Substrate specificity of acetylxylan esterase from Schizophyllum commune: mode of action on acetylated carbohydrates.

Substrate specificity of a purified acetylxylan esterase from Schizophyllum commune was investigated on a variety of methyl per-O-acetyl glycopyranosides, methyl di-O-acetyl-beta-D-xylopyranosides and acetylated polysaccharides. The enzyme preferentially deacetylated the 3-position of methyl 2,3,4-tri-O-acetyl-beta-D-xylopyranoside and 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranoside. Removal of the 3-acetyl group from the xylopyranoside was accompanied by a slower deacetylation at positions 2 and 4. A similarly slower, accompanying deacetylation occurred primarily at position 2 with the glucopyranoside. Such specificity corresponds well to the expected function of the esterase in acetylxylan degradation. Of the three possible diacetates of methyl beta-D-xylopyranoside, the 3,4-diacetate was found to be the most rapidly deacetylated. Unexpectedly, products of its deacetylation were a mixture of 2- and 4-monoacetate. The formation of the methyl 2-O-acetyl-beta-D-xylopyranoside involved an enzyme-mediated acetyl group transfer because the rate of the enzyme-catalyzed reaction exceeded the rate of spontaneous migration of acetyl groups. This is the likely mechanism for acetyl removal from position 2 in the native substrate. The enzyme exhibited the highest regioselectivity with methyl 2,3,4,6-tetra-O-acetyl-beta-D-mannopyranoside. An 80% conversion of this substrate to methyl 4,6-di-O-acetyl-beta-D-mannopyranoside, a new mannose derivative, was achieved. In contrast to the majority of lipases and esterases exploited for regioselective deacetylation, the S. commune acetylxylan esterase did not attack the C-6 acetyl linkages in methyl hexopyranosides when other acetyl groups were available.

Synthesis of cephalotaxine esters and correlation of their structures with antitumor activity.

Twenty-two new esters of natural (-)-cephalotaxine with synthetic acids possessing widely divergent structural features have been synthesized. Murinichloroethyl carbonate (27) esters of cephalotaxine are the most active of this group; this activity is less than that of harringtonine and other naturally occurring cephalotaxine esters. Other synthetic esters exhibiting activity are methyl cephalotaxylfumarate (4) and the trichloroethyl carbonate of cephalotaxyl-L-mandelate (21). The specificity of this experimental tumor system apparently requires esters of (-)-cephalotaxine for tumor inhibition because methyl cephalotaxylitaconate (7b) prepared from the synthetic (+) enantiomer of cephalotaxine is inactive.

Cyclic fatty esters: synthesis, characterization, and lipolysis of isomeric triglycerides of 9-(6-propyl-3-cyclohexenyl)-(Z)8-nonenoic acid.

Triglycerides of a model cyclic fatty acid (CFA) 9-(6-propyl-3-cyclohexenyl)-(Z)8-nonenoic acid (Ia) were synthesized for biological and toxicity evaluations. The monoacid triglyceride II (CyCyCy) was interesterified with triolein (OOO) to obtain mixtures of diacid triglycerides: III (OOCy), IV (OCyO), V (OCyCy), and VI (CyOCy). The interesterification mixtures were separated by preparative HPLC into two 'critical pairs' of isomeric triglycerides. Triglycerides III-VI were synthesized and a 13C-NMR method was developed to estimate 'critical pairs'. CFA-triglycerides were characterized by IR, NMR, HPLC and capillary GLC, and their relative rates of hydrolysis by lipase were compared. Although tricyclin (II) was completely resistant to lipolysis, triglycerides III and VI hydrolyzed significantly slower than triolein, and the 'critical pairs' hydrolyzed as readily as triolein. Therefore, partial CFA-triglycerides formed in heat-abused fats can undergo lipolysis and likely be absorbed like normal dietary fats.

The structure of the acidic exopolysaccharide produced by Pseudomonas "gingeri" strain Pf9.

The structure of the acidic exopolysaccharide produced by the mushroom pathogen Pseudomonas "gingeri" strain Pf9, a bacterium which causes ginger blotch, was investigated by chemical analysis, mass spectrometry and 1D and 2D NMR spectroscopy. The polysaccharide consists of the linear trisaccharide repeating unit [formula: see text] where the cyclic pyruvic acetal groups at O-4 and O-6 of the mannopyranosyl residues have the S-configuration. Methylation analysis under neutral conditions and NMR data showed that the mannose residues are acetylated at O-2. This exopolysaccharide has the same structure as the E. coli K55 capsular polysaccharide and differs from the Klebsiella K5 capsular polysaccharide only in the position of acetylation (C-2 of the glucopyranose residue).

