CN112812075A

CN112812075A - Preparation method and application of benzothiazole Schiff base-based fluorescent probe

Info

Publication number: CN112812075A
Application number: CN202011643167.1A
Authority: CN
Inventors: 宋丽雪; 王丽; 韩辉
Original assignee: Shanxi University
Current assignee: Shanxi University
Priority date: 2020-12-30
Filing date: 2020-12-30
Publication date: 2021-05-18
Anticipated expiration: 2040-12-30
Also published as: CN112812075B

Abstract

The invention discloses a preparation method and application of a benzothiazole Schiff base-based fluorescent probe, and belongs to the technical field of fluorescent probes for metal ion detection. The molecular formula of the fluorescent probe is C₃₄H₃₁N₅O₂S₂The preparation method comprises the following steps: (1) reacting 2-hydroxy-5-methylbenzaldehyde and 2-aminothiophenol to obtain a compound a; (2) the compound a and hexamethylenetetramine react through a doffer reaction to generate a compound b; (3) the compound b and diethylenetriamine are subjected to condensation reaction to obtain a probe molecule L. In the presence of absolute ethyl alcohol: in a Tris-HCl-3: 2 solution, probe molecules react with zinc ions, and the concentration of the zinc ions can be detected by using the change of fluorescence intensity. The probe of the invention has simple synthesis, mild reaction conditions, high selectivity to zinc ions,The sensitivity is high, the response range is 0-8.00 mu M, and the detection limit is 60.4 nM. In addition, the novel fluorescent probe is successfully applied to the detection of zinc ions in cells by a laser confocal scanning microscopy technology.

Description

Preparation method and application of benzothiazole Schiff base-based fluorescent probe

Technical Field

The invention belongs to the technical field of fluorescent probes for metal ion detection, and particularly relates to a preparation method and application of a benzothiazole Schiff base-based fluorescent probe.

Background

Zinc is an important trace element required by organisms, exists in the form of divalent cations, is a second most abundant metal element next to iron in human bodies, plays a vital role in various life activities of organisms, and has important influences on neurotransmission, gene expression, cell metabolism, DNA and RNA synthesis and the like. However, the concentration of zinc is constantly rising due to man-made increases, most of which comes from industrial activities such as mining, coal, waste combustion and steel processing. Not only does an excess of zinc ions pollute the environment, but also in human health, an imbalance of zinc ions causes many psychiatric diseases including alzheimer's disease, parkinson's disease, prostate cancer, epilepsy, and the like. Therefore, designing a detection probe with high selectivity for zinc ions is critical to human health and the environment.

The fluorescent probe is one of the most powerful tools for detecting analytes, and most of the reported zinc ion molecular fluorescent probes have the problems of high cost, complex synthesis process, harsh reaction conditions, weak anti-interference capability, long response time, poor sensitivity and the like, so that the fluorescent probe is greatly limited in practical application. Therefore, there is an urgent need for a fluorescent probe with simple synthesis, high sensitivity, good selectivity, low detection limit, and good photostability for detecting zinc ions.

Disclosure of Invention

Aiming at the problems, the invention provides a preparation method and application of a fluorescence probe based on benzothiazole Schiff base.

In order to achieve the purpose, the invention adopts the following technical scheme:

benzothiazole Schiff base-based fluorescent probe with molecular formula of C₃₄H₃₁N₅O₂S₂The structural formula is as follows:

a preparation method of a benzothiazole Schiff base-based fluorescent probe comprises the following steps:

(1) dissolving 2-hydroxy-5-methylbenzaldehyde and 2-aminothiophenol in an anhydrous methanol solvent, adding a catalyst iodine simple substance, heating and refluxing for 4 hours under the protection of nitrogen, cooling to room temperature after the reaction is finished, separating out yellow solid, washing with cold methanol after filtering, and drying in vacuum to obtain light yellow solid, namely a compound a; the reaction process is as follows:

