A hydrogenation-responsive coenzyme NAD(P)H fluorescent probe, preparation method, fluorescent sensor and application

Abstract

The present invention provides a hydrogenation-responsive coenzyme NAD(P)H fluorescent probe, a preparation method, a fluorescent sensor and an application, and belongs to the technical field of fluorescent probes. In the presence of NAD(P)H, the trimethylbenzoquinone functional group in the molecule of the hydrogenation-responsive coenzyme NAD(P)H fluorescent probe prepared by the present invention can be catalytically reduced to a responsive trimethylhydroquinone by an organic metal iridium or organic metal rhodium catalyst. The generated trimethylhydroquinone will rapidly undergo intramolecular cyclization and fall off from the probe molecule, turning on the fluorescence of the probe molecule, thereby achieving a rapid and efficient fluorescent response to NAD(P)H. The hydrogenation-responsive coenzyme NAD(P)H fluorescent probe of the present invention has a stable structure, high fluorescence quantum yield, a wide pH adaptability range, good biocompatibility, and mitochondrial targeting. When combined with highly active organic iridium and rhodium catalysts, it can quickly, sensitively and specifically detect the coenzyme NAD(P)H with a detection limit of 1.0 μM.

Description

Technical Field

[0001] The present invention relates to the technical field of fluorescent probes, and in particular to a hydrogenation-responsive coenzyme NAD(P)H fluorescent probe, a preparation method, a fluorescent sensor and applications. Background Art

[0002] Reduced nicotinamide adenine dinucleotide NAD(P)H is an important coenzyme in cells, involved in multiple physiological processes such as intracellular energy metabolism and gene expression. The content of NAD(P)H in cells is directly related to the health of the organism. It is crucial to study and detect the level of NAD(P)H in cells.

[0003] There are many common methods for detecting NAD(P)H, but many of them have many disadvantages. For example, the enzymatic cycling method is expensive and has strict experimental conditions; although the high-performance liquid chromatography method has good selectivity, it requires specific chromatographic columns and a large amount of eluent, which is expensive; the electrochemical method is complicated to operate and has poor reproducibility; the capillary electrophoresis method has a small injection volume but low sensitivity; although the ultraviolet spectrophotometry method is simple to operate, it has low accuracy; the NAD(P)H autofluorescence method has many interference factors and complex data processing.

[0004] Although many fluorescent probes for NAD(P)H detection have been reported, most have long response times and low sensitivity, limiting their application in biological samples. For example, CHEN H et al. achieved a detection limit of 3.1 μm mol / L (Reversible ratiometric NADH sensing using semiconducting polymer dots); SOUNDHARRAJ P et al. achieved a detection limit of 4.7 μm mol / L (Fluorescence sensing of NADH using silica-zinc nitride nanocomposite for monitoring diabetes); JOO JH et al. achieved a detection limit of 1.2 μm mol / L (Mitochondria-targetable red-emitting probe for real-time fluorescence monitoring of NAD(P)H in live cells); and MAITI M et al. achieved a detection limit of 1.25 μmmol / L (NADH-induced “kick-on” fluorescent probe validates crosstalk with redox regulator GSH).

[0005] Therefore, there is an urgent need to develop a fluorescent probe for rapid detection of NAD(P)H that has a simple synthesis method, high selectivity and high sensitivity and can be used for NAD(P)H detection in vitro or in vivo. Summary of the Invention

[0006] The present invention aims to provide a hydrogenation-responsive coenzyme NAD(P)H fluorescent probe, a preparation method, a fluorescent sensor and applications. The synthesis method of the hydrogenation-responsive coenzyme NAD(P)H fluorescent probe is simple, and the fluorescent sensor composed of the hydrogenation-responsive coenzyme NAD(P)H fluorescent probe and a catalyst has high selectivity and high sensitivity to NAD(P)H.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a hydrogenation-responsive coenzyme NAD(P)H fluorescent probe, the structure of which is shown in Formula 1:

[0009]

[0010] The present invention also provides a method for preparing the above-mentioned hydrogenation-responsive coenzyme NAD(P)H fluorescent probe, comprising the following steps:

[0011] (1) Preparation of 3-bromo-7H-benzo[d]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one:

[0012] 4-Bromo-1,8-naphthalene dicarboxylic anhydride and o-phenylenediamine are dissolved in glacial acetic acid and heated under reflux for reaction. After the reaction, the solution is cooled to room temperature and the solid is collected by filtration to obtain a crude intermediate compound 3-bromo-7H-benzo[d]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one. The collected crude solid product is then transferred to a saturated aqueous solution of sodium bicarbonate and filtered. The resulting solid is rinsed with deionized water and analytical grade ethanol in sequence to obtain a pure bright yellow solid 3-bromo-7H-benzo[d]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one.

