Two-step Synthesis Of Guanine Arabinoside By Microbial Transformation Method

Guanine arabinoside (ara-G) is a kind of nucleoside drug for the treatment of leukemia, which is animportant precursor for synthesis of nucleoside drugs such as nelarabine. The chemical synthesis of ara-Gis extremely complex, and direct biocatalysis is not yet successful. Since guanine has very low water-solubility, using nucleoside phosphorylase or microbial cells with nucleoside phosphorylase to synthesizeara-G is difficult to achieve which use guanine and uracil arabinoside (ara-U) as substrate. So finding ahigher solubility in water of the substrate is the key for the synthesis of ara-G. However,2,6-diaminopurine(DAP) is easily soluble in water and can accept arabinose group under the catalyst of purine nucleosidephosphorylase (PNPase). Therefore, DAP can be used as substrate for synthesis of ara-G.Escherichia sp. AEM0812preserved in our laboratory which has higher nucleoside phosphorylaseactivity has successfully catalyzed ara-U and adenine to synthesize adenine arabinoside. Further studiesshowed that E. sp. AEM0812also has the capable of catalyzing DAP and ara-U for synthesis of2,6-diaminopurine arabinoside (ara-DA). The difference between ara-DA and ara-G are the groups in C6of thepurine ring, it is amino for ara-DA and ketone for ara-G. Therefore, it is possible to use ara-DA throughdeamination reaction to synthesize the disired product ara-G. Based on this, the research work was carriedout as following.At first, ara-DA biosynthesis system was established: using E. sp. AEM0812cells as catalyst whichhas nucleoside phosphorylase activity, the substrates ara-U and DAP were dissolved in Tris-hydrochloricacid buffer, intermediate product ara-DA were synthetized at appropriate temperature. Through thecondition experiments, the optimum conditions for the enzymatic reaction were determined:60mmol/Lara-U and60mmol/L DAP were dissolved in600mmol/L pH7.5Tris-hydrochloric acid buffer, added15%of E. sp. AEM0812bacterial cells as catalyst, the optimum reaction temperature is30℃, the optimumreaction time was48h.Under such condition, the conversion rate of ara-U can achieve95.2%. These worklaid groundwork for the two-step biological synthesis of ara-G.Secondly, using the enrichment medium and adenosine selective medium, a bacteria TX16stain with strong adenosine deaminase activity was isolated and screened from sewage water in a factory. StrainTX16was preliminarily identified as Pseudomonas aeruginosa through16SrDNA sequence analysis.TX16cells could catalyse the deamination reaction of ara-DA and produce ara-G.Then ara-G reaction biosynthesis system were established: using Pseudomonas aeruginosa TX16cellsas catalyst which have deaminase activity, the substrates ara-DA were dissolved in Tris-hydrochloric acidbuffer, disired product ara-G were synthetized at appropriate temperature. Through the conditionalexperiments, the optimum conditions for the enzymatic reaction were determined:30mmol/L ara-DA wasdissolved in1600mmol/L pH7.5Tris-hydrochloric acid buffer, added13%of P. aeruginosa TX16bacterial cells, the catalytic reaction for120h at30℃. Under this condition, the concentration of ara-Greached27.1mmol/L, and the conversion rate of ara-DA was90.0%. After separation and purification, thedisired product were identified by high performance liquid chromatography, mass spectrometry andinfrared spectroscopy, the results showed that the desired product was ara-G.In this study, using ara-U and DAP as substrates and E. sp. AEM0812with nucleoside phosphorylaseactivity as catalyst, ara-DA was synthesized. Then using ara-DA as substrate and P. aeruginosa TX16cellsof high deaminase activity as catalyst, the synthesis of ara-G was successful. The two-step biosynthesis ofara-G was achieved.