Chlorosalicylaldimine-based fluorescent probe and preparation method and application thereof

Abstract

The present application relates to the field of chemical analysis test, specifically relates to a kind of based on chlorosalicyl imine fluorescent probe and its preparation method and application.First, malononitrile and 3-hydroxy-3-methyl-2-butanone are dissolved in ethanol, sodium ethoxide is used as basic condition to promote reaction, and intermediate compound 1 is generated.Then, compound 1 and 4-aminobenzaldehyde are dissolved in ethanol, and ammonium acetate is used as catalyst to react, and compound 2 is obtained.Finally, compound 2 and 4-chloro-2-hydroxybenzaldehyde are dissolved in methanol, and acetic acid is used as acidic reaction condition, and the final purple fluorescent product TCN-1 is generated.The fluorescent probe TCN-1 of the present application can quickly and effectively identify NADH in cetyltrimethylammonium bromide aqueous solution.Through the fluorescence change in the presence of NADH, the concentration of NADH can be quickly detected, which makes the probe have important application prospect in biochemical detection.

Description

Technical Field

[0001] This invention belongs to the field of chemical analysis and testing, specifically relating to a fluorescent probe based on chloresalicylaldehyde imine, its preparation method, and its application. Background Technology

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

[0003] Currently, commonly used methods for NADH detection include enzyme-linked immunosorbent assay (ELISA), nuclear magnetic resonance (NMR), electrochemical methods, and high-performance liquid chromatography (HPLC). Although significant progress has been made in existing detection technologies, many methods are complex to operate and have long detection cycles, making them difficult to meet the needs of real-time detection. In contrast, fluorescence analysis methods, with their advantages of simple operation, rapid detection, and high sensitivity, have become a research hotspot in recent years. Developing efficient, stable, convenient, and real-time suitable fluorescent probe technologies is of great significance. Summary of the Invention

[0004] This invention provides a fluorescent probe TCN-1 based on chloresalidinimine, the structural formula of which is as follows:

[0005]

[0006] This invention also provides a method for preparing a fluorescent probe based on chloresalicylaldehyde imine, wherein the chemical reaction formula for preparing the fluorescent probe is as follows:

[0007]

[0008] The specific preparation method 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. The mixture was refluxed at 80°C for 2.5-3 hours, cooled to room temperature, and the pH of the solution was adjusted to 4-5 with 6N hydrochloric acid, resulting in a large amount of solid. The solid was collected by vacuum filtration, the filter cake was washed with cold ethanol, and dried to obtain light gray crystals, namely intermediate compound 1.

[0010] (2) Compound 1, 4-aminobenzaldehyde, and ammonium acetate were dissolved in ethanol in a molar ratio of 1:1:0.3 to 1:1.1:0.5 and reacted in a single-necked flask at 80°C for 8-12 hours. After the reaction was cooled to room temperature, a large amount of solid precipitated out. The solid was collected by vacuum 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 were dissolved in methanol in 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, a solid precipitated out. The solid was collected by vacuum filtration and the filter cake was washed with cold ethanol and dried to obtain purple crystals, which were the final purple fluorescent product, the fluorescent probe TCN-1.

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

[0013] Beneficial effects

[0014] The raw materials for preparing the fluorescent probe of this invention are readily available, the synthesis method is simple, the reaction conditions are easy to control, and the pure product can be obtained through simple post-processing. The prepared probe TCN-1 solves the problem of rapid recognition of NADH in the CTAB solvent system. In fluorescence detection, after the addition of NADH, the fluorescence intensity of probe TCN-1 at 620 nm significantly decreased, demonstrating its recognition function for NADH in this system; no significant change in fluorescence intensity was observed for other similar substances. Furthermore, probe TCN-1 is relatively sensitive to the solution environment during NADH detection, achieving specific recognition at CTAB aqueous solution concentrations of 0.9 mM–2.0 mM. Attached Figure Description

[0015] Figure 1 The fluorescent probe prepared for Example 1 was used at 1×10 -3 Fluorescence spectra of CTAB at a concentration of mol / L reacting with NADH and analogues in aqueous solution.

