Microwave assisted facile synthesis of amino acid benzyl ester p-toluenesulfonate and hydrochloride salts

Summary A simple method for the synthesis of several amino acid benzyl esterp-toluenesulfonate salts from the corresponding amino acid and benzyl alcohol in presence ofp-toluenesulfonic acid accelerated with microwave irradiation is described. Under similar condition, the amino acid benzyl ester hydrochloride salts have also been obtained by using thionyl chloride instead ofp-toluenesulfonic acid in good yield and purity.     

A new acid hydrolysis method for determining tryptophan in peptides and proteins

Three different methods for hydrolysis and determination of amino acid composition of peptides and proteins were compared. We found, that the method of Matsubara and Sasaki (using 6N HCl and thioglycolic acid) gives comparatively low recoveries for tryptophan, while Liu and Chang's method, using p-toluenesulfonic acid and tryptamine, is more suitable. To eliminate the difficulties of the latter method, we used mercaptoethane-sulfonic acid, which, in the concentration used, results in total hydrolysis of peptide bonds within 22 hr and gives very high tryptophan recoveries. Both sulfonic acid methods were used for hydrolysis of the pentapeptide “pentagastrine” as well as of the proteins lysozyme, cytochrome c, and chymotrypsine. Their amino acid composition was determined using an automatic amino acid analyzer. Similarly to the p-toluenesulfonic acid method, the results of our method are totally reliable only for pure peptides and proteins, though the results obtained with our method using samples containing carbohydrates are better than those of all earlier methods.     

Interactions of D-cellobiose with p-toluenesulfonic acid in aqueous solution: a C NMR study

The effects of adding D₂SO₄, and p-toluenesulfonic acid-d to D-cellobiose dissolved in D₂O were investigated at 23 °C by plotting ¹³C NMR chemical shift changes (Δδ) against the acid to D-cellobiose molar ratio. ¹³C Chemical shifts of all 18 carbon signals from α and β anomers of D-cellobiose showed gradual decreases due to increasing acidity in aqueous D₂SO₄ medium. The C-1 of the α anomer showed a slightly higher response to increasing D⁺ concentration in the surrounding. In the aqueous p-toluenesulfonic acid-d medium, C-6′ and C-4′ carbons of both α, and β anomeric forms of D-cellobiose are significantly affected by increasing the sulfonic acid concentrations, and this may be due to a 1:1 interaction of p-toluenesulfonic acid-d with the C-6′, C-4′ region of the cellobiose molecule.     

Synthesis of daunosamine-containing disaccharides

Treatment of 2,3,6-trideoxy-1,4-di-O-(p-nitrobenzoyl)-3-(trifluoroacetamido)-l-lyxo-hexopyranose (1) with benzyl 2,3-dideoxy-d-glycero-pentopyranoside and p-toluenesulfonic acid gave a mixture of benzyl 2,3,6-trideoxy-4-O-p-nitrobenzoyl-3- (trifluoroacetamido)-l-lyxo-hexopyranoside (49%) and benzyl 2,3-dideoxy-4-O-[2,3,6-trideoxy-4-O-(p-nitrobenzoyl)-3-(trifluoroacetamido)-α-l-lyxo-hexopyranosyl]-d-glycero-pentopyranoside (4, 20 %). The structure of the disaccharide 4 was confirmed by a detailed, mass-spectrometric analysis in three modes, namely, negative- and positive-ion, chemical ionization, and electron impact. Similar treatment of the bis(p-nitrobenzoate) 1 with ethyl 2,3-dideoxy-d-glycero-pentopyranoside gave the ethyl glycoside and the desired disaccharide, showing that the transglycosylation is not restricted to benzyl glycosides. Removal of the p-nitrobenzoyl and the benzyl groups from 4 gave the disaccharide 2,3-dideoxy-4-O-(2,3,6-trideoxy-3-trifluoroacetamido-α-l-lyxo-hexopyranosyl)-d-glycero-pentopyranose.     

