Study On Synthesis Of Thymidine By Combined Nucleoside Phosphorylase-Chemical Technology

Nucleosides and their derivatives have good antiviral,antitumor activity,and have been widely used in clinical application.Nowadays,how to efficiently and environmentally friendly synthesize nucleoside drugs has been the focus of research.This paper combines chemical and biological method to synthesize nucleosides based on wide range of reference literature from domestic and abroad.First,intermediate 2-deoxy-a-D-ribose-1-phosphate cyclohexylamine salt was chemically synthesized from 1-chloro-3',5'-di-(4-chlorobenzoyl)-2-deoxy-D-ribose by crystallization-induced asymmetric transformation.Then thymidine was enzymatically synthesized from substrates 2-deoxy-a-D-ribose-l-phosphate cyclohexyla-mine salt and thymine by using Escherichia coli with nucleoside phosphorylase.The results showed that,the yield of 2-deoxy-a-D-ribose-1-phosph-ate cyclohexylamine salt could rearch 84.9%in chemical synthesis,and the purity was 94.7%.Structure of the chemical synthesis product was consistent with the literature via hydrogen nuclear magnetic spectra and infrared spectra appraisal.Using The intermediate as substrate,thymidine was synthesized with biocatalyst nucleoside phosphorylase,and the biological catalytic process was optimized,the optimum conditions were as follows,100 mmol/L 2-deoxy-a-D-ribose-l-phosphate cyclohexylamine and 400 mmol/L thymine were used as substrates,80 g/L wet whole cell was used as biocatalyst in 30 mmol/L pH 8 PBS buffer,the reaction was incubated at 55 ? for 4 h,and the transformation rate could reach 61.8%in enzymatical synthesis.Finally,the product was separated by column chromatography,high purity thymidine was obtained using silica gel column(particle size distribution of 200?300 mesh),and the eluent composition was:methanol/methylene chloride = 1/15.The stucture was consistent with target product via hydrogen nuclear magnetic spectra and infrared spectra appraisal.It's melting point was measured 184?186 ?,and the yield and purity reached 49.7%and 95.3%respectively.In this paper,chemical and biological method were combined effectively,and complement with each other,which solve the problem of the nucleoside synthesis raw material and chemical synthesis problem of poor stereoselectivity,and reduce the use of poisonous reagent.The combined method possesses the advantages of high selection,high efficiency and less pollution.This method can also be used for other deoxyribonucleoside synthesis,and has positive significance for promoting research on nucleosides.