Determination of the Optical Isomers of Insecticidal Pyrethroids by Gas-liquid Chromatography

Determination of the optical isomers of insecticidal pyrethroids was carried out by gas-liquid chromatography. Chrysanthemic acid, which was obtained during hydrolysis of the corresponding pyrethroids, was esterifled with d- or l-2-octanol and the diastereoisomer ester derivatives were resolved on a column of Chromosorb W coated with 10% QF–1. All four isomers, d-trans, l-trans, d-cis and l-cis chrysanthemic acids, could be separated. These compounds were not isomerized during hydrolysis, esteriflcation or gasliquid chromatographic operations and their proportions were accurately determined from their peak area ratios.     

The Occurrence and Some Properties of a Spawning Inducing Substance in the Testis of the Hydromedusan Spirocodon saltatrix

Gas chromatographic methods were developed for the determination of optical isomers of 2-(4-chlorophenyl)isovaleric acid (CPIA) which constituted the acid moiety of the insecticide Fenvalerate molecule. The enantiomers of CPIA were derivatized into diastereoisomeric l-menthyl esters quantitatively via their acid chlorides and separated from each other on a column of 10% silicone DC QF-1 (3 mm i.d. × 2.25m). They were also derivatized into isopropylamides in the presence of N,N′-dicyclohexylcarbodiimide and were resolved on an open tubular glass capillary column (0.25 mm i.d. × 40 m) coated with optically active N,N′-[2,4-(6-ethoxy-1,3,5-triazine)diyl] bis(l-valyl-l-valyl-l-valine isopropyl ester) (OA-300). The ratios of optical isomers were determined from their separated peak areas and analytical values obtained by two methods were in good agreement with each other.

The chemical purity of CPIA was also determined by gas chromatography after derivatization to its methyl ester on a column of 2% DEGS (3 mm i.d. × 3 m) using m-nitroanisole as an internal standard.     

Optically Active Trimethylcyclopropanecarboxylic Acids

Four optically active trimethylcyclopropanecarboxylic acids were prepared from chrysanthemic acids, and their absolute configurations were determined on the basis of chrysanthemic acids whose configuration were already known. Toxicities of several rethronyl esters towards housflies were compared with those of chrysanthemic acids and the cis-acids were proved to be more effective than the trans-acids.     

Studies of Chrysanthemic Acid

(±) -cis and trans-2,2-dimethyl-3- (2′-cyano-l′-propenyl) cyclopropane carboxylic acids and their optically active forms were synthesized starting from chrysanthemic acids via oximes of 2,2-dimethyl3- (2′-formyl-l′-propenyl) cyclopropane carboxylates. Their allethrolone esters were also prepared which showed insecticidal activity.     

The Collection and Elution of Radioactive Fatty Acid Methyl Esters During Quantitative Gas-Liquid Chromatography

Abstract

An improved procedure is described for the collection and elution of low levels of radioactive fatty acid methyl esters separated by gas-liquid chromatography. A gas chromatographic effluent splitter was employed to partition fatty acid methyl ester samples in the column effluent, Condensation of a portion of the eluted fatty acids was accomplished in borosilicate glass tubing collectors maintained at ?70°C, Quantitation of nanomolar levels of fatty acid methyl esters was accomplished by calibrating the gas chromatographic flame ionization detectors with the splitters opened or closed. The elution of condensed radioactive fatty acid methyl esters from the glass collectors was complete when benzene followed by a toluene based scintillation fluid were employed as solvents. The method described may be applicable to the analysis of cis-trans isomers of unsaturated fatty acids.     

13C-NMR Studies on Halomethylvinyl Chrysanthemic Acids and Esters-Spin-lattice Relaxation Time and 13C-1H Coupling Constants

New types of stable chrysanthemic acids and esters were synthesized, and their ¹³C-NMR were examined and fully analyzed. The configurations of the cyclopropanecarboxylic acid and halomethylvinyl group were reflected on the spin-lattice relaxation time of the substituted methyl carbon involved in their structure. The long-range spin-spin coupling constants (³J_CH) correlated well to the NOE and T₁ measurements, which can generally be used to distinguish the geometry of the substituted double bond.     

