bluuebunny/crossref_metadata_2025 huggingface dataset:
Conformational studies of synthetic tripeptide chemoattractants
Authors: VERTUANI G. , SPISANI S. , BOGGIAN M. , TRANIELLO S. , SCATTURIN A.
Published: August 2010

The conformational behavior of the chemotactic peptide analogs formylmethionylleucylphenylalanine methyl ester (CHO‐Met‐Leu‐Phe‐OMe) and formylmethionylleucylcyclohexylalanine methyl ester (CHO‐Met‐Leu‐Cha‐OMe) has been studied in solvents of different polarity by circular dichroism and infrared absorption. Both analogs and very probably the chemotactic peptide formylmethionylleucylphenylalanine (CHO‐Met‐Leu‐Phe‐OH) preferably adopt in solution a folded “active” conformation which allows a strong interaction with the receptor on the human polymorphonuclear leukocyte surface.
Conformational study of O‐glycosylated threonine containing peptide models
Authors: MAEJI N. JOE , INOUE YOSHIO , CHÜJÖ RIICHIRO
Published: August 2010

1H n.m.r. studies of Z‐Thr‐OMe, Z‐Thr‐Ala‐OMe, Z‐Ala‐Thr‐OMe and their glycosylated derivatives indicate the possibility of an intramolecular hydrogen bond between Thr NαH and the N‐acetyl carbonyl of the carbohydrate moiety, 3,4,6‐tri‐O‐acetyl‐2‐acetamido‐2‐deoxy‐α‐d‐galactopyranose(AcGalNAc). This is especially true in the case of Z‐Thr(AcGalNAc)‐Ala‐OMe, suggesting that the strength of this hydrogen bond is dependent on the neighboring amino acids on the carbonyl terminal side of Thr. The existence of such a hydrogen bond implies a conformation in which the carbohydrate moiety is restricted to an orientation with its plane roughly perpendicular to the peptide backbone. In such an orientation, steric problems will be minimized in the case of clustered O‐glycosidically linked Thr(Ser) residues as found in human erythrocyte glycophorin. A locked orientation of the carbohydrate moiety with respect to the peptide backbone may also play a conformational role in antifreeze glycoproteins.
Practical preparation and deblocking conditions for N‐α‐(2‐(P‐biphenylyl)‐2‐propyloxycarbonyl)‐amino acid (N‐α‐Bpoc‐Xxx‐OH) derivatives
Authors: KEMP D.S. , FOTOUHI NADER , BOYD JAMES G. , CAREY ROBERT I. , ASHTON CHRISTOPHER , HOARE JOHN
Published: August 2010

Reproducible preparations are given for salts of the following L‐amino acid derivatives: Bpoc‐Ala‐OH, Bpoc‐Arg(Mtr)‐OH, Bpoc‐Asn‐OH, Bpoc‐Asp(OtBu)‐OH, Bpoc‐Cys(Acm)‐OH, Bpoc‐Cys(S‐tBu)‐OH, Bpoc‐Gln‐OH, Bpoc‐Glu(OtBu)‐OH, Bpoc‐Gly‐OH, Bpoc‐Ile‐OH, Bpoc‐Leu‐OH, N‐α‐Bpoc‐Lys(ε‐Boc)‐OH, Bpoc‐Met‐OH, Bpoc‐Phe‐OH, Bpoc‐Pro‐OH, Bpoc‐Ser(OtBu)‐OH, Bpoc‐Thr(OtBu)‐OH, Bpoc‐Tyr‐OH, Bpoc‐Val‐OH. A study of the deblocking of N‐α‐Bpoc peptides in dichloromethane containing 0.5% trifluoroacetic acid revealed that a rapid equilb‐rium is established between the first‐formed monomeric alkene 2‐p‐biphenylylpropene and the hindered dimer 2,4‐bis(p‐biphenylyl)‐4‐methyl‐l‐pentene. Thioethers were found to be inefficient carbocation scavengers for the deblocking reaction. The most efficient scavengers were found to be thiophenol and benzyl mercaptan, and the following approximate reactivity order was established: benzyl mercaptan ∼ thiophenol 〉 indole 1,3‐dimethoxybenzene ∼ resorcinol 〉1,3,5‐trimethoxybenzene ∼ dimethyl sulfide ∼ thioanisole.
β‐Endorphin
Authors: HO CHEWN LANG , KO JANE LING , LI CHOH HAO
Published: August 2010

Three βh‐EP analogs which show different extents of alteration in analgesic potency by substitution of a single amino acid residue were assayed for their peripheral opioid activity and the binding to opioid μ‐receptor to determine the relationships among the opioid activities obtained from different assays. In the guinea pig ileum assay, [Gln8]‐βh‐EP showed a higher inhibitory activity than the parent peptide. [Tyr31]‐analog had the same potency as βh‐EP, while [Trp27]‐analog retained only one fourth the potency of βh‐EP. Assayed on the vas deferens of the mouse and the rat, all three substituted βh‐EP analogs exhibited a lower potency than their parent peptide. Receptor binding assay using [3H]‐dihydromorphine as the primary ligand showed that [Gln8]‐analog had a binding potency 1.5‐fold that of βh‐EP, while the potencies of [Tyr31]‐ and [Trp27]‐analogs were not significantly different from that of the parent peptide. No correlation in relative potency was found between vas deferens assays and their μ‐receptor binding or analgesic activity. However, the relative potencies of binding to μ‐receptor in [Gln8]‐ and [Tyr31]‐analogs were found to be consistent with those of analgesic and guinea pig ileum assays, whereas the binding to β‐EP receptor of all analogs appeared to be related to the charge properties of β‐EP molecule.
Structural stability of β‐lactoglobulin in the presence of kosmotropic salts A kinetic and thermodynamic study
Authors: KELLA NAVIN KUMAR D. , KINSELLA JOHN E.
Published: August 2010