(2) dissolving a compound a and hexamethylenetetramine in trifluoroacetic acid, and heating and refluxing for 12h under the protection of nitrogen; after the reaction is finished, cooling to room temperature, adding a hydrochloric acid solution, stirring for 5 hours under an ice bath condition, extracting the mixture with dichloromethane, drying with anhydrous magnesium sulfate, filtering, concentrating under reduced pressure, and performing column chromatography to obtain a light yellow solid, namely a compound b; the reaction process is as follows:

(3) dropwise adding a diethylenetriamine solution into a dichloromethane solution dissolved with a compound b, dropwise adding 3 drops of glacial acetic acid, stirring at room temperature for 30 minutes, performing reflux reaction for 8 hours, cooling to room temperature, filtering, washing with a cold methanol solution to obtain a dark orange solid, namely a probe L, namely the benzothiazole Schiff base-based fluorescent probe, wherein the reaction process is as follows:

further, the molar ratio of 2-hydroxy-5-methylbenzaldehyde to 2-aminothiophenol in said step 1 is 1:1.

Further, the molar ratio of the 2-hydroxy-5-methylbenzaldehyde to the iodine elementary substance as the catalyst in the step 1 is 1: 0.5.

Further, the molar ratio of the compound a to the hexamethylenetetramine in the step 2 is 1: 1.3.

Further, the molar ratio of diethylenetriamine to the compound b in the step 3 is 1: 2.2.

Application of a benzothiazole Schiff base fluorescent probe in the preparation of absolute ethyl alcohol: detection of zinc ions in Tris-HCl ═ 3:2 solution.

Further, the reaction is carried out in the presence of absolute ethyl alcohol: the method for detecting zinc ions in a Tris-HCl (3: 2) solution comprises the following steps: in the presence of absolute ethyl alcohol: reacting a fluorescent probe with zinc ions in a Tris-HCl (3: 2) solution, and detecting whether the zinc ions exist by using the change of fluorescence intensity at 480 nm; the probe solution has a fluorescence signal at 552nm before the zinc ions are added, and when the zinc ions are added, the fluorescence emission is blue-shifted, and the fluorescence signal at 480nm is enhanced.

Further, the linear range of the detected zinc ions is 0-8.00 multiplied by 10^-6M, detection limit of 6.04X 10^-8M。

An application of a fluorescence probe based on benzothiazole Schiff base is used for detecting zinc ions in cells.

Compared with the prior art, the invention has the following advantages:

1. the fluorescent probe has the advantages of simple synthesis steps, low cost, short reaction time and mild conditions.

2. The detection means is simple and can be realized only by means of a fluorescence spectrometer;

3. the fluorescent probe compound can identify Zn with high sensitivity and high selectivity²⁺And short response time in Zn² ⁺Has good application prospect in the detection.

4. The fluorescent probe compound can be used for qualitative or quantitative detection of zinc ions in water bodies, soil and biological systems.

Drawings

FIG. 1 example 2 fluorescent Probe vs. Zn²⁺A varying ultraviolet absorption spectrum;

FIG. 2 example 3 Selective assay of fluorescent probes for Metal ions;

FIG. 3 example 4 fluorescent Probe pairs Zn²⁺A responsive operating curve;

FIG. 4 example 5 competitive assay of fluorescent probes for metal ions;

FIG. 5 image of Hella cells of example 6.

Detailed Description

Example 1

Synthesis of Compound a: 2-Aminothiophenol (2g, 16.1mmol) and 2-hydroxy-5-methylbenzaldehyde (2.45g, 16.1mmol) were added to a round bottom flask containing 20mL of anhydrous methanol, and I was added to the solution₂(2.04g, 8.05mmol), the mixture was heated to reflux under nitrogen for 4 h. After the reaction was complete, it was cooled to room temperature and a yellow solid precipitated, which was collected, filtered on a buchner funnel and washed with cold methanol. Further drying in vacuo afforded compound a (1.63g, 42.1%) as a pale yellow solid. The characterization results were as follows:¹H NMR(600MHz,CDCl₃)δ12.21(s,1H),7.92(d,J＝8.2Hz,1H),7.84(d,J＝8.0Hz,1H),7.54–7.43(m,2H),7.34(t,J＝7.2Hz,1H),7.19(d,J＝8.4Hz,1H),7.13(d,J＝8.4Hz,1H),2.29(s,3H)。

