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Glycosylnaphthalimide-containing fluorescent probe and application thereof

A technology based on naphthalimide and naphthalimide, applied in the field of novel glycosyl naphthalimide derivatives, which can solve the problems of low solubility, measurement results easily affected by the environment, and the inability to visually evaluate enzyme activity, etc. problem, to achieve the effect of high research value, excellent optical and physical properties

Active Publication Date: 2018-12-21
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, the glycosylated p-nitrophenol substrate is determined by ultraviolet absorption method, which has weak sensitivity, narrow detection range (0-0.5), high substrate concentration (500μM), compound color interferes with reaction determination, and is easy to decompose at room temperature ; After enzymatic hydrolysis of the glycosylated 4-methylumbelliferone substrate, the solubility is low, and saturated sodium carbonate needs to be added to amplify the fluorescence signal. live, which leads to certain limitations in their application

Method used

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  • Glycosylnaphthalimide-containing fluorescent probe and application thereof
  • Glycosylnaphthalimide-containing fluorescent probe and application thereof
  • Glycosylnaphthalimide-containing fluorescent probe and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0024] Embodiment 1, R in formula I 1 =H,R 2 =NHAc,R 3 =H,R 4 =OH, the compound preparation of n=4, concrete steps are as follows:

[0025]

[0026] (1) Add N-acetylglucosamine (10g, 40.3mmol) and acetyl chloride (40ml, 186.8mmol) into a 250mL round-bottomed flask, react at room temperature for 48h, and monitor the completion of the reaction by TLC. The reaction solution was added to 500mL ice water, extracted with 200mL DCM to obtain an organic layer, and then saturated NaHCO 3 Aqueous solution (100mL), water (100mL) washing, anhydrous Na 2 SO 4 After drying and concentration, 2-acetylamino-3,4,6-tri-O-acetyl-2-deoxy-α-D-glucopyranoside chloride was obtained, which was directly carried out to the next reaction without further purification.

[0027] Add 4-hydroxybenzaldehyde (2.5g, 20.5mmol) in the reaction bottle of 500mL, Na 2 CO 3 (2.2g, 20.5mmol), tetrabutylammonium bromide (6.6g, 20.5mmol), 100mL DCM and 150mL H 2 O, 2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-α-D-...

Embodiment 3

[0038] The effect of embodiment 3 compound I and Hex and the relation of time

[0039]On the basis of Example 2, we studied the relationship between the action of Compound I and Hex and time. Add 160 μL of 200 μM compound I and 1440 μL of PBS buffer solution containing Hex to the cuvette in sequence, and measure the change of the fluorescence spectrum of the reaction in 0-80 minutes at room temperature. We found that the fluorescence intensity with a wavelength of 438nm has been decreasing in the first 10 minutes, and a new fluorescence emission peak was added at 540nm, and the fluorescence emission peak at 540nm increased rapidly in the last 70 minutes, far greater than the fluorescence intensity at 438nm. It shows that the probe and the enzyme have a good response over time.

Embodiment 4

[0040] The kinetic parameter of embodiment 4 compound I and Hex

[0041] Further, we explored the kinetic parameters of compound I and Hex. First configure a series of concentrations of Compound I solutions in a cuvette, measure the relationship between its fluorescence intensity and concentration, and obtain y=166.95x-1.5388 (R 2 =0.9994), and then configure compound I solutions with different concentrations containing Hex, incubate at 37° C. for 10 minutes, and measure the fluorescence value. Get the linear curve y=16.18x+0.40677(R 2 =0.9995), and then get K m =39.76nm( Figure 4 ), indicating that compound I has a good affinity with the enzyme.

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Abstract

The invention discloses a glycosylnaphthalimide-containing fluorescent probe and application thereof in screening and cell imaging of a glycosidase inhibitor. The structure formula of the glycosylnaphthalimide-containing fluorescent probe is shown as formula I, a glycosyl part is beta-glucose, beta-N-acetylglucosamine, beta-galactose, beta-N-acetyllactosamine, and n is equal to 1 to 6. The glycosylnaphthalimide-containing fluorescent probe designed and synthesized in the invention has good water solubility and fluorescent performance, can be applied to the molecular screening of the glycosidase inhibitor, and can rapidly screen the inhibitor molecules with the inhibition rate greater than 70 percent by virtue of eyes; and the probe can be further used for the fluorescent imaging of the glycosidase in living cells.

Description

technical field [0001] The invention relates to a novel glycosylnaphthalimide derivative, its preparation method and its application as a fluorescent probe in the screening of glycosidase inhibitor molecules and cell imaging. Background technique [0002] Glycosidase is a glycosyl hydrolase that can hydrolyze glycosidic bonds between glycogens or between sugars and non-sugars. It is one of the important enzymes to maintain the basic functions of organisms. It is widely distributed in nature and exists in almost all types of animals, plants and microorganisms. At the same time, glycosidase plays an important role in biotechnology, human diseases, control of fungi and insects, and is a good target enzyme. Its small-molecule inhibitor not only acts as a diabetes treatment and insecticide, but is also a useful biological tool. [0003] Glycosylated p-nitrophenol substrates and 4-methylumbelliferone substrates are commonly used in the detection of glycosidases, but both substra...

Claims

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Application Information

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IPC IPC(8): C07H1/00C07H15/203C09K11/06G01N21/64
CPCC07H1/00C07H15/203C09K11/06C09K2211/1029C09K2211/1088G01N21/6428Y02P20/55
Inventor 张建军董利利沈生强路慧哲金淑惠王道全
Owner CHINA AGRI UNIV
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