Studies of lipase-catalyzed esterification reactions of some acetylenic fatty acids

Esterification of five positional isomers of acetylenic fatty acids [viz. 9:1(2a), 11:1(10a), 18:1(6a), 18:1(9a) and 22:1(13a)] of different chain lengths with n-butanol in n-hexane in the presence of eight different lipases [Lipozyme IM 20 (Rhizomucor miehei), Lipolase 100T (R. miehei), Novozyme 435 (Candida antarctica), PPL (porcine pancreatic lipase), CCL (C. cylindracea), PS-D (Pseudomonas cepacia), Lipase A-12 (Aspergillus niger) and Lipase AY-30 (C. rugosa)] was studied. 2-Nonynoic acid was not esterified except when catalyzed by the lipase from C. antarctica (Novozyme 435) to give 42% butyl ester after 48 h. The lipases from A. niger (Lipase A-12) and C. rugosa (Lipase AY-30) showed poor biocatalytic behavior in the esterification of the acetylenic fatty acids studied. 10-Undecynoic acid gave the highest conversion rate of esterification with each kind of lipase used. 6-Octadecynoic acid showed a marked degree of resistance to esterification carried out in the presence of C. cylindracea (CCL), P. cepacia (PS-D), or porcine pancreatic (PPL) lipase but not significantly in the presence of the lipases of R. miehei (Lipozyme IM 20), R. miehei (Lipolase 100T), or Novozyme 435. 9-Octadecynoic acid and 13-docosynoic acid were not discriminated and were readily esterified by the remaining six lipases, but when compared to oleic acid the acetylenic fatty acids were comparatively much slower in conversion to the esters.     

Aggregation of unsaturated long-chain fatty alcohols in nonaqueous systems

Aggregation and related phenomena in nonaqueous binary and ternary solutions containing unsaturated long-chain fatty alcohol amphiphiles have been studied. Six C₁₈ fatty alcohols were studied—oleyl alcohol (9Z-octadecen-1-ol), elaidyl alcohol (9E-octadecen-1-ol), linoleyl alcohol (9Z, 12Z-octadecadien-1-ol), elaidolinoleyl alcohol (9E, 12E-octadecadien-1-ol), linolenyl alcohol (9Z, 12Z, 15Z-octadecatrien-1-ol) and elaidolinolenyl alcohol (9E, 12E, 15E-octadecatrien-1-ol). Equivalent conductivity and photon correlation spectroscopy confirmed that unsaturated long-chain fatty alcohols form large and polydisperse aggregates in methanol. Critical micelle concentration (CMC) results showed that the degree of unsaturation and configuration of the double bonds in the fatty alcohol significantly influences aggregation. Aggregation of oleyl alcohol in a series of straight and branched medium-chainlength (C₃-C₈) alkanol solvents was studied. For shorter-chained alkanols (C₁-C₄), decreasing solvent dielectric constant decreases the CMC; however, for longer-chained alkanols (C₄-C₈), no significant effects occurred on the CMC. The effect of solubilized soybean oil on the viscosity of long-chain fatty alcohol/methanol solutions was also analyzed. Relative viscosity results were consistent with those expected for microemulsions. Although preliminary in nature, these results generally support the notion that soybean oil is solubilized by incorporation into large soybean oil-in-fatty alcohol aggregates in methanol solvent, resembling a nonaqueous detergentless microemulsion. Presented at the 67th Colloid and Surface Science Symposium, Toronto, Canada, June 20–23, 1993.     

