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Fluorescent probe GH and preparation method and application thereof

A fluorescent probe and fluorescence technology, applied in the field of fluorescent probes, can solve the problem of few fluorescent probes

Active Publication Date: 2016-04-27
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The most widely used detection method is to detect the absorption spectrum of the dephosphorylated product of pnpp (p-nitrophenol phosphate) at 405 nm. Although there are some new developments in the detection of fluorescent probes for alkaline phosphatase, fluorescent probes with good selectivity needles are still few
In particular, alkaline phosphatase, acid phosphatase, phosphodiesterase, and adenylate cyclase have many substrate crossover phenomena for dephosphorylation, and it is very challenging to develop selective probes for them.

Method used

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  • Fluorescent probe GH and preparation method and application thereof
  • Fluorescent probe GH and preparation method and application thereof
  • Fluorescent probe GH and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Embodiment 1 (synthesis of probe):

[0032] Such as figure 1 As shown, the synthesis of GH: a 50mL two-necked flask was replaced with vacuum / nitrogen three times, 20mL of dry pyridine and 2mL of dry phosphorus oxychloride were added, HBT1.81g was added slowly, and stirred at room temperature for 1h. After 1 h, it was heated and evaporated to dryness at 70°C under vacuum to obtain a light green solid. Stop heating and wait for cooling, reconnect the vacuum / nitrogen replacement three times, add 20 mL of pyridine again, vortex to dissolve all the light green precipitate, add 2.58 g of 2'3'-O-isopropyguanosine (2',3'-O-isopropyguanosine (2',3'-O-isopropyguanosine) Propylene guanosine), stirred, and reacted at room temperature for 10 h. After 10 hours, heat and spin dry at 70°C under vacuum. Add 20 mL of distilled water, stir for 1 h, filter with suction, and dry by infrared to obtain 2.05 g of solid. The obtained solid was washed three times with methanol (50 mL each ti...

Embodiment 2

[0034] Such as Figure 4 Shown, (UV-Vis absorption spectrum (a), fluorescence excitation spectrum (b), emission spectrum (c) of fluorescent probe GH and fluorophore HBT aqueous solution):

[0035] Dissolve GH in 100 mM PB buffer to make a 20 μM solution. Take 3mL of the solution and add it to a cuvette of 1cmⅹ1cmⅹ4cm, measure the UV-visible absorption spectrum (a) of the working solution, the fluorescence excitation spectrum (b) and emission spectrum (c) of the fluorophore HBT aqueous solution produced after the action of GH and alkaline phosphatase )); The results show that the maximum excitation wavelength of the probe GH / HBT is 318nm / 330nm. The maximum emission wavelength of the probe GH is 370nm, and the fluorophore HBT has two emission peaks at 460nm / 512nm with different concentrations in aqueous solution.

Embodiment 3

[0036]Embodiment 3 (GH is to the selectivity of alkaline phosphatase):

[0037] Alkaline phosphatase, acid phosphatase, adenylyl cyclase, 3'-5' phosphodiesterase (four kinds of enzyme concentrations are all 10μM) aqueous solution, the four kinds of enzymes are divided into several parts, each part The same amount was stored at -20°C. Thaw slowly on ice before use and use each aliquot only once. Configure GH (20 mM) in DMSO (an important polar aprotic solvent). Add 2 μLGH (20 mM) and 2 μL enzyme to 196 μL of 100 mM PB buffer for each reaction, and use a full-wavelength scanning multifunctional reader and a 96-well microplate plate for measurement. The final concentrations of the probe and enzyme are 20 μM and 100 nM, respectively. Measure the fluorescence emission spectrum of the working solution, λex=330nm, the grating width is 5nm, λem=512nm.

[0038] The results of the selectivity experiment of GH to alkaline phosphatase are as follows: Figure 5 As shown, the ordinate r...

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Abstract

The invention discloses a fluorescent probe GH and a preparation method and application thereof. The fluorescent probe can be used for selectively detecting intracellular alkaline phosphatase (ALP). The main synthetic method includes the steps that 2'2-(hydroxy-phenyl)benzothiazole (HBT) is introduced to micromolecule 5'guanosine monophosphate (GMP) of an analog substrate of the alkaline phosphatase, so that a compound of the structure as shown in the specification is formed. Under the action of the alkaline phosphatase, the HBT is generated. By the aid of the difference of fluorescent properties before and after reacting, selective detection of the alkaline phosphatase can be achieved.

Description

technical field [0001] The invention relates to the field of fluorescent probes, in particular to a fluorescent probe that can be used for selectively detecting alkaline phosphatase. Introduce the fluorophore 2'2 hydroxyphenylbenzothiazole (HBT) on the common small molecule 5' phosphoribosine guanosine (GMP) in the cell, and use the fluorescence difference between the reactant and the product after reacting with alkaline phosphatase Achieve selective detection of alkaline phosphatase in living cells. Background technique [0002] Alkaline phosphatase (ALP, ALKP, ALPase, AlkPhos) (EC3.1.3.1) is widely distributed in various organs of the human body, among which the liver is the most, followed by the kidney, bone, intestine, and placenta and other tissues. ALP can mediate the dephosphorylation of biological macromolecules such as proteins, nucleic acids, and alkaloids. This dephosphorylation is closely related to the phosphorylation of kinases, and is a necessary regulatory ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C09K11/06C07H19/207C07H1/00G01N33/52G01N21/64
Inventor 贾燕韩克利李鹏
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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