A Modified,High Yield Procedure for the Synthesis of Unlabeled and 14C-Labeled 4-Methylene-DL-glutamic Acid

This paper describes the complete chemical synthesis of 4-methylene-DL-glutamic acid from diethylmalonate, formaldehyde and diethyl acetamidomalonate. The amino acid was obtained pure following ion-exchange chromatography and/or crystallization from hot water in an overall yield of 30% based on the amount of diethylmalonate used. Several physico-chemical characteristics of the synthetic compound were determined, including ir and pmr spectra, chromatography on paper, retention time on an amino acid analyzer, pK values and melting point; all properties cf the synthetic material were found to be identical to those seen with the naturally occurring L-isomer. The procedure for obtaining gram quantities of the unlabeled compound has also been modified for the synthesis of high specific activity (10.6 mCi/nmol) 4-methylene-[2-¹⁴C]-DL-glutamic acid.     

Solid-phase synthesis of thymosin beta 4: chemical and biological characterization of the synthetic peptide

The chemical synthesis of thymosin beta 4 using a solid-phase procedure has been accomplished. The synthetic product was found to be homogeneous on paper electrophoresis at pH 6.5, high-performance liquid chromatography on a reversed-phase column, and isoelectric focusing using polyacrylamide gels. The synthetic material was also shown to be identical with the natural thymosin beta 4 by tryptic peptide mapping, amino acid compositional analyses, and polyacrylamide gel isoelectric focusing. Biologically, synthetic thymosin beta 4 was found to be as active as the natural compound in a terminal deoxynucleotidyltransferase induction assay and in a macrophage migration inhibition assay. The proposed structure of the peptide hormone was thus confirmed by a chemical synthesis.     

N5-(1-carboxyethyl)-ornithine, a new amino acid from the intracellular pool of Streptococcus lactis.

Intracellular concentrations of amino acids were determined in cells of Streptococcus lactis 133 during growth in complex, spent, and chemically defined media. Glutamic and aspartic acids represented the major constituents of the amino acid pool. However, organisms grown in spent medium or in defined medium supplemented with ornithine also contained unusually high levels of two additional amino acids. One of these amino acids was ornithine. The second compound exhibited properties of a neutral amino acid by coelution with valine from the amino acid analyzer. The compound did not, however, comigrate with valine or any other standard amino acid by two-dimensional thin-layer chromatography. The unknown amino acid was purified by paper and thin-layer chromatography, and its molecular structure was determined by 1H and 13C nuclear magnetic resonance spectroscopy. This new amino acid was shown to be N5-(1-carboxyethyl)-ornithine. The 14C-labeled compound was formed by cells of S. lactis 133 during growth in spent medium or defined medium containing [14C]ornithine. Formation of the derivative by resting cells required ornithine and the presence of a metabolizable sugar. N5-(1-Carboxyethyl)-ornithine was synthesized chemically from both poly-S-ornithine and (2S)-N2-carbobenzyloxy-ornithine as a 1:1 mixture of two diastereomers. The physical and chemical properties of the amino acid purified from S. lactis 133 were identical to those of one of the synthetic diastereomers. The bis-N-trifluoroacetyl-di-n-butyl esters of the natural and synthetic compounds generated identical gas chromatography-mass spectrometry spectra. A mechanism is suggested for the in vivo synthesis of N5-(1-carboxyethyl)-ornithine, and the possible functions of this new amino acid are discussed.     

3-hydroxy-5-methylproline, a new amino acid identified as a component of actinomycin Z1.

3-Hydroxy-5-methylproline has been identified in hydrolysates of actinomycin Z₁ by ion-exchange and paper chromatography, paper electrophoresis, gas chromatography and mass spectrometry in comparison with the synthetic compound. The stereochemistry of this amino acid is under investigation. The amino acid composition of actinomycin Z₁ thus consists of threonine, hydroxythreonine, D-valine(2), 4-oxo-5-methylproline, 3-hydroxy-5-methylproline, sarcosine(2), N-methylalanine and N-methylvaline.     

A toxic amino acid, 2(S)3(R)-2-amino-3-hydroxypent-4-ynoic acid from the fungus Sclerotium rolfsii

An amino acid, lethal to New Hampshire chickens (LD₅₀, 150 mg/kg) was isolated from dried sclerotia of the fungus Sclerotium rolfsii (Sacc.). Purification of the rather unstable compound was effected on a cation exchange column by means of displacement chromatography and the amino acid was crystallised from 80% methanol. A structure was assigned to the compound on the basis of available chemical and physical data, namely 2(S),3(R)-2- amino-3-hydroxypent-4-ynoic acid. Confirmation of this structure was gained by direct and indirect synthetic procedures.     

