Synthèses d’un des Diastéréo-isoméres des Acides Dithio-4,4 Bis(Amino-2 Méthyl-3)Butyriques et des DL-Amino-2 Méthyl-3 Butyrothiolactones

2-Methyl-3-benzylmercaptopropanal, obtained by condensation of methacrolein with benzylmercaptan, was treated with a mixture of sodium cyanide, ammonium carbonate and triethylamine to give two diastereomeric hydantoins. Each diastereomer gave the same S-benzylmercapto-valine on treatment with alkali, which shows that the epimerization occured in this condition and the more stable one was formed. This compound was treated with sodium in liquid ammonia to give the corresponding disulfide. S-benzylmercapto-valine gave 2-amino-3-methyl-butyrothiolactone when refluxed under inert atmosphere after the reduction. These compounds were derived to 2-methylhomocysteic acid by treatment with bromine and valine by treatment with Raney nickel.     

Optical Rotatory Dispersion and Circular Dichroism of Amino Acid Hydantoins

Optical rotatory dispersion (ORD) and circular dichroism (CD) of 17 amino acid hydantoins were measured between 190 and 600 nm. Most of hydantoins exhibited the negative Cotton effect which showed the trough between 238 and 245 nm. The negative trough of CD was also observed between 212 and 236 nm. The Cotton effect of hydantoins was attributable to n→π^* transition of carbonyl group at C-4 of hydantoin ring.     

Complete conversion of D,L-5-monosubstituted hydantoins with a low velocity of chemical racemization into D-amino acids using whole cells of recombinant Escherichia coli

A reaction system was developed for the production of D-amino acids from D,L-5-monosubstituted hydantoins with a very slow rate of spontaneous racemization. For this purpose the D-hydantoinase and D-carbamoylase from Agrobacterium radiobacter NRRL B11291 were cloned in separate plasmids and expressed in Escherichia coli. The third enzyme, hydantoin racemase, was cloned from Agrobacterium tumefaciens C58. The hydantoin racemase amino acid sequence was significantly similar to those previously described. A reaction system consisting of recombinant Escherichia coli whole cell biocatalysts containing separately expressed D-hydantoinase, D-carbamoylase, and hydantoin recemase showed high substrate specificity and was effective toward both aliphatic and aromatic D,L-5-monosubstituted hydantoins. After optimizing reaction conditions (pH 8 and 50 degrees C), 100% conversion of D,L-5-(2-methylthioethyl)-hydantoin (15 mM) into D-methionine was obtained in 30 min.     

The Synthesis of Some 5-Vinyluracil-Nucleoside Analogues

Abstract

The reaction of 5-iodouridine with esters of acrylic acid in the palladium-catalysed Heck reaction was used to generate a series of esters of the acid (E) -5- (2-carboxyvinyl)uridine in poor to moderate yield. An alternative method starts from the acid by protection of the sugar moiety followed by in situ generation of the acid chloride then ester formation followed by simultaneous sugar deprotection. Treatment of the methyl ester with ammonia and methylamine gave the corresponding amides, while treatment with dimethylamine gave 5-(1-dimethylamino-2-carboxy)ethyluridine as the major product.     

Racemization in the synthesis of polytripeptide models of a collagen.

Racemization in the synthesis of tripeptide intermediates and their polymers was investigated, using L -amino acid oxidase. Stepwise investigation of peptide intermediates showed no racemization during peptide coupling steps or deprotection of benzyl esters by hydrogenolysis. Saponification of one of the methyl esters produced some racemization. Preparation of active esters from N-protected tripeptide acids containing optically active C-terminal amino acid, with one exception, produced racemization. The fractionated polymers were found to contain less racemized amino acids than the crude products or starting monomeric tripeptides, indicating that the racemized sequences gave rise to lower molecular-weight oligomers. The sequences investigated were -Pro-Pro-Ala-, -Ala-Pro-Pro-, -Val-Pro-Pro-, -Pro-Pro-Leu-, -Pro-Gly-Leu-, -Pro-Gly-Phe-, -Pro-Gly-Val-, -Gly-Val-Pro-, -Phe-Pro-Gly-, -Leu-Pro-Gly-, and Ile-Pro-Gly-.     

