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Subcellular area targeting, long-wavelength and dual-wavelength ratio method Ca<2+> fluorescent probe and synthesis method thereof

A technology of fluorescent probes and synthesis methods, applied in the field of biochemical sensing research, can solve the problems of non-targeting, difficult dual-wavelength ratiometric measurement, and inability to use subcellular measurement, etc.

Inactive Publication Date: 2010-01-06
BEIJING INSTITUTE OF TECHNOLOGYGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The purpose of the present invention is to provide a class of subcellular region-targeted, long-wavelength, dual-wavelength ratio method Ca 2+ The fluorescent probe and its synthesis method solve the problem that the existing calcium fluorescent probe needs to be excited in the ultraviolet region, and it is difficult to measure it by the dual-wavelength ratio method. It is not targeted and cannot be used for subcellular Ca levels. 2+ issues such as the determination of

Method used

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  • Subcellular area targeting, long-wavelength and dual-wavelength ratio method Ca&lt;2+&gt; fluorescent probe and synthesis method thereof
  • Subcellular area targeting, long-wavelength and dual-wavelength ratio method Ca&lt;2+&gt; fluorescent probe and synthesis method thereof
  • Subcellular area targeting, long-wavelength and dual-wavelength ratio method Ca&lt;2+&gt; fluorescent probe and synthesis method thereof

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Experimental program
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Effect test

Embodiment 1

[0030] (1) Weigh 29.42g (50mmol) of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraethyl acetate into a 250mL conical flask, add 150mL to dry Dimethylformamide, stirring to dissolve 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraethyl acetate. Cool to -2℃, add POCl dropwise 3 18.14mL (200mmol), need 2h, then stir at room temperature for about 40h. After the reaction, the reaction mixture was poured into 500 mL of deionized water at 0°C under stirring, and there was precipitation. Adjust the suspension to neutral with 5% KOH solution, filter with suction, and redissolve the resulting crude product in ethyl acetate. In the ester, wash twice with deionized water and saturated NaCl solution respectively. Anhydrous Na for organic phase 2 SO 4 After drying, filtering, and evaporating ethyl acetate under reduced pressure on a rotary evaporator to obtain a brown oil, the pure product 5,5'-dialdehyde-BAPTA-tetraethyl ester is obtained by column chromatography. The reaction formula of this...

Embodiment 2

[0037] (1) Weigh 14.71g (25mmol) of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraethyl acetate into a 250mL conical flask, add 150mL to dry Dimethylformamide, stirring to dissolve 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraethyl acetate. Cool to -2℃, add POCl dropwise 3 18.14mL (200mmol), need 2h, then stir at room temperature for about 40h. After the reaction, the reaction mixture was poured into 500 mL of deionized water at 0°C under stirring, and there was precipitation. Adjust the suspension to neutral with 5% KOH solution, filter with suction, and redissolve the resulting crude product in ethyl acetate. In the ester, wash twice with deionized water and saturated NaCl solution respectively. Anhydrous Na for organic phase 2 SO 4 After drying, filtering, and evaporating ethyl acetate under reduced pressure on a rotary evaporator to obtain a brown oil, the pure product 5,5'-dialdehyde-BAPTA-tetraethyl ester is obtained by column chromatography. The reaction formula of this...

Embodiment 3

[0044] (1) Weigh 11.77g (20mmol) of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraethyl acetate into a 250mL conical flask, add 70mL to dry Dimethylformamide, stirring to dissolve 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraethyl acetate. Cool to -2℃, add POCl dropwise 3 9.07mL (100mmol), need 2h, then stir at room temperature for 40h. After the reaction, the reaction mixture was poured into 250 mL of deionized water at 0°C under stirring, and precipitation was precipitated. Adjust the suspension to neutral with 5% KOH solution, filter with suction, and redissolve the resulting crude product in ethyl acetate. In, and wash with deionized water and saturated NaCl solution twice. Anhydrous Na for organic phase 2 SO 4 After drying, filtering, and evaporating ethyl acetate under reduced pressure on a rotary evaporator to obtain a brown oil, the pure product 5,5'-dialdehyde-BAPTA-tetraethyl ester is obtained by column chromatography. The reaction formula of this step is as follows:...

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Abstract

The invention relates to design and synthesis of subcellular area targeting, long-wavelength and dual-wavelength ratio method Ca<2+> fluorescent probe. In the synthesis of the probes, 1, 2-di(2-aminophenoxy)ethane-N, N, N', N'-tetraacetic acid tetraethyl ester is taken as the initial material to first synthesize Ca<2+> receptor (5, 5'-dicarboxo-BAPTA) with two formyl active groups as the key intermediate of the Ca<2+> fluorescent probes. Then, the key intermediate is condensed with the substrate containing active methyl in agreeable organic solvent to obtain a series of Ca<2+> fluorescent probes excited by visible light, fluorescence excitation and emission peak as well as position all change before and after the probes combine with Ca<2+>, and Ca<2+> can be measured by dual-wavelength ratio method. The probes can carry out targeting marking on the specified subcellular area after entering cells. With the probes adopted, the real-time dynamic measurement of Ca<2+> content in the cells and space-time distribution condition can be realized in the subcellular level, thereby, an efficient means for cell calcium signal transduction research is provided.

Description

Technical field [0001] The invention relates to a type of subcellular region targeting, long-wavelength, dual-wavelength ratio method. 2+ The design and synthesis of fluorescent probes belong to the field of biochemical sensing research. Background technique [0002] Ca 2+ As the second messenger in the cell, it participates in and controls a series of important life processes. It not only directly activates the mechanical activities in the cell with important physiological significance (such as muscle contraction), but also acts as the second messenger in the cell to mediate the first messenger (Extracellular signals such as hormones) The reaction to the cell, its impact almost involves all the physiological and biochemical processes of the cell, such as material metabolism, muscle contraction, endocytosis, pinocytosis, neurotransmitter release, chromosome movement, DNA synthesis , Cell division and cell apoptosis. Cellular Ca 2+ His research has always been a hot topic in the ...

Claims

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Application Information

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IPC IPC(8): G01N33/52G01N21/64
Inventor 张小玲贾宏瑛朱宝存
Owner BEIJING INSTITUTE OF TECHNOLOGYGY
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