Preparation method of BODIPY-tetrazine bio-orthogonal probe

A bioorthogonal, fluoroboron fluorescence technology, applied in the field of biological probes, can solve the problems of complex synthesis steps, poor water solubility, low yield, etc., and achieve the effects of improving fluorescence intensity, enhancing fluorescence intensity and reducing fluorescence background signal.

Inactive Publication Date: 2018-12-14
WEST CHINA HOSPITAL SICHUAN UNIV
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  • Application Information

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Problems solved by technology

[0005] The invention provides a method for preparing a bodipyr-tetrazine bio-orthogonal probe, which uses a BODIPY compound to react with a tetrazine compound to generate a bodipyr-tetrazine bio-orthogonal probe, which solves the problem of fluorescence in existing cell imaging Probes are expensive, complex synthesis steps, low yield, poor water solubility and other technical problems. At the same time, the fluorescence intensity of fluorescent probes is greatly improved and the fluorescence background signal is reduced.

Method used

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  • Preparation method of BODIPY-tetrazine bio-orthogonal probe
  • Preparation method of BODIPY-tetrazine bio-orthogonal probe

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Embodiment 1

[0042] The preparation method of flubofluorine-tetrazine bio-orthogonal probe, the reaction structural formula is as follows, such as Figure 18 ,

[0043]

[0044] The specific operation method is: add 5 mmol of TMSOTf to 9 ml of anhydrous acetonitrile solution containing 1 mmol of BODIPY1-2, stir and react at 0°C for 30 minutes to activate BODIPY1-2. Then add 4 mmol t-BuOH to quench TMSOTf, quickly add 5 mmol 2,6-lutidine and 5 mmol tetrazine Tza-Tzg mixed anhydrous acetonitrile solution, and continue stirring for 10 min at 0°C. The reaction was quenched with water, and the reaction product was extracted with DCM. The organic phase was dried with anhydrous sodium sulfate, spin-dried, and separated on a silica gel column to obtain a fluboron-tetrazine bioorthogonal probe. The reaction equation and yield are as follows.

[0045] In this example, in the reaction structure formula, the synthesis method using Tza is obtained by using the prior art method.

[0046] In the pre...

Embodiment 2

[0126] Fluorescence performance test of the flubofluorine-tetrazine bio-orthogonal fluorescent probe prepared in Example 1:

[0127] The fluborine-tetrazine bio-orthogonal fluorescent probe prepared above was prepared into a fluborine-tetrazine bio-orthogonal fluorescent probe solution (concentration is 5 μm / ml, solvent is 20% DMF and 80% mixed solvent ), under the conditions of 495nm excitation light irradiation at room temperature, continuously irradiated for 10min, the maximum fluorescence intensity of the flubororine-tetrazine bioorthogonal fluorescent probe still remained above 90% of the initial value, as Figure 15 As shown, it shows that the boron-tetrazine bioorthogonal fluorescent probe can be applied to long-term cell imaging.

[0128] The live cell imaging of the boron-tetrazine bio-orthogonal fluorescent probe, the specific operation method is: the above-prepared boron-tetrazine bio-orthogonal fluorescent probe 7 (0.5% cross) is dissolved in In the PBS solution, ...

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Abstract

The invention discloses a BODIPY-tetrazine bio-orthogonal probe and a preparation method thereof. The preparation method comprises the steps: 1) dissolving BODIPY1-2 and TMSOTf in an anhydrous acetonitrile-dichloromethane mixed solvent, mixing evenly, stirring the reaction solution for 30 min under a condition of the temperature of 0 DEG C, and activating BODIPY1-2; 2) adding tert-butanol quenchedTMSOTf to the reaction solution, then dropwise adding 2,6-dimethylpyridine and a tetrazine Tza-Tzg pre-mixed acetonitrile solution, and carrying out a stirring reaction of the mixed solution under acondition of the temperature of 0 DEG C for 10 min; and 3) quenching the reaction in the step 2 by water, then extracting by DCM, separating an organic layer, drying the organic phase by Na2SO4, concentrating in vacuum, carrying out separated purification of the reaction product by a silica gel column, and thus obtaining the BODIPY-tetrazine bio-orthogonal fluorescent probe. The BODIPY-tetrazine bio-orthogonal probe is synthesized by the reaction of the BODIPY compound with the tetrazines. The yield in the synthesis process is greatly increased, the water solubility of the probe is greatly improved, the probe has the fluorescent opening properties, and the improvements are conducive to an application of the probe in cell imaging.

Description

technical field [0001] The invention relates to the technical field of biological probes, in particular to a preparation method of a boron-tetrazine bio-orthogonal probe. Background technique [0002] Labeling bioactive substances with fluorescent probes can realize real-time visualization of vital activities and pathological processes in vivo. Bioorthogonal chemistry is a class of chemical reactions that can occur in the environment of organisms with sufficient kinetics and high specificity, and the reaction process does not interfere with the life process of the organism itself. Because of its good biocompatibility, strong specificity, and high reaction efficiency, it has attracted extensive attention from scientists in chemical biology, medical imaging, and pharmacy. [0003] BODIPY is a commonly used fluorescent reporter group in the field of biomedical research. Due to the excellent optical properties of this type of molecule, it is often used in the selective fluoresc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07F5/02C09K11/06G01N21/64A61K49/00
CPCA61K49/0021C07F5/022C09K11/06C09K2211/1029C09K2211/1055C09K2211/1074G01N21/6428G01N21/6486
Inventor 龚启勇吴昊星董萍
Owner WEST CHINA HOSPITAL SICHUAN UNIV
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