Acid-catalyzed conversion of epoxyesters to hydroxyesters

Esters of 9,10-epoxystearic aeid (epoxidized oleic acid), dissolved in 1,4-dioxane, were treated at 15C, first with aqueoiis acid and then with water to convert them to 9,10-dihydroxystearates in high yields. Ester functions remained intact. Glycidyl 9,10-epoxystearate, ethylene glycolbis-9,10-epoxystearate and catecholbis-9,10-epoxystearate were converted to the corresponding tetrahydroxy esters by this method. Treatment of methyl 9,10-epoxystearate with diluted (24%) fluoboric acid gave methyl 9,10-dihydroxystearate in 89% yield. Under similar conditions glycidyl stearate did not react and the internal epoxy group of glycidyl 9,10-epoxystea-rate was hydrated preferentially. Hydration of methyl 9,10-epoxystearate with coned H₂SO₄ led to the formation of considerable amt of byproducts, principally methyl 9(10)-ketostearate. Side reactions were inhibited by diluting the acid-catalyst. Presented at the AOCS Meeting, Minneapolis, 1963. A laboratory of the B. Utiliz. Res.& Der. Dir., ABS, USDA.     

Studies on the nuclear magnetic resonance spectra of olefinic protons of conjugated fatty acid methyl esters

The NMR spectra of olefinic protons in the four representative conjugated fatty acid methyl esters, methylcis-9,trans-11-octadecadienoate, methyltrans-9,trans-11-octadecadienoate, methyl α eleostearate, and methyl β eleostearate, were studied. The chemical shift of each olefinic proton in these compounds was determined by considering their intramolecular environment. Coupling constants were also obtained as the results of spectral analysis.     

The absolute optical configurations of the isomeric 9,10-epoxystearic, 9,10-dihydroxystearic and 9,10,12-trihydroxystearic acids

The absolute optical configuration of (−)-cis-9,10-epoxystearic acid has been verified as being L. Here (−)-erythro-9,10-dihydroxystearic acid, isolated from castor oil, was converted by stereospecific reactions to (+)-cis-9,10-epoxystearic acid and was thereby proved to be D-9,D-10-dihydroxystearic acid. Removal of the D-12-hydroxy group from the higher meltingerythro-9,10,12-trihydroxystearate derived from ricinoleic acid, after protection of the glycol group, gave the L-9,L-10-dihydroxystearate derivative. This proved the high melting diastereoisomer to be L-9,L-10,D-12-trihydroxystearate and directly verified the supposition that the higher melting, arsenite-complexing diastereoisomer of such oxidation pairs has thetrans-10,12-diol grouping. On this basis, the higher meltingthreo-trihydroxystearate from ricinoleate must be D-9,L-10,D-12-trihydroxystearate and removal of the 12-hydroxy group must give D-9,L-10-dihydroxystearate which proved to be the levorotatory enantiomer. The dextrorotatory L-9,D-10-dihydroxystearate was transformed by stereospecific reactions to (+)-trans-9,10-epoxystearic acid, thereby defining the absolute configurations oftrans-9,10-epoxystearic acids. On the basis of these results conclusions may be drawn as to the stereospecificity and site of action of enzymes which hydrate 9,10-epoxystearic acids.     

Cyclopropane fatty acid metabolism: Physical and chemical identification of propane ring metabolic products in the adipose tissue