The thiol group of β‐lactoglobulin reacted very sluggishly with dithio‐bis‐nitro‐benzoic acid as compared to that of glutathione at pH 6.85. The pKapp value of the thiol group of the protein was 9.35. In the presence of 3 M urea, the thiol group reacted completely with dithio‐bis‐nitrobenzoic acid at pH 6.85. Heating (from 50° to 80°) increased the exposure of the thiol by dissociating the dimer unit. From the pseudo‐first order rate constants of heat‐exposure of thiol, thermodynamic activation parameters, ΔG++, ΔH++, and ΔS++, for the heat‐dissociation of β‐lactoglobulin dimer were estimated to be 23 290 cal/mol, 31 160cal/mol, and 22.9 e.u. (at 70°), respectively. Addition of kosmotropic salts, chloride, tartrate, sulfate, phosphate, and citrate (0.2 m) decreased the heat‐induced exposure of the thiol group (at 70°), probably by decreasing the dissociation of the dimer at pH 6.85. The relative change in free energy of activation for the dissociation of the dimer, Δ(ΔG++dimer), in the presence of the salts was positive, suggesting that these additives increase the stability of the dimer against heat. These salts also increased the conformational stability of β‐lactoglobulin as revealed by an increase in ‐ Δ(ΔG0conf) values in their presence. Both Δ(ΔG++dimer) and ‐ Δ(ΔG0conf) values followed the order, chloride < tartrate < sulfate < phosphate < citrate. These salts seem to manifest their structure‐stabilizing effect by increasing both inter‐ and intramolecular hydrophobic interactions via changes in structure of water.
Substance P‐like immunoreactivity in human cerebrospinal fluid
Authors: HIGA TOSHINOBU , WOOD GEORGE , DESIDERIO DOMINIC M.
Published: August 2010

In a study to elucidate molecular mechanisms in pain, substance P‐like immunoreactivity (SP‐LI) was measured in lumbar CSF from 75 patients with lower back pain. Two samples – one before and one after lidocaine treatment – were obtained from each patient, and total SP‐LI was measured in unfractionated (no HPLC) samples. SP‐LI data were separated into three categories: placebo responders, pharmacological responders, and pharmacological non‐responders. A significant difference was observed between the total SP‐LI measurement of first and second samples of pharmacological non‐responders. Distribution of SP‐LI immunoreactive molecular species in two CSF patient samples (no ODS) was analyzed with a combination of reversed phase (RP) HPLC and RIA. Immunoreactivity in collected HPLC fractions was measured at calibrated retention times of synthetic SP‐sulfoxide (SP‐O), SP, and SP5–11. Qualitative and quantitative differences in those HPLC‐RIA metabolic profiles were observed within and between those two patients' samples. These data indicate that the type and amount of SP metabolism and SP precursor‐processing differs in CSF between these two representative patients and within the short amount of time elapsed between acquiring these two samples.
Synthesis of an actinomycin‐related peptide lactone from the corresponding cyclic pentapeptide by NO‐acyl shift
Authors: MAUGER ANTHONY B. , STUART OSWALD A.
Published: August 2010

The N,O‐acyl shift was investigated as a method for the synthesis of an O‐peptide or peptide lactone from a linear or cyclic peptide respectively. Protected derivatives of glycyl‐l‐threonine could be converted to O‐peptides by the action of HCl/dioxane at room temperature and N‐acylated under conditions which precluded a reverse O,N‐acyl shift. For effecting N,O‐acyl shift in cyclo(Thr‐d‐Val‐Pro‐Sar‐MeAla) this reagent was unsatisfactory and p‐toluenesulfonic acid in dioxane at 80° was used instead. The resulting crystalline peptide lactone p‐toluenesulfonate salt was N‐acylated with 3‐benzyloxy‐4‐methyl‐2‐nitroben‐zoyl chloride to afford a known intermediate in the synthesis of 5,5′‐MeAla actinomycin D. This approach constitutes a novel synthetic route to actinomycins and potentially to other peptide lactone antibiotics.
Solid phase synthesis of a trypsin inhibitor isolated from the Cucurbitaceae Ecballium elaterium
Authors: LE‐NGUYEN DUNG , NALIS DOMINIQUE , CASTRO BERTRAND
Published: August 2010

The synthesis of a 28‐residue peptide isolated from Ecballium elaterium of the Cucurbitaceae family which strongly inhibits trypsin activities (Ka = 8·10−11 M), using BOP as the coupling reagent in a solid phase procedure is presented. This micro protein contains three disulfide bridges in its sequence and was obtained after oxidation of the six half‐cystine residues either by air or with the use of carboethoxysulfenyl chloride. After purification by semi‐preparative HPLC, the synthetic product was shown by trypsin inhibition tests to be identical with the trypsin inhibitor EETI II isolated from Ecballium elaterium.
Peptide substrate specificity of the α‐amidating enzyme isolated from rat medullary thyroid CA‐77 cells
Authors: TAMBURINI PAUL P. , YOUNG STANLEY D. , JONES BARRY N. , PALMESINO RANDI A. , CONSALVO ANGELO P.
Published: August 2010

The kinetic parameters were obtained for enzymatic α‐amidation of peptides of the form N‐dansyl‐(Gly)4‐X‐Gly‐OH, in which the amino acid at position × was substituted with each of the 20 natural amino acids. The enzyme used in these studies was a highly enriched preparation of α‐amidating enzyme secreted by a clonal (CA‐77) cell line which actively expresses mature α‐amidated peptides. A 130‐fold and 11‐fold variation respectively in apparent Km and Vmax values was observed. The effect of the amino acid side chain at position × in stabilization of the enzyme‐substrate complex decreased through the series ×= planar aromatic or sulfur containing > neutral aliphatic > polar and basic > cyclic aliphatic or acidic.
Semisynthesis of carboxy‐terminal fragments of thermolysin
Authors: DE FILIPPIS VINCENZO , FONTANA ANGELO
Published: August 2010

