The first total synthesis of the marine fatty acid (+/-)-2-methoxy-13-methyltetradecanoic acid: a cytotoxic fatty acid to leukemia cells

The recently discovered marine fatty acid (+/-)-2-methoxy-13-methyltetradecanoic acid was synthesized for the first time in six steps (26% overall yield) starting from commercially available methyl 12-methyltridecanoate. The synthetic approach provided enough material to corroborate the structure of the acid, which was recently identified in the sponge Amphimedon complanata from Aguadilla, Puerto Rico, and to test its cytotoxicity to three leukemia cell lines. The key step in the synthesis was the addition of trimethylsilyl cyanide to 12-methyltridecanal under triethylamine catalysis. Nuclear magnetic resonance data are provided for the first time for this methoxylated fatty acid and the synthetic approach utilized is of general applicability since it can be used in the synthesis of other methyl-branched 2-methoxylated fatty acids. We also report that the acid (+/-)-2-methoxy-13-methyltetradecanoic acid is cytotoxic to human chronic myelogenous leukemia K-562 (EC50=238 microM), histiocytic lymphoma U-937 (EC50=250 microM), and promielocytic leukemia HL-60 (EC50=476 microM) in RPMI 1640 medium.     

Facile syntheses for (5Z,9Z)-5,9-hexadecadienoic acid, (5Z,9Z)-5,9-nonadecadienoic acid, and (5Z,9Z)-5,9-eicosadienoic acid through a common synthetic route

The delta 5,9 fatty acids (5Z,9Z)-5,9-hexadecadienoic acid, (5Z,9Z)-5,9-nonadecadienoic acid, and (5Z,9Z)-5,9-eicosadienoic acid were synthesized for the first time in four steps (9-12% overall yield) starting from commercially available 2-(2-bromoethyl)-1,3-dioxolane. The synthetic approach provided enough material to corroborate the structure and stereochemistry of (5Z,9Z)-5,9-nonadecadienoic acid which was recently identified in the flowers of Malvaviscus arboreus (Malvaceae). The novel phospholipids 1-hexadecanoyl-2-[(5Z,9Z)-5,9-eicosadienoyl]-sn-glycer o-3-phosphocholine and 1-octadecanoyl-2-[(5Z,9Z)-5,9-eicosadienoyl]-sn- glycero-3-phosphocholine were also synthesized from commercially available L-alpha-phosphatidylcholine (egg yolk) and characterized by positive ion electrospray mass spectrometry. These are the first examples of unsymmetrical phospholipids with saturated fatty acids at the sn-1 position and delta 5,9 fatty acids at the sn-2 position.     

First total synthesis of (5Z,9Z)-(+/-)-2-methoxy-5,9-octadecadienoic acid, a marine derived methoxylated analog of taxoleic acid

The first total synthesis for the sponge derived (5Z,9Z)-(+/-)-2-methoxy-5,9-octadecadienoic acid, an analog of taxoleic acid, was accomplished in seven steps and in a 10% overall yield. It was again corroborated that the best strategy to prepare these cis,cis dimethylene interrupted double bonds is the double-alkyne bromide coupling reaction of 1,5-hexadiyne, which provides the advantage of achieving a 100% cis stereochemical purity for both double bonds after hydrogenation under Lindlar conditions. The alpha-methoxy functionality was best prepared via the Mukaiyama reaction of (4Z,8Z)-heptadecadienal with trimethylsilyl cyanide and triethylamine followed by acid hydrolysis. Selective methylation of the hydroxyl group of (5Z,9Z)-(+/-)-2-hydroxy-5,9-octadecadienoic acid was achieved with sodium hydride/methyl iodide when tetrahydrofuran was used as solvent. Complete spectral data is presented, for the first time, for this unusual marine alpha-methoxylated fatty acid.     