[0013] (2) Preparation of 3-(2-(methylamino)ethyl)amino)-7H-benzo[d]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one:

[0014] The 3-bromo-7H-benzo[d]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one obtained above is dissolved in ethylene glycol monoethyl ether solvent, and then an excess amount of the compound N-methylethane-1,2-diamine is added and refluxed. After the reaction, the reaction solution is cooled to room temperature, and then an appropriate amount of ice water is added and extracted with dichloromethane. The organic phase is collected and the organic solvent is removed by distillation under reduced pressure to obtain a crude product of the intermediate product 3-(2-(methylamino)ethyl)amino)-7H-benzo[d]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one. The obtained crude product is further separated and purified by column chromatography to obtain a pure intermediate product 3-(2-(methylamino)ethyl)amino)-7H-benzo[d]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one.

[0015] (3) Preparation of hydrogenation-responsive coenzyme NAD(P)H fluorescent probe:

[0016] 3-(2-(methylamino)ethyl)amino)-7H-benzo[d]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one and 2-methyl-2-(2,4,5-trimethyl-3,6-dioxocyclohexyl-1,4-dien-1-yl)propionic acid, EDCI, and HOBT are dissolved in dry Et3N solvent and DMF solvent, and reacted under light-proof and nitrogen protection conditions; after the reaction is completed, an appropriate amount of ice water is added to the reaction liquid, followed by extraction with dichloromethane, the organic phase is collected, dried with anhydrous sodium sulfate, and then the organic solvent is distilled off under reduced pressure to obtain a hydrogenation-responsive coenzyme NAD(P)H fluorescent crude product; the obtained crude product is further separated and purified by column chromatography to obtain a pure red powdery hydrogenation-responsive coenzyme NAD(P)H fluorescent probe.

[0017] Preferably, in step (1), the molar ratio of 4-bromo-1,8-naphthalene dicarboxylic anhydride to o-phenylenediamine is 1:1-5, the molar volume ratio of o-phenylenediamine to glacial acetic acid is 1 mmol:10-100 mL; the reaction temperature is 100-120° C., and the reaction time is 3-24 h.

[0018] Preferably, in step (2), the molar ratio of 3-bromo-7H-benzo[d]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one to N-methylethane-1,2-diamine is 1:1-5; the molar volume ratio of 3-bromo-7H-benzo[d]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one to ethylene glycol monoethyl ether is 1 mmol:5-20 mL; the reflux reaction time is 5-10 h, the temperature is 100-150° C., and the reflux reaction conditions are to avoid light; the conditions for chromatographic column separation are: silica gel column chromatography, and the mobile phase is methanol:dichloromethane = 1:5.

[0019] Preferably, in step (3), the molar ratio of 3-(2-(methylamino)ethyl)amino)-7H-benzo[d]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one, 2-methyl-2-(2,4,5-trimethyl-3,6-di-oxocyclohexyl-1,4-di-en-1-yl)propionic acid, Et3N, EDCI, and HOBT is 1:1~2:2~4:2~4, and the reaction time is 5~24h; the number of dichloromethane extractions is 2~5 times; and the column chromatography purification conditions are: silica gel column chromatography, and the mobile phase is methanol:dichloromethane = 1:10.

[0020] The present invention also provides a hydrogenation-responsive coenzyme NAD(P)H fluorescence sensor, which is prepared by combining a hydrogenation-responsive coenzyme NAD(P)H fluorescent probe and a catalyst.

[0021] Preferably, the catalyst is an organometallic iridium or organometallic rhodium catalyst.

[0022] Preferably, the organometallic iridium or organometallic rhodium catalyst comprises the structure shown below:

[0023]

[0024] Wherein M is iridium or rhodium;

[0025] R is one of -COOH, -OCH3, -CH3, -H, -CN, and adamantane.

[0026] The present invention also provides an application of the above-mentioned hydrogenation-responsive coenzyme NAD(P)H fluorescence sensor in fluorescence imaging analysis of hydrogenation-responsive coenzyme NAD(P)H in cells.

