Fluorescent probe based on chlorosalicylaldehyde imine as well as preparation method and application of fluorescent probe

Abstract

The invention relates to the field of chemical analysis and testing, in particular to a fluorescent probe based on chlorosalicylaldehyde imine as well as a preparation method and application of the fluorescent probe. The preparation method comprises the following steps: firstly, dissolving malononitrile and 3-hydroxy-3-methyl-2-butanone in ethanol, and promoting reaction by using sodium ethoxide as an alkaline condition to generate an intermediate compound 1; then, dissolving the compound 1 and 4-aminobenzaldehyde in ethanol, and reacting by taking ammonium acetate as a catalyst to obtain a compound 2; and finally, dissolving the compound 2 and 4-chloro-2-hydroxybenzaldehyde in methanol, and generating a final purple fluorescent product TCN-1 by using acetic acid as an acidic reaction condition. The fluorescent probe TCN-1 disclosed by the invention can be used for quickly and effectively identifying NADH (Nicotinamide Adenine Dinucleotide Hormone) in a hexadecyl trimethyl ammonium bromide aqueous solution. The concentration of the NADH can be rapidly detected through fluorescence change in the presence of the NADH, so that the probe has an important application prospect in the aspect of biochemical detection.

Description

Technical Field

[0001] The invention belongs to the field of chemical analysis and testing, and specifically relates to a chlorosalicylaldehyde imine-based fluorescent probe and a preparation method and application thereof. Background Art

[0002] Reduced coenzyme I (NADH) is an important coenzyme that plays a key role in redox reactions in cells. NADH is the reduced form of nicotinamide adenine dinucleotide (NAD+), which is produced through metabolic pathways such as the tricarboxylic acid cycle and glycolysis. It is an important carrier of intracellular electron transfer and a key molecule that drives oxidative phosphorylation to generate ATP in the mitochondrial respiratory chain. Therefore, rapid and sensitive detection of NADH concentration is of great significance for studying cell metabolic mechanisms and disease diagnosis.

[0003] At present, commonly used NADH detection methods include: enzyme-linked immunosorbent assay, nuclear magnetic resonance, electrochemical method, and high-performance liquid chromatography. Although existing detection technologies have made significant progress, many methods are complex to operate and have long detection cycles, making it difficult to meet the needs of real-time detection. In contrast, fluorescence analysis has become a research hotspot in recent years due to its advantages such as simple operation, rapid and sensitive detection. It is of great significance to develop efficient, stable, convenient and suitable fluorescent probe technology for real-time detection. Summary of the invention

[0004] The present invention provides a chlorosalicylaldehyde imine fluorescent probe TCN-1, the structural formula of the fluorescent probe is:

[0005]

[0006] The present invention also provides a method for preparing a chlorosalicylaldehyde imine fluorescent probe, and the chemical reaction formula for preparing the fluorescent probe is:

[0007]

[0008] The specific preparation steps are as follows:

[0009] (1) Malononitrile, 3-hydroxy-3-methyl-2-butanone and sodium ethoxide were dissolved in ethanol in a molar ratio of 1:1:0.15, and refluxed at 80°C for 2.5-3 hours, then cooled to room temperature, and the pH of the solution was adjusted to 4-5 with 6N hydrochloric acid to produce a large amount of solid. The solid was collected by suction filtration, and the filter cake was washed with cold ethanol and dried to obtain light gray crystals, namely the intermediate compound 1.

[0010] (2) Compound 1, 4-aminobenzaldehyde and ammonium acetate were dissolved in ethanol at a molar ratio of 1:1:0.3 to 1:1.1:0.5, and reacted in a single-mouth bottle at 80°C for 8-12 hours. After the reaction was cooled to room temperature, a large amount of solid precipitated, which was collected by suction filtration, and the filter cake was washed with cold ethanol and dried to obtain purple crystals, namely compound 2.

