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
复制标题
新型脂肪酸探针 ω-乙炔基二十碳五烯酸类似物的合成和功能评估，用于分析二十碳五烯酸的体内行为
DOI：
10.1021/acs.bioconjchem.7b00235
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发表时间：
2017
影响因子：
4.7
通讯作者：
Kurihara Tatsuo
中科院分区：
文献类型：
--
作者：
Tokunaga Tomohisa;Watanabe Bunta;Sato Sho;Kawamoto Jun;Kurihara Tatsuo
二十碳五烯酸（Eicosapentaenoic acid， EPA）是一种ω-3多不饱和脂肪酸，在从细菌到人类的生物体中发挥着多种有益作用。虽然其有益的生理功能已被公认，但一种能够监测其体内行为而不取消其天然功能的分子探针尚未开发出来。本文设计并合成了ω-乙基EPA类似物（eEPA），作为分析EPA在体内行为和功能的工具。eEPA有一个ω-乙基标签代替EPA的ω-甲基。乙基具有典型的拉曼信号，可以用拉曼散射显微镜观察。此外，该基团可以通过点击化学与叠氮化合物（如具有荧光基团的叠氮化合物）在原位特异性反应。在本研究中，我们首先基于以下众所周知的策略高效地合成了eEPA。为了引入4个C-C双键，采用末端乙炔与丙炔卤化物或戊磺酸盐的偶联反应，然后与P-2镍同时进行立体选择性部分加氢，将三键转化为双键。与含乙基的磷盐通过Wittig反应引入了一个双键和一个ω端C-C三键。然后，我们利用产生epa的希瓦氏菌（Shewanella livingstonensisAc10）来评估所得探针的体内功能。这种冷适应细菌在低温下诱导产生EPA， EPA缺陷突变体（ΔEPA）在低温下表现出生长迟缓和形态异常。当外源补充eEPA至ΔEPA时，eEPA以酰基链的形式掺入到膜磷脂中，其含量约为膜中总脂肪酸的5%，与亲本菌株的膜中EPA含量相当。值得注意的是，添加eEPA后，ΔEPA的生长迟缓和形态异常几乎完全被抑制。这些结果表明，eEPA能很好地模拟EPA，为分析EPA在体内的行为提供了依据。
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 tocisdouble 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 livingstonensisAc10. 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.