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Method for synthesizing fludarabine phosphate through biological catalysis

A technology of fludarabine phosphate and catalyzed enzymes, applied in biochemical equipment and methods, botany equipment and methods, oxidoreductase, etc., can solve the problem of high cost, step-by-step operation of excessive materials, and preparation of enzyme-containing wet bacteria Complicated issues

Pending Publication Date: 2021-11-02
JIANGSU OCEAN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0012] Compared with the chemical synthesis method, this method has improved in terms of safety, operation and yield; but there are still disadvantages, the addition of too many materials in the catalytic reaction system requires step-by-step operation, and the reaction time is also longer; And the preparation of enzyme-containing wet cells is complicated and the cost is high

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  • Method for synthesizing fludarabine phosphate through biological catalysis
  • Method for synthesizing fludarabine phosphate through biological catalysis
  • Method for synthesizing fludarabine phosphate through biological catalysis

Examples

Experimental program
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Embodiment 1

[0027] The nucleotide sequence shown in the sequence list was selected to be ligated with the vector pBAD-D, and 20 μl of the ligated product was added to 200 μl of Escherichia coli TOP10 competent cells in ice bath, followed by ice bath for 30 min, heat shock at 42°C for 60 s, and ice bath for 5 min. Add 300 μl of 37°C non-antibiotic culture solution to the tube, repair it on a 200r shaker at 37°C for 1 hour, then spread it on an ampicillin-resistant solid LB plate and incubate at 37°C, and pick a single colony with an autoclaved toothpick after the colonies grow. First draw a line on the ampicillin-resistant plate for seed preservation, mark the corresponding bacteria and the line area on the plate, and then put the toothpick in the 20μl PCRMix system that has been added to the primer and mix it for a while. PCR amplification, PCR reaction conditions: 95°C, 15min, denaturation at 94°C for 15s, annealing at 55°C for 15s, extension at 72°C for 1min, 30 cycles, and finally 72°C,...

Embodiment 2

[0030] The nucleotide sequence shown in the sequence list was selected to be ligated with the carrier pBAD-D, and 20 μl of the ligated product was added to 200 μl of Escherichia coli TOP10 competent cells in ice bath, followed by ice bath for 30 min, heat shock at 42°C for 60 s, and ice bath for 5 min. Add 300 μl of 37°C non-antibiotic culture solution to the tube, repair it on a 200r shaker at 37°C for 1 hour, spread it on an ampicillin-resistant solid LB plate and incubate at 37°C, and pick a single colony with an autoclaved toothpick after the colonies grow. First draw a line on the ampicillin-resistant plate for seed preservation, mark the corresponding bacteria and the line area on the plate, and then put the toothpick in the 20μl PCR Mix system that has been added to the primer and stir it. Carry out PCR amplification, the PCR reaction conditions are: 95°C, 15min, denaturation at 94°C for 15s, annealing at 55°C for 15s, extension at 72°C for 1min, 30 cycles, and finally 7...

Embodiment 3

[0033] The nucleotide sequence shown in the sequence list was selected to be ligated with the carrier pBAD-D, and 20 μl of the ligated product was added to 200 μl of Escherichia coli TOP10 competent cells in ice bath, followed by ice bath for 30 min, heat shock at 42°C for 60 s, and ice bath for 5 min. Add 300 μl of 37°C non-antibiotic culture solution to the tube, repair it on a 200r shaker at 37°C for 1 hour, spread it on an ampicillin-resistant solid LB plate and incubate at 37°C, and pick a single colony with an autoclaved toothpick after the colonies grow. First draw a line on the ampicillin-resistant plate for seed preservation, mark the corresponding bacteria and the line area on the plate, and then put the toothpick in the 20μl PCR Mix system that has been added to the primer and stir it. Carry out PCR amplification, the PCR reaction conditions are: 95°C, 15min, denaturation at 94°C for 15s, annealing at 55°C for 15s, extension at 72°C for 1min, 30 cycles, and finally 7...

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Abstract

The invention relates to a method for preparing fludarabine phosphate by an enzyme method. Fludarabine phosphate is prepared by taking fludarabine as a raw material through an enzyme catalytic reaction in the presence of inorganic salt and coenzyme; an enzyme used in the enzyme catalysis reaction is selected from at least one of phosphotransferase, aldoketoreductase, alcohol oxidase or alcohol dehydrogenase; the pH value of the enzyme catalytic reaction is 7.3-7.6, the reaction temperature is 37 DEG C, and the reaction time is 1-4 hours; the coenzyme is ATP. According to the invention, fludarabine phosphate is prepared by adopting a novel enzymatic process, so that the technical problems existing in some existing chemical processes are solved, and the process is improved compared with the existing enzymatic preparation process; the production cost is reduced; the process is environment-friendly and reaction conditions are mild.

Description

technical field [0001] The invention relates to the technical field of compound synthesis, in particular to a preparation method of fludarabine phosphate. [0002] technical background [0003] Fludarabine phosphate (9-β-D-arabinofuranosyl-2-fluoroadenine-5'-phosphate), English name; fludarabine phosphate, molecular formula: C 10 h 13 FN 5 o 7 P, molecular weight: 365.2117, its structural formula is: [0004] [0005] It is an anti-metabolite fluorinated purine nucleoside analog, which was first launched in the United States in 1991. It is the treatment of leukemia and lymphoma (including chronic lymphocytic leukemia, non-Hodgkin's lymphoma, acute myeloid leukemia and acute Lymphoblastic leukemia) chemotherapy drugs are given intravenously or by mouth. After this product enters the body, it undergoes dephosphorylation to generate the metabolite 9-β-fluoroarabinoadenosine (F-Ara-A), and then becomes F-Ara-ATP with anti-tumor activity after phosphorylation. The anti-tu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/32C12N15/70C12N15/54C12N15/53
CPCC12P19/32C12N15/70C12N9/12C12N9/0006C12Y101/01C12Y101/01184C12Y101/01001
Inventor 张坤晓毛联岗侯学雯王媛王睿君杨玉梅
Owner JIANGSU OCEAN UNIV
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