[0011] (3) Compound 2, 4-chloro-2-hydroxybenzaldehyde and acetic acid are dissolved in methanol at a molar ratio of 1:1:1.1 to 1:1.1:1.5, and reacted at 70°C for 8-12 hours. After cooling to room temperature, solids precipitate, which are collected by suction filtration and the filter cake is washed with cold ethanol, and dried to obtain purple crystals, i.e., the final purple fluorescent product fluorescent probe TCN-1.

[0012] The present invention also provides an application of the fluorescent probe: the prepared fluorescent probe TCN-1 can quickly identify NADH in a CTAB aqueous solution (0.9 mM-2.0 mM).

[0013] Beneficial Effects

[0014] The raw materials for preparing the fluorescent probe of the present invention are easy to obtain, the synthesis method is simple, the reaction conditions are easy to control, and the pure product can be obtained by simple post-treatment. The prepared probe TCN-1 solves the problem of rapid identification of NADH in the CTAB solvent system. In the fluorescence detection, after adding NADH, the fluorescence intensity of the probe TCN-1 at 620nm is significantly reduced, reflecting its recognition effect on NADH in the system, and no significant change in fluorescence intensity is observed for other analogs. At the same time, the probe TCN-1 is sensitive to the solution environment during the detection of NADH, and specific identification can be achieved when the concentration of the CTAB aqueous solution is 0.9mM-2.0mM. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The fluorescent probe prepared in Example 1 was 1×10 -3 mol / L concentration of fluorescence spectra after the reaction with NADH and analogues in CTAB aqueous solution.

[0016] Figure 2 The fluorescent probe prepared in Example 1 was 1×10 -3 mol / L concentration in CTAB aqueous solution after the reaction with different concentrations of NADH.

[0017] Figure 3 The fluorescent probe prepared in Example 1 was 1×10 -3 mol / L concentration of NADH in CTAB aqueous solution and in the presence of interfering substances.

[0018] Figure 4 This is the hydrogen spectrum of the fluorescent probe prepared in Example 1.

[0019] Figure 5 The fluorescence intensity changes of the fluorescent probe prepared in Example 1 at different CTAB aqueous solution concentrations and after adding NADH. DETAILED DESCRIPTION

[0020] The present invention is further described in detail below in conjunction with specific embodiments:

[0021] Example 1

[0022] (1) Preparation of Intermediate 1

[0023] Using ethanol (4.0mL) as solvent, dissolve malononitrile (100mg, 1.51mmol) and 3-hydroxy-3-methyl-2-butanone (154mg, 1.51mmol) in a single-mouth bottle, and add sodium ethoxide (15.7mg, 0.23mmol) as an alkaline reagent. Heat the reaction system to 80°C and reflux for 2.5 hours. After the reaction is completed, cool to room temperature, adjust the solution pH to 4-5 with 6N hydrochloric acid, and a large amount of solid precipitates. Collect the precipitate by suction filtration, wash the filter cake with cold ethanol, and dry to obtain 216.6mg of light gray crystals, namely intermediate 1, with a yield of 72%.

[0024] (2) Intermediate 2

[0025] Using ethanol (4.0 mL) as solvent, intermediate 1 (100 mg, 0.5 mmol) and 4-aminobenzaldehyde (60.6 mg, 0.5 mmol) were dissolved in a single-mouth bottle, and ammonium acetate (11.6 mg, 0.15 mmol) was added as a catalyst. The reaction system was heated to 80 ° C and reacted for 8 hours. After cooling to room temperature, a large amount of solid precipitated. The precipitate was collected by suction filtration, and the filter cake was washed with cold ethanol. After drying, 119.4 mg of purple crystals, namely intermediate 2, were obtained, with a yield of 79%.

[0026] (3) Preparation of TCN-1

[0027] Using methanol (4.0 mL) as solvent, compound 2 (100 mg, 0.33 mmol) and 4-chloro-2-hydroxybenzaldehyde (51.8 mg, 0.33 mmol) were dissolved in a single-mouth bottle, and acetic acid (21.6 mg, 0.36 mmol) was added as an acidic reagent. The reaction system was heated to 70 ° C and reacted for 8 hours. After the reaction was cooled to room temperature, a large amount of solid precipitated. The precipitate was collected by suction filtration, the filter cake was washed with cold ethanol, and 112 mg of purple crystals were obtained after drying, i.e., the final purple fluorescent product TCN-1, with a yield of 77%.