Reduction of Carpophilus freemani dobson (Coleoptera: nitidulidae) aggregation pheromone response by synthetic analogues.

Analogues of (2E,4E,6E)-5-ethyl-3-methyl-2,4,6-nonatriene, the major component of the aggregation pheromone of Carpophilus freemani Dobson (Coleoptera: Nitidulidae), were synthesized and the potency of these compounds in suppressing the response of C. freemani to its pheromone in a wind tunnel bioassay was determined. The most potent compounds reduced behavioral response to pheromone 83-96% when the inhibitors were present in 10-fold excess. These compounds are (1Z, 3E,5E)-1-methoxy-3-ethyl-5-methyl-1,3,5-heptatriene, (1E,3E, 5E)-1-cyclopropyl-3-ethyl-5-methyl-1,3,5-heptatriene, and (1Z,3E, 5E)-1-cyclopropyl-3-ethyl-5-methyl-1,3,5-heptatriene. In the presence of fermenting bread dough (a pheromone synergist), the most potent inhibitory compound, (1Z,3E, 5E)-1-cyclopropyl-3-ethyl-5-methyl-1,3,5-heptatriene, was less effective in reducing mean landings (69% vs 99%) than when dough was absent. This inhibitory compound causes a reduction of response to pheromone but does not cause a reduction of response to fermenting food-type volatiles such as fermenting bread dough. Analogues of pheromones that strongly reduce response to pheromones by insects might be useful as biochemical probes to study the pharmacophoric (three-dimensional structure) requirements for pheromone perception.

Addition of N-acetylcysteine to linoleic acid hydroperoxide.

Catalyzed by 10(-5)M ionic iron in 80% ethanol, N-acetylcysteine added to linoleic acid hydroperoxide, forming a thiobond. Reaction of a specific isomer of hydroperoxide, 13-hydroperoxy-trans-11, cis-9-octadecadienoic acid, and N-acetylcysteine, forms a number of products, of which two were identified as addition compounds. One addition product was 9-S-(N-acetylcysteine)-13-hydroxy-10-ethoxy-trans-11-octadecenoic acid, and the other was 9-S-(N-acetylcysteine)-10, 13-dihydroxy-trans-11-octadecenoic acid.

Synthesis and characterization of triacylglycerols containing linoleate and linolenate.

Triacylglycerols containing linoleate and linolenate found in vegetable oils were synthesized in gram quantities for oxidation studies. Two acylation methods were examined to convert diacylglycerols or monoacylglycerols to the desired triacylglycerols. Acylations with fatty acid and 1,1'-dicyclohexylcarbodiimide in the presence of 4-dimethylaminopyridine were more rapid, gave triacylglycerols of better isomeric purities and generally better overall yields than the acylations with acid chloride in pyridine. The functional and isomeric purity of the synthetic triacylglycerols were investigated by thin-layer and gas-liquid chromatography of the methyl esters, by lipase hydrolysis, and by(13)C NMR. Quantitative(13)C NMR provided a valuable tool to determine isomeric structures of the unsaturated triacylglycerols and complemented the lipase hydrolysis method. The triacylglycerols purified by dry column chromatography were obtained in the following respective percent yields, functional and isomeric purities: LLLn, 92.1, 99.4, 98.1; LLnLn, 91.7, 99.2, 97.4; LLnL, 84.2, 99.7, 98.9; and LnLLn 77.5, 97.8, 99.0 (where L=linoleoyl and Ln=linolenoyl glycerol residues). These synthetic triacylglycerols are valuable models to elucidate the interrelationship of unsaturated fatty acids on the oxidative stability of polyunraturated vegetable oils.

Cyclic fatty esters: hydroperoxides from autoxidation of methyl 9-(6-propyl-3-cyclohexenyl)-(Z)8-nonenoate.

