Preparation method and applications of oriented immobilized PEGA composite resin

A composite resin technology, applied in the field of biomedicine, can solve the problems of active site influence, protein loss, weak interaction between protein and solid materials, etc., and achieve the effect of simple reaction steps and stable reaction

Active Publication Date: 2015-08-19
NORTHWEST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the non-specificity of the method used and the weak interaction between the protein and the solid material, the immobilized protein obtained by this method is prone to loss during use, which also affects the accuracy of the research results
The chemical bonding method can be divided into random immobilization and directional immobilization. The random immobilization method generally uses g

Method used

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  • Preparation method and applications of oriented immobilized PEGA composite resin
  • Preparation method and applications of oriented immobilized PEGA composite resin
  • Preparation method and applications of oriented immobilized PEGA composite resin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] a. Add 2-amino-6-chloropurine (compound A) and 4-nitrobenzyl alcohol (compound B) into methanol, the reaction temperature is 80°C, the molar ratio of the reaction substrate is 1:1, and the heating and reflux time is 2 The Williamson ether formation reaction occurred under alkaline conditions for 4 hours to generate 4-nitro-benzylguanine; the yield was 95%. The following reaction formula is shown:

[0026]

[0027] b, 4-nitro-benzylguanine is protected by tert-butoxycarbonyl group, hydrogenation under normal pressure under the catalysis of palladium carbon consumption being 5% of the reaction product, dehydrated tetrahydrofuran is added dropwise, in 4-dimethylaminopyridine Under the condition of existence, di-tert-butyl dicarbonate was added, the reaction temperature was 25° C., and the stirring time was 6 hours, and the compound (3) was reduced to generate compound (3); the yield was 97%. The following reaction formula is shown:

[0028]

[0029] c. Compound (3)...

Embodiment 2

[0037] a. Add 2-amino-6-chloropurine (compound A) and 4-nitrobenzyl alcohol (compound B) into ethanol, the reaction temperature is 80°C, the molar ratio of the reaction substrate is 1:1, and the heating time is 6 The Williamson ether formation reaction occurred under alkaline conditions for 4 hours to generate 4-nitro-benzylguanine; the yield was 96%. The following reaction formula is shown:

[0038]

[0039] b, 4-nitro-benzylguanine is protected by tert-butoxycarbonyl group, hydrogenation under normal pressure under the catalysis of palladium carbon consumption being 5% of the reaction product, dehydrated tetrahydrofuran is added dropwise, in 4-dimethylaminopyridine Under the condition of existence, di-tert-butyl dicarbonate was added, the reaction temperature was 25° C., and the stirring time was 7 hours, and the compound (3) was reduced; the yield was 98%. The following reaction formula is shown:

[0040]

[0041] c. Compound (3), the γ-aminobutyric acid protected b...

Embodiment 3

[0048] a. Add 2-amino-6-chloropurine (compound A) and 4-nitrobenzyl alcohol (compound B) into isopropanol, the reaction temperature is 80°C, the molar ratio of the reaction substrate is 1:1, and the heating time is reflux The Williamson ether formation reaction occurred under alkaline conditions for 12 hours to generate 4-nitro-benzylguanine; the yield was 98%. The following reaction formula is shown:

[0049]

[0050] b, 4-nitro-benzylguanine is protected by tert-butoxycarbonyl group, hydrogenation under normal pressure under the catalysis of palladium carbon consumption being 5% of the reaction product, dehydrated tetrahydrofuran is added dropwise, in 4-dimethylaminopyridine Under the condition of existence, di-tert-butyl dicarbonate was added, the reaction temperature was 25° C., and the stirring time was 8 hours, and the compound (3) was reduced; the yield was 99%. The following reaction formula is shown:

[0051]

[0052] c. Compound (3), the γ-aminobutyric acid p...

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Abstract

The invention discloses a preparation method and applications of an oriented immobilized PEGA composite resin. The preparation method comprises following steps: 2-amino-6-chloropurine and 4-nitrobenzyl alcohol are taken as raw materials for Williamson ether synthesis so as to obtain 4-nitryl-O6-benzylguanine; 4-nitryl- O6-benzylguanine is subjected to reduction reaction with protection of t-butyloxycarboryl; coupling reaction of an obtained compound with gamma-aminobutyric acid protected by carbobenzoxy chloride is carried out in the presence of ethyl dimethyl carbodiimide and 1-hydroxybenzotriazole; O<6>-benzylguanine derivative modified PEGA resin is obtained via reduction, separation and purification, reaction with PEGA, and deprotection; and transalkylation reaction of the O<6>-benzylguanine derivative modified PEGA resin with a protein containing MGMT label is carried out, so that oriented immobilization on PEGA resin surface via thioether covalent bonds is realized. Reaction of the preparation method is stable; the steps are simple; a stable uniform protein coating with uniform orientation can be formed on solid material surface by a prepared product; and the preparation method is used for preparing reagent kit with specific recognition effects.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to immobilizing a recombinant protein containing O6-alkylguanine-DNA methyltransferase (MGMT) on the surface of a solid material to establish a method for immobilizing the MGMT recombinant protein. Background technique [0002] Technologies such as enzyme reactors, biosensors, and biochips centered on immobilized proteins are of great significance for biocatalysis, proteomics, clinical diagnosis, and new drug development. The strength of the immobilized protein bioactivity is the prerequisite and determinant factor for the successful construction of the above-mentioned immobilized protein interface, and it is also the most critical factor affecting the results of later catalysis, identification, diagnosis and screening. Therefore, the research on the high activity immobilization method of protein can not only improve the accuracy of the above-mentioned technical research results, but also...

Claims

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Application Information

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IPC IPC(8): C07K17/08
Inventor 赵新锋郑晓晖张亚军肖超妮王静
Owner NORTHWEST UNIV
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