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Method for regenerating reducing sugar from hydrazine chromophoric reagent derivative of reducing sugar

A chromogenic reagent and derivative technology, applied in the field of biochemistry, can solve the problems of high yield of by-products, harsh reaction conditions, low yield of reducing sugar chains, etc., and achieves high recovery rate, mild reaction and simple operation. Effect

Active Publication Date: 2018-09-04
NORTHWEST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, when these sugar chain derivatives are separated by chromatography, it is usually difficult to recover the reducing sugar chains by removing reagents
Since the structure of the secondary amine (-C-NH-) is very stable, it is generally necessary to oxidize its imino group with hydrogen peroxide to achieve the purpose of cleavage. The reaction conditions are harsh, and the yield of by-products is high. very low rate
1-phenyl-3-methyl-5-pyrazolone (PMP) and 1,3-substituted pyrazolone compounds such as PMPMP are also commonly used in the pre-column derivatization of sugar chains by HPLC. The methyl group and the aldehyde group at the reducing end of the sugar chain undergo a condensation reaction under alkaline conditions to form a derivative with a -C=C- or -C-C- structure. This structure is very stable and it is difficult to achieve the regeneration of the reducing sugar chain

Method used

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  • Method for regenerating reducing sugar from hydrazine chromophoric reagent derivative of reducing sugar
  • Method for regenerating reducing sugar from hydrazine chromophoric reagent derivative of reducing sugar
  • Method for regenerating reducing sugar from hydrazine chromophoric reagent derivative of reducing sugar

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Example 1: Regeneration of free reduced maltooligosaccharide monomers from BSH derivatives of maltodextrin

[0064] Specific steps are as follows:

[0065] (1) Take 5 mg of standard maltodextrin, take BSH (10 mg) according to the molar ratio of not less than 1:10, and dissolve maltodextrin and BSH in 1 mL of weak acid aqueous solution of organic solvent (500 μL ethanol + 50 μL acetic acid + 450 μL Water=1mL, containing 50% ethanol and 5% acetic acid), shake well, react at 60°C for 45min, then concentrate and dry.

[0066] (2) The obtained sample was dissolved in water, then extracted with an organic solvent (dichloromethane), centrifuged, and the supernatant was taken, and repeated 3-5 times, and finally the obtained supernatant was concentrated and dried.

[0067] (3) Dissolve the obtained sample with acetonitrile, and then perform HILIC-HPLC separation. The liquid phase conditions are: when performing HILIC-HPLC separation, use a 4.6mm×250mm TSK-GEL Amide-80 column, ...

Embodiment 2

[0070] Example 2: Regeneration of free reduced maltooligosaccharide monomers from TSH derivatives of maltodextrin

[0071] Specific steps are as follows:

[0072] (1) Take 5 mg of standard maltodextrin, take TSH (10 mg) according to the molar ratio of not less than 1:10, dissolve maltodextrin and TSH in 1 mL of weak acid aqueous solution of organic solvent (500 μL ethanol + 50 μL acetic acid + 450 μL Water=1mL, containing 50% ethanol and 5% acetic acid), shake well, react at 60°C for 45min, then concentrate and dry.

[0073] (2) The obtained sample was dissolved in water, then extracted with an organic solvent (dichloromethane), centrifuged, and the supernatant was taken, and repeated 3-5 times, and finally the obtained supernatant was concentrated and dried.

[0074] (3) Dissolve the obtained sample with acetonitrile, and then perform HILIC-HPLC separation. The liquid phase conditions are: when performing HILIC-HPLC separation, use a 4.6mm×250mm TSK-GEL Amide-80 column, the ...

Embodiment 3

[0077] Example 3: Regeneration of free reduced maltooligosaccharide monomers from BZH derivatives of maltodextrin

[0078] Specific steps are as follows:

[0079] (1) Take 5 mg of standard maltodextrin, take BZH (10 mg) according to the molar ratio of not less than 1:10, dissolve maltodextrin and BZH in 1 mL of weak acid aqueous solution of organic solvent (500 μL ethanol + 50 μL acetic acid + 450 μL Water=1mL, containing 50% ethanol and 5% acetic acid), shake well, react at 60°C for 45min, then concentrate and dry.

[0080] (2) Dissolve the obtained sample in water, then pass through C18 and PGC solid-phase extraction column to purify the sample, first pass through the C18 column, the specific operation is: 3mL acetonitrile activation, 12mL water balance, sample loading, then 0%, 5%, 10%, 15% acetonitrile washes 3mL every 5% gradient to remove impurities, 3mL 20% acetonitrile elutes the sugar chain, and then passes through the PGC column. The specific operation is: 3mL aceto...

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Abstract

The invention discloses a method for regenerating reducing sugar from a hydrazine chromophoric reagent derivative of the reducing sugar. The method comprises the following steps: 1) taking the reducing sugar, adding a hydrazine chromophoric reagent with an amount of a mole number being no less than 10 times of the reducing sugar, then dissolving the materials in a weak acid aqueous solution of anorganic solvent, uniformly shaking the materials, heating the materials for a reaction, after the reaction is completed, performing condensation drying; 2) adding water in a sample obtained in the step 1) for dissolving, then using the organic solvent for extraction, or passing the material through a C18 column and / or a PGC solid-phase extraction column for purification; 3) separating the sample obtained in the step 2) through a chromatography; and 4) separating a single carbohydrate derivative collected in the step 3) for drying, then dissolving the material in the weak acid aqueous solution,removing a hydrazine chromophoric reagent tag under heating condition, and regenerating a reducing sugar chain. The method has the beneficial effects that the operation is simple, the reaction is mild and fast, and the generated reducing sugar chain monomer has no generated side reaction products, and the recovery rate is high.

Description

technical field [0001] The invention relates to a method for regenerating reducing sugars, in particular to a method for regenerating reducing sugar monomers from hydrazine chromogenic reagent derivatives of reducing sugars, and belongs to the technical field of biochemistry. Background technique [0002] Glycosylation is one of the common post-translational modifications and plays an important role in the regulation of cell life activities. Studies have reported that sugar chain conjugates such as glycoproteins, glycolipids, and proteoglycans participate in various molecular interactions in vivo, and sugar chains, as an information molecule on the conjugates, not only participate in cell recognition, adhesion , Signal transduction and immune response between cells, also play an important role in inflammation and autoimmune diseases, aging, abnormal proliferation and metastasis of cancer cells, pathogen infection and other processes. Therefore, it is of great significance t...

Claims

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Application Information

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IPC IPC(8): C07H1/00C07H3/06C07H5/04G01N30/02G01N30/06
CPCC07H1/00C07H3/06C07H5/04G01N30/02G01N30/06
Inventor 王仲孚王承键郜茜龚桂萍黄琳娟
Owner NORTHWEST UNIV
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