Full Library of (<i>Bis–</i>allyl)‐deuterated Arachidonic Acids: Synthesis and Analytical Verification

Fomich, Maksim A.; Bekish, Andrei V.; Vidović, Dragoslav; Lamberson, Connor R.; Lysenko, Ivan L.; Lawrence, Peter; Brenna, J. Thomas; Шарко, О. Л.; Shmanai, Vadim V.; Shchepinov, Mikhail S.

doi:10.1002/slct.201600955

Cited by 13 publications

(10 citation statements)

References 49 publications

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“… 16 Following on this observation, if LOX-catalyzed oxidation of AA—its native substrate—plays a key role in ferroptosis execution, cells grown in media supplemented with AA labeled at the bis-allylic positions from which each LOX isoform regiospecifically abstracts a H-atom should be protected from ferroptosis. The use of regiospecifically labeled substrates 39 as isoform-selective inhibitors of LOX ( Figure 4 A, Figure S4A,B ) removes any uncertainty regarding off-target effects of inhibitors or interactions of inhibitors with the agents used to induce ferroptosis. As expected, 7,7- d 2 -AA inhibited 5-LOX activity, but not p12-LOX or 15-LOX-1 activity ( Figure 4 B), 10,10- d 2 -AA inhibited p12-LOX activity, but not 5-LOX or 15-LOX-1 activity ( Figure 4 C), and 13,13- d 2 -AA inhibited 15-LOX-1 activity, but not 5-LOX or p12-LOX activity ( Figure 4 D).…”

Section: Resultsmentioning

confidence: 99%

Resolving the Role of Lipoxygenases in the Initiation and Execution of Ferroptosis

2018

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Lipoxygenases (LOXs) have been implicated as central players in ferroptosis, a recently characterized cell death modality associated with the accumulation of lipid hydroperoxides: the products of LOX catalysis. To provide insight on their role, human embryonic kidney cells were transfected to overexpress each of the human isoforms associated with disease, 5-LOX, p12-LOX, and 15-LOX-1, which yielded stable cell lines that were demonstrably sensitized to ferroptosis. Interestingly, the cells could be rescued by less than half of a diverse collection of known LOX inhibitors. Furthermore, the cytoprotective compounds were similarly potent in each of the cell lines even though some were clearly isoform-selective LOX inhibitors. The cytoprotective compounds were subsequently demonstrated to be effective radical-trapping antioxidants, which protect lipids from autoxidation, the autocatalytic radical chain reaction that produces lipid hydroperoxides. From these data (and others reported herein), a picture emerges wherein LOX activity may contribute to the cellular pool of lipid hydroperoxides that initiate ferroptosis, but lipid autoxidation drives the cell death process.

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Section: Resultsmentioning

confidence: 99%

Resolving the Role of Lipoxygenases in the Initiation and Execution of Ferroptosis

2018

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“…D2‐Lin, D4‐Lnn , and D6‐Ara were prepared as described previously by assembling corresponding polyyne chains and subjecting them to catalytic hydrogenation. Although a similar convergent synthetic strategy, based on a Wittig olefination, was developed to obtain selectively deuterated DHA species , D10‐DHA could be prepared using the less laborious approach of catalytic H/D exchange .…”

Section: Resultsmentioning

confidence: 99%

Threshold protective effect of deuterated polyunsaturated fatty acids on peroxidation of lipid bilayers

