Electrophilic Cyclopentenone Neuroprostanes Are Anti-inflammatory Mediators Formed from the Peroxidation of the ω -3 Polyunsaturated Fatty Acid Docosahexaenoic Acid.

The omega-3 polyunsaturated fatty acid docosahexaenoic acid (DHA) possesses potent anti-inflammatory properties and has shown therapeutic benefit in numerous inflammatory diseases. However, the molecular mechanisms of these anti-inflamma- tory properties are poorly understood. DHA is highly suscepti- ble to peroxidation, which yields an array of potentially bioac- tive lipid species. One class of compounds are cyclopentenone neuroprostanes (A4/J4-NPs), which are highly reactive and similar in structure to anti-inflammatory cyclopentenone prostaglandins. Here we show that a synthetic A4/J4-NP, 14-A4-NP (A4-NP), potently suppresses lipopolysaccharide- induced expression of inducible nitric-oxide synthase and cyclooxygenase-2 in macrophages. Furthermore, A4-NP blocks lipopolysaccharide-induced NF-KB activation via inhibition of I kinase-mediated phosphorylation of IKB. Mutation on Ik kinase b-cysteine 179 markedly diminishes the effect of A4-NP, suggesting that A4-NP acts via thiol mod- ification at this residue. Accordingly, the effects of A4-NP are independent of peroxisome proliferator-activated receptor-gamma and are dependent on an intact reactive cyclopentenone ring. Interestingly, free radical-mediated oxidation of DHA greatly enhances its anti-inflammatory potency, an effect that closely parallels the formation of A4/J4-NPs. Furthermore, chemical reduction or conjugation to glutathione, both of which elim- inate the bioactivity of A4-NP, also abrogate the anti-inflam- matory effects of oxidized DHA. Thus, we have demonstrated that A4/J4-NPs, formed via the oxidation of DHA, are potent inhibitors of NF-kB signaling and may contribute to the anti- inflammatory actions of DHA. These findings have implica- tions for understanding the anti-inflammatory properties of omega-3 fatty acids, and elucidate novel interactions between lipid peroxidation products and inflammation

First total synthesis of labeled EPA and DHA-derived A-type cyclopentenone isoprostanoids: [D2]-15-A3t-IsoP and [D2]-17-A4t-NeuroP

A-type cyclopentenone isoprostanoids are abundantly formed in vivo by radical peroxidation of eico- sapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which are consumed daily for the prevention of cardiovascular and neurological pathologies. To facilitate in depth studies concerning the effects of these oxidized isoprostanoids on human health, labeled derivatives are necessary. In this paper, we have accomplished the ␣rst total synthesis of labeled A-type cyclopentenone isoprostanoids, namely 17,18- [D2]-15-A3t-IsoP and 19,20-[D2]-17-A4t-NeuroP. The two enantioselective routes are highly convergent, stemming from a common intermediate, readily available by a JuliaeKocienski reaction, and feature the semihydrogenation of an alkyne moiety for the installation of the labeled lower side chain

Asymmetric synthesis of 14-A(4t)-neuroprostane: hunting for a suitable biomarker for neurodegenerative diseases

Oxidative stress has long been associated with aging and age-related pathologies, such as neurodegenerative diseases. One of the direct effects of oxidative stress in vivo is the formation of prostaglandin-like compounds, named isoprostanes, by the action of reactive oxygen species on membrane phospholipids. A particular subclass of isoprostanes, named neuroprostanes, is formed from docosahexaenoic acid (C22: 6ω3, DHA) and is considered to be specific for neuronal oxidative stress. Since isoprostanes are considered as golden standards for oxidative stress, and due to the specificity of neuroprostanes for this condition in neurons and their relation with Alzheimer’s and Parkinson’s diseases, they are envisioned to be suitable biomarkers for these pathologies. Herein we describe the first total synthesis of 14-A4t-NeuroP in an enantioselective and stereoselective fashion, by means of a new and rapid approach for the installation of the ω chain based on a chemoselective Julia-Kocienski olefination. Furthermore, the construction of the 4,5-cis-disubstituted cyclopentenone moiety characteristic of class A neuroprostanes is achieved in a stereospecific fashion, and suitable reaction conditions have been tuned to avoid epimerization of the labile stereogenic centers