synthesis of Compound b: compound a (1.40g,5.82mmol), hexamethylenetetramine (1.22g,8.73mmol) was added to a round bottom flask containing 8mL trifluoroacetic acid (TFA) and heated to reflux under nitrogen for 15h (110 ℃). After completion of the reaction, it was cooled to room temperature, acidified (35mL,4mmol) by addition of hydrochloric acid solution and stirred for 5h under ice-bath conditions. The mixture was then extracted with dichloromethane, washed with saturated aqueous sodium bicarbonate, dried over anhydrous magnesium sulfate, and finally concentrated under reduced pressure and purified by silica gel column (ethyl acetate: petroleum ether ═ 1:5) to give compound b (0.970g, 62.0%) as a pale yellow solid. The characterization results were as follows:¹H NMR(600MHz,CDCl₃)δ13.06(s,1H),10.52(s,1H),8.06(s,1H),7.97(s,1H),7.92(s,1H),7.74(s,1H),7.57(s,1H),7.47(s,1H),2.43(s,3H).¹³C NMR(151MHz,CDCl₃)δ190.88(s),158.58(s),151.52(s),135.23(s),133.13(s),132.64(s),128.96(s),126.90(s),125.86(s),123.74(s),122.42(s),121.63(s),118.71(s),20.37(s)。

synthesis of Probe L: diethylenetriamine (6.50. mu.L, 99%) was added dropwise to dissolved compound b (30.2mg,0.112 mmol)) Then 3 drops of glacial acetic acid were added dropwise to the dichloromethane (5mL) solution, and after stirring at room temperature for 30min, the reaction was refluxed for 8h, after completion of the reaction, the reaction was cooled to room temperature, the solid was collected, filtered through a buchner funnel and washed with cold methanol solution, and dried to give a dark orange solid, probe L (22.3mg, 65.4%). The characterization results were as follows:¹H NMR(600MHz,CDCl₃)δ8.42(s,1H),8.33(s,1H),8.05(s,1H),7.90(s,1H),7.49(s,1H),7.35(s,1H),7.26(s,1H),7.12(s,1H),5.32(s,1H),3.77(s,2H),3.11(s,2H),2.29(s,3H).¹³C NMR(151MHz,CDCl₃)δ165.78(s),163.20(s),162.11(s),151.93(s),135.91(s),134.34(s),133.71(s),126.51(s),125.91(s),124.40(s),122.39(s),121.80(s),121.37(s),117.96(s),57.07(s),49.18(s),20.27(s).HRMS(ESI)：C₃₄H₃₁N₅O₂S₂ for[L+H]⁺，calculated 606.1999,found 606.1989。

example 2

Probe molecule with Zn²⁺Ultraviolet absorption spectrum chart with concentration change

For testing probe molecule pairs with different Zn concentrations²⁺Study of ultraviolet absorption Spectroscopy under the same conditions of experiment, 1mL of a buffer solution (CH) of absolute ethanol and Tris-HCl₃CH₂To Tris-HCl 3:2, V/V,20mmol, pH 7.4, 10. mu.L of a stock solution of probe molecules (1mmol/L) was added Zn²⁺Uv titration experiments and uv absorption spectra were tested (figure 1). As can be seen from FIG. 1, Zn was added²⁺Then, a new absorption peak appears at 421nm of the probe molecule, the absorption peak at 421nm gradually increases with the gradually increasing concentration of zinc ions in the solution, and meanwhile, the absorption peak at 363nm of the probe molecule is red-shifted to 373nm, and an isoabsorption point appears at 383 nm.

Example 3

Probe molecule and Zn²⁺Fluorescence spectrum study before and after action

For testing probe molecules with Zn²⁺Fluorescence spectra before and after the action, in the same experimental conditions, to 1mL of a buffer solution (CH) of absolute ethanol and Tris-HCl₃CH₂Tris-HCl 3:2, V/V,20mmol, pH 7.4 to 1. mu.L of stock probe molecule (1mmol/L)Subsequently, 1. mu.L of a stock solution of different metal ions (10mmol/L) was added dropwise and the fluorescence spectrum was measured (FIG. 2). As can be seen from FIG. 2, only Zn was added to the probe molecule solution²⁺The emission peak at 480nm then increased significantly and changed from yellow to bluish with a significant change in fluorescence color, indicating that the probe molecule can selectively detect zinc ions.

