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Tetrazine compound as well as preparation method and application thereof

A compound and reaction solvent technology, applied in the field of tetrazine labeling, can solve problems such as poor selectivity, greater influence on the activity of biomolecules itself, and poor stability of coupling products

Active Publication Date: 2021-05-18
WEST CHINA HOSPITAL SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to provide tetrazine compounds and their preparation methods and applications, to achieve selective tetrazine labeling of amino acids, polypeptides, proteins and biological materials containing sulfhydryl groups, and to use bioorthogonal reactions to label biological compounds with tetrazine labels. Fast, efficient and stable functional derivatization application of functional molecules, so as to solve the selectivity of labeling reagents used in the prior art for labeling sulfhydryl-containing amino acids, polypeptides, proteins, and sulfhydryl-containing biological materials, drugs, and functional imaging molecules Poor, it is difficult to functionalize biomolecules in the later stage, the stability of coupling products is poor, and it has a great impact on the activity of biomolecules themselves.

Method used

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  • Tetrazine compound as well as preparation method and application thereof
  • Tetrazine compound as well as preparation method and application thereof
  • Tetrazine compound as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0089] Synthesis of 3-phenyl-6-vinyl-1,2,4,5-tetrazine:

[0090]

[0091] Benzonitrile (206 mg, 2 mmol), 3-hydroxypropionitrile (548 μL, 8 mmol), 3-mercaptopropionic acid (174 μL, 3 mmol), ethanol (0.6 mL) and hydrazine hydrate (1.2 mL, 24 mmol) were added to a A 10 mL reaction vessel with a magnetic stir bar. Under the protection of argon, the reaction solution was stirred overnight at 40°C. After that, the reaction solution was cooled to 0° C., and sodium nitrite (1.38 g, 20 mmol) dissolved in ice water (50 mL) was slowly added to the reaction solution. Then slowly add 1M HCl under an ice-water bath, the reaction solution turns bright red and gas is generated, then continue to add HCl until the generation of gas stops, the pH value is 1-6, preferably, the pH value is 1-4, more preferably 2 ~3. The resulting mixture was further stirred for about 5 minutes, extracted with dichloromethane (50 mL x 3), dried over anhydrous sodium sulfate, concentrated in vacuo, and purifie...

Embodiment 2

[0100]Synthesis of 3-(2-methoxyethyl)-6-vinyl-1,2,4,5-tetrazine:

[0101]

[0102] 3-Methoxypropionitrile (190 mg, 2 mmol), 3-hydroxypropionitrile (548 μL, 8 mmol), 3-mercaptopropionic acid (174 μL, 3 mmol), ethanol (0.6 mL) and hydrazine hydrate (1.2 mL, 24 mmol) Add to a 10 mL reaction vessel equipped with a magnetic stir bar. Under the protection of argon, the reaction solution was stirred at room temperature for 16 hours. After that, the reaction solution was cooled to 0° C., and sodium nitrite (1.38 g, 20 mmol) dissolved in ice water (50 mL) was slowly added to the reaction solution. Then slowly add 1M HCl under an ice-water bath, the reaction solution turns bright red and gas is generated, then continue to add HCl until the generation of gas stops, the pH value is 1-6, preferably, the pH value is 1-4, more preferably 2 ~3. The mixture was further stirred for about 5 minutes, extracted with dichloromethane (50mL x6), dried over anhydrous sodium sulfate, concentrated...

Embodiment 3

[0111] Synthesis of 3-isopropyl-6-vinyl-1,2,4,5-tetrazine:

[0112]

[0113] 2-Methylalanine hydrochloride (292 mg, 2.6 mmol), 3-hydroxypropionitrile (712 μL, 10.4 mmol), 3-mercaptopropionic acid (226 μL, 2.6 mmol), ethanol (0.7 mL) and hydrazine hydrate (1.5 mL, 30 mmol) was added to a 10 mL reaction vessel equipped with a magnetic stir bar. Under the protection of argon, the reaction solution was stirred overnight at room temperature. After that, the reaction solution was cooled to 0° C., and sodium nitrite (1.8 g, 26 mmol) dissolved in ice water (50 mL) was slowly added to the reaction solution. Then slowly add 1M HCl under an ice-water bath, the reaction solution turns bright red and gas is generated, then continue to add HCl until the gas generation stops, the pH value is 1-6, preferably, the pH value is 1-4, more preferably 2~3. The mixture was further stirred for about 5 minutes, extracted with dichloromethane (50 mL x 5), dried over anhydrous sodium sulfate, conc...

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Abstract

The invention discloses a tetrazine compound as well as a preparation method and application thereof. The tetrazine compound provided by the invention can realize selective tetrazine labeling of sulfydryl-containing polypeptides, proteins and biological materials, and quick, efficient and stable functional derivatization application of tetrazine labeled substances by utilizing a biological orthogonal reaction. Besides, the tetrazine compound has high selectivity, when the tetrazine compound is rich in other functional groups in vivo, the tetrazine compound can be selectively combined with cysteine to generate a stable connection product, no by-product is detected, after the tetrazine compound and sulfydryl form the connection product, the reaction power of the dienophile and the connection product is rapid, and is obviously faster than that of a manner in the prior art that thiotetrazine is used as a tetrazine marker, and it is indicated that the tetrazine compound can be applied to in-vivo biological orthogonal pre-targeting and radiolabeling with nano-molar concentration.

Description

technical field [0001] The invention relates to the field of tetrazine labeling, in particular to tetrazine compounds used for selective tetrazine labeling of sulfhydryl-containing amino acids, polypeptides, proteins and biological materials, as well as the preparation method and application of the tetrazine compounds. Background technique [0002] Bioconjugation of proteins with cytotoxic drugs, imaging agents, and small molecule probes, bionanomaterials, etc. has emerged as an attractive strategy for targeted chemotherapy, molecular imaging, and chemical biology. Because inappropriate protein modification can have a significant impact on the tertiary structure and downstream activities of the protein, such as biodistribution, affinity and drug release rate. To address this issue, several practical chemical and regioselective bioconjugation methods have been developed that allow for site-specific conjugation and controlled loading. In recent years, studies have reported th...

Claims

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Application Information

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IPC IPC(8): C07D257/08C07D403/04C07D401/04G01N33/68
CPCC07D257/08C07D403/04C07D401/04G01N33/68
Inventor 吴昊星毛梧宇孙洪宝贺欣羽杨豪杰
Owner WEST CHINA HOSPITAL SICHUAN UNIV
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