Microbial Biotrsformation Of14-deoxy-14-methylenetriptolide

Tripterygium wilfordii Hook.f was used in the clinical treatment of glomerulonephritis, rheumatoid arthritis,eprosy,systemic lupus erythematosus, and skin diseases and autoimmune diseases as a new drug resources in recent year. Triptolide, diterpenoid triepoxide, are the major active constituents of Tripterygium wilfordii Hook.f. Previous studies showed that triptolide is effective in the treatment of autoimmune diseases and have potent antileukemic and antitumor activities. As the biologically active agents, the application of triptolide and triptonide were limited due to their strong toxicity.To find more effective compounds with less toxicity, scientists have been searching for the approach to modify the structures of triptolide by efficient methods.Biotransformation is now becoming a useful tool for structural modifications of bioactive natural products. It is a kind of chemical reaction of structure modification by enzymes of plant cell suspension cultures and microorganisms, which modifies the structures of specific substrates. It has such advantages over chemical synthesis as high stereo-and region-selectivity, as well as mild reaction conditions, lower cost, less by-product and pollution. Some reactions which can’t be fulfilled in chemical approach are facile process by biotransformation. This paper presents the biotransformation of T-144, which was carried out for the first time.14-Deoxy-14-methylenetriptolide(T-144) is obtained from triptonide, after a series of chemical reactions, the chemical structural formula is C21H24O5, molecular weight is356.41. The pharmacodynamic evaluation found that it could inhibit human ovarian cancer cells SK-OV-3, human breast cancer cells MDA-MB-468and human prostate cancer cells PC-3significantly.Our target is to find the compounds with higher activity through the biotransformation of T-144. The biotransformation products were obtained and identified by employing spectroscopic methodsIn this study, we screened sixteen microbial strains by using resting cells method and PDA culture method. Ultimately, we chose the PDA culture method and Cunninghamella blakesleana (AS3.970) for the transformation of T-144. and its transformation products B1, B2. B3were detected by LC-MS as molecular weight of372,374,390.After a series of testes for the transformation conditions about AS3.970. we found the best transformation conditions:dissolved T-144in ethanol containing10%DMSO. transformed at25℃for certain times. The best transformation time for B1was24hours, the best transformation time for B2and B3were96hours. According to the changes of the products in the process of transformation, we also discovered that:T-144was first transformed to B1and B2,as the transformation continued, B1was transformed to B3.Expanded biological transformation experiments were arranged to obtain the crude extract of the transformation products. We used silica gel to remove some impurities, and the products were initially separated as a mixture of T-144and B1and a mixture of B2and B3.The transformation products were separated and purified by high performance liquid chromatography, and the conditions were optimized. Finally, we determined the best conditions as follows:volume ratio of methanol and water about70:30to separate T-144and B1,50:50to separate B2and B3; a flow rate of1.5mL/min. a sample volume of80μL/0.32mg.. The three products, purities of which are all more than97.5%, were analyzed by the NMR to determine their structures. We found that B1was added a hydroxyl group at C19compared with T-144; and B2was added a water between C14and C21. and there were two structures of B2as B2-a and B2-b; B3was added a water between.C14and C21based on B1. B1, B3and B2-b were new compounds, which might be used as the lead compounds for drug development.