Biotransformation Process Of 4′-d-β-demethylepipodophyllotoxin To 4′-d-α-demethylepipodophyllotoxin

C-S bond is the key step in the biosynthesis natural products of antimicrobial Epidithiodiketopiperazine(ETP),anti-tumor inhibitor diallyl sulfanilamide(DAS)and antibodies LC214-HC220 natural products.Laboratory early stage,using 4′-demethylepipodophyllotoxin,2-mercapto-1,3,4-thiadiazole and 3-mercapto-1,2,4-triazole as the substrate,employing Penicillium purpurogenum as the biocatalyst to achieve C-S bond formation,synthesis of target products 4β-S-(1,2,4-triazole-3)-4-deox-4′-demethylepipodophyllotoxin and 4β-S-(1,3,4-thiadiazole-2)-4-deoxy-4’-demethylepipodophyllotoxin.Compared with the positive reference etoposide,the activity of the target product against lung cancer cells increased by 42.62 and 558.90 times,indicating that it has the value of patent medicine.This paper mainly through screening with efficient synthesis target products strains,optimizing transformation process,identification of new product of optimization process to reveal the enzymatic catalysis mechamism of C-S bond formation employing Penicillium purpurogenum to provides a theoretical basis for the biosynthesis of other C-S linked natural products.Screening of microorganisms for biosynthesis of C-S bond products.Obtaining the 17 strains Penicillium purpurogenum from the microbial species preservation center to analyze function of catalyzing substrates 4′-demethylepipodophyllotoxin 2-mercapto-1,3,4-thiadiazole and 3-mercapto-1,2,4-triazole to generate the products.It was found that Penicillium purpurogenum ACCC 32170,P.purpurogenum CGMCC 3.5188,P.purpurogenum CGMCC 3.8023,P.purpurogenum CGMCC 3.7915,P.purpurogenum CGMCC 3.7800 has conversion ability and among them P.purpurogenum ACCC 32170 has high conversion which conversion rate reache 0.16%.Optimizing the biotransformation process.The effects of glucose concentration,the addition time of the functional module 2-mercapto-1,3,4-thiadiazole,the inoculation amount on the bioconversion process of p.purpurogenum ACCC 32170 were analyzed.It was found that these had no significant effect.And P.purpurogenum ACCC 32170 synthesis the product the yield of 4β-S-(1,3,4-thiadiazole-2)-4-deoxy-4’-demethylepipodophyllotoxin was increased by 6.74 times to 4.644 mg/L.Identification of new products in biotransformation process.The bioconversion samples of P.purpurogenum ACCC 3.7915 obtained were isolated and purified,and identified by NMR and mass spectrometry.The new products were identified as 4’-demethylepipodophyllotone and 4’-d-α-demethylepipodophyllotoxin.The reaction mechanism of C-S bond formation was analyzed,and it was speculated that the intermediate product of C-S bond formation might be 4’-demethylepipodophyllotone.Through the above studies,The product of 4β-S-(1,3,4-thiadiazole-2)-4-deoxy-4’-demethylepipodophyllotoxin biotransformation process was speculated.The results provide a theoretical basis for biosynthesis of other natural products of C-S bond.