A six-membered spirocyclic rhodamine copper ion fluorescent probe containing a benzothiazole hydrazine Schiff base structure, and its preparation method and application

Abstract

The present invention discloses a six-membered spirocyclic rhodamine copper ion fluorescent probe containing a benzothiazole hydrazine Schiff base structure, a preparation method thereof, and an application thereof. The six-membered spirocyclic rhodamine copper ion fluorescent probe containing a benzothiazole hydrazine Schiff base structure has the structural formula shown in (I). The preparation method comprises the following steps: weighing equimolar amounts of 2-aldehyde rhodamine B and 2-hydrazine benzothiazole, dissolving them in dry ethanol, heating to 80°C for reaction for 4 hours, cooling to room temperature, and distilling under reduced pressure. The obtained solid is purified by column chromatography to obtain a light pink solid, which is the six-membered spirocyclic rhodamine copper ion fluorescent probe containing a benzothiazole hydrazine Schiff base structure. The fluorescent probe of the present invention has good sensitivity to copper ions and can qualitatively and quantitatively detect copper ions in a solution.

Description

Technical Field

[0001] The invention belongs to the technical field of probe materials, and in particular relates to a six-membered spirocyclic rhodamine copper ion fluorescent probe containing a benzothiazole hydrazine Schiff base structure, and a preparation method and application thereof. Background Art

[0002] Copper (Cu) is the third most abundant transition metal in the human body and plays an important role in many physiological processes. 2+ ) plays an important role in biological processes such as hemoglobin synthesis, oxygen binding, nerve function and electron transport. 2+ Abnormal homeostasis can lead to various diseases such as renal failure, liver disease, Wilson disease, etc. Compared with traditional detection methods, fluorescent probes are more effective in detecting Cu 2+ It has the advantages of low cost, easy operation, high sensitivity and good selectivity. Therefore, in recent years, many researchers have combined small molecule fluorescent probes based on xanthene structure with Cu 2+ To investigate the origins of diseases and evaluate the clinical application of potential drugs, information such as the cellular concentration and distribution of copper is crucial. Therefore, the development of copper ion fluorescent probes that are simple to operate, highly selective, sensitive, and low-cost is of great significance.

[0003] Fluorescence signals exhibit fast response times, excellent spatial resolution, and a high signal-to-noise ratio. Fluorescent staining offers high sensitivity and the advantages of being non-destructive to samples and minimally damaging to cells under microscopy. Rhodamine fluorescent dyes are widely used as fluorescent probes for detecting heavy metal ions due to their high absorption coefficient, broad fluorescence in the visible light region, high fluorescence quantum yield, and photostability. Previous studies have shown that rhodamine fluorescent probes almost always contain a five-membered spirolactam ring structure, while six-membered rhodamine fluorescent probes have rarely been designed and synthesized. However, the five-membered spirolactam moiety has poor water solubility, short fluorescence stability, and strong pH dependence. Reported six-membered rhodamine probes have demonstrated higher sensitivity and advantages over five-membered rings in bioimaging. Therefore, six-membered rhodamine fluorescent indicators have broad application prospects. Summary of the Invention

[0004] The present invention aims to provide a six-membered spirocyclic rhodamine copper ion fluorescent probe containing a benzothiazole hydrazine Schiff base structure, which can quickly respond and detect copper ions with high sensitivity.

[0005] The technical solution adopted in the present invention is:

[0006] A six-membered spirocyclic rhodamine copper ion fluorescent probe containing a benzothiazole hydrazine Schiff base structure, wherein the six-membered spirocyclic rhodamine copper ion fluorescent probe containing a benzothiazole hydrazine Schiff base structure is SRh-BT, having the structural formula shown in (I):

[0007]

[0008] A method for preparing a six-membered spirocyclic rhodamine copper ion fluorescent probe containing a benzothiazole hydrazine Schiff base structure comprises the following steps: dissolving a rhodamine compound and 2-hydrazinobenzothiazole (benzothiazole hydrazine) in dry ethanol, heating for reaction, cooling to room temperature, performing reduced pressure distillation, and purifying the obtained solid by column chromatography to obtain a light pink solid, which is the six-membered spirocyclic rhodamine copper ion fluorescent probe containing a benzothiazole hydrazine Schiff base structure.