Enzyme‐catalyzed synthesis of two polypeptide fragments, one of which is obtained by chemical synthesis, in the presence of proteolytic enzymes and in aqueous organic solvents constitutes a convenient procedure for the synthesis of proteins and their analogs. This novel semisynthetic procedure was investigated for preparingCOOH‐terminal fragments of the metallo‐protease thermolysin. Fragment 205–316, obtained by autolysis of the protein in the presence of EDTA, was first cleaved selectively withStaphylococcus aureusV8 protease at the level of the single Glu302residue into fragments 205–302 and 303–316. Upon incubation for 2–5 days of fragment 205–302 with a 5–fold excess of peptide 303–316, prepared by solid phase synthesis, with V8‐protease in 0.1M ammonium acetate, pH6.0, containing 50% glycerol as organic cosolvent, enzyme‐catalyzed reformation of the peptide bond was achieved in yields up to ñ90% (based on fragment 205–302). The same procedure was used to prepare also the thermolysin fragments 205–315 and 205–311 by enzymatic coupling of fragment 205–302 to peptide 303–315 or 303–311, these last prepared by proteolytic digestion of the synthetic peptide 303–316. This procedure of semisynthesis opens up an approach for the site‐directed modification of the tetrahelicalCOOH‐terminal fragment 205–316 of thermolysin at the level of its helical segment encompassing residues 301–312 in the native, intact protein. Such analogs will be useful for examining structure‐folding‐stability relationships in this folded fragment possessing domain‐like characteristics.
Synthesis of biologically active analogs of the dodecapeptide α‐factor mating pheromone of Saccharomyces cerevisiae
Authors: EWENSON ARIEL , MARCUS STEVAN , BECKER JEFFREY M. , NAIDER FRED
Published: August 2010

A number of dodecapeptides with the sequence YIIKGVFWDPAC were synthesized using solid phase peptide synthesis. The purity of the crude cleavage product was found to be directly related to the cysteine protecting group and the conditions employed for cleavage of the peptide from the resin. When 4‐methyl‐benzyl cysteine was used, complete deprotection was only achieved with low‐high HF conditions at temperatures of 10°‐25°, whereas milder conditions could be used for dodecapeptides containing ethyl cysteine or acetamidomethyl cysteine. In several syntheses the biological activity of the crude cleavage product greatly exceeded the biological activity of a purified major peptide component. The high activity found in the crude cleavage peptide was probably due to minor peptide side products in which the cysteine sulfur was alkylated by hydrophobic species during HF treatment. Two dodecapeptides, YIIKGV‐FWDPAC and YIIKGFWDPAC(Ethyl), had significant α‐factor activity against MATα strains of Saccharomyces cerevisiae. These peptides represent the first synthetic analogs with α‐factor activity.
Synthesis of oligophosphoseryl sequences occurring in casein
Authors: PAQUET ALENKA , JOHNS MICHAEL
Published: August 2010

The protected oligophosphoseryl peptides from bovine caseins, Z‐Xxx‐{Ser[PO(OPh)2]}3‐Glu(OBzl)‐OBzl for Xxx = Ile, Val, Gly, Leu and Ph = phenyl, were synthesized in high yields by stepwise lengthening using Boc‐Ser[PO(OPh)2]‐OH as acylating carboxyl component and N‐ethyl‐N′‐(3‐dimethylaminopropyl)‐carbodiimide hydrochloride as coupling reagent. The hydrogenolytic deprotection (PtO2) was carried out with the valine derivative and with the tetrapeptide Ser[PO(OPh)2]3‐Glu(OBzl)‐OBzl. Phosphorylation of oligoseryl peptides failed to give the expected products. Large scale phosphorylation of protected serine was carried out in the presence of triethylamine using absolute ether as a solvent. 2,2,2‐Trichloroethyl group (Tc) was shown to be a useful phosphorus protecting moiety in phosphopeptide synthesis: Boc‐Ser[PO(OTc)2]‐OBzl, Z‐Ser[PO(OTc)2]‐OBzl and Boc‐Glu(OBzl)‐Ser[PO(OTc)2]‐OBzl were synthesized in high yields using bis‐(2,2,2‐trichloroethyl) phosphochloridate.
Synthesis of glycosylated tuftsins and tuftsin‐containing IgG fragment undecapeptide
Authors: BIONDI L. , FILIRA F. , GOBBO M. , SCOLARO B. , ROCCHI R. , CAVAGGION F.
Published: August 2010

Syntheses are described of two new tuftsin derivatives containing a 2‐acetamido‐2‐deoxy‐D‐galac‐topyranosyl unit α‐ or β‐glycosidically linked to the threonine's hydroxy side chain function and of the glycosylated undecapeptide corresponding to the tuftsin region of the heavy chain of IgG (amino acid sequence 289–299). The glycosylated tuftsins were synthesized by the solution procedure. Fmoc‐[Gal NAc(Ac)3α]Thr‐OH and Fmoc‐[GalNAc(Ac)3β]Thr‐OH were allowed to react with H‐Lys(Z)‐Pro‐Arg(NO2)‐OBzl by the mixed anhydride procedure and the resulting glycosylated tetrapeptides were fully deblocked by catalytic hydrogenation followed by treatment with potassium cyanide, purified by ion exchange chromatography and characterized by analytical HPLC, elemental and amino acid analyses, optical rotation, and proton NMR spectroscopy. Synthesis of the glycosylated undecapeptide was achieved by the continuous flow solid phase procedure on 4‐hydroxymethylphenoxyacetyl‐norleucyl derivatized Kieselguhr‐supported resin. Fmoc‐amino acid symmetrical anhydrides or pentafluorophenyl esters, in the presence of N‐hydroxybenzotriazole, were used as the acylating agents. To mimic the native sequence of the tuftsin region at the Fc‐domain of immunoglobulin G a 2‐acetam∼do‐2‐deoxy‐β‐D‐glucopyranosyl unit was N‐glycosidically linked to the amide side chain of Asn 297. The glycosylated asparagine residue was introduced as N2‐fluorenylmethyloxycarbonyl‐N4‐(2‐acetamido‐3,4,6‐tri‐O‐acetyl‐2‐deoxy‐β‐D‐glucopy‐ranosyl)‐asparagine pentafluorophenyl ester. After cleavage from the resin the glycopeptide was deprotect‐ed, purified by ion exchange chromatography, and characterized by analytical HPLC, amino acid analysis, high voltage electrophoresis, and proton NMR. The conformational features of the glyco‐undecapeptide were determined by circular dichroism measurements both in water and in 98% trifluoroethanol. Results of biological assays will be published elsewhere.
Synthesis and properties of chemotactic peptide analogs I. Crystal structure and molecular conformation of HCO‐Met‐Leu‐Ain‐OMe
Authors: GAVUZZO E. , LUCENTE G. , MAZZA F. , ZECCHINI G. PAGANI , PARADISI M. PAGLIALUNGA , POCHETTI G. , TORRINI I.
Published: August 2010

