bluuebunny/crossref_metadata_2025 huggingface dataset:
Synthesis and biological activity of new conformationally restricted analogues of pepstatin*
Authors: SZEWCZUK ZBIGNIEW , REBHOLZ KAREN L. , RICH DANIEL H.
Published: August 2010

A new statine derivative, 3‐hydroxy‐4‐amino‐5‐mercaptopentanoic acid; cysteinylstatine (CySta), was synthesized and used to prepare a series of conformationally restricted analogues of pepstatin (Iva‐Val‐Val‐Sta‐Ala‐Sta) in which the conformational constraint was introduced via a bis‐sulfide connecting the appropriately substituted residues in the P1 and the P3 inhibitor side chains. The precursor peptide, Iva‐Cys‐Val‐CySta‐Ala‐Iaa, was synthesized and alkylated with a series of dibromoalkanes and alkenes to produce the cyclic structures. This strategy permitted the carbon atom spacing between the P1 and the P3 inhibitor side chains to be systematically varied so as to produce inhibitors with 15‐, 16‐, and 17‐membered ring systems. Additional non‐cyclic analogues were synthesized as controls by alkylating the bisthiol intermediates with methyl iodide. The inhibitory potency of the analogues were determined against porcine pepsin and penicillopepsin by using standard enzyme kinetic assays. The cyclic inhibitor were found to be potent inhibitors of both aspartic proteases; inhibitor that contained a trans‐2‐butene link between the two sulfur atoms was found to be the most potent inhibitor with a Ki less than 1 nm against pepsin and 3.94 nm against penicillopepsin. This series of compounds illustrates a new type of conformational restriction that can be used to probe for the bioactive conformation of peptides.
Influence of glycine residues on peptide conformation in membran environments
Authors: LI SHUN‐CHENG , DEBER CHARLES M.
Published: August 2010

Transmembrane (TM) segments of integral membrane proteins are putatively α‐helical in conformation, yet their primary sequences are rich in residues known in globular proteins as helix‐breakers (Gly) and β‐sheet promoters (Ile, Val, Thr). To examine the specific 2° structure propensities of such residues in membrane environments, we have now designed and synthesized a series of model 20‐residue peptides with “guest” hydrophobic segments embedded in “host”N‐ and C‐terminal hydrophilic matrices. Molecular design was based on the prototypical sequence NH2‐(Ser‐Lys)2‐Ala5‐Leu6‐x7‐A1a8‐Leu9‐y10‐Trp11‐A1a12‐Leu13‐z14‐(Lys‐Ser)3‐OH. The 10‐residue hydrophobic mid‐segment 5–14 is expected to act as ca. three turns of an α‐helix. In the present work, we compare the 20‐residue peptide having three “helix‐forming” Ala residues [x = y = z = Ala (peptide 3A)] to the corresponding peptide 3G (x = y = z = Gly) which contains three “helix‐breaking” Gly residues. Trp was inserted to provide a measure of aromatic character typical of TM segments; Ser and Lys enhanced solubility in aqueous media. Circular dichroism studies in water, in a membrane‐mimetic [sodium dodecylsulfate (SDS)] medium, and in methanol solutions, demonstrated the exquisite sensitivity of the conformations of these peptides to environment, and proved that despite its backbone flexibility, Gly can be accommodated as readily as Ala into a hydrophobic a‐helix in a membrane. Nevertheless, the relative stability of Ala‐ vs. Gly‐containing helices emerged in methanol solvent titration and temperature dependence experiments in SDS. The overall results conjure a dual functional role for Gly residues in hydrophobic segments, i.e., “helix‐formation” as well as “helix‐destabilization”, and lead us to propose that the helical propensity of a given residue in a peptide must be specified by the contexts ‐ local segment primary sequence and environmental media ‐ in which it occurs.
Semisynthesis of human growth hormone‐releasing factor by trypsin catalyzed coupling of leucine amide to a C‐terminal acid precursor
Authors: BONGERS JACOB , OFFORD ROBIN E. , FELIX ARTHUR M. , CAMPBELL ROBERT M. , HEIMER* EDGAR P.
Published: August 2010

Human growth hormone‐releasing factor, GRF(1–44)‐NH2, was synthesized by trypsin catalyzed coupling of Leu‐NH2 to Arg43 of the precursor, GRF(1–43)‐OH, prepared by solid phase peptide synthesis. The semisynthetic GRF(1–44)‐NH2 was fully characterized and showed full potency in the rat pituitary in vitro bioassay. Conversion to GRF(1–44)‐NH2 was limited to 60–70% in both 75% v:v N,N′‐dimethylacetamide and 95% v:v 1,4‐butanediol due to competing transpeptidations at Arg41 and Arg38 generating [Leu42]‐GRF(1–42)‐NH2 and [Leu39]‐GRF(1–39)–NH2 side‐products, respectively. The rates of formation and yields of GRF(1–44)‐NH2 versus pH, Leu‐NH2 concentration, and solvent composition were also studied.
Comparative study of methods to couple hindered peptides
Authors: SPENCER JEFFREY R. , ANTONENKO VALERY V. , DELAET NANCY G.J. , GOODMAN MURRAY
Published: August 2010

A comparative study of modern coupling reactions involving Boc‐protected amino acid derivatives and dipeptides with N‐terminal α,α–dialkylation and N‐methylation was carried out. The coupling reactions were run using either equimolar amounts of the amino and activated carboxyl components or an excess of the activated carboxyl component. Yields of the target tripeptide Boc‐Phe‐Xaa‐Phe‐OBzl (Xaa = (NMe)Ala, (NMe)Alb, or (NMe)αAc5c) were compared. Less than 10% of the product was obtained from methods utilizing pivaloyl mixed anhydride, pentafluorophenyl ester or acyl fluoride activation when Xaa = (NMe)Aib and (NMe)αcAc5c. At room temperature, significant yields of these two products were obtained from reactions which utilized an excess of the HBTU reagent (O‐benzotriazol‐1‐yl‐N,N,N′,N′‐tetramethyluronium hexafluorophosphate), the PyBroP reagent (bromo‐tris‐pyrrolidino‐phosphonium hexafluorophosphate) or Boc‐Phe‐NCA (Boc‐protected phenylalanine N‐carboxyanhydride). Moreover, the Boc‐Phe‐NCA method was superior when used over a prolonged reaction time or at elevated temperature.
Cleavage and deprotection of peptides on MBHA‐resin with hydrogen bromide
Authors: WANG S.S. , WANG B.S.H. , HUGHES J.L. , LEOPOLD E.J. , WU C.R. , TAM J.P.
Published: August 2010

