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Application of asymmetrical 1,2,4,5-tetrazine molecule

An asymmetric and molecular technology, applied in 1 field, can solve the problems of limited application prospects, low efficiency, slow activation rate, etc., and achieve high activation efficiency and broad application prospects

Inactive Publication Date: 2017-10-24
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, the existing activation methods of prodrugs or proteins have the problems of slow activation rate and low efficiency
Therefore, it cannot be used for the efficient activation of functional molecules in living systems, which greatly limits the application prospects of these methods in the precise treatment of actual diseases.

Method used

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  • Application of asymmetrical 1,2,4,5-tetrazine molecule
  • Application of asymmetrical 1,2,4,5-tetrazine molecule
  • Application of asymmetrical 1,2,4,5-tetrazine molecule

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] 1. Preparation of 3-(2-pyrimidine)-6-methyl-1,2,4,5-tetrazine molecule

[0032] Mix 1.0mmol cyano-based substrate 2-cyanopyrimidine, 10.0mmol cyano-based substrate acetonitrile, 25mmol hydrazine hydrate, and 0.5mmol zinc trifluoromethanesulfonate in a round bottom flask and stir with a magnetic stirrer. The reaction conditions are vacuum nitrogen protection, The reaction was carried out at 60°C for 24 hours, then cooled to room temperature, slowly added dropwise a 1M sodium nitrite solution to the reaction system in an ice-water bath, adjusted the pH to 3 with a 1M aqueous hydrochloric acid solution, and then added 20 mL of dichloromethane Extracted three times, combined the organic phase, dried with anhydrous sodium sulfate, removed the organic solvent with a rotary evaporator, and finally separated and purified by column chromatography to obtain a red solid 3-(2-pyrimidine)-6-methyl-1, 2,4,5-tetrazine molecule, 40% yield. Characterization data: 1 H NMR (CDCl 3 ,400...

Embodiment 2

[0039] Example 2: In vitro activation of luciferase fLuc

[0040] fLuc is a firefly luciferase that converts luciferin to oxyluciferin and emits fluorescence at the same time. First, trans-cyclooctene-protected lysine (TCOK) was inserted into the 529 position of fLuc to obtain the protein fLuc-K529TCOK to inactivate fLuc, and then after the cells expressed the fLuc-K529TCOK protein, the cells were broken and purified to obtain pure fLuc- K529TCOK protein, followed by adding 50 μM of 3-(2-pyrimidine)-6-methyl-1,2,4,5-tetrazine molecules (the preparation method is the same as in Example 1) to the protein solution to make fLuc- K529TCOK is deprotected, and the activated fLuc catalyzes the conversion of luciferin to oxyluciferin, and produces fluorescence at the same time, and can activate fLuc-K529TCOK protein into wild-type protein fLuc within 4 minutes, and the activation efficiency can reach more than 90%.

Embodiment 3

[0041] Example 3: Activation of luciferase fLuc

[0042] First, insert trans-cyclooctene-protected lysine (TCOK) at position 529 of fLuc to obtain protein fLuc-K529TCOK can inactivate fLuc, and the activity of fLuc is completely inhibited. Add 50uM 3 to living HEK293T cells -(2-pyrimidine)-6-methyl-1,2,4,5-tetrazine molecule (the preparation method is the same as in Example 1) deprotects fLuc-K529TCOK, and the activated fLuc catalyzes the conversion of luciferin to oxidation Luciferin produces fluorescence at the same time, and can fully activate fLuc-K529TCOK within 4 minutes, and the activation efficiency can reach more than 95%.

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Abstract

The invention discloses application of an asymmetrical 1,2,4,5-tetrazine activating agent to a prodrug or rapid activation of protein in the bioorthogonal field based on an inverse electron Diels-Alder reaction. The application disclosed by the invention has the advantages that compared with a symmetrical tetrazine molecule, the asymmetrical 1,2,4,5-tetrazine activating agent has the advantages of extremely high activating efficiency and no toxicity to cells in the asymmetrical 1,2,4,5-tetrazine activating agent and the bioorthogonal reaction process, can be used for rapidly activating the prodrug, in particular to proteins taking lysine, tyrosine, serine or threonine as active residues in vitro or vivo, can be used for the aspects such as research on life course and treatment on diseases, and has an extremely wide application prospect.

Description

technical field [0001] The invention relates to the application of an activator, in particular to the application of an asymmetric 1,2,4,5-tetrazine molecule. Background technique [0002] The key groups of many prodrugs or proteins are active groups such as hydroxyl or amino groups. Therefore, the activity of these molecules can be regulated by protecting and deprotecting their key active groups. However, in many cases, the activation of these molecules needs to be carried out in a living system, which requires that the deprotection reaction must be bioorthogonal and have high reaction efficiency. Bioorthogonal reaction (Bioorthogonal reaction) refers to the chemical reaction that can be carried out in living cells or tissues without interfering with the biochemical reaction of the organism itself, which is of great significance to the basic research and clinical application of life sciences. At the same time, the Diels-Alder reaction is a classic diene addition reaction, ...

Claims

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Application Information

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IPC IPC(8): A61K31/704A61K31/395A61K31/506C07D257/08C07D401/04C07D403/04A61K47/54C12N9/22C12N9/12C12N9/02C12N9/88
Inventor 陈鹏樊新元林锋
Owner PEKING UNIV
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