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Amino acid having p-nitrophenol chromogenic group, and preparation method and applications thereof

A technology of p-nitrophenol and p-nitrophenol, which is applied in the preparation of sulfide, material analysis by observing the influence on chemical indicators, and analysis by making materials undergo chemical reactions, can solve the problem of immobilized chain affinity. Problems such as unsuitable, time-consuming, poor stability of probes, etc.

Active Publication Date: 2019-04-19
CHONGQING FARSIGHTED BLUE DRAGON FBD BIOTECH CO LTD CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It has been reported that biotin derivatives of ligases, fluorophores, and luminescent groups are probes for the determination of immobilized streptavidin, but their use in the measurement process is time-consuming, especially the stability of the probes used is poor, and their affinity for immobilized streptavidin Factors not suitable for production quality control of biotechnology products

Method used

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  • Amino acid having p-nitrophenol chromogenic group, and preparation method and applications thereof
  • Amino acid having p-nitrophenol chromogenic group, and preparation method and applications thereof
  • Amino acid having p-nitrophenol chromogenic group, and preparation method and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Add 5g of 2-hydroxy-5-nitrobenzyl alcohol into a 250ml three-necked flask, add 100 10% sulfuric acid and 10 g KBr, and react at 60°C for 6 hours; vacuum dry it with a rotary evaporator, and collect 2-hydroxy-5-nitrobenzyl alcohol Benzyl bromide, purified by silica gel column.

[0031] Add 2 g of the obtained product into a 250 ml three-necked flask, add 100 ml of dimethylformamide DMF, dissolve with magnetic stirring, then add 3 g of N-Boc cysteine, and reflux the reaction in the three-necked flask for 6 hours, and monitor to The chromophore has reacted. The crude product was vacuum-dried by a rotary evaporator, purified by silica gel column chromatography; deprotected by Boc with 1.0 M HCl and 10% trifluoroacetic acid to obtain the crude product of the candidate aminated small molecule probe; recrystallized twice with acetone to obtain Small molecule aminated chromogenic probe.

Embodiment 2

[0033] Add 1 g of biotin into a 50 ml flask, add 10 ml of DMF, stir magnetically at 80 degrees and heat to dissolve. Cool to room temperature, then add 1.1 times the mass of NHS and 2.0 times the mass of DCC, stir to dissolve, and react at room temperature overnight; filter, add 5 times the amount of ether to the supernatant to precipitate, and obtain the crude biotin active ester; reconstitute with 80ml of isopropanol Crystallized twice to obtain biotin active ester.

[0034] Add 1.0 g of the small-molecule amination probe obtained in Example 1 into a 250 ml three-necked flask, add 100 ml of DMF, and dissolve with magnetic stirring at 50 degrees. Add 5 ml of 0.5 g biotin active ester DMF solution, and stir for 1 hour at room temperature with magnetic stirring. Concentrate to the minimum volume with a rotary evaporator, add 5 times the volume of ether for precipitation; dissolve with DMF, and wash with ether for three cycles to obtain small molecule biotinylated chromogenic p...

Embodiment 3

[0035]Example 3 Determination of activated carboxyl groups in carboxyl magnetic beads MSP-COOH-F1 by small molecule chromogenic probes

[0036] (1) A small molecule aminated chromogenic probe, prepared according to Example 1;

[0037] (2) When pH~8.0, the extinction coefficient of this chromogenic probe is about 16.8 (mM) at 405 nm -1 cm -1 ;

[0038] (3) Dissolve EDC and NHS with pH 6.0 25mM MES solution at a concentration of 25mg / ml, activate carboxyl magnetic beads MSP-COOH-F1 for 30min at a constant temperature of 27 degrees, and wash the activated magnetic beads with ice pH 6.0 25mM MES buffer 2 Repeat, and finally suspended in this pH 6.0 25mM MES buffer; store at 4 degrees and measure within 20 min;

[0039] (4) Dissolve and dilute the small molecule aminated chromogenic probe into pH 6.0 25mM MES buffer, the final concentration is 20.0μM, and its initial absorption is 0.350; prepare 15 ml chromogenic probe solution for each batch of dilution;

[0040] (5) Test tube...

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Abstract

The invention discloses an amino acid having a p-nitrophenol chromogenic group, and a preparation method and applications thereof. The amino acid whose structure has a p-nitrophenol chromogenic groupcontains p-nitrophenol or a 4-nitro-1-naphthol chromogenic group, aliphatic primary amine and an aliphatic carboxyl group; the amino acid is connected to a chromogenic group through a saturated carbonatom; when the chromogenic group is the p-nitrophenol, the amino acid is connected to the ortho-position or meta-position of a phenolic hydroxyl group; when the chromogenic group is 4-nitro-1-naphthol, the amino acid is connected to the other alpha or beta position that does not contain p-nitrophenol aromatic rings; the amino acid contains at least one primary amino group and one carboxyl group,and also contains the other sulfur atom which is used for connecting with the saturated carbon atom and comes from mercaptan or an N atom of the aliphatic primary amine; and the amino acid specifically includes cysteine, lysine, citrulline natural amino acid, and unnatural amino acid which does not contain an aromatic ring and unsaturated bond, has a molecular weight not exceeding 300 Dalton, andcontains at least one aliphatic primary amine, one aliphatic carboxyl group and a required heteroatomic acid connected to the saturated carbon atom on the aromatic ring. The amino acid is used for determining the active group quantity and reversible binding active molecular weight of the covalent reaction of the surface energy of a micro-nano material and a small molecule chromogenic probe.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to the chromogenic probe required for the quantitative determination of the content of highly reactive groups and biologically active molecules on the surface of micro-nano materials, its preparation and application, and is corresponding to the quality control of micro-nano materials and the guidance of preparation process optimization key technologies. Background technique [0002] Active groups on the surface of insoluble micro-nano materials include hydroxyl, amino, carboxyl, aldehyde, p-toluenesulfonate and other active groups. The available content determines the application performance of the corresponding micro-nano materials. The determination of the available content is currently in the national The world is a problem. Existing methods for determining such groups are cumbersome and have low precision. Especially, most of the available probes are fluorescent probes, which have hi...

Claims

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Application Information

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IPC IPC(8): C07C323/56C07C323/58C07C319/14G01N33/68G01N21/78
CPCC07C323/56C07C323/58G01N21/78G01N33/68
Inventor 廖飞龙高波谢万军
Owner CHONGQING FARSIGHTED BLUE DRAGON FBD BIOTECH CO LTD CHINA
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