Biosynthesis of sterols and ecdysteroids in Ajuga hairy roots

Hairy roofs of Ajuga reptans var, atropurpurea produce clerosterol, 22-dehydroclerosterol, and cholesterol as sterol constituents, and 20-hydroxyecdysone, cyasterone, isocyasterone, and 29-norcyasterone as ecdysteroid constituents. To better understand the biosynthesis of these steroidal compounds, we carried out feeding studies of variously H-2- and C-13-labeled sterol substrates with Ajuga hairy roots. In this article, we review our studies in this field. Feeding of labeled desmosterols, 24-methylenecholesterol, and C-13(2)-acetate established the mechanism of the biosynthesis of the two C-29-sterols and a newly accumulated codisterol, including the metabolic correlation of C-26 and C-27 methyl groups. in Ajuga hairy roots, 3 alpha-, 4 alpha-, and 4 beta-hydrogens of cholesterol were all retained at their original positions after conversion into 20-hydroxyecdysone, in contrast to the observations in a fern and an insect. Furthermore, the origin of 5 beta-H of 20-hydroxyecdysone was found to be C-6 hydrogen of cholesterol exclusively, which is inconsistent with the results in the fern and the insect. These data strongly support the intermediacy of 7-dehydrocholesterol 5 alpha,6 alpha-epoxide. Moreover, 7-dehydrocholesterol, 3 beta-hydroxy-5 beta-cholest-7-en-6-one (5 beta-ketol), and 3 beta,14 alpha-dihydroxy-5 beta-cholest-7-en-6-one (5 beta-ketodiol) were converted into 20-hydroxyecdysone. Thus, the pathway cholesterol --> 7-dehydrocholesterol --> 7-dehydrocholesterol 5 alpha,6 alpha-epoxide --> 5 beta-ketol --> 5 beta-ketodiol is proposed for the early stages of 20-hydroxyecdysone biosynthesis. 3 beta-Hydroxy-5 beta-cholestan-6-one was also incorporated into 20-hydroxyecdysone, suggesting that the introduction of a 7-ene function is not necessarily next to cholesterol. C-25 Hydroxylation during 20-hydroxyecdysone biosynthesis was found to proceed with ca. 70% retention and 30% inversion. Finally, clerosterol was shown to be a precursor of cyasterone and isocyasterone.