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Derivatization and separation analysis method of amino pyrazolone iso-bifunctional reagents of reducible carbohydrate chains and glycoprotein O-carbohydrate chains

A technology of aminopyrazolone and two functional groups, applied in the field of glycobiology, can solve the problems of low derivatization efficiency, inability to realize further analysis of sugar chains, harsh reaction conditions, etc., achieve short reaction time, strong versatility, The effect of high derivatization efficiency

Active Publication Date: 2018-03-13
NORTHWEST UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] In order to solve the disadvantages of the derivatization method for reducing sugar chains in the prior art, the reaction conditions are harsh, the derivatization efficiency is not high, and the derivatization reagent only contains one active functional group, and further analysis of sugar chains cannot be realized. In one aspect of the present invention A method for derivatizing aminopyrazolone heterodifunctional reagents of reducing sugar chains is provided, which is achieved through the following technical scheme:

Method used

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  • Derivatization and separation analysis method of amino pyrazolone iso-bifunctional reagents of reducible carbohydrate chains and glycoprotein O-carbohydrate chains
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  • Derivatization and separation analysis method of amino pyrazolone iso-bifunctional reagents of reducible carbohydrate chains and glycoprotein O-carbohydrate chains

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specific Embodiment

[0094] The examples in the present invention are about the derivatization method of aminopyrazolone heterobifunctional reagents of reducing sugar chains, and the preliminary purification, separation and identification methods of derivatized products. The examples in the present invention also describe the derivatization method of aminopyrazolone-based heterobifunctional reagents of glycoprotein O-sugar chains.

[0095] Taking PAP as an example, the process of two test reactions for the derivatization of reducing sugar chains by aminopyrazolone heterobifunctional reagents is as follows: figure 1 shown. First, for the free reducing sugar chains, taking maltodextrin-oligosaccharide mixture as an example, derivatize through two different reaction modes. First, under acidic conditions, the sugar chains are first dissolved in PAP / DMF and a weak acid system for reaction, and then NaBH is added to the above system 3 After the CN reaction is completed, after purification, the active ...

Embodiment 1

[0098] Example 1: Derivatization of maltodextrin (Maltodextrin) under acidic conditions and preliminary purification and detection of derivatives

[0099] (1) Derivatization

[0100] Dissolve PAP in DMF to prepare a solution with a concentration of 0.625 mol / L, weigh 5 mg of maltodextrin (Maltodextrin (Maltodextrin) is a macromolecular reducing oligosaccharide) and add it to 500 μL of PAP solution, and then add to the reaction 150 μL of glacial acetic acid was added to the system, and the reaction mixture was reacted at 70° C. for 2 hours. After the reaction is over, add NaBH with a concentration of 0.75mol / L to the mixture 3 The aqueous solution of CN was 500 μL, and the obtained reaction system was continued to react at 70° C. for 1 hour. After the reaction, cool to room temperature, wash with dichloromethane three times, 1.5mL-2mL each time, and collect the crude product after the aqueous phase is concentrated.

[0101] (2) The crude product of the derivative is prelimin...

Embodiment 2

[0118] Example 2: Derivatization of maltodextrin (Maltodextrin) under alkaline conditions and preliminary purification and detection of derivatives

[0119] (1) Derivatization

[0120] Dissolve PAP in DMF to prepare a solution with a concentration of 0.625 mol / L, weigh 5 mg of maltodextrin (maltodextrin is a macromolecular reducing oligosaccharide) and add it to 500 μL of PAP solution, and then add to the reaction system 500 μL of 0.4 mol / L sodium hydroxide aqueous solution was added, and the resulting reaction system was reacted at 50° C. for 1.5 h. After the reaction, cool to room temperature, adjust the pH to 7 with 1M hydrochloric acid aqueous solution, then wash once with dichloromethane, collect the aqueous phase and add 3-5 drops of glacial acetic acid, and then wash with 1.5mL-2mL dichloromethane. The aqueous phase was collected, concentrated under reduced pressure, dried, dissolved in water, and washed once with dichloromethane to obtain a crude product of the deriva...

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Abstract

The invention relates to a derivatization and separation analysis method of amino pyrazolone iso-bifunctional reagents of reducible carbohydrate chains and glycoprotein O-carbohydrate chains. The method particularly includes the steps: performing derivatization on the reducible carbohydrate chains and the amino pyrazolone iso-bifunctional reagents by two reaction modes; performing reductive amination to generate carbohydrate chain derivatives with active methylene groups under acidic conditions; performing Michael addition reaction to generate carbohydrate chain derivatives with primary aminegroups under alkaline conditions. The two derivatives generated by the derivatization method have different active groups, the carbohydrate chains can further be analyzed and researched, and the derivatization method is high in derivatization efficiency and high in universality. The invention further provides releasing and marking method of the amino pyrazolone iso-bifunctional reagents for glycoprotein O-carbohydrate chains under alkaline conditions. According to the method, O-carbohydrate chain derivatives with primary amine groups are acquired, and sample preparation processes are greatly simplified.

Description

technical field [0001] The invention belongs to the technical field of glycobiology, and in particular relates to derivatization and separation analysis methods of reducing sugar chains and glycoprotein O-sugar chains with aminopyrazolone heterobifunctional reagents. Background technique [0002] As the third type of biomacromolecules that build living organisms, carbohydrates have very important biological functions, such as providing energy substances for organisms, and are the main components of plant cell walls. Intercellular signal transduction, recognition, immune response, etc. play an important role. Glycosylation is one of the most important post-translational modifications of proteins, and plays a very important role in protein translation regulation and protein degradation. More than 50% of proteins exist in the form of glycoproteins, and more and more studies have shown that abnormal glycosylation can lead to human diseases. In the course of clinical treatment,...

Claims

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Application Information

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IPC IPC(8): G01N30/02
CPCG01N30/02
Inventor 王仲孚王承健路宇黄琳娟柳人丹温娅楠
Owner NORTHWEST UNIV
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