Total synthesis and bioactivity of 18(R)-hydroxyeicosapentaenoic acid

Resolvins are family of lipid mediators derived from omega-3 polyunsaturated fatty acids, which are generated during the resolution phase of acute inflammation. Resolvin E1 is biosynthesized from eicosapentaenoic acid via 18(R)-hydroxyeicosapentaenoic acid (18R-HEPE) in the Cox-2 and lipoxygenase mediated pathway and has proven to exhibit potent anti-inflammatory activity. We report herein the first total chemical synthesis of 18R-HEPE and demonstrate that this compound displays in vivo bioactivity by blocking neutrophil infiltration in a murine model of zymosan-induced peritonitis.     

Identification of endogenous resolvin E1 and other lipid mediators derived from eicosapentaenoic acid via electrospray low-energy tandem mass spectrometry: spectra and fragmentation mechanisms

Resolvin E1 (RvE1, 5S,12R,18R-trihydroxy-6Z,8E,10E,14Z,16E-eicosapentaenoic acid) is a novel anti-inflammatory lipid mediator recently found in humans, mice, and fish in vivo. To identify endogenous RvE1 and other eicosapentaenoic acid (EPA)-derived lipid mediators using electrospray low-energy collision-induced dissociation tandem mass spectrometry (MS/MS), the MS/MS product ion spectra of these compounds were correlated with their structures, and the MS/MS fragmentation mechanisms were studied. Deuterium labeling confirmed the proposed correlations and the fragmentation mechanisms. beta-cleavage was observed for RvE1, and beta and gamma cleavages were seen for leukotriene B5; however, alpha-cleavage was more common. The positions and numbers of hydroxyls and double bonds of these lipid mediators can be deduced from the MS/MS spectra. The MS/MS fragmentation generating chain-cut ions involved beta-ene, gamma-ene, or alpha-H-beta-ene rearrangement, depending on the specific structure. The m/z value of a detected chain-cut ion from RvE1 or from an EPA-derived product is equal to the corresponding hypothetical homolytic segment (cc, cm, mc, or mm) with the addition or extraction of up to two hydrogen atoms (H) from hydroxyls or an alpha-carbon; namely, the m/z value of an alpha-cleavage-generated ion is equal to [cc+H], [cm-2H], [mc-H], or [mm]. Wideband activation increased the signal intensities of chain-cut ions, and therefore was better for trace analysis of RvE1 in biological samples. RvE1, LTB5, PGE3, and other EPA-derived lipid mediators were found in trout brain or head-kidney via this approach on the basis of MS/MS spectra and fragmentation mechanisms. Negative ion electrospray low-collision-energy MS/MS spectra provide adequate data to elucidate and identify the structures of RvE1 and other EPA-derived lipid mediators at levels below a few picomoles in trout samples.     

Resolvins,Protectins, and Maresins: DHA-Derived Specialized Pro-Resolving Mediators,Biosynthetic Pathways,Synthetic Approaches,and Their Role in Inflammation

Marine organisms are an important source of natural products with unique and diverse chemical structures that may hold the key for the development of novel drugs. Docosahexaenoic acid (DHA) is an omega-3 fatty acid marine natural product playing a crucial regulatory role in the resolution of inflammation and acting as a precursor for the biosynthesis of the anti-inflammatory specialized pro-resolving mediators (SPMs) resolvins, protectins, and maresins. These metabolites exert many beneficial actions including neuroprotection, anti-hypertension, or anti-tumorigenesis. As dysregulation of SPMs is associated with diseases of prolonged inflammation, the disclosure of their bioactivities may be correlated with anti-inflammatory and pro-resolving capabilities, offering new targets for drug design. The availability of these SPMs from natural resources is very low, but the evaluation of their pharmacological properties requires their access in larger amounts, as achieved by synthetic routes. In this report, the first review of the total organic syntheses carried out for resolvins, protectins, and maresins is presented. Recently, it was proposed that DHA-derived pro-resolving mediators play a key role in the treatment of COVID-19. In this work we also review the current evidence on the structures, biosynthesis, and functional and new-found roles of these novel lipid mediators of disease resolution.     

Convergent oxygenation of arachidonic acid by 5-lipoxygenase and cyclooxygenase-2

5-Lipoxygenase and cyclooxygenase-2 (COX-2) initiate the biosynthesis of distinct families of mediators from arachidonic acid (leukotrienes and prostaglandins, respectively) and are each the target of anti-inflammatory medications. Here we show that the product of 5-lipoxygenase, 5S-hydroxyeicosatetraenoic acid, is selectively and efficiently triply oxygenated by COX-2, implicating a cross-pathway interaction. The product is a unique diendoperoxide, potentially representing the parent compound of a novel group of lipid mediators.     

