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Non-natural difunctional saccharides, and preparing methods and applications thereof

A non-natural, dual-functional technology, applied in the field of chemical glycobiology, can solve problems such as the inability to separate and identify carbohydrate-binding proteins, and the inability to achieve high-throughput and real-time capture of carbohydrates and carbohydrate-binding proteins

Active Publication Date: 2014-12-03
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this unnatural sugar containing only one group cannot achieve high-throughput and real-time capture of the interaction between sugars and sugar-binding proteins, nor can it achieve high-throughput and unbiased separation and identification of sugar-binding proteins.

Method used

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  • Non-natural difunctional saccharides, and preparing methods and applications thereof
  • Non-natural difunctional saccharides, and preparing methods and applications thereof
  • Non-natural difunctional saccharides, and preparing methods and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Synthesis of 9-AzSiaDAz (N-(4-diaziridine)valerylneuraminic acid)

[0048] SiaDAz (N-(4-diaziridine)pentanoylneuraminic acid) was used as the starting material to synthesize in three steps, and all reactions were carried out under dark conditions.

[0049] In the first step, add 1.38g SiaDAz (N-(4-diaziridine)pentanoylneuraminic acid), 50mL methanol and 0.5mL trifluoroacetic acid into a reaction vessel wrapped in aluminum foil, stir at room temperature for 12h, concentrate, and the residue Using dichloromethane and methanol (volume ratio 4:1) as the eluent, it was separated by chromatographic column to obtain 977 mg of 1-MeSiaDAz (N-(4-diaziridine)pentanoylneuraminic acid-1-methyl ester).

[0050] The characterization data of 1-MeSiaDAz (N-(4-diaziridine)pentanoylneuraminic acid-1-methyl ester) are as follows: 1 H NMR (500MHz,D 2 O)δ1.05(s,3H),1.73-1.78(m,2H),1.94(dd,J=12.0,12.5Hz,1H),2.22-2.27(m,2H),2.34(dd,J=4.5 ,13.0Hz,1H),3.62-3.67(m,2H),3.75-3.77(m,1...

Embodiment 2

[0055] Example 2 Synthesis of 9-AzSiaNAl (9-azido-N-(4-alkynyl)pentanoylneuraminic acid)

[0056] Using SiaNAl (N-(4-alkynyl)pentanoylneuraminic acid) as the starting material, it was synthesized through three-step reactions.

[0057] In the first step, 1.80g SiaNAl (N-(4-alkynyl)pentanoylneuraminic acid), 50ml methanol and 0.5ml trifluoroacetic acid were added to the reaction vessel, stirred at room temperature for 12h, concentrated, and the residue was dichloromethane and Methanol (volume ratio 4:1) was used as the eluent for chromatographic column separation to obtain 1.41 g of 1-MeSiaNAl (N-(4-alkynyl)pentanoylneuraminic acid-1-methyl ester).

[0058] The characterization data of 1-MeSiaNAl (N-(4-alkynyl)pentanoylneuraminic acid-1-methyl ester) are as follows: 1 H NMR (500MHz,D 2 O)δ1.94(dd, J=11.5,12.5Hz,1H),2.35(dd,J=5.0,13.0Hz,1H),2.43(br,1H),2.53-2.56(m,4H),3.62( dd,J=6.5,12.0Hz,1H),3.69(d,J=9.0Hz,1H),3.74-3.77(m,1H),3.85-3.88(m,4H),3.96-4.00(app t,1H ),4.08-4.12...

Embodiment 3

[0063] Example 3 Modification of unnatural bifunctional sugars to the cell surface

[0064] The non-natural bifunctional sugar 9-AzSiaNAl(9-azido-N-(4-alkynyl)pentanoyl neuron containing 0mM, 0.1mM, 0.25mM, 0.5mM, 1mM, 2mM and 3mM was used in concentration gradient expression experiments Azine) or 9-AzSiaDAz (9-azido-N-(4-diazido) valerylneuraminic acid) culture medium, with monofunctional unnatural sugar 9AzSia (9-azidoneuraminic acid ) as the control, the non-natural bifunctional sugar was expressed on the cell surface after 24 hours. In time expression experiments, 3 mM 9-AzSiaNAl (9-azido-N-(4-alkynyl)pentanoylneuraminic acid) and 9-AzSiaDAz (9-azido-N-(4-diaziridine ) valerylneuraminic acid) and 9-AzSia (9-azideneuraminic acid) were added to the cell culture medium at 0h, 4h, 8h, 12h, 16h, 20h and 24h, all the cells were collected and washed with PBS , let the cells perform a click reaction with sulfo-DBCO-Biotin to detect whether the cell surface contains azide group...

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Abstract

The invention provides a non-natural difunctional saccharide 9-AzSiaDAz shown as a formula I and / or a non-natural difunctional saccharide 9-AzSiaNAl shown as a formula II, and preparing methods and applications thereof. The non-natural difunctional saccharides 9-AzSiaDAz and the 9-AzSiaNAl can be adopted as probes and used for cell labeling. A single function group cannot achieve high-throughput and real-time capturing of interactions of saccharides and saccharide-binding proteins and cannot perform double-color imaging. Difunctional non-natural saccharide probes developed from the non-natural difunctional saccharides, which contain orthogonal reaction groups and photo-crosslinking groups, can crosslink glycoprotein in a covalent manner to the non-natural difunctional saccharides through UV irradiation without the need of modifying the protein and can purify the protein through affinity capture. The difunctional non-natural saccharide probes with the two different orthogonal reaction groups can perform double-color imaging by a two-step Click reaction.

Description

technical field [0001] The invention relates to the field of chemical glycobiology, in particular to the synthesis of unnatural bifunctional sugar and its application in cell surface markers. Background technique [0002] Sialic acid has important biological functions and plays an extremely important role in anti-inflammation, anti-tumor, anti-viral therapy, etc. However, due to the limitations of traditional biochemical methods and gene technology, people's research on sialic acid at the cellular level relatively lagging behind. In recent years, the strategy of introducing chemical probes through the biological metabolic pathway of sugar has been favored by scientists. It uses chemical biology methods as an entry point, through the design of unnatural groups, bio-orthogonal reactive groups can be introduced on the cell surface, and probe molecules can be introduced under living cell conditions through bio-orthogonal reactions, and then in living cells The mechanism of spe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07H7/027C07H1/00C07K1/22G01N21/64
Inventor 陈兴冯连顺
Owner PEKING UNIV
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