bluuebunny/crossref_metadata_2025 huggingface dataset:
Novel cyclization chemistry especially suited for biologically derive unprotected peptides
Authors: WOOD STEPHEN J. , WETZEL RONALD
Published: August 2010

A novel method is described for the cyclization of peptides ‐ or segments of polypeptides ‐ which requires a free A′‐terminal α‐amino group and a distal amino acid residue containing a nucleophilic side chain. The reaction is conducted in two steps, both in the aqueous phase. The first step involves acylation of the N‐terminal α‐amino group with iodoacetic anhydride at pH 6. This acylation reaction has >90% specificity for peptide α‐amino groups and gives no alkylation of Arg, His, Lys or Met by the iodoacetate side product (R. Wetzel et al, Bioconjugate Chem., 1, 114‐122, 1990). In the second step, the acylation reaction mixture or the isolated iodoacetyl‐peptide is incubated at room temperature to give the cyclic peptide formed by reaction of the nucleophilic side chain with the iodoacetyl moiety. The pH dependence of the cyclization reaction by Met, Lys, Arg or His is consistent with the pKa of the nucleophilic side chain. Thus, peptides containing Met plus other nucleophilic amino acids should preferentially cyclize via Met at low pH. In this paper, preparation of cyclic peptides containing 3‐6 amino acids is described; the full range of ring sizes and sequences which can undergo this cyclization has not been further explored. Preliminary results suggest that this method is also fairly general with respect to the amino acid sequence being cyclized. The reaction appears to be particularly suited for cyclization via Lys and Met side chains. All of the cyclized products are sufficiently stable for many biological applications. However, peptides cyclized via Met slowly decompose at 37° with ring cleavage to yield a C‐terminal lactone peptide in which the Met methylthio‐group is effectively transferred to the JV‐terminus. The reactions described here have several applications which take advantage of their unique ability to efficiently cyclize naturally‐derived (as well as chemically synthesized) peptides in aqueous solution with little or no protection/deprotection chemistry.
Affinity enhancement of complementary peptide recognition
Authors: FASSINA GIORGIO , CORTI ANGELO , CASSANI GIOVANNI
Published: August 2010

A peptide hydropathically complementary to Big Endothelin [Big ET] residues 16‐29 has been synthesized in a multimeric form starting from an octadentate poiylysine core, essentially in a way similar to the procedure used for the production of multiple antigenic peptides [MAP's], Interaction between the multimeric complementary peptide [8δET] and the Big ET fragment 16‐32 containing the target complementary region, also synthesized in a multimeric form [8ET], was evaluated by analytical high performance affinity chromatography and solid phase binding assays. While the binding interaction between the monomerics peptide pair was in the micromolar range, the recognition between the corresponding multimeric form was characterized by enhanced binding affinity of at least two orders of magnitude. In solution, complex formation between multimeric complementary peptide and target Big ET sequence in the monomeric and multimeric form was accompanied by precipitation at concentrations higher than 0.5 mg/mL and 0.1 mg/mL, respectively. Polyclonal antibodies raised against the multimeric target sequence recognized multimeric and monomeric ET target sequences with binding affinities similar to binding affinities exhibited by the multimeric complementary peptide. Multimerization of hydropathically complementary peptides could provide an improved opportunity to measure and thus probe quantitative binding properties of complementary peptides.
In situ neutralization in Boc‐chemistry solid phase peptide synthesis
Authors: SCHNölzer MARTINA , ALEWOOD PAUL , JONES ALUN , ALEWOOD DIANNE , KENT STEPHEN B.H.
Published: August 2010

Simple, effective protocols have been developed for manual and machine‐assisted Boc‐chemistry solid phase peptide synthesis on polystyrene resins. These use in situ neutralization [i.e. neutralization simultaneous with coupling], high concentrations (> 0.2 M) of Boc‐amino acid‐OBt esters plus base for rapid coupling, 100% TFA for rapid Boc group removal, and a single short (30 s) DMF flow wash between deprotection/coupling and between coupling/deprotection. Single 10 min coupling times were used throughout. Overall cycle times were 15 min for manual and 19 min for machine‐assisted synthesis (75 residues per day). No racemization was detected in the base‐catalyzed coupling step. Several side reactions were studied, and eliminated. These included: pyrrolidonecarboxylic acid formation from Gln in hot TFA‐DMF; chain‐termination by reaction with excess HBTU; and, chain termination by acetylation (from HOAc in commercial Boc‐amino acids). The in situ neutralization protocols gave a significant increase in the efficiency of chain assembly, especially for “difficult” sequences arising from sequence‐dependent peptide chain aggregation in standard (neutralization prior to coupling) Boc‐chemistry SPPS protocols or in Fmoc‐chemistry SPPS. Reported syntheses include HIV‐1 protease(1–50,Cys.amide), HIV‐1 protease(53–99), and the full length HIV‐1 protease(1–99).
A rational design of synthetic peptide vaccine with a built‐in adjuvant
Authors: DEFOORT JEAN‐PHILIPPE , NARDELLI BERNARDETTA , HUANG WOLIN , TAM JAMES P.
Published: August 2010

We describe a peptide vaccine model containing a built‐in adjuvant. This model used a multiple antigen peptide system (MAPS) to amplify peptide antigens and a lipoamino acid, tripalmitoyl glyceryl cysteine (P3C), as a built‐in adjuvant. An 18–residue peptide antigen (B2) derived from the third variable domain (amino acid 312–329) of the glycoprotein gp120 of type I human immunodeficiency virus (HIV‐1) was used in this model. This peptide antigen is a suitable target since it consists of neutralizing, T‐helper, and T‐cytotoxic epitopes. The peptide antigen in a tetravalent MAPS format (B2M‐P3C) with a lipophilic attachment was synthesized by two routes for comparison: a direct stepwise approach and an indirect modular approach. In the stepwise approach, each residue was sequentially added to the peptide resin to give B2M‐P3C and the P3C was incorporated to the side chain of a carboxyl terminal lysine as Fmoc‐Lys(P3C). In the modular approach, a module containing a chloroacetylated core matrix of MAPS (M‐P3C) with a carboxyl tetrapeptide bearing Lys(P3C) and a second module containing the peptide antigen B2 with a cysteine at its terminus were synthesized and purified separately, and then coupled to each other to form B2M‐P3C. In the modular approach, the molecular ion of B2M‐P3C was unambiguously identified by ion‐spray mass spectrometry. B2M‐P3C, administered in liposomes without any adjuvant such as Freund's complete adjuvant, was used to immunize mice and found to induce gp120‐specific antibodies in vitro, and prime cytotoxic T lymphocytes in vivo. In contrast, the tetravalent B2 on the MAPS (B2M) without a built‐in adjuvant was non‐immunogenic and required Freund's complete adjuvant to elicit a low‐titered antibody response. Our results show that our model with a built‐in adjuvant may lead to useful designs of vaccines suitable for humans.
Molecular dynamics simulations of opioid peptide analogs containing multiple conformational restrictions
Authors: WILKES BRIAN C. , SCHILLER PETER W.
Published: August 2010