Lipase-catalyzed hydrolysis of TG containing acetylenic FA

Hydrolysis of symmetrical acetylenic TG of type AAA [viz., glycerol tri-(4-decynoate), glycerol tri-(6-octadecynoate), glycerol tri-(9-octadecynoate), glycerol tri-(10-undecynoate), and glycerol tri-(13-docosynoate)] in the presence of eight microbial lipases was studied. Novozyme 435 (Candida antarctica), an efficient enzyme for esterification, showed a significant resistance in the hydrolysis of glycerol tri-(9-octadecynoate) and glycerol tri-(13-docosynoate). Hydrolysis of acetylenic TG with Lipolase 100T (Humicola lanuginosa) was rapidly accomplished. Lipase PS-D (Pseudomonas cepacia) showed a fair resistance toward the hydrolysis of glycerol tri-(6-octadecynoate) only, which reflected its ability to recognize the Δ⁶ positional isomer of 18∶1. Lipase CCL (Candida cylindracea, syn. C. rugosa) and AY-30 (C. rugosa) were able to catalyze the release of 10-undecynoic acid and 9-octadecynoic acid from the corresponding TG, but less readily the 13-docosynoic acid in the case of glycerol tri-(13-docosynoate). The two lipases CCL and AY-30 were able to distinguish the small difference in structure of fatty acyl moieties in the TG substrate. To confirm this trend, three regioisomers of mixed acetylenic TG of type ABC (containing one each of Δ⁶, Δ⁹, and Δ¹³ acetylenic FA in various positions) were prepared and hydrolyzed with CCL and AY-40. The results reconfirmed the observation that AY-30 and CCL were able to distinguish the slight differences in the molecular structure (position of the acetylenic bond and chain length) of the acyl groups in the TG during the hydrolysis of such TG substrates.     

Separation and identification of ximenynic acid isomers in the seed oil ofSantalum spicatum R.Br. as their 4,4-dimethyloxazoline derivatives

The seed oil ofSantalum spicatum contains a significant amount of ximenynic acid,trans-11-octadecen-9-ynoic acid, a long-chain acetylenic fatty acid, as a major component (34%). The identity oftrans-ximenynic acid was confirmed after isolation by ultraviolet, infrared, and nuclear magnetic resonance (NMR) (¹H- and¹³C-) spectroscopy and by gas chromatography/mass spectrometry (GC/MS). Thecis isomer of ximenynic acid was also found (<1%) in some samples. Thecis andtrans isomers were characterized by GC/MS comparison of their methyl esters and 4,4-dimethyloxazoline derivatives.     

Enzymatic Incorporation of Selected Long-Chain Fatty Acids into Triolein

Acidolysis of triolein (tri C18:1) with selected long-chain fatty acids (LCFA) was carried out using Candida antarctica (Novozym 435), Rhizomucor miehei (Lipozyme RM IM), Pseudomonas sp. (PS-30), Aspergillus niger (AP-12), and Candida rugosa (AY-30). A better incorporation of stearic acid (SA), α-linolenic acid (ALA), γ-linolenic acid (GLA), arachidonic acid (AA), and docosapentaenoic acid (DPA) was achieved using lipase from Rhizomucor miehei. Lipase from Pseudomonas sp. catalyzed a better incorporation of linoleic acid (LA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) into triolein. Thus, Rhizomucor miehei and to a lesser extent Pseudomonas sp. might be considered as providing the most effective enzymes for acidolysis of triolein with selected LCFA. In general, incorporation of LCFA into triolein (tri C18:1) may be affected by chain length, number of double bonds, and the location and geometry of the double bonds as well as reaction conditions and reactivity and specificity of lipases used. As the ratio of the number of moles of a mixture of equimole quantities of C18 FA to triolein changed from 1 to 3, incorporation of C18 FA into triolein increased accordingly with Rhizomucor miehei lipase. Similarly, incorporation of n-3 FA into triolein increased when ALA, DPA, DHA, and EPA were used. The same trend was noticed for a mixture of n-6 FA (LA + GLA + AA) and triolein.     

Specificity of papaya lipase in esterification of aliphatic alcohols a comparison with microbial lipases

Straight-chain saturated C4 to C18 alcohols and unsaturated C18 alcohols such as cis-9-octadecenyl (oleyl) cis-6-octadecenyl (petroselinyl), cis-9, cis-12-octadecadienyl (linoleyl), all-cis-9,12,15-octadecatrienyl (α-linolenyl), and all-cis-6,9,12-octadecatrienyl (γ-linolenyl) alcohols, were esterified with caprylic acid using papaya (Carica papaya) latex lipase (CPL) and immobilized lipase from Candida antarctica (Lipase B, Novozym, NOV) and Rhizomucor miehei (Lipozyme, LIP) as biocatalysts. With CPL, highest activity was found for octyl and decyl caprylate syntheses, whereas both NOV and LIP showed a broad chain-length specificity toward the alcohol substrates. CPL strongly discriminated against all C18 alcohols studied, relative to n-hexanol, whereas the microbial lipases accepted the C18 alcohols as substrates nearly as well as n-hexanol. Both petroselinyl and γ-linolenyl alcohol were very well accepted as substrates by NOV as well as LIP, although the corresponding fatty acids, i.e., petroselinic and γ-linolenic acid, are strongly discriminated against by several microbial and plant lipases, including LIP and CPL.     

