Fluorescent probe for detecting NADH (nicotinamide adenine dinucleotide) as well as preparation method and application of fluorescent probe

Abstract

The invention discloses a fluorescent probe for detecting NADH (Nicotinamide Adenine Dinucleotide) as well as a preparation method and application thereof, and provides a structural formula of the fluorescent probe and the preparation method of the fluorescent probe for detecting NADH, and the preparation method comprises the following steps: (1) carrying out condensation reaction on 6-[bis (4-methoxyphenyl) amino]-1-ethyl-2-methylbenzo [cd] indole and quinoline-3-formaldehyde in ethanol; carrying out post-treatment to obtain 6-[bis (4-methoxyphenyl) amino]-1-ethyl-2-[(1E)-2-(quinoline-3-yl) vinyl] benzo [cd] indole; and (2) carrying out substitution reaction on the compound obtained in the step (1) and methyl trifluoromethanesulfonate in dichloromethane, and carrying out post-treatment to obtain the fluorescent probe HCY-N. The detection method disclosed by the invention has the advantages of simplicity and convenience in operation, rapidness, accuracy, high sensitivity and the like, and is suitable for biomedical research, clinical diagnosis and other related fields.

Description

Technical Field

[0001] The invention relates to biological fluorescence imaging contrast agent technology, and in particular to a fluorescent probe for detecting NADH and a preparation method and application thereof. Background Art

[0002] Nicotinamide adenine dinucleotide (NADH) is an indispensable redox regulator in all living cells. It participates in various physiological processes such as glycolysis and oxidative phosphorylation as a carrier of hydrogen ions. At the same time, an imbalance in the NADH / NAD+ ratio often indicates energy metabolism disorders under pathological conditions, such as aging, cancer, Parkinson's disease, diabetes, etc. For example, cancer cells can produce 5-10 times more NADH than normal cells. Especially in solid tumors, mitochondrial dysfunction also has an important impact on the production and consumption of NADH. Therefore, accurate detection of NADH / NAD+ is conducive to understanding and regulating the metabolic process of the disease and providing more reliable data for diagnosis and treatment.

[0003] Optical imaging technology is an emerging imaging technology that is widely used in biomedical research for its advantages such as low investment and simple operation. To date, a large number of fluorescent probes have been developed for optical imaging of cells and living animals. For example, the Liang group synthesized the probe Rh-QL to achieve imaging of overexpression of NADH in mouse tumors, but the reaction time exceeded 30min or longer. The Wang group developed a small molecule probe (QB) that can quickly and sensitively detect intracellular NADH with a maximum emission wavelength at 686nm. Despite the great success of previous studies, traditional fluorescence imaging still has problems such as limited tissue penetration, poor spatial resolution in deep biological tissues, and high tissue strength.

[0004] Recent studies have shown that fluorescence imaging in the second near-infrared window (NIR-II, 900-1700nm) is superior to imaging in the visible and NIR-I regions, significantly improving the spatial resolution and penetration depth in vivo. Therefore, various NIR-II probes have been designed for imaging of tumors, brain injuries, etc. However, most of these NIR-II probes are "always on" type probes, and due to the high background, they still face the critical problem of low signal-to-noise ratio, which greatly affects the specificity of early diagnosis. Therefore, smart responsive fluorescent probes that turn on fluorescence according to different analyte specificities have received widespread attention. Although many activatable NIR-II fluorescent probes have been developed, smart NIR-II responsive probes for NADH detection are far from enough. Summary of the invention

[0005] In view of this, the present invention provides a fluorescent probe for detecting NADH and a preparation method and application thereof.

[0006] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0007] A fluorescent probe for detecting NADH, wherein the fluorescent probe has the following structural formula:

[0008]

[0009] A method for preparing a fluorescent probe for detecting NADH comprises the following steps:

[0010] (1) 6-[bis(4-methoxyphenyl)amino]-1-ethyl-2-methylbenz[cd]indole is subjected to a condensation reaction with quinoline-3-carboxaldehyde in ethanol, and after post-treatment, 6-[bis(4-methoxyphenyl)amino]-1-ethyl-2-[(1E)-2-(quinolin-3-yl)vinyl]benz[cd]indole is obtained;

[0011] (2) The compound obtained in step (1) is subjected to a substitution reaction with methyl trifluoromethanesulfonate in chloroform, and the fluorescent probe HCY-N is obtained after post-treatment.