Twelve male weanling rats were distributed equally into 3 groups and placed on fat-free diets. The diets of groups 1 and 2 were supplemented with 0.54% of recemic methylcis-9,10-methylene octadecanoate (CMO) and racemic methyltrans-9,10-methylene octadecanoate (TMO), respectively. Group 3 served as a control. Gas liquid chromatography (GLC) analyses of the adipose tissue methyl esters indicated at the level fed, that cyclopropane fatty acids do not affect normal fatty acid metabolism as has been shown for cyclopropene fatty acids. GLC analyses of groups 1 and 2 revealed the presence of a different unidentified fatty acid for each of the acids fed in addition to the CMO and TMO acids themselves. Each of the unidentified acids and the CMO and TMO acids were isolated and purified by preparative GLC. The absolute identity of the CMO and TMO acids fed and isolated from body fat was established by IR, NMR, and mass spectra. The biodegradation products of the CMO and TMO esters were shown to becis- andtrans-3,4-methylene dodecanoic acid, respectively. Unequivocal proof of structure was established through synthesis followed by comparison of IR, NMR, and mass spectra and melting points, GLC retention times, and elemental analyses with those obtained for the degradation products. Neither member of the racemic mixtures of either thecis or thetrans cyclopropane acids was preferentially utilized by the rat as shown by the lack of octical activity in the degradation products and the CMO and TMO acids isolated from the body fat. The accumulations of the 3,4-methylene dodecanoic acids in the adipose tissue of the rats fed CMO and TMO cyclopropane fatty acids suggest the inability of the beta oxidation enzyme system to proceed past the cyclopropane ring in a fatty acid chain. The synthesis ofcis- andtrans-3-dodecenoic acids, intermediates in the synthesis of the 3,4-methylene dodecanoic acids, and the geometrical cyclopropane isomers are discussed. This work to be submitted in partial fulfillment of the requirement for Ph.D.     

Stereospecific synthesis ofcis andtrans fatty esters

Theerythro andthreo isomers of methyl 9,10-dihydroxyoctadecanoate and thethreo isomer of methyl 12,13-dihydroxy-cis-9-octadecenoate were converted into methylcis- andtrans-9-octadecenoate and methylcis-9,rans-12-octadecadienoate, respectively, by reaction of the dihydroxy ester with triethyl orthoformate to give the 2-ethoxy-1,3-dioxolane which was thermally decomposed to the unsaturated ester.     

The GLC and TLC resolution of diastereoisomeric polyhydroxystearates and assignment of configurations

Trifluoroacetate (TFA) derivatives of methyl 12-hydroxystearate, methyl ricinoleate, five positional isomers of methylthreo- anderythro-dihydroxystearate, four diastereoisomeric methyl 9,10–12-trihydroxystearates, and four racemic diastereoisomeric methyl 9,10–12,13-tetrahydroxystearates were prepared and analyzed by gas-liquid chromatography (GLC). The isomericthreo- anderythro-dihydroxystearates that had not previously been resolved by GLC were separated. Excellent resolution of the diastereoisomeric pairs of methylthreo- anderythro-9,10–12-triand and methylerythro, erythro- andthreo, threo-9,10–12,13-tetrahydroxystearates was obtained by GLC of their TFA derivatives. Analyses of these high-molecular-weight compounds were carried out on polar and nonpolar packed columns used routinely for methyl ester analysis. The various methyl mono-, di-, tri-, and tetrahydroxysterate esters were also analyzed by thin-layer chromatography (TLC) on Silica Gel G adsorbent layers and on Silica Gel G impregnated with sodium arsenite. Relative and absolute configurations were assigned to the various diastereoisomeric tri- and tetrahydroxysterates based on information obtained from GLC, TLC, synthetic ratios, and molecular-models. A micro hydroxylation method that gives quantitative yields ofthreo- anderythro-dihydroxy acids from various concentrations of C₁₈ monoene geometrical isomers was developed. Subsequent GLC analysis of the isomeric methyl dihydroxy TFA derivatives allows the quantitative determination of double-bond configuration on small samples without expensive or specialized equipment. Presented at the AOCS Meeting, Los Angeles, April 1966. under appointment from Oak Ridge Associated Universities. An operating unit of the Oak Ridge Associated Universities, under contract with the US Atomic Energy Commission.     