Hydrogenation of oleic acid to 9-octadecen-1-ol with rhenium-tin catalyst

A new supported bimetallic catalyst, rhenium-tin, is able to reduce oleic acid to 9-octadecen-1-ol (cis andtrans isomers) with appreciable yield under mild hydrogenation conditions. This paper reports investigations on the effects of catalyst preparation methods, types of support, catalyst raw materials, mole ratio of the metals, activation and reaction conditions on the activity and selectivity of the catalyst. Catalyst derived from the combination of ammonium perrhenate and stannic chloride on alumina gave the best performance, and the presence of tin in the catalyst is essential for the preservation of the olefinic bond of the oleic acid during hydrogenation.     

Enzymatic hydrolysis of long-chainN-heterocyclic fatty esters

Incubation of a 1-pyrroline ester [viz. methyl 8-(5-hexyl-1-pyrroline-2-yl)octanoate,1] with bakers' yeast (Saccharomyces cerevisiae) gave the corresponding free fatty acid (1a, 52%). The C=N bond of the 1-pyrroline was not reduced by the yeast. Complete hydrolysis of compound1 was successful using lipase ofCandida cylindracea (CCL) or Lipolase (Rhizomucor miehei) under stirred or ultrasound condition. Fatty esters containing a pyrrolidine [viz. methyl 8-(cis/trans-5-hexyl-pyrrolidine-2-) octanoate,2] orN-methyl pyrrolidine [viz. methyl 8-(cis-5-hexyl-N-methyl-pyrrolidine-2-)octanoate,3] system in the alkyl chain were not hydrolyzed by either CCL or Lipolase, unless conducted in an ultrasonic bath. The hydrolytic activities of the enzymes appeared to be strongly affected by the stereochemistry of theN-heterocyclic ring system. Chemical hydrolysis of compounds1–3 gave the corresponding fatty acidN-HCl salts.     

Separation of saturated,unsaturated, and acetylenic fatty acid isomers by silver resin chromatography

A column packed with silver-saturated ion exchange resin (Amberlyst XN 1010) was found to have lipid separation capabilities superior to Amberlyst XN 1005 and similar to Amberlite XE 284. The separation of unsaturated fatty methyl ester isomers by silver resin chromatography using methanol as the eluting solvent has been extended to mixtures containing polyunsaturate and acetylenic fatty esters. Separations are possible on the basis of both total number of double bonds and the geometric configuration. Mixtures containing saturates, elaidate, oleate, linoleate, and linolenate can be separated, but 10% 1-hexene must be added to the methanol to elute the linolenate. Mixtures containingtrans,trans-;trans,cis-; andcis,cis-octadecadienoate isomers have also been separated, and partial resolution ofcis-9,cis-12- andcis-2,cis-15-octadecadienoate isomers was obtained. Sterolate, a monounsaturated acetylenic fatty ester was eluted at the same time as oleate. Crepenynate (cis-9-octadecen-12-ynoate) can be separated from linoleate but not fromcis,trans-octadecadienoate. Employed at the Northern Regional Research Center under a USDA cooperative education program with Purdue University.     

Chemical conversion of 9-tetradecen-1-ol acetates to 3,13-octadecadien-1-ol acetates,sex attractants for male clearwing moths (Lepidoptera: Sesiidae)

Males of many species of clearwing moths are attracted by one of the geometrical isomers of 3,13-octadecadien-1-ol acetate or by a mixture of isomers. The synthesis of (E,Z)-, (E,E)-, and (Z,E)-3,13-octadecadien-1-ol acetate is described starting with the (Z)- and (E)-9-tetraceden-1-ol acetates, which are commercially obtainable.     

