Fluorescent probe for identifying reduced coenzyme I and preparation method and application thereof
By developing a coumarin-based fluorescent probe based on aminoindole, the problem of dependence on reducing Coenzyme I detection equipment and high sample pretreatment requirements in the prior art is solved, and the effect of high sensitivity and selective recognition of reducing Coenzyme I in PBS phosphate buffer solution is achieved.
Patent Information
- Application Number
- CN202310006002.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-01-04
- Publication Date
- 2025-05-02
- Estimated Expiration
- 2043-01-04
AI Technical Summary
The existing detection methods are equipment-dependent for reducing Coenzyme I (NADH) detection, high sample pretreatment requirements, and lack of high sensitivity and selectivity fluorescence analysis methods.
A fluorescent probe based on aminoindole was developed. By reacting 7-(diethylamino)coumarin-3-formic acid with compounds such as 5-aminoindole under specific conditions, a fluorescent probe capable of specifically identifying reducing Coenzyme I in PBS phosphate buffer solution was prepared.
The fluorescent probe exhibits high sensitivity and selectivity in PBS phosphate buffered solution, which significantly enhances fluorescence intensity and specifically recognizes reducing Coenzyme I, simplifying the detection process and reducing the requirements for equipment and sample pretreatment.
Smart Images
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Abstract
Description
Technical Field
[0001] The invention belongs to the field of chemical analysis and testing, and in particular relates to a fluorescent probe for identifying reduced coenzyme I and a preparation method and application thereof. Background Art
[0002] Reduced coenzyme I (NADH) is the structure of reduced nicotinamide adenine dinucleotide, which plays the role of hydrogen donor in many chemical reactions in organisms and is of great significance. NADH is produced in the citric acid cycle in glycolysis and cellular respiration, and is a control marker in the energy production chain in mitochondria. It has the functions of improving energy levels, protecting cells, and promoting the production of neurotransmitters.
[0003] At present, the commonly used detection methods of NADH include: ultraviolet spectrophotometry, electrochemical analysis, chromatography-mass spectrometry, etc. These methods require large-scale instruments and equipment, and have high requirements for sample pretreatment and require more professional personnel. Fluorescence analysis methods have the advantages of high sensitivity, good selectivity, simple operation, and easy visualization, and have received widespread attention. However, there are currently no reports on the application of fluorescence analysis to detect reduced coenzyme I. Summary of the invention
[0004] The invention provides an aminoindole-based coumarin fluorescent probe capable of identifying reduced coenzyme I, and a preparation method and application thereof.
[0005] The structural formula of the fluorescent probe is:
[0006]
[0007] The preparation method of the aminoindole-based coumarin fluorescent probe is as follows: 7-(diethylamino)coumarin-3-carboxylic acid is dissolved in dichloromethane, and a condensation agent 1-hydroxybenzotriazole (HOBT), 1-ethyl-(3-dimethylaminoaldehyde)carbodiimide hydrochloride (EDCI) and a trace amount of triethylamine are added under ice bath conditions and stirred for 10 minutes, followed by adding 5-aminoindole and stirring at room temperature for 10-20 hours. After the reaction is completed, the solvent is removed under reduced pressure and a yellow solid is obtained by column chromatography.
[0008] The molar equivalent ratio of 7-(diethylamino)coumarin-3-carboxylic acid, condensation agent 1-hydroxybenzotriazole (HOBT), 1-ethyl-(3-dimethylaminoaldehyde)carbodiimide hydrochloride (EDCI), triethylamine and 5-aminoindole is 1:1.2-1.5:1.0 -1.5:0.1-0.2:1.2.
[0009] The aminoindole-based coumarin fluorescent probe specifically recognizes reduced coenzyme I in a PBS phosphate buffer solution.
[0010] The concentration of the fluorescent probe in PBS phosphate buffer solution is 10 μM, and the pH of the PBS phosphate buffer solution is 7.2.
[0011] The beneficial effects of the present invention are as follows: the raw materials of the present invention are easily available, the synthesis method is relatively simple, the reaction conditions are easy to control, and after the reaction is completed, a pure product can be obtained through simple post-treatment.
[0012] In the PBS phosphate buffer solution system, coumarin fluorescent probes have high sensitivity and good selectivity and can specifically recognize reduced coenzyme I. BRIEF DESCRIPTION OF THE DRAWINGS
[0013] Figure 1 This is the absorption spectrum of the coumarin fluorescent probe prepared in Example 1 in different solvents.
[0014] Figure 2 The fluorescence spectra of the coumarin fluorescent probe prepared in Example 1 after reacting with different proteins in ethanol solution: hemoglobin (Hb), oxidized coenzyme (NAD), reduced coenzyme I (NADH), cyclic adenosine monophosphate (cAMP), adenosine triphosphate (ATP), guanosine triphosphate (GTP), oxidized glutathione (GSSG), glutathione (GSH), insulin, and protamine.
