The Hydroxylation of Plant Diterpene Analogues by the Fungus Syncephalastrum racemosum

Triepoxide diterpenes isolated from the perennial herb, Tripterygium wilfordii, have shown promising anti-leukemic and anti-inflammatory activity in vitro and in vivo. However, the toxicity of these epoxides has limited their clinical usefulness and analogues are sought which possess the pharmacological actions but have a lower toxicity. In this study, we have generated oxidized analogues of these diterpenes from a synthetic lactone precursor (19-hydroxy-18 (4-3) abeo-abieta-3,8,11,13-tetraen-18-oic acid lactone) by incubating the lactone with the Zygomycete fungus, Syncephalastrum racemosum.Incubation of lactone with S. racemosum in liquid media yielded four distinct polar metabolites as determined by thin-layer chromatography of ethyl acetate extracts. Column purification followed by structural analysis by 1H NMR and mass spectroscopy revealed these compounds to be 7-β-hydroxylactone, 7-α-hydroxylactone, 15-hydroxylactone, and 7-ketolactone. When lactone was added to 48-h-old cultures, 7-β-hydroxylactone appeared after 30 min whereas there was a 4-h lag period before the 7-α-hydroxy and 15-hydroxy metabolites were detected suggesting that enzyme induction was necessary before metabolism occurred. The 7-ketolactone was not detected until 24 h of incubation and this may represent a nonenzymatic oxidation of 7-hydroxylactone. The addition of various cytochrome P450 inhibitors (ketoconazole, miconazole, α-naphthoflavone, aminobenzothiazole, and carbon monoxide) to liquid cultures prevented the 15-hydroxylation of lactone. Interestingly, in contrast to the other agents, ketoconazole and miconazole (20 μM) were not able to inhibit 7-hydroxylation even though 15-hydroxylation was inhibited by these agents. With the exception of carbon monoxide, cell growth was unaffected by these agents. These data suggest that the biotransformation of diterpenes by S. racemosum was mediated by an inducible hydroxylase system which is sensitive to cytochrome P450 inhibitors and that more than one hydroxylase enzyme may be responsible for the observed products.