSON KEITH S. , BETZEL CHRISTIAN , GENOV NICOLAY
Published: August 2010

Circular dichroic spectroscopy has been used to study the effect of pH, guanidinium hydrochloride concentration and temperature on the conformation of the fungal subtilisin‐like proteinase K and the bacterial DY. The ellipticity of the bands in the far ultraviolet region remains almost unchanged in the pH range 3.0‐11.0 (PMS‐proteinase K) and 5.0‐10.0 (PMS‐subtilisin DY). The same ranges of pH stability were determined from the pH dependence of the near ultraviolet dichroic spectra. Hence the changes in the tertiary and secondary structure occur in parallel. Proteinase K is considerably more stable at acidic and somewhat more stable at alkaline pH than subtilisin DY. At neutral pH proteinase K is more resistant to denaturation by guanidinium hydrochloride than is subtilisin DY. The midpoints of the denaturation curves were 6.2 M and 3.2 M guanidinium, respectively. The thermal unfolding of proteinase K occurred at a higher temperature than for subtilisin DY, the transition midpoints being 65° and 48°, respectively. Thus proteinase K is overall a much more robust molecule than subtilisin DY, showing greater resistance to all three forms of denaturation. The differences in the stability of the two proteinases can be partly explained by differences in their calcium binding sites.
Mass balance strategy for protein sequencing
Authors: HUA SUMING , VESTLING MARTHA M. , MURPHY CONSTANCE M. , BRYAYT DUNCAN K. , HEIGHT JUDE J. , FENSELAU CATHERINE , THEIBERT JANET , COLLINS JOHN H.
Published: August 2010

The value of the mass balance strategy is illustrated in the sequence determination of S. aureus V8 protease. Capillary electrophoresis, electrospray mass spectrometry, and high performance tandem mass spectrometry are used as well as proteolysis and Edman degradation. The carboxy terminus is found to contain 17 irregularly repeating units of the triptych motifs NNP and DNP, which provide n challenge to any strategy involving mapping, sequencing, and overlapping of hydrolytic peptides.
An equine growth hormone molecular species lacking the 76‐92 peptidic fragment
Authors: CASCONE OSVALDO , FUKUSHIMA JORGE G. , MIHAJLOVICH VERA , SANTOMÉ JOSÉ A. , DE JIMÉNEZ BONINO MIRTHA RISCOGLIO
Published: August 2010

A new eGH molecular species was isolated and purified by reverse phase HPLC. SDS‐polyacrylamide gel electrophoresis, amino acid composition, and C‐ and N‐terminal determinations support a primary structure identical to that described by Zakin el al. (1976), except for the lack of the 76‐92 peptidic fragment and the maintaining of 30% of its biological activity.
Synthesis of optically pure Cα‐methyl‐arginine
Authors: TIAN ZHENPING , EDWARDS PATRICK , ROESKE ROGER W.
Published: August 2010

Optically pure L‐(+)‐Cα‐methyl‐arginine and D‐(‐)‐C α‐methyl‐arginine were synthesized. Experimental results indicated that DL‐ Cα‐methyl‐arginine methyl ester could be resolved by trypsin, but workup posed a technical difficulty. Chemical resolution at the stage of DL‐Cα‐methyl‐ornithine, followed by selective guanid‐ination using N,N′‐di‐Cbz‐S‐methylisothiourea and hydrogenolysis provided a effective and practical method for the synthesis of optically pure Cα‐methyl‐arginine.
Phosphoryl group participation leads to peptide formation from N‐phosphorylamino acids
Authors: LI YAN‐MEI , YIN YING‐WU , ZHAO YU‐FEN
Published: August 2010

N‐phosphorylated amino acids without a side chain functional group can transfer themselves into peptides after prolonged standing in solvents at warm temperatures. Seven N‐phosphorylpeptides and free peptides were isolated and their structures determined. The phosphoryl group participation is the key to the peptide formation. An intramolecular mixed carboxylic‐phosphoric anhydride intermediate was proposed for this type of reaction which might provide a clue to the function of the phosphoryl group in the phosphorylated enzymes and in the prebiotic synthesis of protein.
Studies on the delineation of the antigenic determinants of chicken riboflavin carrier protein (cRCP)
Authors: MAHALE S.D. , KADAM L.R. , PEREIRA J. , NATRAJ U. , IYER K.S.N.
Published: August 2010