[0016] Figure 2 The fluorescent probe prepared for Example 1 was used at 1×10 -3 Fluorescence spectra of CTAB at a concentration of mol / L reacting with different concentrations of NADH in aqueous solution.

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

[0018] Figure 4 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 under different CTAB aqueous solution concentrations and after the addition of NADH. Detailed Implementation

[0020] The present invention will be further described in detail below with reference to specific embodiments:

[0021] Example 1

[0022] (1) Preparation of intermediate 1

[0023] Using ethanol (4.0 mL) as solvent, malononitrile (100 mg, 1.51 mmol) and 3-hydroxy-3-methyl-2-butanone (154 mg, 1.51 mmol) were dissolved in a single-necked flask, and sodium ethoxide (15.7 mg, 0.23 mmol) was added as a basic reagent. The reaction system was heated to 80 °C and refluxed for 2.5 hours. After the reaction was completed, the mixture was cooled to room temperature, and the pH of the solution was adjusted to 4-5 with 6N hydrochloric acid, resulting in the precipitation of a large amount of solid. The precipitate was collected by vacuum filtration, the filter cake was washed with cold ethanol, and dried to obtain 216.6 mg of light gray crystals, i.e., intermediate 1, with a yield of 72%.

[0024] (2) Intermediate 2

[0025] Intermediate 1 (100 mg, 0.5 mmol) and 4-aminobenzaldehyde (60.6 mg, 0.5 mmol) were dissolved in ethanol (4.0 mL) in a single-necked flask, 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 vacuum filtration, the filter cake was washed with cold ethanol, and dried to obtain 119.4 mg of purple crystals, namely intermediate 2, with a yield of 79%.

[0026] (3) Preparation of TCN-1

[0027] Compound 2 (100 mg, 0.33 mmol) and 4-chloro-2-hydroxybenzaldehyde (51.8 mg, 0.33 mmol) were dissolved in methanol (4.0 mL) in a single-necked flask, 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 cooling to room temperature, a large amount of solid precipitated. The precipitate was collected by vacuum filtration, the filter cake was washed with cold ethanol, and dried to obtain 112 mg of purple crystals, which was the final purple fluorescent product TCN-1, with a yield of 77%.

[0028] The application method is as follows: Add 2 μL of a 10 mM NADH aqueous solution or its analogues, 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), and Fe... 2+ Fe 3 + K + Ca 2+ Zn 2+ The solutions were added to 96-well plates, followed by 196 μL of 0.9 mM CTAB aqueous solution and 2 μL of 1×10⁻⁶ CTAB aqueous solution. -3 A DMSO solution of probe TCN-1 at a concentration of mol / L.

[0029] Meanwhile, a probe solution without NADH was used as a blank control. After thorough mixing, the fluorescence intensity of each well was measured using a microplate reader.

[0030] Figure 1 The experiment demonstrated the effectiveness of the probe in reacting with NADH and its analogues (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), and Fe) in CTAB aqueous solution. 2+ Fe 3+ K + Ca 2+ Zn 2+ The fluorescence spectrum after the reaction showed the changes in probe fluorescence intensity. The results indicated that when 10 mM NADH aqueous solution was added, the fluorescence intensity of the probe solution at 620 nm decreased significantly, only 1 / 30 of the blank control. In contrast, the fluorescence intensity of the probe in CTAB aqueous solution did not change significantly for other analogs. This indicates that the probe has a high selective recognition effect on NADH in the CTAB aqueous solution system.

