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Application of alpha-L-rhamnosidase to preparing hydroxycarbamide and glycoside derivatives

A rhamnosidase amino acid and hydroxyurea glycoside technology, which is applied in the field of glycoengineering, can solve the problems of no rhamnosylation modification of hydroxyurea, and achieve the goal of improving anti-tumor activity, increasing targeting, and reducing toxic and side effects Effect

Inactive Publication Date: 2016-03-23
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the glycosylation modification of hydroxyurea is only one case of N-(β-D-glucopyranosyloxyside) urea modified by glucose through chemical methods, and there is no report of rhamnosylation modification of hydroxyurea

Method used

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  • Application of alpha-L-rhamnosidase to preparing hydroxycarbamide and glycoside derivatives
  • Application of alpha-L-rhamnosidase to preparing hydroxycarbamide and glycoside derivatives
  • Application of alpha-L-rhamnosidase to preparing hydroxycarbamide and glycoside derivatives

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Experimental program
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Effect test

Embodiment 1

[0039] Novel hydroxyurea rhamnoside derivatives, the preparation steps are as follows:

[0040] 1. Preparation of α-L-rhamnosidase

[0041] The α-L-rhamnosidase sequence with GenBank accession number JN704640 (encoded protein GenBank accession number is AFA41506.1, nucleotide sequence as SEQ ID NO.2) was artificially synthesized, ligated to pPIC9K plasmid, and transformed into PichiapastorisGS115. The α-L-rhamnosidase was prepared according to the instructions of the Pichia pastoris expression operation manual provided by Invitrogen, and the content of the enzyme protein was determined by the Coomassie brilliant blue method. After detection, the amino acid sequence was shown in SEQ ID NO.1.

[0042] 2. α-L-rhamnosidase catalyzes the synthesis of hydroxyurea rhamnoside

[0043] 1 mL of the reaction system was prepared with pH 6.5, 50 mM potassium sodium phosphate buffer solution, the final concentration of rhamnose was 0.4 M, the final concentration of hydroxyurea was 0.4 M, a...

Embodiment 2

[0052] A method for enzymatically preparing hydroxyurea rhamnoside, comprising the steps of:

[0053] (1) Use sodium phosphate buffer to prepare a reaction system in which the concentration of rhamnose is 0.3M, the concentration of hydroxyurea is 0.3M, and the amount of α-L-rhamnosidase shown in SEQ ID NO.1 is 5 μg / mL. ;

[0054] The buffer in the step (1) is a sodium phosphate buffer with a concentration of 10mM and pH6;

[0055] (2) React the reaction system prepared in step (1) in a water bath at 45°C for 24 hours, boil at 100°C for 5 minutes to terminate the reaction, centrifuge at 10,000 rpm for 30 minutes, and take the supernatant;

[0056] (3) Separate the supernatant obtained in step (2) with a Bio-gelP2 chromatographic column with a specification of 15mm×100cm, use water as the mobile phase, and a flow rate of 0.3mL / min, collect the eluted samples, and detect them by thin-layer chromatography , combine products with the same migration distance, freeze-dry and make pow...

Embodiment 3

[0061] A method for enzymatically preparing hydroxyurea rhamnoside, comprising the steps of:

[0062] (1) Use sodium phosphate buffer to prepare a reaction system in which the concentration of rhamnose is 0.5M, the concentration of hydroxyurea is 0.5M, and the amount of α-L-rhamnosidase shown in SEQ ID NO.1 is 10 μg / mL. ;

[0063] The buffer in the step (1) is a sodium phosphate buffer with a concentration of 100mM and a pH of 8;

[0064] (2) React the reaction system prepared in step (1) in a 60°C water bath for 48 hours, boil at 100°C for 5 minutes to terminate the reaction, centrifuge at 12000 rpm for 30 minutes, and take the supernatant;

[0065] (3) Separate the supernatant obtained in step (2) with a Bio-gelP2 chromatographic column with a specification of 15mm×100cm, use water as the mobile phase, and a flow rate of 0.3mL / min, collect the eluted samples, and detect them by thin-layer chromatography , combine products with the same migration distance, freeze-dry and ma...

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Abstract

The invention relates to application of alpha-L-rhamnosidase to preparing hydroxycarbamide and glycoside derivatives. The hydroxycarbamide and glycoside derivatives are hydroxyurea and rhamnoside, and a molecular formula of the hydroxycarbamide and glycoside derivatives is C7H14N2O6. Amino acid sequences of the alpha-L-rhamnosidase are shown as SEQ ID NO.1. The application has the advantages that hydroxycarbamide is used as a raw material, rhamnose is used as a glycosyl donor, and the hydroxycarbamide and rhamnoside can be synthesized from the alpha-L-rhamnosidase of a microbial source by means of one-step transglycosylation reaction; a method has simple steps, is low in cost and mild in condition and is environmental friendly, and the alpha-L-rhamnosidase has a potential application prospect; products of the alpha-L-rhamnosidase contain rhamanopyranosyl as compared with the hydroxycarbamide which is the raw material, accordingly, the stability and the targeting can be improved, and the products have broad application prospects for being used as tumor targeted medicines.

Description

technical field [0001] The invention relates to the application of α-L-rhamnosidase in the preparation of hydroxyurea glucoside derivatives, in particular to a method for synthesizing hydroxyurea rhamnoside with the original enzyme of α-L-rhamnosidase, which belongs to sugar field of engineering technology. technical background [0002] Hydroxyurea (Hydroxyurea) is a nucleoside diphosphate reductase inhibitor, as a specific antitumor drug in the S phase of the cell cycle, mainly used for the treatment of chronic myeloid leukemia, melanoma, head and neck cancer, ovarian cancer, etc. In recent years, it has been found that it has a certain effect on sickle cell anemia, beta thalassemia, psoriasis, AIDS and so on. Hydroxyurea has a definite curative effect and a wide range of uses, but its clinical efficacy is greatly limited due to its shortcomings such as high polarity and short half-life. Molecular modification of hydroxyurea can improve its bioavailability and reduce side...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07H99/00C12P19/26A61P35/00
CPCC07H99/00C12P19/26
Inventor 肖敏刘晓红卢丽丽徐莉殷振豪刘倩
Owner SHANDONG UNIV
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