Targeting lipoxygenases with care

Though fish oils possess cardio-protective, anti-inflammatory, and anti-cancer properties, their molecular and biochemical mechanism of action is lacking. In this issue of Chemistry & Biology, Tjonahen and colleagues identify a new metabolite of eicosapentanoic acid, resolvin E2, produced by 5-lipoxygenase.     

Total synthesis of Resolvin E1

The enantioselective total synthesis of Resolvin E1 (RvE1), a naturally occurring small molecule mediator of inflammation resolution, is reported. Two routes are presented, both modular and convergent in nature, with an excellent control of all stereocenters. The C12- and C18-hydroxy groups are derived from (S)-glycidol while the C5-hydroxy group is installed via enantioselective reduction of a ketone precursor. Both the cis-alkenes are introduced with excellent control by the reduction of a late-stage bis-alkyne intermediate. The synthetic disconnections are very amenable to analog preparation, and further modifications to the chemistry have allowed for scale-up and First in Man testing of this novel pro-resolution molecule.     

In vivo and in vitro lipid peroxidation of arachidonate esters: the effect of fish oil omega-3 lipids on product distribution

The effect of lipid composition on the distribution of free radical oxidation products derived from arachidonic acid (20:4) esters has been studied in vitro and in vivo. Pro-inflammatory prostaglandin (PG) F2-like compounds, termed F2-isoprostanes (IsoPs), are produced in vivo and in vitro by the free radical-catalyzed peroxidation of arachidonic acid. Controlled free radical oxidation of mixtures of fatty acid esters in vitro showed that the formation of IsoPs from arachidonate is dramatically influenced by the presence of other fatty acid esters in the reaction mixture. Thus, three lipid mixtures containing the same arachidonate concentration but different amounts of other fatty esters (16:0; 18:1; 18:2; 20:5, and 22:6) were oxidized, and the product yields were determined by GC and LC/MS/MS analysis. The yield of F2-IsoP formed after 1 h of oxidation was 18% (based on arachidonate consumed) for mixtures containing arachidonate as the only oxidizable PUFA, but yields of these biologically active compounds dropped to 6% in polyunsaturated fatty acid (PUFA) mixtures typical of those found in tissues of fish oil-fed animals. F2-IsoP levels were also monitored in the livers of mice on diets supplemented with eicosapentaenoic acid (C20:5 omega-3; EPA), the PUFA most abundant in fish oil. While the level of arachidonic acid present in livers was not significantly different from that in control animals, levels of IsoPs in the liver were reduced in the EPA-fed mice compared to those in controls under conditions of oxidative stress (60 +/- 25% reduction, n = 5) or at baseline (48 +/- 14% reduction, n = 5). These results suggest that dietary omega-3 PUFAs may influence the formation of bio-active peroxidation products derived from omega-6 PUFAs by channeling the free radical pathway away from the F2-IsoPs.     

An investigation of frequently consumed edible oils in Turkey in terms of omega fatty acids

The fatty acid profiles of frequently consumed oils and crops cultivated in Turkey were investigated in regard to omega fatty acids. Analyses were carried out on commercially sold oils, sunflower, olive, and fish oils, and oils extracted from fatty seeds of hazelnut, walnut, olive, sunflower, poppy, sesame, and pumpkin, and butter produced in Turkey. Hazelnut and olive oils were found to be rich in omega-9 (oleic acid 18:1), walnut, poppy seed, sesame, and pumpkin seed were rich in omega-6 (linoleic acid 18:2), and butter was rich in short chain fatty acids and omega-9. Fish oil, from mackerel, was the richest in omega-3 fatty acids and fatty acid diversity. There were some alterations between commercially sold oils and oils extracted from seeds in regard to fatty acid percentages and variety.     