[0028] The application method is as follows: 2 μL of 10 mM aqueous solution of NADH and its analogs, oxidized nicotinamide adenine dinucleotide (NAD+), human serum albumin (HSA), adenosine triphosphate (ATP), guanosine triphosphate (GTP), reduced glutathione (GSH), oxidized glutathione (GSSG), glutamine (Gln), cysteine ​​(Cys), arginine (Arg), Fe 2+ , Fe 3 + , K + , Ca 2+ 、Zn 2+ The solutions were added to the 96-well plate, followed by 196 μL of 0.9 mM CTAB aqueous solution and 2 μL of 1×10 -3 mol / L DMSO solution of probe TCN-1.

[0029] At the same time, a probe solution without NADH was used as a blank control (Blank). After mixing evenly, the fluorescence intensity of each well solution was detected by an ELISA reader.

[0030] Figure 1 The probes were shown to bind to NADH and its analogs (adenine dinucleotide (NAD+), human serum albumin (HSA), adenosine triphosphate (ATP), guanosine triphosphate (GTP), reduced glutathione (GSH), oxidized glutathione (GSSG), glutamine (Gln), cysteine ​​(Cys), arginine (Arg), Fe 2+ , Fe 3+ , K + , Ca 2+ 、Zn 2+ ) The fluorescence spectrum after the reaction shows the change of the fluorescence intensity of the probe. The results show that when 10mM NADH aqueous solution is added, the fluorescence intensity of the probe solution at 620nm is significantly reduced, which is only 1 / 30 of the blank control. In contrast, the fluorescence intensity of the probe for other analogs in CTAB aqueous solution does not change significantly. This shows that the probe has a highly selective recognition effect on NADH in the CTAB aqueous solution system.

[0031] Figure 2 The fluorescent probe prepared in Example 1 (concentration of 1×10 -3The fluorescence spectra of the probe solution after the reaction of NADH with different concentrations of NADH in a 0.9 mM CTAB aqueous solution were shown in Figure 2. The results show that the fluorescence intensity of the probe solution gradually decreases with the increase of NADH concentration. When the NADH concentration is as low as 5 μM, the fluorescence peak at 620 nm can still be significantly distinguished from the background fluorescence curve without the addition of NADH. This shows that the fluorescent probe has a low detection limit for NADH and high sensitivity.

[0032] Figure 3 The fluorescent probe prepared in Example 1 (concentration of 1×10 -3 NADH (final concentration: 100 nM) and analogs (final concentration: 1 mM) were added to 0.9 mM CTAB aqueous solution, as well as the changes in fluorescence intensity at 620 nm under the influence of other interfering substances. When only NADH solution was added, the fluorescence intensity of the probe at 620 nm was significantly reduced compared with the blank control. For other analogs (adenine dinucleotide (NAD+), human serum albumin (HSA), adenosine triphosphate (ATP), guanosine triphosphate (GTP), reduced glutathione (GSH), oxidized glutathione (GSSG), glutamine (Gln), cysteine ​​(Cys), arginine (Arg), Fe 2+ , Fe 3+ , K + , Ca 2+ 、Zn 2+ ) after the action of the probe. In addition, when the interfering substance was added to the detection system, the fluorescence intensity of the probe at 620nm remained stable, indicating that the detection result of the probe was not significantly affected.

[0033] Figure 4 This is the hydrogen spectrum of the fluorescent probe prepared in Example 1.

[0034] 1 H NMR (300MHz, DMSO-d6) δ13.13(s,1H),9.06(s,1H),8.03(d,J=8.3Hz,2H),7.97(d,J=16.4Hz,1H),7.75(d,J=8.9Hz,1 H), 7.56 (d, J = 8.2Hz, 2H), 7.26 (d, J = 16.4Hz, 1H), 7.09 (s, 1H), 7.06 (d, J = 2.1Hz, 1H), 6.88-6.77 (m, 1H), 1.82 (s, 6H).