Photodecomposition of S-Benzyl O,O-Diisopropyl Phosphorothiolate (Kitazin P®)

Two main steps of photodecomposition were observed at the initial stage on the irradiation of ultraviolet light to Kitazin P® in a thin film. One was the isomerization to a thionate, O-benzyl O, O-diisopropyl phosphorothionate, which was gradually hydrolyzed or oxidized to its oxygen analogue. The other step was the cleavage of P-S bond to produce O, O-diisopropyl phosphonate and α-toluenethiol, the latter of which was degraded to produce α-toluenesulfonic acid via dibenzyl disulfide, and finally sulfuric acid and benzoic acid. O, O-Diisopropyl hydrogen phosphorothioate, O, O-diisopropyl hydrogen phosphate and benzyl alcohol were detected as hydrolyzates. Benzyl alcohol was further oxidized to benzoic acid via benzaldehyde. In addition to these compounds, O, O, S-triisopropyl phosphorothiolate, O, O, O-triisopropyl phosphorothionate, O, O, O-triisopropyl phosphate and benzyl isopropyl sulfide were also detected.     

Some reactions of a furanoid 2-aminoglycal derivative

Some reactions, catalyzed by p-toluenesulfonic acid, of 2-acetamido-1,4-anhydro-2-deoxy-5,6-O-isopropylidene-d-arabino-hex-1-enitol (1), a furanoid 2-aminoglycal derivative, were examined. Reaction with methyl and with benzyl alcohol gave the corresponding furanoid 2,3-unsaturated glycosides (2 and3) in good yield. Similar reaction with water, followed by acetylation, gave 2-acetamido-1,4,6-tri-O-acetyl-2,3-dideoxy-d-ribo-hex-2-enopyranose, which was hydrogenated to 2-acetamido-1,4,6-tri-O-acetyl-2,3-dideoxy-d-ribo-hexopyranose (an N-acetyllividosamine derivative) and its arabino analog. Addition of a catalytic amount of p-toluenesulfonic acid to a solution of 1 in dry 1,4-dioxane afforded furanoid, (1→3)-disaccharides in high yield. Tosylation of 1 to yield a furan derivative was, however, unsuccessful. Hydrogenation of methyl 2-acetamido-2,3-dideoxy-5,6-O-isopropylidene-d-erythro-hex-2-enofuranoside (2) was examined by use of palladium-on-carbon, as well as platinum oxide, as the catalyst     

Studies on Chrysanthemic Acid

(±) -trans-2,2-Dimethyl-3- (2′-methyl-2′-propenyl) cyclopropan-l-carboxylic acid (VII) was obtained by the treatment of (±) -pyrocin (IV) with thionyl chloride and absolute ethanol saturated with dry hydrogen chloride followed by the cyclization action of sodium tert-amylate in dry benzene and alkaline hydrolysis. This was converted into (±) -trans-chrysanthemic acid (VIII) by the catalytic action of p-toluenesulfonic acid.     

Synthesis of Two 3-(7′-Theophyllyl)glycals, 3-Deoxy-3-(7′-theophyllyl)-d-xylo-hex-1-enopyranose and Methyl 3-Deoxy-3-(7′-theophyllyl)-d-xylo-hex-1-enopyranuronate,and their Derivatives

Two 3-(7′-theophyllyl)glycals, (IV) and (V), were synthesized by fusion of theophylline and the appropriate glycals in the presence of p-toluenesulfonic acid. The structure and stereochemistry of the glycals were determined mainly from NMR analysis of their dihydro and 1,6-anhydro derivatives.     

Umsetzung von D-glucose mit diethylammoniumtartrat: isolierung von 3,5-dihydroxy-6-methyl-2H-pyran-2-on sowie nachweis cyclischer aminoreduktone als reduktonmethyläther

Heating of D-glucose with diethylammonium tartrate to about 130° gave a dark-brown reaction mixture from which 3,5- dihydroxy-6-mothyl-2H-pyran-2-one and the cyclic amino reductones were isolated. The major amino reductone 4-(diethylamino)-1,3-dihydroxy-1-methyl-3-cyclopenten-2-one was determined by g.l.c. after treatment with methanol-p-toluenesulfonic acid to give 3-hydroxy-1,4-dimethoxy-1-methyl-3-cyclopenten-2-one and 1,3,4-trimethoxy-1-methyl-3-cyclopenten-2-one.     

