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Synthetic method of citicoline sodium

A technology of citicoline sodium and its synthesis method, which is applied in the field of synthesis of citicoline sodium, and can solve problems such as high synthesis cost and limited enzyme source

Inactive Publication Date: 2020-10-23
ENZYMASTER NINGBO BIO ENG CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] For the various technical defects existing in the prior art, the present invention aims to provide a new preparation method of citicoline sodium, which uses the free enzyme expressed by Escherichia coli as a biocatalyst, and 5'-cytidylic acid, phosphorylcholine, ATP is used as the raw material for biotransformation, and the target product is successfully produced; at the same time, the technical solution provided by the invention also solves the technical problems of limited enzyme sources and high synthesis costs

Method used

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  • Synthetic method of citicoline sodium
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  • Synthetic method of citicoline sodium

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preparation example Construction

[0036] According to the synthetic method of citicoline sodium described in the preferred embodiment, it mainly comprises the following steps: utilize the Escherichia coli engineering bacteria of molecular cloning CMP phosphokinase, nucleoside diphosphate kinase, phosphorylcholine-cytidylyl transferase and prepare The above 3 kinds of enzymes, and provide the supernatant of the enzyme liquid of the 3 kinds of enzymes; using phosphorylcholine chloride calcium salt, ATP, and CMP as raw materials, the above three kinds of enzymes are jointly catalyzed to generate citicoline sodium, and the used The amino acid sequence of the CMP phosphokinase shown in SEQ ID NO.1, the amino acid sequence of the nucleoside diphosphate kinase used is shown in SEQ ID NO.2, the amino acid sequence of the phosphorylcholine-cytidylyl transferase used The sequence is shown in SEQ ID NO.3.

[0037] In a preferred embodiment, the synthetic method of described citicoline sodium comprises the following steps...

Embodiment

[0051] Prepare citicoline sodium according to the following specific steps successively:

[0052] I construct 3 types of strains respectively :

[0053] Three genes (genes of these three types of enzymes) that are active against specific substrates were found through gene search, and the corresponding base sequences were synthesized by a gene company and cloned into the pACYC-Duet-1 vector. We transformed the received plasmid into Escherichia coli BL21 (DE3) (laboratory preservation host), and in chloramphenicol (Cam + )-LB plates were screened to get positive colonies.

[0054] Use a single colony as a template to perform PCR amplification with primer 1 and primer 2. The amplification conditions include: initial denaturation at 95°C for 5 minutes, and then perform the following reaction conditions: 95°C for 30s, 55°C for 30s, 72°C for 90s, 30 cycles , and finally extended at 72°C for 3 minutes to obtain a PCR product of the expected size; the sequencing of the PCR product ...

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Abstract

The invention discloses a synthetic method of citicoline sodium. The synthetic method comprises the following steps: adding 5'-cytidylic acid, calcium phosphorylcholine chloride, ATP, an aqueous magnesium chloride solution and a Tris-HCl buffer solution into a reaction vessel, and carrying out uniform mixing; then, adjusting a pH value to 6.0-8.0, adding CMP phosphokinase, nucleoside diphosphate kinase and choline phosphate-cytidyltransferase, and carrying out a stirring reaction for 4-10 hours at a temperature of 25-50 DEG C; and conducting high-speed centrifugation, and taking a supernatantto obtain citicoline sodium. Compared with the traditional citicoline sodium synthesis process, the synthetic method provided by the invention is environment-friendly in a synthesis route, stable in yield and is beneficial for reducing energy consumption and protecting the environment, so the synthetic method is suitable for large-scale industrial production.

Description

technical field [0001] The invention relates to the technical field of enzyme catalysis, in particular to a synthesis method of citicoline sodium. Background technique [0002] Citicoline sodium, also known as citicoline sodium, is a brain metabolism activator, which is a nucleoside derivative, a precursor of phosphatidylcholine, and a coenzyme necessary for lecithin synthesis. Studies have shown that citicoline sodium has the functions of repairing brain damage, resisting hypoxia, improving memory and enhancing intelligence, and is widely used in clinical practice. [0003] Therefore, the preparation method of citicoline sodium is a research subject that researchers are more concerned about. [0004] In the prior art, the method for preparing citicoline sodium comprises: [0005] ①Chemical synthesis method: CMP (5'-cytidylic acid) and phosphorylcholine are used as reactants, p-toluenesulfonyl chloride is used as a condensation agent, and citicoline is condensed in the pre...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/30C12N15/54C12N9/12C12N15/70
CPCC12N9/1229C12N9/1241C12N15/52C12N15/70C12P19/305C12Y207/04006C12Y207/04025C12Y207/07015
Inventor 何魁芳胡东晓马克·博科拉蔡宝琴张城孝胡虎罗霄彭沁利崔琴燕刘思彤余梦娇
Owner ENZYMASTER NINGBO BIO ENG CO LTD
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