Studies on Biosynthesis of Ecdysteroids with Plant Tissue Culture

植物组织培养脱皮素生物合成研究

• 批准号: 02640422
• 负责人: FUJIMOTO Yoshinori
• 金额: $ 1.34万
• 依托单位: Tokyo Institute of Technology
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1990
• 资助国家: 日本
• 起止时间: 1990 至 1991
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-02640422/
• 关键词: ecdysteroid; 20-hydroxyecdysone; plant tissue culture; stable isotope; ^2H-NMR; biosynthetic pathway; 生合成機構; 安定同位体標識化合物; 毛状根; Ajuga reptans; 生合成メカニズム

1. Feeding experiment of (2- ^<13>C) acetate with the tissue culture of Ajuga hairy root revealed that it was incorporated into ecdysteroids efficiently. It was found that the 24-alkyl group of C-28 and C-29 ecdysteroids was not derived from acetate.2. Feeding of synthetic (26, 27- ^<13>C_2) cholesterol as an emulsion into Tween80 to the tissue culture was allowed to incorporate into 20-hydroxy-ecdysone efficiently, but not into C-28 and C-29 ecdysteroids.3. More importantly, the above experiment has demonstrated that stable isotopes such as ^1H and ^<13>C can be utilized in biosynthetic studies of ecdysteroids.4. (3 - ^2H)- and (4 - ^2H) Cholesterols were chemically synthesized.5. ^2H-NMR analysis of 20-hydroxyecdysones derived from the ^2H-labeled cholesterols revealed that the 3alpha- and 4beta-hydrogens were incorporated into 20-hydroxyecdysone without change of the location and stereochemistry.6.5beta- ^2H derivative of an intermediate, 3beta-hydroxy-5beta-cholest-7-en-6-one, which was postulated by the aforementioned results, was synthesized and fed with the culture system. The feeding experiment demonstrated that the intermediate was metabolized into 20-hydroxyecdysone much better than cholesterol.7. We now propose 3beta-hydroxy-5beta-cholest-7-en-6-one as a key intermediate in the earlier stage of ecdysone biosynthesis. Our present studies are focused on the identification of a precursor of this intermediate.

1. （2- ^<13>C）乙酸酯与牛毛根组织培养的饲喂试验表明，乙酸酯能有效地融入体外甾体。结果表明，C-28和C-29外甾体的24-烷基并非来源于乙酸酯。将合成的（26,27 - ^<13>C_2）胆固醇作为乳化液投喂到Tween80中进行组织培养，可以有效地融入20-羟基蜕皮激素，但不能融入C-28和C-29蜕皮激素。更重要的是，上述实验证明了^1H和^<13>C等稳定同位素可用于体外甾体激素的生物合成研究。（3 - ^2H）-和（4 - ^2H）胆固醇被化学合成。^2H- nmr分析表明，由^2H标记的胆固醇衍生的20-羟基蜕皮酮的3 -和4 -氢被纳入20-羟基蜕皮酮中，而位置和立体化学没有改变。根据上述结果，合成了中间产物3 -羟基-5 - β -胆-7-烯-6- 1的6.5 β - ^2H衍生物，并在培养体系中进行了培养。饲养试验表明，该中间体代谢成20-羟基蜕皮激素的能力明显优于胆固醇。我们现在提出3 - β -羟基-5 - β -胆-7-en-6- 1是蜕皮激素生物合成早期阶段的关键中间体。我们目前的研究主要集中在鉴定这种中间体的前体。