First total synthesis and antileishmanial activity of (Z)-16-methyl-11-heptadecenoic acid, a new marine fatty acid from the sponge Dragmaxia undata

The first total synthesis for the (Z)-16-methyl-11-heptadecenoic acid, a novel fatty acid from the sponge Dragmaxia undata, was accomplished in seven steps and in a 44% overall yield. The use of (trimethylsilyl)acetylene was key in the synthesis. Based on a previous developed strategy in our laboratory the best synthetic route towards the title compound was first acetylide coupling of (trimethylsilyl)acetylene to the long-chain protected 10-bromo-1-decanol followed by a second acetylide coupling to the short-chain 1-bromo-4-methylpentane, which resulted in higher yields. Complete spectral data is also presented for the first time for this recently discovered fatty acid and the cis double bond stereochemistry of the natural acid was established. The title compound displayed antiprotozoal activity against Leishmania donovani (IC₅₀ = 165.5 ± 23.4 μM) and inhibited the leishmania DNA topoisomerase IB enzyme (LdTopIB) with an IC₅₀ = 62.3 ± 0.7 μM.     

First total synthesis and antiprotozoal activity of (Z)-17-methyl-13-octadecenoic acid, a new marine fatty acid from the sponge Polymastia penicillus

The first total synthesis for the (Z)-17-methyl-13-octadecenoic acid was accomplished in seven steps and in a 45% overall yield. The use of (trimethylsilyl)acetylene was key in the synthesis. Based on a previous developed strategy in our laboratory the best synthetic route towards the title compound was first acetylide coupling of (trimethylsilyl)acetylene to the long-chain protected 12-bromo-1-dodecanol followed by a second acetylide coupling to the short-chain 3-methyl-1-bromobutane, which resulted in higher yields. Complete spectral data is also presented for the first time for this recently discovered fatty acid. The title compound displayed antiprotozoal activity against Leishmania donovani (EC₅₀ = 19.8 μg/ml) and inhibited the leishmania DNA topoisomerase IB at concentrations of 50 μM.     

Microwave-assisted synthesis of deuterium labeled estrogen fatty acid esters

Deuterated analogs of estrogen fatty acid esters are needed as internal standards for isotope dilution GC/MS analyses. We have developed a rapid and efficient synthesis for 2,4,16,16-D4-estrone palmitate, stearate, oleate, linoleate, and linolenate and the corresponding 2,4,16,16,17alpha-D5-estradiol fatty acid 17-mono and 3,17-diesters using analogous fatty acid chlorides or fatty acid anhydrides and 4-(dimethylamino)pyridine under microwave irradiation. Chemoselective hydrolysis of fatty acid diesters was carried out by KOH in t-BuOH.     

Fatty acid synthesis is a target for antibacterial activity of unsaturated fatty acids

Long-chain unsaturated fatty acids, such as linoleic acid, show antibacterial activity and are the key ingredients of antimicrobial food additives and some antibacterial herbs. However, the precise mechanism for this antimicrobial activity remains unclear. We found that linoleic acid inhibited bacterial enoyl-acyl carrier protein reductase (FabI), an essential component of bacterial fatty acid synthesis, which has served as a promising target for antibacterial drugs. Additional unsaturated fatty acids including palmitoleic acid, oleic acid, linolenic acid, and arachidonic acid also exhibited the inhibition of FabI. However, neither the saturated form (stearic acid) nor the methyl ester of linoleic acid inhibited FabI. These FabI-inhibitory activities of various fatty acids and their derivatives very well correlated with the inhibition of fatty acid biosynthesis using [(14)C] acetate incorporation assay, and importantly, also correlated with antibacterial activity. Furthermore, the supplementation with exogenous fatty acids reversed the antibacterial effect of linoleic acid, which showing that it target fatty acid synthesis. Our data demonstrate for the first time that the antibacterial action of unsaturated fatty acids is mediated by the inhibition of fatty acid synthesis.     

Rapid stable isotope dilution analysis of very-long-chain fatty acids, pristanic acid and phytanic acid using gas chromatography–electron impact mass spectrometry

A common feature of most peroxisomal disorders is the accumulation of very-long-chain fatty acids (VLCFAs) and/or pristanic and phytanic acid in plasma. Previously described methods utilizing either gas chromatography alone or gas chromatography–mass spectrometry are, in general, time-consuming and unable to analyze VLCFAs, pristanic and phytanic acid within a single analysis. We describe a simple, reproducible and rapid method using gas chromatography/mass spectrometry with deuterated internal standards. The method was evaluated by analysing 30 control samples and samples from 35 patients with defined peroxisomal disorders and showed good discrimination between controls and patients. This method is suitable for routine screening for peroxisomal disorders.     

