Efficient Strategy for Characterization and Quantification of Polyunsaturated Lipids by Microwave-Assisted MMPP Epoxidation.

Lipids play integral roles in biological processes, with carbon-carbon double bonds (C═C) markedly influencing their structure and function. Precise characterization and quantification of unsaturated lipids are crucial for understanding lipid physiology and discovering disease biomarkers. However, using mass spectrometry for these purposes presents significant challenges. In this study, we developed a microwave-assisted magnesium monoperoxyphthalate hexahydrate (MMPP) epoxidation reaction, coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS), to analyze unsaturated lipids. Microwave irradiation expedited the MMPP epoxidation, achieving complete derivatization in 10 min without byproducts. A diagnostic ion pair, displaying a 16 Da mass difference, effectively identified the location of the C═C bond in mass spectra. Microwave irradiation also significantly facilitated the epoxidation reaction of polyunsaturated lipids, achieving yields greater than 85% and yielding a complete epoxidation product. This simplifies chromatographic separation and aids in accurate quantification. Additionally, a purification process was implemented to remove excess derivatization reagents, significantly reducing mass spectrometry response suppression and enhancing analytical reproducibility. The method's effectiveness was validated by analyzing unsaturated lipids in rat plasma from a type I diabetes model. We quantified nine unsaturated lipids and characterized 42 fatty acids and glycerophospholipids. The results indicated that unsaturated fatty acids increased in diabetic plasma while unsaturated glycerophospholipids decreased. Furthermore, the relative abundances of Δ9/Δ11 isomer pairs also exhibited a close association with diabetes. In conclusion, microwave-assisted MMPP epoxidation coupled with LC-MS/MS provides an effective strategy for characterization and quantification of polyunsaturated lipids, offering deeper insight into the physiological impact of unsaturated lipids in related diseases.

Collective Total Synthesis of Ecklonialactones, Eiseniachlorides and Analogs.

Ecklonialactones, Eiseniachlorides, and Egregiachlorides are synthesized in living organisms via the lipoxygenase-mediated oxidation of polyunsaturated fatty acids. Originally isolated and identified from brown seaweed (Ecklonia stolonifera, Eisenia bicyclis, and Egregia menziesii), and later replicated on milligram scale through chemical synthesis, the full biological activities of these compounds remain to be elucidated. To bridge this gap in knowledge, we propose a unified methodology to synthesize the 14-membered macrocyclic structures of Ecklonialactones, Eiseniachlorides and analogs using a versatile and convergent approach. This study delineates the synthesis of Ecklonialactone A, B, C, D, and Eiseniachlorides A and B, as well as ent-Ecklonialactone B, 16-epi-Ecklonialactone B and 12,13-diepi-Ecklonialactone B.

Stereoselective Syntheses of Polyunsaturated Fatty Acids, 13-(S)-HODE and 15-(S)-HETE.

Polyunsaturated fatty acids and their metabolites have been reported in which their pathway has potential for the modulation of cancer cell growth. 13-(S)-HODE and 15-(S)-HETE, both of which are main metabolites of 15-LOXs, play an important role as endogenous ligands in biological systems. However, the modification of 13-(S)-HODE and 15-(S)-HETE in pharmaceutical applications has not been explored widely. Herein, we report the stereoselective syntheses of 13-(S)-HODE, 15-(S)-HETE, and its derivatives to enable the synthesis of bioactive fatty acid analogues.

A gram-scale synthesis of very-long chain polyunsaturated fatty acids (VLC-PUFAs).

This manuscript describes our third generation, gram-scale synthesis of very long chain-polyunsaturated fatty acids (VLC-PUFAs), a unique and increasingly important class of lipids. Critical to this work and what makes it different from our previous approach to this family was the avoidance of oxidation sequences. Central to accomplishing this involved the use of a Negishi coupling reaction between the acid chloride derived from DHA and a saturated alkyl zinc reaction. Overall, the general approach required 6 synthetic transformations from DHA and was accomplished with an overall yield of 40%.

Development of an efficient and scalable bioprocess for the plant hormone 12-OPDA: Overcoming the hurdles of nature's biosynthesis.

