Studies on Biosynthesis of Ecdysteroids with Plant Tissue Culture

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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.