et al. 2019

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Autoxidation of polyunsaturated fatty acids (PUFAs) damages lipid membranes and generates numerous toxic by‐products implicated in neurodegeneration, aging, and other pathologies. Abstraction of bis‐allylic hydrogen atoms is the rate‐limiting step of PUFA autoxidation, which is inhibited by replacing bis‐allylic hydrogens with deuterium atoms (D‐PUFAs). In cells, the presence of a relatively small fraction of D‐PUFAs among natural PUFAs is sufficient to effectively inhibit lipid peroxidation (LPO). Here, we investigate the effect of various D‐PUFAs on the stability of liposomes under oxidative stress conditions. The permeability of vesicle membranes to fluorescent dyes was measured as a proxy for bilayer integrity, and the formation of conjugated dienes was monitored as a proxy for LPO. Remarkably, both approaches reveal a similar threshold for the protective effect of D‐PUFAs in liposomes. We show that protection rendered by D‐PUFAs depends on the structure of the deuterated fatty acid. Our findings suggest that protection of PUFAs against autoxidation depends on the total level of deuterated bi‐sallylic (CD2) groups present in the lipid bilayer. However, the phospholipid containing 6,6,9,9,12,12,15,15,18,18‐d10‐docosahexaenoic acid exerts a stronger protective effect than should be expected from its deuteration level. These findings further support the application of D‐PUFAs as preventive/therapeutic agents in numerous pathologies that involve LPO.

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“…Shchepinov and collegues; the synthesis and analytical verification is described elsewhere. 8 Isotopologues of arachidonic acid (AA) used in this study: 7,7-(D 2 )-AA, 10,10-(D 2 )-AA, 13,13-(D 2 )-AA, 7,7,10,10-(D 4 )-AA, 7,7,13,13-(D 4 )-AA, 10,10,13,13-(D 2 )-AA, and 7,7,10,10,13,13-(D 6 )-AA. These AA compounds were synthesized as ethyl esters for improved stability during storage.…”

Section: Methodsmentioning

confidence: 99%

Lipidomics Reveals Dramatic Physiological Kinetic Isotope Effects during the Enzymatic Oxygenation of Polyunsaturated Fatty Acids Ex Vivo

2017

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Arachidonic acid (AA, 20:4) is an omega-6 polyunsaturated fatty acid (PUFA) and the main precursor to the class of lipid mediators known as eicosanoids. The enzymes that catalyze the oxygenation of AA begin by abstracting hydrogen from one of three bis-allylic carbons within 1,4-cis,cis-diene units. Substitution of deuterium for hydrogen has been shown to lead to massive kinetic isotope effects (KIE) for soybean lipoxygenase (sLOX) oxygenation of linoleic acid (LA, 18:2). Yet, experimental determination of the KIE during oxygenation of AA and LA by mammalian enzymes including cyclooxygenase (COX) and lipoxygenase (LOX) has revealed far lower values. All prior studies investigating the KIE of PUFA oxygenation have relied on in vitro systems using purified enzymes and were limited by availability of deuterated substrates. Here we demonstrate the use of macrophages as an ex vivo model system to study the physiological KIE (PKIE) during enzymatic AA oxygenation by living cells using a newly synthesized library of deuterated AA isotopologues. By extending lipidomic UPLC-MS/MS approaches to simultaneously quantify native and deuterated lipid products, we were able to demonstrate that the magnitude of the PKIE measured in macrophages for COX and LOX oxygenation of AA is similar to KIEs determined in previous reports using the AA isotopologue deuterated at carbon 13 (C13). However, for the first time we show that increasing the number of deuterated bis-allylic carbons to include both C10 and C13 leads to a massive increase in the PKIE for COX oxygenation of AA. We provide evidence that hydrogen(s) present at C10 of AA play a critical role in the catalysis of prostaglandin and thromboxane synthesis. Furthermore, we discovered that deuteration of C10 promotes the formation of the resolving lipid mediator lipoxin B4, likely by interfering with AA cyclization and shunting AA to the LOX pathway under physiological conditions.

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Full Library of (Bis–allyl)‐deuterated Arachidonic Acids: Synthesis and Analytical Verification

Cited by 13 publications

References 49 publications

Resolving the Role of Lipoxygenases in the Initiation and Execution of Ferroptosis

Resolving the Role of Lipoxygenases in the Initiation and Execution of Ferroptosis

Threshold protective effect of deuterated polyunsaturated fatty acids on peroxidation of lipid bilayers

Lipidomics Reveals Dramatic Physiological Kinetic Isotope Effects during the Enzymatic Oxygenation of Polyunsaturated Fatty Acids Ex Vivo

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