Improved synthesis and characterization of saturated branched‐chain fatty acid isomers

The development of viable technologies for producing green products from renewable fats and oils is highly desirable since such materials can serve as replacements for non‐renewable and poorly biodegradable petroleum‐based products. Mixtures of saturated branched‐chain fatty acid isomers (sbc‐FAs), commonly referred to as isostearic acid, are important intermediates for the production of biodegradable lubricants, cosmetics, emollients, and hydraulic fluids. Present methods for producing sbc‐FAs, however, often give low yields of sbc‐FAs or sbc‐FA preparations with a high content of dimer acid fatty acid co‐products. This study reports an improved route to synthesizing sbc‐FAs from monounsaturated fatty acids using a modified H‐Ferrierite zeolite catalyst in conjunction with small amounts of triphenylphosphine additive. The yields of sbc‐FAs (up to 80 wt%) and co‐products (up to15 wt%) were determined using a modification of a previously reported GC method. A more detailed analysis of the distribution of sbc‐FA isomers in the products was made by the combined use of GC × GC‐TOF‐MS. Additionally, it was found that the H‐Ferrierite zeolite catalyst was recyclable and reusable up to 10 times without significant loss of activity and selectivity for sbc‐FAs.     

Impact of Fatty Acid Structure on CALB‐Catalyzed Esterification of Glucose

Enzymatic synthesis of fatty acid glucose esters from different fatty acyl donors are performed via enzymatic catalysis in the presence of Candida antarctica lipase B (CALB), using acetonitrile as the solvent. The acyl donor nature (fatty acid or fatty acid vinyl ester) and structure are varied. Lower reaction rates and lower conversions are obtained with fatty acids in comparison to their corresponding vinyl esters. Moreover, the acyl donor with the longest chain length gives the highest conversions. The presence of unsaturation on the acyl donor chain is also shown to be detrimental to the conversion. Practical Applications: The practical applications of the present work are related to the production of gluco‐esters that could be used as nonionic surfactants as detergents, cosmetics and food emulsifiers, emollients or conservatives, respectively. In this study, it is shown that in order to get high production yields, each reaction parameter has to be tuned properly.     

Elongation of very Long-Chain (>C<Subscript>24</Subscript>) Fatty Acids in <Emphasis Type="Italic">Clarias gariepinus</Emphasis>: Cloning,Functional Characterization and Tissue Expression of <Emphasis Type="Italic">elovl4</Emphasis> Elongases

Elongation of very long‐chain fatty acid 4 (Elovl4) proteins participate in the biosynthesis of very long‐chain (>C₂₄) saturated and polyunsaturated fatty acids (FA). Previous studies have shown that fish possess two different forms of Elovl4, termed Elovl4a and Elovl4b. The present study aimed to characterize both molecularly and functionally two elovl4 cDNA from the African catfish Clarias gariepinus. The results confirmed that C. gariepinus possessed two elovl4‐like elongases with high homology to two previously characterized Elovl4 from Danio rerio, and thus they were termed accordingly as Elovl4a and Elovl4b. The C. gariepinus Elovl4a and Elovl4b have open reading frames (ORF) of 945 and 915 base pairs, respectively, encoding putative proteins of 314 and 304 amino acids, respectively. Functional characterization in yeast showed both Elovl4 enzymes have activity towards all the PUFA substrates assayed (18:4n‐3, 18:3n‐6, 20:5n‐3, 20:4n‐6, 22:5n‐3, 22:4n‐6 and 22:6n‐3), producing elongated products of up to C₃₆. Moreover, the C. gariepinus Elovl4a and Elovl4b were able to elongate very long‐chain saturated FA (VLC‐SFA) as denoted by increased levels of 28:0 and longer FA in yeast transformed with elovl4 ORF compared to control yeast. These results confirmed that C. gariepinus Elovl4 play important roles in the biosynthesis of very long‐chain FA. Tissue distribution analysis of elovl4 mRNAs showed both genes were widely expressed in all tissues analyzed, with high expression of elovl4a in pituitary and brain, whereas female gonad and pituitary had the highest expression levels for elovl4b.     

