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Cell membrane fluorescent probe based on SNAP-tag technology as well as preparation and application of the cell membrane fluorescent probe

A fluorescent probe and cell membrane technology, applied in the field of fluorescence imaging, can solve the problems of limited application and inability to artificially control cell membrane permeability, and achieve the effects of simple operation, excellent photostability, and high impermeability.

Active Publication Date: 2020-06-26
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, this method also has some shortcomings: in order to achieve higher signal intensity and improve signal-to-noise ratio, sufficient washing is required to remove non-specific staining, which also limits its application in real-time imaging of live cells
However, the current fluorescent probes for cell membrane imaging are mainly inserted into the cell membrane with molecular amphiphilicity (hydrophilic and lipophilic), and the permeability of the cell membrane cannot be artificially controlled for long-term imaging observation.

Method used

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  • Cell membrane fluorescent probe based on SNAP-tag technology as well as preparation and application of the cell membrane fluorescent probe
  • Cell membrane fluorescent probe based on SNAP-tag technology as well as preparation and application of the cell membrane fluorescent probe
  • Cell membrane fluorescent probe based on SNAP-tag technology as well as preparation and application of the cell membrane fluorescent probe

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Synthesis of small molecule fluorescent probe Rho6G-SNAP for cell membrane imaging.

[0036] Synthesis of intermediate rhodamine 6G-carboxylic acid:

[0037]

[0038] 1 g of rhodamine 6G was dissolved in 30 mL of ethanol, 300 mg of sodium hydroxide was dissolved in 5 mL of water, and slowly added to the reaction mixture, heated and stirred to reflux. After 6 hours, spot the plate for monitoring, and stop the reaction after the reaction raw materials are completely reacted. The solvent was removed by rotary evaporation, separated on a silica gel column, using dichloromethane:methanol=5:1 (volume ratio) as the eluent, and the solvent was removed under reduced pressure to obtain 700 mg of a purple solid with a yield of 80%. Its nuclear magnetic spectrum hydrogen spectrum data are as follows:

[0039] 1 H NMR (400MHz, CD3OD), δ8.22(d, J=6.4Hz, 1H), 7.80(m, 2H), 7.36(d, J=6.8Hz, 1H), 6.89(d, J=10.4Hz, 4H), 3.50(q, J=7.2Hz, 4H), 2.15(s, 6H), 1.35(t, J=6.8Hz, 6H).

[0...

Embodiment 2

[0057] Synthesis of small molecule fluorescent probe Rho6G-SNAP for cell membrane imaging.

[0058] Synthesis of intermediate rhodamine 6G-carboxylic acid:

[0059]

[0060] 1 g of rhodamine 6G was dissolved in 20 mL of ethanol, 200 mg of sodium hydroxide was dissolved in 5 mL of water, and slowly added to the reaction mixture, heated and stirred to reflux. After 5 hours, spot the plate for monitoring, and stop the reaction after the reaction raw materials are completely reacted. The solvent was removed by rotary evaporation, separated on a silica gel column, using dichloromethane:methanol=5:1 (volume ratio) as the eluent, and the solvent was removed under reduced pressure to obtain 600 mg of a purple solid with a yield of 69%.

[0061] Synthesis of intermediate rhodamine 6G-succinimide carboxylate:

[0062]

[0063] Weigh 500mg of rhodamine 6G-carboxylic acid and 300mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride in 20mL of dichloromethane, then add ...

Embodiment 3

[0073] Synthesis of small molecule fluorescent probe Rho6G-SNAP for cell membrane imaging.

[0074] Synthesis of intermediate rhodamine 6G-carboxylic acid:

[0075]

[0076] 1 g of rhodamine 6G was dissolved in 40 mL of ethanol, 400 mg of sodium hydroxide was dissolved in 5 mL of water, and then slowly added to the reaction mixture, heated and stirred to reflux. After 6 hours, spot the plate for monitoring, and stop the reaction after the reaction raw materials are completely reacted. The solvent was removed by rotary evaporation, separated on a silica gel column, using dichloromethane:methanol=5:1 (volume ratio) as the eluent, and the solvent was removed under reduced pressure to obtain 672 mg of a purple solid with a yield of 77%.

[0077] Synthesis of intermediate rhodamine 6G-succinimide carboxylate:

[0078]

[0079] Weigh 500mg of rhodamine 6G-carboxylic acid and 400mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride in 30mL of dichloromethane, then...

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Abstract

The invention provides a cell membrane fluorescent probe based on SNAP-tag technology and preparation and application thereof. The fluorescent probe is based on rhodamine 6G micromolecular fluorescentdye, is connected with an SNAP protein label BG group, and has a structural formula as shown in (1). The fluorescent probe provided by the invention has the advantages of excellent light stability and fluorescence brightness of a small molecular fluorescent dye, and is different from the existing method for identifying a cell membrane by utilizing amphipathicity (hydrophilicity and lipophilicity)of the small molecular fluorescent dye, the probe can be specifically covalently bonded with SNAP protein of the cell membrane, the bonding is more stable, and long-time imaging observation can be realized; synthesis steps are simple, raw materials are cheap and easy to obtain, and the cell membrane fluorescent probe has a huge application prospect in the field of cell membrane imaging.

Description

technical field [0001] The invention belongs to the field of fluorescence imaging, and in particular relates to a cell membrane fluorescent probe based on SNAP-tag technology and its preparation and application. Background technique [0002] The SNAP-tag protein labeling technology based on organic small molecule fluorescent dyes has unique advantages: the reaction between protein labels and substrates is fast and specific; the binding protein labeling through covalent bonds has good stability, even in vitro analysis Stable labeling is still possible under the denaturing conditions of SDS-PAGE; the label-specific substrate BG group is combined with functional dyes through a modular design, which has high flexibility; it is easy to derivate a variety of fluorescent dyes, which is versatile and can meet various requirements. The needs of this kind of fluorescence imaging research. At present, SNAP-tag has been widely used in the research of intracellular protein labeling, in ...

Claims

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Application Information

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IPC IPC(8): C07D473/18C09K11/06G01N21/64
CPCC07D473/18C09K11/06G01N21/6486A61K49/0021C09K2211/1007C09K2211/1044C09K2211/1088Y02P20/55
Inventor 徐兆超周伟乔庆龙
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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