Taxol biosynthesis: Differential transformations of taxadien-5 alpha-ol and its acetate ester by cytochrome P450 hydroxylases from Taxus suspension cells

The biosynthesis of the diterpenoid antineoplastic drug Taxol in Taxus spec ies involves the cyclization of the ubiquitous isoprenoid intermediate gera nylgeranyl diphosphate to taxa-4(5),11(12)-diene followed by cytochrome P45 0-mediated hydroxylation (with allylic rearrangement) of this olefin precur sor to taxa-4(20),11(12)-dien-5 alpha -ol, and further oxygenation and acyl ation reactions. Based on the abundances of naturally occurring taxoids, th e subsequent order of oxygenation of the taxane core is considered to occur at C10, then C2 and C9, followed by C13, and finally C7 and C1, Circumstan tial evidence suggests that the acetylation of taxadien-5 alpha -ol may con stitute the third specific step of Taxol biosynthesis, To determine whether taxadienol or the corresponding acetate eater serves as the direct precurs or of subsequent oxygenation reactions, microsomal preparations isolated fr om induced Taxus cells and optimized for cytochrome P450 catalysis were inc ubated with each potential substrate. Both taxadienol and taxadienyl acetat e were oxygenated to the level of a diol and to higher polyols at comparabl e rates by cytochrome P450 enzymes of the microsomal preparation. Preparati ve-scale incubation allowed the isolation of sufficient quantities of the d iol derived from taxadienol to permit the NMR-based structural elucidation of this metabolite as taxa-4(20),11(12)-dien-5 alpha ,13 alpha -diol, which may represent an alternate route of taxoid metabolism in induced cells. GC -MS-based structural definition of the diol monoacetate derived in microsom es from taxadienyl acetate confirmed this metabolite as taxa-4(20),11(12)-d ien-5 alpha -acetoxy-10 beta -ol, thereby indicating that acetylation at C5 of taxadienol precedes the cytochrome P450-mediated insertion of the C10-b eta -hydroxyl group of Taxol, (C) 2001 Academic Press.