Molecular dynamics simulations were performed on the potent and slightly μ‐receptor selective cyclic dermorphin analog as well as on analogs containing a conformationally restricted phenylalanine derivative in place of Phe in the 3 position of the peptide sequence. Peptides studied included the potent and highly μ‐selective analogs (Aic = 2‐aminoindan‐2‐carboxylic acid), (Atc = 2‐aminotetralin‐2‐carboxylic acid) and and the weakly active analog (Tic = tetrahydroisoquinoline‐3‐carboxylic acid). Four different starting conformations were chosen for each peptide, and after equilibration each simulation was allowed to proceed for 100 picoseconds at 600°K. The 14‐membered ring structures in the Phe‐, Aic‐, l‐ and d‐Atc‐containing analogs showed moderate structural flexibility, while the peptide ring in the Tic‐containing analog was more rigid. As theoretically predicted, the φ3 and Ψ3 angles of the Aic‐, l‐ and d‐Atc‐containing analogs were limited to values of either about +50° or ‐50° during almost the entire period of the simulations. In the Tic‐containing analog the φ3 and Ψ33 angles were 0° and 90°, respectively, and did not change for the entire duration of the simulation. The side chains of the constrained amino acids showed limited movement, but transitions between the allowed conformations did occur on the time scale of the simulations. One interesting aspect of the 5‐membered ring of the Aic side chain was that it underwent a greater number of conformational transitions than the 6‐membered rings contained in Atc and Tic, but covered a smaller volume of conformational space. Thus, the relative flexibility of these constrained amino acids was Phe > Aic > l‐ and d‐Atc = Tic, but the relative volume of conformational space visited by these same residues was Phe > l‐ and d‐Atc = Tic > Aic. In all compounds studied the exocyclic Tyr1 residue had the greatest flexibility and covered a large volume of conformational space.
Solid phase peptide synthesis of lipopeptide vaccines eliciting epitope‐specific B‐, T‐helper and T‐killer cell response
Authors: WJESMÜLLER KARL‐HEINZ , BESSLER WOLFGANG G. , JUNG GÜNTHER
Published: August 2010

Lymphocyte subpopulations involved in the self and nonself recognition processes are antibody producing cells, T‐helper cells and T‐killer cells. By using lipopeptide adjuvants and lipopeptide–antigen conjugates each of the major pathways of immune response can be specifically addressed on the molecular level of minimized synthetic lipopeptide vaccines. The immunologically active principle of the lipopeptide constructs is the synthetic N‐terminus of bacterial lipoprotein, tripalmitoyl‐S‐glycerylcysteine, which can be covalently linked to B‐, T‐helper and CTL epitopes. Methods of multiple peptide synthesis based on Merrifield's solid‐phase synthesis allow the economic production of the high numbers of overlapping lipopeptides required for the complete immunological screening of viral proteins.
Amino acid structure and “difficult sequences” in solid phase peptide synthesis
Authors: BEDFORD JOY , HYDE CAROLYN , JOHNSON T. , JUN WEN , OWEN D. , QUIBELL M. , SHEPPARD R.C.
Published: August 2010

Deprotection peak profiles have been determined as a measure of internal aggregation during Fmoc‐polyamide continuous flow solid phase synthesis. The results have been correlated with amino‐acid structure and discussed in terms of minimising aggregation during synthesis.
Solid phase peptide synthesis on hydrophilic supports
Authors: ENGLEBRETSEN DARREN R. , HARDING DAVID R.K.
Published: August 2010

Beaded cellulose (Perloza) was modified with acrylonitrile followed by reduction with diborane to give a functionalised support containing aminopropyl groups. This spacer arm was then further extended with a glycolamido or an Fmoc‐amino acid‐4‐oxymethylphenoxyacetyl moiety. A number of peptides, including the Merrifield test peptide leucyl‐alanyl‐glycyl‐valine, leucine‐enkephalin, Acyl Carrier Protein (65–74). angiotensin I and II, ACTH(4‐11) and LHRH were synthesised on the aminopropyl beaded cellulose support using modified t‐butyloxycarbonyl (Boc) or fluorenylmethoxycarbonyl (Fmoc) synthesis protocols. The peptides were cleaved from the support and further purified.
Design, construction and application of a fully automated equimolar peptide mixture synthesizer
Authors: ZUCKERMANN RONALD N. , KERR JANICE M. , SIANI MICHAEL A. , BANVILLE STEVEN C.
Published: August 2010

A fully automated peptide synthesizer has been constructed that is capable of the synthesis of equimolar peptide mixtures and the simultaneous synthesis of 36 individual peptides. The synthesizer was constructed from a workstation of our own design utilizing a Zymark robot arm. A Macintosh II computer coordinates the movements of the robotic arm, the switching of over 40 solenoid valves and the monitoring of sensors in the workstation. The robot hands are used to deliver solvents from pressurized spigot lines and to pipet amino acid solutions from reservoirs to an array of reaction vessels. Liquid dispensing, reagent mixing and solvent removal are controlled from a multifunction I/O board in the computer. The design features of the synthesizer are presented, as well as the characterization of multiple individual peptides, a simple mixture of 19 components, and a complex mixture of 15625 components.
Hydrogen fluoride catalyzed migration of side chain protecting groups onto Fmoc during solid phase peptide synthesis
Authors: GRODE STEPHEN H. , STROTHER DIANE S. , RUNGE THOMAS A. , DOBROWOLSKI PAUL J.
Published: August 2010