Synthetic hFSH peptide constructs in the evaluation of previous studies on the hFSH receptor interaction

The human follicle-stimulating hormone (hFSH) belongs to a family of glycoprotein hormones which contains two non-identical subunits. This paper describes the design and synthesis of a series of synthetic hFSH constructs as putative ligands for the receptor. The design of these constructs is based on the crystal structure of hCG and molecular modelling using the program package Insight II/Discover. The designed constructs contain peptides ranging from 7 to 48 amino acid residues, disulphide bridges and glycan residues. All the synthetic peptides were synthesized by the stepwise solid-phase method using Fmoc chemistry. Two of the synthetic peptides contain the glycosylated amino acid, Asn(GlcNAc-GlcNAc) and both were prepared using fully protected glycosylated building blocks in the solid-phase peptide synthesis. The disulphide bridges were formed from acetamidomethyl-protected glycopeptides and peptides by a direct deprotection/oxidation method using thallium(III) trifluoroacetate. Mass spectroscopy and amino acid analysis were used for characterization of the synthetic hFSH glycopeptides and peptides. The synthetic hFSH constructs were tested for binding activity on FSH receptor assays but none showed improved binding properties compared with the naturally occurring hormone. It was finally demonstrated that non-related peptides showed non-specific binding at the same level as reported for specific peptides. © 1997 European Peptide Society and John Wiley & Sons, Ltd.     

Isolation and partial characterization of glabrin, a neurotoxin from Cnestis glabra (Connaraceae) root barks

A neurotoxic compound, inhibiting protein synthesis in cell culture, was isolated in a yield of about 0.4 per cent from Cnestis glabra root barks (Connaraceae) by a five-step fractionation procedure (filtration on activated charcoal, treatment by neutral lead acetate and fractionations on Dowex 50 X 8 in H+ and NH+4 forms). The purified toxin appeared homogeneous on thin-layer and in gas chromatography. The compound has a low molecular weight (less than 500). It is heat-stable, insoluble in usual organic solvents and gives a positive reaction with ninhydrin. Acidic hydrolysis does not change its behaviour on an amino acid analyzer. Its possible amino acid nature is discussed. It is temporarily named glabrin.     

The isolation and partial characterization of trypsinogen, pancreatic secretory trypsin inhibitor and multiple forms of chymotrypsinogen and trypsin from the pancreas of the ostrich (Struthio camelus).

1. PSTI, two chymotrypsinogens and two trypsins were purified to homogeneity by acid extraction, salt fractionation, SP-Sephadex C-50 chromatography and RP-HPLC. 2. A third chymotrypsinogen, a trypsinogen and another trypsin were purified using an alkaline extraction procedure, followed by Trasylol- and Benzamidine-Sepharose affinity chromatography and hydroxylapatite chromatography. 3. The enzymes differed in amino acid composition as well as in specific activities towards synthetic amidase and esterase substrates. 4. N-terminal amino acid sequences were determined for one chymotrypsinogen and one trypsin.     

Syntheses of biotinylated and dethiobiotinylated insulins

The 600-MHz proton spectrum of dethiobiotin (prepared from d-biotin with Raney nickel) was measured in order to gain information pertaining to its stereochemical homogeneity. The spectrum demonstrated clearly that the material is a 6:1 mixture of two stereoisomers. The cis compound, corresponding to the stereochemistry of d-biotin, is the major isomer. Two biotinyl- and two dethiobiotinylinsulins were prepared in which the distance between the biotins and insulin was varied by interposition of spacer arms. The synthesis of these compounds involved repeated N-hydroxysuccinimido ester condensations. Biotin N-hydroxysuccinimido ester, dethiobiotin N-hydroxysuccinimido ester, 6-aminohexanoic acid, and N-[3-[(3-aminopropyl)carboxyamino]-propyl]succinamic acid N-tert-butyl ester served as the building blocks for the spacers. The latter compound was prepared from N-[3-[(3-aminopropyl)amino]propyl]succinamic acid sulfate by the use of a selective amino-protecting method based on the differential stability toward acid of citraconyl and tert-butoxycarbonyl amino-protecting groups. The structure of N-[3-[(3-aminopropyl)amino]propyl]succinamic acid sulfate was established unequivocally by X-ray diffraction. The attachment of the biotinylated spacers to the insulin was exclusively at the N alpha, B1 position. Homogeneity of the final products as well as of the intermediates used in their synthesis was established by thin-layer chromatography, by high-pressure liquid chromatography, and in most instances by elemental analysis. The ratio of 6-aminohexanoic acid to lysine in hydrolysates of the insulin derivatives was in agreement with theory. The insulin derivatives were required for a study on the effect of avidin on their ability to interact with insulin receptors on rat epididymal adipocytes, which is described in the accompanying paper.     