As riboflavin carrier proteins play a critical role in the maintenance of pregnancy, studies on the immunology of these proteins have become very relevant. This paper describes our attempts to identify the sequential antigenic determinants of chicken riboflavin carrier protein (cRCP). Potential surface oriented regions of cRCP were identified by constructing acrophilicity and hydrophilicity profiles of the protein. Of the three regons identified, the pentadecapeptide 10–24 of cRCP forms the subject of the present study. The pentadecapeptide amide was synthesized by solid phase synthesis using Fmoc chemistry. Immunization of rabbits with the peptide‐diphtheria toxoid conjugate elicited high titres of the antipeptide antibodies, revealing the high immunogenicity of the peptide. The antipeptide antibodies bound to both cRCP and reduced carboxymethylated RCP (RCM‐RCP). Antisera to RCM‐RCP showed significant binding to the peptide showing thereby that the region 10–24 of cRCP is perhaps one of the major epitopes of RCM‐RCP. Thus, the studies have resulted in the identification of the region 10–24 as an antigenic determinant of cRCP.
Total synthesis of horse heart cytochrome c
Authors: DI BELLO CARLO , GOZZINI LUIGIA
Published: August 2010

A peptide corresponding to the native (1–66) sequence of horse heart cytochrome c has been synthesized by stepwise automated solid‐phase methods on PAM resin. The course of the synthesis has been monitored by several analytical methods including quantitative ninhydrin and Edman degradation. After HF cleavage, the peptide has been purified by a combination of semipreparative ion‐exchange and RP‐HPLC. The homogeneity of the purified synthetic peptide has been determined by different criteria including HPLC, amino‐acid composition, electrophoresis, antibody binding, tryptic and chymotryptic peptide mapping. After deprotection of the Acm‐Cys residues and CNBr cleavage of the Met65‐Glu66 peptide bond with simultaneous transformation of the Met65 residue into the activated C‐terminal [Hse65lactone, this purified synthetic peptide has been utilized for conformation‐assisted joining experiments in combination with synthetic (66–104) to produce fully synthetic [Hse65]apocytochrome c. This latter, after mitochondria‐mediated stereospecific heme insertion, has given a functional molecule corresponding to native horse heart holocytochrome c.
Efficient solution phase synthesis and use of multiple O‐phosphothreonyl‐containing peptides for calcium phosphate binding studies
Authors: PERICH JOHN W. , And DAVID P. KELLY , REYNOLDS ERIC C.
Published: August 2010

The protected phosphothreonine derivative Boc‐Thr(PO3Ph2)‐OH was prepared in high yield from Boc‐Thr‐OH by a simple three‐step procedure which involved 4‐nitrobenzylcarboxyl protection, either phosphorotriester (diphenyl phosphorochloridate) or “phosphite‐triester” (diphenyl N.N‐diethylphosphoramidite) phosphorylation of the thrconine hydroxyl group of Boc‐Thr‐ONb followed by hydrogenolytic carboxyl deprotection. The three Thr(P)‐containing peptides, H‐Thr(P)‐Glu‐Glu‐NHMe.TFA, H‐Thr(P)‐Thr(P)‐Glu‐Glu‐NHMe.TFA and H‐Thr(P)‐Thr(P)‐Thr(P)‐Glu‐Glu‐NHMe.TFA, were prepared in high yield by the use of Boc‐Thr(PO3Ph2)‐OH in the Boc mode of peptide synthesis (mixed anhydride method) followed by platinum‐mediated hydrogenolytic deprotection of the Thr(PO33Ph2)‐containing peptides. The use of the phosphopeptides in calcium phosphate binding studies showed that the triple Thr(P)‐cluster was a basic structural requirement, since only the pentapeptide was able to bind calcium phosphate efficiently.
Importance of lysine and arginine residues to the biological activity of trichosanthin, a ribosome‐inactivating protein from Trichosanthes kirilowii tubers
Authors: KEUNG W.M. , YEUNG H.W. , FENG Z. , NG T.B.
Published: August 2010