Example 4

Probe molecule pairs with different concentrations of Zn²⁺Investigation of fluorescence intensity Change under conditions

For testing probe molecule pairs with different Zn concentrations²⁺Study of fluorescence intensity Change under the same Experimental conditions, 1mL of a buffer solution (CH) of absolute ethanol and Tris-HCl was added₃CH₂Tris-HCl 3:2, V/V,20mmol, pH 7.4) to 1. mu.L of a stock solution of probe molecules (1mmol/L) was added and Zn was performed²⁺Fluorescence titration experiments and their fluorescence spectra were tested (fig. 3). As can be seen from FIG. 3, the fluorescence intensity at 480nm gradually increased with the increase of the concentration of zinc ions, and when the fluorescence emission intensity of the solution at 480nm was plotted against the concentration of zinc ions, a good linear relationship (inset) was observed between the zinc ion concentration and the solution in the range of 0-8. mu.M, and quantitative detection of zinc ions in this concentration range was achieved. The detection limit is 60.4nM, which is lower than the maximum Zn in WHO specified drinking water²⁺The concentration (76 mu M) shows that the fluorescent probe molecule of the invention has higher sensitivity to zinc ions with different concentrations.

Example 5

Probe molecule pair Zn²⁺Interference study of

Detection of Zn as a fluorescence sensor for testing probe molecules²⁺Under the same experimental conditions, 1mL of a buffer solution (CH) of absolute ethanol and Tris-HCl is added₃CH₂Tris-HCl 3:2, V/V,20mmol, pH 7.4) was added to 1. mu.L of a stock solution of probe molecules (1mmol/L) and 1. mu.L of a stock solution of zinc ions (10mmol/L), followed by dropwise addition of 1. mu.L of each of the different metal ion stock solutions (10mmol/L) thereto and fluorescence spectra thereof were measured (FIG. 4). Drawing a graph with the fluorescence intensity at 480nm as the ordinate and different metal ions as the abscissa, and obtaining the probe molecule pair Zn from the graph²⁺Is not detectedBy interference of other potentially competing metal ions, e.g. K⁺,Na⁺,Ca²⁺,Mg²⁺,Ba²⁺,Fe³⁺,Al³⁺,Zn²⁺,Li⁺,Cr³⁺,Cd²⁺,Co²⁺,Pb²⁺,Cu²⁺,Ag⁺,Hg²⁺Under the condition of existence of the metal ions, the detection of the system on the zinc ions is still not interfered.

Example 6

Imaging Hella cell

Fluorescent probe stock solutions were prepared at 1mM concentration by dissolving in DMSO before the experiments. The cultured Hella cells were washed with PBS buffer pH 7.40, and then 1mL of PBS buffer (pH 7.40) containing 10. mu.L of the probe was added to both dishes, and the mixture was incubated at 37 ℃ in 5% CO₂After incubation in the incubator for 15min, the medium was gently washed three times with PBS buffer to remove excess stock of probes that did not enter the cells. To one of the dishes incubated with the probe, a solution containing 9. mu.L (1X 10)^-2M)Zn²⁺1mL of the buffer solution of PBS (pH 7.40), and incubation and washing were carried out in the same manner.

Finally, the cells which only incubate the probe and those which incubate the probe and also incubate the zinc ions are respectively placed in a culture dish, and 1.0mL of PBS solution with the pH value of 7.40 is added to the culture dish for imaging observation under a laser confocal microscope. The fixed excitation wavelength is 412nm, and the collection emission bands are a blue channel (420-490nm) and a yellow channel (500-600 nm). As can be seen from FIG. 5, when only the probe is added, the cell shows strong yellow fluorescence, the blue channel does not fluoresce, and Zn is added²⁺The post-yellow fluorescence decreased, while the blue fluorescence increased.