Autoxidation of the cyclic fatty acid ester, methyl 9-(6-propyl-3-cyclohexenyl)-(Z)8-nonenoate (I) was investigated to characterize the hydroperoxide isomers formed and to provide basic information on their chemistry, detection and effect on the quality of polyunsaturated cooking oils. Oxidation at 60 C with 1% hydroperoxide initiator produced a monohydroperoxide fraction containing five positional isomers (7-, 11-, 12-, 13- and 14-OOH), resolved by high performance liquid chromatography, as their allylic hydroxy esters. Their structures were established by 1H- and 13C-NMR spectroscopy and by capillary gas chromatography-mass spectrometry (GC-MS) as trimethylsilyl ether derivatives. Two additional isomers (8- and 9-OOH) were detected by GC-MS in only trace (less than 1%) quantities. Capillary GC resolved some geometric and stereoisomers, as well as positional isomers. Compared to photosensitized oxidation, two additional positional isomers (11- and 14-OOH) were produced by autoxidation. More stereoisomers were formed, and oxidation of the ring double bond was favored 8:1 over that of the side chain. This selectivity may be attributed to greater steric hindrance for oxygen attack at the side-chain double bond. A free radical mechanism is proposed to explain the greater isomeric complexity of the hydroperoxide products compared to photosensitized oxidation.

Positional specificity of gamma-ketol formation from linoleic acid hydroperoxides by a corn germ enzyme.

We have shown unequivocally that the positional specificity of gamma-ketol formation by a corn germ enzyme was different from that observed previously by others with an alfalfa seedling enzyme. When the pure positional isomers of linoleic acid hydroperoxide served as substrates, the corn germ enzyme formed one of two gamma-ketols: 12-oxo-9-hydroxy-trans-10-octadeconoic acid from 13-hydroperoxys-10-octadecenoic acid from 13-hydroperoxy-cis-9,trans-11-octadecadienoic acid (99+% pure) and 10-oxo-13-hydroxy-trans-11-octadecenoic acid from 9-hydroperoxy-trans-10,cis-12-octadecadienoic acid (96% pure). Also isolated from these reactions was one of two alpha-ketols commonly found as a result of catalysis by linoleic acid hydroperoxide isomerase: 12-oxo-13-hydroxy-cis-9-octadecenoic acid from the 13-hydroperoxide and 10-oxo-9-hydroxy-cis-12-octadecenoic acid from the 9-hydroperoxide. Evidence is offered that gamma-ketol formation is catalyzed by linoleic acid hydroperoxide isomerase, the same enzyme responsible for alpha-ketol production.

Biotransformation of linoleic acid by Clavibacter sp. ALA2: heterocyclic and heterobicyclic fatty acids.

Clavibacter sp. ALA2 transformed linoleic acid into a variety of oxylipins. In previous work, three novel fatty acids were identified, (9Z)-12, 13, 17-trihydroxy-9-octadecenoic acid and two tetrahydrofuran-(di)hydroxy fatty acids. In this report, we confirm the structures of the tetrahydrofuran-(di)hydroxy fatty acids by nuclear magnetic resonance as (9Z)-12-hydroxy-13,16-epoxy-9-octadecenoic acid and (9Z)-7,12-dihydroxy-13,16-epoxy-9-octadecenoic acid. Three other products of the biotransformation were identified as novel heterobicyclic fatty acids, (9Z)-12,17;13, 17-diepoxy-9-octadecenoic acid, (9Z)-7-hydroxy-12,17;13,17-diepoxy-9-octadecenoic acid, and (9Z)-12,17;13,17-diepoxy-16-hydroxy-9-octadecenoic acid. Thus, Clavibacter ALA2 effectively oxidized linoleic acid at C-7, -12, -13, -16, and/or -17.

Cysteine adds to liquid hydroperoxide.

Cysteine reacts with linoleic acid hydroperoxide to yield several products, some of which were identified as fatty acid-cysteine adducts. The addition was catalyzed by ferric chloride (10(-5) M) by initiating free radical reactions. When isomerically pure 13-hydroperoxy-cis-9,trans-11-octadecadienoic acid and cysteine were reacted in 80% ethanol under N2, the major adducts were 9-S-cysteine-13-hydroxy-10-ethoxy-trans-11-octadecenoic acid (I) and 9-S-cysteine-10,13-dihydroxy-trans-11-octadecenoic acid (II). When the reaction included both isomers of the hydroperoxide (13- and 9-hydroperoxide) and air, an adduct of 9-oxononanoic acid and cysteine also was isolated. Additional experiments gave information about possible mechanisms of I and II formation.

Photosensitized oxidation of methyl linoleate: Secondary and volatile thermal decomposition products.