[0012] Preferably, in step 2, the chemical reaction formula of HCY-N is:

[0013]

[0014] Preferably, in step (1), the molar concentration of 6-[bis(4-methoxyphenyl)amino]-1-ethyl-2-methylbenz[cd]indole is 0.47 mmol, and the added amount is 200 mg; the molar concentration of quinoline-3-carboxaldehyde is 1.27 mmol, and the added amount is 200 mg;

[0015] Preferably, the molar concentration of 6-[bis(4-methoxyphenyl)amino]-1-ethyl-2-[(1E)-2-(quinolin-3-yl)vinyl]benz[cd]indole is 0.21 mmol, and the added amount is 120 mg; the molar concentration of methyl trifluoromethanesulfonate is 0.78 mmol, and the added amount is 127 mg; and the added amount of CH2Cl2 is 10 ml.

[0016] Preferably, in step (1), the post-treatment comprises: stirring the mixture at 50°C for 5 hours, cooling to room temperature, and filtering the precipitate; in step (2), the reaction conditions are: stirring at 25°C for 48 hours; and the post-treatment comprises: removing the solvent by suction filtration and purifying the product by column chromatography.

[0017] The invention discloses an application of a fluorescent probe for detecting NADH in detecting NADH in a living body.

[0018] Preferably, the NADH detection reagent is used for NADH spectral response and NIR-II fluorescence imaging.

[0019] Preferably, the NADH detection reagent is also used for qualitative detection of NADH;

[0020] The specific method is as follows: the near-infrared fluorescent probe HCY-N is prepared into a test solution, and then the sample to be tested is added, and the change in the NIR-II fluorescence emission intensity is measured under 808nm excitation. If the NIR-II fluorescence becomes stronger, it is determined that the sample to be tested contains NADH.

[0021] Compared with the prior art, the present invention has achieved the following technical effects:

[0022] The detection method of the present invention has the advantages of simple operation, rapidity, accuracy, high sensitivity, etc., and is suitable for biomedical research, clinical diagnosis and other related fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The present invention provides a fluorescent probe for detecting NADH and a preparation method and application thereof;

[0024] Figure 2 A fluorescent probe for detecting NADH and a preparation method and application thereof, and a high-resolution mass spectrum of the probe HCY-N;

[0025] Figure 3 The present invention provides a fluorescent probe for detecting NADH, a preparation method thereof, and a curve of fluorescence intensity and NADH response time of the probe HCY-N used;

[0026] Figure 4 A fluorescent probe for detecting NADH and a preparation method thereof and a response curve of the fluorescence intensity and NADH concentration of the probe HCY-N used in the present invention;

[0027] Figure 5 A fluorescent probe for detecting NADH and a preparation method thereof and a fluorescence change spectrum of the probe HCY-N used in the present invention after acting on different species;

[0028] Figure 6 The present invention is a fluorescent probe for detecting NADH, and its preparation method and application probe HCY-N for detecting NIR-II fluorescence imaging of NADH in a solution; DETAILED DESCRIPTION

[0029] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] like Figure 1 As shown, the present invention discloses a fluorescent probe for detecting NADH, and the fluorescent probe has the following structural formula:

[0031] .

[0033] The present invention also discloses a method for preparing a fluorescent probe for detecting NADH, comprising the following steps:

[0034] (1) 6-[bis(4-methoxyphenyl)amino]-1-ethyl-2-methylbenz[cd]indole with a molar concentration of 0.47 mmol and an added amount of 200 mg was subjected to a condensation reaction with quinoline-3-carboxaldehyde with a molar concentration of 1.27 mmol and an added amount of 200 mg in ethanol, and the mixture was stirred at 50° C. for 5 h. After cooling to room temperature, the precipitate was filtered to obtain 6-[bis(4-methoxyphenyl)amino]-1-ethyl-2-[(1E)-2-(quinolin-3-yl)vinyl]benz[cd]indole;

[0035] (2) A 0.21 mmol, 120 mg, 6-[bis(4-methoxyphenyl)amino]-1-ethyl-2-[(1E)-2-(quinolin-3-yl)vinyl]benz[cd]indole and a 0.78 mmol, 127 mg, methyl trifluoromethanesulfonate were subjected to substitution reaction in 10 ml of dichloromethane, and the mixture was stirred at 25° C. for 48 hours. The solvent was removed by suction filtration, and the product was purified by column chromatography to obtain a fluorescent probe HCY-N.

[0036] The chemical reaction formula of HCY-N is:

[0037]

[0038] The invention also discloses the application of a fluorescent probe for detecting NADH in detecting NADH in a living body.