The solid‐phase synthesis of the Nα‐Fmoc analog of protein kinase C substrate (PKCS, Lys‐Arg‐Ala‐Lys‐Ala‐Lys‐Thr‐Thr‐Lys‐Lys‐Arg) was characterized by low recovery from the resin and the concomitant appearance of four impurities. FAB‐MS revealed molecular weights for two of these impurities that corresponded to the desired peptide plus Tos or Bzl. The other two were justified by invoking a CO2 elimination of the Clz protecting group to yield: 1) peptide plus 2‐chlorobenzyl (CIBzl) and 2) peptide plus ClBzl and Tos. A CF‐FAB analysis of carboxypeptidase digestions allowed observation of peptide cleavage down to an ion corresponding to lysine, Fmoc, and the corresponding protecting group(s). These data revealed that the impurities were not the result of incomplete deprotection but the result of migration of the protecting groups to the N‐terminal end of the peptide. NMR experiments were subsequently performed and revealed the exact site of substitution: the meta positions of the N‐terminal Fmoc. These impurities are presumed to arise by electrophilic aromatic substitution of the fluorene group during HF treatment. The desired Fmoc analog served as a convenient, albeit low‐yielding, intermediate for purification of the highly charged PKCS by preparative self‐displacement HPLC.
Structure‐toxicity relationships in the amatoxin series
Authors: ZANOTTI GIANCARLO , PETERSEN GABRIELE , WIELAND THEODOR
Published: August 2010

The amatoxins, highly toxic components of death cap Amanita mushrooms, bind strongly to RNA polymerase II (or B) in cell nuclei thus preventing the transcription of DNAs to hn‐RNAs (Pre‐mRNAs), the precursors of messenger RNAs. Three of the binding sites of the bicyclic octapeptides have been identified: an isoleucine side chain in position 6, a trans‐4‐hydroxyl group at proline in position 2 and a hydroxylated l‐isoleucine side chain in position 3. No information exists about the stereochemical conditions at the β‐C‐atom (C‐atom 3) of this side chain. We have now synthesized the diastereomeric S‐deoxoamaninamides (Fig. 1) containing, in position 3, l‐allo‐isoleucine (analog 1), (2S, 3R)‐2‐amino‐4‐hydroxy‐3‐methyl butyric acid (analog 2), the diastereomer (2S, 3S)‐2‐amino‐4‐hydroxy‐3‐methylbutyric acid (analog 3) and d‐isoleucine (analog 4). In the last synthesis, besides the “normal” bicyclic octapeptide 4, an isomeric Iso‐4 was formed. The affinities for Drosophila RNA polymerase II were 100 times weaker as compared to γ‐amanitin for 1, 10 times weaker for 2, 200 times weaker for 3, 100 times weaker for 4, and more than 1000 times weaker for Iso‐4 The results point to the importance of a methyl group in (R)‐configuration at the β‐C atom of side chain 3.
Site‐specific biotinylation
Authors: NATARAJAN SESHA , FESTIN STEPHEN M. , HEDBERG ANDERS , LIU EDDIE C.‐K. , FLOYD DAVID M. , HUNT JOHN T.
Published: August 2010

We have developed an expeditious method for the incorporation of the biotinylaminocaproyl moiety on the ε‐amino group of a lysine residue within a peptide chain in a site‐specific manner. Using t‐Boc chemistry for the solid phase synthesis approach and a base labile, acid stable protecting group (Fmoc‐) for the ε‐amino group of the target lysine, we prepared fully protected resin bound peptides which are site‐specifically biotinylated. Following HF cleavage, the uniquely biotinylated peptides were obtained in a high degree of purity. Using this approach, a number of biotinylaminocaproyllysyl derivatives of a monocyclic Endothelin‐1 analog were prepared. Synthesis of selected bicyclic analogs of high affinity monocycles led to the preparation of the bicyclic [Nle7]ET‐1 analog containing ε‐biotinylaminocaproyllysine at position‐9. This peptide, with Kd= 0.08 nM, has 1000‐fold higher affinity for the ETA receptor than the commercially available Nα‐biotinylated Endothelin‐1. The general utility of this biotinylation methodology was demonstrated by the synthesis of a site‐specifically biotinylated PTH analog which contained several side chain functionalized amino acid residues in its sequence. The synthetic method reported here is convergent in that it allows the facile variation of the length of the spacer and also offers the potential to introduce in a site specific manner other groups such as affinity labels and fluorescent tags.
Continuous flow synthesis of peptides using a polyacrylamide gel resin (Expansin™)
Authors: MENDRE CHRISTIANE , SARRADE VERONIQUE , CALAS BERNARD
Published: August 2010

The continuous flow syntheses of endothelin 1, proendothelin 2. ATP binding site of the CDC2 kinase 3, and fragment 18‐30 of an actin 4, have been performed by using a polyacrylamide gel resin Expansin™ (about 0.6 mmol NH2/g) with the glycolamidic ester handle as labile anchorage. In addition, we report here a method of air oxidation which reduces the formation of side‐products related to the formation of intermolecular disulfide bridges.
Proteinase‐catalyzed conversion of a substance P‐precursor peptide
Authors: SCHELLENBERGER VOLKER , KLÖPPEL WERNER TEGGE. KLAUS‐DIETER , FRANK RONALD
Published: August 2010

The protease‐catalyzed conversion of peptides and proteins produced by recombinant DNA technology is a promising method for large‐scale production of peptides including those with non‐proteinogenic structural elements. As a model system we have investigated the proteinase‐catalyzed modification of a chemically synthesized substance P‐precursor. In the precursor peptide the residues of substance P(1‐8) were flanked by tripeptide linkers on both sides. In the first step the C‐terminal tripeptide amide was replaced by the authentic C‐terminal tripeptide amide of substance P via α‐chyniotrypsin‐catalyzed transpeptidation. The enzyme simultaneously attacks two peptide bonds in the precursor molecule leading to the formation of several side‐products. The desired peptide was obtained with 25%, yield. In the second step the other tripeptide linker was selectively and almost quantitatively removed from the N‐terminus of the precursor via trypsin‐catalyzed hydrolysis. This study demonstrates that substance P can be obtained from an engineered protein by proteinase‐catalyzed processing.
Design of potent substrate‐analogue inhibitors of canine renin
Authors: HUI K.Y. , SIRAGY H.M. , HABER E.
Published: August 2010