Dilute hydrogen bromide in trifluoroacetic acid containing pentamethylbenzene and thioanisole was used in the cleavage and deprotection of peptides on MBHA‐resin. Particular attention was paid to potential applicability of the method to kilogram scale synthesis of thymosin α1. In the HPLC purification of the peptides, acetonitrile was replaced by relatively nontoxic isopropanol. The change should be economically and environmentally very attractive.
Incorporation of azaglutamine residues into peptides synthesised by the ultra‐high load solid (gel)‐phase technique
Authors: GRAY C.J. , QUIBELL M. , BAGGETT N. , HAMMERLE T.
Published: August 2010

The solid (gel)‐phase peptide synthesis of peptides, each containing an azaglutamine residue, has been examined. Procedures using various mono‐, di‐ and tripeptide and carbazate fragments containing or relating to an azaglutamine (1) residue have been evaluated. N‐Activation of the amino‐terminus of a resin‐bound peptide with bis‐(2,4‐dinitrophenyl)carbonate (2) yielded the terminal isocyanate species, which reacted with protected carbazates to give resin‐bound protected peptides containing the aza‐residue. By contrast, coupling of activated amino‐acid derivates to the free amino‐group of a resin‐bound peptide with an aza‐residue at the N‐terminus was a slow and unsatisfactory process. It is concluded that the route yielding the best results involves the reaction of a protected amino‐acyl carbazate to a resin‐bound isocyanate‐activated peptide.
Synthesis and biological activity of analogues of theC‐terminal hexapeptide of substance P with modifications at glutaminyl and methioninyl residues
Authors: ANTONIOU MARIA , POULOS CONSTANTINE , TSEGENIDIS THEODORE
Published: August 2010

Analogues of [Orn6]‐SP6‐11have been synthesized in which the methionyl residue is replaced by glutamine γ‐carboxamide substituted derivatives. These analogues where tested in threein vitropreparations representative of NK‐1, NK‐2 and NK‐3 receptor types. Substitution of the SCH3group of the Met11side chain by CONHCH3, CON(CH3)2, CONHPh and CONCH3Ph groups results in analogues which are full agonists in NK‐1 and NK‐2 preparations with the exception of the Glu[N(CH3)2]11and the Glu(NHCH3)11analogues, which are partial agonists at NK‐1 and NK‐2 receptors respectively. The Glu(NHCH3)11analogue shows selectivity for the NK‐1 receptor type and is equipotent to the Glu(NCH3Ph)11analogue in the same receptor type. The latter analogue is 2.84 times more potent than the parent hexapeptide in the NK‐2 preparation. The Glu(NHPh)11analogue is a full agonist in the NK‐3 preparation and equipotent to the parent hexapeptide, in contrast to the other analogues in which Met has been replaced by glutamine γ‐carboxamide substituted residues. It is concluded that for NK‐1 receptor type the lipophilic character of Met11side chain is not a determining factor for activity but it is an important factor for activity in the NK‐2 receptor type and has a stronger effect when a phenyl group is present, thus leading to analogues which are full agonists and more potent than the parent hexapeptide.
Fatty acid and drug binding to a low‐affinity component of human serum albumin, purified by affinity chromatography
Authors: VORUM HENRIK , PEDERSEN ANDERS O. , HONORÉ BENT
Published: August 2010

Binding equilibria for decanoate to a defatted, commercially available human serum albumin preparation were investigated by dialysis exchange rate determinations. The binding isotherm could not be fitted by the general binding equation. It was necessary to assume that the preparation was a mixture of two albumin components about 40% of the albumin having high affinity and about 60% having low affinity. By affinity chromatography we succeeded in purifying the low‐affinity component from the mixture. The high‐affinity component, however, could not be isolated. We further analyzed the fatty acid and drug binding abilities of the low‐affinity component. The fatty acids decanoate, laurate, myristate and palmitate were bound with higher affinity to the mixture than to the low‐affinity component. Diazepam was bound with nearly the same affinity to the low‐affinity component as to the albumin mixture, whereas warfarin was not bound at all to the low‐affinity component.
Pituitary adenylate cyclase activating polypeptide (PACAP) with 27 residues
Authors: INOOKA HIROSHI , ENDO SATOSHI , KITADA CHIEKO , MIZUTA EIJI , FUJINO MASAHIKO
Published: August 2010

The conformation of pituitary adenylate cyclase activating polypeptide with 27 residues (PACAP27) has been determined by two‐dimensional NMR and CD spectroscopies and distance geometry in 25% methanol. Residues 9‐20 and 22‐25 have well‐defined conformations but other residues do not show ordered conformations. The conformation of residues 9‐20 is composed of three distinct regions of β turn‐like conformation (residues 9‐12), α helix (residues 12‐14) and the looser helical conformation (residues 15‐20), while residues 22‐24 form α helix. PACAP27 has a 2 helices separated by a disordered region similar to a VIP analog reported by Fry et al. but is distinct from the VIP analog in the position of the first helix, which is shifted by 2 residues toward the C‐terminus, and in the form of the second helix [Fry, D.C., Madison, V.S., Bolin, D.R., Greeley, D.N., Toome, V. and Wegrzynski, B.B. (1989) Biochemistry28, 2399‐2409].
Structural variations in the crystal structures of two homologous DL‐Leu and Δ‐Leu containing peptides+
Authors: EL‐MASDOURI LARBI , AUBRY ANDRÉ , BOUSSARD GUY , MARRAUD MICHEL
Published: August 2010

