Probe as well as synthesis method and application thereof

A synthesis method and probe technology, applied in material separation, instruments, analysis materials, etc., can solve problems such as damage to protein complexes, the study of interactions that cannot be used, and the study of interactions between proteins that cannot be directly applied, and achieves easy operation. , Improve enrichment and identification ability, environment-friendly effect

Active Publication Date: 2020-03-10
SOUTH UNIVERSITY OF SCIENCE AND TECHNOLOGY OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, currently reported probe technologies often require exposure of cells or proteins to oxidative or short-wavelength ultraviolet (254 nm) environments, causing irreversible damage to interacting protein complexes.
[0005] US005532379A discloses a Sulfo-SBED probe, which includes a biotin group, an NHS group and an aryl azide photoreactive group, wherein the aryl azide group has obvious non-specific labeling function, and cannot be directly applied to the study of protein-protein interactions; US20140011212A1 discloses a trifunctional probe for the study of interactions between glycoprotein receptors and their ligands, but cannot be used for the study of other protein-protein interactions

Method used

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  • Probe as well as synthesis method and application thereof
  • Probe as well as synthesis method and application thereof
  • Probe as well as synthesis method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] The synthesis of embodiment 1 probe skeleton 1

[0065]

[0066] According to literature [Ouchi T, Yamabe E, Hara K, Hirai M, Ohya Y (2004) Design of attachment type of drug delivery system by complex formation of avidin with biotinyl drug model and biotinyl saccharide. J Control Release 94 (2-3): 281 -291; Frei AP, Wollscheid B, Jeon OY, Carreira EM US 2014 / 0011212A1] Synthesis of compound c1;

[0067] A mixture of c1 (896 mg, 1 mmol) and diethylamine (2.19 g, 30 mmol) in acetonitrile was stirred at room temperature, and when the starting material c1 disappeared (monitored by TLC), the solvent was removed; the residue was dissolved in dimethylformamide (DMF , 10 mL), added N,N-diisopropylethylamine (258 mg, 2 mmol) and succinic anhydride (200 mg, 2 mmol), stirred for more than 12 hours, removed DMF, separated the residue by silica gel, and obtained in 50% yield c2 (387 mg);

[0068] HRMS (m / z): [M-H] - C 35 h 61 o 11 N 6 The calculated value of S is 773.4125,...

Embodiment 2

[0075] The synthesis of embodiment 2 probe skeleton 2

[0076]

[0077]According to literature [Ouchi T, Yamabe E, Hara K, Hirai M, Ohya Y (2004) Design of attachment type of drug delivery system by complex formation of avidin with biotinyl drug model and biotinyl saccharide. J Control Release 94 (2-3): 281 -291; Frei AP, Wollscheid B, Jeon OY, Carreira EM US 2014 / 0011212A1; Srinivasan B, Huang X (2008) Functionalization of magnetic nanoparticles with organic molecules: loading level determination and evaluation of linker length effect on immobilization. Chirality 20( 4): 265-277] synthesis of compound c4;

[0078] At 0°C, TFA (1.026g, 9mmol) was added to a solution of c4 (100mg, 0.09mmol) in dichloromethane (5mL), stirred for 2 hours, TLC monitoring found that the raw material c4 disappeared; after the mixture was evaporated in vacuo, the dissolved In DMF (5 mL), triethylamine was added to adjust the pH to greater than 9; 4-benzoylbenzoic acid-2,5-dioxopyrrolidin-1-yl est...

Embodiment 3

[0084] Synthesis of Example 3 Probe Skeleton 3

[0085]

[0086] At 0°C, TFA (1.026g, 9mmol) was added to a solution of c4 (100mg, 0.09mmol) in dichloromethane (5mL), stirred for 2 hours, TLC monitoring found that the raw material c4 disappeared; after the mixture was evaporated in vacuo, the dissolved In DMF (5 mL), add triethylamine to adjust the pH to greater than 9; add 2,5-dioxopyrrolidin-1-yl 4-azidobenzoate (35.2 mg, 0.14 mmol), and stir overnight , the solvent was removed in vacuo and the residue was separated through silica gel to afford c6 (63 mg) in 61% yield;

[0087] HRMS (m / z): [M-H] - C 55 h 89 o 13 N 12 The calculated value of S is 1157.6398, and the measured value is 1157.6417;

[0088] Add EDCI (30mg, 0.158mmol) to the DMF (5mL) mixed solution of compound c6 (61mg, 0.053mmol) and hydroxysuccinimide (12mg, 0.106mmol), stir at room temperature overnight, when compound c6 disappeared (by TLC monitoring), the solvent was removed under vacuum, the residu...

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Abstract

The invention provides a probe as well as a synthesis method and application thereof. The method comprises the following steps: (1) removing Boc from a compound A, and reacting with a compound B to obtain a compound C; (2) condensing the compound C obtained in the step (1) with N-hydroxysuccinimide to obtain a probe skeleton; and (3) mixing bait protein with the obtained probe skeleton, connecting, adding a glycine solution to terminate the reaction, and filtering to obtain the probe. The synthesis method disclosed by the invention is simple and convenient to operate, low in cost and environment-friendly; according to the prepared probe, an SH2 structural domain is used as bait protein; analysis of weak interaction and instantaneous interaction between proteins is facilitated; especially,enrichment and identification of the SH2 structural domain and tyrosine phosphorylated protein are facilitated, the enrichment and identification capacity of target proteins and compounds thereof nearhuman or animal tissue samples and cell membranes is remarkably improved, and background interference of other non-specific adsorption proteins is reduced.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to a probe and its synthesis method and application, in particular to a probe containing three functional groups and its synthesis method and application. Background technique [0002] Proteins are the building blocks of living organisms, usually composed of modular domains that can fold independently and perform different biological functions. Protein interaction domains play an important role in protein interactions and the formation of protein complexes. Protein-protein interactions are often discovered during the study of post-translational modifications of proteins, such as phosphorylation, acetylation, methylation, and ubiquitination. Src homology 2 (SH2) domain is a representative example of protein interaction domain, and SH2 domain can specifically recognize phosphorylated tyrosine (pTyr) proteins in various signal transduction pathways. Currently, more than 120 different SH2 do...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/06G01N30/88G01N30/90C07K5/027
CPCG01N30/06G01N30/88G01N30/90C07K5/0205Y02P20/55
Inventor 田瑞军李鹏飞初碧珠何岸
Owner SOUTH UNIVERSITY OF SCIENCE AND TECHNOLOGY OF CHINA
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