Fluorescent probe AH and preparation 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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  • 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 AH and preparation and application thereof
  • Fluorescent probe AH and preparation and application thereof
  • Fluorescent probe AH and preparation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 1 (synthesis of probe):

[0033] Such as figure 1 As shown, the synthesis of AH: a 50mL two-necked flask was replaced with vacuum / nitrogen three times, 20mL of dry pyridine and 2mL of dry phosphorus oxychloride were added, HBT0.82g 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 1.2 g of 2',3'-O-Isopropylideneadenosine (2',3'-O- isopropylidene adenosine), stirred, and reacted at room temperature for 10 h, and after 10 h, heated and evaporated to dryness at 70° C. under vacuum. Add a small amount of water, stir for 1h, and spin dry with n-butanol azeotropically. The resulting product was purified by silica gel column chromatography (using dichloromethane and methanol as elue...

Embodiment 2

[0035] Such as Figure 4 Shown, (UV-Vis absorption spectrum (a), fluorescence excitation spectrum (b), emission spectrum (c) of fluorescent probe AH and fluorophore HBT aqueous solution):

[0036]Dissolve AH in 100mM PB buffer to make a 20uM solution. Take 3mL of the solution and add it to a 1cmⅹ1cmⅹ4cm cuvette, 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 AH and alkaline phosphatase )); The results show that the maximum excitation wavelength of the probe AH / HBT is 318nm / 330nm. The maximum emission wavelength of the probe AH is 370nm, and the fluorophore HBT has two emission peaks at 460nm / 512nm with different concentrations in aqueous solution.

Embodiment 3

[0037] Embodiment 3 (AH is to the selectivity of alkaline phosphatase):

[0038] Alkaline phosphatase, acid phosphatase, adenylyl cyclase, 3'-5' phosphodiesterase (four kinds of enzyme concentrations are all 10uM) aqueous solution, the four 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 AH (20mM) in DMSO (an important polar aprotic solvent). Add 2ulAH (20mM) and 2ul enzyme to 196ul of 100mM PB buffer for each reaction, use a full-wavelength scanning multifunctional reader and a 96-well microplate plate for measurement, and the final concentrations of the probe and enzyme are 20uM and 100nM, respectively. Measure the fluorescence emission spectrum of the working solution, λex=330nm, the grating width is 5nm, λem=512nm.

[0039] The experimental results of AH to alkaline phosphatase selectivity are as follows: Figure 5 As shown, the ordinate represents the fluores...

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Abstract

The invention provides a fluorescent probe AH and preparation and application thereof and specifically provides a fluorescent probe capable of being used for selectively detecting intracellular alkaline phosphatase. The main synthesis method comprises the step of introducing 2',2-hydroxyphenyl benzothiazole (HBT) to an alkaline phosphatase similar substrate micromolecular 5'-adenosine monophosphate (AMP) so as to produce a compound with a structure represented by a formula defined in the description. HBT is produced under the action of the alkaline phosphatase, the selective detection on the alkaline phosphatase is achieved by using difference of a fluorescent property before and after a reaction, and a coloration action on the alkaline phosphatase of a zebra fish body can be exerted.

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 adenosine (AMP) in the cell, and use the fluorescence difference between the reactant and the product after reacting with alkaline phosphatase It achieves selective detection of alkaline phosphatase in living cells, and has achieved good application in live imaging of zebrafish. 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 relat...

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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