The similar conformations and interaction modes of Ac‐DL‐Leu‐Nme2 and Ac‐Δ‐Leu‐NMe2 molecules in the solid state allow the comparison of their geometrical parameters. The most evident variations are essentially restricted to the α,β‐unsaturated side‐chain which adopts the Z‐disposition. The dimensions of the peptide backbone are much less sensitive to α,β‐unsaturation, with a small shortening by 0.04 Å and 0.02 Å of the N‐Cα and Cα‐C′ bonds, respectively, and an increase by 6° of the N‐Cα‐ C′ bond angle. The ethylenic and amide groups in the Δ‐Leu derivative are far from coplanarity, and a significant electronic conjugation of the π‐orbital is likely to be rejected.
Studies on sensitivity to racemization of activated residues in couplings of N‐benzyloxycarbonyldipeptides
Authors: BENOITON N. LEO , LEE YOUNG C. , STEINAUR RENE , CHEN FRANCIS M.F.
Published: August 2010

A series of 24 peptides Z‐Gly‐Xaa(R)‐OH where Xaa= 15 different residues and R= H, NH2, tBu, Bzl, Trt, Mtr, and StBu were coupled with valine benzyl ester in dimethylformamide or dichloromethane at +5°. The accompanying racemization was determined by analysis of the epimeric products by normal phase high‐performance liquid chromatography (HPLC) for Xaa(R) = Met, Cys(StBu) and Lys(Z) and by reversed‐phase HPLC after removal of benzyl‐based protecting groups for Xaa(R) = Ser(tBu), Thr(tBu) and Arg(Mtr). The coupling methods examined included mixed anhydride (MxAn) at ‐5°, and N,N′‐dicyclohexylcarbodiimide (DCC), benzotriazol‐1‐yl‐tris(dimethylamino)phosphonium hexafluorophosphate (BOP) and O‐benzotriazol‐1‐yl‐N,N,N′,N′‐tetramethyluroniumhexafluorophosphate (HBTU) in the presence of 1‐hydroxybenzotriazole (HOBt). Very few couplings gave stereochemically pure products. The order of sensitivity to racemization of residues depended on the method of coupling and the solvent. It varied most when comparing MxAn to HOBt‐assisted reactions; it varied moderately when comparing HOBt‐assisted reactions. There was less variation in comparing BOP and HBTU reactions that are initiated by the same mechanism. Leu, Nle, Phe, Asn, Lys(Z) and Asp(OBzl) are identified as the residues least sensitive to racemization. DCC‐HOBt generally led to less epimerization than the other methods.
Isolation and primary structure of a potent toxin from the venom of the scorpionCentruroides sculpturatusEwing
Authors: PETE MATTHEW J. , CONLON J. MICHAEL , MURPHY RICHARD F.
Published: August 2010

A potent toxin has been purified from the venom of the scorpionCentruroides sculpturatusEwing using the ion‐exchange resin CM‐Sepharose CL‐6B at basic pH. The toxin, designated CsE M1, comprised 65 amino acid residues and its primary structure was established as: Lys‐Glu‐Gly‐Tyr‐Leu‐Val‐Asn‐Ser‐Tyr‐Thr10‐Gly‐Cys‐Lys‐Tyr‐Glu‐Cys‐Leu‐Lys‐Leu‐Gly20‐Asp‐Asn‐Asp‐Tyr‐Cys‐Leu‐Arg‐Glu‐Cys‐Arg30‐Gln‐Gln‐Tyr‐Gly‐Lys‐Ser‐Gly‐Gly‐Tyr‐Cys40‐Tyr‐Ala‐Phe‐Ala‐Cys‐Trp‐Cys‐Thr‐His‐Leu50‐Tyr‐Glu‐Gln‐Ala‐Val‐Val‐Trp‐Pro‐Leu‐Pro60‐Asn‐Lys‐Thr‐Cys‐Asn. CsE M1is the most lethal protein to be identified inC. sculpturatusvenom and the LD50of the toxin, determined by subcutaneous injection into Swiss mice, is 87 μg/kg. CsE M1shows strong structural similarity (92% positional identity) to the most potent β‐toxin, Css II, from the Mexican scorpion,Centruroides suffusus suffususbut is quite dissimilar to the previously characterized toxins with low potency isolated fromC. sculpturarusEwing.
14‐Membered cyclic opioids related to dermorphin and their partially retro‐inverso modified analogues II. Preferred conformations in solution as studied by1H‐NMR spectroscopy
Authors: YAMAZAKI TOSHIMASA , MIERKE DALE F. , SAID‐NEJAD ODILE E. , FELDER EDUARD R. , GOODMAN MURRAY
Published: August 2010

The1H‐NMR studies were extensively carried out to elucidate preferred conformations of a series of 14‐membered cyclic dermorphin analogues containing two phenylalanines at both the third and fourth positions, e.g., Tyr‐c[D‐A2bu‐Phe‐Phe‐(landd)‐Leu], Tyr‐c[d‐A2bu‐Phe‐gPhe‐(S and R)‐mLeu], and Tyr‐c[d‐Glu‐Phe‐gPhe‐(L and D)‐rLeu]. The temperature coefficients of the amide proton chemical shifts, vicinal1H‐1H coupling constants for the NH‐CH groupings, and nuclear Overhauser effects provided information regarding the preferred conformations of the backbones. The conformational preferences and flexibility of the side chains were also estimated from the vicinal1H‐1H coupling constants around the C‐Cβ and C‐Cβ bonds in the articulated side chains. A comparison of the results obtained was made with the results previously obtained for the corresponding enkephalin analogues containing a glycine at the third position. It was found that the replacement of the glycine with the phenylalanine at the third position increases the conformational flexibility of the molecules with anl‐, or S‐, residue at the fifth position but reduces the flexibility of the molecules withd‐, or R‐, residue at the same position. The rotating frame nuclear Overhauser experiments gave direct evidence for compact conformations, with the Tyr side chain folding back over the 14‐membered ring in Tyr‐c[d‐Glu‐Phe‐gPhe‐rLeu], which displays relatively high selectivity for the δ‐receptor over the μ‐receptor. This observation is in agreement with our model proposed for the cyclic enkephalin analogues: folded forms with close aromatic ring placement are required for the activity at the δ‐receptor.
Circular dichroism of reduced and oxidized recombinant human epidermal growth factor
Authors: NARHI LINDA OWERS , ARAKAWA TSUTOMU , MCGINLEY MICHAEL D. , ROHDE MICHAEL F. , WESTCOTT KEITH R.
Published: August 2010

