4 years ago

Synthesis and Functional Assessment of a Novel Fatty Acid Probe, ω-Ethynyl Eicosapentaenoic Acid Analog, to Analyze the in Vivo Behavior of Eicosapentaenoic Acid

Synthesis and Functional Assessment of a Novel Fatty Acid Probe, ω-Ethynyl Eicosapentaenoic Acid Analog, to Analyze the in Vivo Behavior of Eicosapentaenoic Acid
Jun Kawamoto, Tatsuo Kurihara, Bunta Watanabe, Sho Sato, Tomohisa Tokunaga
Eicosapentaenoic acid (EPA) is an ω-3 polyunsaturated fatty acid that plays various beneficial roles in organisms from bacteria to humans. Although its beneficial physiological functions are well-recognized, a molecular probe that enables the monitoring of its in vivo behavior without abolishing its native functions has not yet been developed. Here, we designed and synthesized an ω-ethynyl EPA analog (eEPA) as a tool for analyzing the in vivo behavior and function of EPA. eEPA has an ω-ethynyl group tag in place of the ω-methyl group of EPA. An ethynyl group has a characteristic Raman signal and can be visualized by Raman scattering microscopy. Moreover, this group can specifically react in situ with azide compounds, such as those with fluorescent group, via click chemistry. In this study, we first synthesized eEPA efficiently based on the following well-known strategies. To introduce four C–C double bonds, a coupling reaction between terminal acetylene and propargylic halide or tosylate was employed, and then, by simultaneous and stereoselective partial hydrogenation with P-2 nickel, the triple bonds were converted to cis double bonds. One double bond and an ω-terminal C–C triple bond were introduced by Wittig reaction with a phosphonium salt harboring an ethynyl group. Then, we evaluated the in vivo function of the resulting probe by using an EPA-producing bacterium, Shewanella livingstonensis Ac10. This cold-adapted bacterium inducibly produces EPA at low temperatures, and the EPA-deficient mutant (ΔEPA) shows growth retardation and abnormal morphology at low temperatures. When eEPA was exogenously supplemented to ΔEPA, eEPA was incorporated into the membrane phospholipids as an acyl chain, and the amount of eEPA was about 5% of the total fatty acids in the membrane, which is comparable to the amount of EPA in the membrane of the parent strain. Notably, by supplementation with eEPA, the growth retardation and abnormal morphology of ΔEPA were almost completely suppressed. These results indicated that eEPA mimics EPA well and is useful for analyzing the in vivo behavior of EPA.

Publisher URL: http://dx.doi.org/10.1021/acs.bioconjchem.7b00235

DOI: 10.1021/acs.bioconjchem.7b00235

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