Lysme and arginine residues in trichosanthin were modified with ethyl acetimidate and phenylglyoxal, respectively. The effects of these chemical modifications on the cell‐free protein‐synthesis‐inhibitory activity and the antigen–antibody (Ag–Ab) interaction between trichosanthin and its antibody were examined. Ethyl acetimidate modification abolished the protein‐synthesis‐inhibitory activity of trichosanthin. The inactivation process followed simple first‐order kinetics with an inactivation half‐life of about 18.5 min. Modification of one lysine residue seemed to be responsible for such inactivation. Arginine modification also inactivated trichosanthin. The inactivation process, however, was biphasic with inactivation half‐lives of approximately 15 and 540 min. Arginine modification of trichosanthin had little or no effect on Ag–Ab interaction, whereas lysine modification slightly but significantly weakened Ag–Ab binding.
Synthesis and properties of the peptide corresponding to the ACTH‐like sequence of human immunoglobulin G1
Authors: MITIN Y.V. , NAVOLOTSKAYA E.V. , VASILENKO R.N. , ABRAMOV V.M. , ZAV'YALOV V.P.
Published: August 2010

The decapeptide Val‐Lys‐Lys‐Pro‐Gly–Ser‐Ser‐Val‐Lys‐Val (termed immunocorticotropin) corresponding to the sequence 11–20 of the variable part of the human immunoglobulin G1 heavy chain was synthesized. The ACTH‐like peptide was found to have an ability to increase body temperature. Intracerebroventricular injection of 5–10 μg of the peptide to rabbits induced an elevation of body temperature by 0.7–1.3°C. The ACTH‐like peptide was shown to compete with [125I]‐ACTH (13–24) for binding to receptors on murine brain synaptic membranes and to activate adenylate cyclase of these membranes.
H‐Gly‐Hisψ(NHCO)Lys‐OH, partially modified retro‐inverso analogue of the growth factor glycyl‐L‐histidyl‐L‐lysine with enhanced enzymatic stability
Authors: DALPOZZO ALMA , KANAI KAROLY , KERESZTURI GÉZA , CALABRESE GERARDO
Published: August 2010

A partially retro‐inverso analogue of the natural growth factor GHK was synthesized, in which the —CONH— bond between histidine and lysine was modified as —NHCO—. This modification is not expected to perturb the spatial distribution of the side‐chains, and therefore the binding processes, compared to the native peptide.

In the synthesis of the analogue two possible systems for deblocking of Nπ‐Born group of histidine have been applied and compared. An alternative method is also described for the incorporation of malonyllysine into the peptide chain.

When evaluated with respect to resistance toward degradation by human plasma in vitro, the new peptide analogue showed approximately a ten‐fold increase in stability versus the parent peptide.
Studies on the carboxyl terminal peptides of human seminal plasma inhibin (HSPI)
Authors: MAHALE S.D. , SHETH A.R , IYER K.S.N.
Published: August 2010

Observation of contradictory results with the in vitro assays for inhibin‐like activity of the carboxyl terminal 28 amino acid peptide 67–94 with a disulfide loop, of human seminal plasma inhibin (HSPI), prompted us to synthesize both the linear and the cyclic peptides and test their ability to suppress the circulating levels of follicle stimulating hormone (FSH) in vivo in adult male rats. The linear peptide [Cys(Acm)73,87] 67–94 of HSPI was synthesized by solid‐phase peptide synthesis using fluorenylmethyloxycarbonyl (Fmoc) chemistry and a continuous‐flow technology. The peptide was cyclized by direct iodine oxidation of the S‐diacetamidomethyl peptide in dilute solution. In the in vivo assay the linear peptide did not affect the levels of FSH, whereas the cyclic peptide suppressed the levels of FSH significantly. Thus, the carboxyl terminal region of HSPI does have inhibin‐like activity and perhaps has the active core of the protein.
Secondary structure prediction from multiple sequence data: blood clotting factor XIII and Yersinia protein‐tyrosine phosphatase
Authors: LIVINGSTONE CRAIG D. , BARTON GEOFFREY J.
Published: August 2010