HCO‐Met‐Leu‐Ain‐OMe (2), an analog of the chemotactic peptide HCO‐Met‐Leu‐Phe‐OH, containing the conformationally blocked residue of the 2‐aminoindane‐2‐carboxylic acid (Ain) has been synthesized and its crystal and molecular conformation has been determined. Crystals of 2 are monoclinic, space group P21, with a = 15.059(7), b = 18.548(7), c = 9.600(4)Å;β= 85.04(3). The structure has been solved by direct methods and refined to R = 0.069 for 2813 independent reflections with I > 2.5σ(I). Two independent molecules A and B have been found in the asymmetric unit of the crystal of 2. Their conformation can be described as extended at the Met and Leu residues, but folded at the C‐terminal Ain residue. The helical folding is left‐ and right‐handed in the A and B molecule, respectively. The crystal packing is characterized by ribbons of intermolecular hydrogen bonded molecules extended along the c direction. The constrained analog 2 is highly active in the superoxide production, thus indicating that a stabilization of a helical folding at the C‐terminal region of chemotactic tripeptides maintains the activity. The orientation of the aromatic ring, with respect to its adjacent backbone atoms, does not seem critical for the activity.
Solid phase synthesis of a fully active analogue of cholecystokinin using the acid‐stable Boc‐Phe (p‐CH2) SO3H as a substitute for Boc‐Tyr(SO3H) in CCK8
Authors: GONZALEZ‐MUNIZ ROSARIO , CORNILLE FABRICE , BERGERON FLORENCE , FICHEUX DAMIEN , POTHIER JOËL , DURIEUX CHRISTIANE , ROQUES BERNARD P.
Published: August 2010

Substitution of the ‐OSO3H group in the sulfated‐tyrosine by the non‐hydrolyzable ‐CH2SO3H group was the first described modification of the sulfate ester that does not affect CCK8 activity. In addition to its capacity to mimic the sulfated tyrosine residue, the amino acid Phe(p‐CH2SO3Na) was shown to be stable in acidic media, including HF containing mixtures. The synthesis of Boc‐Phe(p‐CH2SO3Na)‐OH in racemic and resolved forms and its introduction into the sequence of CCK8 by solid phase using standard Boc/benzyl synthesis conditions and BOP as coupling reagent is now reported. The two CCK8 analogues containing the l‐ or the d‐Phe(p‐CH2SO3Na) residue, obtained in satisfactory yields, were separated by HPLC and the stereochemistry of Phe(p‐CH2SO3Na) residue in each peptide was established by NMR spectroscopy and confirmed by a separate solid phase synthesis in which the pure l isomer was used. Both CCK8 analogues displayed high affinities for peripheral and central receptors (KI ∼ 1 nm) and proved to be full agonists in the stimulation of pancreatic amylase secretion. The „stabilized‐CCK8 peptide”, easily prepared by solid phase, could replace the native peptide in biochemical and pharmacological studies. Moreover the modified amino acid Phe (p‐CH2SO3Na) could also be used in solid phase synthesis to prepare a wide variety of CCK analogues and more generally, peptides analogues containing the acid‐labile O‐sulfated tyrosine.
Isolation of a ribosome‐inactivating and abortifacient protein from seeds of Luff a acutangula
Authors: YEUNG H. W. , LI W.W. , NG T. B.
Published: August 2010

A glycoprotein with a molecular weight of 28 000 as estimated by SDS‐polyacrylamide gel electrophoresis was isolated from seeds of Luffa acutangula using a procedure that involved acetone precipitation, ion exchange chromatography on CM Sepharose CL‐6B and gel filtration on Sephadex G‐50. In immunodiffusion studies it was found to be immunologically distinct from abortifacient proteins isolated from other members of the Cucurbitaceae family including Momordica charantia, Momordica cochinchinensis, Trichosanthes kirilowii and Trichosanthes cucumeroides. There were some differences in amino acid composition among the proteins although there was a gross similarity. The protein from L. acutangula was capable of inducing mid‐term abortion in mice and inhibiting protein synthesis in a cell‐free system.
Design of chimeric peptide ligands to galanin receptors and substance P receptors
Authors: LANGEL ÜLO , LAND TIIT , BARTFAI TAMAS
Published: August 2010

Several chimeric peptides were synthesized and found to be high‐affinity ligands for both galanin and substance P receptors in membranes from the rat hypothalamus. The peptide galantide, composed of the N‐terminal part of galanin and C‐terminal part of substance P (SP), galanin‐(1‐12)‐Pro‐SP‐(5‐11) amide, which is the first galanin antagonist to be reported, recognizes two classes of galanin binding sites (KD(1)<0.1 nM and KD(2)∼ 6 nM) in the rat hypothalamus, while it appears to bind to a single population of SP receptors (KD∼ 40 nM). The chimeric peptide has higher affinity towards galanin receptors than the endogenous peptide galanin‐(1‐29) (KD ∼ 1 nm) or its N‐terminal fragment galanin‐(1‐13) (KD∼ 1,nm), which constitutes the A′‐terminus of the chimeric peptide. Galantide has also higher affinity for the SP receptors than the C‐terminal SP fragment‐(4‐11) amide (KD= 0.4μm), which constitutes its C‐terminal portion. Substitution of amino acid residues, which is of importance for recognition of galanin by galanin receptors, such as [Trp2], in the galanin portion of the chimeric peptide or substitution of ([Phe7] or [Met11]‐amide) in the SP portion of chimeric peptide both cause significant loss in affinity of the analogs of galantide for both the galanin‐ and the SP‐receptors. These results suggest that the high affinity of the chimeric peptide, galantide, may in part be accounted for by simultaneous recognition/binding to both receptors. In line with this suggestion is the finding that the binding of the chimeric ligands to the galanin receptor is strongly influenced by the presence of SP (1 μm) or spantide (1 μm). We have performed the synthesis and binding studies with 11 chimeric peptides, all composed of the N‐terminal galanin‐(1‐13) fragment or of its analogs, linked to the C‐terminal portion of SP or its peptide antagonist, spantide. Our results, similar to earlier reports on chimeric peptides, suggest that high‐affinity ligands to peptide receptors can be produced by linking biologically active N‐terminal and C‐terminal portions of peptides via linkers, enabling a) independent recognition of the chimeric peptide by the relevant receptors and b) intramolecular interactions between the joined N‐ and C‐terminal peptide fragments. These two phenomena may also explain why some of the chimeric peptides have higher affinity than the endogenous peptide(s) and why galantide, and some of its analogs presented here, behave(s) as a galanin receptor antagonist(s).
Design of hydropathically complementary peptides for Big Endothelin affinity purification
Authors: FASSINA GIORGIO , CONSONNI ROBERTO , ZETTA LUCIA , CASSANI GIOVANNI
Published: August 2010