[0027] The hydrogenation-responsive coenzyme NAD(P)H fluorescent probe of the present invention has the following advantages compared with the prior art:

[0028] (1) In the presence of NAD(P)H, the trimethylbenzoquinone functional group in the hydrogenation-responsive coenzyme NAD(P)H fluorescent probe prepared by the present invention can be rapidly catalytically reduced to the responsive trimethylhydroquinone by an organometallic iridium or organometallic rhodium catalyst. The generated trimethylhydroquinone will rapidly undergo intramolecular cyclization and fall off from the probe molecule, thereby turning on the fluorescence of the probe molecule and achieving a rapid and efficient fluorescent response to the coenzyme NAD(P)H.

[0029] (2) The hydrogenation-responsive coenzyme NAD(P)H fluorescent probe prepared by the present invention has a stable structure, high fluorescence quantum yield, a wide pH adaptability range, good biocompatibility, and cell mitochondrial targeting. When it is combined with highly active organic iridium and rhodium catalysts, it can quickly, sensitively and specifically detect the coenzyme NAD(P)H, with a detection limit of 1.0 μM. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0031] Figure 1 This is the synthesis route of the hydrogenation-responsive coenzyme NAD(P)H fluorescent probe in Example 1;

[0032] Figure 2 The fluorescence spectra and absorbance-fluorescence intensity linear fitting graphs of the hydrogenation-responsive coenzyme NAD(P)H fluorescent probe and the fluorophore compound of formula 3 shown in Example 1 are shown, wherein A is the fluorescence spectrum and B is the absorbance-fluorescence intensity linear fitting graph;

[0033] Figure 3 The detection mechanism of the hydrogenation-responsive coenzyme NAD(P)H fluorescent probe for detecting coenzyme NADH in Example 2 is as follows;

[0034] Figure 4 The fluorescence response spectra and fluorescence detection standard curve of the hydrogenation-responsive coenzyme NAD(P)H fluorescent probe detecting different concentrations of NADH in Example 2, wherein A is the fluorescence response spectra; B is the fluorescence detection standard curve;

[0035] Figure 5 Comparison of fluorescence detection spectral intensities of the hydrogenation-responsive coenzyme NAD(P)H fluorescent probe for different biomolecules including coenzyme NADH in Example 3;

[0036] Figure 6 This is a fluorescence imaging image of human non-small cell lung cancer cells (A459) using the 2-pyridinebenzamide pentamethylcyclopentadienyl organoiridium catalyst as the organometallic catalyst in Example 4 in combination with the hydrogenation-responsive coenzyme NAD(P)H fluorescent probe prepared in Example 1. DETAILED DESCRIPTION

[0037] The following are detailed descriptions of the embodiments of the present invention. The embodiments are intended to explain the present invention and are not to be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, the techniques or conditions described in the literature within the art or the product specifications are used. Reagents or instruments used without manufacturer's indication are commercially available conventional products.

[0038] 4-Bromo-1,8-naphthalene dicarboxylic anhydride was purchased from Shanghai MacLean Biochemical Technology Co., Ltd., CAS No.: 21563-29-1;

[0039] N-Methylethylenediamine, purchased from Shanghai MacLean Biochemical Technology Co., Ltd., CAS No.: 109-81-9:

[0040] Et3N was purchased from Shanghai MacLean Biochemical Technology Co., Ltd., CAS No.: 121-44-8;

[0041] 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) was purchased from Shanghai MacLean Biochemical Technology Co., Ltd., CAS No.: 25952-53-8;

[0042] 1-Hydroxybenzotriazole (HOBT) was purchased from Shanghai MacLean Biochemical Technology Co., Ltd., CAS No.: 2592-95-2.

[0043] In the embodiment of the present invention, a hydrogenation-responsive coenzyme NAD(P)H fluorescent probe capable of specifically detecting the coenzyme NAD(P)H is prepared, and its structure is shown in Formula 1:

[0044]

[0045] The preparation method of the hydrogenation-responsive coenzyme NAD(P)H fluorescent probe comprises the following steps:

[0046] (1) Preparation of 3-bromo-7H-benzo[d]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one:

[0047] 4-Bromo-1,8-naphthalene dicarboxylic anhydride and o-phenylenediamine are dissolved in glacial acetic acid and heated under reflux for reaction. After the reaction, the solution is cooled to room temperature and the solid is collected by filtration to obtain a crude intermediate compound 3-bromo-7H-benzo[d]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one. The collected crude solid product is then transferred to a saturated aqueous solution of sodium bicarbonate and filtered. The obtained solid is rinsed with deionized water and analytical grade ethanol in sequence to obtain a pure bright yellow solid 3-bromo-7H-benzo[d]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one.