[0035] Example 2

[0036] Add 2 μL of 1×10 -2196 μL of 12 groups of CTAB aqueous solutions with different concentrations (0 mM, 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1.0 mM, 2.0 mM) and 2 μL of fluorescent probe TCN-1 DMSO solution (concentration of 1.0 × 10 -3 mol / L), mix the solutions in each well evenly, and detect the fluorescence intensity of the solution in each well by an enzyme-labeled instrument.

[0037] Figure 5 The fluorescence intensity change of the fluorescent probe TCN-1 prepared in Example 1 at 620nm in different concentrations of CTAB aqueous solution and after adding NADH is shown. The results show that the fluorescence intensity of the fluorescent probe TCN-1 gradually increases with the increase of the concentration of the CTAB aqueous solution, and a significant increase occurs after 0.9mM, indicating that the concentration of the CTAB aqueous solution has an important influence on the fluorescence response of the probe. Before and after the addition of NADH, the intensity of TCN-1 fluorescence changes significantly in the range of 0.9mM-2.0mM of the CTAB aqueous solution concentration before and after the addition of NADH, where the horizontal axis is the concentration of the CTAB aqueous solution and the vertical axis is the TCN-1 fluorescence intensity.

[0038] Example 3

[0039] (1) Preparation of Compound 1

[0040] Using ethanol (8.0mL) as solvent, dissolve malononitrile (200mg, 3.02mmol) and 3-hydroxy-3-methyl-2-butanone (308mg, 3.02mmol) in a single-mouth bottle, and add sodium ethoxide (31.4mg, 0.46mmol) as an alkaline reagent. Heat the reaction system to 80°C and reflux for 3 hours. After the reaction is completed, cool to room temperature, adjust the solution pH to 4-5 with 6N hydrochloric acid, and a large amount of solid precipitates. Collect the precipitate by suction filtration, wash the filter cake with cold ethanol, and dry to obtain 457.2mg of light gray crystals, namely intermediate 1, with a yield of 76%.

[0041] (2) Preparation of Compound 2

[0042] Using ethanol (8.0 mL) as solvent, intermediate 1 (200 mg, 1.0 mmol) and 4-aminobenzaldehyde (133.2 mg, 1.1 mmol) were dissolved in a single-mouth bottle, and ammonium acetate (25.4 mg, 0.33 mmol) was added as a catalyst. The reaction system was heated to 80 ° C and reacted for 10 hours. After cooling to room temperature, a large amount of solid precipitated. The precipitate was collected by suction filtration, the filter cake was washed with cold ethanol, and 244.7 mg of purple crystals, i.e., compound 2, were obtained after drying, with a yield of 81%.

[0043] (3) Preparation of TCN-1

[0044] Using methanol (8.0mL) as solvent, compound 2 (200mg, 0.66mmol) and 4-chloro-2-hydroxybenzaldehyde (114.3mg, 0.73mmol) were dissolved in a single-mouth bottle, and acetic acid (43.8mg, 0.73mmol) was added as an acidic reagent. The reaction system was heated to 70°C and reacted for 10 hours. After the reaction was cooled to room temperature, a large amount of solid precipitated. The precipitate was collected by suction filtration, the filter cake was washed with cold ethanol, and 241.5mg of purple crystals were obtained after drying, i.e., the final purple fluorescent product TCN-1, with a yield of 83%.

[0045] Example 4

[0046] (1) Preparation of Compound 1

[0047] This embodiment is the same as (1) in Embodiment 3.

