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Multifunctional chemical crosslinking agent, preparation method and application thereof

A chemical cross-linking agent, multifunctional technology, applied in the field of protein and its function research

Active Publication Date: 2017-09-05
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to provide a multifunctional chemical cross-linking agent that can solve complex biological sample analysis

Method used

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  • Multifunctional chemical crosslinking agent, preparation method and application thereof
  • Multifunctional chemical crosslinking agent, preparation method and application thereof
  • Multifunctional chemical crosslinking agent, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1: Synthesis of Leiker 1

[0063] (1) Synthesis of compound 2

[0064]

[0065] Compound 1 (23.2 mg, 0.1 mmol), sulfo-NHS (71.6 mg, 0.33 mmol) and EDCI (67.1 mg, 0.35 mmol) were dissolved in anhydrous DMSO (5 mL), and the reaction mixture was stirred at room temperature for 24 hours. Then 40 mL of anhydrous THF was added, the solution became turbid, and after standing for 12 hours, the viscous solid settled at the bottom of the round bottom flask and the solvent became clear. Carefully pour off the supernatant, and then wash the viscous solid twice with 5 mL of anhydrous THF, and finally obtain the white viscous crude product 2, which is directly used in the next reaction without further purification;

[0066] (2) Synthesis of compound 4

[0067]

[0068] Dissolve compound 3 (500mg, 3.6mmol) in anhydrous DMF (7.5mL), then add anhydrous potassium carbonate (745mg, 5.4mmol), stir at 30°C for 30 minutes, and then slowly add 3-bromopropyne (462 μL, 5.4 mmol...

Embodiment 2

[0096] Embodiment 2: the synthesis of Leiker 2

[0097] (1) Synthesis of Compound 11

[0098]

[0099] Compound 7 (69.2 mg, 0.181 mmol) obtained in Example 1 was dissolved in DMF / DCM / H 2 O (2mL / 2mL / 2mL), followed by adding azide biotin 10 (77mg, 0.236mmol), CuSO 4 ·5H 2 O (2.3mg, 0.009mmol) and sodium ascorbate (5.4mg, 0.027mmol), the reaction mixture was stirred at room temperature for 24 hours. After the reaction, 4 mL of water was added, and extracted three times with ethyl acetate (12 mL×3). The combined organic phases were washed with saturated brine (5 mL), and finally washed with anhydrous Na 2 SO 4 Dry, filter, and concentrate the filtrate under reduced pressure. The residue was purified by flash silica gel column chromatography (6%~10% methanol in dichloromethane solution) to give bright yellow solid 11 (117mg, 91%);

[0100] The characterization data of the obtained compound 11 are as follows:

[0101] 1 H NMR (400MHz Methanol-d 4 ): δ1.44(m, 2H), 1.48(s...

Embodiment 3

[0118] Embodiment 3: the synthesis of Leiker 3

[0119] (1) Synthesis of compound 15

[0120]

[0121] 3-Amino-1-propanol (300mg, 4mmol) was dissolved in anhydrous dichloromethane (15mL), the reaction solution was placed in an ice bath, and a dichloromethane solution (12mL) of FmocCl (528mg, 2mmol) was added dropwise to The reaction solution lasted for 30 minutes. The temperature was raised to room temperature and stirring was continued for 1.5 hours. Wash three times with 0.5M HCl solution (5mL×3), combine the organic phase and wash with saturated brine (5mL), and finally wash with anhydrous Na 2 SO 4 Dry, filter, and concentrate the filtrate under reduced pressure. The residue was purified by flash silica gel column chromatography (50% ethyl acetate petroleum ether solution) to give white solid 15 (585 mg, 98%);

[0122] (2) Synthesis of compound 16

[0123]

[0124] p-Nitrophenol (211mg, 1.52mmol) was dissolved in anhydrous THF (37mL), then compound 15 (519mg, 1...

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Abstract

The invention relates to a multifunctional chemical crosslinking agent. The multifunctional chemical crosslinking agent is a three-arm type structure consisting of two reaction functional group arms and an arm containing an affine functional group and a cleavage site. The invention further provides a preparation method of the multifunctional chemical crosslinking agent. The multifunctional crosslinking agent is very excellent in both of water solubility and stability, and high in crosslinking efficiency; when the multifunctional crosslinking agent is applied to protein analysis; its good crosslinking peptide fragment identification ability is presented in samples of simple systems such as BSA, and ten standard protein mixtures; the crosslinking agent also can identify considerate crosslinking site in complex biological samples such as escherichia coli 70S ribosome, escherichia coli full cell lysis buffer and caenorhabditis elegans full cell lysis buffer, and present very good application value; thereby providing a powerful tool for the future study of interaction of complex biological system proteins.

Description

technical field [0001] The invention relates to the field of protein and its function research, in particular to a multifunctional chemical cross-linking agent. Background technique [0002] As the material basis of life and the main bearer of life activities, the study of protein functions is of great significance to the understanding of life activities. With the completion of the Human Genome Project, the number of proteins identified has increased dramatically over the past decade. However, the physiological functions of many newly discovered proteins are still unclear, and the three-dimensional structure of proteins and protein-protein interactions are closely related to the study of protein functions. The resolution of traditional protein structure analysis methods such as X-ray crystallography and nuclear magnetic resonance spectroscopy can reach the atomic level. However, both of these methods require a large amount of protein, and it is very difficult to obtain hig...

Claims

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Application Information

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IPC IPC(8): C07D207/416C07D495/04G01N27/62
CPCC07D207/416C07D495/04G01N33/6848
Inventor 雷晓光李强董梦秋谭丹
Owner PEKING UNIV
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