Glucose stimulates a decrease of the fatty acid saturation degree in Acinetobacter calcoaceticus

The fatty acid composition of Acinetobacter calcoaceticus 69-V was determined under various growth conditions. Saturated, unsaturated, and hydroxy fatty acids with chain lengths of 12–18 carbon atoms predominated in the fatty acid profile. With acetate or propanol as growth substrates, the ratio of saturated to unsaturated fatty acids varied with changes in the temperature. This was the only adaptive mechanism detected that compensated for the physical effects of temperature alterations on the cell membranes. The fatty acid composition of A. calcoaceticus grown at 40 °C had a saturation degree of approximately 50%; after growth at 20 °C it was approximately 35%. In the presence of a carbon and energy source, A. calcoaceticus was able to respond to temperature reductions under oxic conditions regardless of whether fatty acid biosynthesis was inhibited or not. This suggests an aerobic mechanism of fatty acid biosynthesis and the involvement of a fatty acid desaturase system. Addition of the non-growth substrate, glucose, helped the organism to adapt to lower temperature. The molecular mechanism of the aid is not really understood. The oxidation of glucose could provide the desaturase either with electrons directly via a pyrrolo-quinoline-quinone-linked glucose dehydrogenase or with NADH after fatty acid degradation has been initiated by ATP generated by the oxidation of glucose. Received: 19 June 1998 / Accepted: 28 December 1998     

Carotenoid-to-chlorophyll ratio as a proxy for assay of total fatty acids and arachidonic acid content in the green microalga Parietochloris incisa

The relationships between pigment (carotenoid and chlorophyll) content with accumulation of total fatty acids (TFA) and arachidonic acid (AA) were studied in the green microalga Parietochloris incisa (Trebouxiophyceae, Chlorophyta) grown under different PFDs (35, 200, and 400 μmol photons m⁻² s⁻¹) and nitrogen availabilities. The growth of P. incisa under higher light and nitrogen deficiency was accompanied by accumulation of FA, an increase in carotenoid and a decline in chlorophyll content. It was found that the carotenoid-to-chlorophyll ratio (but not the individual pigment content) correlates closely with the volumetric content of both TFA and AA. Analysis of scattering-compensated absorption spectra of P. incisa suspensions revealed their tight relationship in the blue-green range of the spectrum with the carotenoid-to-chlorophyll ratio, TFA, and AA content. These findings allowed the development of algorithms for the non-destructive assay of TFA and AA in cell suspensions in the ranges of 0.09–3.04 and 0.04–1.7 μg mL⁻¹, with accuracy of 0.06 and 0.01 μg mL⁻¹, respectively, via analytically measured carotenoid-to-chlorophyll ratio and using the ratio of absorption coefficients at 510 and 678 nm, with accuracy of 0.07 and 0.02 μg mL⁻¹, respectively. The feasibility of obtaining essential spectral information concerning the physiological condition of P. incisa using a standard spectrophotometer is also shown.     

Dynamics of blood chylomicron fatty acids in a marine carnivore: implications for lipid metabolism and quantitative estimation of predator diets

Blubber fatty acid(s) (FA) signatures can provide accurate estimates of predator diets using quantitative FA signature analysis, provided that aspects of predator FA metabolism are taken into account. Because the intestinal absorption of dietary FA and their incorporation into chylomicrons (the primary transport lipoproteins for dietary FA in the blood) may influence the relationship between FA composition in the diet and adipose tissue, we investigated the metabolism of individual FA at these early stages of assimilation. We also investigated the capacity of chylomicron signatures to provide quantitative estimates of prey composition of an experimental meal. Six captive juvenile grey seals (Halichoerus grypus) were fed either 2.3 kg (n=3) or 4.6 kg (n=3) of Atlantic herring (Clupea harengus). Although chylomicron FA signatures resembled diet signatures at all samplings, absolute differences were smallest at 3-h post-feeding, when chylomicrons were likely largest and had the greatest ratio of triacylglycerol to phospholipid FA. Specific FA that differed significantly between diet and chylomicron signatures reflected either input from endogenous sources or loss through peroxisomal -oxidation. When these aspects of metabolism were accounted for, the quantitative predictions of diet composition generated using chylomicron signatures were extremely accurate, even when tested against 28 other prey items.     

