Synthesis of cis-12, 13-epoxy-cis-9-octadecenol and 12(13)-hydroxy-cis-9-octadecenol from vernonia oil using lithium aluminum hydride

Reduction of vernonia oil methyl esters (VOME) into epoxy fatty alcohol and diols was achieved with lithium aluminum hydride (LAH), under reflux and room temperature conditions, by using hexane and tetrahydrofuran (THF) as solvents. The reactions of VOME with LAH in hexane produced cis-12,13-epoxy-cis-9-octadecenol as a major product with an isolated yield of 73.6%, whereas the reaction with LAH in THF gave isomers of 12(13)-hydroxy-cis-9-octadecenol as the major products with an isolated yield of 95.1%. LAH was similarly reacted with vernonia oil (VO) to give the same products in lower yields. ¹H nuclear magnetic resonance (NMR), ¹³C NMR, gas chromatography-mass spectrometry, and infrared were used to characterize these products. This study demonstrates the ability to control the reactivity of the epoxy functionality in VO or VOME with the choice of polar or nonpolar solvents, and extends the range of oleochemicals that can be derived from vernonia oil.     

Survey of the fatty acid composition of peanut (arachis hypogaea) germplasm and characterization of their epoxy and eicosenoic acids

Peanut (Arachis hypogaea) plant introductions (732) were analyzed for fatty acid composition. Palmitate varied from 8.2 to 15.1%, stearate 1.1 to 7.2%, oleate 31.5 to 60.2%, linoleate 19.9 to 45.4%, arachidate 0.8 to 3.2%, eicosenoate 0.6 to 2.6%, behenate 1.8 to 5.4%, and lignocerate 0.5 to 2.5%. The eicosenoate was shown to be cis-11-eicosenoate. In addition, epoxy fatty acids were found in many plant introductions in percentages ranging as high as 2.5%. These were tentatively identified as chiefly 9,10-epoxy stearate and coronarate with smaller amounts of vernolate. The percentage of palmitate was shown to be correlated positively with linoleate and negatively with oleate, eicosenoate, and lignocerate. Stearate was highly correlated with arachidate and negatively with eicosenoate and lignocerate. Oleate and linoleate, the two major fatty acids, were negatively correlated. Arachidate was negatively correlated with eicosenoate, and eicosenoate was positively correlated with lignocerate. Behenate and lignocerate were positively correlated. Epoxy esters were positively correlated with palmitate and negatively with oleate. Segregation of the plant introductions by axis flower, growth habit, and pod types showed significant differences that reflected the same fatty acid groupings revealed by the correlations.     

Cryptolepis buchnani seed oil: A rich source of keto fatty acid

A keto fatty acid (9-oxo-cis-12-octadecenoic acid) has been isolated in appreciable amounts (45.9%) fromCryptolepis buchnani seed oil. The identification was based on chemical and spectroscopic methods.     

A minor source of vernolic,malvalic, and sterculic acids inPithecollobium dulce (syn.Inga dulcis) seed oil

Pithecollobium dulce, Benth (syn.Inga dulcis, Willd) seed oil, belonging to the Leguminosae plant family, contains minor amounts of vernolic acid (12,13-epoxy-octadec-cis-9-enoic acid, 10.0%), malvalic acid [7-(2-octacyclopropen-1-yl)heptanoic acid, 3.2%], and sterculic acid [8-(2-octacyclopropen-1-yl)octanoic acid, 2.0%]. The other normal fatty acids are palmitic (12.1%), stearic (4.2%), behenic (10.6%), oleic (34.1%), and linoleic (23.8%). These fatty acids have been characterized by Fourier transform infrared,¹H nuclear magnetic resonance, mass spectrometry and gas-liquid chromatography techniques and by chemical degradations.     

Epoxy oleic acid inQuamoclit seed oils

Quamoclit phoenicea Choisy andQuamoclit coccinea Moench. (Syn.Ipomoea coccinea Linn), belonging to the Convolvulaceae plant family, was found to contain palmitic (22.2%, 33.3%), stearic (11.3%, 1.7%) oleic (13.5%, 14.6%), linoleic (40.1%, 30.8%), vernolic (6.4%, 10.2%), arachidic (3.5%, 6.8%) and behenic (3.8%, 2.6%) acids, respectively.     