Fatty Acid-Specific Alterations in Leptin,PPARα, and CPT-1 Gene Expression in the Rainbow Trout

It is known that fatty acids (FA) regulate lipid metabolism by modulating the expression of numerous genes. In order to gain a better understanding of the effect of individual FA on lipid metabolism related genes in rainbow trout (Oncorhynchus mykiss), an in vitro time‐course study was implemented where twelve individual FA (butyric 4:0; caprylic 8:0; palmitic (PAM) 16:0; stearic (STA) 18:0; palmitoleic16:1n‐7; oleic 18:1n‐9; 11‐cis‐eicosenoic 20:1n‐9; linoleic (LNA) 18:2n‐6; α‐linolenic (ALA) 18:3n‐3; eicosapentenoic (EPA) 20:5n‐3; docosahexaenoic (DHA) 22:6n‐3; arachidonic (ARA) 20:4n‐6) were incubated in rainbow trout liver slices. The effect of FA administration over time was evaluated on the expression of leptin, PPARα and CPT‐1 (lipid oxidative related genes). Leptin mRNA expression was down regulated by saturated fatty acids (SFA) and LNA, and was up regulated by monounsaturated fatty acids (MUFA) and long chain PUFA, whilst STA and ALA had no effect. PPARα and CPT‐1mRNA expression were up regulated by SFA, MUFA, ALA, ARA and DHA; and down regulated by LNA and EPA. These results suggest that there are individual and specific FA induced modifications of leptin, PPARα and CPT‐1 gene expression in rainbow trout, and it is envisaged that such results may provide highly valuable information for future practical applications in fish nutrition.     

Synthesis of alkyl‐branched fatty acids

Alkyl‐branched fatty compounds are of interest for industrial products in the cosmetics and lubricant areas. In this review, clay‐ and zeolite‐catalyzed isomerizations of unsaturated fatty compounds, especially of oleic acid, are discussed. While clay‐catalyzed reactions give most complex mixtures of dimeric fatty acids and of monomeric so‐called “isostearic acid”, the zeolite‐catalyzed process yields preferentially an isomeric mixture of isostearic acids having the methyl branch on the 8–14 positions of the alkyl chain. Synthetically useful additions of alkyl radicals can only be performed on ω‐unsaturated fatty compounds, whereas perfluoroalkyl iodides were added to fatty compounds with terminal as well as internal double bonds using electron transfer‐initiated radical addition reactions. Electrophilic additions of alkyl carbenium ions generated by decomposition of alkyl chloroformates by ethylaluminum sesquichloride give well‐defined alkyl‐branched oleochemicals with good yields.     

Phospholipid synthesis inS. cerevisiae strain GL7 grown without unsaturated fatty acid supplements

In the absence of exogenous unsaturated fatty acids (UFA),Saccharomyces cerevisiae strain GL7 synthesizes low levels of UFA and large amounts of decanoic, dodecanoic and tetradecanoic fatty acids. Supplementation with hemin leads to slightly higher levels of UFA, but synthesis of the medium-chain saturated fatty acids (SFA) continues. Under these conditions of limited UFA availability, strain GL7 incorporates most of its UFA into phosphatidylethanolamine (PE), whereas phosphatidylcholine (PC) and phosphatidylserine+phosphatidylinositol (PS+PI) are enriched with the mediumchain SFA. The association of specific fatty acids with the various phospholipids is not accompanied by changes in the proportions of newly synthesized phospholipids, demonstrating that the fatty acid composition of PE can be modulated independently of the other phospholipids. The effect of sterol structure on the fatty acid composition of cells grown with limiting UFA was also examined. Yeast cells grown with either ergosterol or stigmasterol contained less UFA and more medium-chain SFA in their phospholipids than did cholesterol-grown cells, suggesting that the former sterols allow strain GL7 to grow with a lower UFA content. Portions of this paper were presented at the AOCS 72nd Annual Meeting, New Orleans, May 1981.     