First total syntheses of (Z)-15-methyl-10-hexadecenoic acid and the (Z)-13-methyl-8-tetradecenoic acid

The first total syntheses for the (Z)-15-methyl-10-hexadecenoic acid and the (Z)-13-methyl-8-tetradecenoic acid were accomplished in seven steps and in 31-32% overall yields. The (trimethylsilyl)acetylene was the key reagent in both syntheses. It is proposed that the best synthetic strategy towards monounsaturated iso methyl-branched fatty acids with double bonds close to the omega end of the acyl chain is first acetylide coupling of (trimethylsilyl)acetylene to a long-chain bifunctional bromoalkane followed by a second acetylide coupling to a short-chain iso bromoalkane, since higher yields are thus obtained. Spectral data is also presented for the first time for these two unusual fatty acids with potential as biomarkers and as topoisomerase I inhibitors.     

Cell culture models of fatty acid overload: Problems and solutions

High plasma levels of fatty acids occur in a variety of metabolic diseases. Cellular effects of fatty acid overload resulting in negative cellular responses (lipotoxicity) are often studied in vitro, in an attempt to understand mechanisms involved in these diseases. Fatty acids are poorly soluble, and thus usually studied when complexed to albumins such as bovine serum albumin (BSA). The conjugation of fatty acids to albumin requires care pertaining to preparation of the solutions, effective free fatty acid concentrations, use of different fatty acid species, types of BSA, appropriate controls and ensuring cellular fatty acid uptake. This review discusses lipotoxicity models, the potential problems encountered when using these cellular models, as well as practical solutions for difficulties encountered.     

Metabolism of polyunsaturated fatty acids and their toxicity to the microflora of the rumen

Ruminal microorganisms hydrogenate polyunsaturated fatty acids (PUFA) present in forages and thereby restrict the availability of health-promoting PUFA in meat and milk. The aim of this study was to investigate PUFA metabolism and the influence of PUFA on members of the ruminal microflora. Eleven of 26 predominant species of ruminal bacteria metabolised linoleic acid (LA; cis-9,cis-12–18:2) substantially. The most common product was vaccenic acid (trans-11–18:1), produced by species related to Butyrivibrio fibrisolvens. α-Linolenic acid (LNA; cis-9,cis-12,cis-15–18:3) was metabolised mostly by the same species. The fish oil fatty acids, eicosapentaenoic acid (EPA; 20:5(n − 3)) and docosahexaenoic acid (DHA; 22:6(n − 3)) were not metabolised. Cellulolytic bacteria did not grow in the presence of any PUFA at 50 μg ml⁻¹, nor did some butyrate-producing bacteria, including the stearate producer Clostridium proteoclasticum, Butyrivibrio hungatei and Eubacterium ruminantium. Toxicity to growth was ranked EPA > DHA > LNA > LA. Cell integrity, as measured using propidium iodide, was damaged by LA in all 26 bacteria, but to different extents. Correlations between its effects on growth and apparent effects on cell integrity in different bacteria were low. Combined effects of LA and sodium lactate in E. ruminantium and C. proteoclasticum indicated that LA toxicity is linked to metabolism in butyrate-producing bacteria. PUFA also inhibited the growth of the cellulolytic ruminal fungi, with Neocallimastix frontalis producing small amounts of cis-9,trans-11–18:2 (CLA) from LA. Thus, while dietary PUFA might be useful in suppressing the numbers of biohydrogenating ruminal bacteria, particularly C. proteoclasticum, care should be taken to avoid unwanted effects in suppressing cellulolysis.     

Development of Escherichia coli MG1655 strains to produce long chain fatty acids by engineering fatty acid synthesis (FAS) metabolism

The goal of this research was to develop recombinant Escherichia coli to improve fatty acid synthesis (FAS). Genes encoding acetyl-CoA carboxylase (accA, accB, accC), malonyl-CoA-[acyl-carrier-protein] transacylase (fabD), and acyl-acyl carrier protein thioesterase (EC 3.1.2.14 gene), which are all enzymes that catalyze key steps in the synthesis of fatty acids, were cloned and over-expressed in E. coli MG1655. The acetyl-CoA carboxylase (ACC) enzyme catalyzes the addition of CO₂ to acetyl-CoA to generate malonyl-CoA. The enzyme encoded by the fabD gene converts malonyl-CoA to malonyl-[acp], and the EC 3.1.2.14 gene converts fatty acyl-ACP chains to long chain fatty acids. All the genes except for the EC 3.1.2.14 gene were homologous to E. coli genes and were used to improve the enzymatic activities to over-express components of the FAS pathway through metabolic engineering. All recombinant E. coli MG1655 strains containing various gene combinations were developed using the pTrc99A expression vector. To observe changes in metabolism, the in vitro metabolites and fatty acids produced by the recombinants were analyzed. The fatty acids (C16) from recombinant strains were produced 1.23-2.41 times higher than that from the wild type.     