To further elucidate the role of the disulfide bonds in determining the protein folding of recombinant human epidermal growth factor (r‐HuEGF) we studied the structure of reduced and oxidized r‐HuEGF using circular dichroism (CD). The far UV CD spectrum of reduced r‐HuEGF in 10 mm sodium phosphate pH 3.0 is very different from that of the oxidized molecule. The spectrum of the reduced molecule consists of a plateau from 225 to 200 nm, consistent with the presence of α‐helix, β‐sheet, and unordered structure. The addition of the α‐helix inducer trifluoroethanol to the reduced molecule resulted in an enhancement of α‐helix, at the apparent expense of β‐sheet, while the oxidized molecule was unaffected by the presence of this reagent. Secondary structure predictions based on the amino acid sequence of EGF correlate most closely with the structure of the reduced molecule. From these results, it appears that the r‐HuEGF has a more regular secondary structure in the absence of the disulfide bonds than in their presence. This suggests that the folding of EGF occurs by destroying the regular secondary structure that was present in the reduced state, and that the structure of the native molecule is dictated largely by disulfide bonding.
Fmoc‐amino acid chlorides in solid phase synthesis of opioid peptides
Authors: SIVANANDAIAH KADLEBAL M. , BABU VOMMINA V. SURESH , RENUKESHWAR CHANDRAMOWLY
Published: August 2010

Fmoc‐amino acid chlorides were employed in the solid phase synthesis of the opioid peptides [Leu]enkephalin, [Leu]enkephalin amide, and dermorphin. The conventional polystyrene‐based Merrifield resin or Wang's resin served as solid support. A binary salt of either triethylamine or diisopropylethylamine in the presence of 1‐hydroxybenzotriazole or pivalic acid was used for acylation. The coupling rates were quite fast, being comparatively faster when 1‐hydroxybenzotriazole was used along with triethylamine or diisopropylethylamine. The peptides obtained in good yields showed, after purification, biological and spectral properties identical with those of the natural peptides.
Conformational investigation of α, β‐dehydropeptides
Authors: CISZAK EWA , PIETRZYÑSKI GRZEGORZ , RZESZOTARSKA BARBARA
Published: August 2010

The crystal structure of Ac‐Pro‐ΔVal‐NHCH3 was examined to determine the influence of the α,β‐dehydrovaline residue on the nature of peptide conformation. The peptide crystallizes from methanol‐diethyl ether solution at 4° in needle‐shaped form in orthorhombic space group P212121 with a= 11.384(2) Å, b = 13.277(2) Å, c = 9.942(1) Å. V = 1502.7(4) Å3 Z = 4, Dm= 1.17 g cm−3 and Dc=1.18 g cm−3 The structure was solved by direct methods using SHELXS‐86 and refined to an R value of 0.057 for 1922 observed reflections. The peptide is found to adopt a β‐bend between the type I and the type III conformation with φ1=−68.3(4)°, ψ1=− 20.1(4)°, φ2=−73.5(4)°= and Ψ2=−14.1(4)°=. An intramolecular hydrogen bond between the carbonyl oxygen of ith residue and the NH of (i+ 3)th residue stabilizes the β‐bend. An additional intermolecular N.,.O hydrogen bond joins molecules into infinite chains. In the literature described crystal structures of peptides having a single α,β‐dehydroamino acid residue in the (i+ 2) position and forming a β‐bend reveal a type II conformation.
Isolation and characterization of glycosylated and non‐glycosylated prolactins from alligator and crocodile
Authors: NOSO TOYOHIKO , SWANSON PENNY , LANCE VALENTINE A. , KAWAUCHI HIROSHI
Published: August 2010

Two molecular forms of prolactin (PRL). glycosylated and non‐glycosylated, were isolated from pituitary glands of two reptiles, alligator and crocodile. The reptilian PRLs were extracted under alkaline conditions from the precipitate obtained after pituitaries were first extracted with 0.25 m sucrose, 1 mM NH4HCO3, pH 6.3. Purification was performed by ion exchange chromatography on DE‐52, gel filtration on Sephadex G‐75 superfine, and reversed phase high performance liquid chromatography. Two forms of both alligator and crocodile PRL, designated PRLI and PRLII, with molecular weights of 26000 and 24000 were isolated. Alligator and crocodile PRLI and PRLII were stained specifically in immunoblots with anti‐sea turtle PRL and anti‐ostrich PRL. Sequence analysis revealed that both forms of alligator and crocodile PRLs consisted of 199 amino acid residues with a glycosylation consensus sequence (Asn‐Ala‐Ser) at position 60 in alligator and crocodile PRLs with a molecular weight of 26000 (PRLI). In contrast, Thr was substituted for Asn at position 60 in the PRLs with a molecular weight of 24000 (PRLII). The sequences of alligator PRLs differed from crocodile PRLs only in position 134: Val for alligator PRLs and He for crocodile PRLs. There is a high degree of structural conservation between the reptilian PRLs isolated in this study and avian PRL; each showed 92% sequence identity with chicken PRL and 89% with turkey PRL.
Studies on cell‐clearing activity in α‐lactalbumin
Authors: WHITE FREDERICK H.
Published: August 2010

Effects of calcium removal on the cell‐clearing activity of α‐lactalbumin (α‐LA) and concomitant changes in conformational structure have been investigated as part of a continuing study of the activity found earlier [McKenzie, H.A. & White, F.H., Jr. (1987) Biochem. Int. 14, 347]. This activity is similar to that of lysozyme, whereby lysis of the bacterial cell wall is catalyzed. However, the specific activity of α‐LA is on the order of 10−6 that of lysozyme. Under conditions where activities of apo and native α‐LA were approximately linear functions of the protein concentration, the maximal ratio of apo to native activity was 5.7:1, determined by comparison of second order velocity constants. The CD spectrum of apo α‐LA is intermediate between that of the A state and the native protein. By NMR, the conformation of apo α‐LA is similar to, but distinctly different from, that of the native protein. The apo form did not revert completely to the native state when Ca(II) was resupplied, consistent with a role for this cation in folding. It is suggested that the activity increase may result from a diminished constriction of the “cleft” region in α‐LA.
“Carba” peptide bond surrogates Different approaches to Gly‐Ψ(CH2‐CH2)‐d,l‐Xaa pseudo‐dipeptide units
Authors: RODRIGUEZ MARC , HEITZ ANNIE , MARTINEZ JEAN
Published: August 2010