Molecular recognition between Big Endothelin (Big ET) and a computer generated peptide hydropathically complementary to Big ET[16‐29] sequence has been studied by analytical high performance liquid affinity chromatography (HPLAC), circular dichroism (CD) and nuclear magnetic resonance (NMR) experiments. Specific binding was observed between solid support immobilized complementary peptide and Big ET[1‐38], [1‐32], and [16‐32], but not with Big ET fragments [1‐21], [16‐21], [22‐32], and [22‐38], obtained by chymotrypsin proteolytic degradation. Selectivity in the recognition process was clearly demonstrated by the ability of complementary peptide affinity column to purify the Big ET molecule from complex peptide mixtures, even when present in very low concentrations. Similar selectivity was evidenced with the Big ET fragment [16‐32], [NH2‐HLDIIWVNTPEHIVPYG‐COOH] containing the entire hydropathically complementary sequence. Binding was followed by marked spectroscopic changes, as monitored by circular dichroism and one‐ and two‐dimensional nuclear magnetic resonance experiments. The NMR spectra of the complementary peptides 1:1 mixture showed variations in the chemical shifts of proton resonances in several residues, both in the main chain (amide protons) and in the side chains (aliphatic and aromatic protons). These data support the hypothesis of a multilocalized type of interaction between complementary peptides, where many residues along the peptide chains participate in co‐operative stabilizing contacts in the forming complex.
Review of the Fogarty International Center Conference, NIH: Peptides in Health and Disease*
Authors: HIRSCHMANN RALPH
Published: August 2010

This article represents the closing remarks by the author summarizing the lectures presented at the Fogarty International Center Conference on Peptides in Health and Disease held at the National Institutes of Health, Bethesda, MD, USA, January 6–8, 1992.
Complete structure of eclosion hormone of Manduca sexta
Authors: KATAOKA HIROSHI , LI JORGE P. , LUI ALFRED S.T. , KRAMER STEVEN J. , SCHOOLEYS DAVID A.
Published: August 2010

The locations of the three disulfide bonds of eclosion hormone (EH) isolated from Manduca sexta were assigned by sequence analysis of thermolysin fragments and by comparison of a key heterodimeric fragment to regiospecifically synthesized parallel and antiparallel isomers. We elucidated the complete structure of Manduca EH as a 62‐residue peptide which has three disulfide bonds between Cys14‐Cys38, Cys18‐Cys34, and Cys21‐Cys49.
Alkylation of histidine with maleimido‐compounds
Authors: PAPINI A. , RUDOLPH S. , SIGLMÜLLER G. , MUSIOL H.‐J. , GÖHRING W. , MORODER L.
Published: August 2010

Introduction of the maleimide function via a spacer into histidine‐containing peptides was found to produce ring closure by nucleophilic addition of the Nim‐imino function of the histidine side‐chain to the activated double bond of the maleimide. As an intramolecular cyciization reaction it proceeds at remarkably higher rates than the bimolecular alkylation of histidine derivatives with N‐ethyl‐maleimide. Correspondingly, in the case of the histidine‐peptides examined only mixtures of the cyclic isomeric compounds were isolated and structurally characterized by 1H‐NMR analysis. As expected, prevention of this reaction in histidine‐containing maleoyl‐peptides can be achieved by Nim‐protection of the imidazole group. However, upon removal of this protection, the reaction takes place again, thus remarkably hampering the usefulness of the maleimide/thiol addition principle in conjugate chemistry for peptides. On the other hand this reaction could represent an interesting new approach for the design of cyclic peptidomimetic analogs.
Binding and spectroscopic properties of ostrich neurophysins
Authors: BRESLOW ESTHER , LABORDE THIRLEEN , SAAYMAN HESTA S. , OELOFSEN WILLEM , NAUDÉ RYNO J.
Published: August 2010

Binding and spectroscopic properties of ostrich neurophysins were examined with emphasis on the behavior of Tyr‐35, a residue that provides a potential probe of the monomer‐monomer interface and of allosteric interrelationshíps between this region and the binding site. Mesotocin‐associated ostrich neurophysin was found to bind oxytocin and related peptides with affinities comparable to the mammalian proteins, but induced a significantly different optical activity in bound peptides than the mammalian proteins. Gel‐filtration studies indicated higher dimerization constants for the ostrich neurophysins than for the bovine neurophysins. Consistent with this, Tyr‐35 was found to be largely buried, as monitored by tyrosine titration and lack of reactivity towards tetranitromethane under non‐denaturing conditions. Reaction of Tyr‐35 of the mesotocin‐associated protein with tetranitromethane under denaturing conditions, followed by refolding, allowed isolation of an active product with an altered interface region as partially evidenced by its titration properties and consistent with its markedly altered CD spectrum. Comparison of the CD spectra of the modified and native proteins and analysis of pH effects indicated the contribution of Tyr‐35 to an unusual 237 nm band in the mesotocin‐associated protein. Small shifts in the 350 nm CD band of nitrated Tyr‐35 on binding peptide and apparent effects of nitration on the induced optical activity in bound peptide provided evidence of at least weak structural communication between Tyr‐35 and the binding site. However, no significant effect of nitration on binding affinity was observed, suggesting that, in the mesotocin‐associated protein, the region around residue 35 is not a stringent modulator of the thermodynamic behavior of the binding site.
Partial synthesis of five new analogues of the peptido‐lactone Virginiamycine S1, modified in the fifth and/or sixth position ([Xxx5]‐VS1 with Xxx = Ala, Asp, Asn and Lys and [Ala5,Gly6]‐VS1)
Authors: MOERMAN MARC C. , ANTEUNIS MARC J.O.
Published: August 2010