Besides its native biological function as a plant hormone, cis-(+)-12-oxo-phytodienoic acid (12-OPDA) serves as a metabolite for the cellular formation of (-)-jasmonic acid and has also been shown to have an influence on mammalian cells. In order to make this biologically active, but at the same time very expensive natural product 12-OPDA broadly accessible for further biological and medicinal research, we developed an efficient bioprocess based on the utilization of a tailor-made whole-cell catalyst by following the principles of its biosynthesis in nature. After process optimization, the three-step one-pot synthesis of 12-OPDA starting from readily accessible α-linolenic acid could be conducted at appropriate technically relevant substrate loadings in the range of 5-20 g L-1. The desired 12-OPDA was obtained with an excellent conversion efficiency, and by means of the developed, efficient downstream-processing, this emulsifying as well as stereochemically labile biosynthetic metabolite 12-OPDA was then obtained with very high chemical purity (>99%) and enantio- and diastereomeric excess (>99% ee, 96% de) as well as negligible side-product formation (<1%). With respect to future technical applications, we also demonstrated the scalability of the production of the whole cell-biocatalyst in a high cell-density fermentation process.

Screening for the production of polyunsaturated fatty acids and cerebrosides in fungi.

AIMS: To investigate fatty acid, including polyunsaturated fatty acids (PUFA), and cerebroside production of a large diversity of fungi from the Ascomycota, Basidiomycota, and Mucoromycota phyla. METHODS AND RESULTS: Seventy-nine fungal strains were grown in Kavadia medium using a microcultivation system, i.e. Duetz microtiter plates. Following cultivation, fatty acid and cerebroside contents were analyzed by gas chromatography-flame ionization detection (GC-FID) and high performance thin-layer chromatography (HPTLC), respectively. Mucoromycota fungi appeared as the most promising candidates for omega-6 PUFA production. The best omega-6 producer, including γ-linolenic acid (GLA, 18:3n-6), was Mucor fragilis UBOCC-A109196 with a concentration of 647 mg L-1 total omega-6 PUFA (representing 35% of total fatty acids) and 225 mg L-1 GLA (representing 12% of total fatty acids). Arachidonic acid concentration (20:4n-6) was the highest in Mortierella alpina UBOCC-A-112046, reaching 255 mg L-1 and 18.56% of total fatty acids. Interestingly, several fungal strains were shown to produce omega-7 monounsaturated fatty acids. Indeed, Torulaspora delbrueckii strains accumulated palmitoleic acid (16:1n-7) up to 20% of total fatty acids, reaching 114 mg L-1 in T. delbrueckii UBOCC-A-214128, while C. elegans UBOCC-A-102008 produced mainly paullinic acid (20:1n-7) with concentrations up to 100 mg L-1. Concerning cerebroside production, HPTLC appeared as a relevant approach for their detection and quantification. Promising candidates belonging to the Mucoromycota phylum were found, especially in the Absidia genus with A. spinosa UBOCC-A-101332 as the best producer (12.7 mg L-1). CONCLUSIONS: The present study highlighted PUFA and cerebroside production in a large diversity of fungi and the fact that members of the Mucoromycota phylum are good producers of PUFA as well as cerebrosides.

Isolation and Structure Determination of cis-OPDA-α-Monoglyceride from Arabidopsis thaliana.

cis-12-oxo-Phytodieneoic acid-α-monoglyceride (1) was isolated from Arabidopsis thaliana. The chemical structure of 1 was elucidated based on exhaustive 1D and 2D NMR spectroscopic measurements and supported by FDMS and HRFDMS data. The absolute configuration of the cis-OPDA moiety in 1 was determined by comparison of 1H NMR spectra and ECD measurements. With respect to the absolute configuration of the ß-position of the glycerol backbone, the 2:3 ratio of (S) to (R) was determined by making ester-bonded derivatives with (R)-(+)-α-methoxy-α-trifluoromethylphenylacetyl chloride and comparing 1H NMR spectra. Wounding stress did not increase endogenous levels of 1, and it was revealed 1 had an inhibitory effect of A. thaliana post germination growth. Notably, the endogenous amount of 1 was higher than the amounts of (+)-7-iso-jasmonic acid and (+)-cis-OPDA in intact plants. 1 also showed antimicrobial activity against Gram-positive bacteria, but jasmonic acid did not. It was also found that α-linolenic acid-α-monoglyceride was converted into 1 in the A. thaliana plant, which implied α-linolenic acid-α-monoglyceride was a biosynthetic intermediate of 1.