Racemic “carba” pseudo‐dipeptide units such as Gly‐Ψ(CH2‐CH2)‐d,l‐Xaa were obtained either through the Horner‐Emmons condensation of N‐tert.‐butyloxycarbonyl‐βalaninal with the appropriate substituted triethyl phosphonoacetate, or from commercially available 3‐carbethoxy‐2‐piperidone.
Apparent specific volumes of some dipeptides (containing L‐valine and L‐leucine in aqueous alkylurea solutions)
Authors: POKLAR NATAŠA , SENEGAČNIK MARJAN M. , ŠVEGLJ FRANCI , LAPANJE SAVO
Published: August 2010

The apparent molal volumes of nine dipeptides containing glycine, l‐valine, and l‐leucine have been determined in methyl, N,N′‐dimethyl and ethylurea solutions from precise density measurements. Limiting partial molal volumes. 02, at various solute concentrations have also been calculated. The experimental values of 02 in water agree reasonably well with those calculated as the sum of 02 of both acids after accounting for the electrostrictive effect and loss of water. There is no correlation between the values of 02 of individual dipeptides in alkylureas which means that the intrinsic volume and the electrostrictive effect make the largest contribution to 02. The contribution from other effects is within the limit of experimental error. The volumes of transfer from water to alkylurea solutions are all positive and reflect by and large the electrostrictive effect.
Crystal structure of tert.‐butyloxycarbonyl‐L‐prolyl‐D‐alanyl‐D‐alanyl‐N‐methylamide Dimeric β‐sheet formation
Authors: ANANTHANARAYANAN V.S. , BOULAY GASTON , MARY J. SAHAYA , SUBRAMANIAN E. , MICHEL ANDRÉ G.
Published: August 2010

The crystal structure of the tripeptide t‐Boc‐L‐Pro‐D‐Ala‐D‐Ala‐NHCH3, monohydrate, (C17H30N4O5·H2O, molecular weight = 404.44) has been determined by single crystal X‐ray diffraction. The crystals are mono‐clinic, space group P21, a = 9.2585(4), b = 9.3541(5). c = 12.4529(4) Å, β= 96.449(3)°, Z = 2. The peptide units are in the trans and the tBoc‐Pro bond in the cis orientation. The first and third peptide units show significant deviations from planarity (Δω=5.2° and Δω=3.7°, respectively). The backbone torsion angles are: φ1, = ‐60°, ψ1/= 143.3°, ω1= ‐174.8°, φ2= 148.4°, ψ2= ‐143.1°, ω2= ‐179.7°, φ3= 151.4°, ψ3= ‐151.9°, ω3= ‐176.3°. The pyrrolidine ring of the proline residue adopts the C2— Cγ conformation. The molecular packing gives rise to an antiparallel β‐sheet structure formed of dimeric repeating units of the peptide. The surface of the dimeric β‐sheet is hydrophobic. Water molecules are found systematically at the edges of the sheets interacting with the urethane oxygen and terminal amino groups. Surface catalysis of an L‐Ala to D‐Ala epimerization process by water molecules adsorbed on to an incipient β‐sheet is suggested as a mechanism whereby crystals of the title peptide were obtained from a solution of tBoc‐Pro‐D‐Ala‐Ala‐NHCH3.
Efficient solution‐phase synthesis of multiple O‐phosphoseryl‐containing peptides related to casein and statherin
Authors: PERICH JOHN W. , KELLY DAVID P. , REYNOLDS ERIC C.
Published: August 2010

The multiple Ser(P)‐containing peptides, H‐Ser(P)‐Ser(P)‐Ser(P)‐Glu‐Glu‐NHMe‐TFA, H‐Asp‐Ser(P)‐Ser(P)‐Glu‐Glu‐NHMe‐TFA and H‐Glu‐Ser(P)‐Ser(P)‐Glu‐Glu‐NHMe‐TFA were prepared by the use of Boc‐Ser(PO3Ph2)‐OH in the Boc mode of solution phase peptide synthesis followed by platinum‐mediated hydrogenolytic de‐protection of the Ser(PO3Ph2)‐containing peptides. The protected peptides were assembled using the mixed anhydride coupling methods with 40% TFA/CH2C12 used for removal of the Boc group from intermediate Boc‐protected peptides.
Unexpected racemization of proline or hydroxy‐proline phenacyl ester during coupling reactions with Boc‐amino acids
Authors: KURODA HISAYA , KUBO SHIGERU , CHINO NAOYOSHI , KIMURA TERUTOSHI , SAKAKIBARA SHUMPEI
Published: August 2010

When L‐proline or O‐benzyl‐trans‐4–hydroxy‐L‐proline phenacyl ester was coupled with Boc‐amino acids in dimethylformamide using water‐soluble carbodiimide (WSCI) in the presence of anhydrous 1‐hydroxybenzotriazole (HOBt) as coupling reagents, extensive racemization was observed at the Cα of the proline or hydroxy‐proline residue. The extent of racemization was measured by HPLC after the coupling with Boc‐L‐Leu‐OH in the presence or absence of HOBt. The extent of racemization increased when HOBt was added to the reaction mixture, but greatly decreased when it was not, indicating that HOBt was needed for inducing racemization. Almost no racemization was observed when the coupling reaction was carried out by the mixed anhydride procedure in tetrahydrofuran or by the carbodiimide method in dichloromethane without using HOBt. In the case of coupling reactions with ordinary L‐amino acid phenacyl esters, no racemization was observed. Examination of some model systems yielded sufficient evidence to prove that HOBt is an efficient catalyst for racemizing proline or hydroxy‐proline phenacyl ester not only in the stage of cyclic intermediate formation but also in the opening of the ring structure. Thus, the racemization reaction was found to be closely related to the formation of the cyclic carbinol‐amine derivative.
Conformationally biased analogs of oxytocin
Authors: LEBL MICHAL , TOTH GEZA , SLANINOVÁ JIŘINA , HRUBY VICTOR J.
Published: August 2010

