CONVERSION OF 24-METHYLCHOLESTEROL TO 6-OXO-24-METHYLCHOLESTANOL, A PUTATIVE INTERMEDIATE OF THE BIOSYNTHESIS OF BRASSINOSTEROIDS, IN CULTURED-CELLS OF CATHARANTHUS-ROSEUS

Early steps of brassinosteroid biosynthesis were investigated using cultured cells of Catharanthus roseus. In the cells, 24-methylcholesterol was the major sterol component which accounted for 50% of total sterols and was assumed to be the possible biosynthetic precursor of brassinolide. [C-13(5)] and/or [C-14]24-methylcholesterol were prepared by the cultured cells in the presence of compactin, a mevalonic acid biosynthetic inhibitor. The endogenous metabolites of the labelled 24-methylcholesterol, 24-methylcholestanol, 6 alpha-hydroxy-24-methyIcholestanol (24-methyl-5 alpha-cholestane-3 beta,6 alpha-diol), and 6-oxo-24-methylcholestanol (3 beta-hydroxy-24-methyl-5 alpha-cholestan-6-one were identified by full-scan GC-mass spectrometry. The biosynthetic sequence of 24-methylcholesterol-->24-methylcholestanol--> 6 alpha-hydroxy-24-methylcholestanol-->6-oxo-24-methylcholestanol was established in cultured cells of C. roseus by feeding experiments using labelled 24-methylcholestanol and 6 alpha-hydroxy-24-methylcholestanol. This sequence probably constitutes an early part of the biosynthetic pathway of brassinolide, as introduction of vicinal hydroxyls in the side chain (C-22R and C-23R) of 6-oxo-24-methylcholestanol will yield teasterone. This is the first report of the natural occurrence of 6 alpha-hydroxy-24-methylcholestanol.