[0009] The reaction formula is as follows:

[0010]

[0011] Furthermore, in the above-mentioned preparation method, the rhodamine compound is 2-aldehyde rhodamine B, and its preparation method is based on invention patent ZL2018109542954.

[0012] Preferably, in the above preparation method, the molar ratio of 2-aldehyde rhodamine B:2-diaminobenzothiazole is 1:1.

[0013] Preferably, in the above preparation method, the heating reaction condition is: 80° C. reaction for 4 hours.

[0014] The above-mentioned six-membered spirocyclic rhodamine copper ion fluorescent probe containing a benzothiazole hydrazine Schiff base structure is used in the detection of copper ions. The mechanism of the six-membered spirocyclic rhodamine copper ion fluorescent probe containing a benzothiazole hydrazine Schiff base structure of the present invention is that copper ion-induced complexation causes the six-membered spirocyclic ring to open to generate bright red and highly fluorescent rhodamine, thereby achieving rapid detection of copper ions.

[0015] Preferably, the application of the six-membered spirocyclic rhodamine copper ion fluorescent probe containing a benzothiazole hydrazine Schiff base structure in the qualitative detection of copper ions is as follows: the six-membered spirocyclic rhodamine copper ion fluorescent probe containing a benzothiazole hydrazine Schiff base structure is dissolved in an acetonitrile aqueous solution to prepare a concentration of 2×10 -5 ~3×10 -4 mol / L, pH 7.4 test solution, add the solution to be tested into the test solution, and observe the change in solution color.

[0016] Preferably, the application of the six-membered spirocyclic rhodamine copper ion fluorescent probe containing a benzothiazole hydrazine Schiff base structure in the quantitative detection of copper ions is as follows:

[0017] 1) Preparation of test solution: Dissolve the six-membered spirocyclic rhodamine copper ion fluorescent probe containing benzothiazole hydrazine Schiff base structure in acetonitrile aqueous solution to a concentration of 2×10 -5 ~3×10-4 mol / L, pH 7.4 test solution;

[0018] 2) Drawing a standard curve: Take the test solution and copper ion solutions of different known concentrations, mix them evenly, and perform UV-visible absorption and fluorescence spectrum tests respectively, and draw standard curves of the UV-visible absorption and fluorescence spectra of copper ions;

[0019] 3) Testing: Take the test solution and the solution to be tested, mix them evenly, and then perform UV-visible absorption spectroscopy and fluorescence spectroscopy tests respectively. Use the standard curves of the UV-visible absorption spectrum and fluorescence spectrum of copper ions to calculate the concentration of copper ions in the solution to be tested.

[0020] More preferably, in the above application method, the volume ratio of acetonitrile to water in the acetonitrile aqueous solution is 5:5.

[0021] The beneficial effects of the present invention are as follows: the six-membered spirocyclic rhodamine copper ion fluorescent probe containing a benzothiazole hydrazine Schiff base structure constructed by the present invention can undergo a complexation reaction upon encountering copper ions to generate strongly fluorescent bright red rhodamine, and the solution changes from colorless and non-fluorescent to bright red and strongly fluorescent visible to the naked eye, thereby realizing ultraviolet-visible spectrophotometry and fluorescence spectrophotometry detection of metallic copper ions. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The SRh-BT prepared in Example 1 is Cu 2+ UV-visible absorption and fluorescence color response diagram, where a: UV-visible absorption color change; b: fluorescence color change before and after adding copper ions.

[0023] Figure 2 The selectivity of the UV-visible absorption spectrum of SRh-BT prepared in Example 1 to different cations.

[0024] Figure 3 The selectivity of the fluorescence spectrum of SRh-BT prepared in Example 1 to different cations.