[0031] Figure 2 The fluorescent probe prepared in Example 1 (concentration of 1×10⁻⁶) is shown. -3The fluorescence spectra of the probe solution (0.9 mM CTAB aqueous solution, mol / L) after interaction with different concentrations of NADH were obtained. The results showed that the fluorescence intensity of the probe solution gradually decreased with increasing NADH concentration. Even at a NADH concentration as low as 5 μM, the fluorescence peak at 620 nm was still significantly different from the background fluorescence curve without added NADH. This indicates that the fluorescent probe has a low detection limit and high sensitivity for NADH.

[0032] Figure 3 The fluorescent probe prepared in Example 1 (concentration of 1×10⁻⁶) is shown. -3 The fluorescence intensity at 620 nm was observed after adding NADH (final concentration: 100 nM) and its analogues (final concentration: 1 mM) to a 0.9 mM CTAB aqueous solution, and after considering the interference of NADH with other interfering substances. When only NADH solution was added, the fluorescence intensity of the probe at 620 nm was significantly reduced compared to the blank control. However, the fluorescence intensity of other analogues (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...) was significantly reduced. 2+ Fe 3+ K + Ca 2+ Zn 2+ The fluorescence intensity did not change significantly after the action. Furthermore, when interfering substances were added to the detection system, the fluorescence intensity of the probe at 620 nm remained stable, indicating that the detection results of the probe were not significantly affected.

[0033] Figure 4 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 a 1×10⁻⁶ solution to each well of a 96-well plate. -2Twelve parallel groups were prepared using 196 μL of 12 different concentrations of CTAB aqueous solution (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-1DMSO solution (concentration 1.0 × 10⁻⁶). -3 The solution in each well was mixed thoroughly (mol / L), and the fluorescence intensity of the solution in each well was detected by an ELISA reader.

[0037] Figure 5 The changes in fluorescence intensity at 620 nm of the fluorescent probe TCN-1 prepared in Example 1 in different concentrations of CTAB aqueous solution and after the addition of NADH are shown. The results show that the fluorescence intensity of the fluorescent probe TCN-1 gradually increases with increasing CTAB aqueous solution concentration, and shows a significant increase after 0.9 mM, indicating that the concentration of CTAB aqueous solution has a significant impact on the fluorescence response of the probe. Before and after the addition of NADH, the fluorescence intensity of TCN-1 changes significantly within the range of CTAB aqueous solution concentration from 0.9 mM to 2.0 mM. The x-axis represents the CTAB aqueous solution concentration, and the y-axis represents the TCN-1 fluorescence intensity.

[0038] Example 3

[0039] (1) Preparation of compound 1

[0040] Using ethanol (8.0 mL) as solvent, malononitrile (200 mg, 3.02 mmol) and 3-hydroxy-3-methyl-2-butanone (308 mg, 3.02 mmol) were dissolved in a single-necked flask, and sodium ethoxide (31.4 mg, 0.46 mmol) was added as a basic reagent. The reaction system was heated to 80 °C and refluxed for 3 hours. After the reaction was completed, the mixture was cooled to room temperature, and the pH of the solution was adjusted to 4-5 with 6N hydrochloric acid, resulting in the precipitation of a large amount of solid. The precipitate was collected by vacuum filtration, the filter cake was washed with cold ethanol, and dried to obtain 457.2 mg of light gray crystals, which is intermediate 1, with a yield of 76%.

[0041] (2) Preparation of compound 2

[0042] Intermediate 1 (200 mg, 1.0 mmol) and 4-aminobenzaldehyde (133.2 mg, 1.1 mmol) were dissolved in ethanol (8.0 mL) in a single-necked flask, with ammonium acetate (25.4 mg, 0.33 mmol) 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 vacuum filtration, the filter cake was washed with cold ethanol, and dried to give 244.7 mg of purple crystals, namely compound 2, with a yield of 81%.

