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Biotin labeled ligustrazine and preparation method thereof

A biotin-labeled, ligustrazine technology, applied in the biological field, achieves the effects of protecting and stabilizing, promoting recovery and reducing the accumulation of free radicals

Inactive Publication Date: 2014-07-23
WENZHOU MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But so far, there is no report on substances targeting TFAM on mitochondrial DNA but not degraded by Lon protease

Method used

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  • Biotin labeled ligustrazine and preparation method thereof
  • Biotin labeled ligustrazine and preparation method thereof
  • Biotin labeled ligustrazine and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 Preparation of biotin-labeled Ligustrazine: first Ligustrazine is oxidized to 2-hydroxyl-3,5,6-trimethylpyrazine, and D-biotin, EDCI, and DMAP according to 12:29:30 Dissolve in anhydrous DMF at a mass ratio of : 1, stir and react at 60°C for 72h, monitor the completion of the reaction by TLC, pour the reaction solution into 30mL of ether, stir at 4°C for 4h, a white precipitate precipitates, filters, washes with ether, The crude product was purified by column chromatography to obtain biotin-labeled ligustrazine with a yield of about 78%.

[0044] (1) Synthesis of 2-hydroxy-3,5,6-trimethylpyrazine, namely intermediate

[0045] With 6.8g, the ligustrazine that molar concentration is 50mmoL, i.e. substance 1, 12mL glacial acetic acid and 6mL mass concentration are 30% H 2 o 2 Add it into a three-necked flask, heat and react at 70°C for 4 hours, then add 6 mL of 30% H 2 o 2 , Continue to reflux for 4h. TLC monitors that the reaction is complete, cool to room ...

Embodiment 2

[0051] Example 2 Ligustrazine inhibits the degradation of TFAM in vitro

[0052] Use 20 μM bortezomib (Velcade) as a positive control, DMSO as a negative control, and 20 μL of 0.4 mM ligustrazine for doubling dilution; take the centrifuge tube for labeling, add reaction buffer, Lon, BSA, TMP (DMSO or Velcade), mix thoroughly; incubate at 37°C for 1 hour; add TFAM and ATP in turn to make the final concentration of Lon, TFAM, and BSA 300nM, 150nM, 0.1μg / μL, mix well, and incubate at 37°C for 1h; At the time point of 0 and 60 minutes, draw 20 μL samples from each tube, add 5 μL 5X sample buffer; heat at 95°C for 5 minutes to fully denature the protein; load 8 μl standard protein Maker and 20 μL denatured protein samples to 12% SDS-PAGE, and Western blot analysis.

[0053] The results showed that TFAM remained stable after 1 hour of reaction without adding ATP and was not degraded by Lon; in the positive control group, Velcade inhibited the activity of Lon, and TFAM remained stab...

Embodiment 3

[0054] Ligustrazine in embodiment 3 promotes Hela low Recovery of TFAM in cells

[0055] 1. Cultivate HeLaρ with EB complete medium containing 50 ng / mL + Eight days after the cells, EB was removed, and the cells were divided into two groups. One group was treated with 10M ligustrazine, and the other group was treated with DMSO as a negative control. The cells were harvested at 1, 3, and 4 days, and the protein was extracted and determined by BCA method. After the protein concentration, the protein concentration was adjusted to 1g / μL, heated at 95°C for 5min, and the sample was loaded on 12% SDS-PAGE for western blot analysis ( image 3 , Figure 4 ).

[0056] 2. Treatment of HeLaρ with different concentrations (2.5, 5, 10 μM) of ligustrazine low After 24 hours of cells, no ligustrazine or DMSO was added as a negative control, the protein was extracted, the protein concentration was determined by the BCA method, and the protein concentration was adjusted to 1 μg / μL, heated ...

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Abstract

The invention belongs to the biotechnical field, and concretely relates to a mitochondrial TFAM target conjugate and its application in mitochondrial function stabilization maintenance. A biotin labeled ligustrazine molecule is designed in the invention, and the affinity between streptavidin and biotin is high, so a stable labeled probe-ligustrazine compound plays a role in clinic monitoring and treatment. The TFAM targeting conjugate can be specifically bound with TFAM to protect the TFAM from being degraded by Lon protease, and the mitochondrial stabilization is maintained by reducing the level of ROS in cells and reducing cell apoptosis.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a compound targeting and binding to TFAM on mitochondrial DNA and an application for maintaining the stability of mitochondrial function. Background technique [0002] Biotin (Biotin, B), also known as vitamin H and coenzyme R, is a water-soluble vitamin and also belongs to the vitamin B family. Part; II is a thiophene ring, which contains a pentanoic acid side chain, and its terminal carboxyl group can be connected with biological macromolecules to form biotin-labeled antigens, antibodies, enzymes, etc. There is an imidazolone ring in its chemical structure, which can specifically bind to avidin (AV) and streptavidin (SA). The binding between biotin and avidin has high affinity and strong specificity, each of them can bind to various types of large and small molecules, and the advantages of the multi-stage amplification effect of the two in the binding reaction, and its...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07D495/04A61K31/497A61K47/48A61P39/06A61P43/00
CPCC07D495/04
Inventor 吕斌张奕华刘永章艾勇
Owner WENZHOU MEDICAL UNIV
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