Predictions of protein structure are best tested without prior knowledge of the protein three‐dimensional structure. Three‐dimensional atomic models will soon be determined by X‐ray crystallography for the α‐subunit of human blood clotting factor XIII and members of the family of protein tyrosine specific phosphatases. Accordingly, we here present secondary structure predictions for each of these proteins. The secondary structure predictions were generated from aligned sets of protein sequences. This technique has previously provided reliable predictions for the Annexins and the SH2 domains. The factor XIIIα prediction contains 39 regions predicted in strand conformation (34% of the protein) with only 3 helices (4%). The protein tyrosine phosphatases have 12 predicted strands and 5 helices (30 and 17%, respectively). We expect greater reliability from regions of alignments that show clear patterns of residue conservation (61% of factor XIIIα and 57% of the protein tyrosine phosphatases). The aligned protein tyrosine phosphatases show two regions (L39‐L80 and 1138‐E253) with clear patterns of residue conservation separated by a region of variable amino acid composition. We suggest this indicates that the tyrosine phosphatase fold comprises two domains separated by an exposed linker. Potential phosphate binding sites are identified in the protein tyrosine phosphatases.
Spontaneous hydrolysis of vasoactive intestinal peptide in neutral aqueous solution
Authors: MODY RUSTOM , TRAMONTANO ALFONSO , PAUL SUDHIR
Published: August 2010

Hydrolysis of radioiodinated vasoactive intestinal peptide (VIP) was observed in buffered aqueous solution at neutral pH and 38 °C. The reaction displayed apparent first‐order kinetics at initial peptide concentrations below 3 nM (Kobs= 1.5 × 10−5s−1), but the rate deviated below predicted values at higher peptide concentrations. The rate constant derived from the reaction progress curve over three half lives, starting at a concentration of 82 PM peptide, was also consistent with a first‐order process. The reaction results in several products that were isolated and characterized as peptide fragments. Based on the identity of these fragments, we deduced hydrolysis at five different peptide bonds clustered between residues 17–25 of VIP. Control experiments were devised to eliminate trivial explanations for the peptide hydrolysis. Peptides representing the C‐terminal segment 15–28 and the internal segment 14–22 assayed by analogous methods and under identical conditions were not degraded at a measurable rate. Sodium dodecyl sulfate and acetonitrile, agents known to influence the secondary structure of VIP, inhibited its spontaneous hydrolysis, as did chloride salts of sodium and calcium, albumin and a peptide unrelated to VIP. The rate and product distribution are inconsistent with known pathways of peptide degradation involving cyclic imide or anhydride formation at asparagine or aspartate residues. We suggest that the breakdown of VIP in dilute solutions represents an autolytic process.
Theoretical conformational analysis and synthesis of analogues of the heptapeptide antibiotic K‐582 A
Authors: COEN J.‐L. DE , WATHELET B. , DEMEUSE F. , MAYON C. , CULOT M. , BRAKEL J.
Published: August 2010

A detailed theoretical conformational analysis of the linear heptapeptide antibiotic [Arg2]K‐582 A (Arg‐Arg‐D‐Orn‐Thr‐D‐Om‐Lys‐D‐Tyr) was carried out. The results of the computer simulation suggest that the linear peptide has a high propensity to fold in solution into a quasi‐cyclic conformation in equilibrium with π(L‐D) helices. The synthesis of two inactive analogues with an L‐Lys in place of D‐Orn3 or D‐Orn5 confirms the importance of the proposed folding pattern for the occurence of the antimicrobial activity of K‐582 A.
Conformational characteristics of alternating stereo‐co‐oligopeptides of D‐ and L‐norleucine: influence of an N‐methyl group
Authors: SCHOCH EMMA FENUDE , RÖMER DANIEL URS , LORENZI GIAN PAOLO
Published: August 2010