Berberine synthesis by callus and cell suspension cultures of Coscinium fenestratum

Callus and cell suspension cultures of Coscinium fenestratum were established from sterile petiole segments on Murashige & Skoog (MS) medium, supplemented with 2,4-dichlorophenoxy acetic acid (2,4-D) and benzyl amino purine (BAP). The cells in the culture produced berberine as the major compound. NAA stimulated the product synthesis over 2,4-D. Presence of light inhibited the growth and enhanced the berberine synthesis.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxy acetic acid - HPLC high pressure liquid chromatography - IAA indole-3-acetic acid - NAA -naphthaleneacetic acid - TLC thin layer chromatography     

Peptide-Induced Morphogenesis in the Nematode-Trapping Fungus Arthrobotrys oligospora

The present investigation shows the ability of peptides to induce capture organ formation in Arthrobotrys oligospora when applied in a synthetic low nutrient medium. Under certain conditions casitone was shown to induce capture organ formation. The active principle in casitone was concentrated and purified by alternating procedures of ion exchange chromatography and gel chromatography in pyridine-acetic acid buffers. Crude casitone solutions were applied to columns of Dowex 50 W-X2 and eluted stepwise with 0.1–1.0 M pyridine-acetic acid pH 3.2–5.1. Active portions, free from most acid and neutral amino acids, were further purified on columns of Sephadex G-10 in 0.1 M pyridine-acetic acid pH 4.6. Aromatic amino acids and large molecules in the void volume could be separated from an active peptide mixture which was subjected to renewed ion exchange chromatography on Bio-Rad AG 50 W-X2. By stepwise and/or gradient elution in 0.1–0.5 M pyridine-acetic acid pH 3.2 fairly purified peptides were obtained. The composition of the test medium is an important factor in spontaneous capture organ formation. The peptides isolated from casitone induced capture organ formation, when given to the fungus in a synthetic mineral salt medium supplied with thiamin and biotin. Similar effects were obtained with small synthetic peptides in the same concentration (0.1 mg/ml). A large variety of peptides seem to be active when applied in a suitable medium. This was especially true for peptides with Rf > Rf_leu on thin layers of cellulose developed with butanol-acetic acid-water (4: 1: 1). Of the peptides investigated valyl-peptides exerted the most drastic effect.     

Regulation of fatty acid synthetase activity. The 4'-phosphopantetheine hydrolase of rat liver.

The 4'-phosphopantetheine hydrolase of rat liver, partially purified by ammonium sulfate precipitation, catalyzes the hydrolysis of the prosthetic group 4'-phosphopantetheine from the holo-fatty acid synthetase. The two products of the action of this enzyme, 4'-phosphopantetheine and apo-fatty acid synthetase, were isolated by DEAE-cellulose chromatography and by chromatography on a Sepharose epsilon-aminocaproyl pantetheine column, respectively. The resultant apo-fatty acid synthetase was quantitated by immunoprecipitation and it was also converted to the holoprotein with a crude preparation of rat liver 4'-phosphopantetheine transferase. Quantitative determination of the hydrolase reaction product, 4'-phosphopantetheine, by amino acid analysis and microbiological assays confirmed the presence of 1 mol of this compound/mol of holo-fatty acid synthetase.     

Preparation and characterization of [N alpha-(4-azido-2-nitrophenyl)Ala1,Tyr36]-parathyroid hormone related peptide (1-36)amide: a high-affinity, partial agonist having high cross-linking efficiency with its receptor on ROS 17/2.8 cells

The synthesis, purification, and structural analysis of the major compounds resulting from photoderivatization of [Tyr36]-parathyroid hormone related peptide (1-36)amide [[Tyr36]PTHrP(1-36)amide] are described. The reaction of the synthetic peptide with 4-fluoro-3-nitrophenyl azide under nonaqueous conditions yields three major products (peaks D-1, D-2, and G), which were purified to homogeneity by reverse-phase high-performance liquid chromatography. Subsequent amino acid analysis showed that the peptides of peaks D-1 and G each lack one lysine residue, while the peptide in peak D-2 lacks one alanine residue, suggesting that these residues are chemically modified by photoderivatization. Sequence analysis of the photoderivatized peptides revealed that compounds D-1 and G were derivatized on Lys13 and Lys11, respectively. Compound D-2 was N-blocked, indicating that this compound is derivatized on the alpha-amino function of Ala1. Both Lys residues of D-2 were quantitatively recovered upon sequencing after digestion with endoproteinase Glu-C. Compounds D-2 and G had apparent KdS of 1 X 10(-9) M and 0.6 X 10(-9) M, respectively, for their receptors on ROS 17/2.8 cells, which are identical with or similar to that of the underivatized [Tyr36]PTHrP(1-36)amide. Compound G had the same adenylate cyclase stimulating potency as the underivatized, synthetic [Tyr36]PTHrP(1-36)amide, whereas compound D-2 was only a partial agonist, having about 25% of the maximal cAMP production. Compound D-1, which is modified on Lys13, retained only 2-4% of its receptor binding affinity and biological activity relative to that of its parent compound.(ABSTRACT TRUNCATED AT 250 WORDS)     