Anti-Inflammatory and Anti-Platelet Properties of Lipid Bioactives from Apple Cider By-Products

The valorization of food industry by-products as sources of bioactive compounds is at the forefront of research in functional foods and nutraceuticals. This study focuses on bioactives of apple cider by-products (ACBPs) with putative cardio-protective properties. Total lipids (TLs) were extracted from ACBPs of apple varieties that are low (ACBP1), medium (ACBP2), and high (ACBP3) in tannins and were further separated into polar lipids (PLs) and neutral lipids (NLs). The functionality of these lipid extracts and of their HPLC-derived lipid fractions/PL subclasses were assessed in vitro against human platelet aggregation induced by the thrombotic and inflammatory platelet agonists platelet-activating factor (PAF) and adenosine diphosphate (ADP). The fatty acid profile of PLs and their most bioactive lipid fractions were evaluated by GC–MS analysis. The PL extracts exhibited higher specificity against the PAF-induced platelet aggregation compared to their anti-ADP effects, while TL and NL showed lower bioactivities in all ACBPs. HPLC analysis unveiled that the most bioactive PL from all ACBPs were those in PL fraction 3 containing phosphatidylcholines (PCs). PLs from all ACBPs and their PC bioactives were rich in polyunsaturated fatty acids (PUFAs) and especially in the essential omega-6 (n-6) linoleic acid (LA) and omega-3 (n-3) alpha linolenic acid (ALA), with favorably low values of the n-6/n-3 PUFA ratio, thus providing a rationale for their higher anti-inflammatory bioactivities. Within this study, highly bioactive PL compounds with strong anti-inflammatory and anti-platelet properties were identified in ACBPs, which can be potentially utilized for producing cardio-protective functional foods and/or nutraceuticals.     

Fatty-acid composition of rat brain lipids. Determined by support-coated open-tubular gas chromatography.

The nonhydroxy fatty acid composition of rat brain lipids (except gangliosides) was determined by support-coated open-tubular (SCOT) gas chromatography. Fatty acids of both odd and even chain lengths ranging from C14 to C26 were detected. Brain lipids contained 49% saturated, 29% monounsaturated, and 22% polyunsaturated fatty acids. Monoenoic fatty acids were mainly of the omega-9 and omega-7 series with minor amounts of omega-10 and amega-11 isomers. Dienes and trienes consisted of omega-6, amega-7, and omega-9 series. Tetraenes were of the omega-6 series. Small amounts of omega-6 and omega-3 pentaenes were detected. The most abundant polyunsaturated fatty acid was 22:6omega-3. The advantages of support-coated open-tubular columns over wall-coated open-tubular columns for the analysis of brain lipid fatty acids are discussed.     

Synthesis and inhibitory effect of novel glycyrrhetinic acid derivatives on IL-1 beta-induced prostaglandin E(2) production in normal human dermal fibroblasts

Olean-11,13(18)-dien-3beta,30-diol dihemiphthalate (3), which was derived from glycyrrhetinic acid (GA), has been reported to produce a potent of anti-inflammatory effect in in vivo assays. Using 3 as a lead compound, we attempted to synthesize some modified compounds which varied in the following; i) the position of a carboxyl group in the phthalate moiety, ii) the number of carboxyls attached to the benzoyl group, iii) conversion of benzene ring to another ring system, iv) the linkage form between the benzene ring and oleanene skeleton at position 3 and/or 30. These were screened for their inhibitory activity against interleukin-1 beta (IL-1 beta)-induced prostaglandin E(2) (PGE(2)) production in normal human dermal fibroblasts (NHDF). Although conversion of the ortho-carboxyl group of 3 into the meta-position or the para-position led to an increase in inhibitory activity, the elimination or increase of the carboxyl group resulted in loss of the inhibitory activity. Conversion of the ester bond to the amide bond at position 3 and/or 30 of 3 did not contribute to a significant increase in inhibitory activity. On the other hand, among the derivatives possessing an anthranilic acid moiety at position 30 of 3beta-O-acetyl-olean-11,13(18)-dien-30-oic acid (20), 3beta-hydroxy-30-nor-olean-11,13(18)-dien-20 beta-[N-(2-carboxyphenyl)]carboxamide (30) showed the most potent inhibitory activity (IC(50) 1.0 microM) in this series.     

Simultaneous lipidomic analysis of three families of bioactive lipid mediators leukotrienes, resolvins, protectins and related hydroxy-fatty acids by liquid chromatography/electrospray ionisation tandem mass spectrometry

Bioactive lipid mediators derived from polyunsaturated fatty acids (PUFA) exhibit a range of tissue- and cell-specific activities in many physiological and pathological processes. Electrospray ionisation tandem mass spectrometry coupled to liquid chromatography (LC/ESI-MS/MS) is a sensitive, versatile analytical methodology for the qualitative and quantitative analysis of lipid mediators. Here we present an LC/ESI-MS/MS assay for the simultaneous analysis of twenty mono- and poly-hydroxy-fatty acid derivatives of linoleic, arachidonic, eicosapentaenoic and docosahexaenoic acids. The assay was linear over the concentration range 1-100 pg/microL, whilst the limits of detection and quantitation were 10-20 and 20-50 pg, respectively. The recovery of the extraction methodology varied from 76-122% depending on the metabolite. This system is useful for profiling a range of biochemically related potent mediators including the newly discovered resolvins and protectins, and their precursor hydroxyeicosapentaenoic and hydroxydocosahexaenoic acids, and, consequently, advance our understanding of the role of PUFA in health and disease.     