Four diastereomeric analogs of oxytocin containing substituted phenylalanine in position 2 were synthesized. This modified phenylalanine side chain contained one methyl group attached to the β‐carbon and the second one at the 2′ position of the aromatic ring. All analogs were found to be inhibitors of uterotonic activity of oxytocin with pA2 values ranging from 6.0 to 8.3; the most potent one (pA2= 8.3) contained dimethylphenylalanine of the D‐erythro configuration
Studies on antigenic variability of C strains of foot‐and‐mouth disease virus by means of synthetic peptides and monoclonal antibodies
Authors: CARRENO CRISTINA , ROIG XAVIER , CAIRO JORDI , CAMARERO JULIO , MATEU MAURICIO G. , DOMINGO ESTEBAN , GIRALT ERNEST , ANDREU DAVID
Published: August 2010

Peptides representing the sequence of the immunodominant loop of foot‐and‐mouth disease virus strain C‐S8 (YTASARGDLAHLTTTHARHLP, residues 136–156 of VP1) and of several variant viruses have been prepared by solid phase methods. In addition, five peptides with single‐residue replacements at Leu147 (Ile, Nle, Val, Ala, Gly) have been synthesized. Tosyl and dinitrophenyl protections for histidine have been compared, the latter being found to give better synthetic products. The peptides have been tested in an immunodot assay against a panel of monoclonal antibodies directed towards the VP1 loop. Immunochemical results are discussed on the basis of the mobility of the region reproduced by the peptides and the nature of the side chain of residue 147.
Further studies on the use of 2,2,2‐trichloroethyl groups for phosphate protection in phosphoserine peptide synthesis
Authors: PAQUET ALENKA
Published: August 2010

Boc‐Ser(PO3Tc2)‐OH, Z‐Ser(PO3Tc2)‐OH and Fmoc‐Ser(PO3Tc2)‐OH, derivatives useful for peptide synthesis, have been obtained in high yields by acylation of H‐Ser(PO3Tc2)‐OHCF3COOH. The latter was obtained from Boc‐ or Z‐Ser(PO3Tc2)‐OBzl by simultaneous removal of the amino‐ and carboxy‐protecting groups by Pd‐catalyzed hydrogenolysis in acetic acid‐trifiuoroacetic acid solution. Removal of the Tc‐protecting group was efficiently achieved by hydrogenolysis in aqueous ethanol.
Simultaneous use of 1‐hydroxybenzotriazole and copper(II) chloride as additives for racemization‐free and efficient peptide synthesis by the carbodiimide method
Authors: MIYAZAWA TOSHIFUMI , OTOMATSU TOSHIHIKO , FUKUI YOSHIMASA , YAMADA TAKASHI , KUWATA SHIGERU
Published: August 2010

In the carbodiimide mediated coupling of Z‐Gly‐l‐Val‐OH with H‐l‐Val‐OMe in DMF, the simultaneous use of HOBt and copper(II) chloride as additives was found to give the desired peptide in a high yield without racemization. In the presence of HOBt, reducing the amount of copper(II) chloride produced a higher yield. Besides improving the coupling efficiency as compared with the case using copper(II) chloride alone as an additive, the present procedure offered another advantage for racemization suppression. Thus, even for the couplings where a low level of racemization was observed in the presence of copper(II) chloride, the simultaneous addition of HOBt and copper(II) chloride resulted in the elimination of racemization. The effectiveness of this new procedure using the two carbodiimide additives in the synthesis of biologically active peptides was assessed by the preparation of a protected Leuenkephalin. In the 4+1 segment condensation using HOBt and copper(II) chloride simultaneously as additives, no racemization was detected and the yield was high enough. The elimination of racemization and improvement of coupling efficiency produced by the present procedure can be attributable to a reduced tendency for the activated forms of the carboxyl component to form a 5(4H)‐oxazolone by the action of HOBt, and to the prevention of racemization by copper(II) chloride of the small amount of the oxazolone formed which is not eliminated by the action of HOBt alone.
Structure activity of C‐terminal modified analogs of Ac‐CCK‐7
Authors: TILLEY JEFFERSON W. , DANHO WALEED , SHIUEY SHIAN‐JAN , KULESHA IRINA , SARABU RAMAKANTH , SWISTOK JOSEPH , MAKOFSKE RAYMOND , OLSON GARY L. , CHIANG ELLIOT , RUSIECKT VICTORIA K. , WAGNER ROLF , MICHALEWSKY JOSEPH , TRISCARI JOSEPH , NELSON DAVID , CHIRUZZO FRANCISCA Y. , WEATHERFORD SALLY
Published: August 2010

Previous work indicates that both the C‐terminal phenylalanine amide and the tryptophan moieties of chole‐cystokinin (CCK) are critical pharmacophores for interaction with either the A or B receptor subtypes. We have examined a series of analogs of Ac‐CCK‐7 [Ac‐Tyr(SO3H)‐Met‐Gly‐Trp‐Met‐Asp‐Phe33‐NH2] (2) in which the phenyl ring of the C‐terminal Phe‐NH2 has been modified. Compounds were assessed in binding assays using homogenated rat pancreatic membranes and bovine striatum as the source of CCK‐A and CCK‐B receptors respectively and for anorectic activity after intraperitoneal administration to rats. Substitution of a number of cycloalkyi or bicyclic aryl moieties for the phenyl ring of phenylalanine33 including cyclopentyl (20), cyclohexyl (21), cyclooctyl (23), 2‐(5,6,7,8‐tetrahydro)naphthyl (26), 2‐naphthyl (27), and 1‐naphthyl (29) led to analogs with 10–70 times the anorectic potency of 2. The anorectic activity of 21 was blocked by the specific CCK‐A receptor antagonist MK‐329. Other bulky aliphatic groups in place of the phenylalanine33 aromatic ring such as isopropyl, 2‐adamantyl and cyclohexylmethyl gave derivatives similar to 2 in potency. While most of the new compounds were comparable to CCK in binding assays, 23, 26, 27 and 29 were exceptionally potent with IC50s 10‐11‐10‐14 M in the pancreas. Compounds 23 and 29 were further evaluated for their ability to stimulate amylase secretion and found to have potencies similar to that of CCK. The dissociation between potency in the binding and amylase secretion assays suggests that they may interact with a high affinity binding site which is not coupled to amylase secretion. We conclude that CCK receptors possess a generous hydrophobic pocket capable of accommodating large alkyl groups in place of the side chain of phenylalanine33 and that the pharmacological profile of CCK analogs can be tailored by appropriate exploitation of this finding.
Structure activity studies of tryptophan30 modified analogs of Ac‐CCK‐7
Authors: DANHO WALEED , TILLEY JEFFERSON W. , SHIUEY SHIAN‐JAN , KULESHA IRINA , SWISTOK JOSEPH , MAKOFSKE RAYMOND , MICHALEWSKY JOSEPH , WAGNER ROLF , TRISCARI JOSEPH , NELSON DAVID , CHIRUZZO FRANCISCA Y , WEATHERFORD SALLY
Published: August 2010