We achieved the reconstruction of VS1‐analogues containing a substitute for the fifth residue, γ‐oxo‐Pip (Pip = pipecolic acid), starting from VS1‐pentapeptide (VS5P; 3) the latter being prepared by a two‐step degradation process of the native antibiotic VS1 (1a). Protecting groups during the procedure were chosen in order to realize a minimal number of steps. Most of these gave excellent yields, including final cyclization between the fourth and fifth residue. In total, four analogues were synthesized with Ala, Asp, Asn and Lys (1b) replacing γ‐oxo‐Pip. Among these, [Lys5(Tfa salt)]‐VS1 is water‐soluble, which is an important characteristic for eventual application of VS1 as a pharmaceutical agent. In the proposed reaction sequence, we made sure that residues 4 (MePhe) and 6 (Phg) became partially epimerised. We therefore obtained each time after cyclization a total of four epimers that have been separated by preparative TLC. The chiral identity of the final residues was realized by GC (Chirasil Val®‐III) on the total hydrolysates.
Racemization of α‐amino acid esters by aliphatic ketones in the presence of carboxylic acids
Authors: HARRY L.G. , PUGNIÈRE M. , CASTRO B. , PREVIERO A.
Published: August 2010

Amino acid esters are racemized by dissolution in a mixture of aliphatic ketones and carboxylic acids. The racemization rate mainly depends on the structure of the amino acid and on the kind of ketone and carboxylic acid used, the best racemizing medium being acetone containing 15% acetic acid. The mechanism of the racemization and the practical consequences of this study in the optical resolution field are discussed.
The β‐bend ribbon spiral
Authors: CRISMA M. , VALLE G. , BONORA G.M. , TONIOLO C. , CAVICCHIONI G.
Published: August 2010

The synthesis and conformational analysis in solution (by FTIR absorption and 1H NMR) and in the crystal state (by X‐ray diffraction) of three Hib‐containing depsipeptides have been performed. In the crystal state Z‐Aib‐Hib‐Aib‐OMe is folded into a type‐III β‐bend, while the conformation adopted by Z‐(Aib‐Hib)2‐Aib‐OMe is a β‐bend ribbon spiral, characterized by two type‐III β‐bends with Aib(1)‐Hib(2) and Aib(3)‐Hib(4) as corner residues, respectively. Both independent molecules in the asymmetric unit of t‐Boc‐L‐Ala‐Hib‐L‐Ala‐OMe crystals are folded into a type‐II β‐bend. For the Aib‐Hib depsipeptides the conformation adopted in the crystal state is also that largely prevailing in solution, whereas for t‐Boc‐L‐Ala‐Hib‐L‐Ala‐OMe the β‐bend conformation is significantly less populated in solution. A comparison is also made with: (i) the published crystal‐state conformations of fully protected ‐(Aib)3−, ‐(Aib)5−, and ‐L‐Ala‐Aib‐L‐Ala‐ sequences and the β‐bend ribbon spiral generated by (Aib‐L‐Pro)n oligomers, and (ii) with the herewith described solution preferred conformation of Z‐L‐Ala‐Aib‐L‐Ala‐OMe. The possible use of Hib as an isosteric replacement for Aib in the design of conformation ally constrained depsipeptides is briefly discussed.
Synthesis and stability of 3‐nitro‐2‐pyridinesulfenyl chloride (NpysCl)
Authors: PUGH KATHLEEN C. , GERA LAJOS , STEWART JOHN M.
Published: August 2010

3‐Nitro‐2‐pyridinesulfenyl chloride (NpysCl) is the starting material for the synthesis of N‐, O‐ and S‐Npys‐protected amino acids. Two efficient, novel synthetic routes to NpysCl are described. The stability of NpysCl was determined in a variety of solvents, with and without base, to determine the most suitable solvent and base for the synthesis of N‐Npys amino acids. The syntheses of Npys‐Ala and Boc‐Lys(Npys) tert‐butylammonium salt are also described.
Diketopiperazine formation and N‐terminal degradation in recombinant human growth hormone
Authors: BATTERSBY J.E. , HANCOCK W.S. , CANOVA‐DAVIS E. , OESWEIN J. , O'ONNOR B.
Published: August 2010

A new degradation process has been identified that occurs in recombinant DNA‐derived human growth hormone. Non‐enzymatic cyclization of the first two amino acids from the N‐terminus and subsequent cleavage results in the formation of a diketopiperazine and a truncated variant of rhGH. The truncated protein was separated using hydrophobic interaction chromatography and identified as desPhe1Pro2‐rhGH using N‐terminal sequence analysis, tryptic mapping, and mass spectrometry.
Kinetics of peptide synthesis studied by fluorescence of fluorophenyl esters
Authors: PERMYAKOV EUGENE A. , MEDVEDKIN VYACHESLAV N. , KLIMENKO LYCBOV V. , MITIN YURI V. , PERMYAKOV SERGE E.
Published: August 2010

The kinetics of the reaction of Boc‐alanine‐trifluorophenyl, Boc‐alanine‐tetrafluorophenyl, Boc‐alanine‐pentafluoropbenyl, and Boc‐alanine‐p‐chlorotetrafluorophenyl esters (BocAlaOTrf, BocAlaOTfp, BocAlaOPfp, and BocAlaTfc, respectively) with leucine amide and with valine methyl ester have been measured using changes in fluorophenyl chromophore emission at 375 nm. The kinetic data cannot be well fit with a simple second‐order reaction scheme. Measurements of the reaction kinetics at different concentrations of the reagents showed that the expression for the reaction rate is in which k is the reaction rate constant, CN is the concentration of either LeuNH2 or ValOCH3, and CAE is the concentration of the fluorophenyl ester. This reaction equation indicates a complex, probably chain‐like, reaction mechanism. The order of reactivity for these active esters with ValOCH3 is BocAlaOTfc > BocAlaOPfp > BocAlaOTfp > BocAlaTrf. The apparent rate constant, k, for the reaction with LeuNH2 is higher than that for the reaction with ValOCH3.
Interaction with model membrane systems induces secondary structure in amino‐terminal fragments of parathyroid hormone related protein
Authors: WILLIS K. J.
Published: August 2010