Metabolism of S-Benzyl O-Ethyl Phenylphosphonothioate (Inezin®) by Mycelial Cells of Pyricularia oryzae

Gas-liquid chromatographic study revealed that organophosphorus fungicide Inezin® (S-benzyl O-ethyl phenylphosphonothioate) was metabolized to O-ethyl hydrogen phenylphosphonothioate or ethyl hydrogen phenylphosphonate by mycelial cells of Pyricularia oryzae. Metabolic fate of the removed benzyl or benzylthio group was further studied by labeling benzene ring of benzyl radical of the fungicide with ¹⁴C, and dibenzyl disulfide, benzyl alcohol, toluene-α-sulfonic acid and benzoic acid were found as metabolites by ion exchange chromatography and thin-layer chromatography. Gas-liquid chromatographic study also ascertained that a part of Inezin was hydroxylated at m-position of the benzene ring of benzyl radical, but o- or p-hydroxylation of benzyl radical was not seemed to occur.

No significant difference was found in metabolism of the fungicide between susceptible and resistant clones of the fungi.     

Metabolism of Hydrocarbons in Microorganisms

The crude extract obtained from Pseudomonas aeruginosa grown on xylene as sole carbon source converted p-xylene-methyl-¹⁴C or toluene-methyl-¹⁴C to p-toluic or benzoic acid, respectively. However, addition of p-methylbenzyl or benzyl alcohol to the reaction mixture resulted in accumulation of p-methylbenzyl or benzyl alcohol. This indicates that in the crude extract, p-xylene or toluene is metabolized via its corresponding alcohol to p-toluic or benzoic acid, respectively. The enzyme system responsible for these reactions required NAD or NADH and FAD, and could be stabilized by the presence of glutathione. When the crude extract was fractionated by the use of DEAE-cellulose chromatography, it was demonstrated that two distinct protein fractions and two cofactors (NADH and FAD) were required for the step of the hydroxylation of p-xylene or toluene to its corresponding alcohol. NAD, NADP or NADPH had very few or little activity. FMN partially replaced FAD.     

New Photosensitizers of Bacteriochlorin Series for Photodynamic Cancer Therapy

New derivatives of bacteriochlorophyll a bearing an extra glutarimide exocycle were synthesized, and their reactivity was studied. Acetyl group in 3-acetyl-2,7,12,18-tetramethyl-8-ethyl-13,15-dicarboxy-17-carboxyethyl-7,8,17,18-tetrahydroporphyrin (bacteriochlorin p) was chemically modified into -hydroxyethyl and vinyl groups. A simple method of preparation of vinylbacteriopurpurin esters under the catalysis by p-toluenesulfonic acid was proposed. The resulting compounds exhibit a high adsorption in the visible and near IR areas of electronic spectra, a reasonable stability, and amphiphilic properties and, therefore, may be regarded as promising photosensitizers for the photodynamic cancer therapy.     

Diastereomeric resolution of p‐chloromandelic acid with (R)‐phenylethylamine

The optical resolution of p‐chloromandelic acid using (R)‐α‐phenylethylamine as resolving agent was presented. The effect of solvents, molar ratio of racemate to the resolving agent, filtration temperature as well as the amount of solvent on resolution was investigated by orthogonal experimentation. The binary melting point phase diagram and crystal structure analysis of diastereomeric salts rationalized the success of the resolution. Chirality, 2010. © 2009 Wiley‐Liss, Inc.     