Amino acid metabolism of preimplantation bovine embryos cultured with bovine serum albumin or polyvinyl alcohol

Bovine serum albumin (BSA) is an embryotrophic macromolecule used in embryo culture media, which is commonly replaced with synthetic compounds, such as polyvinyl alcohol (PVA). This study compared the effect of BSA and PVA on the development, blastocyst cell number and amino acid metabolism of preimplantation bovine embryos in vitro. Embryos were produced by in vitro maturation and fertilization of immature oocytes from abattoir-derived ovaries. Zygotes were cultured in synthetic oviduct fluid with either 4 mg/ml BSA (SOFaaBSA) or 1 mg/ml PVA (SOFaaPVA) in microdrops with a mineral oil overlay at 39 degrees C under a 5% O2/5% CO2/90% N2 atmosphere. Blastocyst rate and cell numbers were determined after 123 h of culture. In parallel, single expanding blastocysts grown in either medium were incubated in microdrops for 12 h. Amino acid profile of spent drops was determined by high performance liquid chromatography. Replacing BSA with PVA depressed blastocyst rate and cell numbers, and led to quantitative and qualitative differences in amino acid appearance, disappearance and turnover. These differences could partly be due to an increase in free intracellular amino acid concentration in SOFaaBSA embryos derived from hydrolysis of endocytosed BSA, and argue against the inclusion of PVA in bovine embryo culture media.     

Novel Synthesis of <Emphasis Type="Italic">N</Emphasis>-Glycosyl Amino Acids Using T3P<Superscript>®</Superscript>: Propylphosphonic Acid Cyclic Anhydride as Coupling Reagent

In this paper is presented a novel and simple synthetic pathway for obtaining new protected and unprotected N-glucosyl amino acids from 2,3,4,6-tetra-O-acetyl-β-d-glucopyranosyl amine and Fmoc-l-amino acids. Three methodologies were evaluated, using the coupling reagents: N,N,N′,N′-Tetramethyl-O-(benzotriazol-1-yl)uronium tetrafluoroborate, diisopropylcarbodiimide and propylphosphonic acid cyclic anhydride. The obtained products using propylphosphonic acid cyclic anhydride showed less undesired species, easy purification and higher yields than the other two methodologies. Deprotection strategies widely used in solid phase peptide synthesis were applied to develop the synthetic pathway reported and achieve the final products. The protected and unprotected N-glucosyl amino acids were purified using solid phase extraction chromatography and characterized by high performance liquid Chromatography and nuclear magnetic resonance spectroscopy. Different amino acids (Fmoc-l-Asp(OtBu)OH, Fmoc-l-Phe(OH) and Fmoc-l-Lys(Boc)-OH) have been employed to demonstrate the simple and reproducible coupling methodology using propylphosphonic acid cyclic anhydride. The results showed that new protected and unprotected N-glucosyl amino acids can be obtained with high purity and the methodology could be used with any Fmoc-amino acid. The methodology developed could be considered as a synthetic tool for obtaining building blocks for glycopeptide synthesis and potential drugs candidates based on glycoconjugates.     

Automated solid-phase synthesis of linear nitrogen-linked compounds

A synthetic library motif has been developed to create linear, nitrogen-linked compounds as screening libraries to target structured RNA for drug discovery. Scaffolds were created in situ from suitably protected bifunctional compounds linked together either by acyl or amine links. Acyl links were created from amino acids, which also introduce one degree of functionality. Amine links from the amino acid nitrogen were created from an N-protected amino alcohol via Fukuyama Mitsunobu alkylation. Each amine site can then be used for introducing functionality or extending the scaffold. This synthetic scheme can be used to create a wide variety of modified-backbone PNA in situ, as shown by the synthesis of a PNA-type monomer. The synthesis steps have been enabled on a 96-well parallel-array synthesizer for high-throughput synthesis. The present study represents a versatile synthetic approach to a wide variety of potential RNA-binding molecules.     

