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20-hydroxycholesterol-Biotin molecular probe and preparation method and application thereof

A technology of hydroxycholesterol and molecular probe, which is applied in the field of biology, medicine and its preparation and application, can solve the problem of not identifying 20-hydroxycholesterol binding protein well, and achieves simple and easy operation, mild reaction conditions and high yield high effect

Active Publication Date: 2017-05-24
EAST CHINA NORMAL UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the bipolar molecular characteristics of 20-hydroxycholesterol molecules, there has been no good method for identifying 20-hydroxycholesterol-binding proteins.

Method used

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  • 20-hydroxycholesterol-Biotin molecular probe and preparation method and application thereof
  • 20-hydroxycholesterol-Biotin molecular probe and preparation method and application thereof
  • 20-hydroxycholesterol-Biotin molecular probe and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Embodiment 1: Preparation of compound formula (1) 20-hydroxycholesterol-Biotin molecular probe

[0043] 20-hydroxycholesterol (20(S)-amine) (200mg, 0.53mmol) and Biotin-OSu (280mg, 0.8mmol) were dissolved in anhydrous DMF (10ml), triethylamine (107mg, 1.06mmol) was added, N 2 Stir at room temperature under protection for 12 hours. After the completion of the reaction as detected by TLC, add water and extract with ethyl acetate, combine the organic phases, wash with a large amount of water, wash with saturated sodium chloride solution (30mL×3), dry over anhydrous sodium sulfate, and concentrate. Silica gel column chromatography (DCM:MeOH=10:1) gave a white solid (280 mg, 88%). 1 H NMR (500MHz, DMSO-d6) δ7.72(t, J=5.0Hz, 1H), 6.42(s, 1H), 6.35(s, 1H), 5.25(d, J=5.0Hz, 1H), 4.58 (d,J=5Hz,1H),4.32–4.28(m,1H),4.15–4.10(m,1H),3.71(s,1H),3.29–3.20(m,1H),3.09(m,1H) ,2.92-2.99(m,2H),2.83-2.92(m,1H),2.57(d,J=15.0Hz,1H),2.19-2.06(m,2H),2.06-1.98(m,3H),1.93 –1.85(m,1H),1.81–1....

Embodiment 2

[0044] Example 2: Application of 20-hydroxycholesterol-Biotin molecular probe in identifying 20-hydroxycholesterol-binding protein.

[0045] Smoothened proteins can bind 20-hydroxycholesterol molecules at the cysteine-rich region of their N-terminus. First, at 4°C, the 20-hydroxycholesterol-Biotin molecular probe prepared in Example 1 was incubated with Avidin beads for 4 hours, and the 20-hydroxycholesterol-Biotin molecular probe was coated on the Avidin beads . Then the free 20-hydroxycholesterol-Biotin molecular probe was washed to obtain Avidin beads coated with 20-hydroxycholesterol-Biotin molecular probe. After the Smoothened protein was transfected in 293T cells, cultured at 37°C for 48 hours, the cells were collected, and treated with 10mM HEPESpH=7.5, 300mM NaCl and 1.3% (mass / volume) n-dodecyl-β-d-maltopyranoside (DDM, Anatrace) to lyse the cells, use Flag beads to specifically immunoprecipitate the Smoothened protein, 4 degrees Celsius for 3 hours, then use 150 μg / m...

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Abstract

The invention discloses a 20-hydroxycholesterol-Biotin molecular probe and a preparation method and application thereof. 20-hydroxycholesterol is used as a raw material and performs condensation reaction with Biotin-OSu to obtain the 20-hydroxycholesterol-Biotin molecular probe shown as a formula (1). The preparation method has the advantages of mild reaction conditions, simple and easy operation and high yield. The invention further discloses application of the 20-hydroxycholesterol-Biotin molecular probe in identification of 20-hydroxycholesterol binding proteins.

Description

technical field [0001] The invention belongs to the technical field of biology, medicine and its preparation and application, and specifically relates to a 20-hydroxycholesterol-Biotin molecular probe, its preparation, and its application in the process of identifying 20-hydroxycholesterol-binding protein. Background technique [0002] As an important cholesterol derivative, 20-hydroxycholesterol can activate the hedgehog pathway by binding smoothened protein. Abnormal activation of the hedgehog pathway often leads to tumors and other diseases, while abnormal silencing can lead to developmental disorders and other diseases. The study of the combination of 20-hydroxycholesterol and smoothened protein is of great significance to the study of diseases related to the hedgehog pathway, and the identification of new 20-hydroxycholesterol-binding proteins also has important scientific research and clinical value. However, due to the bipolar molecular characteristics of 20-hydroxyc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07J9/00G01N33/92G01N33/68
CPCC07J9/00G01N33/6803G01N33/92
Inventor 仇文卫宋保亮杨连芳唐静洁肖旭肖洁心赵青玲
Owner EAST CHINA NORMAL UNIV
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