Estolides from meadowfoam oil fatty acids and other monounsaturated fatty acids

The formation of estolides was detected during the studies on dimerization of meadowfoam oil fatty acids. By adjusting the reaction conditions, it was possible to produce monoestolides with little dimer or trimer formations. Estolides have potential use in lubricant, cosmetic and ink formulations and in plasticizers. This paper reports the conditions for production of estolides from mixed meadow-foam fatty acids, commercial oleic acid, high-oleic sun-flower oil fatty acids,cis-5,cis-13-docosadienoic acid, petroselinic acid and linoleic acid.     

Syntheses of pure (9Z, 11Z), (9E, 11E), (9E, 11Z), and (9Z,11E)-9,11-hexadecadienals-possible candidate pheromones

The title compounds were prepared by six different routes, and recommendations are given for the more convenient procedures in laboratory-scale syntheses. Modifications in the literature preparations of the 9E,11E and 9E,11Z isomers are described. Baseline separation of a prepared mixture of all four isomers of the (9Z, 11Z), (9E, 11E), (9E, 11Z), and (9Z, 11E)-9,11-hexadecadienals was achieved using GC methods with standard capillary columns. [¹³C]NMR spectroscopy of the alkene carbon atoms clearly differentiates between theZ,Z, E,E and eitherE,Z orZ,E isomers of the precursor dienols and thus of the dienals.     

Ricinoleic Acid in Common Vegetable Oils and Oil Seeds

An original gas chromatography/mass spectrometry method for quantifying trace amounts of ricinoleic acid (12-hydroxy-cis-9-octadecenoic acid) is detailed. Data are presented on trace amounts of ricinoleic acid found in several common vegetable oils and oils extracted from common oil seeds: e.g., ca. 30 ppm in commercial olive oil was the lowest amount; and ca. 2,690 ppm in oil extracted from cottonseeds was the highest amount.     

The seed oil ofBernardia pulchella (Euphorbiaceae) —A rich source of vernolic acid

The fatty acids from the seed oil ofBernardia pulchella (Euphorbiaceae) have been analyzed by gas chromatography (GC) and GC-mass spectrometry (MS) analysis of their methyl esters. Vernolic acid is the main compound (91%), along with other usual fatty acids. In addition to the quantitation by GC analysis,¹H-nuclear magnetic resonance (NMR) signals from the seed oil have been used to estimate the total epoxy fatty acid content. The structure of vernolic acid has been proven by spectroscopic methods (infrared,¹H, and¹³C-NMR) and by GC-MS analysis of the corresponding silylated hydroxy-methoxy derivative. The 4,4-dimethyloxazoline derivatives of the fatty acid mixture have also been examined by GC-MS, and it was shown that this derivazation reaction is not suitable for the structure analysis of vernolic acid.     

Biosynthesis of tetrahydrofuranyl fatty acids from linoleic acid by <Emphasis Type="Italic">clavibacter</Emphasis> sp. ALA2

Clavibacter sp. ALA2 converts linoleic acid into many novel oxygenated products including hydroxy FA and tetrahydrofuranyl unsaturated FA (THFA). One of them was tentatively identified by GC-MS as 12,13,16-trihydroxy-9(Z)-octadecenoic acid (12,13,16-THOA) (Hou, C.T., H.W. Gardner, and W. Brown, J Am. Oil Chem. Soc. 78∶1167–1169, 2001). We have separated and purified 12,13,16-THOA from its isomer, 12,13,17-THOA, by silica gel column chromatography and by preparative TLC. Its structure was then confirmed by proton and ¹³C NMR analyses. Purified 12,13,16-THOA was used as a substrate to study the biosynthesis of THFA. Within 24 h of incubation, cells of strain ALA2 converted 12,13,16-THOA to both 12-hydroxy-13,16-epoxy-9(Z)-octadecenoic acid (12-hydroxy-THFA) and 7,12-dihydroxy-13,16-epoxy-9(Z)-octadecenoic acid (7,12-dihydroxy-THFA). The relative abundance of 7,12-dihydroxy-THFA increased with incubation time, whereas that of 12,13,16-THOA and of 12-hydroxy-THFA decreased. Therefore, the biosynthetic pathway of THFA from linoleic acid by strain ALA2 is as follows: linoleic acid→12,13-dihydroxy-9(Z)-octadecenoic acid→12,13,16-THOA→12-hydroxy-THEA→7,12-dihydroxy-THFA.     