Stereospecific synthesis of (Z,Z)-3,5-tetradecadienoic acid,a component ofAttagenus elongatulus (Casey) pheromone

A simple stereospecific synthesis of (Z,Z)-3,5-tetradecadienoic acid is described based upon coupling of 1-decyne and 3-butyn-1-ol to give 3,5-tetradecadiyn-1-ol. Subsequent reduction of the diyne to (Z,Z)-3,5-tetradecadien-1-ol and oxidation of the dienol gave the desired acid.Attagenus elongatulus were strongly attracted to the pure acid.     

Sex attractant for currant clearwing mothSynanthedon tipuliformis (Clerck) (Lepidoptera: Sesiidae)

The currant clearwing moth,Synanthedon tipuliformis (Clerck) (Lepidoptera: Sesiidae), is a pest in many parts of the world. In field tests it was found that (E,Z)-2,13-octadecadien-1-ol acetate attracts males of this species. The synthesis of this compound and of its geometrical isomer (Z,Z)-2,13-octadecadien-1-ol acetate is described.     

Curupira tefeensis II: Occurrence of Acetylenic Fatty Acids

Four acetylenic fatty acids [11-octadecen-9-ynoic acid (E) (ximenynic acid), 11-octadecen-9-ynoic acid (Z), 9,11-octadecadiynoic acid and 13-octadecen-9,11-diynoic acid (E) (exocarpic acid)] were found in the seed oil of Curupira tefeensis (Olacaceae) as minor compounds. The mass spectra of the methyl esters (EI and CI mode), picolinyl esters and 4,4-dimethyloxazoline derivatives of these acids are discussed in detail. Furthermore the NMR data of 9,11-octadecadiynoic acid and 13-octadecen-9,11-diynoic acid are presented.     

Utilization of polyunsaturated long-chain alcohols byClostridium butyricum

When a trace amount of linolenyl alcohol (cis,cis,cis-9,12,15-octadecatriene-1-ol) was added to the culture media ofClostridium butyricum, the polyunsaturated alcohol was readily assimilated. After an initial 18-hr exposure interval, there was a large increase in the amount and a change in the composition of thelong-chain alcohols present. However, this alteration was temporary. As the duration of the exposure interval increased, the alcohol content and composition of the treated bacteria returned toward control values. This return of the long-chain alcohol composition toward control values was associated with an increase in the proportion of linolenic acid in the phospholipid fraction. The ether moiety of the alk-1-enyl phosphoglycerides was unchanged in amount or composition upon exposure to linolenyl alcohol.     

Acetylenic acid biosynthesis inCrepis rubra

Time studies of crepenynic acid synthesis inCrepis rubra show that this acid is not present in the seed for several days after, flowering commences but builds up rapidly between the 14th and 28th days to become the major fatty acid of the seed oil. Radioactive tracer studies clearly demonstrate that the acetylenic bond is introduced into the carbon chain of a preformed long-chain fatty acid rather than built in during formation of the carbon chain. The nearest precursor found is oleic acid. There is no conversion to crepenynic acid by seed preparations ofcis,cis-linoleic acid,cis,trans (trans,cis)-linoleic acid, orcis-12,13-epoxy-oleic acid. Possible biosynthetic pathways to explain these results are suggested. The crepenynic acid is chiefly, but not entirely, in triglycerides in the seed oil, and it has been shown to be esterified in the 2- and 3- positions of the triglyceride.     

Analysis of the seed oil of Heisteria silvanii (Olacaceae)—A rich source of a novel C18 acetylenic fatty acid

