Fatty acids. Part 50. 13C nuclear magnetic resonance studies of olefinic fatty acids and esters

The ¹³C-NMR spectra of 48 cis alkenoic acids and esters (C₈–C₂₀), 18 trans alkenoic acids and esters (C₉–C₁₈), and 26 polyenoic acids and esters (C₁₈–C₂₂) are reported and interpreted. The characteristic features of such spectra which permit structural assignments to be made are discussed.     

Free and bound sterols in seedlings of Cucumis sativus

Free sterols, sterol esters, sterol monoglucosides and sterol acylmonoglucosides have been obtained from 10 days old seedlings of Cucumis sativus. Free sterols and sterol esters consist mainly of Δ⁷ di- and triunsaturated sterols, whereas Δ⁷ mono-unsaturated and Δ⁵ mono- and diunsaturated sterols predominate in the glucosides and acylglucosides. Both acetates and derivatives of higher fatty acids, mainly linoleic and linolenic acids, have been found in the sterol esters. Sterol acylglucosides contain mostly saturated fatty acids, palmitic and stearic acids being the main components.     

Early alterations induced by carbon tetrachloride in the lipids of the membranes of the endoplasmic reticulum of the liver cell I. Separation and partial characterization of altered lipids

Alterations induced by carbon tetrachloride poisoning in fatty acids of liver microsomal lipids were studied. Thin layer chromatography of fatty acid methyl esters prepared from liver microsomal lipids, revealed, in the CCl₄-treated rats, the presence of a component (the “D” spot) with an R_f value lower than that of the methyl esters. The lipids recovered from this component showed a marked diene conjugation absorption when examined spectrophotometrically over the UV range, while the lipids recovered from the spot of the methyl esters showed no absorption of conjugated dienes.Studies carried out with labelled carbon tetrachloride indicated that compounds present in the “D” spot contained 28% of ¹⁴C applied to the chromatoplate. The spot of the methyl esters (the “M” spot) contained 42% of ¹⁴C applied to the chromatoplate. However, specific activity of the “D” spot was about 1000 times greater than specific activity of the “M” spot.The lipids recovered from either the “D” spot or the spot of the methyl esters were analyzed separately by gas-liquid chromatography (GLC) with an electron capture detector (ECD). It was found that the lipids recovered from the “D” spot showed no response, while those recovered from the spot of the methyl esters exhibited the response of the ECD, which was similar to that observed with the unfractionated fatty acid methyl esters. The lack of the response of the ECD for compounds in the “D” spot appears to be due to the fact that they cannot be eluted from the column.On the basis of the analytical results, it can be postulated that the “D” spot contains compounds formed by a chain termination addition reaction of free radicals derived from CCl₄ (probably trichloromethyl free radicals) to fatty acid free radicals containing conjugated dienes. On the other hand, the spot of the methyl esters appears to contain also, together with unmodified fatty acids, the fatty acids in which a simple addition of CCl₄ free radicals to double bonds has occurred.     

Halo lipids. V. Synthesis,nuclear magnetic resonance spectra and cytostatic properties of halo analogues of alkyllysophospholipids

A series of fluorine- and chlorine-containing analogues of alkyllysophospholipids has been synthesised as potential antimetabolites of phospholipids. These compounds include various structural isomers of racemic long-chain alkyldeoxyhaloglycerophosphocholines and N,N-dimethylethanolamines, alkyldeoxyhaloglycerophosphoric acids, and alkyl esters. ¹H- and ¹⁹F-NMR spectroscopic data are presented and analysed. Alkyldeoxyhaloglycerophosphocholines were found to exhibit a strong inhibitory effect on the proliferation of Ehrlich ascites carcinoma cells in vitro.     

Expansion of the ω‐oxidation system AlkBGTL of Pseudomonas putida GPo1 with AlkJ and AlkH results in exclusive mono‐esterified dicarboxylic acid production in E. coli

The AlkBGTL proteins coded on the alk operon from Pseudomonas putida GPo1 can selectively ω‐oxidize ethyl esters of C6 to C10 fatty acids in whole‐cell conversions with Escherichia coli. The major product in these conversions is the ω‐alcohol. However, AlkB also has the capacity to overoxidize the substrate to the ω‐aldehyde and ω‐acid. In this study, we show that alcohol dehydrogenase AlkJ and aldehyde dehydrogenase AlkH are able to oxidize ω‐alcohols and ω‐aldehydes of esterified fatty acids respectively. Resting E. coli expressing AlkBGTHJL enabled exclusive mono‐ethyl azelate production from ethyl nonanoate, with an initial specific activity of 61 U g_cdw^?1. Within 2 h, this strain produced 3.53 mM mono‐ethyl azelate, with a yield of 0.68 mol mol^?1. This strain also produced mono‐ethyl dicarboxylic acids from ethyl esters of C6 to C10 fatty acids and mono‐methyl azelate from methyl nonanoate. Adding ethyl nonanoate dissolved in carrier solvent bis‐(2‐ethylhexyl) phthalate enabled an increase in product titres to 15.55 mM in two‐liquid phase conversions. These findings indicate that E. coli expressing AlkBGTHJL is an effective producer of mono‐esterified dicarboxylic acids from fatty acid esters.     

