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Small-molecular fluorescent probe for SNAP protein labeling as well as synthesis method and application of small-molecular fluorescent probe

A technology of fluorescent probe and synthesis method, applied in the field of biological analysis and detection, can solve the problems of single fluorescence spectrum, slow response, low sensitivity, etc., and achieve the effects of simple synthesis route, mild reaction conditions, and simple and convenient post-processing

Active Publication Date: 2018-05-25
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the fluorescence spectrum of GFP is relatively simple, the luminescence is oxygen-dependent, and there are shortcomings such as slow response and low sensitivity, which make GFP have certain limitations in the research of biophysics and mechanism.

Method used

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  • Small-molecular fluorescent probe for SNAP protein labeling as well as synthesis method and application of small-molecular fluorescent probe
  • Small-molecular fluorescent probe for SNAP protein labeling as well as synthesis method and application of small-molecular fluorescent probe
  • Small-molecular fluorescent probe for SNAP protein labeling as well as synthesis method and application of small-molecular fluorescent probe

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1: Synthesis of small molecule fluorescent probes for protein labeling

[0040] (1) Synthesis of intermediates:

[0041]

[0042] 4-Bromo-1,8-naphthalene anhydride (2.77 g, 10 mmol) was dissolved in 25 mL of absolute ethanol, and 4-aminomethylbenzyl alcohol (1.37 g, 10 mmol) was added to the reaction solution. The reaction solution was heated to reflux. After 6 hours, ethanol was removed under reduced pressure, and column chromatography (dichloromethane: methanol = 100:1) gave 3.08 g of white solid (intermediate compound 1), with a yield of 78%.

[0043]

[0044] Compound 1 (200 mg, 0.51 mmol) was dissolved in 5 mL of ethylene glycol methyl ether, and 100 microliters of 70% ethylamine aqueous solution was added thereto under nitrogen protection. The reaction solution was slowly heated to 120° C. and stirred for 2 days. Ethylene glycol methyl ether was removed under reduced pressure, and the residue was separated through a silica gel column (dichlorometh...

Embodiment 2

[0048] Example 2: Fluorescence changes after fluorescent probe reacts with SNAP

[0049] The probe was dissolved in DMSO solution to prepare 2 mM stock solution, and 1 μM probe and 5 μM SNAP were respectively prepared in 4 mL of 20 mM PBS solution with pH=7.4. A 1 μM probe was additionally prepared with 5 μM SNAP. Stir at 37 °C for 1 h. Measure the fluorescence to get Figure 5 .

[0050] Figure 5 In the presence of only the probe (1μM) showed weak fluorescence. When the probe reacts with SNAP, the fluorescence is significantly enhanced, several times stronger, and the emission wavelength is about 530nm.

Embodiment 3

[0051] Embodiment 3: the kinetics of fluorescent probe and SNAP reaction

[0052] Add 1μM probe to 4mL, 2μM SNAP solution, measure the fluorescence change at 530nm wavelength (excitation wavelength is 460nm) to get Image 6 , the results showed that the fluorescence increased rapidly after adding the probe, indicating that the probe could specifically recognize and react rapidly with SNAP.

[0053] Fluorescence is significantly increased by about 30 times, through which the interference of background light can be excluded.

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Abstract

The invention discloses a small-molecular fluorescent probe for SNAP protein labeling as well as a synthesis method and an application of the small-molecular fluorescent probe. The probe molecule synthesis route is simple, the reaction conditions are mild, aftertreatment is simple and convenient, and the probe has specific protein recognition capability and higher response speed. In a water solution, the probe has slight fluorescence signals, is specifically bound with SNAP protein and has the fluorescence enhanced by about 30 times, and the emission wavelength of the probe has blue shift about 530 nm after binding. Compared with existing SNAP fluorescent probes, the probe has high selectivity, can specifically recognize SNAP in a biosystem in the complicated environment and has very important application value in the biological and medical fields.

Description

technical field [0001] The invention belongs to the field of biological analysis and detection, and in particular relates to a small molecule fluorescent probe for SNAP protein labeling and its synthesis method and application. Background technique [0002] Proteins are the basic components of cells and the embodiment of life functions. Studying the structure of proteins, the movement characteristics of functional machinery in cells, interactions and chemical microenvironments, etc., is crucial for understanding the complex life processes of cells. In recent years, the continuous development of protein-specific labeling technology, especially gene-encoded fusion tag technology, has met the needs of these studies. The discovery and medical use of green fluorescent protein (GFP) is a milestone in the development of protein-specific labeling methods in living cells. Visual monitoring. However, the fluorescence spectrum of GFP is relatively simple, the luminescence is oxygen-...

Claims

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Application Information

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IPC IPC(8): C07D473/18C09K11/06G01N21/64
CPCC07D473/18C09K11/06C09K2211/1029C09K2211/1044G01N21/6486
Inventor 徐兆超冷双乔庆龙苗露
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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