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Preparation method of cyclobutene derivative and its application in preparation of fluorescent labeling reagent
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A technology of cyclobutene and derivatives, applied in the field of biomarkers, can solve the problems of unsuitable industrial production and high cost, and achieve the effect of mild conditions and simple operation
Active Publication Date: 2022-04-15
WEST CHINA HOSPITAL SICHUAN UNIV
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[0005] Moreover, the currently reported method for the preparation of dienophiles that undergo Diels-Alder reactions with tetrazine compounds is relatively expensive and requires the use of heavy metal catalysts, which is not suitable for industrial production
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Embodiment 1
[0054] Example 1, Synthetic Compound 1
[0055] Compound 1:
[0056]
[0057] 1,3-cyclobus (15 mg, 0.17 mmol, 1 eq) and triethylamine (47 ul, 0.34 mmol, 2 eq) were dissolved in 0.3 ml of dichloromethane under argon, and the reaction liquid was cooled to 0 ° C; p-toluenesulfonyl chloride (22 mg, 0.12 mmol, 0.7 eq) was dissolved in 0.5 ml of dichloromethane, and the reaction was added dropwise to the reaction liquid under stirring conditions. After the reaction was reacted at room temperature, the thin layerchromatography (TLC) was monitored, and the reaction was carried out by drying the reactive system, and the residue was separated from the silica gel column (PE: EA volume ratio = 2: 1). Purification, drying, Yellow oil compound 1, yield of 38%.
Embodiment 2
[0058] Example 2, Synthetic Compounds 3A to 3D
[0059] Synthesis Compounds 3A ~ 3D are synthesized according to the following synthesis route:
[0060]
[0061] (1) Synthetic Compound 3A
[0062] The compound 1 (1 eq) and compound 2a (270 mg, 1.2 eq) were dissolved in 5 ml of tert-butanol under argon conditions, and the reaction liquid was obtained, and then potassium t-butoxide (1.3 eq) was added to the reaction solution under stirring. The reaction was reacted at 80 ° C for 7 hours, and the reaction was monitored through TLC. The reaction was quenched with water, and the reaction system was extracted 3 times with ethyl acetate, and the organic phase was mixed with saturated saline water, dried over anhydroussodiumsulfate, dried liquid decompression, residual silica gel column (PE: EA volume ratio = 3: 1) Separated purification, dried to give Compound 3a, yield 86%. . 1 H NMR (400MHz, CDCL 3 Δ7.16 (D, J = 8.1 Hz, 2H), 7.10 (D, J = 8.0 Hz, 2H), 4.59 (p, j = 6.4 Hz, 1H), 3.82 ...
Embodiment 3
[0069] Example 3, Synthetic Compounds 4A ~ 4D
[0070] According to the following synthesis route, the compounds 4a to 4d are synthesized:
[0071]
[0072] (1) Synthetic Compound 4A
[0073] The compound 3a (186 mg, 1eq), triethylamine (3eq) and DMAP (0.2 eq) were dissolved in 5 ml of dichloromethane under argon conditions, and the reaction liquid was cooled to 0 ° C. The toluenesulfonyl chloride (2 eq) was dissolved in 5 ml of dichloromethane, and the reaction was added dropwise to the reaction liquid under stirring conditions. The reaction was reacted at room temperature overnight, and the reaction was monitored through the TLC, and the reaction was quenched with water. The reaction system was extracted 3 times with ethyl acetate, combined with the organic phase, washed twice with saturated saline, dry alhydrate sodiumsulfate, dried liquid decompression, residual material with silica gel column (PE: EA volume ratio = 2: 1 Isolated and purified, dried, and obtained a light ye...
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Abstract
The invention provides a preparation method of a cyclobutene derivative and its application in the preparation of a fluorescent labeling reagent. The structure of the cyclobutene derivative is shown in formula I. Experiments have shown that the cyclobutene derivatives provided by the present invention have excellent stability and have a fast reaction speed when bioorthogonal reactions occur with tetrazine compounds. Therefore, the cyclobutene derivatives of the present invention can It can maintain good stability in complex biological systems before the activation of the cross-reaction, and after the activation of the bio-orthogonal reaction, it can quickly undergo a bio-orthogonal reaction with the tetrazine structure on the target protein with a fluorescent group to turn on the fluorescence. Achieve fluorescent labeling of the target protein. The cyclobutene derivatives provided by the invention can be used to prepare fluorescent labeling reagents, play the role of fluorescent labels, and have broad application prospects in the fields of living body tracking, fluorescent imaging and the like.
Description
Technical field [0001] The present invention belongs to the field of biomarkers, and is specifically related to the preparation method of cyclobutylene derivatives and its application in the preparation of fluorescent labeling reagents. Background technique [0002] Development of real-time visualization of protein molecules in cells has important significance for understanding the molecular basis of life. The label of the protein currently uses fluorescent proteins, self-labeled proteins (such as SNAP tags), enzymes (such as sulfic acid ligation enzymes, and self-labeled labels (such as cysteine). Although these methods can achieve rapid markers, but Introducing additional sequences in the proteins that are concerned may interfere with the structure and function of the protein, thereby impact on biology research; in addition, it is very challenge of inserting these probe molecules into the protein. Sexual. [0003] Bioorthogonal Reaction is a class of high-speed, high-efficiency...
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