The impact of dietary mono‐ and poly‐unsaturated fatty acids on risk factors for atherosclerosis in humans

The fatty acid composition of the diet has various effects on atherosclerosis risk factors. Dietary saturated fatty acids (SFA) and trans‐unsaturated fatty acids increase the low‐density lipoprotein (LDL)‐/high‐density lipoprotein (HDL)‐cholesterol ratio in serum, while these fats do not have a significant bearing on serum triglyceride levels. By contrast, dietary monounsaturated fatty acids (MUFA), n‐6 polyunsaturated fatty acids (PUFA), and α‐linolenic acid (C18:3n‐3) similarly reduce LDL cholesterol concentrations, while their influence on serum HDL cholesterol and triglycerides is not appreciable. Dietary long‐chain n‐3 PUFA slightly increase serum LDL cholesterol concentrations, but are nevertheless considered salubrious with regard to serum lipids due to the distinct triglyceride‐lowering effects. MUFA‐rich compared to n‐6 PUFA‐rich diets strongly reduce the in vitro oxidizability of LDL. The available studies on this subject also suggest that n‐3 PUFA in the small amounts usually present in the diet are not unduly harmful. These findings are consistent with reports from observational studies: the amount of SFA is positively and the amount of MUFA and n‐6 PUFA in the diet is inversely associated with the risk of cardiovascular disease in most epidemiological studies. The available studies have had an impact on current dietary guidelines, which unanimously recommend that most of the dietary fat should be in the form of MUFA, while the amount of SFA and trans fatty acids in the diet should be as low as possible.     

“Click Chemistry” for the Simple Determination of Fatty‐Acid Uptake and Degradation: Revising the Role of Fatty‐Acid Transporters

Fatty acids (FAs) have numerous functions in all living organisms, ranging from structural roles and energy production to the biosynthesis of secondary metabolites. Because of the high energy content of exogenous FAs, their acquisition is central of metabolism, and several biological systems are known, although their precise roles are not yet entirely clear. We investigated the roles of FadD (CoA ligase) and FadL (FA transporter) in different bacterial strains by using an improved version of click‐chemistry‐assisted labelling of azido‐FAs. The high sensitivity of this method allows a direct and precise assessment of FA metabolism, and is thus far better suited than growth experiments. Our results show that although FA activation is indeed essential for FA degradation, their transport can be independent of transporters like FadL.     

Polyoxyethylene esters of fatty acids: an alternative synthetic route for high selectivity of monoesters

Esterification reaction of fatty acids with polyoxyethylenes has been studied in the presence of solid acid catalysts and the results are compared with those obtained with a classical homogeneous catalyst, p-toluene sulphonic acid. Solid acid catalysts showed very high selectivities for monoesters even under less favorable reaction conditions of 1:1 molar ratios of oleic acid to PEG. Excess amount of PEG is normally required to ensure high selectivity for monoesters in the presence of homogeneous catalysts. The yields for monoesters given by heterogeneous catalysts such as zeolites, HPA and nafion were comparable to that with p-toluene sulphonic acid after 24 h of reaction. Results suggest that solid acid catalysts are potential catalysts for selective synthesis of monoesters from oleic acids and PEG even under severe reaction conditions. A screening study of different solid acid catalysts showed ZSM-5, zeolite-β, and nafion to be better catalysts than zeolite USY and mordenite, the former giving higher selectivity and yield to monoesters. The results suggest that the most important parameters in optimization catalyst performance for monoesters production is the acid strength and less so on other properties such as shape selectivity and concentration of active sites.     

Synthesis and physical properties of isostearic acids and their esters

Saturated branched‐chain fatty acids (sbc‐FAs) are found as minor constituents in several natural fats and oils. Sbc‐FAs are of interest since they have lower melting points than their linear counterparts and exhibit good oxidative stability; properties that make them ideally suited in a number of applications. We (and others) have previously synthesized sbc‐FAs by clay‐ or zeolite‐catalyzed isomerization of unsaturated linear‐chain fatty acids (ulc‐FAs) to unsaturated branched‐chain fatty acids (ubc‐FAs) that were subsequently hydrogenated to the desired sbc‐FAs. These acid‐catalyzed isomerization reactions, however, proceed in moderate conversion and selectivity. Recently, our group found that H‐Ferrierite zeolite catalyst isomerized ulc‐FAs to their branched‐chain counterparts in high conversion (>95%) and selectivity (85%). This paper reports the use of this type of catalyst for the preparation of a series of sbc‐FAs and their ester derivatives. Selected physical properties of these branched acids and esters such as cloud point (CP) and pour point (PP), cold filter plugging point (CFPP), viscosity index (VI), thermo‐oxidation stability, iodine value, and lubricity are also reported.     

