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Application of alpha-L-rhamnosidase to preparation of 2'-deoxy-5-fluorouridine derivative

A technology of ‐deoxyuridine and rhamnosidase, which is applied in the field of synthesizing 5-fluoro-2'-deoxyuridine rhamnoside, can solve the problems of low selectivity toxicity and side effects, limited and other problems, and achieve increased stability and target tropism, broad application prospects

Active Publication Date: 2016-02-10
SHANDONG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0002] 5-Fluoro-2'-deoxyuridine (2'-Deoxy-5-fluorouridine) is a fluorodeoxynucleoside analog, which is widely used in the treatment of various tumors, such as liver cancer, rectal cancer, colon cancer, esophagus Cancer, gastric cancer and lung cancer have good curative effect, but the drug shows low selectivity and various side effects clinically
The enzymatic modification of 5-fluoro-2'-deoxyuridine has simple reactions, mild conditions, and environmental friendliness, but the natural Enzymes are very limited, and there is no report on enzymatic rhamnosylation of 5-fluoro-2'-deoxyuridine

Method used

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  • Application of alpha-L-rhamnosidase to preparation of 2'-deoxy-5-fluorouridine derivative
  • Application of alpha-L-rhamnosidase to preparation of 2'-deoxy-5-fluorouridine derivative
  • Application of alpha-L-rhamnosidase to preparation of 2'-deoxy-5-fluorouridine derivative

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] The preparation method of 5-fluoro-2'-deoxyuridine rhamnoside, the steps are as follows:

[0048] 1. Preparation of α-L-rhamnosidase

[0049] Artificially synthesized α-L-rhamnosidase sequence with GenBank accession number JN704640 (encoded protein GenBank accession number is AFA41506.1, nucleotide sequence as SEQ ID NO.2), ligated it to pPIC9K plasmid, and transformed PichiapastorisGS115. The α-L-rhamnosidase was prepared according to the Pichia pastoris expression operation manual of Invitrogen, and the protein content of the enzyme was determined by the Coomassie brilliant blue method. After detection, the amino acid sequence was shown in SEQ ID NO.1.

[0050] 2. Synthesis of 5-fluoro-2'-deoxyuridine rhamnoside catalyzed by α-L-rhamnosidase

[0051] 10 mL of the reaction system was prepared with pH 6.5, 50 mM sodium phosphate buffer solution, the final concentration of rhamnose was 0.4 M, and the amount of enzyme added was 8 μg / mL. After reacting at 55°C for 48 hou...

Embodiment 2

[0063] A method for enzymatically preparing 5-fluoro-2'-deoxyuridine rhamnoside, comprising the steps of:

[0064] (1) Use phosphate buffer to prepare the concentration of rhamnose at 0.3M, the concentration of 5-fluoro-2'-deoxyuridine at 0.4M, and the amino acid sequence as shown in SEQ ID NO.1. 5μg / mL reaction system;

[0065] The phosphate buffer in the step (1) is a sodium phosphate buffer with a concentration of 10mM and pH6;

[0066] (2) React the reaction system prepared in step (1) in a water bath at 45°C for 40 hours, boil at 100°C for 5 minutes to terminate the reaction, centrifuge at 10,000 rpm for 30 minutes, and take the supernatant;

[0067] (3) The supernatant obtained in step (2) is separated and detected by thin-layer chromatography, and the product with the same migration distance and color development under ultraviolet light is freeze-dried to obtain 5-fluoro-2' - deoxyuridine rhamnoside;

[0068] The separation in the step (3) adopts TLC recovery, and th...

Embodiment 3

[0074] A method for enzymatically preparing 5-fluoro-2'-deoxyuridine rhamnoside, comprising the steps of:

[0075] (1) Use phosphate buffer to prepare rhamnose concentration of 0.5M, 5-fluoro-2'-deoxyuridine concentration of 0.6M, amino acid sequence as shown in SEQ ID NO.1 α-L-rhamnosidase addition amount 10μg / mL reaction system;

[0076] The phosphate buffer in the step (1) is a sodium phosphate buffer with a concentration of 100mM and a pH of 8;

[0077] (2) React the reaction system prepared in step (1) in a water bath at 60°C for 60 hours, boil at 100°C for 5 minutes to terminate the reaction, centrifuge at 12,000 rpm for 30 minutes, and take the supernatant;

[0078] (3) The supernatant obtained in step (2) is separated and detected by thin-layer chromatography, and the product with the same migration distance and color development under ultraviolet light is freeze-dried to obtain 5-fluoro-2' - deoxyuridine rhamnoside;

[0079] The separation in the described step (3)...

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Abstract

The invention relates to application of alpha-L-rhamnosidase to preparation of a 2'-deoxy-5-fluorouridine derivative. The 2'-deoxy-5-fluorouridine derivative is 2'-deoxy-5-fluorouridine rhamnoside which comprises 5-flurouracil alpha-L-rhamnopyranosyl-(1->3)-1-beta-D-2'-deoxyribofuranosyl and 5-flurouracil alpha-L-rhamnopyranosyl-(1->5)-1-beta-D-2'-deoxyribofuranosyl; the molecular formula of 2'-deoxy-5-fluorouridine rhamnoside is C15H21FN2O9. According to the application, 2'-deoxy-5-fluorouridine is taken as a raw material, cheap rhamnose is taken as a glycosyl donor, and alpha-L-rhamnosidase sourced from microbes is taken as an original enzyme, so as to synthesize 2'-deoxy-5-fluorouridine rhamnoside. The application is simple in step, low in cost, mild in condition and environmentally friendly; compared with 2'-deoxy-5-fluorouridine, the product contains rhamnopyranosyl, so that the stability and the targeting property are improved, and the application prospect in tumor targeting medicine is wide.

Description

technical field [0001] The present invention relates to the application of a α-L-rhamnosidase in the preparation of 5-fluoro-2'-deoxyuridine derivatives, in particular to a method of using α-L-rhamnosidase original enzyme to synthesize 5-fluoro A method for 2'-deoxyuridine rhamnoside, which belongs to the technical field of sugar engineering. technical background [0002] 5-Fluoro-2'-deoxyuridine (2'-Deoxy-5-fluorouridine) is a fluorodeoxynucleoside analog, which is widely used in the treatment of various tumors, such as liver cancer, rectal cancer, colon cancer, esophagus Cancer, stomach cancer and lung cancer have better curative effect, but this medicine shows lower selectivity and various side effects clinically. In order to improve the targeting of 5-fluoro-2'-deoxyuridine, enhance the curative effect, and reduce the side effects, research on its derivatives has become a hot spot in the development of new drugs in recent years. It has been proved that the glycosylated...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07H19/06C07H1/06C12P19/38A61P35/00
Inventor 肖敏殷振豪徐莉卢丽丽刘晓红刘倩
Owner SHANDONG UNIV
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