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Protein chemical crosslinking agent and preparation method and application thereof

A chemical cross-linking agent and protein technology, applied in the field of protein structure and function research, can solve the problem of low proportion of cross-linked peptides, difficulty in identifying cross-linked peptides, complex sample analysis does not have much effect, etc. problems to achieve the effect of enriching structural information

Active Publication Date: 2018-02-02
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] In addition, although many chemical cross-linking agents have been commercialized, for more complex protein systems, since most of the protein products after enzymatic digestion are ordinary peptides without cross-linking, the proportion of cross-linked peptides is Very low, it is very difficult to identify cross-linked peptides in complex systems
Most of the cross-linking reagents are currently only limited to the analysis of relatively simple protein samples, and do not have much effect on the analysis of complex samples

Method used

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  • Protein chemical crosslinking agent and preparation method and application thereof
  • Protein chemical crosslinking agent and preparation method and application thereof
  • Protein chemical crosslinking agent and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Step (1): The starting material polyethylene glycol (14.62 mmol) was dissolved in dichloromethane (10 mL). Meanwhile, iodine (8.91 g, 35.09 mmol), triphenylphosphine (9.20 g, 35.09 mmol) and imidazole (2.99 g, 43.86 mmol) were dissolved in dichloromethane (90 mL) and stirred for 5 minutes. Then the polyethylene glycol dichloromethane solution was added dropwise to the mixture. The reaction was stirred at room temperature for 3 hours. TLC monitored the complete conversion of starting material. Saturated sodium thiosulfate was added to the reaction solution to quench the reaction. The organic phase was separated off and extracted again with dichloromethane. The combined organic phases were washed with saturated brine and dried over anhydrous sodium sulfate. The obtained residue was separated and purified by silica gel column chromatography (5%-10% ethyl acetate / petroleum ether) to obtain light yellow oily liquid diiodide.

[0065] Step (2): Add 2M trimethylsilyldiazo...

Embodiment 2

[0077] The present embodiment adopts the step (1) of the method described in Example 1, and carries out the iodination reaction with tetraethylene glycol as a raw material to obtain the following product:

[0078]

[0079] Pale yellow oily liquid; 2.39g, yield 88% (calculated using 6.56mmol tetraethylene glycol as raw material). The NMR spectrum of the product is consistent with the literature report.

[0080] Present embodiment is on the basis of gained diiodide 2, through embodiment 1 described step (2) reaction, obtains following product:

[0081]

[0082] The characterization information is as follows:

[0083] Orange oily liquid; 993mg, quantitative yield (calculated with 2.46mmol diiodide 2 as raw material):

[0084] IR(neat)ν max 2954,2896,2863,2030,1248,1110,835,751,625cm -1 .

[0085] 1 H NMR (400MHz CDCl 3 ): δ0.14(s, 18H), 2.21(t, J=6.8Hz, 4H), 3.54(t, J=6.8Hz, 4H), 3.62(m, 8H);

[0086] 13 C NMR (100MHz CDCl 3 ): δ-1.7, 25.7, 70.3, 70.6, 70.7;

[...

experiment example 1

[0088] Experimental example 1: Reaction of Diazoker 1 crosslinker with N-(Boc) methyl glutamate

[0089] The present invention utilizes Diazoker 1 and N-(Boc) methyl glutamate to test the reactivity of this cross-linking agent with carboxylic acid-containing amino acid cross-linking ( image 3 ). Methyl N-(Boc)glutamate (50.0 mg, 0.191 mmol) was dissolved in water (3 mL). Then the cross-linking agent Diazoker 1 (131.0mg, 0.383mmol) was dissolved in acetonitrile (1mL) and added to the reaction solution openly at room temperature with bubbles coming out. The reaction was stirred at room temperature for 1 hour. TLC monitored the complete conversion of the raw material N-(Boc)methyl glutamate. The reaction solution was quickly extracted with ethyl acetate. After combining the organic phases, drying over anhydrous sodium sulfate and filtering, the filtrate was concentrated under reduced pressure. The obtained residue was separated and purified by silica gel column chromatograp...

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Abstract

The invention relates to a protein chemical crosslinking agent which is as indicated in the formula I and meanwhile, relates to a preparation method of the protein chemical crosslinking agent. The multifunctional crosslinking agent is good in both crosslinking efficiency and stability, and in particular, the crosslinking agent based on trimethyl-silicyl diazomethyl functional group types is high in crosslinking efficiency for side chain carboxyl groups of aspartic acid and glutamic acid and carbon-end carboxyl of protein and is the crosslinking agent, of the first case, based on carboxyl-containing amino acid residue selectivity and with no need of activation by adding a condensating agent. Crosslinking reaction of N-(Boc) glutamate methyl ester and polypeptide containing multi-amino acidsproves that the crosslinking agent has high crosslinking efficiency and selectivity on carboxylic acid amino acid residues, and crosslinking reaction with nine kinds of the proteins and one mixture containing eight kinds of proteins proves that the crosslinking agent, a simple pure protein body containing the carboxylic acid amino acid residues and a complicated protein system are all good in crosslinking efficiency. R1-X-R2--I.

Description

technical field [0001] The invention relates to the field of protein structure and function research, in particular to a crosslinking application of a protein chemical crosslinking agent and a preparation method thereof. Background technique [0002] With the completion of the Human Genome Project, it is far from enough to complete the research at the genetic level for an in-depth understanding of the various physiological functions and mechanisms of organisms. As the material basis of living organisms and the main bearer of life activities, the number of proteins discovered has increased significantly in the past ten years, and the study of their functions is of great significance for understanding the nature of various life activities. At present, the function and mechanism of action of a large number of newly discovered proteins are still unclear, and the essence of protein function research is to analyze its three-dimensional structure and the proteins that interact with...

Claims

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Application Information

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IPC IPC(8): C07F7/08C07C269/06C07C271/22G01N33/68
CPCC07C269/06C07F7/081C07F7/083G01N33/6848C07C271/22
Inventor 雷晓光张小昀董梦秋王建华
Owner PEKING UNIV
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