Total synthesis of resolvin E1

Resolvin E1 (RvE1), which is an endogenous mediator to resolve inflammation, was synthesized by Wittig reaction between the C15-C20 aldehyde and the C10-C14 phosphonium salt possessing the vinyl iodo moiety followed by Suzuki-Miyaura coupling of the resulting vinyl iodide with the vinyl borane of the C1-C9 part, which was derived from the corresponding acetylene by hydroboration. The C5 and C18 chiral centers in these parts were created by the kinetic resolution of the racemic γ-TMS allylic alcohols using the asymmetric epoxidation, while that of the C10-C14 part was constructed by the asymmetric hydrogen transfer reaction of the corresponding γ-TMS acetylene ketone.     

Separation of Hydroxylated Metabolites of Fatty Acids (C10-C18) on a μPorasil Silica Column Using an Isocratic HPLC System

Abstract

A simple, versatile, and rapid normal-phase isocratic HLPC system is described for the analysis of the major (omega and omega-1) metabolites of C₁₀-C₁₈ chain length fatty acids formed upon incubation with rat liver microsomes and NADPH. Quantitation was achieved by radiometric detection. Chromatographic separation was performed by elution of the fatty acids and their omega and omega-1 metabolites from a 10μ silica column (μPorasil) with hexane:2-propanol:acetic acid (96.5:2.5:1.0). Retention times for these metabolites ranged from 10 to 13 minutes for stearic acid and from 16 to 21 minutes for capric acid. Recovery of the fatty acids and their metabolites from the column was greater than 95 percent. Relative quantitative conversion of the fatty acid substrates to omega and omega-1 metabolites was in the following order: myristic acid > capric acid=lauric acid=palmitic acid ? stearic acid. The omega products were formed preferentially over the omega-1 products of all the fatty acids except lauric acid. The method proved suitable for routine determination of NADPH-dependent fatty acid hydroxylase activities in rat liver microsomes.     

Cold-Pressed Pomegranate Seed Oil: Study of Punicic Acid Properties by Coupling of GC/FID and FTIR

Over the last decades, we have witnessed an increasing interest in food-related products containing vegetable oils. These oils can be obtained either by extraction or by mechanical pressing of different parts of plants (e.g., seeds, fruit, and drupels). Producers of nutraceuticals have ceaselessly searched for unique and effective natural ingredients. The enormous success of argan oil has been followed by discoveries of other interesting vegetable oils (e.g., pomegranate oil) containing several bioactives. This work describes the pomegranate fruit extract and seed oil as a rich source of conjugated linolenic acid as a metabolite of punicic acid (PA), deriving from the omega-5 family (ω-5). Through the chemical characterization of PA, its nutritional and therapeutic properties are highlighted together with the physiological properties that encourage its use in human nutrition. We analyzed the composition of all fatty acids with beneficial properties occurring in pomegranate seed oil using gas chromatography (GC) with flame-ionization detection (FID) analysis combined with Fourier transform infrared spectroscopy (FTIR). Pomegranate seed oil mainly consists of 9,11,13-octadic-trienoic acid (18:3), corresponding to 73 wt % of the total fatty acids. Nine components were identified by GC in PSO, varying between 0.58 and 73.19 wt %. Using midinfrared (MIR) spectroscopy, we compared the composition of pomegranate seed oil with that of meadowfoam seed oil (MSO), which is also becoming increasingly popular in the food industry due to its high content of long chain fatty acids (C20-22), providing increased oil stability. From the results of FTIR and MIR spectroscopy, we found that punicic acid is unique in PSO (73.19 wt %) but not in MSO.     

