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A novel monosaccharide fluorescence derivatization method and application

A monosaccharide and fluorescent technology, applied in the direction of fluorescence/phosphorescence, chemical instruments and methods, luminescent materials, etc., can solve the problems of a large number of time samples, unreliable sugar quantitative effect, loss, etc.

Inactive Publication Date: 2017-06-06
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, people have developed a variety of monosaccharide-derived labeling compounds such as 2-aminopyridine (2-PA), 2-anthranilic acid (2-AA) and 1-phenyl-3-methyl-5-pyrazole However, these methods have certain limitations: for example, when using 2-PA, it is necessary to pass through the Sephadex column to remove excess 2-PA, which requires a lot of time for sample preparation and causes sample loss ; When 2-AA is used to label glucosamine, 15% of glucosamine will be converted to mannosamine; when PMP is labeled, it also needs to undergo multiple ether extractions to remove excess PMP, and the quantitative effect of PMP-labeled sugar is not reliable

Method used

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  • A novel monosaccharide fluorescence derivatization method and application
  • A novel monosaccharide fluorescence derivatization method and application
  • A novel monosaccharide fluorescence derivatization method and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Take galactose 0.25g, NaHCO 3 67mg, then add 0.67g of 1,3-bis(2-pyridine)-1,3-propanedione, then add 40ml of methanol aqueous solution (3:1, v / v), mix well, and react overnight at 110°C. After methanol was removed under reduced pressure, it was dissolved in an appropriate amount of water, and further purified by a C18 solid-phase extraction column (elution with 10% acetonitrile) and a silica gel column (10% methanol in chloroform solution).

[0029] The resulting labeled product was assayed by HPLC. Measurement was performed at an excitation wavelength of 330 nm and an absorption wavelength of 390 nm using a C18 reversed-phase column (HyperClone5uODS120A250x4.60 mm).

[0030] HPLC analysis results such as figure 1 As shown, the ESI MS results are as figure 2 shown.

Embodiment 2

[0032] Take D-galactose (D-Galactose), D-mannose (D-Mannose), D-gulose (D-Gulose), D-arabinose (D-Arabinose), D-celery sugar (D-Apiose ), D-xylose (D-Xylose), L-fucose (L-Fucose), L-rhamnose (L-Rhamnose) aqueous solution 8ul (20mM), add 2ul 0.4M sodium bicarbonate aqueous solution, Then add 30ul of 0.1M 1,3-bis(2-pyridine)-1,3-propanedione methanol solution, seal and mix well, then react overnight at 110°C. After the reaction was completed, they were diluted with water to 2 ml, centrifuged at 13000 r / m for 1 minute, and the supernatant was taken for HPLC analysis.

[0033] HPLC analysis results such as image 3 , wherein peak numbers 1-8 are D-galactose, D-gulose, D-mannose, D-arabinose, D-apiose, D-xylose, L-fucose and L - Derivative products of rhamnose.

Embodiment 3

[0035] Weigh 100ug of glycoprotein horseradish peroxidase (HRP), add 40ul 4M trifluoroacetic acid (TFA) aqueous solution, hydrolyze at 115°C for 3 hours, remove TFA by vacuum rotary evaporator, in order to remove TFA as much as possible, add 100ul 50% methanol aqueous solution and spin dry, repeat 3 times. HPLC analysis was performed after labeling with 1,3-bis(2-pyridine)-1,3-propanedione.

[0036] The result is as Figure 4 , where peak numbers 1-4 are derivatives of D-mannose, D-arabinose, D-xylose and L-fucose, respectively.

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Abstract

The present invention discloses a novel monosaccharide fluorescence derivation method and applications thereof. According to the present invention, the non-fluorescent compound represented by a formula (I) can be used for the field of the fluorescence derivation reagent of the reducing sugar, wherein Ar is aryl or heteroaryl; and the used fluorescence derivation reagent of the present invention does not have fluorescence property, and can make the sugar have the fluorescence property after the used fluorescence derivation reagent and the reducing sugar are subjected to a chemical reaction, such that the removal of the excess derivative is not required after the reaction so as to rapidly carry out HPLC analysis. In addition, in the method, the derivative can react with the sugar according to the molecular weight ratio of 1:1 so as to achieve the quantitative analysis on the sugar. The formula (I) is defined in the instruction.

Description

technical field [0001] The invention relates to a novel fluorescent derivatization reagent and its application method, and relates to its application in the analysis of monosaccharide components of biological samples. Background technique [0002] With the development of scientific research, people have gradually realized that sugar chains composed of different monosaccharides are the third type of biological macromolecules besides proteins and nucleic acids, and the research on sugar chains has been recognized as the successor to the research on proteins and nucleic acids. After exploring the third milestone of the mystery of life. [0003] Sugar chains play important roles in organisms, such as cell recognition, cell interaction, inflammation and disease progression, etc. In addition, sugar chains are receptors for many viruses, bacteria, etc., and have received increasing attention as tumor markers. [0004] However, due to the complexity and diversity of sugar chain st...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07D405/04C09K11/06G01N21/64
CPCC07D405/04C09K11/06C09K2211/1029C09K2211/1088G01N21/6486G01N30/74
Inventor 约瑟夫·弗戈迈尔刘丽蔡志鹏安德鲁·黑根
Owner NANJING AGRICULTURAL UNIVERSITY