Isolation of 4-Hydroxy-5-methyl-3(2H)-furanone,a Flavor Component in Shoyu (Soy Sauce)

An analytical method was developed for determining the four optical isomers of a new synthetic pyrethroidal insecticide, α-cyano-3-phenoxy-benzyl 2-(4-chlorophenyl)isovalerate (Fenvalerate, S–5602). The cyano group in S–5602 molecule was converted into l-menthyloxycarbonyl group by heating S–5602 with l-menthol and hydrochloric acid. The obtained diastereoisomer derivative (l-menthyl ester deriv.) was chromatographed on a μPorasil column (4 mm i.d. × 0.3 m) using a solvent system of hexane-ethyl acetate (500+7, v/v) as a mobile phase, and the four isomers of S–5602 were separated from one another within 20 min. The compositions of S–5602 isomers were determined from their peak areas.     

Determination of erythrocyte amino acids by gas chromatography

Erythrocyte amino acid levels were determined, by gas chromatography, in a group of 34 normal human adults. No significant sex or age correlations were noted.A method for the quantitative gas chromatographic analysis of free amino acids in erythrocytes is described. Following hemolysis and deproteinization the amino acids were isolated on a cation-exchange resin. Glutathione was removed from the amino acid mixture by adsorption on an anion-exchange resin. Following conversion to their N-acetyl-n-propyl esters, 19 amino acids were separated and quantitated by gas chromatography on a single column in 18 min. Typical reproducibility data indicate that a coefficient of variation of 2–5% is attainable.     

Studies on Moss Spores. IV. Mass Spectrometric Identification of Saturated and Monoenoic Long Chain Fatty Acids in the Triglyceride Fraction of Polytrichum commune Spores

The saturated long chain fatty acid methyl esters of the triglyceride fraction of Polytrichum commune spores were separated by silver nitrate TLC and identified by a combination of gas chromatographic-mass spectrometric technique. The saturated fatty acid methyl esters were straight-chained, and even-numbered with carbon numbers ranging from 12 to 26 or odd-numbered with carbon numbers ranging from 13 to 25. The major components of the fraction containing saturated fatty acid methyl esters were methyl palmitate and methyl stearate. The fatty acid methyl esters of the monoenoic fraction isolated by silver nitrate TLC were converted to TMSO derivates which were analysed by gas chromatography-mass spectrometry. The analysis gave evidence of positional isomers. The fraction contained the following straight chain monoenoic fatty acid methyl ester isomers: methyl 7-cis-hexadecenoate, methyl 9-cis-hexadecenoate, methyl 9-cis-heptadecenoate, methyl 9-cis-octadecenoate, methyl 11-cis-octadecenoate, and methyl 11-cis-eicosenoate. The major components were methyl 9-cis-octadecenoate and methyl 7-cis-hexadecenoate.     

Relatively simple methodology for the determination of configuration of unsaturation of bacterial monounsaturated fatty acids: application to the unsaturates of Legionella spp.

Unsaturated fatty acids of known degree, position, and configuration of unsturation were esterified, and stereospecifically dihydroxylated at the double bond(s). cis-Hydroxylation was effected using Woodward's reagent (silver acetate/iodine/acetic acid), while trans-hydroxylation was effected using Fenton's reagent (hydrogen peroxide/ferrous sulfate/acetic acid). Diols derived from monounsaturated esters were recovered quantitatively, while tetraols derived from diunsaturates were lost, presumably during extensive washing. The stereospecifically dihydroxylated esters were analyzed by thin-layer chromatography on borate-impregnated silica gel plates, and by gas-liquid chromatography, on a nonpolar capillary column, as the trimethylsilyl, acetyl, n-butylboronyl, isopropylidene, and trifluoroacetyl derivatives. For each derivative, the erythro and threo diols were readily separable, and some resolution of positional isomers was observed. Thus, the cis/trans configuration, and in some instances, the position of unsaturation of the original monounsaturated fatty acid may be deduced. However, gas chromatography-mass spectrometry of appropriately derivatized diol esters is required for unambiguous determination of position of unsaturation in most cases. These reactions are simple, use readily available reagents, and require relatively little operator attention. Further, they do not require specialized apparatus, as do hydrogenation and ozonolysis, or potentially toxic chemicals, such as osmium tetroxide. This series of analyses was applied to the unsaturated non-hydroxylated fatty acids of Legionella species, which were shown to be monounsaturated and of the cis Δ9 family. Position of unsaturation was confirmed by gas chromatography-mass spectrometry.     