[0025] Figure 4 This is a trend diagram of the change of the ultraviolet-visible absorption peak with the copper ion concentration in the copper ion concentration determination of SRh-BT prepared in Example 1 (standard curve of the ultraviolet-visible absorption spectrum of copper ions).

[0026] Figure 5 This is the variation trend of the fluorescence peak along with the copper ion concentration in the copper ion concentration determination of SRh-BT prepared in Example 1 (standard curve of the fluorescence spectrum of copper ions). Specific implementation methods

[0027] Example 1 Six-membered spirocyclic rhodamine copper ion fluorescent probe containing benzothiazole hydrazine Schiff base structure - SRh-BT

[0028] The reaction formula is as follows:

[0029]

[0030] 2-Formylrhodamine B was prepared according to the preparation method provided by invention patent ZL2018109542954.

[0031] In a round-bottom flask, 1 mol of 2-formylrhodamine B and 1 mol of 2-hydrazinebenzothiazole were added to 120 mL of dry ethanol. The mixture was reacted in an 80°C oil bath for 4 hours. After cooling to room temperature, the solvent was removed by distillation under reduced pressure. The resulting solid was dissolved in dichloromethane and purified by silica gel column chromatography with a mobile phase of CH2Cl2:CH3OH = 1:15 to obtain a pale pink solid. This is the six-membered spirorhodamine copper ion fluorescent probe SRh-BT containing a benzothiazole hydrazine Schiff base structure. HRMS: 573.2594.

[0032] Example 2 Copper ion detection test

[0033] This application test was carried out using the SRh-BT fluorescent probe prepared in Example 1.

[0034] (1) Qualitative testing

[0035] 1) Preparation of SRh-BT test solution: Dissolve SRh-BT in acetonitrile aqueous solution (acetonitrile: water = 5:5 by volume) to a concentration of 2×10 -5 SRh-BT test solution of 100 mol / L and pH=7.4.

[0036] 2) Take 3 mL of SRh-BT test solution and add 30 μL of 2×10 -2 mol / L of different cations Fe 2 + 、Fe 3+ 、Al 3+ Mg 2+ 、Na + , Ca 2+ 、Hg 2+ , K + 、Mn 2+ Cr 3+ 、Zn 2+ , Pb 2+ 、Cd 2+ UV-visible spectroscopy and fluorescence spectroscopy were performed respectively.

[0037] like Figure 1As shown in (a), when SRh-BT encounters copper ions, the solution color changes from colorless to red. However, the addition of other cations has no effect on the UV-visible absorption spectrum, such as Figure 2 shown.

[0038] like Figure 1 As shown in (b), when SRh-BT encounters copper ions, the fluorescence color of the solution changes from colorless to red. However, the addition of other cations has no effect on the fluorescence spectrum, such as Figure 3 shown.

[0039] The copper ion detection mechanism of the six-membered spirocyclic rhodamine copper ion fluorescent probe containing a benzothiazole hydrazine Schiff base structure is complexation, which causes the six-membered spirocyclic ring to open to generate bright red and highly fluorescent rhodamine, thereby enabling rapid detection of copper ions. SRh-BT can achieve visible, rapid, and highly sensitive detection of copper ions. Other cations did not cause changes in the UV-visible and fluorescence spectra.

[0040] (2) Quantitative detection

[0041] 1) Preparation of SRh-BT test solution: Dissolve SRh-BT in acetonitrile aqueous solution (acetonitrile: water = 5:5 by volume) to a concentration of 2×10 -5 SRh-BT test solution of 100 mol / L and pH=7.4.

[0042] 2) Draw the standard curve: take 3mL SRh-BT test solution and 30μL 0~4×10 -3 Copper ion solutions with different known concentrations within the mol / L concentration range were mixed evenly and then subjected to UV-visible spectroscopy and fluorescence spectroscopy detection respectively.

[0043] The absorbance at the maximum absorption peak of 560nm in the UV-visible spectrum was selected as the ordinate and the copper ion concentration was selected as the abscissa to draw a standard curve. Figure 4 As shown in the absorption standard curve, the copper ion concentration ranges from 0 to 2.0×10 -5 The relationship is linear in the mol / L range.