Through a systematic study of structure‐activity relationships, we designed potent renin inhibitors for use in dog models. In assays against dog plasma renin at neutral pH, we found that, as in previous studies of rat renin inhibitors, the structure at the P2 position appears to be important for potency. The substitution of Val for His at this position increases potency by one order of magnitude. At the P3 position, potency appears to depend on a hydrophobic side chain that does not necessarily have to be aromatic. Our results also support the approach of optimizing potency in a renin inhibitor by introducing a moiety that promotes aqueous solubility (an amino group) at the C‐terminus of the substrate analogue. In the design of potent dog plasma renin inhibitors, the influence of the transition‐state residue 4(S)‐amino‐3(S)‐hydroxy‐5‐cyclohexylpentanoic acid (ACHPA) ‐ commonly used as a substitute for the scissile‐bond dipeptide to boost potency ‐ is not obvious, and appears to be sequence dependent. The canine renin inhibitor Ac‐paF‐Pro‐Phe‐Val‐statine‐Leu‐Phe‐paF‐NH2 (compound 15; IC50 of 1.7 nM against dog plasma renin at pH 7.4; statine, 4(S)‐amino‐3(S)‐hydroxy‐6‐methylheptanoic acid; paF, para‐aminophenylalanine) had a potent hypotensive effect when infused intravenously into conscious, sodium‐depleted, normotensive dogs. Also, compound 15 concurrently inhibited plasma renin activity and had a profound diuretic effect.
Glycoconjugates of opioid peptides
Authors: VARGA‐DEFTERDAROVIĆ L. , HORVAT Š. , CHUNG N.N. , SCHILLER P.W.
Published: August 2010

ThreeN‐glycoconjugates of the general formula H‐Tyr‐Gly‐Gly‐Phe‐Leu‐NH‐R (R = carbohydrate residue) were synthesized in order to determine the influence of some carbohydrate molecules (6‐amino‐6‐deoxy‐d‐glucopyranose, 2‐amino‐2‐deoxy‐d‐glucopyranose, β‐d‐glucopyranosylamine) on the biological activity, conformation, and stability of the opioid pentapeptide [Leu5]enkephalin. For the preparation of this compound different methods of peptide synthesis (active ester and mixed anhydride) were investigated. In comparison with [Leu5]enkephalin, all threeN‐glycoconjugates showed higher potency in the guinea pig ileum assay and lower potency in the mouse vas deferens assay, indicating a decrease inbopioid receptor selectivity.
Cyclic hexapeptides derived from the human thymopoietin III
Authors: KESSLER HORST , HAASE BURKHARD
Published: August 2010

Six diastereoisomeric cyclic hexapeptides of the partial sequence 39‐44 of human thymopoietin III in which each residue was replaced with the d‐amino acid were synthesized. In contrast to expectations, the yields from cyclization were not influenced by the location of the d‐residue.
Replacement of Tyr‐SO3H by ap‐carboxymethyl‐phenylalanine in a CCK8‐derivative preserves its high affinity for CCK‐B receptor
Authors: MCCORT‐TRANCHEPAIN I. , FICHEUX D. , DURIEUX C. , ROQUES B.P.
Published: August 2010

The sulfated tyrosine present in the sequence of CCK8Asp26‐Tyr(SO3H)‐Met‐Gly‐Trp‐Met‐Asp‐PheNH2, seems to play a critical role in the recognition of CCK‐A binding sites. In this work, we have investigated whether the presence of an anionic charge on the tyrosine moiety is strictly necessary and whether the sulfate moiety interacts with a divalent cation in the receptor subsite. For this purpose, the novel amino acids (l,d) Phe(p‐CH2CO2H) and (l,d) Phe(p‐CH2CONHOH), as well as theirl‐resolved forms were introduced into the sequence of Ac[X27, Nle28, Nle31]‐CCK27‐33by solid phase method. The biological activities of these new derivatives were compared to two almost equiactive analogues of CCK8, Ac[Phe(p‐CH2SO3H)27, Nle28, Nle31]‐CCK27‐33and Boc[Nle28, Nle31]‐CCK27‐33(BDNL) and to the nonsulfated analogue of the latter peptide (BDNL NS). All these new CCK‐related analogues behave as agonists in stimulating pancreatic amylase release and display high affinity for brain binding sites (K1∼ 3‐11 nm) but the only peptides which retain affinity for CCK‐A receptors (K1∼ 20 nm) are those containing ap‐caiboxymethyl phenylalanine. Thus, introduction of this amino acid under an esterified form on the side chain, into specific and potent CCK‐B agonists could allow compounds endowed with good bioavailabilities to be obtained.
Precursors of molecules related to mammalian opioid peptides in brain of a marine worm
Authors: DUGIMONT THIERRY , GUISSI‐KADRI SANAE , CURGY JEAN‐JACQUES
Published: August 2010

Total mRNA were extracted from brain of Nereis diversicolor (Annelida, Polychaeta) and were translated in vitro or in ovo. The newly synthesized polypeptides were analyzed through electrophoresis of immunoprecipitated products or the Western blotting technique using polyclonal antibodies raised against mammalian dynorphin 1–17 and mammalian α‐neo‐endorphin. Among the products translated in vitro, only one class of polypeptide of 70 kDa was recognized by anti‐dynorphin 1–17 antibodies. Furthermore, some in ovo translated products as well as proteins extracted from brain of worms showed identical immunoreactivity. These polypeptides, 60–70 kDa, reacted with anti‐dynorphin 1–17 and anti α‐neo‐endorphin antibodies. These results suggest the existence of epitopes common to in ovo and in vitro translated products, to polypeptides extracted from the brain and to some mammalian opioid peptides of the prodynorphin family. We postulate the presence, in the brain of N. diversicolor, of precursors of peptides related to mammalian dynorphin 1–17 and α‐neo‐endorphin. Data reported in this investigation do not allow us to propose or even postulate the presence, in the brain of the worm, of one precursor molecule common to polypeptides related to mammalian dynorphin 1–17 and α‐neo‐endorphin. Furthermore, the Nereis precursor molecules exhibit a clear‐cut difference in molecular mass with the mammalian prodynorphin: 70 kDa versus 30 kDa.
Degradation of aspartic acid and asparagine residues in human growth hormone‐releasing factor
Authors: BONGERS JACOB , HEIMER EDGAR P. , LAMBROS THEODORE , PAN YU‐CHING E. , CAMPBELL ROBERT M. , FELIX ARTHUR M.
Published: August 2010