2H- and13C-labeled amino acids generated by obligate methylotrophs Biosynthesis and MS monitoring

Summary Biosynthetic preparation of²H- and¹³C- labeled amino acids was studied using a leucine-producing mutant of the obligate methylotroph,Methylobacillus flagellatum. The strain was cultivated in various media containing¹³C- or²H-analogs of methanol. The total protein from each experiment was subjected to acid hydrolysis and converted into a mixture of dansyl amino acid methyl esters. The samples of excreted leucine were converted into methyl esters of dansyl and benzyloxycarbonyl derivatives. Electron impact mass spectrometry was performed to detect stable isotope enrichment of the amino acids. According to the mass spectrometric analysis it is feasible to use methylotrophic microorganisms for the preparation of²H- and¹³C- analogs of amino acids by labeled methanol bioconversion; the excreted amino acids can be convenient for express analysis as an indicator of isotopic enrichment of the total protein. The data obtained testified to a high efficiency of dansyl derivatization for mass spectrometric analysis of complex amino acid mixtures.     

A search for an ‘Ouabain-like’ substance from the electric organ of Electrophorus electricus which led to arachidonic acid and related fatty acids

The electric organ of Electrophorus electricus contains substances which inhibit (Na⁺ + K⁺)-ATPase activity, the specific binding of [³H]ouabain to purified (Na⁺ + K⁺)-ATPase and ⁸⁶Rb⁺ uptake by chick cardiac cells in culture. The active organic material was extracted from microsomal membranes. Its purification was carried out by chromatography on Sep-Pak C-18 and thin-layer chromatography. Reverse-phase liquid chromatography and mass spectrometry identified the active material as a mixture of unsaturated fatty acids. Linoleic (18:2), arachidonic (20:4), linolenic (18:3) and docosahexaenoic acids (22:6) contributed to about 60% of the total activity of the active material. The other active substances could be arachidonic analogs, since they have both a lipophilic and carboxylic character. Pure unsaturated fatty acids have been shown to be active in the different biological assays used to analyze the endogenous ‘ouabain-like’ activity. Linolenic, arachidonic and docosahexaenoic acids were the most active, whereas saturated fatty acids and glyceryl esters or methyl esters of unsaturated fatty acids were inactive. It is possible that in pathological situations in which the level of unsaturated fatty acids increases, these molecules may then act as physiological inhibitors of the sodium pump.     

The interaction of amino acids with o-phthaldialdehyde: A kinetic study and spectrophotometric assay of the reaction product

A detailed study has been made of the kinetics of interaction between amino acids and esters of amino acids and o-phthaldialdehyde in the presence of mercaptoethanol. The reaction products have been characterized. A spectrophotometric method for quantitative analysis of all amino acids, except proline and hydroxyproline, has been developed. The possibility of determination of amino acid esters in mixtures containing free amino acids has been demonstrated. It is noted that determination of glycine and histidine with the help of o-phthaldialdehyde has certain specificities associated with faster, compared to other amino acids, degradation of their derivatives. Optimal conditions for quantitative analysis of amino acids in solutions of higher than 10^?5m concentration are recommended. The reproducibility of the determination was ±2%.     

Branched Chain Amino Acid Metabolism in the Biosynthesis of Lycopersicon pennellii Glucose Esters

Lycopersicon pennellii Corr. (D'Arcy) an insect-resistant, wild tomato possesses high densities of glandular trichomes which exude a mixture of 2,3,4-tri-O-acylated glucose esters that function as a physical impediment and feeding deterrent to small arthropod pests. The acyl moieties are branched C₄ and C₅ acids, and branched and straight chain C₁₀, C₁₁, and C₁₂ acids. The structure of the branched acyl constituents suggests that the branched chain amino acid biosynthetic pathway participates in their biosynthesis. [¹⁴C]Valine and deuterated branched chain amino acids (and their oxo-acid derivatives) were incorporated into branched C₄ and C₅ acid groups of glucose esters by a process of transamination, oxidative decarboxylation and subsequent acylation. C₄ and C₅ branched acids were elongated by two carbon units to produce the branched C₁₀-C₁₂ groups. Norvaline, norleucine, allylglycine, and methionine also were processed into acyl moieties and secreted from the trichomes as glucose esters. Changes in the acyl composition of the glucose esters following sulfonylurea herbicide administration support the participation of acetohydroxyacid synthetase and the other enzymes of branched amino acid biosynthesis in the production of glucose esters.     