[0043] (3) Preparation of TCN-1

[0044] Compound 2 (200 mg, 0.66 mmol) and 4-chloro-2-hydroxybenzaldehyde (114.3 mg, 0.73 mmol) were dissolved in methanol (8.0 mL) in a single-necked flask, and acetic acid (43.8 mg, 0.73 mmol) was added as an acidic reagent. The reaction system was heated to 70 °C and reacted for 10 hours. After cooling to room temperature, a large amount of solid precipitated. The precipitate was collected by vacuum filtration, the filter cake was washed with cold ethanol, and dried to obtain 241.5 mg of purple crystals, which was 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] Intermediate 1 (200 mg, 1.0 mmol) and 4-aminobenzaldehyde (133.2 mg, 1.1 mmol) were dissolved in ethanol (8.0 mL) in a single-necked flask, 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 vacuum filtration, the filter cake was washed with cold ethanol, and dried to give 241.8 mg of purple crystals, namely compound 2, with a yield of 80%.

[0050] (3) Preparation of TCN-1

[0051] Compound 2 (200 mg, 0.66 mmol) and 4-chloro-2-hydroxybenzaldehyde (114.3 mg, 0.73 mmol) were dissolved in methanol (8.0 mL) in a single-necked flask, 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 cooling to room temperature, a large amount of solid precipitated. The precipitate was collected by vacuum filtration, the filter cake was washed with cold ethanol, and dried to obtain 230.0 mg of purple crystals, which was the final purple fluorescent product TCN-1, with a yield of 79%.

Claims

1. A fluorescent probe based on chloresalicylaldehyde imine, characterized in that: The structural formula of the chloresalidin imine-based fluorescent probe is as follows: 。 2. A method for preparing a fluorescent probe based on chloresalidin and imine as described in claim 1, characterized in that: The preparation method steps are as follows: (1) Using ethanol as a solvent, malononitrile and 3-hydroxy-3-methyl-2-butanone were dissolved in a single-necked flask. Sodium ethoxide was added as an alkaline condition, and the mixture was heated to 80°C and refluxed for 2.5-3 hours. After cooling to room temperature, the solution was adjusted to pH 4-5 with 6N HCl. The solid precipitated out. After filtration, the filter cake was washed with cold ethanol and dried to obtain light gray crystals, i.e., intermediate compound 1, whose structural formula is as follows: ； (2) Using ethanol as a solvent, compound 1 and 4-aminobenzaldehyde were dissolved in a single-necked flask. Ammonium acetate was added as a catalyst and the mixture was heated to 80°C for 8-12 hours. After cooling to room temperature, a solid precipitated out. The solid was filtered and the filter cake was washed with cold ethanol. After drying, purple crystals, namely compound 2, were obtained. Its structural formula is as follows: ； (3) Using methanol as solvent, compound 2 and 4-chloro-2-hydroxybenzaldehyde were dissolved in a single-necked flask. Acetic acid was added as an acidic reaction condition. The mixture was heated to 70°C and reacted for 8-12 hours. After cooling to room temperature, a solid precipitated out. The filter cake was washed with cold ethanol after filtration and dried to obtain purple crystals, which is the final purple fluorescent product TCN-1. Its structural formula is as follows: 。 3. The preparation method of the fluorescent probe based on chloresalicylaldehyde imine as described in 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 preparation method of the fluorescent probe based on chloresalicylaldehyde imine as described in claim 2, characterized in that: In step (2), the molar ratio of compounds 1, 4-aminobenzaldehyde and ammonium acetate is 1:1:0.3 to 1:1.1:0.

5.

5. The preparation method of the fluorescent probe based on chloresalicylaldehyde imine as described in 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 chloresalidin-imine fluorescent probe as described in claim 1, characterized in that: The fluorescent probe is used to prepare a reagent for the in vitro detection of reduced coenzyme I NADH.

7. The application of the chloresalicylaldehyde imine-based fluorescent probe as described in claim 6, characterized in that: The fluorescent probe is used to prepare a reagent for detecting NADH in CTAB aqueous solution by fluorescence quenching.

8. The application of the chloresalidin-based imine fluorescent probe as described in claim 7, characterized in that: The concentration of the CTAB aqueous solution is 0.9 mM-2.0 mM.

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