[0048] (2) Preparation of Compound 2

[0049] Using ethanol (8.0 mL) as solvent, intermediate 1 (200 mg, 1.0 mmol) and 4-aminobenzaldehyde (133.2 mg, 1.1 mmol) were dissolved in a single-mouth bottle, and ammonium acetate (38.5 mg, 0.5 mmol) was added as a catalyst. The reaction system was heated to 80 ° C and reacted for 12 hours. After cooling to room temperature, a large amount of solid precipitated. The precipitate was collected by suction filtration, the filter cake was washed with cold ethanol, and 241.8 mg of purple crystals, i.e., compound 2, were obtained after drying, with a yield of 80%.

[0050] (3) Preparation of TCN-1

[0051] Using methanol (8.0 mL) as solvent, compound 2 (200 mg, 0.66 mmol) and 4-chloro-2-hydroxybenzaldehyde (114.3 mg, 0.73 mmol) were dissolved in a single-mouth bottle, and acetic acid (59.4 mg, 0.99 mmol) was added as an acidic reagent. The reaction system was heated to 70 ° C and reacted for 12 hours. After the reaction was cooled to room temperature, a large amount of solid precipitated. The precipitate was collected by suction filtration, the filter cake was washed with cold ethanol, and 230.0 mg of purple crystals were obtained after drying, i.e., the final purple fluorescent product TCN-1, with a yield of 79%.

Claims

1. A fluorescent probe based on chlorosalicylaldehyde imine, characterized in that: The structural formula of the chlorosalicylaldehyde imine fluorescent probe is as follows:

2. A method for preparing a chlorosalicylaldehyde imine fluorescent probe according to claim 1, characterized in that: The preparation method steps are as follows: (1) Using ethanol as solvent, dissolving malononitrile and 3-hydroxy-3-methyl-2-butanone in a single-mouth bottle, adding sodium ethoxide as alkaline condition, heating to 80° C. and reflux for 2.5-3 hours, then cooling to room temperature, adjusting the solution to pH 4-5 with 6N HCl, solid precipitated, and after suction filtration, eluting the filter cake with cold ethanol, and drying to obtain light gray crystals, namely intermediate compound 1; (2) Using ethanol as solvent, compound 1 and 4-aminobenzaldehyde were dissolved in a single-mouth bottle, ammonium acetate was added as a catalyst, and the mixture was heated to 80°C for reaction for 8-12 hours. The mixture was cooled to room temperature, and solid was precipitated. After filtration, the filter cake was rinsed with cold ethanol and dried to obtain purple crystals, namely compound 2; (3) Using methanol as solvent, compound 2 and 4-chloro-2-hydroxybenzaldehyde were dissolved in a single-mouth bottle, acetic acid was added as an acidic reaction condition, heated to 70°C for reaction for 8-12 hours, cooled to room temperature, solid precipitated, filtered and then rinsed with cold ethanol to obtain purple crystals, i.e., the final purple fluorescent product TCN-1.

3. The method for preparing a chlorosalicylaldehyde imine fluorescent probe according to claim 2, characterized in that: In step (1), the molar ratio of malononitrile, 3-hydroxy-3-methyl-2-butanone and sodium ethoxide is 1:1:0.

15.

4. The method for preparing a chlorosalicylaldehyde imine fluorescent probe according to claim 2, characterized in that: In step (2), the molar ratio of compound 1, 4-aminobenzaldehyde and ammonium acetate is 1:1:0.3 to 1:1.1:0.

5.

5. The method for preparing a chlorosalicylaldehyde imine fluorescent probe according to claim 2, characterized in that: In step (3), the molar ratio of compound 2, 4-chloro-2-hydroxybenzaldehyde and acetic acid is 1:1:1.1 to 1:1.1:1.

5.

6. An application of the chlorosalicylaldehyde imine fluorescent probe as claimed in claim 1, characterized in that: The fluorescent probe is used for detecting reduced coenzyme I NADH in vitro.

7. The use of the chlorosalicylaldehyde imine fluorescent probe according to claim 6, characterized in that: The fluorescent probe detects NADH in a CTAB aqueous solution by means of fluorescence quenching.

8. The use of the chlorosalicylaldehyde imine fluorescent probe according to claim 7, characterized in that: The concentration of the CTAB aqueous solution is 0.9 mM-2.0 mM.

Images