"Sweet space" strategy for solvent system selection in countercurrent chromatography: A case study on separation of polyunsaturated fatty acids from borage oil.

This study presents a comprehensive strategy for the selection and optimization of solvent systems in countercurrent chromatography (CCC) for the effective separation of compounds. With a focus on traditional organic solvent systems, the research introduces a "sweet space" strategy that merges intuitive understanding with mathematical accuracy, addressing the significant challenges in solvent system selection, a critical bottleneck in the widespread application of CCC. By employing a combination of volume ratios and graphical representations, including both regular and trirectangular tetrahedron models, the proposed approach facilitates a more inclusive and user-friendly strategy for solvent system selection. This study demonstrates the potential of the proposed strategy through the successful separation of gamma-linolenic acid, oleic acid, and linoleic acid from borage oil, highlighting the strategy's effectiveness and practical applicability in CCC separations.

Identification of "modified OPDA (mo-OPDA)" as a Michael adduct of cis-OPDA.

cis-(+)-12-Oxo-phytodienoic acid (cis-OPDA) is a significant plant oxylipin, known as a biosynthetic precursor of the plant hormone jasmonoyl-l-isoleucine (JA-Ile), and a bioactive substance in plant environmental stresses. A recent study showed that a plant dioxygenase, Jasmonate Induced Dioxygenase 1 (JID1), converts cis-OPDA into an unidentified metabolite termed "modified-OPDA (mo-OPDA)" in Arabidopsis thaliana. Here, using ultra-performance liquid chromatography coupled with triple quad mass spectrometry (UPLC-MS/MS) experiment, the chemical identity of "mo-OPDA" was demonstrated and identified as a conjugate between cis-OPDA and 2-mercaptoethanol (cis-OPDA-2ME), an artifact produced by Michael addition during the JID1 digestion of cis-OPDA. However, previous reports demonstrated a decreased accumulation of cis-OPDA in the JID1-OE line, suggesting the existence of an unknown JID1-mediated mechanism regulating the level of cis-OPDA in A. thaliana.

Study on the Mechanism of Lipid Peroxidation Induced by Carbonate Radicals.

Based on the reported research, hydroxyl radicals can be rapidly transformed into carbonate radicals in the carbonate-bicarbonate buffering system in vivo. Many of the processes considered to be initiated by hydroxyl radicals may be caused by carbonate radicals, which indicates that lipid peroxidation initiated by hydroxyl radicals can also be caused by carbonate radicals. To date, theoretical research on reactions of hydrogen abstraction from and radical addition to polyunsaturated fatty acids (PUFAs) of carbonate radicals has not been carried out systematically. This paper employs (3Z,6Z)-nona-3,6-diene (NDE) as a model for polyunsaturated fatty acids (PUFAs). Density functional theory (DFT) with the CAM-B3LYP method at the 6-311+g(d,p) level was used to calculate the differences in reactivity of carbonate radicals abstracting hydrogen from different positions of NDE and their addition to the double bonds of NDE under lipid solvent conditions with a dielectric constant of 4.0 (CPCM model). Grimme's empirical dispersion correction was taken into account through the D3 scheme. The energy barrier, reaction rate constants, internal energy, enthalpy and Gibbs free energy changes in these reactions were calculated With zero-point vibrational energy (ZPVE) corrections. The results indicated that carbonate radicals initiate lipid peroxidation primarily through hydrogen abstraction from diallyl carbon atoms. The reaction of hydrogen abstraction from diallyl carbon atoms exhibits the highest reaction rate, with a reaction rate constant approximately 43-fold greater than the second-ranked hydrogen abstraction from allyl carbon atoms. This process has the lowest energy barrier, internal energy, enthalpy, and Gibbs free energy changes, indicating that it is also the most spontaneous process.

Biology and Total Synthesis of n-3 Docosapentaenoic Acid-Derived Specialized Pro-Resolving Mediators.