Biology and chemistry of neuroprostanes. First total synthesis of 17-A4-NeuroP: Validation of a convergent strategy to a number of cyclopentenone neuroprostanes

In a process associated with ageing and neurodegeneration, radical peroxidation of docosahexaenoic acid (DHA) in neurons affords a multitude of prostaglandin-like neuroprostanes in a non-regioselective and non-stereoselective manner. In this paper, the synthesis of racemic 17-A4 -NeuroP and 14-A4 -NeuroP validated a general approach to several regioisomeric cyclopentenone A4 - and J4 -NeuroPs needed for bio- logical tests. In preliminary experiments 17-A4 -NeuroP, in analogy with 14-A4 -NeuroP, readily adducted GSH free thiol, suggesting a similar mechanism of action for biological activity

Cyclopentenone prostaglandin, 15-deoxy-Δ12,14-PGJ2, is metabolized by HepG2 cells via conjugation with Glutathione

15-Deoxy-Delta(12,14)-prostaglandin J(2) (15-d-PGJ(2)) is a dehydration product of PGD(2). This compound possesses a highly reactive polyunsaturated carbonyl moiety that is a substrate for Michael addition with thiol-containing biomolecules such as glutathione and cysteine. residues on proteins. By reacting with glutathione and proteins, 15-d-PGJ(2) is believed to exert potent biological activity. Despite the large number of publications that have ascribed bioactivity to this molecule, it is not known to what extent 15-d-PGJ(2) is formed in vivo. Levels of free 15-d-PGJ(2) measured in human biological fluids such as urine are low, and the biological importance of this compound has thus been questioned. Because of its reactivity, we hypothesized that 15-d-PGJ(2) is present in vivo primarily as a Michael conjugate. Therefore, we undertook a detailed study of the metabolism of this compound in HepG2 cells that are known to metabolize other cyclopentenone eicosanoids. We report that HepG2 cells primarily convert 15-d-PGJ(2) to a glutathione conjugate in which the carbonyl at C-11 is reduced to a hydroxyl. Subsequently, the glutathione portion of the molecule is hydrolyzed with loss of glutamic acid and glycine resulting in a cysteine conjugate. These findings confirm a general route for the metabolism of cyclopentenone eicosanoids in HepG2 cells and may pave the way for new insights regarding the formation of 15-d-PGJ(2) in vivo

The fatty acid oxidation product 15-A3t-Isoprostane is a potent inhibitor of NFκB transcription and macrophage transformation

Fatty acids such as eicosapentaenoic acid (EPA) have been shown to be beneficial for neurological function and human health. It is widely thought that oxidation products of EPA are responsible for biological activity, although the specific EPA peroxidation product(s) which exert these responses have not yet been identified. In this work we provide the first evidence that the synthesized representative cyclopentenone IsoP, 15-A3t-IsoP, serves as a potent inhibitor of lipopolysaccharide- stimulated macrophage activation. The anti-inflammatory activities of 15-A3t-IsoP were observed in response not only to lipopolysaccharide, but also to tumor necrosis factor alpha and IL-1b stimulation. Subsequently, this response blocked the ability of these compounds to stimulate nuclear factor kappa b (NFjB) activation and production of proinflammatory cytokines. The bioactivity of 15-A3t-IsoP was shown to be dependent upon an unsaturated carbonyl residue which transiently adducts to free thiols. Site directed mutagenesis of the redox sensitive C179 site of the Ikappa kinase beta sub- unit, blocked the biological activity of 15-A3t-IsoP and NFjB activation. The vasoprotective potential of 15-A3t-IsoP was underscored by the ability of this compound to block oxidized lipid accumulation, a critical step in foam cell transformation and atherosclerotic plaque formation. Taken together, these are the first data identifying the biological activity of a specific product of EPA peroxidation, which is formed in abundance in vivo. The clear mechanism linking 15-A3t-IsoP to redox control of NFjB transcription, and the compound’s ability to block foam cell transformation suggest that 15-A3t-IsoP pro- vides a unique and potent tool to provide vaso- and cytopro- tection under conditions of oxidative stress