Photo-activated metal carbonyl complexes as stereoselective catalysts for the homogeneous hydrogenation of dienes

Significantly increased activity of Cr(CO)₆ was achieved for the stereoselective homogeneous hydrogenation of methyl sorbate andtrans,trans-conjugated fatty esters at ambient temperature and pressure by exposing the catalyst to UV irradiation (3500 Å) in a solvent mixture of cyclohexane-acetonitrile (20:1). In this solvent mixture, methyl sorbate was converted quantitatively at ambient conditions into methylcis-3-hexenoate, and methyltrans-9,trans-11-octadecadienoate into methylcis-10-octadecenoate (99.9%). These products are expected by 1,4-addition of hydrogen. Under these conditions no hydrogenation of methyl linoleate occurred. Under the same conditions, cycloheptatriene-Cr(CO)₃ showed lower activity than Cr(CO)₆, and Mo(CO)₆ and mesitylene-Mo(CO)₃ showed no significant activity toward conjugated substrates. When Cr(CO)₆ and Mo(CO)₆ were irradiated at 2537 Å they caused the geometric isomerization of methyl sorbate without hydrogenation, but had no effect on methyl linoleate. A hydrogenation mechanism is proposed for Cr(CO)₆ that involves CH₃CN- and H₂-Cr(CO)₃ complexes as intermediates for the stereoselective 1,4-addition of hydrogen totrans,trans-conjugated dienes.     

Ring location in cyclopropane fatty acid esters by boron trifluoride-catalyzed methoxylation followed by mass spectroscopy

Methyl esters of methylcis- andtrans-9,10-methyleneoctadecanoic acids react with 50% boron trifluoride-methanol to produce unsaturated and methoxy-esters; both products are shown by gas chromatography to be a mixture of several isomers. Mass spectra of the methoxylated esters are characterized by intense peaks due to cleavage adjacent to methoxy-functions which allow the position of the ring in the original cyclopropane ester to be easily assigned. Methyl oleate is also partially attacked by 50% BF₃−MeOH to produce a mixture of methyl 9- and 10-methoxyoctadecanoates. 14% BF₃−MeOH does not react with cyclopropane and olefinic esters under the reaction conditions employed.     

Synthesis of phenyl substituted C18 furanoid fatty esters

Methyl 9,12-epoxy-10-phenyl-9,11-octadecadienoate was prepared by acid catalyzed cyclization of methyl 9,12-dioxo-10-phenyloctadecanoate, which was derived from the oxidation of methyl 9-hydroxy-12-oxo-10-phenyloctadecanoate. The latter was exclusively obtained from methylcis-9,10-epoxy-12-oxooctadecanoate with phenyllithium in the presence of copper (I) bromide. A mixture of positional isomers, methyl 9,12-epoxy-10(11)-phenyl-9,11-octadecadienoates, was also prepared by another route. The spectroscopic properties of the various intermediates and products were studied. The positional isomers of the phenyl substituted furanoid fatty esters were characterized by¹³C nuclear magnetic resonance spectrometry.     

Study on the effect of antioxidants in the autoxidation of methyl nonconjugated octadecadienoates

Many studies have been published on the effect of antioxidants on unsaturated fatty acid esters but the differences of the effects of antioxidants on geometric isomers have never been investigated. In this study, methylcis-9,cis-12-octadecadienoate and itstrans isomer methyltrans-9,trans-12-octadecadienoate were used as methyl nonconjugated dienoates, and BHA, BHT, PG, NDGA, 4,4′-dihydroxy-3,5,3′,5′-tetratert-butyl diphenyl methane, L-thyroxine sodium salt,a-tocopherol and sesamol were used, as antioxidants. The differences of the effects of antioxidants on both geometric isomers were investigated by determining the induction period using the weighing method. Also determined were the infrared and ultraviolet spectra, peroxide values, conjugated diene contents, isolatedtrans double bond contents and molecular weights for the controls and the samples containing antioxidants. Thecis,cis isomer was more easily autoxidized and had a shorter induction period than thetrans,trans form. By the end of the induction period, no isolatedtrans double bond forms in thecis,cis isomer, but a considerable amount of isolatedtrans double bond decreased in thetrans,trans isomer. In general, the effects of antioxidants, except NDGA, on thecis,cis isomer were larger than thetrans,trans form.     

