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Preparation method of intermediate 2'-deoxyguanosine

A technology of deoxyguanosine and intermediates, applied in the field of microorganisms, can solve the problems of low enzyme activity and unfavorable purification in a two-phase system, and achieve the effects of high yield and low cost

Active Publication Date: 2018-05-11
山东格得生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Although the measures adopted in the above reports have achieved certain results in improving the solubility of guanine or the yield of deoxyguanosine, there are still some shortcomings, such as the aqueous two-phase system is not conducive to subsequent purification, and additional guanine precursors are required. Through chemical deprotection group or enzymatic deamination or dechlorination transformation, and the enzyme activity used in biocatalysis is not high, etc.

Method used

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  • Preparation method of intermediate 2'-deoxyguanosine

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Experimental program
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Effect test

Embodiment 1

[0046] (1) The engineered strain of recombinant N-nucleoside deoxyribose transferase Escherichia coli DNTase-1 Escherichia coliDNTase-1, preserved in the China Center for Type Culture Collection, the preservation number is CCTCC No: M 2017060, and the preservation time is February 2017 No. 23, the deposit address is Wuhan University, Wuhan, China, zip code 430072.

[0047] (2) Preparation of recombinant N-nucleoside deoxyribose transferase:

[0048] The obtained recombinant N-nucleoside deoxyribose transferase Escherichia coli engineering strain DNTase-1 (preserved in the China Center for Type Culture Collection, the preservation number is CCTCC No: M 2017060) was inserted under sterile conditions into a 100ml induction In a 500ml Erlenmeyer flask of medium, the composition of the induction medium is: yeast extract powder 20g / L, fish peptone 12.5g / L, dipotassium hydrogen phosphate 2.5g / L, sodium chloride 5.0g / L, magnesium sulfate heptahydrate 1.0g / L, lactose 3.2g / L, glycerin ...

Embodiment 2

[0052] Step (3) of Example 1 When the conversion is terminated, take 1ml of the conversion solution, dilute it to 100 times with 10% methanol solution, and then centrifuge at 12000 rpm for 3 minutes. The supernatant of the centrifugation is filtered through a 0.45 μm filter, and the obtained filtrate is HPLC analysis and determination of 2'-deoxyguanosine. The determination method adopts high performance liquid chromatography (HPLC), and its detection method is as follows:

[0053] Column: Diamonsil C 18 , 5um, 150 x 4.6mm; mobile phase: acetonitrile: methanol: 10mM ammonium dihydrogen phosphate = 1:4:100; flow rate: 0.8ml / min; detection wavelength: 254nm.

[0054] HPLC analysis result shows the following table:

[0055]

Embodiment 3

[0057] 2’-deoxyguanosine prepared with different deoxyribose donor concentrations:

[0058] Get 50ml of the fermented liquid containing recombinant N-nucleoside deoxyribose transferase in Step (2) of Example 1, then add 100mM guanine and add β-thymidine at different concentrations, then adjust the pH value of the transformation system to 5.0, The transformation was carried out in a constant temperature environment at 35°C for 2 hours. When the conversion is terminated, take 1ml of the conversion solution, dilute it to 100 times with 10% methanol solution, then centrifuge at 12000 rpm for 3 minutes, filter the centrifuged supernatant through a 0.45 μm filter, and perform high performance liquid chromatography analysis on the obtained filtrate. '-deoxyguanosine.

[0059] HPLC analysis result shows the following table:

[0060]

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Abstract

The invention discloses a preparation method of intermediate 2'-deoxyguanosine. A novel recombinant strain of N-nucleoside desoxyribose transferase is constructed, recombinant N-nucleoside desoxyribose transferase is synthesized through self-induction expression, a substrate is added in a dry powder feeding manner, 2'-deoxyguanosine is efficiently prepared with a biological catalysis method, the yield of prepared 2'-deoxyguanosine is high, and the cost is low.

Description

technical field [0001] The invention relates to the field of microorganisms, in particular to a method for preparing an intermediate 2'-deoxyguanosine. Background technique [0002] 2'-Deoxyguanosine is a natural deoxynucleoside, which can be directly used for the preparation of combined deoxynucleoside drugs or as a chemical reagent for biochemical research, and its molecular structure can also be appropriately modified to synthesize molecular markers (such as 8-bromo-2'-deoxyguanosine, 8-hydroxy-2'-deoxyguanosine, N7-guanine alkylate) and antitumor derivatives (such as 2'-deoxyguanosine 6-methoxy or 6- amino-derivatives). [0003] At present, the commercial production of 2'-deoxyguanosine mainly adopts chemical synthesis. Due to various shortcomings such as long chemical synthesis steps, many by-products, and low overall yield, researchers began to use biological methods to prepare 2'-deoxyguanosine. guanosine. There are two main ways to prepare 2'-deoxyguanosine by bio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N9/10C12P19/40C12R1/19
CPCC12N9/1077C12P19/40C12Y204/02006
Inventor 李保山李海存王洪钟黄爱清宋长红
Owner 山东格得生物科技有限公司
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