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Two-photon fluorescent dye taking isoquinolinone as parent, preparation method thereof and application

A two-photon fluorescence, isoquinolinone technology, applied in biochemical equipment and methods, luminescent materials, chemical instruments and methods, etc., can solve the problems of reducing the application value of RNA dyes, high phototoxicity, poor photostability, etc. Achieve high-efficiency two-photon absorption ability, improve absorption cross-section, and increase the effect of π-electron conjugated system

Active Publication Date: 2013-01-30
DALIAN UNIV OF TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the dye has the following defects in practical imaging applications: long response time with RNA, high phototoxicity, poor photostability, etc., which greatly reduces the application value of this RNA dye in biological imaging labeling
In the prior art, there is no report that the compound of the isoquinolinone parent is used as a two-photon fluorescent dye for biological staining, especially RNA specific staining

Method used

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  • Two-photon fluorescent dye taking isoquinolinone as parent, preparation method thereof and application
  • Two-photon fluorescent dye taking isoquinolinone as parent, preparation method thereof and application
  • Two-photon fluorescent dye taking isoquinolinone as parent, preparation method thereof and application

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preparation example Construction

[0042] 8. the preparation method of the two-photon fluorescent dye taking isoquinolinone as the parent as claimed in claim 1, comprises the steps: 1) the compound of 4-bromo-1,8-naphthalene anhydride and formula i is according to molar ratio 1: 1-1:5 reaction, preparation of compound B:

[0043]

[0044] The reaction temperature is 70-150°C, the reaction time is 1-12 hours, and the reaction solvent is selected from dichloromethane, ethanol, ethyl acetate, acetic acid or a mixture thereof;

[0045] In a preferred embodiment, the reaction temperature is 80-140°C, the reaction time is 2-10 hours, the reaction solvent is selected from ethanol, ethyl acetate, acetic acid or a mixture thereof, 4-bromo-1,8-naphthalene anhydride and formula i Mole is 1:1-1:4;

[0046] In a further preferred embodiment, the reaction temperature is 90-120°C, the reaction time is 3-10 hours, the reaction solvent is selected from ethyl acetate, acetic acid or a mixture thereof, 4-bromo-1,8-naphthalene...

Embodiment 1

[0063] Embodiment 1. Preparation of dye compound A 1

[0064]

[0065] (1) Synthesis of intermediate 1

[0066] Add 20mmol of 4-bromo-1,8-naphthalene anhydride and 35mmol of 4-nitro-o-phenylenediamine into a round bottom flask containing 20ml of acetic acid solution under nitrogen protection. The reaction was heated to reflux at 105°C for 3 hours and then stopped. The mixture was poured into ice water, precipitated out, and suction filtered to obtain the crude product of yellow solid powder, intermediate product 1, with a yield of 84%.

[0067] (2) Dye A 1 Synthesis

[0068] Add 20 mmol of crude product 1 and 25 mmol of ethanolamine from the previous step into a round-bottomed flask containing 20 ml of ethylene glycol monomethyl ether solution, under nitrogen protection. The reaction was heated to reflux at 125°C for 4 hours and then stopped. The mixture was poured into ice water, an orange-red precipitate was precipitated, and the crude product was obtained by sucti...

Embodiment 2

[0069] Example 2. Dye Compound A 1 Labeling assay on cellular RNA

[0070] Compound A synthesized using Example 1 1 , at a concentration of 5 μM A 1 - Add 4 μL of DMSO solution to the Hela cells at 37°C, 5% CO 2 dye A 1 Hela cells were incubated in culture medium for 30 minutes. Then, shake and rinse with PBS for 5min×3, then add cell culture medium, and conduct two-photon laser confocal imaging. Select a representative area, observe with an oil lens (60×), and repeat three times. Experimental results such as figure 2 As shown, the imaging shows that there is a strong fluorescent signal in the cellular RNA of Hela cells, while there is no fluorescent signal in other regions of the cell. figure 2 For adding dye A 1 Focused image of posterior HeLa cells. The excitation wavelength is 800nm, and the collection wavelength range is 520-570nm. The scale bar in the figure is 20 μm.

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Abstract

A two-photon fluorescent dye taking isoquinolinone as the parent is provided with a structure presented in formula I, wherein R1 is selected from -H, -CN, -OCH3, -COOH, -OH, -NO2, -NH2, -NHCH3, -N(CH3)2, -OCOCH3 and -SH; R2 is selected from -N-, -C-, -S- and -O-; and R3 is selected from -NHCH2OH, -NH(CH2)2OH, -NH(CH2)3OH, -NH(CH2)4OH,-N(CH3)CH2OH, - N(CH3)(CH2)2OH, -N(CH3)(CH2)3OH and -N(CH3)(CH2)4OH. The fluorescent dye is efficient in two-photon absorptive capacity, low in photo-induced toxicity and photobleaching, and strong in specificity for ribonucleic acid (RNA) in cells; and can perform specificity marking on the RHA in living cells and fixed cells simultaneously. The formula I is shown in the description.

Description

technical field [0001] The invention relates to a class of two-photon fluorescent dyes with isoquinolone as a parent, a preparation method thereof, and the application of the fluorescent dye compounds in labeling cell RNA. Background technique [0002] Ribonucleic acid (abbreviated as RNA, that is, Ribonucleic Acid), is a genetic information carrier that exists in biological cells and some viruses and viroids. A type of nucleic acid composed of at least dozens of ribonucleotides linked by phosphodiester bonds, named for its ribose sugar, referred to as RNA. RNA is ubiquitous in animals, plants, microorganisms, and some viruses and bacteriophages. RNA and protein biosynthesis are closely related. In RNA viruses and bacteriophages, RNA is the carrier of genetic information. RNA is generally a single-stranded linear molecule; there are also double-stranded ones such as reovirus RNA; circular single-stranded ones such as viroid RNA; in 1983, branched-chain RNA molecules were ...

Claims

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Application Information

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IPC IPC(8): C09B57/08C09K11/06C12Q1/68
Inventor 彭孝军仉华樊江莉王静云孙世国宋锋玲
Owner DALIAN UNIV OF TECH
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