Total syntheses of four possible stereoisomers of resolvin E3

Resolvin E3, 17,18-dihydroxy-5Z,8Z,11Z,13E,15E-eicosapentaenoic acid, is a potent anti-inflammatory lipid mediator. To determine the stereochemistries of the C17- and C18-hydroxy groups of resolvin E3 and to supply a sufficient amount of material for future biological studies, we developed a highly convergent and practical route to its four possible stereoisomers. The key reactions employed here were the Horner–Wadsworth–Emmons coupling, the two copper(I)-mediated reactions between the alkynes and the propargyl tosylates, and the simultaneous reduction of the three triple bonds to the three Z-olefins.     

Fatty acids and beta-carotene in australian purslane (Portulaca oleracea) varieties

The fatty acid profile and beta-carotene content of a number of Australian varieties of purslane (Portulaca oleracea) were determined by GC and HPLC. The total fatty acid content ranged from 1.5 to 2.5 mg/g of fresh mass in leaves, 0.6 to 0.9 mg/g in stems and 80 to 170 mg/g in seeds. alpha-Linolenic acid (C18:3omega3) accounted for around 60% and 40% of the total fatty acid content in leaves and seeds, respectively. Longer-chain omega-3 fatty acids were not detected. The beta-carotene content ranged from 22 to 30 mg/g fresh mass in leaves. These results indicate that Australian purslane varieties are a rich source of alpha-linolenic acid and beta-carotene.     

Synthesis and antiallergic activity of N-[4-(4-diphenylmethyl-1-piperazinyl)butyl]-1,4-dihydro-4-oxopyridine-3 - carboxamides

A new series of oxopyridinecarboxamide derivatives 3a--g and 5a were synthesized and evaluated for their antiallergic activity. 1,4-Dihydro-7-methyl-4-oxo-1,8-naphthyridine-3-carboxamides 3a and 5a exhibited potent antiallergic activity (inhibitory rates of 80.7 and 88.3%, respectively, at 20 mg/kg, p.o.) in the rat passive cutaneous anaphylaxis (PCA) test and also exhibited much more potent in vitro inhibitory activity than caffeic acid against the enzyme 5-lipoxygenase (5-LO). Their in vitro antihistamine activity, however, was weaker than that of ketotifen. Compounds 3a and 5a are viewed as promising candidates for antiallergic agents.     

Syntheses of (4,1-benzoxazepine-3-ylidene)acetic acid derivatives and their inhibition of squalene synthase

The (3,5-trans)-7-chloro-5-(2-chlorophenyl)-2-oxo-1,2,3,5-tetrahydro-4,1-benzoxazepine-3-acetic acid derivatives 1 have been previously identified as potent squalene synthase inhibitors. A series of (4,1-benzoxazepin-3-ylidene)acetic acid derivatives were synthesized and evaluated for their inhibition of rat and human squalene synthase, and the (E)-isomers were found to exhibit potent inhibitory activity, with the same potency as 4,1-benzoxazepine-3-acetic acid derivatives. In contrast the (Z)-isomers did not exhibit significant inhibitory activity, and the active conformation of the 4,1-benzoxazepine-3-acetic acid derivatives was deduced from the folded conformation of the (E)-isomers.     

Effects of UV Radiation and Diet on Polyunsaturated Fatty Acids in the Skin,Ocular Tissue and Dorsal Muscle of Atlantic Salmon (Salmo salar) Held in Outdoor Rearing Tanks

The effect of UV radiation (UVR) on juvenile Atlantic salmon (Salmo salar) was assessed by measuring the fatty acid (FA) profiles of muscle, dorsal and ventral skin, and ocular tissues following 4-month long exposures to four different UVR treatments in outdoor rearing tanks. Fish were fed two different diets (Anchovy- and Herring-oil based) that differed in polyunsaturated fatty acid (PUFA) concentrations. Anchovy-fed salmon had higher concentrations of ALA (alpha-linoleic acid; 18:3n-3), EPA (eicosapentaenoic acid; 20:5n-3) and DPA (docosapentaenoic acid, 22:5n-3) in their muscle tissues than fish fed the Herring feed. Fish subjected to enhanced UVB levels had higher concentrations of LIN (linolenic acid, 18:2n-6) and ALA, total omega-6 FA and SAFA (saturated fatty acids) in their tissues compared with fish in reduced UV treatments. Concentrations of ALA, LIN, GLA (gamma-linolenic acid; 18:3n-6), EPA, PUFA and total FA were higher in ventral skin of fish exposed to enhanced UVB compared with fish in reduced UV treatments. Salmon exposed to reduced UV weighed more per-unit-length than fish exposed to ambient sunlight. The FA profiles suggest that fish exposed to UV radiation were more quiescent than fish in the reduced UV treatments resulting in a buildup of catabolic substrates.