Microalgal cryo-preservation using dimethyl sulfoxide (Me2SO) coupled with two freezing protocols: Influence on the fatty acid profile

Procedures for determining the optimal pre-freezing protocol for cryo-preservation of microalgae are discussed. Three algal species were used (Chlorella vulgaris, Isochrysis galbana and Dunaliella salina) and cryo-stored using two different methods: the slow cooling and the fast freezing. In the slow cooling, each algae batch was treated with or without cryo-protectant (dimethyl sulfoxide: Me₂SO 5% v/v). After 20 min at 4 °C, the midi-straws were filled and cooled slowly (1.5 °C min⁻¹) to −140 °C, by a programmable freezer (Digitcool—IMV), before putting them directly into liquid nitrogen. Fast freezing was performed with 10% or 15% Me₂SO prior to plunging into liquid nitrogen. The three algal species followed the same re-growth pattern as that of the controls. The post-thawed viability with Me₂SO was good for all the selected algae (C. vulgaris >95%, I. galbana and D. salina >70% of the control), applying the slow cooling. The post-thawed viability without Me₂SO was 60% for I. galbana, 52% for D. salina and 33% for C. vulgaris. Fast freezing was not suitable for cryo-storage of I. galbana but gave good post-thawing viability for D. salina (70%). The decrease in fatty acid content of the cryo-stored algae was influenced by the temperature. The rapid decrease in temperature induced by fast freezing can explain the low level of fatty acid content of the three cryo-stored algae. Fatty acid profiles show that the nutritional values of the three cryo-stored micro-algae were not significantly affected especially when treated with slow cooling protocols.     

Synthesis and properties of methyl 5-(1'R,2'S)-(2-octadecylcycloprop-1-yl)pentanoate and other omega-19 chiral cyclopropane fatty acids and esters related to mycobacterial mycolic acids

A 23-26-carbon chain length range of omega-19 (1'R,2'S) cyclopropane fatty acids, related to mycobacterial mycolic acids, has been prepared. The key cyclopropyl intermediate, (1'R,2'S)-(Z)-1-formyl-2-octadecylcyclopropane, underwent Wittig chemistry with various reagents to provide vinylic precursors, which were selectively reduced to the corresponding saturated omega-19 cyclopropane fatty acids or esters. The 24-carbon omega-19 cyclopropane ester was made by chain elongation of the 23-carbon ester. Saturated and unsaturated chiral cyclopropane acids and esters were assayed, using wall extracts of Mycobacterium smegmatis; the incorporation of 14C-acetate was used to measure inhibition or stimulation of mycolic acid synthesis. Minor inhibition (2-3%) was shown by the 23- and 24-carbon saturated esters; all the other compounds were stimulants. The most effective (38-55%) stimulators of mycolate synthesis were the unsaturated esters with 23- and 26-carbons and the saturated and unsaturated 25-carbon acids.     

De novo bio-production of odd-chain fatty acids in Saccharomyces cerevisiae through a synthetic pathway via 3-hydroxypropionic acid

Odd-chain fatty acids (OCFAs) and their derivatives have attracted increasing attention due to their wide applications in the chemical, fuel, and pharmaceutical industry. However, most natural fatty acids are even-chained, and OCFAs are rare. In this work, a novel pathway was designed and established for de novo synthesis of OCFAs via 3-hydroxypropionic acid (3-HP) as the intermediate in Saccharomyces cerevisiae. First, the OCFAs biosynthesis pathway from 3-HP was confirmed, followed by an optimization of the precursor 3-HP. After combining these strategies, a de novo production of OCFAs at 74.8 mg/L was achieved, and the percentage of OCFAs in total lipids reached 20.3%, reaching the highest ratio of de novo-produced OCFAs. Of the OCFAs produced by the engineered strain, heptadecenoic acid (C17:1) and heptadecanoic acid (C17:0) accounted for 12.1% and 7.6% in total lipid content, respectively. This work provides a new and promising pathway for the de novo bio-production of OCFAs.