Products of the degradation of human growth hormone‐releasing factor (GRF) in aqueous solutions (15–200 μM) have been isolated and fully characterized. The cleavage product, GRF(4–44)‐NH2, and the isomer‐ization product, [β‐Asp3]GRF(1–44)‐NH2, from the degradation of GRF(1–44)‐NH2 in acidic solution and the corresponding products, GRF(4–29)‐NH2 and [β‐Asp3]GRF(1–29)‐NH2, from the degradation of GRF(1–29)‐NH2 have been isolated and characterized. The products, [β‐Asp8]GRF(1–44)‐NH2 and [Asp8]GRF(1–44)‐NH2, from the deamidation of GRF(1–44)‐NH2 at pH 8.0 and the corresponding products, [β‐Asp8]GRF(1–29)‐NH2 and [Asp8]GRF(1–29)‐NH2, from the deamidation of GRF(1–29)‐NH2 have been isolated and characterized. AH the degradation products of GRF(1–44)‐NH2 and GRF(1–29)‐NH2 were evaluated for biological activity and found to have much lower in vitro potencies than the parent peptides. Degradation occurs at Asp3 and Asn8 and the kinetics of these various transformations versus pH and temperature have been studied. GRF is most stable at pH 4–5. At pH below the pKa of the Asp3 side‐chain (pH<4), cleavage at Asp3‐Ala4 is the major route of degradation. For pH>4, isomerization of Asp3 to β‐Asp3 (iso‐Asp3) predominates. The rates of cleavage and isomerization are simple first order and vary with pH, independent of buffer concentration, such that the protonated (COOH) form of Asp3 undergoes cleavage while the ionized (COO‐) form isomerizes. The more rapid deamidation of Asn8 to generate β‐Asp8 and Asp8 in about a 4:1 ratio, presumably via a cyclic imide intermediate, occurs at pH < 5 and is general base‐catalyzed. Evidence was also obtained for direct hydrolysis of protonated Asn8 in GRF(1–29)‐NH2 at pH<2 to give exclusively [Asp8]GRF(1–29)‐NH2. The deamidation of Asn8 in GRF(1–29)‐NH2 at pH 8.0, 22–55°C, is relatively insensitive to temperature for T>37°C, possibly due to conformational constraints. Asp25 and Asn35 are sterically, conformationally, or otherwise hindered with respect to these changes as no degradation at these sites was observed under the conditions employed.
New convenient synthesis of immunostimulating peptides containingmeso‐diaminopimelic acid Syntheses of FK‐565 and FK‐156
Authors: KOLODZIEJCZYK A.M. , KOLODZIEJCZYK A. S. , STOEV S.
Published: August 2010

A new improved synthesis of two immunostimulating peptides: FK‐156 (d‐lactyl‐alanyl‐γ‐d‐glutamyl‐(l)‐meso‐2,6‐diaminopimelyl‐(l)‐glycine)and FK‐565 (heptanoyl‐γ‐d‐glutamyl‐(l)‐meso‐2,6‐diaminopimelyl‐(l)‐d‐alanine) is described. A proper differentiation between the two chiral amino acid moieties of diaminopimelic acid was accomplished by selective enzymatic hydrolysis of one methyl ester group of the l‐centre of Z2‐meso‐A2pm(OMe)2 (2). Utilization of a commercially available protease and diester 2 as an enzyme substrate made possible the relatively simple synthesis of a key intermediate 4 and considerably simplified the final deprotection steps. Aminolysis of the N‐carboxyanhydride (4) with d‐AlaONBzl or GlyONBzl was chosen to obtain the appropriate dipeptides with one free amino group as convenient intermediates for further peptide synthesis. The BOP reagent, used for peptide bond formation, secured good yields and high chemical and chiral purity of the peptides. A modification of alanine deamination procedure leading to a significant increase of d‐Lac(OAc) yield is presented.
Characterization of heptapeptide toxins extracted from Microcystis aeruginosa (Egyptian isolate)
Authors: ABDEL‐RAHMAN S. , EL‐AYOUTY Y.M. , KAMAEL H.A.
Published: August 2010

Four toxic peptides from local fresh water cyanobacterium Microcystis aeruginosa were purified and identified by high performance liquid chromatography (HPLC) and ion spray mass spectroscopic studies as: RR; YR; LR and LA with molecular weights of 1006.8, 1073, 984.8 and 910.6 respectively. Amino acid analysis indicated the presence of equimolar amounts of aspartic acid, glutamic acid, arginine, leucine and tyrosine, in addition to both alanine and dehydroalanine. Mouse assay toxicity indicated that the first two peptides, at the peak area of RR, YR. were highly toxic with LD50 20, 18.2 μg/kg body weight; however, the latter two, at the peak areas LR and I.A. have a lesser toxicity with LD50 36 and 40 μg/kg body weight respectively. Three linear peptide analogs to those naturally found devoid of Adda were synthesized using the continuous flow technique. HPLC pure synthesized analog products were tested for toxicity using male mice (i.p. injection). None of them induced toxic activity.
Synthesis and immunological characterization of a 134‐mer synthetic peptide corresponding to the N‐terminal half of the HIV‐1 nucleoprotein, p24
Authors: SIA PELE CHONG. CHARLES , TAM EILEEN , KANDIL ALI , KLEIN MICHEL
Published: August 2010

