Biosynthesis of the Insect-Moulting Hormone Cyasterone in the Plant Cyathula capitata

Approximately 30 different closely related compounds possessing moulting hormone activity (ecdysones) have been isolated from plants, but such compounds are by no means universally distributed within the plant kingdom (Hikino & Hikino, 1970; Rees, 1971 ; Horn, 1971). They include compounds possessing C2,, CZ8 or CZ9 steroid skeletons, ecdysterone (Cz7) being probably the most widespread insect-moulting hormone in the plants investigated so far. The incorporation of ['4C]cholest-5-en-3~-ol into several Cz, ecdysones, including ecdysterone (compound I in Scheme l), has been demonstrated in various plants (Sauer etul., 1968; Hikino etal., 1970; de Souzaetal., 1970). However, it is conceivable that the ecdysone biosynthetic pathway in plants could branch off the normal phytosterol pathway before the 4-desmethyl sterol stage. We now report studies on the biosynthesis of the Cz9 moulting hormone, cyasterone (11). This is the major moulting hormone in the plant, Cyuthula capitata (Amaranthaceae), where it is accompanied by smaller amounts of other ecdysones, e.g. compounds IV, V and VI (Hikino & Takemoto, 1972). A plausible biosynthetic pathway [(IV) -+ (V) -+ (VI) --f (II)] to cyasterone (11) has been suggested, based on structural considerations of the ecdysones occurring in the plant (Takemoto et al., 1968). As a preliminary part of our biosynthetic studies, the major sterols of Cyathula capitata were identified as 5astigmasta-7,Z-24(28)-dien-3~-01 (VII), 24-ethyl-5a-cholest-7-en-3B-ol (VIII), (248)-24ethylcholesta-5,22-dien-3~-ol (IX), 24-ethylcholest-5-en-3B-01 (X) and 24-methylcholest-5-en-3B-01 (XI) by g.l.c., mass and nuclear magnetic resonance (n.m.r.) spectrometry. The configuration of the (2-24 ethyl group in compound IX was deduced from its 220 MHz n.m.r. spectrum. For investigation of the biosynthetic pathway of cyasterone, [2-'4C,(4R)-4-3H,]mevalonic acid was administered to Cyathula capitata. A solution of [2-14C,(4R)-4-3H1]mevalonic acid (45pCi of 14C; 150pCi of 3H) in ethanol containing 0.01 % Nonidet P42 (non-ionic detergent) was administered to the leaves of young seedlings of Cyathula capitata over a 4 week period. After a further week, the plants were harvested and the leaves washed with light petroleum, to remove any unabsorbed mevalonate. The plants were macerated in ethanol and the slurry heated under reflux for 10h. After cooling, the ethanolic solution was filtered and the filtrate evaporated to dryness yielding a gummy residue, which was suspended in water and extracted with light petroleum. The petroleum extracts were combined and washed with water. The original aqueous layer was re-extracted with n-butanol and the combined butanol extracts were then washed with the above water washings. The light petroleum and butanol extracts were then evaporated to dryness. Purification of sterols. The light petroleum extract was saponified with aqueous ethanolic KOH in the usual manner and the non-saponifiable material was fractionated by alumina column chromatography. The 4-desmethyl sterols were purified by t.1.c. on silica gel, acetylated and the acetates separated by t.1.c. on AgN03-impregnated silica gel H with chloroform (ethanol-free) for development. 24-Methylcholest-5-en-3B-ol (XI) and 24-ethylcholest-5-en-3/?-ol (X) were separated by preparative g.1.c. The latter sterol was further transformed into 24-ethylcholest-4-en-3,6-dione by treatment with Jones reagent (Crabbe et ul., 1961). Steryl acetates (except 24-methylcholest-5-en-3~-ol) were diluted with carrier material and recrystallized to constant specific radioactivity. The crude squalene fraction from the column chromatography was further purified by t.l.c., hexahydrochloride formation after addition of carrier material, and recrystallization. Purification of cyasterone. A portion of the butanol extract was diluted with carrier cyasterone and subjected to two successive separations by silica gel t.1.c. in each of two solvent systems [chloroform-methanol, 3 : 1 (v/v); ethyl acetate-methanol, 10: 1 (vlv)].