The conformational behavior of members of the series Boc‐(L‐Nle)m‐(D‐Nle‐L‐Nle)(n‐m)/2‐OMe (m= 0 or 1; n= total number of residues) with n≤ 12, and of analogs of comparable chain length having a NMe‐group on the (n ‐ 3)th residue has been investigated. The study has shown that D,L‐alternating oligonorleucines behave very differently from stereo‐co‐oligopeptides of D‐alloisoleucine and L‐isoleucine, D‐ and L‐valine, or D‐ and L‐leucine. In particular, it has been found that oligonorleucines do not form β‐helices as do the other oligopeptides. Instead, they form aggregates (very likely of the α‐pleated sheet type), which are insoluble in common organic solvents even at moderate chain lengths. In marked contrast with this behavior, N‐methylated analogs such as those studied, with n from 9 to 15, cannot generate very stable aggregates owing to the N‐methyl group, and they prefer to form β‐helices. These β‐helices have been found by solution 1H NMR techniques to be almost exclusively of the types β4,4 (single‐stranded with about 4.4 residues per turn) and ↓↑5,6 (double‐stranded, antiparallel, with about 5.6 residues per turn).
lH NMR analysis of fibril‐forming peptide fragments of transthyretin
Authors: JARVIS JACQUELINE A. , KIRKPATRICK ALAN , CRAIK DAVID J.
Published: August 2010

Peptide fragments of the protein transthyretin, previously shown to form cross β‐sheet amyloid‐like fibrils in vitro, were investigated using 1H 1D and 2D NMR techniques. TTR 10‐20, TTR 105‐115 as well as a substituted analogue, (TTR 105‐115met111) all formed amyloid‐like fibrils readily in 20‐30% acetonitrile/water at room temperature. It was found that the presence of fibrils in the peptide solutions did not affect the observable NMR spectra, which may have been due to the line‐broadening that would be associated with these macromolecular species. 1H NMR spectra were thus representative of the monomeric form of the peptide in solution. Information from D2O exchange, 3JNH‐xH coupling measurements, temperature coefficients and NOESY experiments suggested that these peptides have some propensity for turn or helix but were predominantly unstructured. There was no indication of the monomeric species existing predominantly in an extended form, suggesting that the formation of β‐sheet based fibrils does not require preformed extended structures. TTR 105‐115Met111 displayed slight structural differences from TTR 105‐115 which may be related to the fibril‐forming propensity of the corresponding mutant TTR. © Munksgaard 1994.
Synthesis and conformation of constrained peptides with hypoglycaemic activity derived from human growth hormone
Authors: EDE NICHOLAS J. , RAE IAN D. , HEARN MILTON T.W.
Published: August 2010

The α‐aminosuccinimide (Asu11) octapeptide analogue of human growth hormone hGH[6‐13] (Leu6‐Ser‐Arg‐Leu‐Phe‐Asu‐Asn‐Ala13) has been reported [Robson et al. (1990) Biol. Chem. Hoppe Seyler371, 423‐431] to have hypoglycaemic activity whilst the corresponding peptide with Asp at position 11 is inactive. In order to determine whether this change in activity is caused by conformational and/or stereo‐electronic effects, the incorporation of two different isomeric γ‐lactam structures at position 11 has been investigated. One lactam structure (i) is of the type developed by Freidinger and coworkers [Freidinger et al. (1982) J. Org. Chem.47, 102‐107], whilst the isomeric γ‐lactam structure (ii) represents a new type of constrained synthon for use in peptide synthesis. The chiral type‐ii γ‐lactam was synthesized via a suitably protected desoxo‐dipeptide prepared in several ways from L‐aspartic acid. The solution conformations of the [Asu11]‐ and the [γ‐lactam11]‐containing hGH[6‐13] peptide analogues were investigated with the aid of two‐dimensional NMR (COSY and NOESY) spectroscopy. Conformational similarities were found for these hGH[6‐13] peptide analogues. For example, for all peptide analogues studied, weak NOEs were evident between the Phelo ring protons and protons of the amino acid residues at the C‐terminus. Overall, however, the NOESY NMR spectra of the [Asu11]‐ and the [γ‐lactam11]‐containing peptides related to hGH[6‐13] suggest the presence of an extended structure in solution with a possible weak type II′β‐turn at position 11. The extent of conformational constraint introduced into these hGH[6‐13] peptide analogues by substitution of the Asu11 residue with either isomeric γ‐lactam structure was reflected as differences in their hypoglycaemic activity. In particular, the hGH[6‐13] peptide analogue derived from the new chiral type‐ii γ‐lactam exhibits both lower activity in intravenous insulin tolerance tests in vivo and weaker NOEs than the isomeric hGH[6‐13] peptide analogue derived from the type (i) γ‐lactam structure. The relative change in blood glucose levels from 20 to 90 min for the racemic (R,S)‐form of the type‐ii γ‐lactam compared to the control values was approximately half that of the (S)‐stereoisomer. © Munksgaard 1994.
The usage of oligopeptides in proteins correlates negatively with molecular weight
Authors: BARRAI I. , VOLINIA S. , SCAPOLI C.
Published: August 2010