Occurrence of Hexacosapolyenoic Acids 26:7(n‐3), 26:6(n‐3), 26:6(n‐6) and 26:5(n‐3) in Deep‐Sea Brittle Stars from Near the Kuril Islands

Significant amounts of polyunsaturated fatty acids (PUFA) with a chain length of 26 carbon atoms were detected in lipids of five deep water species of Ophiuroidea besides common fatty acids with chain lengths between 14 and 24. By means of hydrogenation, GC–MS of the methyl esters, and 4,4‐dimethyloxazoline (DMOX) derivatives of these C26 acids were characterized as 5,8,11,14,17,20,23‐hexacosaheptaenoic [26:7(n‐3)]; 8,11,14,17,20,23‐hexacosahexaenoic [26:6(n‐3)]; 5,8,11,14,17,20‐ hexacosahexaenoic [26:6(n‐6)]; and 11,14,17,20,23‐hexacosapentaenoic [26:5(n‐3)]. Concentrations of these acids varied from 0.3 to 4.5 mol% of the total FA. In all the samples investigated, the main component of C26PUFA was hexacosaheptaenoic acid 26:7(n‐3). These C26PUFA are localized mainly in polar lipids. The presence of the possible biosynthesis precursors suggests that the C26PUFA are produced by the brittle stars, and are not accumulated from food sources. This finding can also explain the presence of small amounts of the 26:7(n‐3) acid detected earlier in flesh lipids of the roughscale sole Clidoderma asperrimum, which feeds on deep water brittle stars. We suggest a possible scheme of the biosynthesis of C26 PUFA.     

Metathesis of unsaturated fatty acids: Synthesis of long-chain unsaturated-α,ω-dicarboxylic acids

The self-metathesis of readily available monounsaturated FA has the potential of being an important pathway for the synthesis of symmetrical long-chain unsaturated-α,ω-dicarboxylic acids (C₁₈−C₂₆). Previous studies on the self-metathesis of monounsaturated FA esters using ruthenium catalysts in solution, however, suffered from low conversions as a result of the thermodynamic control of the reaction. We have found that the second-generation Grubbs catalyst can effectively catalyze the solvent-free self-metathesis of monounsaturated FA of varying purity (from 90 to 99%) to afford two important products—monounsaturated dicarboxylic acids and hydrocarbons—in very high molar conversions (>80%). This solvent-free self-metathesis reaction also works for monounsaturated FA containing additional functional groups. Reactions were conducted at catalyst loadings as low as 0.005 mol%, and turnover numbers as high as 10,800 could be obtained. This discovery represents an attractive approach to the large-scale production of useful monounsaturated-α,ω-dicarboxylic acids and long-chain unsaturated hydrocarbons by means of this solvent-free ruthenium-catalyzed self-metathesis of readily available monounsaturated FA.     

Benzaldehyde Lyase‐Catalyzed Direct Amidation of Aldehydes with Nitroso Compounds

Benzaldehyde lyase from the Pseudomonas fluorescens catalyzes the reaction of aromatic aldehydes with nitroso compounds and furnishes N‐arylhydroxamic acids in high yields. Aromatic aldehydes and benzoins are converted into enamine‐carbanion‐like intermediates prior to their reaction with nitroso compounds. The kinetic resolution of rac‐2‐hydroxy‐1,2‐diphenylethanones furnished (S)‐benzoins and arylhydroxamic acids with high enantioselectivities and conversions.     

Impact of frying on key fatty acid ratios of canola oil

The study was carried out to investigate the changes in saturated (SFA), monoene (MUFA), trans (TFA), and polyunsaturated (PUFA) fatty acids and the key fatty acid ratios (SFA/UFA, cis PUFA/SFA, C18:2/C16:0 and C18:3/C16:0) during potato chips frying in canola oil using single bounce attenuated total reflectance FTIR (SB‐ATR‐FTIR) spectroscopy. The data obtained from GC‐FID were used as reference. The calibration of main fat groups and their key fatty acid ratios were developed by partial least square (PLS) regression coefficients using 4000 to 650 cm^?1 spectral range. FTIR PLS regression for the predicted SFA, MUFA, TFA, and PUFA were found 0.999, 0.998, 0.998, and 0.999, respectively, whereas for SFA/UFA, cis PUFA/SFA, C18:2/C16:0 and C18:3/C16:0 the regression coefficients were 0.991, 0.997, 0.996, and 0.994, respectively. We conclude that FTIR‐PLS could be used for rapid and accurate assessment of changes in the main fat groups and their key fatty acid ratios ratio during the frying process. Practical applications: FTIR‐ATR method is very simple, rapid, and environmentally friendly. No sample preparation is required and one drop of oil is enough for FTIR analysis. The proposed method could be applied for quick determination of key fatty acid ratios in the food processing industry.     