Research over the last 25 years related to structural elucidations and biological investigations of the specialized pro-resolving mediators has spurred great interest in targeting these endogenous products in total synthesis. These lipid mediators govern the resolution of inflammation as potent and stereoselective agonists toward individual G-protein-coupled receptors, resulting in potent anti-inflammatory activities demonstrated in many human disease models. Specialized pro-resolving mediators are oxygenated polyunsaturated products formed in stereoselective and distinct biosynthetic pathways initiated by various lipoxygenase and cyclooxygenase enzymes. In this review, the reported stereoselective total synthesis and biological activities of the specialized pro-resolving mediators biosynthesized from the polyunsaturated fatty acid n-3 docosapentaenoic acid are presented.

OxPAPC stabilizes liquid-ordered domains in biomimetic membranes.

Long-chain polyunsaturated fatty acids (PUFAs) are prone to nonenzymatic oxidation in response to differing environmental stressors and endogenous cellular sources. There is increasing evidence that phospholipids containing oxidized PUFA acyl chains control the inflammatory response. However, the underlying mechanism(s) of action by which oxidized PUFAs exert their functional effects remain unclear. Herein, we tested the hypothesis that replacement of 1-palmitoyl-2-arachidonyl-phosphatidylcholine (PAPC) with oxidized 1-palmitoyl-2-arachidonyl-phosphatidylcholine (oxPAPC) regulates membrane architecture. Specifically, with solid-state 2H NMR of biomimetic membranes, we investigated how substituting oxPAPC for PAPC modulates the molecular organization of liquid-ordered (Lo) domains. 2H NMR spectra for bilayer mixtures of 1,2-dipalmitoylphosphatidylcholine-d62 (an analog of DPPC deuterated throughout sn-1 and -2 chains) and cholesterol to which PAPC or oxPAPC was added revealed that replacing PAPC with oxPAPC disrupted molecular organization, indicating that oxPAPC does not mix favorably in a tightly packed Lo phase. Furthermore, unlike PAPC, adding oxPAPC stabilized 1,2-dipalmitoylphosphatidylcholine-d6-rich/cholesterol-rich Lo domains formed in mixtures with 1,2-dioleoylphosphatidylcholine while decreasing the molecular order within 1,2-dioleoylphosphatidylcholine-rich liquid-disordered regions of the membrane. Collectively, these results suggest a mechanism in which oxPAPC stabilizes Lo domains-by disordering the surrounding liquid-disordered region. Changes in the structure, and thereby functionality, of Lo domains may underly regulation of plasma membrane-based inflammatory signaling by oxPAPC.

Differential Behavior of Eicosapentaenoic and Docosahexaenoic Acids on the Organization, Dynamics, and Fusion of Homogeneous and Heterogeneous Membranes.

Membrane fusion is a common course in innumerable biological processes that helps in the survival of eukaryotes. Enveloped viruses utilize this process to enter the host cells. Generally, the membrane lipid compositions play an important role in membrane fusion by modulating the membrane's physical properties and the behavior of membrane proteins in the cellular milieu. In this work, we have demonstrated the role of polyunsaturated fatty acids (PUFAs), such as eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids, on the organization, dynamics, and fusion of homogeneous and heterogeneous membranes. We have exploited arrays of steady-state and time-resolved fluorescence spectroscopic methods and polyethylene glycol-induced membrane fusion assay to elucidate the behavior of EPA and DHA on dioleoyl phosphatidylcholine (DOPC)/cholesterol (CH) homogeneous and DOPC/sphingomyelin/CH heterogeneous membranes. Our results suggest that EPA and DHA display differential effects on two different membranes. The effects of PUFAs in homogeneous membranes are majorly attributed to their flexible chain dynamics, whereas the ability of PUFA-induced cholesterol transfer from the lo to the ld phase rules their behavior in heterogeneous membranes. Overall, our results provide detailed information on the effect of PUFAs on homogeneous and heterogeneous membranes.

Stereocontrolled total synthesis of Resolvin D4 and 17(R)-Resolvin D4.

The total synthesis of Resolvin D4 and its 17(R)-hydroxy-epimer is reported. These lipid-based natural products are biosynthesized from docosahexaenoic acid (DHA, C22:6) during the body's rapid cellular and chemical response to injurious stimuli and are part of a large class of bioactive molecules that resolve inflammation. Our convergent synthesis employed a chiral pool strategy starting from glycidol derivatives and D-erythrose to introduce stereogenic centers. A copper(I)-mediated cross coupling between propargyl bromide and terminal acetylenic precursors yielded core structures of late-stage key intermediates. A simultaneous Lindlar reduction of the skipped diynyl moiety followed by silyl group cleavage securely completed the synthesis. The synthetic availability of these molecules helped further elucidate their stereoselective biofunctions.