Relationship between Stereoisomerism and Biological Activity of Pyrethroids

Higher homologous acids of chrysanthemic acid described in the previous papers were esterified with (±)-allethrolone. The toxicity of these esters and the related compounds were evaluated by the topical application method to Musca domestica vicina Macq. The allethronyl homochry-santhemate (entry Nos. 4, 5) was shown to be toxic and the dextrorotatory form was far more toxic than the laevorotatory one. Further elongation of the ester linkage resulted in a loss of toxicity. The cyclobutane carboxylic acid ester (entry No. 10) was shown to be toxic and so, the cyclopropane ring might be replaced to some extent by the cyclobutane ring, provided the other requirements were fulfiled. However, further elongation of the ester linkage also reduced the toxicity. The lactones (entry Nos. 12–16) obtained by the hot sulfuric acid treatment were non-toxic.     

Bacterial assimilation of d- and l-2-chloropropionates and occurrence of a new dehalogenase

The occurrence of a new bacterial dehalogenase acting on both the optical isomers of 2-halogenated alkanoic acids was demonstrated. When the haloalkanoic acid-utilizing bacteria were screened in a medium containing dl-2-chloropropionate as a sole carbon source, two types of bacteria were isolated: (1) a few strains utilizing both d- and l-isomers of 2-chloropropionate and (2) strains utilizing only the l-isomer. A dehalogenating enzyme was obtained from the cells of Pseudomonas sp. which is able to utilize both isomers. The crude enzyme catalyzed the dehalogenation of d- and l-2-chloropropionates to yield l- and d-isomers of lactate, respectively. The enzyme showed the same pH optimum and heat inactivation rate for the d- and l-isomers. Apparent K _m values for d- and l-2-chloropropionates were 4.5 and 1.0 mM, respectively. The enzyme acted specifically on 2-haloalkanoic acids. Activity staining of disc-gels electrophoresed witg the crude enzyme preparation showed that the dehalogenation of d- and l-2-chloropropionates, monochloroacetate, dichloroacetate, 2,2-dichloropropionate, and dl-2-chlorobutyrate is due to a single protein.Abbreviations MCA monochloroacetic acid - DCA dichloroacetic acid - TCA trichloroacetic acid - 2 MCPA 2-monochloropropionic acid - 22 DCPA 2,2-dichloropropionic acid - 3 MCPA 3-monochloropropionic acid - 2 MCBA 2-monochlorobutyric acid - 3 MCBA 3-monochlorobutyric acid - 4 MCBA 4-monochlorobutyric acid     

EFFECT OF FATTY-ACID DOUBLE-BOND POSITION ON THE SELECTIVITY OF RAT-BRAIN ENZYMES: THE INCORPORATION OF OLEIC AND CIS-VACCENIC ACIDS INTO LYSOLECITHIN IN VITRO

Abstract— —Selectivity in the esterification of fatty acids to lysolecithin by rat-brain enzymes in vitro was investigated using free fatty acids (activation plus esterification) and CoA esters (esterification) of two naturally-occurring monoenoic fatty-acid isomers, oleic acid [18:1 (n - 9)] and cis-vaccenic acid [18:1 (n - 7)]. Esterification of free acids to l-acyl-sn-glycero-3-phosphorylcholine (1-acyl GPC) was dependent on CoA and ATP, and was stimulated by MgCl₂ and NaF. Under comparable conditions, fatty-acid activation (acyl-CoA synthetase [acid: CoA ligase (AMP)] EC 6.2.1.3.) appeared to be rate-limiting to 1-acyl GPC acyltransferase (acyl-CoA:l-acylglycero-3-phosphocholine O-acyltrans-ferase, EC 2.3.1.23.), since rates were always less with free fatty acids than with the CoA esters. A comparison of substrate curves obtained with free fatty acids and CoA esters suggests a preference for oleic acid during activation. Acyltransferase activity with 2-acyl GPC was similar with both acyl-CoA isomers, whereas with 1-acyl GPC, activity with oleoyl-CoA consistently exceeded that with cis-vaccenoyl-CoA. This difference between patterns of selectivity in esterification of positions 1 and 2 of lecithin suggests that separate enzymes catalyze the two reactions. The transfer of the isomers to the 2 position was affected in a similar manner by changes in pH and temperature, as well as in protein, fatty acid (or acyl-CoA), and 1-acyl GPC concentrations. Patterns of incorporation with simultaneous incubation of both isomers suggests one enzyme. Differences in acyltransferase activity with the two isomerie acyl-CoA's were observed in subcellular distribution, activity changes with brain maturation, and loss of activity on preincubation of microsomes at 45C. From these results it is not certain whether oleic and cis-vaccenic acids are esterified to the 2 position by separate enzymes, or by one enzyme with different affinities for the isomers. However, the investigation clearly indicates that acyltransferases, and possibly acyl-CoA synthetases in brain possess selectivity related to subtle differences in double-bond position. These selectivities probably are important in determining the specific fatty-acid composition of the complex lipids of brain.     