Isolation and characterization of a metallothionein-1 protein in Chloris virgata Swartz that enhances stress tolerances to oxidative, salinity and carbonate stress in Saccharomyces cerevisiae

Chloris virgata Swartz (C. virgata) is a gramineous wild plant that is found in alkaline soil areas in northeast China and is highly tolerant to carbonate stress. We constructed a cDNA library from C. virgata seedlings treated with NaHCO₃, and isolated a type1 metallothionein (MT1) gene (ChlMT1: AB294238) from the library. The amino acid sequence of ChlMT1 contained 12 cysteine residues that constituted the Cys-X-Cys (X = amino acid except Cys) motifs in the N- and C-terminal regions. Northern hybridization showed that expression of ChlMT1 was induced by several abiotic stresses, from salts (NaCl and NaHCO₃), a ROS inducer (paraquat), and metals (CuSO₄, ZnSO₄, and CoCl₂). ChlMT1 expression in leaf was induced by 200 mM NaCl and 100 mM NaHCO₃. About 5 μM Paraquat, 500 μM Zn²⁺, and 500 μM Co²⁺ also induced expression of ChlMT1 in leaf after 6 h, and 100 μM Cu²⁺ induced it after 24 h. Saccharomyces cerevisiae when transformed with the ChlMT1 gene had dramatically increased tolerances to salts (NaCl and NaHCO₃) and ROS.     

Enantiopure O‐Ethyl Phenylphosphonothioic Acid: A Solvating Agent for the Determination of Enantiomeric Excesses

An improved method, which is highly reproducible, was developed for the enantioseparation of racemic O‐ethyl phenylphosphonothioic acid ( 1a ) with brucine by introducing seeding to a supersaturated solution of the diastereomeric salt mixture. The present method gave both diastereomeric salts in high yields with a diastereomeric ratio of >99.5:0.5 upon choosing the crystallization solvent (MeOH for the ( (R)-1a salt and MeOH/H₂O for the ( (S)-1a salt). The enantiopure acid (R)-1a , (S)-1a showed a good chirality recognition ability for not only strong bases, such as amines and amino alcohols, but also weakly basic alcohols and was applicable as a solvating agent to the ¹H NMR determination of the enantiomeric excess of chiral amines, amino alcohols, and alcohols, including aliphatic substrates. Chirality 26:614–619, 2014. © 2014 Wiley Periodicals, Inc.     

The Effects of Serum Albumin and Related Amino Acids on Pancreatic Lipase and Bile Salts Inhibited Microbial Lipases

The activities of microbial lipases were inhibited by bile salts in a non-emulsifying assay system. To protect lipase activities from inactivation, the effects of proteins and amino acids were investigated. Bovine serum albumin (BSA) and α-lactalbumin (α-LA) stored the bile salts inhibited microbial lipases. Among N-end amino groups contained in BSA, L-histidine restored the activities of the bile salts inhibited microbial lipases. On the other hand, pancreatic lipase activity was stimulated by not only BSA, but L-histidine and L-aspartic acid as N-end amino groups of BSA and additionally accelerated it in combination with bile salts.     

Purification and properties of ap-nitrobenzyl esterase fromBacillus subtilis

A procedure for purifying to homogeneity a microbially produced biocatalyst useful for deblocking intermediates in the manufacture of beta-lactam antibiotics is reported. In aqueous solution the purifiedp-nitrobenzyl (PNB) carboxy-esterase was soluble, monomeric (molecular weight: 54 000 by SDS-PAGE or by gel filtration) and exhibited an acidic pl, 4.1. The PNB carboxy-esterase catalyzed rapid ester hydrolysis for simple organic esters such as PNB-acetate, benzyl acetate and -naphthyl acetate and catalyzed deblocking (ester hydrolysis) of beta-lactam antibiotic PNB esters such as cephalexin-PNB and loracarbef-PNB. TheN-terminal amino acid sequence and the amino acid composition are reported. A serine residue is involved in ester hydrolysis: the PNB carboxy esterase was inhibited by phenylmethylsulfonyl fluoride and diethylp-nitrophenyl phosphate; one mole of diisopropyl fluorophosphate titration was required per mole of PNB carboxy-esterase for complete inhibition. When the [³H]-diisopropyl fluorophosphate-treated biocatalyst was digested with Lys C and the resulting peptides separated by HPLC, a single [³H]-labeled peptide was obtained; its amino acid sequence is reported. Inhibition of the PNB carboxy esterase by diethyl pyrocarbonate suggests that a histidinyl residue (or residues) is (are) also involved in the catalytic site of the esterase.Abbreviations used -ME -mercaptoethanol - Cf cefaclor - Cf nucleus-PNB - (6R, 7R) 7-amino-3-chloro-8-oxo-5-thia-1-azabicyclo[4.2.0]-oct-2-ene-2-carboxylic acid, (4-nitrophenyl)methyl ester - Cp cephalexin - Cp-PNB p-nitrobenzyl carboxy-ester of cephalexin - DEPC diethyl, pyrocarbonate - DFP diisopropyl fluorophosphate - DMSO dimethyl sulfoxide - DNP diethylp-nitrophenyl phosphate - EDTA ethylenediaminetetraacetic acid - EGTA ethylene, glycol-bis(aminoethyl ether) - N,N,NN tetracetic acid - Lc loracarbef - Lc-PNB p-nitrobenzyl carboxy-ester of loracarbef - Lc nucleus-PNB - (6R, 7S) 7-amino-3-chloro-8-oxo-1-azabicyclo[4.2.0]-oct-2-ene-2-carboxylic acid, (4-nitrophenyl)methyl ester - Lys C an endoproteinase specifically cleaving at C terminal lysine residues - MW_r relative molecular weight - PAGE polyacrylamide gel electrophoresis - PMSF phenylmethylsulfonylfluoride - PNB p-nitrobenzyl - PNBCE p-nitrobenzyl carboxy-esterase - SDS sodium dodecyl sulfate     