Role of VLC-PUFAs in Retinal and Macular Degeneration.

Very-long-chain polyunsaturated fatty acids (VLC-PUFAs) are a special class of fatty acids that are present in the retina and a few other human tissues. They cannot be synthesized de novo and are rarely present in dietary sources. Structurally, these lipids are composed of a proximal end with a typical saturated fatty acid character and a distal end more characteristic of common PUFAs. They have not been studied in detail until recently due to their low abundance in these tissues and technical difficulties in assaying these lipids by conventional chromatography. This unique class of lipids has chain lengths greater than 24 carbons, with the longest typically 38 carbons long. There is increasing interest in understanding their roles in the maintenance of retinal membrane integrity and the prevention of macular degeneration and inherited retinal diseases.

Epoxidation of Methyl Esters as Valuable Biomolecules: Monitoring of Reaction.

The paper is focused on the epoxidation of methyl esters prepared from oil crops with various profiles of higher fatty acids, especially unsaturated, which are mainly contained in the non-edible linseed and Camelina sativa oil (second generation). The novelty consists in the separation and identification of all products with oxirane ring formed through a reaction and in the determination of time course. Through the epoxidation, many intermediates and final products were formed, i.e., epoxides with different number and/or different position of oxirane rings in carbon chain. For the determination, three main methods (infrared spectroscopy, high-pressure liquid chromatography and gas chromatography with mass spectrometry) were applied. Only gas chromatography enables the separation of individual epoxides, which were identified on the base of the mass spectra, molecule ion and time course of products. The determination of intermediates enables: (i) control of the epoxidation process, (ii) determination of the mixture of epoxides in detail and so the calculation of selectivity of each product. Therefore, the epoxidation will be more environmentally friendly especially for advanced applications of non-edible oil crops containing high amounts of unsaturated fatty acids.

Influence of very-long-chain polyunsaturated fatty acids on membrane structure and dynamics.

The unique attributes of very-long-chain polyunsaturated fatty acids (VLC-PUFAs), their long carbon chains (n > 24) and high degree of unsaturation, impart unique chemical and physical properties to this class of fatty acids. The changes imparted by VLC-PUFA 32:6 n-3 on lipid packing and the compression moduli of model membranes were evaluated from π-A isotherms of VLC-PUFA in 1,2-distearoyl-sn-3-glycero-phosphocholine (DSPC) lipid monolayers. To compare the attractive or repulsive forces between VLC-PUFA and DSPC lipid monolayers, the measured mean molecular areas (MMAs) were compared with the calculated MMAs of an ideal mixture of VLC-PUFA and DSPC. The presence of 0.1, 1, and 10 mol % VLC-PUFA shifted the π-A isotherm to higher MMAs of the lipids comprising the membrane and the observed positive deviations from ideal behavior of the mixed VLC-PUFA:DSPC monolayers correspond to repulsive forces between VLC-PUFAs and DSPC. The MMA of the VLC-PUFA component was estimated using the measured MMAs of DSPC of 47.1 ± 0.7 Å2/molecule, to be 15,000, 1100, and 91 Å2/molecule at 0.1, 1, and 10 mol % VLC-PUFA:DSPC mixtures, respectively. The large MMAs of VLC-PUFA suggest that the docosahexaenoic acid tail reinserts into the membrane and adopts a nonlinear structure in the membrane, which is most pronounced at 0.1 mol % VLC-PUFA. The presence of 0.1 mol % VLC-PUFA:DSPC also significantly increased the compression modulus of the membrane by 28 mN/m compared with a pure DSPC membrane. The influence of VLC-PUFA on lipid "flip-flop" was investigated by sum-frequency vibrational spectroscopy. The incorporation of 0.1 mol % VLC-PUFA increased the DSPC flip-flop rate fourfold. The fact that VLC-PUFA promotes lipid translocation is noteworthy as retinal membranes require a high influx of retinoids which may be facilitated by lipid flip-flop.