2-Keto-l-Gulonic Acid Fermentation

Fractionation of sorbitol metabolites in the culture liquid of Gluconobacter melanogenus IFO 3292 was examined by column chromatographic techniques. Ion exchange column chromatography of the culture supernatant allowed to divide the components of the metabolites into Fractions I, II, III and IV. Paperelectrophoretic and paperchromatographic analyses of these fractions revealed that Fractions I, II, III and IV contained neutral sugar, hexonic acids, 5-ketohexonic acid and 2-ketohexonic acids, respectively.

The neutral sugar in Fraction I, the 5-ketohexonic acid in Fraction III and the 2-ketohexonic acids in Fraction IV were isolated and determined to be l-sorbose, 5-keto-d- mannonic, 2-keto-d-gluconic and 2-keto-l-gulonic acids, respectively, from their physical properties. In Fraction II were contained two different hexonic acids, one of which was identified to be l-idonic acid by the aid of substrate specificity of a hexonic acid dehydrogenase of Pseudomonas aeruginosa, and the other was determined to be d-mannonic acid as the phenylhydrazide derivative.     

Quantitative analysis of histidine and cis and trans isomers of urocanic acid by high-performance liquid chromatography: a new assay method and its application

To elucidate the factors involved in dry skin and the skin damage caused by UV light, it is necessary to analyze small amounts of stratum corneum to determine amino acid contents. A new assay method for this purpose is described. Dabsylated amino acids including histidine and the cis and trans isomers of urocanic acid were analyzed quantitatively by high-performance liquid chromatography (HPLC), using a reversed-phase column. Histidine and the isomers of urocanic acid were separated from 36 other amino acids thought to be present in the extract of stratum corneum. In the presence of the 36 amino acids, standard calibration curves were obtained from 0.25 to 2.5 pmol/μl, for histidine and for both isomers of urocanic acid. The coefficients of variation for the reproducibility of the analysis at 1.0 pmol/μl were 3.8%, 2.9% and 2.5% for the cis and trans isomers of urocanic acid and for histidine, respectively. Amounts of 2 to 50 pmol of cis and trans isomers of urocanic acid and histidine in the stratum corneum were detected. The ratio of the cis to the trans isomer of urocanic acid in sunburned stratum corneum was more than three times that in normal stratum corneum. This method appears to be useful for the determination of small amounts of histidine and of the cis and trans isomers of urocanic acid in the stratum corneum.     

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.     

Purification and Properties of Hydroxycinnamic Acid Ester Hydrolase from Aspergillus japonicus

Hydroxycinnamic acid ester hydrolase from the wheat bran culture medium of Aspergillus japonicus was purified 255-fold by ammonium sulfate fractionation, DEAE-Sephadex treatment and column chromatographies on DEAE-Sephadex, CM-Sephadex and various other Sephadexes. The purified enzyme was free from tannase and found to be homogeneous on polyacrylamide disc gel electrophoresis. Its molecular weight was estimated to be 150,000 by gel filtration and 142,000 by SDS-gel electrophoresis. The isoelectric point of the enzyme was pH 4.80. As to its amino acid composition, aspartic acid and glycine were abundant. The optimum pH and temperature for the enzyme reaction were, respectively, 6.5 and 55°C when chlorogenic acid was used as a substrate. The enzyme was stable between pH 3.0 to 7.5 and inactivated completely by heat treatment at 70°C for 10 min.

