阅读说明：本技术 一种三联8-羟基喹啉及其制备和应用 (Tri-8-hydroxyquinoline and preparation and application thereof ) 是由 张煊 鲁珍妮 于 2020-05-14 设计创作，主要内容包括：本发明涉及一种三联8-羟基喹啉及其制备和应用,所述结构如式I所示。制备包括:将5,7-二甲酰基-8-羟基喹啉与2-甲基-8-羟基喹啉在乙酸酐中回流,经水解后即得；本发明三联8-羟基喹啉化合物作为荧光探针,应用于水溶液中镁离子的高选择性识别和活体细胞中荧光成像。(The invention relates to a tri-8-hydroxyquinoline and a preparation method and application thereof, and the structure is shown as a formula I. The preparation method comprises refluxing 5, 7-diformyl-8-hydroxyquinoline and 2-methyl-8-hydroxyquinoline in acetic anhydride, and hydrolyzing; the tri-8-hydroxyquinoline compound is used as a fluorescent probe and applied to high-selectivity identification of magnesium ions in aqueous solution and fluorescence imaging in living cells.)

1. A tris-8-hydroxyquinoline having the structure of formula I:

2. a method of preparing tris-8-hydroxyquinoline, comprising:

(1) reacting 8-hydroxyquinoline with hexamethylenetetramine in trifluoroacetic acid, cooling to room temperature, filtering, washing and drying to obtain 5, 7-diformyl-8-hydroxyquinoline;

(2) and (2) refluxing the 5, 7-diformyl-8-hydroxyquinoline and 2-methyl-8-hydroxyquinoline in the step (1) in acetic anhydride, cooling to room temperature, extracting and collecting an organic phase, performing rotary evaporation, dissolving the obtained solid product with pyridine, adding water, continuously reacting under heating, purifying, and drying to obtain the terpyridyl-8-hydroxyquinoline.

3. The preparation method according to claim 2, wherein the ratio of 8-hydroxyquinoline to hexamethylenetetramine to trifluoroacetic acid in step (1) is 1mmol:2mmol:2 ml.

4. The method as claimed in claim 2, wherein the reaction temperature in step (1) is 105 ℃ to 115 ℃ and the reaction time is 48 to 72 hours.

5. The preparation method according to claim 2, wherein the ratio of 5, 7-diformyl-8-hydroxyquinoline, 2-methyl-8-hydroxyquinoline, acetic anhydride, pyridine and water in the step (2) is 1mmol:2.4mmol:15ml:10ml:10 ml.

6. The preparation method as claimed in claim 2, wherein the reflux in acetic anhydride in step (2) is 120-130 ℃ for 72-96 h; the reaction was continued with heating as follows: refluxing at 100 ℃ and 120 ℃ for 36-60 h.

7. A fluorescent probe based on the tris-8-hydroxyquinoline of claim 1.

8. The use of the fluorescent probe of claim 7 in selective recognition of magnesium ions and quantitative determination of magnesium ion concentration by fluorescence.

9. The use of claim 8, wherein the fluorescence quantitative determination of the magnesium ion concentration is: and (3) testing the fluorescence intensity at 602nm, establishing a standard curve between the fluorescence intensity and the magnesium ion concentration, wherein the regression equation is that Y is 1.5859X +0.1181, Y is the fluorescence intensity, X is the magnesium ion concentration, the linear regression constant is 0.9905, the detection range of the magnesium ion concentration is 0-8 mu M, and the detection limit is 0.1 mu M.

10. Use of the fluorescent probe of claim 7 in bioluminescent imaging.

Technical Field

The invention belongs to the field of functional organic materials and preparation and application thereof, and particularly relates to a tri-8-hydroxyquinoline and preparation and application thereof.

Background

8-hydroxyquinoline is a complexing agent widely used for metal ion determination, but has poor selectivity, so that the application of the 8-hydroxyquinoline in a fluorescent probe is greatly limited. The selectivity is improved by modifying 8-hydroxyquinoline molecules, and the method is an important strategy for expanding the application of the compounds in fluorescent probes and is widely concerned. The fluorescence light-emitting wavelength of most of the currently reported 8-hydroxyquinoline compounds is similar to that of the 8-hydroxyquinoline parent compound and is about 500 nm. For cellular fluorescence imaging applications, the fluorescence emission of long wavelength (>600nm) has more application prospects. Kao et al have reported that magnesium ion fluorescent probes based on 8-hydroxyquinoline derivatives have a maximum fluorescence emission wavelength of 487nm and are also less selective for magnesium ions (M.H.Kaoet al, J.Lumin.,2016,169,156). Therefore, designing and synthesizing 8-hydroxyquinoline compounds with both long-wavelength fluorescence emission and high-selectivity recognition performance is still a challenging task. On the other hand, magnesium ions have important physiological functions in human bodies and are closely related to various active enzymes, so that the development of a fluorescent probe capable of realizing high selection and high-sensitivity detection of magnesium ions in cells is of great significance.

Disclosure of Invention

The invention aims to solve the technical problem of providing the 8-hydroxyquinoline and the preparation and application thereof, overcoming the defects of poor selectivity and short emission wavelength of an 8-hydroxyquinoline fluorescent probe in the prior art, and the compound related by the invention is characterized in that three 8-hydroxyquinoline molecules are sequentially connected through double bonds, and the obtained 8-hydroxyquinoline is used as the fluorescent probe to realize high-selectivity identification on magnesium ions, and the fluorescent emission wavelength is longer (>600nm), so that the invention has an application prospect on intracellular magnesium ion fluorescent imaging.

