MICROBIAL BIOTRANSFORMATION OF RUSCOGENINS
Project no. 237 · Status: Kapanmış · 2015
Summary
The natural product drug discovery process involves isolation of new metabolites from natural sources, investigation of their biological activities and semi-synthesis of more active analogs. Compared to chemical methods microbial biotransformation is advantageous for preparing new oxygenated steroid derivatives due to the stereo- and regioselective reaction capability and it operates in mild conditions. In addition to overcome the problems confronted in chemical synthesis; this process can be of assistance to establish biosynthetic pathways, increase/decrease the bioactivity/toxicity profiles of the lead molecules or support structure-activity relationship studies. Moreover, it is a useful method to predict drug metabolism due to the similarity of the enzyme systems of fungi and mammalians. Endophytic organisms been attracted a great deal of attention with their ability to produce novel bioactive secondary metabolites for many years. While producing interesting chemistry, they are also able to interact with their hosts via special enzymes and chemicals, which make them useful sources as biocatalysts. The powder roots of Ruscus aculeatus (Butcher’s broom, Rusci rhizome, Asparagaceae), extracts and the mixture of the major bioactive steroidal sapogenins ruscogenins (mixture of two major sapogenin, viz. neoruscogenin and ruscogenin), have been component of various pharmaceutical preparations to treat chronic venous insufficiency, varicose veins, hemorrhoids and orthostatic hypotension due to their pharmacological activites. Biotransformation studies performed on ruscogenin have so far been limited and there has been neither in vitro nor in vivo report on ruscogenins revealing its metabolism. The aim of this thesis is to estimate metabolism of ruscogenins and obtain new analogs deriving from neoruscogenin. In order to shed light into metabolism of ruscogenins, biotransformation studies performed by C. blakeseleena. In addition to this, in order to investigate the potential use of endophytes as biocatalyst; prior to preparative scale studies of neoruscogenin by Alternaria eureka and Neosartorya hiratsukae, analytical scale biotransformation studies performed by endophytes isolated from Astragalus angustifolius. As a result twenty five previously unreported biotransformation product isolated and the chemical structures elucidated by NMR and HRTOFMS. Fermentation of ruscogenins with C. blakesleeana yielded 8 new oxygenated metabolites.Microbial transformation of neoruscogenin by endophytic fungus N. hiratsukae afforded three new neoruscogenin derivatives. Mainly P450 monooxygenase products obtained together with a glycosylated metabolite A. eureka performed mainly hydroxylation, oxidation and epoxidation reactions on the B and C rings of the neoruscogenin. In addition to these, cleavage of the spiroketal system together with further transformations provided unusual metabolites. As far as can be ascertained from the literature, this is the first study reporting conversion of the spirostanol skeleton to cholestane-type. Additionally, the cleavage of C-22/C-26 oxygen bridge yielding furostanol-type steroidal framework, then leading to an epoxy bridge between C-18 and C-22 was encountered for the first time in steroid chemistry.