[0015] Figure 3 The fluorescence spectra of the coumarin fluorescent probe prepared in Example 1 after reacting with different proteins in PBS phosphate buffer solution: hemoglobin (Hb), oxidized coenzyme (NAD), reduced coenzyme I (NADH), cyclic adenosine monophosphate (cAMP), adenosine triphosphate (ATP), guanosine triphosphate (GTP), oxidized glutathione (GSSG), glutathione (GSH), insulin, and protamine.
[0016] Figure 4 Fluorescence spectra of the coumarin fluorescent probe prepared in Example 1 before and after the reaction with reduced coenzyme I (NADH) in PBS phosphate buffer solutions of different pH values (XDS79 is the code of the prepared fluorescent reagent).
[0017] Figure 5 This is the hydrogen spectrum of the aminoindole-based coumarin fluorescent probe prepared in Example 1. DETAILED DESCRIPTION
[0018] The present invention is described in detail below in conjunction with specific implementation modes.
[0019] Example 1
[0020] 7-(Diethylamino)coumarin-3-carboxylic acid (0.2613 g, 1 mmol) was dissolved in dichloromethane (10 mL), and condensing agent 1-hydroxybenzotriazole (HOBT) (0.1621 g, 1.2 mmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) (0.1918 g, 1 mmol) and triethylamine (0.0101 g, 0.1 mmol) were added under ice bath conditions and stirred for 10 minutes, followed by addition of 5-aminoindole (0.1586 g, 1.2 mmol), and stirring at room temperature for 10 hours. After completion of the reaction, the solvent was removed under reduced pressure, and the product was separated and purified by column chromatography, the eluent being a mixed solution of dichloromethane and methanol (volume ratio of DCM:MeOH=20:1), to obtain 0.2326 g of a yellow solid with a yield of 62%.
[0021] The specific application method is: Absorption of coumarin fluorescent probes in different solvents. First, 198 μL of different solvents were added to a 96-well plate in sequence, and then 2 μL of indole-based coumarin fluorescent probe (1 mM concentration, final concentration in the well was 10 μM) was added to each well, so that the volume of the solution in each well was 200 μL, and the absorption wavelength and absorbance intensity of the solution in each well were measured.
[0022] Fluorescence recognition of reduced coenzyme I (NADH) by coumarin fluorescent probe in PBS phosphate buffer solution. First add 196uL PBS phosphate buffer solution (concentration of 10mM, pH = 7.2) to the 96-well plate, then add 2μL of indole-based coumarin fluorescent probe (concentration of 1mM, final concentration in the well is 10μM), mix the solution in each well, and finally add 2μL of different protein solutions (concentration of 10mg / mL, final concentration in the well is 100μg / mL), mix again, and use an enzyme reader to measure the fluorescence intensity of the solution in each well. When the final concentration of the fluorescent probe is less than 10μM, the absorbance intensity and corresponding fluorescence intensity of the probe are weak, which is not conducive to detection; and if the final concentration of the probe is too large, it will cause the absorbance intensity and fluorescence intensity to be oversaturated, affecting the accuracy of the probe detection.
[0023] Effect of PBS phosphate buffer solutions with different pH values on the fluorescent recognition of reduced coenzyme I (NADH) by coumarin fluorescent probes. PBS buffer solutions with different pH values (concentration of 10mM, pH=7.2) were added to the 96-well plate in sequence, and then 2μL of indole-based coumarin fluorescent probes (concentration of 1mM, final concentration of 10μM in the well) were added, and the solution in each well was mixed, and finally 2μL of NADH protein solution (concentration of 10mg / mL, final concentration of 100μg / mL in the well) was added, mixed again, and the fluorescence intensity of the solution in each well was measured. At the same time, the coumarin fluorescent probe solution without NADH protein solution was used as a blank control.
[0024] Figure 1 The absorption spectra of the coumarin fluorescent probe prepared in Example 1 in different solvent systems are shown in the figure. The figure shows that the fluorescent reagent (10 μM) has strong absorption at 425-435 nm in water phase, buffer solution and ethanol.
[0025] Figure 2 Fluorescence spectra of the coumarin fluorescent probe prepared for implementation example 1 before and after the action with different proteins in ethanol solution: hemoglobin (Hb), oxidized coenzyme (NAD), reduced coenzyme I (NADH), cyclic adenosine monophosphate (cAMP), adenosine triphosphate (ATP), guanosine triphosphate (GTP), oxidized glutathione (GSSG), glutathione (GSH), insulin, and protamine. The figure shows the change in the fluorescence intensity of the probe when different proteins are added. When the protein is added to the ethanol solution system containing the fluorescent probe, the fluorescence emission wavelength of the probe does not shift significantly, and at the same time, the fluorescence emission intensity does not change significantly. The results show that the probe has no fluorescent selective recognition effect on reduced coenzyme I (NADH) and other proteins in ethanol solution.