Besides some usual fatty acids (FA), two conjugated ene-yne acetylenic FA [trans-10-heptadecen-8-ynoic acid (pyrulic acid) (7.4%), and trans-11-octadecen-9-ynoic acid (ximenynic acid) (3.5%)], a novel ene-yne-ene acetylenic FA [cis-7, trans-11-octadecadiene-9-ynoic acid (heisteric acid) (22.6%)], and 9,10-epoxystearic acid (0.6%) could be identified in the seed oil of Heisteria silvanii (Olacaceae). Two further conjugated acetylenic FA [9,11-octadecadiynoic acid (0.1%) and 13-octadecene-9,11-diynoic acid (0.4%)] were identified tentatively by their mass spectra. The FA mixture has been analyzed by gas chromatography/mass spectrometry (GC/MS) of their methyl ester and 4,4-dimethyloxazoline derivatives. The structure of heisteric acid was elucidated after isolation via preparative silver ion thin-layer chromatography and by various spectroscopic methods [ultraviolet; infrared; ¹H, ¹³C nuclear magnetic resonance (NMR); ¹H−¹H and ¹H−¹³C correlation spectroscopy]. To determine the position of the conjugated ene-yne-ene system, the NMR spectra were also measured after addition of the lanthanide shift reagent Resolve-Al EuFOD^TM. Furthermore, the triyglyceride mixture was analyzed by high-temperature GC and high-temperature GC coupled with negative chemical ionization MS. A glass capillary column coated with a methoxy-terminated 50%-diphenyl-50%-dimethylpolysiloxane was used for the separation of the triacylglycerol (TAG) species. No evidence of decomposition of the TAG species containing conjugated ene-yne-ene FA was observed. Twenty-six species of the separated TAG were identified by means of their abundant quasi molecular ion [M−H]⁻ and their corresponding carboxylate anions [RCOO]⁻ of the fatty acids, respectively. The major molecular species of the TAG were found to be 16:0/18:1/18:1, 16:0/18:1/18:3 (heisteric acid), 17:2 (pyrulic acid)/18:1/18:1, 18:1/18:1/18:3 (heisteric acid). The TAG containing acetylenic FA showed an unexpected increase of the retention time in comparison to the TAG containing usual FA, thus making the prediction of the elution order of lipid samples containing acetylenic FA difficult.     

Separation of Picric Acid with Trioctyl Amine (TOA) Extractant in Diluents

Separation of 2,4,6-trinitrophenol (Picric acid) from aqueous solution by trioctyl amine (TOA), which is a long-chain aliphatic amine, has been studied at 298 K. Diluents (alcohols) (isoamyl alcohol, octan-1-ol, decan-1-ol) have been used to dissolve TOA. Experimental results of batch extraction are calculated and reported as distribution coefficients (K_D), loading factors (Z), and extraction efficiency (E). The most effective diluent has been found to be isoamyl alcohol with a distribution coefficient value of 19.333. In this article, the linear solvation energy relationship (LSER) modeling (included Hidebrand-Hansen solubility parameters) has been done on the reactive extraction of picric acid to obtain the model parameters. Modeling using an LSER model predict a close resemblance of experimental data.     

Study of female sex pheromone of leopard moth,Zeuzera pyrina L. Isolation and identification of three components

Three compounds have been identified in the abdominal tip extracts from the female leopard moth,Zeuzera pyrina L. Gas-liquid chromatography and mass spectroscopy data showed that (E, Z)-2, 13-octadecadien-1-ol acetate was the main component and that (Z)-13-octadecen-1-ol acetate and octadecan-1-ol acetate were secondary components. The electroanten-nographic responses of maleZ. pyrina to nanogram amounts of all four 2, 13-octadecadien-1-ol acetate isomers indicated that theE, Z isomer had the maximum activity. A strong EAG response was also recorded for (Z)-7-do-decen-1-ol acetate, which was not detected in the female extracts.     

Reduction with zinc of triple bonds tocis double bonds in long chain conjugated fatty acids

The methyl ester of santalbic acid (trans-11-octadecen-9-ynoic acid) was hydrogenated with powdered zinc in 50% propanol to yield 90% of thecis,trans-diene ester; similarly, 7,9-octadecadiynoic methyl ester was converted tocis,cis-diene ester but yields were no better than 75%. Presented in part at the AOCS Meeting, Chicago, October 1967. East. Market. Nutr. Res. Div., ARS, USDA.     

Sodium santalbate-dimethyl sulfate inclusion complex

A molecular inclusion complex has been obtained from the major acetylenic acid, santalbic acid (octadec-11-en-9-ynoic acid ortrans-11-octadecen-9-ynoic acid) of the seed oil ofSantalum album L. by a simple treatment of its sodium salt with dimethyl sulfate. Aqueous solutions (0.5–1%) of the complex produce good lather and have efficient cleansing (detergent) action on grease and dirt particles.