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Regioselective fucosylation modification method of enzymatic nuclear glucoside class medicine

A technology of regioselectivity and fucosylation, which is applied in the direction of fermentation to achieve the effects of easy separation, environmental friendliness and mild reaction conditions

Inactive Publication Date: 2013-03-13
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, the enzyme-catalyzed regioselective fucosylation of nucleosides to synthesize β-fucosyl-containing disaccharide nucleosides has not been achieved

Method used

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  • Regioselective fucosylation modification method of enzymatic nuclear glucoside class medicine
  • Regioselective fucosylation modification method of enzymatic nuclear glucoside class medicine
  • Regioselective fucosylation modification method of enzymatic nuclear glucoside class medicine

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Add 2mL citric acid-Na into a 15mL Erlenmeyer flask with cap 2 HPO 4 Buffer (50mM, pH 7.0), 0.02mmol p-nitrophenyl β-fucoside and 0.04mmol 5-fluoro-2'-deoxyuridine, mix well, then add 0.06U bovine liver β-galactosidase , React at 40℃ and 200r / min. After 90 hours, the reaction solution was heated to 100°C for 10 minutes to quench the enzyme reaction. After centrifugation, the concentration of the product 5'-O-β-fucosyl-5-fluoro-2'-deoxyuridine was determined by liquid chromatography According to the product calibration curve, the yield is 30%. Product NMR hydrogen spectrum and carbon spectrum are as attached figure 1 with 2 Shown.

Embodiment 2

[0039] Add 2mL citric acid-Na into a 15mL Erlenmeyer flask with cap 2 HPO 4 Buffer (50mM, pH 6.0), 0.02mmol p-nitrophenyl β-fucoside and 0.04mmol 5-fluoro-2'-deoxyuridine, mix well, then add 0.21U bovine liver β-galactosidase , React at 40℃ and 200r / min. After 24 hours, the reaction solution was heated to 100°C for 10 minutes to quench the enzyme reaction. After centrifugation, the concentration of the product 5'-O-β-fucosyl-5-fluoro-2'-deoxyuridine was determined by liquid chromatography According to the product calibration curve, the yield was 41%.

Embodiment 3

[0041] Add 2mL citric acid-Na into a 15mL Erlenmeyer flask with cap 2 HPO 4 Buffer (50mM, pH 7.0), 0.02mmol p-nitrophenyl β-fucoside and 0.04mmol 5-fluoro-2'-deoxyuridine, mix well, then add 0.21U bovine liver β-galactosidase , React at 50℃ and 200r / min. After 17h, the reaction solution was heated to 100℃ for 10min to quench the enzyme reaction. After centrifugation, the concentration of the product 5'-O-β-fucosyl-5-fluoro-2'-deoxyuridine was determined by liquid chromatography According to the product calibration curve, the yield is 40%.

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Abstract

The invention discloses a regioselective fucosylation modification method of an enzymatic nuclear glucoside class medicine, and the method comprises the following specific steps of: (1) evenly mixing a buffer solution, nucleoside and p-nitro-phenyl-beta-fucoside together; (2) adding beta-galactosidase in an enzyme dosage of 1-30 U / gram of nucleoside, reacting for 5-100 hours under the conditions of 30-55 DEG C and 200 r / min, treating for 10 minutes at 100 DEG C, and performing quenching enzyme reaction; and (3) obtaining the modified product through column chromatography isolation. The method has the advantages of simple reaction process, high regioselectivity, no complex protection and deprotection steps, moderate reaction conditions, environmental friendliness, easiness for separation of products and the like.

Description

Technical field [0001] The invention belongs to the field of applied biocatalysis, and specifically relates to a method for catalyzing the regioselective fucosylation modification of nucleoside drugs by using β-galactosidase. Background technique [0002] Nucleoside drugs are an important class of anti-tumor and antiviral agents (J Clin Virol, 2004, 30, 115). For example, 5-fluoro-2'-deoxyuridine has been widely used to treat tumors such as colon cancer and liver metastases, and acyclovir is used to treat herpes virus infections. However, nucleoside drugs usually show low oral bioavailability, so large doses and frequent consumption are required to keep the blood concentration above the minimum threshold required for treatment. In addition, these drugs have poor selectivity. While killing diseased cells, they are also highly toxic to normal cells, so they usually show greater toxic side effects in clinical use (Br J Pharmacol, 2006, 147, 1). [0003] Structure-activity relations...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/38C12P19/40
Inventor 李宁宗敏华颜丽强
Owner SOUTH CHINA UNIV OF TECH
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