All metal ions examined did not activate the enzyme, while Hg⁺⁺ reduced its activity. The enzyme was markedly inhibited by diisopropylfluorophosphate and an oxidizing reagent, iodine, although it was not affected so much by metal chelating or reducing reagents. The purified enzyme hydrolyzed not only esters of hydroxycinnamic acids such as chlorogenic acid, caffeoyl tartaric acid and p-coumaroyl tartaric acid, but also ethyl and benzyl esters of cinnamic acid. However, the enzyme did not act on ethyl esters of crotonic acid and acrylic acid or esters of hydroxybenzoic acids.     

Analysis of problems encountered in the determination of amino acid enantiomeric ratios by gas chromatography

A previously described procedure for determining the enantiomeric ratios of amino acids has produced inconsistent results when determining relatively low (≤0.110) d/l ratios. The method involves synthesis of diastereomeric N-trifluoroacetyl-l-prolyl-d/l-amino acid ester dipeptides which are resolved by gas chromatography (GC). We have found that triethylamine, which is added to maintain a basic pH during the coupling reaction, racemizes the chiral reagent N-trifluoroacetyl-l-prolyl chloride (TPC). Coupling of partially racemized TPC to d/l-amino acid esters results in the formation of four dipeptides (two pairs of enantiomers) instead of the expected two diastereomeric dipeptides. The enantiomeric dipeptides coelute on an achiral GC column, resulting in erroneous d/l ratios. More accurate d/l ratios are obtained by preparing the volatile N-trifluoroacetyl-d/l-amino acid isopropyl ester derivative which can be separated into its enantiomers on a chiral GC column such as the Chirasil-Val III (registered trademark of Applied Science Laboratories).     

Purification of fatty acid methyl esters by high-performance liquid chromatography

The determination by gas chromatography (GC) of fatty acid methyl esters (FAMEs) prepared from complex biological samples is subject to interference from cholesterol. During sample injection on the GC system of FAMEs prepared from tissues that contain cholesterol, we observed a major contaminant that co-eluted with docosahexaenoic acid (DHA, 22:6n-3). To address this problem, FAMEs were purified on an amino-phase high-performance liquid chromatography (HPLC) column using a hexane–isopropanol gradient. The HPLC retention times for both the FAME fraction and cholesterol were stable and reproducible when the amino column was used for sample purification. The purified extracts were analyzed by GC without artifacts or impurity peaks after 50 analytical runs. The method described here will be useful for measurement of 22:6n-3 and other fatty acids important for studies of nutrition or pathology.     

Parameters affecting productivity in the lipase-catalysed synthesis of sucrose palmitate

The industrial application of lipases for the synthesis of sucrose esters is usually limited by its low productivity, as we need a medium where a polar reagent (the sugar) and a non-polar fatty acid donor are soluble and able to react in the presence of the biocatalyst. In this work, we have studied the problems encountered when trying to increase the volumetric productivity of sucrose esters. The synthesis of sucrose palmitate was performed in 2-methyl-2-butanol:dimethylsulfoxide mixtures by transesterification of different palmitic acid donors with sucrose, catalysed by the immobilized lipase from Candida antarctica B (Novozym 435). A protocol for substrate preparation different from that previously reported was found to improve the reaction rate. Several parameters, such as sucrose and acyl donor loadings, the percentage of DMSO in the mixture and the nature of acyl donor, were investigated. Under the best experimental conditions (15% DMSO, 0.1 mol l^?1 sucrose, 0.3 mol l^?1 vinyl palmitate), a maximum of 45 g l^?1 sucrose palmitate was obtained in 120 h. Using methyl or ethyl palmitate, the highest productivity was 7.3 g l^?1 in 120 h using 20% DMSO with 0.2 mol l^?1 sucrose and 0.6 mol l^?1 acyl donor. The formation of free fatty acid, and the effect of the percentage of DMSO on the monoester/diester selectivity were also studied. To our knowledge, this is the first report on enzymatic synthesis of sucrose esters of long fatty acids using alkyl esters as acyl donors.