Internally standardized amino acid analysis for determining peptide/carrier protein coupling ratio

A method based on amino acid analysis has been developed for monitoring the covalent conjugation of synthetic peptide haptens to carrier proteins. The marker amino acid, alpha-aminobutyric acid, is included in the sequence during peptide synthesis. Following reaction, the carrier protein-conjugate is freed of excess peptide by two successive rounds of gel filtration chromatography. Amino acid analysis of a hydrolysate of the conjugate allows the calculation of the coupling ratio of the peptide to the carrier protein. Two typical procedures for conjugation, carbodiimide cross-linking and cysteine-thiol reaction with maleimidyl-proteins, have been evaluated.     

Expression of growth hormone-releasing factor analog Leu27GRF(1-44)OH in Escherichia coli: purification and characterization of the expressed protein

A recombinant plasmid has been constructed to direct the synthesis of Leu27GRF(1-44)OH in Escherichia coli as a fusion protein containing a hexa-His tail followed by amino acids 1-99 of interferon-gamma and a methionine residue at the N-terminal. The expression of the 18-kDa fusion protein (H6GAMGRF) was induced by isopropylthiogalactoside treatment and the protein accumulated as insoluble aggregates in inclusion bodies. The protein aggregates were solubilized in 6 M guanidine-HCl and purified directly by affinity chromatography on a Nichelate column. The growth hormone-releasing factor (GRF) moiety was released from the fusion protein by cyanogen bromide cleavage and purified to homogeneity by anion-exchange chromatography followed by reverse-phase chromatography. The identity of the GRF peak was determined by comparing its retention time with that of synthetic Leu27GRF(1-44)OH. The purified material was further characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, N-terminal sequencing, and amino-acid analysis. The recombinant-derived product and the synthetic compound showed identical reactivities toward anti-GRF polyclonal antibodies and were essentially equipotent as determined by an in vitro biological assay for growth hormone-releasing activity.     

Solid phase synthesis of apamin, the principal neurotoxin in bee venom. Isolation and characterization of acetamidomethyl apamin

The synthesis of apamin, the principal neurotoxin in bee venom, has been accomplished by the solid phase method on a benzhydrylamine resin, 2-Phenylisopropyloxycarbonyl amino acids were used throughout the synthesis except for the C-terminal histidine. Improved yields in the coupling steps in the N-terminal part of the molecule were obtained by coupling each amino acid both in dichloromethane and dimethylformamide. The use of acetamidomethyl as an S-protecting group for cysteine made it possible to isolate and purify the linear peptide. The deblocked and oxidized peptide was fractionated by ion-exchange chromatography (Bio-Rex 70) to obtain a highly purified apamin with full biological activity and with the same physical and chemical properties as the natural peptide. Circular dichroism (CD) spectra of the synthetic and natural apamin were identical.     

Application of a fluorogenic substrate in the assay of proteolytic activity and in the discovery of a potent inhibitor of Candida albicans aspartic proteinase.

A fluorescent method for monitoring the activity of the secreted Candida carboxyl (aspartic) proteinase (EC 3.4.23.6) was developed using a fluorogenic substrate based on resonance energy transfer. The fluorescent assay was used to monitor proteinase production, purification, and inhibition. The Km for the fluorogenic substrate, 4-(4-dimethylaminophenylazo)benzoyl-gamma-aminobutyryl-Ile-His-Pro - Phe-His-Leu-Val-Ile-His-Thr- [5-(2-aminoethyl)amino]naphthalene-1-sulfonic acid, was found to be 4.3 microM at the optimum pH of 4.5. Reaction products were separated by reverse-phase high-performance liquid chromatography and identified by amino acid analysis or by 252Cf plasma desorption mass spectrometry. Cleavage of the fluorogenic substrate was between the histidine-threonine residues, releasing the fluorescent product, threonine-[5-(2-aminoethyl)amino]naphthalene-1-sulfonic acid. Proteolytic activity was expressed as nanomoles of fluorescent product released at 22 degrees C/60 min, pH 4.5, and the release of 0.9 nmol product was equivalent to one hemoglobin proteolytic unit (O.D.A700 increase of 0.100) produced at 37 degrees C/60 min, pH 3.5. The aspartic proteinase inhibitor pepstatin had an IC50 of 27 nM when tested in a dose-response study with the purified enzyme. The apparent Ki for pepstatis was 2.9 nM. Several synthetic inhibitors of the enzymes were identified with IC50's in the nanomolar range. The most potent compound, A70450, was characterized as a fast, tight-binding inhibitor having an IC50 of 1.3 nM and apparent Ki of 0.17 nM.