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Novel BODIPY fluorescent probe for detecting carboxylesterase 1, preparation method and application thereof

A fluorescent probe, carboxylesterase technology, applied in fluorescence/phosphorescence, chemical instruments and methods, compounds containing periodic table Group 3/13 elements, etc., can solve background fluorescence interference, poor tissue penetration, emission Short wavelength and other problems, to achieve the effects of easy large-scale production, large molar absorption coefficient, and narrow fluorescence spectrum peaks

Active Publication Date: 2021-06-01
NANJING NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] Purpose of the invention: To solve the problem of short emission wavelength, unavoidable background fluorescence interference, poor water solubility, poor tissue penetration and low sensitivity of the existing probe technology, which greatly limits the application in complex biological systems

Method used

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  • Novel BODIPY fluorescent probe for detecting carboxylesterase 1, preparation method and application thereof
  • Novel BODIPY fluorescent probe for detecting carboxylesterase 1, preparation method and application thereof
  • Novel BODIPY fluorescent probe for detecting carboxylesterase 1, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Preparation of a Novel BODIPY Fluorescent Probe for Detection of Carboxylesterase 1

[0032] Its specific preparation synthetic route is as follows figure 1 Shown:

[0033] Synthesis of Intermediate Compound 1

[0034] Weigh p-hydroxybenzaldehyde (1.00g) into a round bottom flask, dissolve it with dichloromethane (150mL), add 2,4-dimethylpyrrole (1.69mL), and start stirring at a speed of 500rpm. Add 6 drops of trifluoroacetic acid as a catalyst to catalyze the reaction, react for 12 hours, add chlorobenzoquinone (2.01 g) as an oxidant, and react for 5 hours. Triethylamine (10 mL) was added dropwise using a constant pressure dropping funnel, and boron trifluoride diethyl ether (10 mL) was added dropwise after half an hour. React overnight. After the reaction was completed, extract with water, take the organic layer, dry with anhydrous sodium sulfate, then remove the solvent by rotary evaporation, and further purify the compound by column chromatography (PE:EA=2:1) ​​...

Embodiment 2

[0041] Determination of maximum absorption wavelength and maximum emission wavelength of a novel BODIPY fluorescent probe for detection of carboxylesterase 1

[0042]Prepare a 20mM probe stock solution with DMSO as a solvent, dilute to obtain a probe stock solution with a concentration of 2mM, and prepare a mixture of 2.500mL PBS (100mM, pH=7.4), 2.475mL acetonitrile, and 0.025mL probe in a centrifuge tube. The concentration of the probe in the mixed solution was 10 μM, and a control was set. The sample was placed in an ultraviolet spectrophotometer (UV-6100a) for measurement to determine the maximum absorption wavelength of the probe. After obtaining the maximum absorption wavelength of the probe, the sample was placed in a fluorescence spectrophotometer (F97Pro, Shanghai Lenslight) to measure the maximum emission wavelength. The result is as figure 2 As shown, the maximum absorption wavelength is 585nm, and the maximum emission wavelength is 624nm.

Embodiment 3

[0044] Selectivity Test of Carboxylesterase 1 (CES1) with Novel BODIPY Fluorescent Probe for Detecting Carboxylesterase 1

[0045] To determine the selectivity of the probes for carboxylesterase 1, the probes were added to different enzyme solutions. Prepare a mixed solution with a total volume of 100 μL, containing 98 μL PBS buffer solution, 1 μL probe stock solution (2 mM) and 1 μL CES1 (1 mg / mL). The mixture was incubated at 37° C. for 60 min, and 100 μL of acetonitrile was added to quench the reaction. The final concentrations of probe and CES1 were 10 μM and 5 μg / mL, respectively. Replacement of CES1 with lysozyme, trypsin, papain, chymotrypsin, acetylcholinesterase, serum protease, lysozyme, lipase, pepsin, helicase, proteinase K, and bovine serum albumin under unchanged conditions , to obtain different enzyme solutions, using a microplate reader (Synergy H 1 ) to measure the fluorescence intensity. Such as image 3 As shown, the fluorescence intensity of carboxyles...

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Abstract

The invention discloses a novel BODIPY fluorescent probe for detecting carboxylesterase 1, a preparation method and application thereof. According to the invention, the small molecular fluorescent probe is prepared from benzoyl chloride and BODIPY through an organic synthesis reaction; the novel BODIPY fluorescent probe is designed on the basis of a luminous parent nucleus of BODIPY, wherein N-ethyl carbazole-3-formyl is introduced to 3-position active methyl through a Knoevenagel condensation reaction to extend the conjugated structure of BODIPY fluorophore so as to make the emission wavelength be subjected to red shift to a near-infrared region, and then benzoyl chloride is used for replacing the 8-site to form an ester bond, so that the carboxylesterase 1 can be recognized; and the novel BODIPY fluorescent probe disclosed by the invention has relatively good biocompatibility, has excellent performances of high fluorescence quantum yield, large molar absorption coefficient, narrow fluorescence spectrum peak, high sensitivity, good light stability and the like, and can be used for detecting carboxylesterase 1 (CES1) and pesticide residues.

Description

technical field [0001] The invention belongs to the field of biological analysis and detection, and in particular relates to a novel BODIPY fluorescent probe for detecting carboxylesterase 1, a preparation method and application thereof. Background technique [0002] Carboxylesterase 1 (CES1) is a key broad-spectrum serine hydrolase that catalyzes the hydrolysis of a wide range of endogenous and exogenous substances, such as lipids, amides, thioesters, and carbamates. In addition, carboxylesterase 1 is considered to be an important drug target and prodrug activator based on the irreversible binding of drugs to the active site of the enzyme, involved in drug metabolism and pesticide detoxification. CES1 is mainly expressed in the liver. Studies have shown that abnormal levels of CES1 in vivo can lead to diseases such as atherosclerosis, obesity, mesenchymal cell tumors, hyperlipidemia, and liver cancer. Therefore, it is necessary to develop specialized methods to monitor the...

Claims

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Application Information

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IPC IPC(8): C07F5/02C09K11/06G01N21/64
CPCC07F5/022C09K11/06G01N21/6428C09K2211/1029G01N2021/6439
Inventor 黄和沈宝星张幸李昺之王宜峰马成功
Owner NANJING NORMAL UNIVERSITY
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