Proteinogenic amino acids labelled with 15N and/or 13C for application in peptide synthesis: A short review with a comprehensive list of published derivatives

A review is given of the literature dealing with the most common protected derivatives of ¹⁵N- and/or ¹³C-labelled amino acids of interest in peptide synthesis. The list contains all such Boc-, Z- and Fmoc-amino acids as well as published methyl, ethyl, t-butyl and benzyl esters.     

Lipids in digestive gland of Littorina saxatilis rudis (Maton) and in daughter sporocysts of Microphallus similis (Jäg. 1900)

Parasitism by sporocysts was associated with a slight decrease in triglycerides and fatty acids in host digestive gland cells suggesting that the parasite digests, absorbs, and metabolises fats of host origin. An increase in phospholipids and a marked increase in the incorporation of acetate-1-¹⁴C in parasitised digestive glands may be the result of attempted cell regeneration. The parasite had less monoglycerides, triglycerides, and fatty acids but took up more palmitate-1-¹⁴C than the host. Sterols and sterol esters are more concentrated and more readily synthesised in the parasite than in the host. The relatively high level of phospholipids in the former may be related to number of cercariae within the sporocysts.     

Photosynthesis in Rhodospirillum rubrum. I. Autotrophic Carbon Dioxide Fixation

The incorporation and distribution of activity from ¹⁴CO₂ was investigated under autotrophic conditions in the facultative photoautotroph, Rhodospirillum rubrum, with cells cultured on hydrogen, carbon dioxide, and ammonium sulfate. In 1 second ¹⁴CO₂ fixation experiments essentially all of the activity was found in 3-phosphoglyceric acid: plotted against time percent incorporation into phosphate esters has a strikingly negative slope. These results suggest that under autotrophic conditions the reductive pentose phosphate cycle or the key reactions of the cycle play a major role in carbon metabolism in this photosynthetic bacterium. Incorporation into amino acids and into intermediates of the tricarboxylic acid cycle was quite low.     

Biological activity of some synthetic gibberellin glucosyl esters

Four gibberellin (GA₁, GA₃, GA₄ and GA₃₇) glucosyl esters were synthesized and found to be as active as their respective free acids in the rice seedling bioassay. The rapid hydrolysis of the glucosyl esters in rice seedlings was demonstrated by feeding experiments with glucosyl esters of [³H]GA₁ and [³H]GA₄.     

Biosynthesis of wax esters in tissues of Sinapis alba L. seeds

The biosynthesis of wax esters has been investigated in maturing seeds of Sinapis alba. Exogenous long-chain alcohols are incorporated exclusively into alkyl moieties of wax esters. Oxidation of the long-chain alcohols is not detected. Exogenous fatty acids are incorporated into acyl moieties of wax esters to a low extent. A reduction of fatty acids to alcohols is not observed. Synthesis of wax esters is localized exclusively in the testa; both outer and inner integument are equally active in wax ester biosynthesis. The biosynthesis of wax esters is specific with regard to both chain length and degree of unsaturation of long-chain alcohols. Exogenous and endogenous sterols are not esterified.     

Specific features of phase transfer catalytic glycosylation of aromatic hydroxy acids

Reactions of peracetylated α-D-glucosaminyl chloride with isomeric hydroxybenzoic and 1-hydroxy-2-naphthoic acids in a solid phase transfer system of potassium carbonate-acetonitrile were studied. It was found that the nature of carboxylic acids, lipophilicity of the phase transfer catalyst, and reaction temperatures affected the reaction composition and product yields. The O-β-glycosyl esters of ortho-hydroxyaromatic acids were first found to form anomeric 1,2-cis derivatives in the presence of potassium carbonate. The structures of the synthesized compounds were confirmed by ¹H NMR spectroscopy. As was shown in vivo experiments, the analgesic activities of glycosyl esters of salicylic acid and peracetylated 2-carboxyphenylglucosaminide were comparable with that of aspirin.     

Flavaspidic acid: a biochemical probe for the study of metabolic pathway in hepatocytes.