Those skilled in the art will appreciate that the invention may be practiced without these specific details. Although illustrative embodiments of the present invention have been described above to facilitate the understanding of the present invention by those skilled in the art, it should be understood that the present invention is not limited to the scope of the embodiments, and various changes may be made apparent to those skilled in the art as long as they are within the spirit and scope of the present invention as defined and defined by the appended claims, and all matters of the invention which utilize the inventive concepts are protected.

Claims

1. A fluorescence probe based on benzothiazole Schiff base is characterized in that: molecular formula C₃₄H₃₁N₅O₂S₂The structural formula is as follows:

2. a preparation method of the benzothiazole Schiff base-based fluorescent probe of claim 1, which is characterized in that: the method comprises the following steps:

(1) dissolving 2-hydroxy-5-methylbenzaldehyde and 2-aminothiophenol in an anhydrous methanol solvent, adding a catalyst iodine simple substance, heating and refluxing for 4 hours under the protection of nitrogen, cooling to room temperature after the reaction is finished, separating out yellow solid, washing with cold methanol after filtering, and drying in vacuum to obtain light yellow solid, namely a compound a;

(2) dissolving a compound a and hexamethylenetetramine in trifluoroacetic acid, and heating and refluxing for 12h under the protection of nitrogen; after the reaction is finished, cooling to room temperature, adding a hydrochloric acid solution, stirring for 5 hours under an ice bath condition, extracting the mixture with dichloromethane, drying with anhydrous magnesium sulfate, filtering, concentrating under reduced pressure, and performing column chromatography to obtain a light yellow solid, namely a compound b;

(3) dropwise adding a diethylenetriamine solution into a dichloromethane solution dissolved with the compound b, dropwise adding 3 drops of glacial acetic acid, stirring at room temperature for 30min, performing reflux reaction for 8h, cooling to room temperature, filtering, washing with a cold methanol solution to obtain a dark orange solid, namely a probe L, namely the benzothiazole Schiff base-based fluorescent probe.

3. The preparation method of the benzothiazole Schiff base-based fluorescent probe according to claim 2, wherein the preparation method comprises the following steps: the molar ratio of 2-hydroxy-5-methylbenzaldehyde to 2-aminothiophenol in step 1 is 1:1.

4. The preparation method of the benzothiazole Schiff base-based fluorescent probe as claimed in claim 3, wherein: the molar ratio of the 2-hydroxy-5-methylbenzaldehyde to the catalyst iodine in the step 1 is 1: 0.5.

5. The preparation method of the benzothiazole Schiff base-based fluorescent probe according to claim 4, wherein the preparation method comprises the following steps: the molar ratio of the compound a to the hexamethylenetetramine in the step 2 is 1: 1.3.

6. The preparation method of the benzothiazole Schiff base-based fluorescent probe according to claim 5, wherein the preparation method comprises the following steps: the molar ratio of diethylenetriamine to compound b in step 3 is 1: 2.2.

7. The application of the benzothiazole Schiff base-based fluorescent probe in claim 1, wherein the benzothiazole Schiff base-based fluorescent probe comprises the following components in percentage by weight: in the presence of absolute ethyl alcohol: detection of zinc ions in Tris-HCl ═ 3:2 solution.

8. The use of a benzothiazole Schiff base-based fluorescent probe according to claim 7, wherein: the method comprises the following steps of (1) preparing anhydrous ethanol: the method for detecting zinc ions in a Tris-HCl (3: 2) solution comprises the following steps: in the presence of absolute ethyl alcohol: reacting a fluorescent probe with zinc ions in a Tris-HCl (3: 2) solution, and detecting whether the zinc ions exist by using the change of fluorescence intensity at 480 nm; the probe solution has a fluorescence signal at 552nm before the zinc ions are added, and when the zinc ions are added, the fluorescence emission is blue-shifted, and the fluorescence signal at 480nm is enhanced.

9. The use of a benzothiazole Schiff base-based fluorescent probe according to claim 8, wherein: the linear range of the detected zinc ions is 0-8.00 multiplied by 10^-6M, detection limit of 6.04X 10^-8M。

10. The use of the benzothiazole Schiff base-based fluorescent probe of claim 1, wherein: the method is used for detecting zinc ions in cells.