A 134‐mer peptide corresponding to the N‐terminal sequence of p24 (residues 146–279 of the gag gene product of the LAV strain) was chemically synthesized using highly optimised protocols on an ABI 430A synthesizer. The crude peptide was obtained by treating the peptide‐resin with HF, then purified by a combination of size exclusion and RP‐HPLC. One hundred milligram of 90% pure 134‐mer can be obtained within a month. Both mice and rabbit polyclonal antisera raised against a commercial preparation of recombinant p24, and a pooled sera from HIV‐1 infected individuals reacted strongly with the 134‐mer peptide in ELISA. Both mice and rabbits immunized with the free peptide emulsified in Freund's complete adjuvant generated strong anti‐peptide and anti‐p24 antibody responses as judged by immunoblots and ELISAs. Immunodominant epitopes were mapped to residues 201–227 (LKETINEEAAEWDRVHPVHAGPIAPG). These B‐cell epitopes had previously been identified by mouse monoclonal antibodies raised against HIV‐1 virus or gag gene products. Furthermore, murine T‐cell lines generated against the 134‐mer peptide were found to respond to two short peptides, P24B (residues 195–215) and P24D1 (residues 268–279). These two T‐cell epitopes were previously reported as human helper T‐cell and CTL epitopes, respectively. These results clearly indicate that the synthetic 134‐mer peptide could elicit both T‐ and B‐cell responses to HIV‐1 similar to those obtained with the natural viral gag protein, and could be useful for the development of a synthetic HIV vaccine
Isolation and characterization of two distinct gonadotropins, GTHI and GTHII, from bonito (Katsuwonus plelamis) pituitary glands
Authors: KOIDE YOSHIO , ITOH HIROMICHI , KAWAUCHI HIROSHI
Published: August 2010

Two distinct glycoproteins homologous to chum salmon GTHI and GTHII were isolated from the pituitary glands of a marine fish, the bonito (Katsuwonus plelamis), and characterized by amino acid sequence analysis in order to obtain additional evidence for duality of teleost GTHs. Glycoproteins were extracted from the pituitary glands, and intact GTHI and GTHII, consisting of two distinct subunits, were purified by ion‐exchange chromatography on DEAE‐cellulose, rpHPLC an Asahipak C4P‐50 in alkaline buffer, and gel filtration on Superdex 75. The association of the subunits was stable in GTHI (39 kD) and unstable in GTHII (30 kD) in acidic conditions. Immunoblotting revealed that antisera against β subunits of chum salmon GTHs reacted with GTHII, but not with GTHI. In addition, none of the GTHs was stained with antiserum against human TSHβ. Sequence analysis demonstrated that bonito GTHIβ is homologous to salmon GTHIβ with 43% sequence identity, and bonito GTHIIβis homologous to salmon GTHIIβ with 67% identity. Sequence identity between bonito GTHIβ and GTHIIβ was only 28%. Thus, it is evident that the bonito pituitary gland produces two chemically distinct gonadotropins homologous to chum salmon GTHs.
Conformational study of two substance P hexapeptides by two‐dimensional NMR
Authors: WONG TUCK C. , LEE CHI MING , GUO WEI , CHANG DING‐KWO
Published: August 2010

The conformation of two substance P (SP) related hexapeptides. Glp‐Phe‐Phe‐(L‐Pro)‐Leu‐Met.NH2(I) and Glp‐Phe‐Phe‐(D‐Pro)‐Leu‐Met.NH2(II), in two solvents, chloroform‐d and trifluoroethanol(TFE)‐d3/H2O, was studied by two‐dimensional NMR methods, including COSY, TOCSY, ROESY and HMQC. The study shows that these two peptides exist predominantly in the extended form in TFE/H2O, but in general exhibit a reverse‐turn structure in chloroform. I is clearly less ordered than II in both solvents. Furthermore, extensive Phe3‐Pro4cis↔transisomerization was found in I but not in II. The differences in the conformational behavior of these two peptides, which are selective agonists for neurokinin NK1 and NK2 receptors, respectively, are discussed.
Syntheses, opioid binding affinities, and potencies of dynorphin A analogues substituted in positions 1, 6, 7, 8 and 10
Authors: KAWASAKI ANDREW M. , KNAPP RICHARD J. , WALTON AMANDA , WIRE WILLIAM S. , ZALEWSKA TERESA , YAMAMURA HENRY I. , PORRECA FRANK , BURKS THOMAS F. , HRUBY VICTOR J.
Published: August 2010

Structural, stereochemical, stereoelectronic and conformational requirements for biological activity of dynorphin A1–11‐NH2analogues at opioid receptors were explored by substitution of Tyr1, Arg6, Arg7, Ile8and Pro10with other amino acid residues. Interestingly, substitution of Tyr1withNα‐Ac‐Tyrl, D‐Tyr1, Phe1orp‐BrPhe1led to analogues that were quite potent at κ opioid receptors, and additional substitution of Ile8with D‐Ala8and/or Pro10with D‐Pro10retained high potency in brain binding assay: [Nα‐Ac‐Tyr1]‐ (1), [D‐Tyr1]‐ (2) [Phe1]‐ (3), [Phe1. D‐Ala8]‐ (5), [p‐BrPhe1, D‐Alas]‐ (6), [Phe1, D‐Pro10]‐ (7) and [Phe1, D‐Ala8, D‐Pro10]‐Dyn A1–11‐NH2(8) had IC50(nM) binding affinities of 13.2, 18.6, 1.64, 1.26, 1.84, 2.44 and 1.62 nM, respectively. The D‐Phe1analogue 4, however, was only weakly active (610 nM). All of the analogues except 4 were modestly selective for κ vs. μ guinea pig brain opioid receptor (11‐ to 88–fold) and quite selective for κ vs. δ receptors (65–576). However, all of the analogues appeared to have very low or essentially no activity in the guinea pig ileum and mouse vas deference functional bioassays, and one analogue, 5, appeared to have weak antagonist activities. On the other hand, if constrained amino acids such as β‐methylphenylalanine or 1,2,3,4‐tetrahydroisoquinoline carboxylic acid, and hydroxyproline were placed in the 1 position, inactive analogues or analogues with greatly reduced potency and biological activity were obtained (compounds 12–14).