[0039] This application can be used for NADH spectral response and NIR-II fluorescence imaging;

[0040] The specific method is as follows: the near-infrared fluorescent probe HCY-N is prepared into a test solution, and then the sample to be tested is added, and the change in the NIR-II fluorescence emission intensity is measured under 808nm excitation. If the NIR-II fluorescence becomes stronger, it is determined that the sample to be tested contains NADH.

[0041] Embodiment 1:

[0042] 6-[Bis(4-methoxyphenyl)amino]-1-ethyl-2-methylbenzo[cd]indole (200 mg, 0.47 mmol), quinoline-3-carboxaldehyde (200 mg, 1.27 mmol) and a catalytic amount of piperidine were dissolved in 8 mL of acetonitrile; the mixture was stirred at 50° C. for 5 h, cooled to room temperature, and the precipitate was filtered to obtain the desired product, named 6-[bis(4-methoxyphenyl)amino]-1-ethyl-2-[(1E)-2-(quinolin-3-yl)vinyl]benzo[cd]indole (220 mg, 83%);

[0043] Compound 2 (120 mg, 0.21 mmol) and methyl trifluoromethanesulfonate (127 mg, 0.78 mmol) were dissolved in 10 ml CH2Cl2. The reactants were stirred at 25°C for 48 hours. After the reaction was completed, the solvent was removed by suction filtration, and the product was purified by column chromatography and named HCY-N. The eluent was DCM:MeOH=5:1. The product was subjected to high-resolution mass spectrometry to determine the molecular structure. The results are as follows: Figure 2 shown.

[0044] Embodiment 2:

[0045] Spectral changes of probe HCY-N in response to NADH over time: The probe was dissolved in DMSO to prepare a mother solution, which was placed in a 4°C refrigerator for subsequent dilution. The mother solution was added to PBS (pH 7.4, 20mM, v / v) for testing. The probe concentration after dilution was 10μM, and then 20μM NADH was added. The changes in the fluorescence spectrum over time were recorded, as shown in Figure 2. Figure 3 shown.

[0046] Results from Figure 3 It can be seen that after adding 20 μM NADH, under the excitation at 808 nm, the fluorescence intensity at 1050 nm gradually increased with time and reached a peak at about 10 min, indicating that the probe responded quickly to NADH.

[0047] Embodiment 3:

[0048] Spectral changes of probe HCY-N to different concentrations of NADH: The mother solution was added to PBS (pH 7.4, 20mM, v / v) for testing. The probe concentration after dilution was 10μM. Then, different concentrations of NADH aqueous solutions were added. After equilibrium, the fluorescence emission spectrum was measured. The results are shown in Figure 2. Figure 4 shown.

[0049] from Figure 4 It was found that under 808nm excitation, after adding NADH, the fluorescence of the probe gradually increased and stabilized with the increase of NADH concentration. In the range of 0-7μM, the fluorescence intensity of the probe had an obvious linear relationship with the concentration of NADH, indicating that the probe had a significant response to NADH.

[0050] Embodiment 4:

[0051] Selective spectrum of probe HCY-N for NADH: The mother solution was added to PBS (pH 7.4, 20 mM, v / v) for testing. The probe concentration after dilution was 5 μM. + , Ca 2+ Mg 2+ 、Zn 2+ ), anion (OH - , Cl - ,Ac - 、CO3 2- 、ONOO - ), biological thiols (Cys, Hcy, GSH), amino acids (Leu, Prs, Thr, Arg, Gly, Ser, Tyr), 5 μL of the mother solution was added to the probe system. After adding, the concentration of each analyte was 20 μM. They were named 1-19 in the above order. After reacting for 10 minutes, the fluorescence intensity of different reaction systems under 808 nm laser irradiation was recorded, such as Figure 5 shown.

[0052] Depend on Figure 5 It can be seen that the fluorescence intensity of the probe only changes greatly to NADH, and the changes to other substances are negligible, indicating that the probe has high selectivity in response to NADH.

[0053] Example 5: The activated probe is placed in the NIR-II fluorescence imaging system for detection. Figure 6 It can be seen that under 808nm laser irradiation, the probe solution activated by NADH can detect obvious NIR-II fluorescence.

[0054] The novel NIR-II fluorescent probe HCY-N based on hemicyanine detection of NADH in samples of the present invention generates a fluorescence response after adding NADH in PBS (20mM, v / v, pH 7.4). Under 808nm excitation, there is a strong fluorescence signal at NIR-II after adding NADH, and it increases with the increase of NADH concentration, and the fluorescence intensity reaches saturation at 10μM. In addition, the emission intensity has a good linear relationship with the NADH concentration.