The secondary structure of amino terminal fragments of human parathyroid hormone related protein (PTHrP) in aqueous solution, in trifluoroethanol solution and in the presence of model membrane systems has been studied by circular dichroism (CD) spectroscopy. Far‐UV CD spectra of PTHrP 1–40, 1–34 amide, 7–34 amide and 1–16 are consistent with a predominantly unordered structure. Addition of trifluoroethanol stabilized α‐helical structure in the 1–34 amide and 7–34 amide peptides. The effect reached a plateau at a trifluoroethanol concentration of approximately 40%, and a helix content of some 23 residues was determined. PTHrP 1–34 amide interacted with palmitoyloleoylphosphatidyl serine vesicles and exhibited an increased α‐helix content of approximately 12 helical residues. Similar results were observed for monomyristoyllecithin micelles and sodium dodecyl sulfate micelles. No interaction with dimyristoylphosphatidylcholine vesicles was detected by CD. The ability to bind to palmitoyloleoylphosphotidyl serine vesicles was also a feature of the 1–40 and 7–34 fragments, while the 1–16 fragment was apparently unaffected by interaction with this model membrane system. These results indicate that the conformational properties of the functionally significant amino terminal 1–34 region of PTHrP parallel those reported for the corresponding, but largely nonhomologous, region of parathyroid hormone. Conformational similarities may account for the ability of PTHrP to mimic the functional properties of parathyroid hormone.
Synthesis and solution structure of an S‐glycosylated cyclic hexapeptide
Authors: GERZ M. , MATTER H. , KESSLER H.
Published: August 2010

Synthesis and conformational analysis of the S‐glycosylated cyclic hexapeptide cyclo(‐d‐Pro1‐Phe2‐Cys3(tetra‐O‐acetyl‐β‐d‐galactopyranosyl)‐Trp4‐Lys(Z)5‐Phe6‐) I was carried out to examine the influence of a saccharide residue in position i of a standard β‐turn on the formation of reverse turns and on the biological activity. Synthesis was carried out in the liquid phase employing a galactosylated cysteine building block. The cyclization reagents DPPA/NaHCO3 avoided high dilution conditions. Spectroscopic data were extracted from homo‐ and heteronuclear 2D‐NMR techniques (TOCSY, NOESY, HMQC, HMQC‐TOCSY, HMBCS‐270). For structural refinement restrained molecular dynamics (MD) simulations in vacuo and with explicit DMSO as solvent were performed. Finally, simulations in DMSO without experimental restraints provided insight in stability and dynamics of the structural model.

A comparison of the S‐glycosylated Cys3 peptide with the analogous Thr3 peptide exhibits a similar overall conformation of the hexapeptide [βII’d‐Pro‐Phe and another β‐turn about Trp4‐Lys5(Z)]. However, the latter shows a distinct dynamic flip βI, βII in the glycopeptide, whereas the Thr‐analogue only populates βI. This influence is attributed to a βI stabilizing effect of a hydrogen bridge of Thr‐O, in position i to the NH of the amino acid in position i+ 2, which is lacking in the glycosylated compound.
Synthesis and protease‐catalyzed hydrolysis of a novel hydrazinopeptide
Authors: AMOUR AUGUSTIN , COLLET ANDRÉ , DUBAR CATHERINE , REBOUD‐RAVAUX MICHÈLE
Published: August 2010

In order to explore the potentiality of hydrazinopeptides as protease inhibitors, the resistance of the hydrazinopeptidic bond toward proteolysis was investigated. To this end, the novel hydrazinohexapeptide Z‐Ala2‐Pro‐Val‐hIle‐Leu‐OMe (1), where hIle represents hydrazinoisoleucine, was designed and synthesized together with the parent peptide Z‐Ala2‐Pro‐Val‐Ile‐Leu‐OMe (2). The interactions of 1 and 2 with human leukocyte elastase (HLE) and porcine pancreatic elastase (PPE) were analyzed comparatively. We observed that 1 behaved as a substrate for both elastases, without the formation of a stable acyl‐enzyme as in the case of azapeptides. Compounds 1 and 2 were cleaved at the same site (‐Val‐/‐NH‐) with a slight delay of hydrolysis for 1 compared to 2 (kcat/KM for 1 vs. 2 decreased by a factor of 2.7 for the HLE‐catalyzed hydrolysis at pH 8.0 and 25°C). The presence of the hydrazinopeptidic bond (‐CONHNH‐) in 1 reduced by a factor of 4.7 the apparent enzyme affinity without abolishing it. These results indicate that suitably designed hydrazinopeptides may represent interesting targets in the search for protease resisting pseudopeptides.
Comments on the use of a dichromophoric circular dichroism assay for the identification of β‐turns in peptides
Authors: BALDWIN J.E. , CLARIDGE T.D.W. , HULME C. , RODGER A. , SCHOFIELD C.J.
Published: August 2010

Use of the dichromophoric CD assay for β‐turn formation in peptide sequences has been investigated. The assay involves the observation of Cotton effects in CD spectra, originating from the approach of N‐ and C‐terminal aromatic chromophores in tetrapeptides. The approach of the chromophores was believed to be brought about by a beta;‐turn in the peptide structure. Our investigations were paralleled by NMR studies which revealed the presence of a previously unreported hydrogen bond in the β‐turn conformers, which appears to play a role in the generation of the observed Cotton effects. This suggests caution in the use of the CD technique alone as an assay for β‐turn conformers in peptides.
Reinvestigation of the reactions of carbodiimides with alkoxycarbonylamino acid symmetrical anhydrides
Authors: IZDEBSKI JAN , ORLOWSKA ALICJA , ANULEWICZ ROMANA , WITKOWSKA EWA , FIERTEK DARIUSZ
Published: August 2010

Alkoxycarbonylamino acid symmetrical anhydrides were allowed to react with N,N′‐diisopropylcarbodiimide. The determination of the decrease in carbodiiniide concentration indicated that the reaction was slow and proceeded indirectly. It is presumed that symmetrical anhydride in the presence of basic carbodiimide is converted into oxazolone and alkoxycarbonylamino acid. The latter reacts with carbodiimide to give O‐acylisourea which, in turn, rearranges to N‐acylurea. A second minor product was found to be N‐(N1,N2‐bisalkoxycarbonyldipeptidyl)urea. The main pathway leading to the formation of this product starts with rearrangement of the symmetrical anhydride to N1, N2‐bisalkoxycarbonyldipeptide acid. These experiments suggest that the reaction between anhydride and carbodiimide is not the source of N‐acylurea in peptide synthesis.
The conformational stability of a non‐covalent dimer of a platelet‐derived growth factor‐B mutant lacking the two cysteines involved in interchain disulfide bonds
Authors: PRESTRELSKI STEVEN J. , ARAKAWA TSUTOMU , DUKER KAREN , KENNEY WILLIAM C. , NARHI LINDA O.
Published: August 2010