Studies on the metabolism of linoelaidic acid in the essential fatty acid-deficient rat

Male weanling rats of the Sprague-Dawley strain were made essential fatty acid (EFA)-deficient by feeding them a fat-free diet for five months. The animals were then fed a supple-ment of methyltrans-9,trans-12-octadecadienoate (methyl linoelaidate), as 5% of the dietary calo-ries (approximately 400 mg/animal/day) for 19 days, and killed by exsanguination. The com-position of the liver, kidney, epididymal and plasma lipids was determined and compared with that obtained from EPA-deficient rats given oral supplements of methylcis-9,cis-12-octadeca-dienoate (linoleate) and methylcis-9,trans-12- octadecadienoate. Linoelaidic acid was depos-ited in the phospholipids, sterol esters and tri-glycerides in all of the tissues examined. Iso-lation analysis of the fatty acids showed no evidence that linoelaidic acid was converted to higher polyunsaturated fatty acids in the EFA-deficient rat. Supported in part by U. S. Public Health Service Grant No. AM 04942.     

The lindlar-catalyzed reduction of methyl santalbate: a facile preparation of methyl 9-cis,11-trans-Octadecadienoate-9,10-d2

Conjugated linoleic acid (CLA) has been associated with the reduction of chemically induced cancers in mice and rats and the suppression of atherosclerosis in rats. We have found seed oils to be a valuable source of precursors for the rapid preparation of gram quantities of deuterium-labeled fats. Methyl santalbate (methyl 11-trans-octadecen-9-ynoate), obtained from Santalum album (Linn.) seed, was reduced with Lindlar catalyst, quinoline, and deuterium gas to produce, in yields of 65–75%, the gram quantities of methyl 9-cis,11-trans-octadecadienoate-9,10-d₂ (CLA-d₂) we required for metabolism and oxidation studies. Unlike monoacetylenic and methylene-interrupted polyacetylenic fatty acid methyl esters, the conjugated system was reduced with no noticeable break in the rate of deuterium uptake. The quantity of poison (quinoline) present did influence the amount of CLA-d₂ produced, but the production of overreduced fatty acid methyl esters (perhaps because of the conjugated system) could not be prevented. Fractionation of the reaction mixture by silver resin chromatography resulted in the isolation of >99% chemically pure CLA-d₂ in yields of 60–70%.     

Hydrogenation ofcis-9,cis-12-,cis-9,trans-12- andtrans-9,trans-12- Octadecadienoates

The products formed by hydrogenation of methylcis-9,trans-12- andtrans-9,trans-12-octadecadienoates with nickel and platinum catalysts have been compared with those from methyl esters of the naturally occurring all-cis linoleate. Hydrogen uptake is slower for thetrans isomers. Much of the monoene consisted of esters with double bonds at the 9 and 12 positions with their original geometric configurations. Monoenoic esters with double bonds at the 10 and 11 positions were predominatelytrans and apparently formed by conjugation before hydrogenation. Nickel produced more isomerization than platinum but less than previously reported for copper. With both catalysts hydrogenation proceeded both directly and through conjugated intermediates, in contrast to copper in which all hydrogenation is believed to follow conjugation. Presented at the AOCS Meeting, Los Angeles, April 1972. ARS, USDA.     

Silver resin chromatographic separation of methyl cis- and trans- mono- and dihydroxy fatty esters

Column chromatography on silver ion-saturated Amberlyst XN 1010 cation exchange resin gave very good separation of a mixture of methyl 12-hydroxy-cis-andtrans-9-octadecenoates and of methylthreo-12, 13-dihydroxy-cis- andtrans-9-octadecenoates. Comparison of the retention volumes of nonhydroxy, monohydroxy, and dihydroxy saturated and monoenoic methyl esters and of dienoic methyl esters shows that the hydroxy group interacts with the column packing to slow passage of the compound through the column, although the effect of a hydroxy group is less than that of atrans double bond. The effects of the hydroxy groups are additive; the ratio of retention volumes of dihydroxy ester to monohydroxy ester is slightly larger that that of monohydroxy ester to nonhydroxy ester. The retention volume of a cis monoenoic ester is equal to that of a hydroxytrans monoenoic ester and that of a hydroxycis monoenoic ester is equal to that of a dihydroxytrans monoenoic ester.     

