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A method for enzymatically synthesizing vidarabine

A technology for vidarabine and vidarabine, which is applied in the field of enzymatic synthesis of vidarabine, can solve the problems of chemical synthesis pollution, large substrate consumption, low conversion rate of target substances, etc.

Active Publication Date: 2021-02-19
上海鑫欣生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In the above work, two kinds of enzymes are mixed and used to synthesize the target in one step. Although the pollution problem caused by chemical synthesis is solved, there is a reverse reaction in this method—that is, when the substance at one end of the reaction reaches a certain concentration, it will reverse to the reverse reaction. direction, resulting in unequal molar substrate dosage, that is, in the above reaction, the molar concentration of adenine is 3 times that of arabouridine; the substrate consumption is large; the shortcoming of low conversion rate of the target substance cannot fundamentally replace chemical synthesis

Method used

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  • A method for enzymatically synthesizing vidarabine
  • A method for enzymatically synthesizing vidarabine
  • A method for enzymatically synthesizing vidarabine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0090]Example 1, Two-step reaction 1

[0091]The reaction is carried out in two steps, as follows:

[0092]Response 1:

[0093]Add 0.04 mole of arabinouridine to 1000ml of 300mM sodium phosphate solution of pH6.8 to make the concentration reach 40mM, and then add 1.56g of bacteria (32200U per gram containing uridine phosphorylase) to make the concentration of uridine phosphorylase reach 50U / ml; this mixture is placed in a shaker and shaken at 55°C for 8 hours.

[0094]Take out the reaction solution and cool it to 10°C, add 34g of anhydrous calcium chloride solid, and stir for 30 minutes, ultrafiltration to remove the precipitated uracil, calcium phosphate and other insolubles, the insolubles include bacteria containing uridine phosphorylase Keep the ultrafiltration solution at 5-10℃, collect the ultrafiltration solution, wash the membrane with 500ml of double distilled water after completion, wash out the remaining reaction liquid remaining in the membrane bag, combine the ultrafiltration liqui...

Embodiment 2

[0099]Example 2. Two-step reaction 2

[0100]The reaction is carried out in two steps, as follows:

[0101]Response 1:

[0102]Add 0.2 mole of arabinouridine to 1000ml pH6.8 600mM sodium phosphate solution to make the concentration reach 200mM, and then add 6.21g bacteria (containing uridine phosphorylase 32200U per gram) to make the concentration of uridine phosphorylase reach 200U / ml; this mixture is placed in a shaker and shaken at 55°C for 8 hours.

[0103]Take out the reaction solution and cool it to 10°C, add 1000ml of double distilled water to dilute the reaction solution; add 68g of anhydrous calcium chloride solid, and stir for 30 minutes, ultrafiltration to remove the precipitated uracil, calcium phosphate and other insoluble matter, the insoluble Substances include bacteria containing uridine phosphorylase; keep the ultrafiltration solution at 5-10°C, wash the membrane with 500ml double-distilled water after completion, wash out the remaining reaction solution remaining in the membra...

Embodiment 3

[0108]Example 3, Comparative Example 1

[0109]Using the same method in Example 1, the main difference is that in "Reaction 1", after "shaking at 55°C for 8 hours", adenine is directly added for reaction 2, excluding the low-temperature ultrafiltration and "Reaction Two "in the step of double distilled water dilution.

[0110]The reaction is carried out in two steps, as follows:

[0111]Response 1:

[0112]Add 0.04 mole of arabinouridine to 1000ml of 300mM sodium phosphate solution of pH6.8 to make the concentration reach 40mM, and then add 1.56g of bacteria (32200U per gram containing uridine phosphorylase) to make the concentration of uridine phosphorylase reach 50U / ml; this mixture is placed in a shaker and shaken at 55°C for 8 hours.

[0113]The above-obtained mixture was diluted 3 times and then subjected to quantitative analysis by high performance liquid phase (ELSD). The peak area of ​​arabin-1-phosphate was 72974.04. Substituting this number into the standard curve formula was calculated:...

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Abstract

The invention relates to a method for enzymatically synthesizing vidarabine. The invention provides a two-step method for preparing vidarabine adenosine. The method makes full use of the different effects of uridine phosphorylase and purine nucleoside phosphorylase to make the reaction proceed in one direction, eliminate the occurrence of reverse reaction, and significantly improve The conversion rate of vidarabine.

Description

Technical field[0001]The present invention belongs to the field of biochemical industry. More specifically, the present invention relates to a method for enzymatic synthesis of vidarabine.Background technique[0002]Adenosine Arabinoside (araA for short) is a natural antiviral compound with broad-spectrum antiviral activity. It has been widely used clinically to treat diseases caused by cytomegalovirus, hepatitis B virus and herpes virus.[0003]In the prior art, the synthesis method of arabinosine includes chemical synthesis and biotransformation synthesis. The chemical process is complicated, the reaction conditions are harsh, the cost of raw materials is high, and the use of heavy metal reagents pollutes the environment. The basic principle of the biotransformation method is that arabinuridine and phosphate ions generate arabinose 1-phosphate and corresponding bases under the action of uridine phosphorylase (EC 2.4.2.3); arabinose 1-phosphate and adenine are in purine Under the actio...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P19/40
CPCC12P19/40
Inventor 沈波魏民志
Owner 上海鑫欣生物科技有限公司
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