[0026] Figure 3Fluorescence spectra of the coumarin fluorescent probe prepared for implementation example 1 before and after the action with different proteins: hemoglobin (Hb), oxidized coenzyme (NAD), reduced coenzyme I (NADH), cyclic adenosine monophosphate (cAMP), adenosine triphosphate (ATP), guanosine triphosphate (GTP), oxidized glutathione (GSSG), glutathione (GSH), insulin, and protamine in PBS phosphate buffer solution. The figure shows the change in the fluorescence intensity of the probe after adding different proteins. When reduced coenzyme I (NADH) is added to the PBS buffer solution system containing the fluorescent probe, the fluorescence emission wavelength of the probe blue-shifts from 570nm to 480nm, and the fluorescence intensity is significantly enhanced; after adding other proteins, the fluorescence emission wavelength of the probe is still at 570nm, without obvious displacement, and at the same time, the fluorescence emission intensity does not change significantly. The results show that the probe has a fluorescent selective recognition effect on reduced coenzyme I (NADH) in PBS phosphate buffer solution.
[0027] Figure 4 The fluorescence spectra of the coumarin fluorescent probe prepared in Example 1 before and after the action of reduced coenzyme I (NADH) in PBS phosphate buffer solutions of different pH values. The figure shows that when the pH of the PBS phosphate buffer solution changes from 6.2 to 8.2, the fluorescent detection reagent can still respond to reduced coenzyme I (NADH) by fluorescence enhancement. The results show that the pH value of the PBS phosphate buffer solution does not affect the fluorescence recognition of reduced coenzyme I (NADH) by the probe.
[0028] Figure 5 This is the hydrogen spectrum of the aminoindole-based coumarin fluorescent probe compound prepared in Example 1. 1 HNMR(400MHz,DMSO-d6)δ11.11(s,1H),10.71(s,1H),8.79(s,1H),8.06(s,1H),7.75(d,J=9.0Hz,1H),7.44–7.34(m,2H),7.30(dd ,J=8.7,1.9Hz,1H),6.85(dd,J=9.0,2.3Hz,1H),6.68(d,J=2.1Hz,1H),6.45(s,1H),3.51(q,J=7.0Hz,4H),1.17(t,J=7.0Hz,6H).
[0029] Example 2
[0030] 7-(Diethylamino)coumarin-3-carboxylic acid (0.2613 g, 1 mmol) was dissolved in dichloromethane (10 mL), and condensing agent 1-hydroxybenzotriazole (HOBT) (0.2027 g, 1.5 mmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) (0.2876 g, 1.5 mmol) and triethylamine (0.0202 g, 0.2 mmol) were added under ice bath conditions and stirred for 10 minutes, followed by addition of 5-aminoindole (0.1586 g, 1.2 mmol), and stirring at room temperature for 20 hours. After the reaction was completed, the solvent was removed under reduced pressure, and the product was separated and purified by column chromatography, the eluent being a mixed solution of dichloromethane and methanol (volume ratio of DCM:MeOH=20:1), to obtain 0.2851 g of a yellow solid with a yield of 76%.
Claims
1. An application of a fluorescent probe based on aminoindole and coumarin derivatives, characterized in that: The fluorescent probe is used to identify reduced coenzyme I; The structural formula of the fluorescent probe is: 。 2. The use of the fluorescent probe based on aminoindole and coumarin derivatives as claimed in claim 1, characterized in that: The fluorescent probe specifically recognizes reduced coenzyme I in PBS phosphate buffer solution.
3. The use of the fluorescent probe based on aminoindole and coumarin derivatives as claimed in claim 1, characterized in that: The concentration of the fluorescent probe in PBS phosphate buffer solution is 10 μM, and the pH value of PBS phosphate buffer solution is 7.
2.
4. A method for preparing a fluorescent probe based on aminoindole and coumarin derivatives as claimed in claim 1, characterized in that: The preparation method comprises: dissolving 7-(diethylamino)coumarin-3-carboxylic acid in dichloromethane, adding condensing agent 1-hydroxybenzotriazole (HOBT), 1-ethyl-(3-dimethylaminoaldehyde)carbodiimide hydrochloride (EDCI) and triethylamine under ice bath conditions, stirring for 10 minutes, then adding 5-aminoindole, stirring at room temperature for 10-20 hours, and after the reaction is completed, removing the solvent under reduced pressure, and obtaining a yellow solid by column chromatography.
5. The method for preparing a fluorescent probe based on aminoindole and coumarin derivatives according to claim 4, characterized in that: The molar equivalent ratio of 7-(diethylamino)coumarin-3-carboxylic acid, condensation agent 1-hydroxybenzotriazole (HOBT), 1-ethyl-(3-dimethylaminoaldehyde)carbodiimide hydrochloride (EDCI), triethylamine and 5-aminoindole is 1:1.2-1.5:1.0 -1.5:0.1-0.2:1.2.