Native platelet‐derived growth factor‐B (PDGF‐B) forms a covalent dimer through interchain disulfide bonds. In a previous study, an analog of PDGF‐B was produced by replacing cysteine 43 and 52, which are involved in the interchain disulfide bonds, with serine. It was revealed that this analog protein has the dimeric molecular weight at pH 4 to 7, forming a non‐covalent dimer in solution, and its mitogenic activity is similar to the native covalent dimer. However, the analog protein was more labile to pepsin digestion and low pH treatment, indicating that the interchain disulfides contribute to the stability of the protein. It is interesting to see if the conformation of the protein is affected by elimination of the interchain disulfide bonds, and if the interchain disulfides play any role in the stability of the protein. Circular dichroism and Fourier transform infrared spectroscopic analyses of the analog showed that it has a conformation similar to the wild type at pH 7.5, but is unfolded at pH 2.5, while the native PDGF‐B disulfide‐linked dimer shows an apparently unaltered conformation at pH 2.5. The analog is also less stable to sodium dodecylsulfate and guanidine HC1‐induced denaturation at neutral pH. These results indicate that the non‐covalent interactions are sufficient for proper folding and dimer formation at neutral pH, but that the interchain disulfide bonds greatly stabilize the native conformation of PDGF‐B. © Munksgaard 1994.
Peptidyl substrates containing unnatural amino acid at the P′1 position are potent inhibitors of prohormone convertases
Authors: BASAK AJOY , SCHMIDT CATRIN , ISMAIL ASRAF A. , SEIDAH NABIL G. , CHREATIEN MICHEL , LAZCRE CLAUDE
Published: August 2010

In order to study further the importance of the P′ 1 residue upon the activity of human PCl and human furin, two important members of subtilisin/kexin family of enzymes, we have prepared by solid‐phase Fmoc or recently introduced FastMoc chemistry a series of 10 peptidyl substrate analogs. The structures of these analogs are based upon the core sequence of pro‐mPCl(83‐93) namely, D‐Tyr‐Lys‐Glu‐Arg‐Ser‐Lys‐Arg‐Xaa‐Val‐Gln‐Lys‐Asp, where d‐Ty replaces the native l‐Tyr residue and Xaa, representing the P′ 1 position, corresponds to l‐Ser or to nonproteinacous amino acids such as Tle, Sarc, MLeu, Aib, d‐Tic or l‐Tic. Two more analogs with l‐Tic at P′ 1 position but with one amino acid less, namely P5 Glu or P′3 Gln, and one with a Cit residue in place of Arg at P1 site of the dodecapeptide were also obtained. These peptides were all fully characterized by a combination of MS. 1H‐NMR and amino acid analysis. In contrast to the Ser analog, which is an excellent substrate for both hPCl and hfurin, these analogs displayed moderate to strong inhibition of both hPCl and hfurin activity in a reversible competitive manner. They all exhibited higher potency for hfurin than for hPC1, with an inhibition constant (K1,) ranging from 0.8 to 10 μ M and from 1.0 to 170 μ M, respectively. Incorporation of l‐Tic yielded an analog with a two to four‐fold increased inhibition of either enzymes when compared to its d‐Tic counterpart, the effect being more pronounced for hPCl than for hfurin. Comparison of these data with those for the corresponding N‐terminal Fmoc protected peptides revealed that the highly hydrophobic N‐terminal Fmoc function occupying the P8 position can contribute positively or negatively towards proteinase inhibition depending on the nature of the unnatural amino acid at P′1 and the enzyme used. Finally, none of the analogs was significantly cleaved by either enzyme. FTIR data on these analogs revealed some important structural differences between the substrate and inhibitor analogs, as there appears to be a conformational shift from a more β‐sheet‐like structure for the substrates to a more α‐helical‐like structure for the inhibitors.
Solution structure of a human calcitonin analog elucidated by NMR and distance geometry calculations
Authors: KATAHIRA RITSUKO , DOI MASAMITSU , KYOGOKU YOSHIMASA , YAMADA‐NOSAKA ATSUKO , YAMASAKI KEIKO , TAKAI MICHIHIRO , KOBAYASHI YUJI
Published: August 2010

Three‐dimensional structure of a human calcitonin analog (abbreviated as hCTa) in which the amino acids of the wild type are replaced at positions 12, 16 and 19 by leucine residues and further at position 22 by a tyrosine residue was studied in TFE solution by 1H‐NMR and distance geometry calculations. This analog has a 15‐20 times activity as compared with the wild type. The amino acid replacements resulted in formation of an amphiphilic α‐helix in the region between the residues 4‐20. The overall three‐dimensional structure is similar to that of the wild type. The conformational feature of hCTa with a hydrophobic face composed with a Met and four Leu residues may be related to its higher hypocalcemic potency.
Solution and solid‐state structure of the diketopiperazine of tyrosyl‐tetrahydroisoquinoline‐3‐carboxylic acid
Authors: CIAJOLO M.R. , BALBONI G. , PICONE D. , SALVADORI S. , TANCREDI T. , TEMUSSI P.A. , TUZI A.
Published: August 2010

δSelective antagonism of [L‐Tic2]‐peptides, including the simple dipeptide Tyr‐L‐Tic‐NH2, is linked to the Tyr‐Tic‐“recognition site”. In order to gain further information on the conformational preferences of the Tyr‐Tic‐moiety we have undertaken a structural study of a cyclic analog, the diketopiperazine of Tyr‐Tic. A conformational study of cycle[‐Tyr‐Tic‐], that is almost devoid of opioid activity, can also be useful to discriminate between the role of the two aromatic rings and of the basic nitrogen in determining antagonism. The structure of cycle[‐Tyr‐Tic‐] has been solved in a DMSO/water solution at 278 K by NMR spectros‐copy and in the solid state by X‐ray diffraction methods. The two informations are almost identical, with an arrangement of the aromatic rings rather different from that of the putative bioactive conformation of the parent linear dipeptide. This difference points to the importance of coformational effects and is in agreement with the hypothesis that the positive center may be not essential for antogonism. © Munksgaard 1995.