We scanned the Swissprot databank to study the distribution of oligopeptides in sequenced proteins. We observed that in 7839000 amino acids in the bank, the frequency is strongly and negatively correlated with molecular weight. The correlation coefficient is ‐0.63 for single amino acids, ‐0.58 for dipeptides, ‐0.54 for tri‐ and ‐0.46 for tetrapeptides. In subsets of proteins of man, mouse, rat, drosophila, yeast and E. coli, the correlations were very similar to those for the total database. The intensity of the correlation diminishes linearly with the length of the peptide. We attributed the loss of dominance of molecular weight in determining the frequency of oligopeptides of higher order, to the emergence of function in longer oligopeptides. In the case of dipeptides, we observed that those composed of the same amino acid are in great excess in comparison with their expectation. The WW dipeptide is singular, in the sense that its observed/expected ratio is 13.5 standard deviations above the average ratio.
Simulations of conformers of tuftsin and a cyclic tuftsin analog
Authors: VALDEAVELLA C.V. , BLATT H.D. , PETTITT B. MONTGOMERY
Published: August 2010

The conformational properties of the configurational isomers of tuftsin, a linear tetrapeptide with the sequence Thr‐Lys‐Pro‐Arg, were investigated with six 1 ns molecular dynamics simulations in explicit water and in a 1.0 M NaCl solution. The average conformation of the cis isomer is a type VI β‐turn. Our results indicate that water‐peptide hydrogen bonding, in addition to intramolecular hydrogen bonds, stabilizes the cis conformer. The trans isomer is neither a β‐ nor a γ‐turn. Results are compared with parallel studies on a cyclic analog of tuftsin, cyclo(Thr‐Lys‐Pro‐Arg‐Gly). The addition of salt does not influence the backbone conformation of the peptide. Differences between the structures are confined to the side‐chain orientations of the Lys and Arg residues. © Munksgaard 1995.
Synthesis and activity of the glutathione analogue γ‐(L‐γ‐azaglutamyl)‐L‐cysteinyl‐glycine
Authors: CALCAGNI A. , DUPRÉ S. , LUCENTE G. , LUISI G. , PINNEN F. , ROSSI D.
Published: August 2010

The backbone‐modified glutathione analogue γ‐(L‐γ‐azaglutamyl)‐L‐cysteinyl‐glycine 7, characterized by the presence of a NHCONH urea linkage deriving from the replacement of the native Glu γ‐CH2 with the aza (NH) group, was synthesized and fully characterized by FAB‐MS, 1H‐ and 13C‐NMR. Potential of 7 and its oxidized form 6 as γ‐glutamyltransferase inhibitors was investigated. Both compounds 7 and 6 were found to be competitive inhibitors of hog kidney y‐glutamyltransferase (EC 2.3.2.2.) by binding at the donor site: the reduced analogue is a more efficient inhibitor than glutathione of the γ‐glutamyl transfer reaction. Inhibition at the acceptor site, which is also present, appears to be more complex. In particular, un‐competitive inhibition is observed for compound 7. The results indicate that γ‐azapeptides of type 7 may represent interesting targets in the search for stable inhibitors of γ‐glutamyltransferases. © Munksgaard 1995.