[0044] The intensity of the maximum fluorescence emission peak at 596 nm in the fluorescence spectrum was selected as the ordinate and the copper ion concentration was selected as the abscissa to draw a standard curve. Figure 5 As shown in the fluorescence standard curve, the copper ion concentration ranges from 0 to 1.5×10 -5 The relationship is linear in the range of mol / L.

[0045] 3) Mix 3 mL of SRh-BT test solution and 30 μL of a test solution containing copper ions of unknown concentration. Measure the absorbance at the maximum absorption peak of 560 nm in the UV-visible spectrum and the intensity at the maximum fluorescence emission peak of 596 nm in the fluorescence spectrum. Calculate the copper ion concentration in the test solution using the standard curve.

Claims

1. A six-membered spirocyclic rhodamine copper ion fluorescent probe containing a benzothiazole hydrazine Schiff base structure, characterized in that: The six-membered spirocyclic rhodamine copper ion fluorescent probe containing a benzothiazole hydrazine Schiff base structure is SRh-BT, which has the structural formula shown in (I): (Ⅰ).

2. The method for preparing the six-membered spirocyclic rhodamine copper ion fluorescent probe containing a benzothiazole hydrazine Schiff base structure according to claim 1, characterized in that: The method comprises the following steps: dissolving 2-aldehyde rhodamine B and 2-hydrazinebenzothiazole in dry ethanol, heating for reaction, cooling to room temperature, performing reduced pressure distillation, and purifying the obtained solid by column chromatography to obtain a light pink solid, which is a six-membered spirocyclic rhodamine copper ion fluorescent probe containing a benzothiazole hydrazine Schiff base structure.

3. The preparation method according to claim 2, wherein In terms of molar ratio, 2-aldehyde rhodamine B: 2-diaminobenzothiazole = 1:

1.

4. The preparation method according to claim 2, wherein The heating reaction conditions are: 80° C. for 4 h.

5. Use of the six-membered spirocyclic rhodamine copper ion fluorescent probe containing a benzothiazole hydrazine Schiff base structure according to claim 1 in non-disease diagnosis and treatment methods for detecting copper ions.

6. The use according to claim 5, characterized in that The application of the six-membered spirocyclic rhodamine copper ion fluorescent probe containing a benzothiazole hydrazine Schiff base structure in the qualitative detection of copper ions is as follows: the six-membered spirocyclic rhodamine copper ion fluorescent probe containing a benzothiazole hydrazine Schiff base structure is dissolved in an acetonitrile aqueous solution to prepare a concentration of 2×10 -5 ~ 3×10 -4 mol / L, pH 7.4 test solution, add the solution to be tested into the test solution, and observe the change in solution color.

7. The use according to claim 5, characterized in that The application of the six-membered spirocyclic rhodamine copper ion fluorescent probe containing a benzothiazole hydrazine Schiff base structure in the quantitative detection of copper ions is as follows: 1) Prepare the test solution: dissolve the six-membered spirocyclic rhodamine copper ion fluorescent probe containing benzothiazole hydrazine Schiff base structure in acetonitrile aqueous solution to a concentration of 2×10 -5 ~ 3×10 -4 mol / L, pH 7.4 test solution; 2) Draw a standard curve: Take the test solution and copper ion solutions of different known concentrations, mix them evenly, and perform UV-visible absorption and fluorescence spectrum tests respectively. Draw the standard curves of the UV-visible absorption and fluorescence spectra of copper ions; 3) Testing: Take the test solution and the solution to be tested, mix them evenly, and then perform UV-Vis absorption spectroscopy and fluorescence spectroscopy tests respectively. Use the standard curves of the UV-Vis absorption spectrum and fluorescence spectrum of copper ions to calculate the concentration of copper ions in the solution to be tested.

8. The use according to claim 6 or 7, characterized in that In the acetonitrile aqueous solution, the volume ratio of acetonitrile:water=5:5.

Images