New 2-Methyl-13-Icosenoic Acid from the Temperate Calcisponge <Emphasis Type="Italic">Leuconia johnstoni</Emphasis>

The fatty acid composition of the temperate calcareous marine sponge Leuconia johnstoni Carter 1871 (Calcaronea, Calcarea) was characterized for the first time in specimens collected off the Brittany coast of France over four years from October 2005 to September 2008. Forty-one fatty acids (FA) with chain lengths ranging from C₁₄ to C₂₂ were identified as fatty methyl esters (FAME) and N-acyl pyrrolidide (NAP) derivatives by gas chromatography–mass spectrometry (GC–MS). Twenty-two saturated fatty acids (SFA) were identified accounting for 52.1–59.0% of the total FA and dimethylacetals (DMA). In addition, among the SFA, we noticed the presence of numerous methyl-branched iso and anteiso FA, suggesting a large number of associated bacteria within L. johnstoni. Thirteen monounsaturated fatty acids (MUFA, 28.0–36.0% of total FA + DMA) were also identified as well as six polyunsaturated fatty acids (PUFA, 4.0–8.2%). A noticeable DMA was detected at a high level, particularly in September 2008 (11.8%), indicating the presence of plasmalogens in this sponge species. This calcareous sponge lacked the non-methylene-interrupted FA (NMI FA) with a Δ5,9 system typical of siliceous Demosponges and Hexactinellids. The occurrence of the unusual 8,13-octadecadienoic acid was reported for the first time as a minor PUFA in a calcareous sponge. The major FA, representing 20–25% of this sponge FA, was identified as the new 2-methyl-13-icosenoic acid from mass spectra of its methyl ester and its corresponding N-acyl pyrrolidide derivatives as well as a dimethyl disulfide adduct.     

Identification and Characterization of Phospholipids with Very Long Chain Fatty Acids in Brewer’s Yeast

Yeast lipids and fatty acids (FA) were analyzed in Saccharomyces pastorianus from seven breweries and in the dietary yeast supplement Pangamin. GC–MS identified more than 30 FA, half of which were very‐long chain fatty acids (VLCFA) with hydrocarbon chain lengths of ≥22 C atoms. Positional isomers ω‐9 and ω‐7 were identified in FA with C18–C28 even‐numbered alkyl chains. The most abundant ω‐7 isomer was cis‐vaccenic acid. The structure of monounsaturated FA was proved by dimethyl disulfide adducts (position of double bonds and cis geometric configuration) and by GC–MS of pyridyl carbinol esters. Ultra‐high performance liquid chromatography‐tandem mass spectrometry with negative electrospray ionization identified the phospholipids phosphatidylethanolamine, phosphatidylinositol and phosphatidylcholine, with more than 150 molecular species. Wild‐type unmutated brewer's yeast strains conventionally used for the manufacture of food supplements were found to contain VLCFA.     

Bio‐Oil as a Potential Biomass‐Derived Renewable Raw Material for Bio‐Phenol Production

A route for directional conversion of bio‐oil into phenol by means of coupling the catalytic cracking of the bio‐oil with the hydroxylation of the bio‐oil‐based benzene‐rich aromatics is proposed. High selectivity for phenol in the resulting organic liquid was achieved, with an almost complete conversion of the bio‐oil. Co‐cracking of the bio‐oil with methanol over a Zn‐modified zeolite significantly enhanced the yields of aromatics and decreased the deactivation of the catalyst during the catalytic cracking of the bio‐oil. The phenol yield depended on the metal oxide catalysts, the temperature, and the reaction time during hydroxylation of the benzene‐rich aromatics. The reaction pathway of converting bio‐oil into phenol was elucidated based on the products identified and the characterization of the catalysts.     