Structure and mechanism of Staphylococcus aureus oleate hydratase (OhyA).

Flavin adenine dinucleotide (FAD)-dependent bacterial oleate hydratases (OhyAs) catalyze the addition of water to isolated fatty acid carbon-carbon double bonds. Staphylococcus aureus uses OhyA to counteract the host innate immune response by inactivating antimicrobial unsaturated fatty acids. Mechanistic information explaining how OhyAs catalyze regiospecific and stereospecific hydration is required to understand their biological functions and the potential for engineering new products. In this study, we deduced the catalytic mechanism of OhyA from multiple structures of S. aureus OhyA in binary and ternary complexes with combinations of ligands along with biochemical analyses of relevant mutants. The substrate-free state shows Arg81 is the gatekeeper that controls fatty acid entrance to the active site. FAD binding engages the catalytic loop to simultaneously rotate Glu82 into its active conformation and Arg81 out of the hydrophobic substrate tunnel, allowing the fatty acid to rotate into the active site. FAD binding also dehydrates the active site, leaving a single water molecule connected to Glu82. This active site water is a hydronium ion based on the analysis of its hydrogen bond network in the OhyA•PEG400•FAD complex. We conclude that OhyA accelerates acid-catalyzed alkene hydration by positioning the fatty acid double bond to attack the active site hydronium ion, followed by the addition of water to the transient carbocation intermediate. Structural transitions within S. aureus OhyA channel oleate to the active site, curl oleate around the substrate water, and stabilize the hydroxylated product to inactivate antimicrobial fatty acids.

Reactive Thread Spray Mass Spectrometry for Localization of C═C Bonds in Free Fatty Acids: Applications for Obesity Diagnosis.

Cellulose thread substrates offer a platform for microsampling and reactive ionization of free fatty acid (FFA) isomers for direct differentiation by mass spectrometry. Ambient corona discharge forms when direct current high voltage is applied to the tiny subfibers on the thread substrate in the presence of a polar spray solvent (MeOH/H2O, 2:1, v/v), facilitating chemical reactions across a C═C bond of unsaturated fatty acids. The process was applied for diagnosis of obesity, which we observed to show better discriminatory power when compared to determinations based on body mass index. Overall, the integrated reactive thread-based platform is capable of (i) microsampling and dry-state, room-temperature storage (>30 days) of the biofluids, (ii) in-capillary liquid/liquid extraction, and (iii) in situ epoxidation reactions to locate the C═C bond position in unsaturated fatty acids via reactions with reactive oxygen species present in ambient corona discharge. The study showcased the ability to correctly characterize FFAs, including degree of unsaturation, and the determination of their relative concentrations in clinical biofluid samples.

Identification of Carbon-Carbon Double Bond Stereochemistry in Unsaturated Fatty Acids by Charge-Remote Fragmentation of Fixed-Charge Derivatives.

Separation and identification of fatty acid (FA) isomers in biological samples represents a challenging problem for lipid chemists. Notably, FA regio- and stereo-isomers differing in the location or (cis/trans) geometry of carbon-carbon double bonds are often incompletely separated and ambiguously assigned in conventional chromatography-mass spectrometry analyses. To address this challenge, FAs have been derivatized with the charge-switch derivatization reagents N-methyl-pyridinium-3-methanamine and N-(4-aminomethylphenyl)pyridinium and subjected to reversed-phase liquid chromatography-tandem mass spectrometry. Charge-remote fragmentation of the fixed-charge derivatives leads to characteristic product ions arising from dissociation at allylic positions that enable assignment of position(s) of unsaturation, while a newly discovered dihydrogen neutral loss was found to be dominant for double bonds with cis-stereochemistry. The structure of the [M - 2]+ product ions was probed by gas-phase ozonolysis revealing the presence of two new carbon-carbon bonds on either side of the initial position of unsaturation consistent with an electrocyclic mechanism of 1,4-dihydrogen elimination. Charge-remote fragmentation pathways diagnostic of double bond position and stereochemistry were found to be generalized for FAs of different carbon-chain lengths, double bond positions, and degrees of unsaturation and were effective in the unequivocal assignment of the FA structure in complex mixtures of FA isomers, including bovine milk powder.