On the Acaciin

Synthesis of N-2,2,3,3-tetrafluoropropionyl-amino acids (abbreviated as tetrafluoropropionyl- or Tfp-amino acids) by the reaction of amino acids with tetrafluoropropionyl anhydride was described, Racemization was observed to occur to a considerable extent during the process of the introduction of this group. Optically active Tfp-amino acids were able to be prepared free from racemization by transesterification of methyl tetrafluoropropionate with amino acid tert-butyl esters, followed by treatment of the resulting Tfp-amino acid tert-butyl esters with trifluoroacetic acid. Some Tfp-dipeptide esters were prepared from the corresponding dipeptide tert-butyl esters in this way. This group was cleavable by mild alkaline hydrolysis or by reduction with sodium borohydrde as well as the Tfa-group.     

Synthesis and application of chiral triazine condensing reagents prepared from esters of amino acids

Treatment of cyanuric chloride with chiral amines or esters of chiral amino acids gave chiral 2,4-dichloro-6-alkylamino-1,3,5-triazines (2-5) in 49-69% yield, which were found useful as coupling reagents. Enantioselective activation and enantioselective aminolysis in the presence of 2-5 was observed.     

Synthesis and solid state 13C and 1H NMR analysis of new oxamide derivatives of methyl 2-amino-2-deoxy-alpha-D-glucopyranoside and ester of amino acids or dipeptides

The syntheses of new oxamide derivatives of methyl 2-amino-2-deoxy-alpha-D-glucopyranoside and amino acid or peptide esters are presented. The reaction of methyl 3,4,6-tri-O-acetyl-2-acetamido-2-deoxy-alpha-D-glucopyranoside and oxalyl chloride gave N-(methyl 3,4,6-tri-O-acetyl-2-deoxy-alpha-D-glucopyranosid-2-yl) oxamic acid chloride which on reaction with the ester of Gly, L-Ala, L-Phe, GlyGly, Gly-L-Phe and Gly-L-Ala afforded N-(methyl 3,4,6-tri-O-acetyl-2-deoxy-alpha-D-glucopyranosid-2-yl), N'-oxalyl-amino acid or dipeptide esters. The structure of the oxamides was studied using 1H, 13C NMR in solution and solid state.     

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.     

N-Carbamoyl-2-(p-hydroxyphenyl)glycine from leaves of broad bean (Vicia faba L.)

1. dl-2-(p-Hydroxyphenyl)glycine was resolved through the bromocamphorsulphonate to give its d-isomer. The N-carbamoyl derivatives of these amino acids were synthesized. Circular-dichroism studies on these and related compounds, reported in a deposited Annex, helped to establish the optical configuration. 2. N-Carbamoyl-dl-2-(p-hydroxyphenyl)glycine was isolated from broad-bean leaves. It amounted to about 0.1% of the leaf dry matter. Racemization may or may not have occurred during the isolation. There were indications of the same compound in chicory and in savoy cabbage. Under weakly acidic conditions it was converted gradually into 5-(p-hydroxyphenyl)hydantoin. Both these compounds yielded 2-(p-hydroxyphenyl)glycine on acid hydrolysis. 3. The occurrence is discussed of 2-phenylglycine derivatives in Nature and of N-carbamoyl-amino acids and hydantoins in plants. 4. Gradient elution from anion-exchange resin with acetic acid, besides proving useful for the present work, gave useful separations of pyrrolidonecarboxylic acid and of some N-acetyl-amino acids. 5. Supplementary material (Annex 1: details of experimental work other than ultraviolet and circular-dichroism spectra; Annex 2: ultraviolet absorption and circular dichroism of d-2-phenylglycine and some related compounds) has been deposited as Supplementary Publication SUP 50003 at the National Lending Library for Science and Technology, Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1971), 121, 7.     

Immunological Abnormalities and Hydantoins

Various immunological features were measured in 20 patients with epilepsy who had received prolonged treatment with hydantoins. Immunoglobulin A (IgA) was shown to be absent or low in five patients, β₁C/A was low in 10 patients. Five patients showed negative reactions to skin tests, and two could not be sensitized to dinitrochlorobenzene. The corresponding features were normal in 14 control patients with epilepsy but without hydantoins. It is suggested that the hydantoins influence humoral immunity, whereas other immunosuppressant agents have been found to affect cellular immunity.     

The synthesis of 5-amino-2,6-anhydro-5-deoxy-D-glycero-D-gulo-Heptonic acid and its polycondensation to oligomers

5-Amino-2,6-anhydro-5-deoxy-D-glycero-D-gulo-heptonic acid has been synthesized by conventional introduction of an amino function via azide displacement, starting with a suitable derivative of 2,6-anhydro-D-glycero-L-manno-heptonic acid. The amino acid was converted into the methyl ester hydrochloride which, in methanolic sodium methoxide, gave oligomeric and polymeric amides, depending on the conditions applied. Four oligomeric esters, as well as the corresponding N-(2,4-dinitrophenyl) derivatives of the amino acids, could be separated by paper chromatography. The oligomers could be saponified under mild, basic conditions.     