Vernonia galamensis: A rich source of epoxy acid

A percolation extraction ofVernonia galamensis seed, affording 38.6% of crude vernonia oil is described. The dark colored crude oil was degummed with water, treated with activated charcoal and bleached with a neutral agent, to give a light colored oil (Lovibond: 0.9 red, 3.5 yellow). Gas chromatographic/mass spectrometric analysis of the refined oil indicates a relative fatty acid composition of 79–81% vernolic (cis-12,13-epoxy-cis-9-octadecenoic) acid, 11–12% linoleic acid, 4–6% oleic acid, 2–3% stearic acid, 2–4% palmitic acid, and a trace amount of arachidic acid.     

Monooxygenase system of Bacillus megaterium ALA2: Studies on linoleic acid epoxidation products

Bacillus megaterium ALA2 produces many oxygenated FA from linoleic acid: 12,13-dihydroxy-9(Z)-octadecenoic acid; 12,13,17-trihydroxy-9(Z)-octadecenoic acid; 12,13,16-trihydroxy-9(Z)-octadecenoic acid; 12-hydroxy-13,16-epoxy-9 (Z)-octadecenoic acid; and 12,17;13,17-diepoxy-16-hydroxy-9 (Z)-octadecenoic acid. Recently, we studied the monooxygenase system of B. megaterium ALA2 by comparing its palmitic acid oxidation products with those of the well-studied catalytically self-sufficient P450 monooxygenase of B. megaterium ATCC 14581 (NRRL B-3712) and of B. subtilis strain 168 (NRRI B-4219). We found that their oxidation products are identical, indicating that their monooxygenase systems (hydroxylation) are similar. Now, we report that strain ALA2 epoxidizes linoleic acid to 12,13-epoxy-9(Z)-octadecenoic acid and 9,10-epoxy-12 (Z)-octadecenoic acid, the initial products in the linoleic acid oxidation. The epoxidation enzyme did not oxidize specific double bond of the linoleic acid. The epoxidation activity of strain ALA2 was compared with the above-mentioned two Bacillus strains. These two Bacillus strain also produced 12,13-expoxy-9 (Z)-octadecenoic acid and 9,10-epoxy-12(Z)-octadecenoic acid, indicating that their epoxidation enzyme systems might be similar. The ratios of epoxy FA production by these three strains (A1 A2, NRRI B-3712, and NRRI B-4219) were, respectively, 5.56∶0.66∶0.18 for 12,13-epoxy-9(Z)-octadecenoic acid and 2.43∶0.41∶0.57 for 9,10-epoxy-12(Z)-octadecenoic acid per 50 mL medium per 48 h.     

Conversion of linoleic acid to 10-hydroxy-12(Z)-octadecenoic acid byFlavobacterium sp. (NRRL B-14859)

A new microbial isolate,Flavobacterium sp. strain DS5, converts linoleic acid into 10-hydroxy-12(Z)-octadecenoic acid (10-HOA) with 55% yield. The product was characterized by gas chromatography (GC), GC/mass spectrometry, nuclear magnetic resonance and Fourier transform infrared spectroscopy. The specific optical rotation of 10-HOA is [α] _D ²⁴ =−5.58 (methanol). The optimum time, pH and temperature for the production of 10-HOA were 36h, 7.5 and 20–35°C, respectively. The enzyme(s) that converts linoleic acid to 10-HOA is soluble and located intracellulary in strain DS5. Two minor products, 10-methoxy-12-octade-cenoic acid and 10-keto-12-octadecenoic acid, were also identified. 10-HOA was further metabolized by strain DS5. Among the unsaturated fatty acids studied, the order of reactivity for the DS5 enzyme(s) is oleic>palmitoleic> linoleic>linolenic>γ-linolenic>myristoleic acid.     