Cholecystokinin represents a family of gut hormones which among other activities, have been proposed to participate in satiety signaling. Ac‐CCK‐7 [Ac‐Tyr(SO3H)‐Met‐Gly‐Trp30‐Met‐Asp‐Phe‐NH2 (2)] possesses the full spectrum of activity and potency of the intact hormone; thus analogs of 2 may be useful as anorectic agents. A series of derivatives has been prepared in which the tryptophan indole moiety of 2 has been modified. The new compounds were assayed in CCK binding assays using homogenated rat pancreatic membranes and bovine striatum as a source of CCK‐A and CCK‐B receptors respectively and in vivo in rats for anorectic activity. Although previous studies have concluded that the indole ring of Trp30 is a critical pharmacophore for the interaction of CCK with both its A and B type receptors, we find 2‐Nal30‐Ac‐CCK‐7 (20) to be nearly equipotent to 2 in both CCK binding and as an anorectic agent sensitive to blockade by the Merck CCK‐A receptor antagonist MK‐329. The extreme structural sensitivity of this anorectic activity is illustrated by the l‐naphthylalanine30 (19) and (benzo[b]thien‐2‐yl)alanine30 (21) analogs which are 30 and 100 times less potent than 2 respectively. Other mono‐ and bicyclic Trp30 replacements, including substituted phenylalanines, 3‐quinolinylalanine, and 2‐(5,6,7,8‐tetrahydro)naphthylalanine, gave inactive compounds.
Structure‐activity studies of vasoactive intestinal peptide (VIP): cyclic disulfide analogs
Authors: BOLIN D.R. , COTTRELL J. , GARIPPA R. , O'NEILL N. , SIMKO B. , O'DONNELL M.
Published: August 2010

Analogs of vasoactive intestinal peptide with cysteine residues incorporated at selected sites within the sequence were prepared by solid phase methods, oxidized to the corresponding cyclic disulfides and purified to homogeneity by preparative HPLC. The cyclic compounds were assayed as smooth muscle relaxants on isolated guinea pig trachea, as bronchodilators in vivo in guinea pigs, and for binding to VIP receptors in guinea pig lung membranes. Of the analogs prepared at the N‐terminus, one compound, Ac‐[D‐C∼S6,D‐Cys11,Lys12,Nle17,Val26,Thr28]‐VIP, was found to be a full agonist with slightly more than one tenth the potency of native VIP. Most other cyclic analogs in the N‐terminal region were found to be inactive. A second analog, Ac‐[Lys12,Cys17,Val26,Cys28]‐VIP, was also found to be a full agonist with potency about one third that of native VIP. Furthermore, this compound was active as a bronchodilator in vivo in guinea pig, but with somewhat diminished potency as compared to native VIP. Strikingly, this cyclic compound was found to have significantly longer duration of action (>40 min) when compared to an analogous acyclic compound (5 min). The conformational restrictions imposed by formation of the cyclic ring structures may have stabilized the molecule to degradation, thus enhancing the effective duration of action. Analysis of this series of cyclic analogs has also yielded information about the requirements for the receptor–active conformation of VIP.
Synthesis and biological activities of ψ(CH2NH) pseudopeptide analogues of the C‐terminal hexapeptide of neurotensin
Authors: COUDER J. , TOURWÉ D. , VAN BINST G. , SCHUURKENS J. , LEYSEN J.E.
Published: August 2010

Each peptide CO‐NH function in the biologically important C‐terminal 8‐13 sequence of neurotensin was replaced by the reduced CH2‐NH isostere using the rapid in situ solid phase procedure developed by Sasaki & Coy. In general this modification resulted in a drop in receptor affinity except for the [Argψ(CH2NH)Arg]‐NT8–13 analogue (PIC50 9.23 vs. NT8–13 PIC50 8.03). This analogue also showed enhanced enzymatic stability, but acted as a full agonist as shown by the observation of relaxations of guinea‐pig colon ascendens.
Modulation of the activity and assessment of the receptor selectivity in a series of new RGD‐containing peptides
Authors: FAUCHÈRE JEAN‐LUC , MORRIS ANGELA D. , THURIEAL CHRISTOPHE , SIMONET SERGE , VERBEUREN TONY J. , KIEFFER NELLY
Published: August 2010

We have investigated the structure‐activity relationship of a series of new synthetic RGD analogs and their potential use as specific platelet aggregation inhibitors. Twelve short linear peptides showed high potency to inhibit aggregation in ADP‐stimulated dog platelets. In order to assess the selectivity of these analogs towards platelet integrin GPIIb‐IIIa, a new cell adhesion inhibition system was devised which was able to discriminate between the two closely related β3‐integrins of the vasculature, GPIIb‐IIIa (αIIbβ3), present in platelets, and the vitronectin receptor (αvβ3), expressed in endothelial cells and platelets. As reported for other peptides by Scarborough et. al. (1993, J. Biol. Chem. 268, 1066), the analogs containing lysine instead of arginine in position 1 showed increased selectivity towards GPIIb‐IIIa. One of them, in which the piperidine carboxylic group was attached to the N‐terminus of KGDW, not only strongly inhibited platelet aggregation, but also selectively abolished cell adhesion mediated by GPIIb‐IIIa without effect on the vitronectin receptor.
Peptides from multiple regions of the lectin domain of P‐selectin inhibiting neutrophil adhesion
Authors: HEAVNER GEORGE A. , FALCONE MARGARET , KRUSZYNSKI MARIAN , EPPS LEON , MERVIC MILJENKO , RIEXINGER DOUGLAS , MCEVER ROGER P.
Published: August 2010