Identification of ornithine and arginine conjugates of cholic acid by mass spectrometry.

Nalpha-Cholylornithine, -arginine, and -histidine were prepared according to a method previously employed for the chemical synthesis of the monoamino acid conjugates of bile acids. The products were shown to involve the alpha amino group of the dibasic amino acids by examination of the mass spectra of the original compounds, their lactams, their methyl esters and the methyl ester acetates. Only the methyl ester acetates gave detectable amounts of molecular ion. The free acids and the methyl esters of Nalpha-cholylornithine and -arginine gave identical lactams upon sublimation from the direct insertion probe. The synthetic Nalpha-cholylarginine was shown to yield a mass spectrum identical to that of an arginocholic acid recovered from the bile of an isolated perfused rat liver.     

Cloning, sequencing, and expression in Escherichia coli of the D-hydantoinase gene from Pseudomonas putida and distribution of homologous genes in other microorganisms.

Pseudomonas putida DSM 84 produces N-carbamyl-D-amino acids from the corresponding D-5-monosubstituted hydantoins. The gene encoding this D-hydantoinase enzyme was cloned and expressed in Escherichia coli. The nucleotide sequence of the 1.8-kb insert of subclone pGES19 was determined. One open reading frame of 1,104 bp was found and was predicted to encode a polypeptide with a molecular size of 40.5 kDa. Local regions of identity between the predicted amino acid sequence and that of other known amidohydrolases (two other D-hydantoinases, allantionase and dihydroorotase) were found. The D-hydantoinase gene was used as a probe to screen DNA isolated from diverse organisms. Within Pseudomonas strains of rRNA group I, the probe was specific. The probe did not detect D-hydantoinase genes in pseudomonads not in rRNA group I, other bacteria, or plants known to express D-hydantoinase activity.     

alpha-Chymotrypsin as the catalyst for peptide synthesis.

alpha-Chymotrypsin (EC 3.4.21.1)-catalysed syntheses of peptides were performed with various N-acylated amino acid or peptide esters as donors, and amino acid derivatives, peptides or their derivatives as acceptors. Under optimal conditions the synthesis was almost quantitative. As acceptor nucleophiles, free amino acids or the ester derivatives were inadequate, but amino acid amides or hydrazides, di- or tri-peptides, or the amides, hydrazides and esters of the peptides were useful. The nucleophile specificity for synthesis was markedly similar to the leaving-group specificity in hydrolysis; hydrophobic or bulky amino acid residues were most effecient at both P1' and P2' positions [notation of Schechter & Berger (1967) Biochem. Biophys. Res. Commun. 27, 157-162], but L-proline as well as D-amino acid residues were the worst choices. The synthesis was further dependent on the solubility of the products synthesized; a higher yield of products was expected with lower solubility. As donor esters, good substrates were all useful. Accordingly, fragment condensation was possible by using N-acylated peptide esters and various peptides. The present study suggested that alpha-chymotrypsin may become a useful tool for peptide synthesis.     

Efficient production of the industrial biocatalysts hydantoinase and N-carbamyl amino acid amidohydrolase: Novel non-metabolizable inducers

Abstract The biosynthesis of the hydantoin-hydrolysing enzymes hydantoinase and N -carbamyl amino acid amidohydrolase from Agrobacterium sp. IP I-671, a Gram-negative bacterium used as a biocatalyst for the production of enantiomerically pure ( R ) amino acids, was found to be highly inducible by the addition to the cultivation medium of different non-metabolizable thiolated hydantoins or pyrimidines. Among these inducers the hexacyclic pyrimidine thioderivatives were more potent than all the pentacyclic thiohydantoin compounds. Addition of 2,4-thiouracil to the cultures, at a rate of 0.1 g (g cell dry mass)⁻¹, led to no appreciable growth inhibition and yielded a biocatalyst exhibiting a 40-fold higher hydantoinase and a 15-fold higher N -carbamyl amino acid amidohydrolase activity than the corresponding inducer-free cultures.     