Biotransformation of 12-hydroxyoctadecanoic acid to 12-hydroxyoctadecanamide by Bacillus cereus 50

A new microbial isolate (Bacillus cereus 50) transformed 12-hydroxyoctadecanoic acid to 12-hydroxyoctadecanamide when grown aerobically in 1% yeast extract medium at 30°C, shaken at 250 rpm for 2 to 5 d. The compound was purified by thin-layer chromatography and characterized by infrared, gas chromatography, mass spectrometry, and nuclear magnetic resonance. The yields of 12-hydroxyoctadecanamide were 9.1 and 21.5% after 2 and 5 d, respectively.     

A novel compound, 12,13,17-trihydroxy-9(Z)-Octadecenoic acid,from linoleic acid by a new microbial isolateClavibacter sp. ALA2

A novel compound, 12,13,17-trihydroxy-9(Z)-octadecenoic acid (THOA), was produced from linoleic acid by microbial transformation at 25% yield. The newly isolated microbial strain that catalyzed this transformation was identified asClavibacter sp. ALA2. The product was purified by high-pressure liquid chromatography, and its structure was determined by¹H and¹³C nuclear magnetic resonance, Fourier transform infrared, and mass spectroscopy. Maximum production of THOA was reached after 85 h of reaction. THOA was not further metabolized by strain ALA2. This is the first report on 12,13,17-trihydroxy unsaturated fatty acid and its production by microbial transformation.     

Microbial transformation of 12-hydroxyoctadecanoic acid to 5-n-hexyl-tetrahydrofuran-2-acetic acid

Stationary-phase cells of a corynebacterium (FUI-2) and a bacillus (NRRL B-14864) isolate, when grown aerobically in 1% YE medium at 25°C, converted 12-hydroxystearic acid to a major compound, 5-n-hexyl-tetrahydrofuran-2-acetic acid, and other intermediate and minor compounds (6-hydroxydodecanoic acid, 4-hydroxydecanoic acid, 4-ketodecanoic acid and γ-decanolactone). The yields of 5-n-hexyl-tetrahydrofuran-2-acetic acid, 4-hydroxydecanoic acid, and γ-decanolactone, byBacillus lentus NRRL B-14864 were 43%, 18% and 5%, respectively, after 2.5 d of incubation. Presented in part at the 3rd Annual Student Research Conference, Board of Governors Universities, University Park, Illinois, April 1992; in part at the 92nd American Society for Microbiology General Meeting, New Orleans, Louisiana, May 1992; and in part at the 35th West Central States Biochemistry Conference Annual Meeting, Manhattan, Kansas, October 1992.     

Fatty acid composition of some pine seed oils

The fatty acid composition of seeds from seven species of the genusPinus (P. pinaster, P. griffithii, P. pinea, P. koraiensis, P. sylvestris, P. mughus, andP. nigra) was established. Pine seeds are rich in oil (31–68% by weight) and contain several unusual polymethylene-interrupted unsaturated fatty acids with acis-5 ethylenic bond. These are thecis-5,cis-9 18:2,cis-5,cis-9,cis-12 18:3,cis-5,cis-11 20:2, andcis-5,cis-11,cis-14 20:3 acids, with a trace ofcis-5,cis-9,cis-12,cis-15 18:4 acid. Their percentage relative to total fatty acids varies from a low of 3.1% (P. pinea) to a high of 30.3% (P. sylvestris), depending on the species. The majorcis-5 double bond-containing acid is generally thecis-5,cis-9,cis-12 18:3 acid (pinolenic acid). In all species, linoleic acid represents approximately one-half the total fatty acids, whereas the content of oleic acid varies in the range 14–36% inversely to the sum of fatty acids containing acis-5 ethylenic bond. The easily available seeds fromP. koraiensis appear to be a good source of pinolenic acid: their oil content isca. 65%, and pinolenic represents about 15% of total fatty acids. These values appear to be rather constant.Pinus pinaster, which is grown on several thousand acres in the southwest of France, is an interesting source ofcis-5,cis-11,cis-14 20:3 acid (7% in the oil, which isca. 35% of the dehulled seed weight), an acid sharing in common three double bonds with arachidonic acid. Apparently,P. sylvestris seed oil contains the highest level ofcis-5 double bond-containing acids among pine seed oils that have ever been analyzed.     