The invention relates to a tri-8-hydroxyquinoline with a structure shown in a formula I:

the invention relates to a preparation method of tri-8-hydroxyquinoline, which comprises the following steps:

(1) reacting 8-hydroxyquinoline with hexamethylenetetramine in trifluoroacetic acid, cooling to room temperature, filtering, washing and drying to obtain 5, 7-diformyl-8-hydroxyquinoline;

(2) and (2) refluxing the 5, 7-diformyl-8-hydroxyquinoline and 2-methyl-8-hydroxyquinoline in the step (1) in acetic anhydride, cooling to room temperature, extracting with dichloromethane, collecting an organic phase, removing the solvent by rotary evaporation to obtain a solid product, dissolving the solid product with pyridine, adding water, continuously reacting under heating, purifying, and drying to obtain a dark red solid, namely the terpyriol 8-hydroxyquinoline.

The preferred mode of the above preparation method is as follows:

the proportion of the 8-hydroxyquinoline, the hexamethylenetetramine and the trifluoroacetic acid in the step (1) is 1mmol to 2 ml.

The reaction temperature in the step (1) is 105-115 ℃, and the reaction time is 48-72 h.

The washing in the step (1) is washing by dilute hydrochloric acid, the concentration of the dilute hydrochloric acid is 0.5M, and the volume of the dilute hydrochloric acid is 50 ml.

In the step (2), the ratio of the 5, 7-diformyl-8-hydroxyquinoline to the 2-methyl-8-hydroxyquinoline to the acetic anhydride to the pyridine to the water is 1mmol:2.4mmol:15ml:10ml:10 ml.

Refluxing in acetic anhydride at the temperature of 120-130 ℃ for 72-96h under the protection of nitrogen in the step (2); the reaction was continued with heating as follows: refluxing in pyridine water solution at 100-120 deg.c for 36-60 hr.

And (3) in the step (2), the purification is implemented by removing pyridine through rotary evaporation, adding water, filtering, washing with water and washing with ethanol.

The invention relates to a fluorescent probe based on the tri-8-hydroxyquinoline.

The fluorescent probe disclosed by the invention is applied to selective identification of magnesium ions and fluorescent quantitative detection of magnesium ion concentration. The fluorescence quantitative determination magnesium ion concentration is as follows: and (3) testing the fluorescence intensity at 602nm, establishing a standard curve between the fluorescence intensity and the magnesium ion concentration, wherein the regression equation is that Y is 1.5859X +0.1181, Y is the fluorescence intensity, X is the magnesium ion concentration, the linear regression constant is 0.9905, the detection range of the magnesium ion concentration is 0-8 mu M, and the detection limit is 0.1 mu M.

The invention also discloses application of the fluorescent probe in biological fluorescence imaging.

Further, the fluorescent probe is cultured together with living cells and magnesium ions, and the magnesium ions in the cells can be subjected to fluorescence imaging analysis by using a fluorescence confocal microscope.

The invention tests the fluorescence spectrum of the tri-8-hydroxyquinoline compound and a metal ion mixed solution (pH is 7.4, and Tris-HCl buffer solution contains 90% dimethyl sulfoxide) by an Edinburgh FS5 fluorometer, and inspects the selective identification performance of the tri-8-hydroxyquinoline compound and the metal ion mixed solution as a fluorescence probe for magnesium ions. And respectively adding the magnesium ion aqueous solution which changes in a gradient manner into the fluorescent probe solution, and drawing by taking the magnesium ion concentration as an abscissa and the change of the fluorescence intensity as an ordinate to obtain a linear working curve. The fluorescence intensity at 602nm was measured and the magnesium ion concentration was calculated using the working curve. The detection range of the magnesium ion concentration is 0-8 mu M, and the detection limit is 0.1 mu M. The excitation wavelength of the fluorescent probe is 475nm, and the emission wavelength is 602 nm.

The fluorescent probe and the living Hela cells are cultured for 5min in a Tris-HCl buffer solution with the pH value of 7.4, and the cell fluorescence imaging observation is carried out by using a Carl Zeiss LSM 700 fluorescence confocal microscope. And adding magnesium ions into the Hela cell solution, culturing for 5min, and observing by using a fluorescence confocal microscope.

Advantageous effects

The raw materials used by the triple 8-hydroxyquinoline are cheap, the preparation method is simple, and the fluorescence emission wavelength is more than 600 nm;

the triple 8-hydroxyquinoline has high selectivity as a fluorescent probe, has high selectivity on magnesium ions, and has no obvious interference to other common ions (as shown in figure 2);

the triple 8-hydroxyquinoline fluorescence probe has the advantages that the detection linear range of magnesium ions is wide and is 0-8 mu M, the detection sensitivity is high, the detection limit is 0.1 mu M, and the detection sensitivity is lower than that of the prior art documents by 0.8 mu M (the comparison documents are M.H.Kao et al, J.Lumin.,2016,169,156).

The fluorescent probe works under the physiological environment condition of pH7.4, and can be applied to cell fluorescence imaging.

Drawings

FIG. 1 is a schematic diagram of the synthetic route of tris-8-hydroxyquinoline according to the present invention;

FIG. 2 is a fluorescence spectrum of a selective response of tris-8-hydroxyquinoline to magnesium ions according to the present invention;

FIG. 3 is a fluorescence spectrum of tris (8-hydroxyquinoline) according to the present invention as a function of magnesium ion concentration;

FIG. 4 is a linear relationship between the change in fluorescence intensity of tris-8-hydroxyquinoline and the concentration of magnesium ions in accordance with the present invention;

FIG. 5 is a confocal fluorescence imaging diagram of triple 8-hydroxyquinoline in Hela cells for detecting magnesium ions, wherein (a) and (c) are bright fields, and (b) and (d) are dark fields.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should be understood that various changes or modifications of the present invention may be made by those skilled in the art after reading the teaching of the present invention, and such equivalents may fall within the scope of the present invention as defined in the appended claims.