Taurine Levels in Some Tissues of Yellowtail (Seriola quinqueradiata) and Mackerel (Scomber japonicus)

The mechanism of stereospecific production of l-amino acids from the corresponding 5-substituted hydantoins by Bacillus brevis AJ-12299 was studied. The enzymes involved in the reaction were partially purified by DEAE-Toyopearl 650M column chromatography and their properties were investigated. The conversion of dl-5-substituted hydantoins to the corresponding l-amino acids consisted of the following two successive reactions. The first step was the ring-opening hydrolysis to N-carbamoyl amino acids catalyzed by an ATP dependent l-5-substituted hydantoin hydrolase. This reaction was stereospecific and the N-carbamoyl amino acid produced was exclusively the l-form. N-Carbamoyl-l-amino acid was also produced from the d-form of 5-substituted hydantoin, which suggests that spontaneous racemization occurred in the reaction mixture. In the second step, N-carbamoyl-l-amino acid was hydrolyzed to l-amino acid by an N-carbamoyl-l-amino acid hydrolase, which was also an l-specific enzyme. The ATP dependency of the l-5-substituted hydantoin hydrolase was supposed to be the limiting factor in the production of l-amino acids from the corresponding 5-substituted hydantoins by this bacterium.     

Improved detection of polyunsaturated fatty acids as phenacyl esters using liquid chromatography-ion trap mass spectrometry

The fatty acid composition of Shewanella pealeana was determined by the analysis of fatty acid methyl esters via gas chromatography-mass spectrometry (GC-MS) and fatty acid 2-oxo-phenylethyl esters via high-performance liquid chromatography-mass spectrometry (LC-MS) combined with ultra violet (UV) detection. There was good agreement between the percentage composition of components determined by GC-MS and LC-UV analyses. However, LC-MS analysis using Atmospheric Pressure Chemical Ionization (APCI) demonstrated dramatically enhanced detection of unsaturated fatty acid 2-oxo-phenylethyl esters. The degree of enhancement was proportional to the degree of unsaturation. Tests with a pure polyunsaturated fatty acid (PUFA) standard gave an absolute detection limit in full scan mode of 200 pg. In samples, the selectivity of MS over UV gave a significantly lower detection limit due to lack of chemical interferences. In 'Selected Reaction Monitoring' (SRM) mode, the detection limit was 5 pg. This was essentially independent of whether the sample is a standard or complex mixture of fatty acids. Tandem mass spectrometry was used to support structural information and to enhance the ability to target specific fatty acids. Several PUFAs which were not evident from GC-MS analysis were detected and identified by APCI LC-MS, including some rare or novel PUFAs from S. pealeana and a menhaden oil standard. Detailed analysis of bacterial fatty acid composition by either GC-MS or APCI LC-MS is highly preferable to analysis systems based solely on retention time identification.     

Synthesis of N-hydroxycinnamoyl amide derivatives and evaluation of their anti-oxidative and anti-tyrosinase activities

Twelve N-hydroxycinnamoyl amino acid amide ethyl esters (CAES) were synthesized by using l-amino acid ethyl ester hydrochloride and corresponding cinnamic acid (ferulic acid, acetylferulic acid and caffeic acid) as raw materials in the presence of a catalytic amount of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide-hydrochloride (EDC) and 1-hydroxybenzotriene (HOBt). The 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activities of CAES were evaluated. The anti-tyrosinase activities of N-feruloyl amino acid ethyl esters and the hydroxyl (OH) free radical scavenging activities of N-caffeoyl amino acid ethyl esters were also examined. DPPH free radical scavenging activity was shown in all CAES, of which N-caffeoyl amino acid ethyl esters demonstrated higher radical scavenging activity than N-feruloyl amide derivatives, and (E) -N-(caffeic acid)-l-glycinate ethyl ester (c₅) had the strongest ability to scavenge free radicals with an IC₅₀ value of 18.6 µM. The acetylferuloyl amino acid esters exhibited the highest tyrosinase inhibition activity among the tested amides.     

Reversed phase chromatography of isoaccepting tRNA''s from healthy and crown gall tissues from Nicotiana tabacum.

RPC 5 (Reversed Phase Chromatography) of aminoacyl-tRNA's from healthy and crown gall (induced by Agrobacterium tume-faciens strain B6) tobacco tissues were compared for eleven amino acids. For ten amino acids: alanine, arginine, glutamic acid, glycine, isoleucine, leucine, lysine, methionine, tyrosine, and valine, no qualitative or quantitative differences could be detected between aminoacyl-tRNA's from both sources. Phenylalanyl-tRNA's from crown gall tissues gave two peaks on RPC 5; the minor early eluting species (peak 1) was always absent in elution profiles of phenylalanyl-tRNA's from healthy tissues or from tobacco leaves. After the "Y" base was removed by pH 2.9 treatment, peak 2 of phenylalanine tRNA was shifted to the position of peak 1.