It had previously been suggested that the Arg6and Arg7residues were critical for biological activity. However, when we replace either one of these residues, [Nle6]Dyn A1–11(9) and [Nle7]Dyn A1–11‐NH2(10) were both highly potent binders in κ receptor binding studies (IC50= 0.95 and 0.43 nM, respectively), and interestingly also were potent in μ and δ binding studies. Furthermore, both of the analogues were modestly potent in the GPI and MVD assays (94, 65 nM; 31, 81 nM, respectively). These results demonstrate that basic residues at positions 6 and 7 in dynorphin are not very important for binding to κ opioid receptors. Finally, many of the compounds reported here showed high selectivity for central vs. peripheral κ opioid receptors, with compound 4 being the most selective (63 000‐fold).
Aggregation of resin‐bound peptides during solid‐phase peptide synthesis
Authors: KRCHŇÁK VIKTOR , FLEGELOVÁ ZUZKA , VÁGNER JOSEF
Published: August 2010

Nonrandom incomplete aminoacylation of a pendent peptide chain on an insoluble polymeric support during solid‐phase peptide synthesis is sequence‐dependent and is caused by aggregation of peptide chains, manifested by a decreased swelling capacity. The volume of the swollen peptidyl‐resin after each coupling during the syntheses of 87 sequence unrelated peptides was measured, and for each amino acid an aggregation parameter, 〈Pa〉, was derived that reflects the propensity of the swollen volume of peptidyl‐resin to decrease during peptide synthesis. These aggregation parameters were used to predict potentially difficult sequences.
Conformations and conformational interconversions of diastereomeric cyclic tetraprolines
Authors: LINK ULRICH , MÄSTLE WERNER , ROTHE MANFRED
Published: August 2010

Cyclic tetrapeptides exclusively composed of L‐ and D‐Pro have been studied by theoretical means (conformational searches and molecular mechanics calculations using the CHARMM program) supported by 1H‐NMR spectroscopy, X‐ray analysis and chiroptical measurements. We explored the entire conformational space of the diastereomers cyclo(LLLL‐Pro4) (I), cyc1O(LDLD‐Pro4) (II) and CYClo(LLDD‐Pro4) (III) including the low‐energy conformations and the related interconversion paths. The conformational interconversions were found to be restricted to cis/trans isomerisations of the amide bonds. Owing to the polycyclic nature of cyclo(Pro4) most of the cis/trans transitions are hindered by energy barriers higher than 30 kcal/mol (up to 150–200 kcal/mol). A few transitions are characterized by computed energy barriers comparable to those found in linear ‐Xxx‐Pro‐ sequences (∼ 18 kcal/mol), and are therefore experimentally significant. Experimental evidence has been obtained in the case Of CyClo(LDLD‐Pro4), where two enantiomers are interconverted by a series of 4 cis/trans isomerisations ctct→cttt→tttt→tctt→tctc. The Eyring activation parameters of this reaction were determined in H2O and in DMF by chiroptical measurements (ΔH#= 44 and 28 kcal/mol; ΔS#=59 and 22 cal K −1 mol−1, respectively), and correlated with the calculated barriers. In I and III comparable series of four cis/trans isomerisations relate two main conformations with the peptide bond sequences ctct and tctc. In compound I pseudorotational images are interconverted via ctct→ccct→cctt→cctc→tctc. The pathway ctct→ccct→cctt→cctc→tctc. that relates diastereomeric main conformations of III involves exclusively low‐energy intermediates; however, the transitions leading to the all‐cis conformation are energetically unfavourable, and the conformational space is divided in three insulated domains.
Conformational analysis of μ‐selective [D‐Ala2,MePhe4]enkephalins
Authors: PENKLER LAWRENCE J. , ROOYEN PETRUS H. VAN , WESSELS PHILLIPPUS L.
Published: August 2010

The conformational space of the potent μ‐selective opioids [D‐Ala2,MePhe4,Gly‐o15]enkephalin (DAGO) and [D‐Ala2,MePhe4,Gly‐o15]enkephalin (FK 33‐824) has been analyzed by1H‐NMR spectroscopy and theoretical calculations involving systematic conformational searching and energy minimizations. Acis‐transequilibrium of the Gly3‐MePhe4amide bond is induced by theN‐methyl group, and the more energetically favouredtransisomer is proposed as the biologically relevant form. A compact interaction between the side chains of Tyr1and D‐Ala2was demonstrated by NOE and ROE effects in both peptides in D2O and DMSO‐d6, further supported by shielding of the D‐Ala2methyl protons in both solvents. Analysis of coupling constants, NOE and ROE data indicated significant restriction of the conformational freedom of the MePhe4side‐chain for both peptides in the two solvents. The NMR results and theoretical calculations point towards folded low energy conformations characterized by a β11‐type turn around Gly3‐MePhe4. For thetransisomer, a Tyr1‐MePhe4phenyl ring separation between 8.5 and 12.5 Å was accompanied by proximity between the D‐Ala2side chain and theC‐terminal in low energy conformations The results are in good agreement with available data on related active enkephalins. The conformational effects induced by simultaneous incorporation of D‐Ala2and MePhe4in enkephalins is discussed in the light of the enhanced μ‐opioid receptor selectivity and activity of these peptides.
Multipin peptide synthesis at the micromole scale using 2‐hydroxyethyl methacryiate grafted polyethylene supports
Authors: VALERIO ROBERT M. , BRAY ANDREW M. , CAMPBELL RHONDA A. , DIPASQUALE ANGELA , MARGELLIS CHRYS , RODDA STEWART J. , GEYSEN H. MARIO , MAEJI N. JOE
Published: August 2010

The multipin peptide synthesis procedure has been adapted to allow the synthesis of peptides at micromole loadings. The original solid pin support was replaced with a detachable crown‐shaped polyethylene support with an increased surface area. In addition, the polyethylene crowns were radiation‐grafted with 2‐hydroxyethyl methacryiate monomer instead of acrylic acid to yield hydroxy functionalised supports with a larger concentration of polymer and hence a larger peptide capacity. Fmoc‐β‐Alanine was directly esterified to the HEMA hydroxy groups with subsequent addition of a diketopiperazine‐forming handle for peptide attachment. Peptides varying in length from 10 to 25 residues were assembled at a number of loadings from 1.0 to 2.2 μmol. Purity of peptides at all loadings was equal to, and in some instances superior to, that achieved on conventional solid‐phase supports.
Kinetic characterization of alkaline mesentericopeptidase
Authors: DOLASCHKA PAVLINA , GENOV NICOLAY , ERMER ANTJE , PETERS KLAUS , FITTKAU SIEGFRIED
Published: August 2010