Displacement of fatty acids or their CoA esters from the cytosolic fatty acid-binding-protein (FABP) by β-flavaspidic acid-N-methyl-glucaminate (flavaspidic acid) shifted the flow of lactate from the lipogenic to the gluconeogenic pathway in hepatocytes isolated from livers of fed rats. Analyses of radiolabel in products of 1 hour incubations utilizing 40 mM DL-sodium lactate (U-¹⁴C sodium lactate or ³H₂O) as substrate showed fatty acid synthesis to be depressed in the presence of 5 mM flavaspidic acid to levels ranging to 28% of control. Glucose synthesis, on the other hand, rose to a level 809% of control, a rate approximately equivalent to the maximal rate of gluconeogenesis found in hepatocytes from diabetic rats. The production of ketone bodies was not influenced by flavaspidic acid whereas CO₂ production decreased reflecting the diversion of the flow of lactate from the lipogenic to the gluconeogenic pathway.These results are presented in support of the hypothesis that unbound long-chain acyl CoA esters act as physiological effectors of the adenine nucleotide translocation and through this action, integrate lipogenic and gluconeogenic activities. Flavaspidic acid thus serves as a useful probe for studies of the role of long chain acyl CoA esters in the intracellular regulation of intermediary metabolism and of the modulation exerted by the FABP.     

Phenolic O-methyltransferase from Lentinus lepideus (basidiomycete)

The fungus, Lentinus lepideus, produces crystalline methyl p-methoxycinnamate in stationary cultures. O-methylation and methyl ester formation of hydroxycinnamic acids were examined with enzyme preparations of the fungus. Using S-adenosylmethionine-¹⁴CH3, it was found that only the methyl esters of the hydroxycinnamic acids are substrates for O-methylation and not the free acids. Benzoic acids and their methyl esters are not substrates. The activity of the enzyme is p-specific and its specific activity decreases with increasing number of hydroxyl groups in the substrate. The same enzyme preparations catalyze the formation of the methyl ester of cinnamic acid from the free acid.     

Dienoic acids,synthesis and 13C NMR spectral analysis

A preparation of E,E-2,4-dienoic acids, together with the assignments of their ¹³C NMR signals and the shifts observed after transformation into their sodium salts, is described. The stereochemistry of the double bonds of 3,5-dienoic esters, obtained from 2,4-dienoic acids, on the basis of ¹³C NMR data, is also presented.     

SIMULTANEOUS INCORPORATION OF ORALLY ADMINISTERED [9, 10-3H2]OLEIC AND [1-14C]LINOLEIC ACIDS INTO LIPIDS OF THE ADULT RAT BRAIN

—The incorporation of an orally administered mixture of [9,10-³H₂joleic acid and [1-¹⁴C]linoleic acid into the brain and spinal cord lipids was maximal after 24 h compared with 4 h for extraneural tissue. In the latter, both acids were utilized equally well for triglyceride biosynthesis, but linoleate entered phosphatidylcholine more rapidly than oleate. Oleic acid was preferentially incorporated into newly synthesized cholesterol esters although 4 h after dosing most cholesterol esters present in serum were formed preferentially from linoleate presumably by the action of lecithin-cholesterol acyl transferase. In neural tissue, a considerable amount of [1-¹⁴C]linoleate was metabolized to higher polyunsaturated fatty acids, whereas in the case of oleate, 90 per cent of the tritium activity remained in monoenic acids at all time periods studied. Both acids were initially incorporated most rapidly into the lecithin fraction of brain and spinal cord, but after 7 days diacyl phosphatidylethanolamine had the highest specific activity. These data are consistent with the view that the uptake of labelled fatty acids by the brain takes place principally as free acids but that some uptake of esterified forms, probably largely as phosphatidylcholine, also occurs. The low linoleate content of the brain and probably also of cerebrospinal fluid cannot be explained on the basis of a selective restriction on the uptake of this lipid from plasma.     

Lipase catalyzed reaction of L-ascorbic acid with cinnamic acid esters and substituted cinnamic acids

To perform the lipase-catalyzed synthesis of L-ascorbic acid derivatives from plant-based compounds such as cinnamic and ferulic acid under mild reaction conditions, the activities of immobilized Candida ntarctica lipase with different cinnamic acid esters and substituted cinnamic acids were compared. As a result, immobilized C. ntarctica lipase was found to prefer vinyl cinnamic acid to other esters such as allyl-, ethyl-, and isobutyl cinnamic acids as well as substituted cinnamic acids such as p-coumaric acid, caffeic acid, ferulic acid, and sinapic acid. Based on these results, large-scale synthesis of 6-O-cinnamyl-L-ascorbic acid ester was performed using immobilized C. ntarctica lipase in dry organic solvent, resulting in 68% yield (493 mg) as confirmed by ¹³C-NMR.