Studies of photosensitized oxidation of methyl linoleate show that the greater relative concentration of 9- and 13-hydroperoxides than 10- and 12-hydroperoxides is characteristic of singlet oxygenation and not due to either simultaneous autoxidation or type 1 photosensitized oxidation. Cyclization of the internal 10- and 12-hydroperoxides accounts for their lower relative concentrations. Secondary products separated by silicic acid and high pressure liquid chromatography were characterized spectrally (IR, UV,(1)H-NMR,(13)C-NMR, GC-MS). Major secondary products included diastereomeric pairs of 13-hydroperoxy-10,12-epidioxy-trans-8-octadecenote (I and III) and 9-hydroperoxy-10,12-epidioxy-trans-13-octadecenoate (II and IV); minor secondary products included hydroperoxy oxy genated and epoxy esters. Thermal decomposition of the hydroperoxy cyclic peroxides produced hexanal and methyl 10-oxo-8-decenoate as major volatiles from I and III and methyl 9-oxo-nonanoate and 2-heptenal from II and IV. Hydroperoxy cyclic peroxides may be important sources of volatile decomposition products of photooxidized fats.

Effects of bis homoallylic and homoallylic hydroxyl substitution on the olefinic 13C resonance shifts in fatty acid methyl esters.

Substitution of a hydroxyl group at the bis homoallylic position (OH group located three carbons away from the olefinic carbon) in C18 unsaturated fatty acid esters (FAE) induces a 0.73 +/- 0.05 ppm upfield and a 0.73 +/- 0.06 ppm downfield shift on the delta and epsilon olefinic 13C resonances relative to the unsubstituted FAE, respectively. If the hydroxyl group is located on the carboxyl side of the double bond of the bis homoallylic hydroxy fatty acid esters (BHAHFA), the olefinic resonances are uniformly shifted apart by [formula: see text] where delta delta dbu represents the absolute value of the double bond resonance separation in the unsubstituted FAE and 1.46 ppm is the sum of the absolute values of the delta and epsilon shift parameters. With hydroxyl substitution on the terminal methyl side of the double bond, the olefinic shift separation is equal to [formula: see text] In homoallylic (OH group located two carbons away from the olefinic carbon) substituted FAE the gamma and delta induced hydroxyl shifts for the cis double bond resonances are +3.08 and -4.63 ppm, respectively while the trans double bond parameters are +4.06 and -4.18 ppm, respectively. The double bond resonance separation in homoallylic hydroxy fatty acid esters (HAHFA) can be calculated from the formula [formula: see text] for cis and [formula: see text] for the trans case when the OH substitution is on the carboxyl side of the double bond. Conversely, when the OH resides on the terminal methyl side, the double bond shift separations for cis and trans isomers are [formula: see text] and [formula: see text] respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Analytical determination of fumonisins and other metabolites produced by Fusarium moniliforme and related species on corn.

Fumonisins, secondary metabolites of the fungus Fusarium moniliforme are potent toxins that can be found in fungal contaminated corn. The detection and measurement of these toxins by HPLC with detection by an evaporative light scattering detector and by electrospray MS is reported. The light scattering detector had enough sensitivity to analyze culture materials, however, clean-up was necessary to detect fumonisins at sub-ppm levels in naturally contaminated corn extracts. The detection limit for FB1 with the light scattering detector was in the low ng range (10-50) while the detection limit of less than 1 ng injected was observed for the electrospray detector. Several previously unreported fumonisin isomers were observed in electrospray chromatograms of culture extracts. Two of these compounds, FA3 and FA4 were isolated and their proposed structure confirmed by NMR experiments.

4-O-(1-carboxyethyl)-L-rhamnose, a second unique acidic sugar found in an extracellular polysaccharide from Butyrivibrio fibrisolvens strain 49.

Butyrivibrio fibrisolvens strain 49 excretes a polysaccharide that contains D-glucose, D-galactose, 4-O-(1-carboxyethyl)-D-galactose, and an acidic component of previously unknown structure. We report here the identity of the unknown as 4-O-(1-carboxyethyl)-L-rhamnose. The structure of this previously unknown compound was deduced from (1) comprehensive electron-impact and chemical-ionization mass-spectroscopic studies of differentially labelled derivatives prepared from the unknown, (2) 13C-n.m.r. and 1H-n.m.r. studies of purified neutral sugars derived from the unknown and (3) chemical degradation experiments.