title: Substrate specificity for the 12beta-hydroxylation of bufadienolides by Alternaria alternata.
abstract: Hydroxylation is an important route to synthesize more hydrophilic compounds of pharmaceutical significance. Microbial hydroxylation offers advantages over chemical means for its high specificity. In this study, a fungal strain Alternaria alternata AS 3.4578 was found to be able to catalyze the specific 12beta-hydroxylation of a variety of cytotoxic bufadienolides. Cinobufagin and resibufogenin could be completely metabolized by A. alternata to generate their 12beta-hydroxylated products in high yields (>90%) within 8 h of incubation. A. alternata could also convert 3-epi-desacetylcinobufagin into 3-epi-12beta-hydroxyl desacetylcinobufagin as the major product (70% yield). C-3 dehydrogenated products were detected in these reactions in fair yields, while their accumulation was relatively slow. The 12beta-hydroxylation of bufadienolides could be significantly inhibited by the substitution of 1beta-, 5-, or 16alpha-hydroxyl groups, and the 14beta,15beta-epoxy ring appeared to be a necessary structural requirement for the specificity. For the biotransformation of bufalin, a 14beta-OH bufadienolide, this reaction was not specific, and accompanied by 7beta-hydroxylation as a parallel and competing metabolic route. The biotransformation products were identified by comparison with authentic samples or tentatively characterized by high-performance liquid chromatography-diode array detection-atmospheric pressure chemical ionization-mass spectrometry analyses.
authors.affiliation: The State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Xueyuan Road 38, Beijing 100083, PR China.
authors.forename: Min
authors.initials: M
authors.lastname: Ye
authors.forename: Dean
authors.initials: D
authors.lastname: Guo
mesh_terms: D000528:Alternaria / Q000378:metabolism*; D001711:Biotransformation; D002018:Bufanolides / Q000737:chemistry / Q000378:metabolism; D002770:Cholenes / Q000737:chemistry / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D006900:Hydroxylation; D007700:Kinetics; D021241:Spectrometry, Mass, Electrospray Ionization; D013379:Substrate Specificity
chemical_list: D002018:Bufanolides; D002770:Cholenes; C087925:bufadienolide; C005734:bufogenin; C002471:cinobufagin; C022777:bufalin