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Disulfide bond containing compound and use and preparation methods thereof

A compound and disulfide bond technology, applied in the field of compounds containing disulfide bonds, can solve the problems of difficulty in separating detergents and extracted substances, and achieve the effects of good film dissolution and protein stabilization.

Active Publication Date: 2018-12-14
SHANGHAI TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In view of the above-mentioned shortcoming of prior art, the object of the present invention is to provide a kind of compound containing disulfide bond, is used to solve the difficult problem of separation of detergent and extracted substance in prior art; Optimized membrane protein Preparation and Functional Studies

Method used

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  • Disulfide bond containing compound and use and preparation methods thereof
  • Disulfide bond containing compound and use and preparation methods thereof
  • Disulfide bond containing compound and use and preparation methods thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1 Disulfide bond bridged cleavable detergent molecule and its synthesis

[0063] 1. Disulfide bridged detergents, the structural formulas of which are the above-mentioned (XII)-(XV).

[0064] 2. The synthesis method is as follows, reagents and reaction conditions: i) sodium thiobenzenesulfonate, tetrabutylammonium bromide, acetonitrile, 70°C; ii) triethylamine, dichloromethane, 0°C to room temperature; iii ) sodium methoxide, methanol, room temperature.

[0065]

[0066] Specific steps are as follows:

[0067] (i) Dissolve 14g of alpha-D-bromomaltose heptaacetate 1 in 20ml of anhydrous acetonitrile, add 644mg of tetrabutylammonium bromide (Titan, product number: G28296B) and 8g of sodium thiobenzenesulfonate (Datang Medicine, Cat. No.: BL-06097). The reaction solution was refluxed at 70° C. for 6 hours (or reacted overnight). After the reaction was completed, the solvent was removed by rotary evaporation. Add 100mL ethyl acetate and 100mL water, separate...

Embodiment 2

[0084] Example 2 Evaluate the micelle properties formed by each detergent in Example 1

[0085] 1. CMC determination

[0086] The critical micelle concentration (CMC) of each detergent was determined by fluorescent dye method. The disulfide bond bridged detergents with structural formulas (XII)-(XV) are formulated into a gradient solution, and the CMC value of the detergent is determined according to the mutation point of the fluorescence curve of the hydrophobic fluorescent dye. For example, prepare concentrations of 0, 42, 84, 126, 168, 204, 240, 360, 480 with double distilled water containing 40uM fluorescent dye 8-amino-1-sulfonic acid naphthalene ammonium salt (Baringwei, product number: 442848). , 600, 800 and 1000uM detergent solutions and incubate for 20 minutes in the dark. Measure the fluorescence curve at 477nm / 388nm, and the abrupt change point of the curve is the CMC value of the detergent.

[0087] 2. Dynamic Light Scattering Experiment

[0088] Structural fo...

Embodiment 3

[0093] Example 3 Evaluation of CCD-2

[0094] The detergent CCD-2 in Example 1 was used to dissolve and purify GPCRs of different families, specifically including adenosine receptor 2A subtype protein (A2a protein, class A GPCR), glucagon-like peptide-1 receptor protein (GLP-1R, class B GPCR) and Smoothened receptor (SMO, class F GPCR).

[0095] 1. Specific steps:

[0096] Expression of GPCRs (A2a, GLP-1R, and SMO) in SF9 cells (invitrogen, Cat. No. 11496-015):

[0097] Cells were cultured at 37°C until the cell density reached 1.0-1.3x10 6 cells / mL were collected and broken to obtain cell membranes. Wash the cell membrane with low-salt buffer (10mM magnesium chloride (Sigma-Aldrich, product number M4880), 20mM potassium chloride (Sigma-Aldrich, P9541), 20mM Hepes (ABCONE, product number H33755) at pH 7.5) (50mL*3 times) and centrifuged, and then washed with high-salt buffer (1M sodium chloride (Shanghai Sangon, product number A610476), 10mM magnesium chloride, 20mM potass...

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Abstract

The invention provides a disulfide bond containing compound and use and preparation methods thereof. The compound has a structural general formula represented by a formula shown in the description, wherein A is a substituted or unsubstituted hydrophilic monosaccharide group, oligosaccharide group or ammonium inner salt group, and B is a hydrophobic aliphatic group with 40 or less carbon atoms. According to the disulfide bond containing compound and the use and preparation methods thereof, through introducing disulfide bonds to corresponding positions of micromolecules, the micromolecules reserve dissolved membrane and protein stabilizing actions, which are equivalent to or even better than those of a prototype descaling agent, complete in-situ degradation can be achieved under the action of a breaking agent, to obtain two mercaptan fragments, and properties of a surfactant are lost. The compound can be used for preparing and researching membrane proteins.

Description

technical field [0001] The invention relates to a compound containing a disulfide bond, its use and preparation method. Background technique [0002] Detergents, also known as surfactants, are amphiphilic molecules with both hydrophilic polar groups and hydrophobic non-polar groups. One end is a hydrophilic group, and the other end is a hydrophobic group; the hydrophilic group is often a polar group, such as carboxylic acid, sulfonic acid, sulfuric acid, amino group or amine group and its salt, hydroxyl group, amide group, ether bond etc. can also be used as polar hydrophilic groups; while hydrophobic groups are often non-polar hydrocarbon chains, such as hydrocarbon chains with more than 8 carbon atoms. According to its dissociation state characteristics in water, it is generally divided into ionic detergents (including anionic and cationic), nonionic detergents and zwitterionic detergents. [0003] In the study of membrane proteins, detergents are used to compete and wea...

Claims

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Application Information

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IPC IPC(8): C07H5/10C07H1/00C07K1/14
CPCC07H1/00C07H5/10C07K1/14
Inventor 陶厚朝薛东香
Owner SHANGHAI TECH UNIV
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