Studies on 2-aziridinecarboxylic acid peptides: Their reaction and uses

The reaction of 2-aziridinecarboxylic acid derivatives with several protic reagents was used to synthesize depsipeptides, dehydroamino acid derivatives, diaminopropionic acid derivatives, and phospho peptide derivatives. The reaction of N-aminoacyl-2-aziridinecarboxylic acid benzyl ester with amino acid ester induced stereoselective transacylation.     

Seasonal patterns of nucleic acid concentrations and amino acid profiles of Parapenaeus longirostris (Crustacea, Decapoda): relation to growth and nutritional condition

The present work describes the seasonal changes in nucleic acid concentrations and amino acid profiles in the muscle of juvenile Parapenaeus longirostris and their relation to growth and nutritional condition. RNA content varied significantly between seasons, being the highest values attained in spring and the lowest in winter (p < 0.05). Similar results were obtained with RNA:protein and RNA:DNA ratios. In respect to total amino acid content (TAA), a significant increase from winter to spring was observed (p < 0.05) and the major essential amino acids (EAA) were arginine, histidine and leucine. Within non-essential amino acids (NEAA) glutamic acid, aspartic acid, glycine and proline were dominant. From winter to spring, a significant variation in NEAA content occurred (26.8; p < 0.05), mainly due to the significant increase of glutamic acid (79.1) and serine (66.7) (p < 0.05). EAA content did not vary significantly between seasons (p > 0.05). In opposition, during this period a significant decrease in the free amino acid content (FAA) was observed (p < 0.05); a higher percentage of decrease was attained in free non-essential (FNEAA – 42.9) in comparison to free essential amino acids (FEAA – 40.2). The significant increase in RNA and TAA contents from winter to spring may be related with protein synthesis. On the other hand, the lowest values obtained in winter may be due to a reduction in feeding activity; in this period the muscle protein must be progressively hydrolysed, which is evident with the higher FAA content. The liberated amino acids enter FAA pool and become available for energy production. In conclusion, it was evident that the seasonal cycle in activities such as feeding and growth with nucleic acids and amino acid analyses was noticed.     

Studies on Phenolic Lactones

Isohibalactone, the geometric isomer of hibalactone, was synthesized by the following route. Piperonylsuccinic acid anhydride was converted into thioethyl methyl ester ans was reduced to piperonylbutyrolactone by Raney nickel catalyst. Piperonylbutyrolactone was also prepared from piperonylsuccinic anhydride by the reduction with amalgamated aluminum. Condensation of piperdnal with the lactone in the presence of potassium amide afforded α-(3,4-methyIenedioxy-phenyl-hydroxymethyl)-β-(3,4-methylenedioxybenzyl)-butyrolactone, m.p. 151~2°C. Dehydration of the hydroxylactone with p-toluenesulfonic acid gave isohibalactone, m.p. 156~6.5°C.