[0055] The novel near-infrared fluorescent probe HCY-N of the present invention, under excitation at 808 nm, after adding 10 μM NADH, the fluorescence intensity at 900 nm gradually increases with time and reaches a peak value at about 10 minutes, indicating that the probe HCY-N responds quickly to NADH.

[0056] The novel fluorescent probe HCY-N for detecting NADH based on hemicyanine dyes of the present invention has high selectivity. The cation (K + , Ca 2+ Mg 2+ 、Zn 2+ ), anion (OH - , Cl - ,Ac - 、CO3 2- 、ONOO - ), biothiols (Cys, Hcy, GSH), and amino acids (Leu, Prs, Thr, Arg, Gly, Ser, Tyr) were used to detect its selectivity, among which NADH caused a significant fluorescence enhancement of the probe, while other interfering substances did not cause obvious changes in fluorescence intensity, indicating that HCY-N has significant selectivity for NADH and strong anti-interference ability.

[0057] The results show that the probe can achieve fluorescence response within 10 minutes after the reaction between the hemicyanine dye and NADH, which is highly selective. In addition, the probe HCY-N can effectively detect NADH in the solution and realize NIR-II fluorescence imaging of NADH in the solution.

[0058] The above description is only a preferred embodiment of the present invention and does not limit the technical scope of the present invention. Therefore, any slight modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A fluorescent probe for detecting NADH, characterized in that: The fluorescent probe has the following structural formula:

2. A method for preparing a fluorescent probe for detecting NADH, characterized in that: The following steps are involved: (1) 6-[bis(4-methoxyphenyl)amino]-1-ethyl-2-methylbenz[cd]indole is subjected to a condensation reaction with quinoline-3-carboxaldehyde in ethanol, and after post-treatment, 6-[bis(4-methoxyphenyl)amino]-1-ethyl-2-[(1E)-2-(quinolin-3-yl)vinyl]benz[cd]indole is obtained; (2) The compound obtained in step (1) is subjected to a substitution reaction with methyl trifluoromethanesulfonate in dichloromethane, and the fluorescent probe HCY-N is obtained after post-treatment.

3. The method for preparing a fluorescent probe for detecting NADH according to claim 2, characterized in that: In step 2, the chemical reaction formula of HCY-N is:

4. The method for preparing a fluorescent probe for detecting NADH according to claim 2, characterized in that: In step (1), the molar concentration of 6-[bis(4-methoxyphenyl)amino]-1-ethyl-2-methylbenz[cd]indole is 0.47 mmol, and the amount added is 200 mg; the molar concentration of quinoline-3-carboxaldehyde is 1.27 mmol, and the amount added is 200 mg.

5. The method for preparing a fluorescent probe for detecting NADH according to claim 2, characterized in that: The molar concentration of 6-[bis(4-methoxyphenyl)amino]-1-ethyl-2-[(1E)-2-(quinolin-3-yl)vinyl]benz[cd]indole was 0.21 mmol, and the amount added was 120 mg. The molar concentration of methyl trifluoromethanesulfonate was 0.78 mmol, and the amount added was 127 mg. The amount of CHCl3 added was 10 ml.

6. The method for preparing a fluorescent probe for detecting NADH according to claim 2, characterized in that: In the step (1), the post-treatment includes: stirring the mixture at 50°C for 5 hours, cooling to room temperature, using ether for precipitation, and filtering the precipitate; in the step (2), the reaction conditions are: stirring at 25°C for 6 hours; the post-treatment includes: removing the solvent by suction filtration and purifying the product by column chromatography.

7. Use of the fluorescent probe for detecting NADH according to claim 1 in detecting NADH in a solution.

8. Use of a fluorescent probe for detecting NADH according to claim 7 in detecting NADH in a solution, characterized in that: The NADH detection reagent is used for NADH spectral response.

9. Use of a fluorescent probe for detecting NADH according to claim 7 in detecting NADH in a solution, characterized in that: The NADH detection reagent is also used for qualitative detection of NADH to achieve NIR-II fluorescence imaging; The specific method is as follows: the near-infrared fluorescent probe HCY-N is prepared into a test solution, and then the sample to be tested is added, and the change in the NIR-II fluorescence emission intensity is measured under 808nm excitation. If the NIR-II fluorescence becomes stronger, it is determined that the sample to be tested contains NADH.

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