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Carboxylesterase fluorescent probe as well as preparation method and application thereof

A carboxylesterase and fluorescent probe technology, applied in the field of carboxylesterase fluorescent probes and their preparation, can solve the problems of shallow tissue penetration depth and imaging depth, short excitation wavelength, cell photodamage, etc. Penetration depth and imaging depth, short response time, light damage reduction effect

Active Publication Date: 2018-06-15
GANNAN NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Most of the fluorescent probes reported in the literature for the detection of carboxylesterase are sensitive to pH, have poor stability in cell culture medium, poor cell penetration ability, and cannot be used for imaging in living cells or tissues.
In addition, the existing carboxylesterase fluorescent probes that can be used for cell imaging require short excitation wavelengths (<500nm), and short-wavelength excitation light can easily cause photodamage to cells, generate reactive oxygen species, and cause tissue damage. Shallow penetration depth and imaging depth limit its application in live-cell and intravital imaging

Method used

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  • Carboxylesterase fluorescent probe as well as preparation method and application thereof
  • Carboxylesterase fluorescent probe as well as preparation method and application thereof
  • Carboxylesterase fluorescent probe as well as preparation method and application thereof

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preparation example Construction

[0041] The present invention provides the preparation method of carboxylesterase fluorescent probe described in above-mentioned technical scheme, comprises the following steps:

[0042] (1) A compound having a structure shown in formula II, acetyl chloride, an alkaline reagent and an organic solvent are mixed for acetylation reaction to obtain a compound having a structure shown in formula III;

[0043] (2) Mix the compound having the structure shown in formula III with 1,2,3,3-tetramethyl-3H-indole iodide and an organic solvent, and carry out condensation reaction under a protective atmosphere to obtain formula I The carboxylesterase fluorescent probe of the shown structure;

[0044]

[0045] In formula II and formula III, R 1 , R 2 , R 3 and R 4 independently hydrogen, amino, alkyl, alkoxy, alkylamino, aryl, aryloxy or arylamino.

[0046]In the present invention, the compound having the structure shown in formula II, acetyl chloride, alkaline reagent and organic solv...

Embodiment 1

[0061] Carboxylesterase fluorescent probes were prepared according to the following reaction scheme:

[0062]

[0063] (1) Compound 1 (172.2mg, 1mmol), triethylamine (121.4mg, 1.2mmol) and dichloromethane (anhydrous grade) were mixed, and acetyl chloride (94.2mg, 1.2mmol) was mixed at 0°C within 2min Drop into the obtained mixed solution, stir at room temperature to carry out the acetylation reaction, monitor the reaction progress with a TCL plate until the compound 1 disappears completely; wash the obtained acetylated material with saturated saline for 3 times, and dry the obtained organic phase with anhydrous sodium sulfate , was concentrated to a solid by rotary evaporation, and then recrystallized with a mixture of ethyl acetate and petroleum ether (the volume ratio of ethyl acetate and petroleum ether was 1:40) to obtain a white solid.

[0064] The calculated yield is 92%;

[0065] The resulting white solid is characterized, and the specific data are as follows:

[0...

Embodiment 2

[0074] Carboxylesterase probe prepared in embodiment 1 is carried out performance test, specifically as follows:

[0075] Stability determination of the carboxylesterase probe: 20 μL of HCyNAc in DMSO (1 mM) was added to 2 mL of PBS buffer solution (pH 7.4) to obtain a 10 μM HCyNAc solution, and the resulting solution was incubated at 37° C. for different times (10 , 15, 20, 25, 30, 40, 50, 60 min), measure the variation of the fluorescence intensity of the solution with the incubation time. figure 1It is the change diagram of the fluorescence intensity of HCyNAc incubated in PBS buffer solution at 37°C for different time, from figure 1 It can be seen that the fluorescence intensity remains basically unchanged, indicating that the stability of the carboxylesterase probe is better.

[0076] Sensitivity determination of carboxylate enzyme probe to pH: Add 20 μL HCyNAc DMSO solution (1 mM) in 2 mL PBS buffer solution (pH values ​​are respectively 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8....

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Abstract

The invention provides a carboxylesterase fluorescent probe of a structure of formula I. The carboxylesterase fluorescent probe provided by the invention is a hemicyanine compound with a naphthalene nuclear two-photon absorption unit and a D-pi-A structure, has the two-photon absorption and emission properties, and can be applied to detection on intracellular carboxylesterase. Experiment results of the embodiment show that the carboxylesterase fluorescent probe provided by the invention is capable of prolonging excitation wavelengths (800nm) of carboxylesterase in cells and tissue, reducing optical damage to cells and tissue and increasing tissue penetration depths and imaging depths; meanwhile, when being adopted to detect carboxylesterase, the carboxylesterase fluorescent probe providedby the invention is good in stability, short in response time (7 minutes), no sensitive to pH values, good in specificity for intracellular carboxylesterase imaging, and capable of detecting carboxylesterase in living cells under a two-photon excitation condition.

Description

technical field [0001] The invention relates to the technical field of biochemical materials, in particular to a carboxylesterase fluorescent probe and its preparation method and application. Background technique [0002] Mammalian carboxylesterase (Carboxylesterase, CaE, E.C.3.1.1.1), also known as aliphatic esterase, is widely distributed in mammalian tissues and organs, and its active center contains serine residues, which can effectively catalyze ester bonds, amides Hydrolysis of endogenous and exogenous substances of bonds and thioester bonds. The main function of mammalian carboxylesterases may be to participate in lipid transport and metabolism, catalyzing the hydrolysis of some endogenous compounds such as short-chain and long-chain acylglycerols, long-chain acylcarnitines, and long-chain acyl CoA esters ; and participate in signal transmembrane transmission and maintain biomembrane integrity; catalyze drugs (including prodrugs) to generate corresponding free acids,...

Claims

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Application Information

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IPC IPC(8): C07D209/12C09K11/06G01N33/533G01N33/573G01N33/574
CPCC07D209/12C09K11/06C09K2211/1011C09K2211/1029G01N33/533G01N33/573G01N33/57411G01N33/57496G01N2333/918G01N2800/36
Inventor 王建国姜国玉陈青青李勋范小林
Owner GANNAN NORMAL UNIV
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