Incorporation of oxygen-18 into the oxirane ring ofcis-9,10-epoxyoctadecanoic acid

The synthesis ofcis-9,10-epoxyoctadecanoic acid by tissue slices of wheat plants infected withPuccinia graminis tritici (Wheat stem rust) has been investigated further. Synthetic methylcis-9,10-epoxyoctadecanoate and the same ester isolated from incubation in an atmosphere containing¹⁸O₂, or a medium containing H₂ ¹⁸O, were analyzed by mass spectrometry. These analyses revealed that molecular oxygen was incorporated into the oxirane ring ofcis-9,10-epoxyoctadecanoic acid. Presented in part at the AOCS Meeting, Minneapolis, October 1969.     

trans Fatty acids in milkfat

Milkfat is extremely complex, with ca. 500 different fatty acids reported in the triglycerides. Seasonal feed variation results in higher unsaturated fatty acid levels in summer than in winter. Rumen microbes hydrogenate unsaturated feed lipids to yield a mixture of geometrical and positional isomers which are transmitted to the milk. Total isolatedtrans fatty acids in milkfat reported in the literature range from 2 to 11% with maximal values in summer and minimal values in winter. A study was undertaken about the use of AOCS Method Cd 14-61 for the determination of isolatedtrans levels in milkfat. The triglycerides of milkfat were analyzed using trielaidin as a standard and milkfat methyl esters were analyzed using methyl elaidate as a standard. Corrections were made, for the loss of the methyl esters of the short-chain fatty acids. The apparent levels oftrans found by using the triglycerides were considerably higher than by using the methyl esters. Infrared spectra of pure triglycerides of saturated fatty acids showed measurable absorption at 10.36 μm, whereas methyl esters of saturated andcis unsaturated fatty acids did not.     

Oxygenated fatty acids of oil from sunflower seeds after prolonged storage

Chemical analysis of a number of sunflower (Helianthus annuus) seed oil samples revealed a low and variable percentage of hydrogen bromide-reactive material. To characterize the compounds responsible for this reactivity, oil was extracted from selected introductions from Uruguay, Turkey, and Yugoslavia that had been subjected to prolonged storage. Two epoxy fatty acids and two conjugated dienolic acids were isolated from the methyl esters derived from these sunflower seed oils by using a combination of column chromatography and countercurrent distribution. The epoxy acids arecis-9,10-epoxystearic acid (0.5%) andcis-9,10-epoxy-cis-12-octadecenoic (coronaric) acid (2.2%). Characterization of the dienols revealed that they are 9-hydroxy-trans-10,cis-12-octadecadienoic acid (1.2%) and 13-hydroxy-cis-9,trans-11-octadecadienoic acid (1.3%). Fresher seed of some of these introductions contained less of the oxygenated components. Oil from recently produced seed of selected high-oil Russian sunflower varieties, including some currently grown in the United States, contained no more than trace amounts of oxygenated acids. Though the relative contributions of genetic and environmental factors toward genesis of oxygenated acids are not established, increase of those acids in some sunflower lines as a result of storage has been demonstrated. Presented at the AOCS-AACC Joint Meeting, Washington, D. C., April 1968. No. Utiliz. Res. Dev. Div., ARS, USDA.     

Synthesis of vinyl threo-9, 10-threo-12, 13-tetrahydroxystearates

Methyl esters from vernonia oil containingcis-12,13-epoxy-cis-9-octadecenoate were reacted with acetone in the presence of a boron trifluoride catalyst to yieldtrans-12,13-0-isopropylidene-9-ene derivative I. Epoxidation of the unsaturation followed by reaction with acetone gave the isomerictrans,trans-di-0-isopropylidene deriva-tivesIII of 9,10 :12,13-tetrahydroxystearates. The carboxylic acids of III were converted to vinyl esters and subsequent hydrolysis of the isopropy-lidene groups with boric acid produced vinylthreo-9,10threo-12,13-tetrahydroxystearates. By the same sequence of reactions monoepoxidized methyl linoleate yielded an optically inactive de-rivative which had identical refractive index, IR spectrum, and GLC data toIII from vernonia methyl esters. No. Utiliz. Res. Dev. Div., ABS, USDA.     

