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A kind of disposable photoaffinity linker and preparation method and application

A link body, wash-free technology, applied in the direction of chemical instruments and methods, organic chemical methods, luminescent materials, etc., can solve the problems of cumbersome operation, prone to false positive results, large background interference, etc., and achieve high tracer accuracy , avoid false positive results, low background interference effect

Active Publication Date: 2021-04-20
XI AN JIAOTONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the following problems often occur when the target separation is carried out by introducing fluorophores: ① large background interference; ② cumbersome operation; ③ prone to false positive results

Method used

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  • A kind of disposable photoaffinity linker and preparation method and application
  • A kind of disposable photoaffinity linker and preparation method and application
  • A kind of disposable photoaffinity linker and preparation method and application

Examples

Experimental program
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Effect test

Embodiment 1

[0051] The preparation process in the wash-free photoaffinity linker is as follows: figure 1 as shown,

[0052] a) 4-[3-(trifluoromethyl)-3H-diaziridin-3-yl]benzylamine hydrochloride and (R)-2-((tert-butoxycarbonyl)amino)-4- Under the condensation of pentenoic acid with EDC·HCl, an intermediate containing a carbon-carbon double bond is obtained, and the specific process is as follows:

[0053] (R)-2-((tert-butoxycarbonyl)amino)-4-pentenoic acid (0.0430g, 0.2000mmol), EDC·HCl (0.0500g, 0.2400mmol), NHS (0.0300g, 0.2400mmol) Dissolve in 3mL of anhydrous dichloromethane, stir at 0°C for a period of time, after the system cools down, slowly add triethylamine (60.00μL, 0.4400mmol) dropwise, after reacting for 2h, add 4-[3-(trifluoromethyl )-3H-diaziridin-3-yl]benzylamine hydrochloride (0.05g, 0.2000mmol) was reacted to obtain a crude product containing a carbon-carbon double bond intermediate, washed three times with saturated sodium bicarbonate, saturated sodium chloride Washin...

Embodiment 2

[0059] The principle of the probe of the present invention for cell tracer positioning or imaging is: bio-orthogonal reaction route such as figure 2 Shown, the specific process of reaction is as follows:

[0060] Dissolve 2-(4-methoxyphenyl)-5-phenyl-2H-tetrazole (0.0800g, 0.3200mmol), no-wash photoaffinity linker (0.0100g, 0.0320mmol) in 6ml of ethyl acetate , 302nm UV irradiation for 30min, the reactant was concentrated in vacuo, and separated by column chromatography (P:E=1:1), the product was about 0.04mg, and the yield was 23.4%.

[0061] LC-MS(ESI,m / z):535.10[M+H] + , 533.35[M-H] — .

Embodiment 3

[0063] Preliminary application of wash-free photoaffinity linkers containing carbon-carbon double bonds in photoaffinity labeling technology:

[0064] In the present invention, cytokines are used as targeted recognition molecules, and photoaffinity probe molecules are constructed by chemically modifying cytokines with wash-free photoaffinity linkers, and the constructed photoaffinity probe molecules are used to treat some highly expressed EGFR receptors. The cells of the body were tracked and positioned for imaging analysis.

[0065] Among them, the cytokines are transforming growth factor-β (transforming growth factor-β, TGF-β), vascular endothelial growth factor (VEGF), epidermal growth factor (Epidermal Growth Factor, EGF) or basic growth factor Fibroblast growth factor (bFGF).

[0066] Specifically, the construction process of the photoaffinity probe for EGF cytokines is as follows: (1) Prepare solution I: dissolve EGF cytokines in 1 mL triple-distilled water, store at 4°...

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Abstract

A kind of wash-free photoaffinity linker and its preparation method and application, 4-[3-(trifluoromethyl)-3H-diaziridine-3-yl]benzylamine hydrochloride and (R)-2 ‑((tert-butoxycarbonyl) amino)‑4‑pentenoic acid is condensed under EDC HCl to obtain a photoaffinity linker intermediate containing a carbon-carbon double bond with a Boc protecting group; the Boc protecting group will be The photoaffinity linker intermediate containing a carbon-carbon double bond was deprotected under the action of trifluoroacetic acid to obtain a wash-free photoaffinity linker. The preparation method of the free-washing photoaffinity linker molecule of the invention is simple, easy to realize, and has a high yield. The wash-free photoaffinity linker molecule in the present invention can chemically covalently modify the target recognition molecule to construct a photoaffinity probe molecule, and then carry out specific covalent labeling of its target molecule by photocrosslinking technology, and finally The fluorescence is illuminated through a bioorthogonal reaction, so as to realize the tracking of the target molecule.

Description

technical field [0001] The invention relates to a wash-free photoaffinity linker, a preparation method and an application. Background technique [0002] Bioorthogonal reactions refer to those that can be carried out in living cells or tissues without interfering with the biochemical reactions of the organism itself. This type of reaction can occur under the physiological conditions in the organism, and will not cause side reactions with various active substances in the biological system, and will not cause damage to the organism and target biomolecules. In recent years, it has been mainly used for specific labeling of living biomacromolecules. First, functional groups that can participate in special reactions are introduced into biomacromolecules, and then by reacting with markers with complementary reactive groups, the labeling of intracellular active biomacromolecules is achieved, extending the ability to detect intracellular biomacromolecules. understanding of biologica...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07D229/02C07D403/12C09K11/06G01N21/64
CPCC07B2200/07C07D229/02C07D403/12C09K11/06C09K2211/1044G01N21/6428G01N21/6458G01N2021/6432G01N2021/6439Y02P20/55
Inventor 张杰单媛媛卢闻王瑾王嗣岑潘晓艳贺浪冲
Owner XI AN JIAOTONG UNIV
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