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Glycoprotein-binding membrane dye based on pyrazolone, its preparation method and application

A pyrazolone-binding technology, applied in the field of cell membrane staining, can solve the problems of short retention time on the membrane, high required concentration, and large difference in specific protein expression, achieving excellent staining effect and low staining concentration limit , a wide range of effects

Active Publication Date: 2022-04-05
DALIAN UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, the fluorescent dyes used for in vitro cell membrane labeling on the market are mainly DiO, DiI and other dyes embedded in phospholipid bilayer structures. The structure is easy to aggregate in water)
Excessive variegation of this type of dye and variegation into the cells will seriously interfere with the continuous observation of the membrane structure when used, and it can only ensure clear imaging within a short period of time (not exceeding 1 hour) after incubation and incubation. Obviously cannot meet more needs
However, the limited use of target-based membrane dyes is mainly due to their high cost. The membrane localization achieved by such dyes often requires specific recognition and binding of proteins on the membrane, and this type of dye requires target macromolecules as carriers. The cost of synthesis is high, and the expression of specific proteins varies greatly between different cells, so the application of this type of dye in vitro is limited

Method used

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  • Glycoprotein-binding membrane dye based on pyrazolone, its preparation method and application
  • Glycoprotein-binding membrane dye based on pyrazolone, its preparation method and application
  • Glycoprotein-binding membrane dye based on pyrazolone, its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] The preparation method of the fast-binding cell membrane surface glycoprotein based on the pyrazolone structure in this embodiment for long-retention cell membrane tracking imaging and as a membrane marker to distinguish co-cultured cells includes the following steps:

[0069] (1) Synthesize the dye parent molecule according to the existing method:

[0070]

[0071] (2) Synthetic intermediate 1: ethyl 2-(3-methyl-5-keto-4,5-dihydro-1H-pyrazoline) acetate

[0072] The synthetic route is as follows:

[0073]

[0074] Dissolve ethyl hydrazinoacetate hydrochloride (10mmol) in ethanol, add sodium acetate (10mmol) to it, stir for 30min, then add ethyl acetoacetate (10mmol) to it, under reflux, stir and react for 18h and then react Finish. After the solvent was removed by distillation under reduced pressure, ethyl acetate (100 mL) was used to redissolve and 100 mL of saturated brine was added thereto, the organic layer was separated, and extraction was continued with e...

Embodiment 2

[0085] Same as Example 1, only the structure of the parent dye is different, and the structure of the intermediate 2 has not changed, and its synthetic route is as follows:

[0086]

[0087] Dissolve intermediate 2 (2mmol) in 5mL DMF, add HATU (2mmol) and TMP (3mmol) to it under stirring at low temperature, add dropwise the DMF solution of dye precursor 2 (1mmol) to it half an hour later, stop after 4h of reaction reaction. After the solvent was removed by distillation under reduced pressure, the crude product was purified by silica gel chromatography, the eluent was dichloromethane:methanol=6:1 (v / v), and red solid Example 1 was obtained with a yield of 55%.

[0088] ESI-HRMS: C 31 h 40 BF 2 N 6 o 3 + [M+H] + 593.3213.

[0089] The high-resolution mass spectrum of the bound cell membrane dye prepared in Example 2 is as follows: image 3 shown.

Embodiment 3

[0091] Same as Example 1, only the structure of the parent dye is different, and the structure of the intermediate 2 has not changed, and its synthetic route is as follows:

[0092]

[0093] Dissolve intermediate 2 (2mmol) in 5mL DMF, add HATU (2mmol) and TMP (3mmol) to it under stirring at low temperature, add dropwise DMF solution of dye precursor 3 (1mmol) to it half an hour later, stop after 4h reaction reaction. After the solvent was removed by distillation under reduced pressure, the crude product was purified by silica gel chromatography, the eluent was dichloromethane:methanol=15:1 (v / v), and the blue solid Example 1 was obtained with a yield of 83%. ESI-HRMS: N 32 h 39 N 6 o 2 S + [M] + 571.2858.

[0094] The high-resolution mass spectrum of the bound cell membrane dye prepared in Example 3 is as follows: Figure 4 shown.

[0095] Further, in the present invention, the comparative example of dye structure design in Example 3 is used to verify the unique cell...

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Abstract

The invention discloses a glycoprotein-binding membrane dye based on pyrazolone. The general structure of the membrane dye is shown in formula (I). In formula (I), R 1 is a fluorescent chromophore dye structure with an O-terminal or an N-terminal; R 2 at least one of ‑O‑, ‑O‑O‑, ‑S‑, ‑NH‑, ‑NHNH‑ or ‑ONH‑; R 3 At least one selected from or in. It is used for long-retention cell membrane tracking imaging, and / or for fast and accurate imaging of cell membranes at low concentrations, and / or for observing cell division, migration processes, and / or for membrane labeling to distinguish co-cultured cells, And / or fluorescent signals for distinguishing different cells, and / or for labeling and grouping of cells, and / or for tracking of passaged cells.

Description

technical field [0001] The present invention relates to the field of photochemistry and biological imaging, especially to cell membrane dyeing technology and application, specifically to a series of cell membrane dyes based on pyrazolone а-H sites that can realize rapid binding at low concentrations and have super long residence time , its preparation method and application. Background technique [0002] The cell membrane (also known as the plasma membrane) is a physiological barrier between the cell and the surrounding physiological environment. It is mainly composed of three main components: the phospholipid bilayer forming the skeleton, the protein embedded in or on the surface of the cell membrane, and the glycoprotein on the surface of the cell membrane. While ensuring the stability of the internal environment of the cell, it can exchange matter, energy and information with the external environment. Cell membranes have been shown to be involved in various biological pr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C09B57/08C09B21/00C09B57/00C07D401/12C07F5/02C07D417/12C09K11/06G01N1/30G01N21/64
CPCC09B57/08C09B21/00C09B57/00C07D401/12C07F5/022C07D417/12C09K11/06G01N1/30G01N21/64C09K2211/1044C09K2211/1029C09K2211/1096C09K2211/1037G01N2001/302
Inventor 彭孝军熊涛樊江莉陈颖超
Owner DALIAN UNIV OF TECH
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