GLC and TLC analysis of isopropylidene derivatives of isomeric polyhydroxy acids derived from positional and geometrical isomers of unsaturated fatty acids

Gas-liquid chromatography (GLC) and thin-layer chromatography (TLC) were used to investigate the isomeric positional geometrical isopropylidene derivatives of nine isomeric dihydroxy esters, four isomeric methyl 9,10-12-trihydroxystearates, and eight isomeric methyl 9,10-12,13-tetrahydroxystearates prepared from unsaturated fatty acids. The isopropylidenes derived fromcis andtrans monounsaturated fatty acids were easily separated on both polar and nonpolar columns. Positional isopropylidenes derived from positional isomers of monounsaturated fatty acids were not separated on either liquid phase but were resolved by TLC. Four of the eight isomeric isopropylidenes derived from the four geometrical isomers of linoleic acid were resolved on the polar column; the other four isomers eluted as a single peak. The four isomeric isopropylidene-trifluoroacetate derivatives derived from ricinoleic and ricinelaidic acids were also resolved on the polar column. GLC analyses were carried out with liquid phases of ethylene glycol succinate methyl silicone polymer (EGSS-X) and methyl silicone polymer (SE-30) packed columns. Isopropylidenes, in addition to their applicability for the resolution of polyhydroxy acid mixtures, are particularly useful for the determination of double bond positions and geometrical configurations of fatty acids without cleavage. Under contract with the U. S. Atomic Energy Commission.     

Identification of nine acetylenic fatty acids, 9-hydroxystearic acid and 9,10-epoxystearic acid in the seed oil ofJodina rhombifolia hook et arn. (Santalaceae)

In addition to some usual fatty acids, the seed oil ofJodina rhombifolia (Santalaceae) contains nine acetylenic fatty acids [9-octadecynoic acid (stearolic acid) (1.1%),trans-10-heptadecen-8-ynoic acid (pyrulic acid) (20.1%), 7-hydroxy-trans-10-heptadecen-8-ynoic acid (2.3%),trans-10,16-heptadecadien-8-ynoic acid (0.7%), 7-hydroxy-trans-10,16-heptadecadien-8-ynoic acid (0.1%),trans-11-octadecen-9-ynoic acid (ximenynic acid) (20.3%), 8-hydroxy-trans-11-octadecen-9-ynoic acid (12.2%),trans-11,17-octadecadien-9-ynoic acid (1.5%), 8-hydroxy-trans-11,17-octadecadien-9-ynoic acid (1.3%), 9-hydroxystearic acid (<0.1%) and 9,10-epoxystearic acid (0.7%)]. The fatty acids have been analyzed by gas chromatography/mass spectrometry of their methyl ester and 4,4-dimethyloxazoline derivatives. The hydroxy fatty acid methyl esters have been examined also as trimethyl-silyl ethers. Furthermore, the fatty acid methyl esters (FAME) have been fractionated according to their polarity (FAME-A: nonhydroxy; FAME-B: hydroxy fatty acids) and to their degree of unsaturation (FAME-A1/A2; FAME-B1/B2) by preparative thin-layer chromatography and argentation chromatography, respectively. All of these fractions have been analyzed by ultraviolet and infrared spectroscopy, and the fractions FAME-A and FAME-B have been analyzed further by nuclear magnetic resonance (¹H,¹³C, 2D H/C, attached proton test) spectroscopy and gas chromatography/mass spectrometry. This work is dedicated to the 65th birthday of Prof. Dr. K. Pfeilsticker, Institut of Food Science, University Bonn (Germany).