The selectins are a family of three structurally related glycoproteins that are integral components of leukocyte adhesion to the vascular endothelium. Their involvement in the recruitment and extravasation of neutrophils is critical in mounting an inflammatory reaction. The carbohydrate nature of the selectin ligands suggests that the binding regions of the selectins are contained within the lectin‐like domains of the selectins. The synthesis and evaluation for inhibition of selectin binding of overlapping peptides of the lectin and adjacent EGF‐like domains of P‐selectin have been used to identify small peptides that completely inhibit P‐selectin‐dependent neutrophil adhesion. These peptides span a region of more than 100 amino acids and may define the carbohydrate recognition domain of P‐selectin.
Stereospecificity of hydrogen transfer by the NADP‐linked alcohol dehydrogenase from the thermophilic bacterium Thermoanaerobium brockii
Authors: PERETZ MOSHE , BOGIN OREN , KEINAN EHUD , BURSTEIN YIGAL
Published: August 2010

Class A and class B NAD(H):NADP(H) coenzyme‐dependent dehydrogenases distinguish between the diastereotopic hydrogens pro‐R and pro‐S at position 4 of the cofactor. We investigated the stereochemistry of hydride transfer in reactions catalyzed by an unusual thermophilic, zinc‐containing, NADP‐linked enzyme Thermoanaerobium brockii alcohol dehydrogenase (TBAD). Using proton NMK spectroscopy of monodeuterated alcohols and coenzymes we found that TBAD is a class A enzyme that transfers the pro‐R hydrogen from the pyridine 4 position of the reduced coenzyme. This stereospecificity is stable over (a) a broad range of temperatures up to 70° C. (b) different concentrations of the coenzyme (catalytic or stoichiometric) and (c) a wide scope of substrates. Although NAD + is not an effective coenzyme for TBAD, NADP + and its synthetic analogs, 3‐acetqlpyridine‐ADP + and thio‐NADP +, can be used successfully.
Synthesis and conformational studies on peptides corresponding to a putative autophosphorylation site of abl TPK*
Authors: RUZZA PAOLO , CALDIIRAN ANDREA , FILIPPI BRUNO , DONELLA‐DEANA ARIANNA , PINNA LORENZO A. , BORIN GIANFRANCO
Published: August 2010

The transforming gene of Abelson murine leukaemia virus (v‐abl) codes for a membrane‐associated tyrosine‐specific protein kinase (abl TPK). Analysis of the v‐abl gene has shown that both the fibroblast‐transforming and tyrosine‐protein kinase activities reside within a minimal region encoding a protein of 43 kDa (p43v‐abl), which represents the most active, isolated form of this enzyme. Since the cellular substrates for p43 v‐abl are yet to be identified, we synthesized by classical solution methods the octapeptide H‐Gly‐Asp‐Thr‐Tyr‐Thr‐Ala‐His‐Ala‐OH, corresponding to the structural sequence of the main putative autophosphorylation site (Tyr 515) of the abl TPK, as well as some of its analogs modified in positions ‐2, ‐1, + 1 and + 3.

The synthetic peptides were tested as substrates for the p43v‐abl. The kinetic data obtained indicate that the rates of their phosphorylation vary considerably depending on the sequence of the peptide, as expected. As a rule, no significant increment of the efficiency results from each substitution in the parent sequence. While the replacement of the two charged residues, namely Asp‐2 and His‐7, with neutral Ala is well tolerated, the substitution with amino acids bearing opposite charges is detrimental. The correlation between secondary structure of our synthetic octapeptides and their substrate recognition by p43v‐abl was studied using CD and fluorescence spectroscopy in 5 mM Tris, in 98% TFE/Tris and in 30 mM SDS solutions. The comparison of the spectroscopic data with the kinetic parameters does not confirm a close relationship between the conformational properties of these peptides and their enzymatic role.
Acidolytic cleavage of tris(alkoxy)benzylamide (PAL) “internal reference” amino acyl (IRAA) anchoring linkages: validation of accepted procedures in solid‐phase peptide synthesis (SPPS)
Authors: ALBERICIO FERNANDO , BARANY GEORGE
Published: August 2010

Under the normal conditions of acidolytic cleavage/deprotection of tris(a1koxy)benzylamide (PAL) anchoring linkages in Fmoc solid‐phase peptide synthesis (SPPS), product release occurs by a straightforward single‐step pathway. A recently reported cleavage of the NH‐VH bond of an amino acyl residue adjacent to PAL [see Int. J. Peptide Protein Res.38, 146–153 (1991)] could not be confirmed in novel experiments incorporating a double “internal reference” amino acid (IRAA) design. The results of the present work revalidate the widely accepted application of IRAAs to monitor yields in SPPS, and confirm the reliability of PAL methodology for the preparation of C‐terminal peptide amides.
Spontaneous degradation via diketopiperazine formation of peptides containing a tetrahydroisoquinoline‐3‐carboxylic acid residue in the 2‐position of the peptide sequence
Authors: MARSDEN BRIAN J. , NGUYEN THI M.‐D. , SCHILLER PETER W.
Published: August 2010

The δ‐opioid antagonists H‐Tyr‐Tic‐Phe‐OH (Tic = tetrahydroisoquinoline‐3‐carboxylic acid) and H‐Tyr‐Tic‐Phe‐Phe‐NH2 were shown to undergo spontaneous Tyr‐Tic diketopiperazine formation with concomitant cleavage of the Tic‐Phe peptide bond in DMSO but not in aqueous solution at pH 7.7.
Formation of pyroglutamylglutamine (or asparagine) diketopiperazine in ‘non‐classical’ conditions: a side reaction in peptide synthesis
Authors: MAZUROV ANATOLY A. , ANDRONATI SERGEI A. , KOROTENKO TAMARA I. , GORBATYUK VITALY YA. , SHAPIRO YURY E.
Published: August 2010

Formation of by‐products pyroglutamylglutamine diketopiperazine (5a) or pyroglutamylasparagine diketopiperazine (5b) were observed during the condensation of pyroglutamic acid active ester with C‐protected glutaminyl(or asparaginyl)‐proline derivatives. Successful synthesis of Glp‐Gln(or Asn)‐Pro‐derivatives is possible after coupling of Glp‐Gln(or Asn)‐OH with proline‐derivatives.