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Multifunctional cross-linking agent as well as preparation method and application thereof

A cross-linking agent and multi-functional technology, applied in organic chemistry methods, chemical process analysis/design, organic chemistry, etc., can solve the problem of few reactive groups and inability to capture the dynamic spatial conformation or interaction of transmembrane proteins in situ Information and other issues, to achieve efficient and gentle release, improve identification accuracy, and achieve the effect of chemical cross-linking

Active Publication Date: 2021-06-18
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, plasma membrane proteins, especially transmembrane proteins, contain more hydrophobic amino acids such as leucine and isoleucine, and have fewer reactive groups. Conventional chemical cross-linking agents cannot capture transmembrane proteins in situ. Dynamic spatial conformation or interaction information

Method used

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  • Multifunctional cross-linking agent as well as preparation method and application thereof
  • Multifunctional cross-linking agent as well as preparation method and application thereof
  • Multifunctional cross-linking agent as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] This example discloses a preparation method of a cross-linking agent in which diaziridine is a photoactive group, including seven reaction steps, and the preparation method is as follows:

[0043] The first step, the preparation of biotin aniline (compound 1). Dissolve biotin (1.22g, 5 mmol), 4-aminomethylaniline (670mg, 5.5mmol), EDCI (1.23g, 6.5mmol), HoBt (988mg, 6.5mmol) in 25ml DMF, add DIEA (2ml , 20 mmol), reacted at 25°C for 24h. After the reaction was completed, the solvent was spin-dried under reduced pressure at 45° C., and excess raw materials were removed by column chromatography to obtain 1.72 g of a white solid with a yield of 98.8%. 1 H NMR (400MHz, DMSO-d6,ppm)δ5.83(s,2H),4.76(s,1H),2.80(s,8H),2.72(d,J=4.8Hz,2H),2.65-2.60( m,2H); HR-MS (C15H16N6O9): theoretical value: 348.1620 measured value [M+H]: 349.1589.

[0044] The second step is the preparation of biotinylated azodiphenyl Boc aminomethylphenol (compound 2). Add compound 2 (1.04g, 3mmol), conc...

Embodiment 2

[0053] This embodiment discloses a preparation method of a crosslinking agent in which benzophenone is a photoactive group, including seven reaction steps, and the preparation method is as follows:

[0054] Wherein, the first, second, third and fourth steps are the same as in embodiment 1.

[0055] The fifth step is the preparation of biotin azodiphenyl monosuccinimide benzophenone ester (compound 5-b). Dissolve compound 4 (204.7mg, 0.23mmol) and 66ul TEA in 20ml DMSO, slowly add benzophenone amino hydrochloride (56.8mg, 0.23mmol) DMSO solution dropwise, react for 0.5h, and undergo semi-preparative liquid phase separation Purification (40% acetonitrile isocratic, effluent was collected for 24-27 min) and vacuum freeze-drying at 25°C to obtain 156 mg of red solid compound 5-b with a yield of 68.9%. 1H NMR (400MHz, DMSO-d6, ppm) δ4.76(s,1H),3.36(t,J=6.4Hz,1H),3.10(s,2H),2.92(s,2H),2.86(m, 4H),2.83(s,1H),2.8(s,8H),2.65-2.60(m,2H),2.05(t,J=6.4Hz,2H), 1.57(m,2H),1.25(m,2H ); 13...

Embodiment 3

[0060] This example discloses a preparation method of a crosslinking agent in which phenyl azide is a photoactive group, including seven reaction steps, and the preparation method is as follows:

[0061]

[0062] Wherein, the first, second, third and fourth steps are the same as in embodiment 1.

[0063] The fifth step, the preparation of biotin azodiphenyl monosuccinimide ester phenyl azide (compound 5-c) Dissolve compound 4 (204.7mg, 0.23mmol), 66ul TEA in 20ml DMSO, slowly Add benzophenone amino hydrochloride (42.3mg, 0.23mmol) DMSO solution dropwise, react for 0.5h, undergo semi-preparative liquid phase separation and purification (30% acetonitrile isocratic, collect effluent for 24-27min) and vacuum freeze-drying, The freeze-drying temperature was 25°C, and 125 mg of red solid compound 5-c was obtained with a yield of 59%. 1 H NMR(400MHz,DMSO-d6,ppm)δ4.76(s,1H),2.92(s,2H),2.83(s,1H),2.80(s,8H),2.79(d,J=4.8Hz, 2H), 2.72(d, J=4.8Hz, 2H), 2.65-2.60(m, 2H); 13 C NMR (40...

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Abstract

The invention relates to a multifunctional cross-linking agent as well as a preparation method and application thereof, and belongs to the technical field of organic synthesis. The cross-linking agent is provided with a biotin enrichment unit, so that high-flux and high-sensitivity analysis of cross-linked peptide is realized; an azobenzene chemical fracture unit is provided, so that efficient and mild release of the cross-linking agent after enrichment is realized, and the identification accuracy of mass spectrum data is improved; the cross-linking agent has a succinimide ester structural unit, and is subjected to amidation reaction with protein and polypeptide lysine residue terminal or N-terminal amino under physiological conditions, so that chemical crosslinking of the protein is realized; and also contains optical active groups, so that analysis of real cell in-situ protein structures and interaction information is realized. The cross-linking agent disclosed by the invention is applied to the field of cytoplasmic membrane proteomics, and provides important technical support for realizing large-scale analysis of a cytoplasmic membrane protein complex, analysis of a three-dimensional space structure of cytoplasmic membrane protein and analysis of interaction of cytoplasmic membrane protein-protein.

Description

technical field [0001] The invention relates to a multifunctional crosslinking agent and a preparation method thereof. The cross-linking agent of the present invention has a biotin enrichment unit, an azodiphenyl chemically cleavable unit, a succinimide ester unit, a sodium sulfonate group and a photoactive group. Wherein, the photoactive group includes one or more of diaziridine, benzophenone, and phenyl azide. The invention belongs to the technical field of organic synthesis. Background technique [0002] Protein, as the main bearer of various life activities in organisms, has attracted extensive attention from researchers. Proteins interact with each other (protein-protein interactions, PPIs), forming a delicate and dynamic regulatory network to complete various complex life processes, accompanied by changes in their own conformation. Therefore, the study of protein-protein interactions and the spatial conformation of protein complexes plays a vital role in understandi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07D495/04G16C20/10
CPCC07D495/04G16C20/10C07B2200/07Y02P20/55
Inventor 张丽华高航赵群赵丽丽张玉奎
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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