Comparative studies of the hydrolysis of succinyl‐Ala2‐Phe‐methylcoumarylamide with mesentericopeptidase, a mesophilic extracellular serine proteinase from Bacillus mesentericus, and proteinases produced by organisms representing different levels of evolutionary development, were performed. Drastic differences in the proteolytic coefficient kcat/Km, were found. As regards their catalytic efficiency, the proteinases studied can be placed in the following order: mesentericopeptidase < subtilisin Novo ≪ subtilisin DY < proteinase K < subtilisin Carlsberg < thermitase < α‐chymotrypsin. The size of the substrate‐binding site of mesentericopeptidase for synthetic peptides was studied by using chloromethyl ketones with the general formula benzyloxycarbonyl‐Ala‐Phe‐CH2Cl (n= 1, 2, 3). The presence of at least five binding subsites (S1… S5) on the S‐side of the hydrolysed bond was suggested. Studies of the primary specificity of mesentericopeptidase with a series of dipeptide chloromethyl ketones having the general formula benzyloxycarbonyl‐Ala‐Aa‐CH2Cl (Aa = Ala, Val, Leu, Phe) revealed the following order of reactivity toward these inhibitors: Aa = Leu ≫ Ala > Phe > Val. Kinetically, mesentericopeptidase is similar to subtilisin BPN′/Novo.
Peptide production by a combination of gene expression, chemical synthesis, and protease‐catalyzed conversion
Authors: SCHELLENBERGER VOLKER , POMPEJUS MARKUS , FRITZ HANS‐JOACHIM
Published: August 2010

We describe a new approach for the production of peptides using a combination of recombinant DNA technology, chemical synthesis, and proteinase‐catalyzed processing. An artificial substance P‐precursor is produced as a β‐galactosidase (1–459) fusion protein containing nine copies of the decapeptide sequence Arg‐Leu‐Arg‐Arg‐Pro‐Lys‐Pro‐Gln‐Gln‐Phe. The fusion protein accumulates in E. coli as insoluble inclusion bodies which are easily isolated and purified. The decapeptide blocks are selectively cleaved from the insoluble fusion protein by α‐chymotrypsin. Alternatively, a dodecapeptide ester is produced when a dipeptide ester is included in the chymotrypsin reaction mixture. This peptide ester is converted to substance P by papain‐catalyzed acyl transfer and subsequent tryptic cleavage. These results demonstrate that peptides can be readily produced by a combination of recombinant DNA technology and proteinase‐catalyzed conversion. The approach allows incorporation of groups other than natural amino acids into oligo‐ and polypeptides.
Cyclic disulfide analogues of the complement component C3a Synthesis and conformational investigations
Authors: POHLS MARTINA , AMBROSIUS DOROTHEE , GRÖTZINGER JOACHIM , KRETZSCHMAR TITUS , SAUNDERS DEREK , WOLLMER AXEL , BRANDENBURG DIETRICH , BITTER‐SUERMANN DIETER , HÖCKER HARTWIG
Published: August 2010

The flexible C‐terminal region of the anaphylatoxic peptide C3a was reported to contain the receptor binding site. To elucidate the receptor binding conformation of the C‐terminus, as well as to examine a synthetic approach to potential C3a‐antagonists, 26 cyclic disulfide bridged C3a analogues were synthesized. Solid phase peptide synthesis was performed on different polymeric supports by individual peptide synthesis, with Fmoc strategy, and simultaneous multiple peptide synthesis, using Boc and Fmoc strategies. Both strategies gave open‐chain peptides in comparable yields. Syntheses using the Boc strategy employed the HF‐labile 4(methoxy)benzyl group (Mob) for β‐thiol protection of cysteine; in contrast, the TFA‐stable protecting groups, acetamidomethyl (Acm) and trityl (Trt), were chosen for syntheses employing Fmoc strategy. Ring closure reactions by iodine oxidation were carried out starting from protected (Acm/Acm, Trt/Acm) or unprotected dithiols. The resulting cyclic C3a analogues were characterized by HPLC, amino acid analysis, and FAB‐MS. Conformational investigations using CD spectroscopy and theoretical structural investigations by means of molecular dynamics calculations revealed that slight variations in sequence result in pronounced conformational consequences. The potential of cyclic C3a analogues to activate or to desensitize guinea pig platelets, a standard test system for biological activities of anaphylatoxic peptides like C3a, revealed relatively low activities for cyclic peptides (<0.1% C3a activity). N‐terminal acylation with cationic, arginine‐rich sequences like YRRGR‐ led to amplified biological effects. Three of the synthesized peptides, namely CAALCLAR (P1), YRRGR°CGGLCLAR (P5) and YRRGRAhx°CGGLCLAR (P8), point in the direction of C3a antagonists.
Application of tryptic condensation strategy for the synthesis of α‐melanocyte stimulating hormone
Authors: MIHARA H. , XU M. , NISHINO N. , FUJIMOTO T.
Published: August 2010

The use of trypsin in 1,1,1,3,3,3‐hexafluoro‐2‐propanol (HFIP) paired with N,N‐dimethylformamide (DMF) has been proposed for the enzymatic condensation of peptides. The tryptic condensation strategy was applied to the synthesis of the 13‐peptide, α‐melanocyte stimulating hormone (α‐MSH), which contains two susceptible points to trypsin, in a low‐water‐containing solvent system, 4% H2O in HFIP/DMF (1/1, v/v). The N‐terminal 8‐peptide segment (S1) ending with Arg and C‐terminal 5‐peptide with the side‐chain protections at Lys and Trp (S2) were prepared by the stepwise coupling on p‐nitrobenzophenone oxime resin and by a conventional method, respectively. Both segments were condensed by the aid of trypsin. The acid component was converted into the partially protected α‐MSH at as high as 95% conversion determined by reversed‐phase HPLC. When the side‐chain of Lys at the 11‐position was not protected, α‐MSH was obtained only in 35% yield, and was contaminated with products of secondary hydrolysis. Although the Lys11‐Pro12 sequence was very poorly susceptible to trypsin, the side‐chain of Lys had to be protected in order to be inert to trypsin under the synthetic conditions. HFIP is demonstrated as a good solvent with DMF to allow the efficient tryptic condensation of peptides. The strategy increased the value of the enzymatic condensation as practical method by avoidance of secondary hydrolysis, high dissolution of peptides and retention of activity of enzyme.