Studies on Biosynthesis of Ecdysteroids with Plant Tissue Culture

1.以毛状根组织培养为试材，研究了(2-β&lt；13-gt；C)乙酸酯对毛状根生长的影响，结果表明，(2-β-lt；13-gt；C)乙酸酯能有效地掺入蜕皮甾体中。结果表明，C-28和C-29蜕皮甾体中的24-烷基不是乙酸酯类化合物。将合成的(26，27-^和13&gt；C_2)胆固醇作为乳状液加入吐温80组织培养中，可以有效地掺入20-羟基蜕皮激素，但不能掺入C-28和C-29蜕皮激素。更重要的是，上述实验证明了稳定同位素^1H和^&lt；13&gt；C可用于蜕皮激素的生物合成研究。化学合成了(3-^2H)-和(4-^2H)-胆固醇。从2H标记的胆固醇衍生的20-羟基蜕皮酮的2H-核磁共振分析表明，3α和4β-氢被结合到20-羟基蜕皮酮中，而不改变位置和立体化学。根据上述结果合成了中间体3β-羟基-5β-胆甾烯-7-烯-6-酮的6.5β-^2H衍生物，并与培养体系进行了比较。饲养实验表明，该中间体代谢为20-羟基蜕皮酮的效果远好于胆固醇。我们现在建议3β-羟基-5β-胆碱-7-烯-6-酮作为蜕皮激素生物合成早期阶段的关键中间体。我们目前的研究集中在该中间体的前体的鉴定上。

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.