Synthesizing 2'-deoxyadenosine By Microbial Transformation Method

2′-Deoxyadenosine is a natural nucleoside which are inseparrable from biological life activities, and it has the medicinal value of inhibition of insulin secretion. 2′-Deoxyadenosine has extraordinary strong potentials as a intermediate compound of anti-cancer and anti-viral nucleoside drugs. Nucleosides are traditionally prepared by nuclease hydrolysis, which is restricted to limited nuturul resource (minely isolated from salmon eggs), and difficulties in isolation of pure. The chemical methods synthesis of 2′-deoxyadenosine have problem of complex reaction steps, diffcult to separate isomers, low yield, high raw material costs and environmental pollution and so on, while the use of microbial cells with nucleoside phosphorylase synthesis of highly efficient selective biotransformation 2′-deoxyadenosine is an ideal method, which has become a popular way to synthesis of nucleoside drug.The method for the determination of deoxyadenosine product quickly and easily in biotransrormation system and the separate method of 2′-deoxyadenosine was established. To establish a paper chromatography-spectrophotometry method for the determination of deoxyadenosine product quickly and easily in biotransrormation system. The deoxyadenosine product from orther components in biotransrormation system was separated by means of the paper chromatography method with different polarity chromatographic solution, and the concentration of separated deoxyadenosine was measured by spectrophotometer. The separation effect of 4 kinds chromatographic solutions were tested. The results showed that the deoxyadenosine could be separated from other components by two-dimensional paper chromatography using solution I (n-butanol:glacial acetic acid:water=6:1:3) and II (isopropyl alcohol : ammonia solution : water=7 : 2 : 1) as mobil phaserespectively. The separation of deoxyadenosine could be achieved effectively, When solution IV (n-butanol:isopropyl alcohol:ammonia solution:water=3:3:2:2) was used as solvent to do single-dimensional paper chromatography. Cut off the separated chromatographic spot of deoxyadenosine and re-dissolved, then measured its absorption value at 258 nm by spectrophotometer. The concentration of deoxyadenosine product in biotransrormation system could be calculated according to the absorption value and standard curve. The deoxyadenosine could be separated from other components by paper chromatography using solution IV, then measured its concentration by spectrophotometer. The measurement result showed that it was the same as the method HPLC and the coincidence rate was 99.4 % or more. This method was simple and effective with a good reproducibility for the determination of deoxyadenosine product in biotransrormation system.Further research in the paper, using E.coli strain free cells synthesis 2′-deoxyadenosine by the biological transformation, and the products were identified by paper chromatography. The results showed that synthetic products were 2′-deoxyadenosine. Conditions on the conversion reaction analysis, and that optimum conversion conditions were 57℃, optimum pH 7.5, optimum reaction time 4 h, potassium phosphate buffer 150 mmol/L, optimum substrate concentration of dTR and adenine were 54 mmol/L and 65 mmol/L respectively. The similar structure between product and substrate led to difficult separation of them, but ion exchange column with paper chromatograph can solve this problem. And through a variety of identification methods (chromogenic method, high performance liquid chromatography, HPLC-MS, infrared spectroscopy, nuclear magnetic resonance law), the results further showed that separated products were 2′-deoxyadenosine.