Production of 14-Oxo-<Emphasis Type="Italic">cis</Emphasis>-11-eicosenoic Acid from Lesquerolic Acid by <Emphasis Type="Italic">Sphingobacterium multivorum</Emphasis> NRRL B-23212

The objective of this study was to explore the extent of microbial conversion of lesquerolic acid (14-hydroxy-cis-11-eicosenoic acid; LQA) by whole cell catalysis and to identify the newly converted products. Among compost isolates including NRRL strains B-23212 (Sphingobacterium multivorum), B-23213 (Acinetobacter sp.), B-23257 (Enterobacter cloacae B), B-23259 (Escherichia sp.) and B-23260 (Pseudomonas aeruginosa) the S. multivorum strain was the only microorganism that converted LQA to produce a new product identified as 14-oxo-cis-11-eicosenoic acid by GC-MS and NMR analyses. The conversion yield was 47.4% in 48 h at 200 rpm and 28°C in small shake flask experiments. In comparison, both Acinetobacter and Pseudomonas strains failed to convert LQA to major new products but used LQA apparently as an energy source during fermentation. For structural analysis, 6.88 g of 14-oxo-cis-11-eicosenoic acid was produced from converting 11 g LQA (a 62% yield) in 72 h at 200 rpm and 28 °C in Fernbach flasks using 18-h-old NRRL B-23212 cultures and an improved medium that also contained EDTA and glycerol in lieu of glucose as carbon source. NRRL B-23212 was further identified by 16S rRNA gene sequence analysis as a unique strain of S. multivorum. Therefore, S. multivorum NRRL B-23212 possesses an enzymatic activity presumably a secondary alcohol dehydrogenase for converting LQA to produce 14-oxo-cis-11-eicosenoic acid, a first report that demonstrates the functional modification of LQA by whole cell catalysis.     

Enantiomers of (Z,Z)-6,9-Heneicosadien-11-OL: Sex Pheromone Components of Orgyia detrita

(6Z,9Z,11S)-6,9-Heneicosadien-11-ol (Z6Z9-11S-ol-C21) and (6Z,9Z,11R)-6,9-heneicosadien-11-ol (Z6Z9-11R-ol-C21) were identified as major sex pheromone components of female tussock moths, Orgyia detritaGuérin-Méneville (Lepidoptera: Lymantriidae), on the basis of (1) analyses of pheromone gland extracts of female O. detrita by coupled gas chromatographic-electroantennographic detection (GC-EAD) and GC mass spectrometry, and (2) field trapping experiments with synthetic standards. Z6Z9-11S-ol-C21 and Z6Z9-11R-ol-C21 in combination, but not singly, attracted significant numbers of male moths. Racemic Z6Z9-11-ol-C21 was more attractive than the 1:3.5 (R:S) blend ratio found in pheromone gland extracts from female moths. Lower and higher homologues of Z6Z9-11-ol-C21 were also detected in GC-EAD recordings of pheromone extracts, and the racemic compounds enhanced attractiveness of Z6Z9-11-ol-C21 in field experiments. Because of trace amounts of these homologues in extracts, their enantiomeric composition could not be determined. This is the first report of secondary alcohols as pheromone components in the ditrysian (advanced) Lepidoptera.     

Quantitative preparation of long chain thioethers from an oxo-trans-2-enoic ester and a 9,12-dioxo-trans-10-enoic acid

Branched-chain thioethers have been prepared from methyl 4-oxo-trans-2-hexadecenoate and 9,12-dioxo-trans-10-octadecenoic acid. The reagents involved in these preparations were mercaptoacetic and mercaptopropionic acids. The yields of these thioethers are almost quantitative.