Extraction of pollen lipids by SFE‐CO2 and determination of free fatty acids by HPLC

Rape bee pollen lipids obtained by petrol ether extraction (PEE) or supercritical fluid (carbon dioxide) extraction (SFE) were compared with regard to their free fatty acid (FFA) components. Optimal SFE conditions were selected by carrying out the Taguchi method with an OA9 (3³) matrix design, and are as follows: extraction pressure at 35 MPa, temperature at 45 °C, and dynamic extraction time at 90 min. The lipid yield based on PEE was 7.42 wt‐% and the extracts of the desired analytes based on SFE varied in the range of 3.23–5.58 wt‐% under different conditions. With the optimized procedure, the lipid yield was 6.09 wt‐%. The FFA in the lipids were separated with a pre‐column derivation method and 1‐[2‐(p‐toluenesulfonate) ethyl]‐2‐phenylimidazole [4,5‐f]9,10‐phenanthrene as labeling regent, followed by high‐pressure liquid chromatography (HPLC) with fluorescence detection. HPLC analysis shows that the lipids contain abundant unsaturated fatty acids (UFA) in high to low concentrations as follows: linolenic acid (18:3), oleic acid (18:1), linoleic acid (18:2), nervonic acid (24:1), and lignoceric acid (20:4). The UFA contents in the SFE extracts were higher than those after PEE. The results indicated that SFE under suitable conditions is more selective than conventional PEE with regard to lipid extraction and preservation of their quality.     

Branched‐chain fatty acids as activators of peroxisome proliferator‐activated receptors

We observed earlier that phytanic acid activated subtype α of the peroxisome proliferator‐activated receptor (PPAR) via the cytosolic liver‐type fatty acid‐binding protein (L‐FABP). In a further search for minor lipid nutrients that interact with genes, we explored here the potential of branched‐chain fatty acids to serve as agonists for the PPAR subtypes α, β and γ in rodent and human molecular test systems. Beyond chlorophyll‐derived pristanic and phytanic acids, bacteria‐derived iso‐ and anteiso‐fatty acids and avian‐derived ‘uropygial’ fatty acids were tested by transactivation assay. In addition, we studied binding of these fatty acids to recombinantly expressed PPAR ligand binding domains (LBDs) and to L‐FABP by competition with fluorescent parinaric acid. In contrast to single methyl‐branched agonists, multi methyl‐branched fatty acids had high transactivation potentials in either test system; in addition, some agonists of the latter were highly subtype selective. Multi methyl‐branched chain fatty acids were superior activators of human PPARγ, a preference not seen in the murine test system. High‐affinity binding of isoprenoid‐derived pristanic and phytanic acids to PPARγ‐LBD and to L‐FABP was observed, and also of pristanic acid to PPARα‐LBD. Polyketidic uropygial fatty acids bound to PPARγ‐LBD only, though weakly. As both isoprenoid and polyketidic fatty acids showed high activation potentials, it became clear that binding data determined in vitro cannot predict biological activity as determined by transactivation assay. For pristanic acid, however, a signalling path similar to that found for phytanic acid can be concluded. Taken together, multi methyl‐branched fatty acids of the human food chain can affect cellular metabolism through regulating gene expression.     

Direct Access to Medium‐Chain α,ω‐Dicarboxylic Acids by Using a Baeyer–Villiger Monooxygenase of Abnormal Regioselectivity

Baeyer–Villiger monooxygenases (BVMOs) are versatile biocatalysts in organic synthesis that can generate esters or lactones by inserting a single oxygen atom adjacent to a carbonyl moiety. The regioselectivity of BVMOs is essential in determining the ratio of two regioisomers for converting asymmetric ketones. Herein, we report a novel BVMO from Pseudomonas aeruginosa (PaBVMO); this has been exploited for the direct synthesis of medium‐chain α,ω‐dicarboxylic acids through a Baeyer–Villiger oxidation–hydrolysis cascade. PaBVMO displayed the highest abnormal regioselectivity toward a variety of long‐chain aliphatic keto acids (C₁₆–C₂₀) to date, affording dicarboxylic monoesters with a ratio of up to 95 %. Upon chemical hydrolysis, α,ω‐dicarboxylic acids and fatty alcohols are readily obtained without further treatment; this significantly reduces the synthetic steps of α,ω‐dicarboxylic acids from renewable oils and fats.