[0048] (2) Preparation of 3-(2-(methylamino)ethyl)amino)-7H-benzo[d]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one:

[0049] The 3-bromo-7H-benzo[d]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one obtained above is dissolved in ethylene glycol monoethyl ether solvent, followed by adding an excess amount of the compound N-methylethane-1,2-diamine and reacting under reflux; after the reaction, the reaction solution is cooled to room temperature, followed by adding an appropriate amount of ice water, and extracted with dichloromethane, the organic phase is collected, and the organic solvent is removed by distillation under reduced pressure to obtain a crude product of the intermediate product 3-(2-(methylamino)ethyl)amino)-7H-benzo[d]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one, and the obtained crude product is further separated and purified by column chromatography to obtain a pure intermediate product 3-(2-(methylamino)ethyl)amino)-7H-benzo[d]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one;

[0050] (3) Preparation of hydrogenation-responsive coenzyme NAD(P)H fluorescent probe:

[0051] 3-(2-(methylamino)ethyl)amino)-7H-benzo[d]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one and a benzoquinone derivative 2-methyl-2-(2,4,5-trimethyl-3,6-dioxocyclohexyl-1,4-dien-1-yl)propionic acid are dissolved in a dry N,N-dimethylformamide (DMF) solvent, followed by the addition of Et3N, EDCI, and HOBT, and the mixture is reacted under light-shielding conditions; after the reaction is completed, an appropriate amount of ice water is added to the reaction solution, followed by extraction with dichloromethane, the organic phase is collected, dried over anhydrous sodium sulfate, and the organic solvent is removed by reduced pressure distillation to obtain a crude hydrogenation-responsive coenzyme NAD(P)H fluorescent product; the crude product is further separated and purified by column chromatography to obtain a pure red powdery hydrogenation-responsive coenzyme NAD(P)H fluorescent probe.

[0052] Example 1

[0053] Embodiment 1 of the present invention is based on Figure 1 The process shown in the figure is to prepare a hydrogenation-responsive coenzyme NAD(P)H fluorescent probe, comprising the following steps:

[0054] Step (1) Biopreparation of 3-bromo-7H-benzo[d]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one (Formula 2): 4-bromo-1,8-naphthalic anhydride (2 g, 7.3 mmol) and o-phenylenediamine (0.79 g, 7.3 mmol) were dissolved in 100 ml of glacial acetic acid and heated under reflux at 120° C. for 6 hours. After the reaction, the reaction solution was filtered and the solid was transferred to a beaker containing 100 mL of saturated NaHCO 3 solution and filtered again. The obtained solid was then rinsed with water and ethanol in turn to obtain a bright yellow solid compound 3-bromo-7H-benzo[d]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one (2.18 g, yield 86%).

[0055] The results of nuclear magnetic resonance hydrogen spectrum, carbon spectrum and mass spectrometry are as follows:

[0056] 1 H NMR (400MHz, CDCl3) δ8.82 (dd, J=17.4, 7.3Hz, 1H), 8.71-8.34 (m, 3H), 8.18-7.98 (m, 1H), 7.96-7.75 (m, 2H), 7.48 (dd, J=5.7, 3.0Hz, 2H).

[0057] 13 C NMR(101MHz, CDCl3)δ176.44(s),160.05(s),149.28(s),148.61(s) , 143.80(s) , 134.59(s), 134.20(s), 132.42(s), 131.57(s), 131.41(s), 131.25(s), 131.20(s), 126.01(s), 125.73(s), 115.87(s).

[0058] MS(ESI): m / z cacld for([M+H + ] + )C 18 H 10 BrN2O348.99; found 348.98.

[0059] It can be seen that the structural formula of 3-bromo-7H-benzo[d]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one is Formula 2 (see Figure 1 ) shown in the compound.

[0060] (2) Preparation of 3-(2-(methylamino)ethyl)amino)-7H-benzo[d]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one (Formula 3): The compound represented by Formula 2 (300 mg, 0.89 mmol) was dissolved in 15 ml of ethylene glycol monoethyl ether, and then a triethylamine solution of N-methylethylenediamine (375 μL, 4.45 mmol) was added. The reaction solution was refluxed at 120° C. in the dark for 5 h. After the reaction was completed, the reaction solution was stirred for 1 h. The mixture was cooled to room temperature, followed by the addition of 20 ml of ice water. The crude product was extracted with dichloromethane (DCM) (20 mL × 3 times) and washed with water (20 mL × 3 times). The organic phase was then collected and dried over anhydrous sodium sulfate (20 g). The solvent was then removed by distillation under reduced pressure to yield a crude intermediate compound, 3-(2-(methylamino)ethyl)amino)-7H-benzo[d]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one. The crude product was further separated and purified by silica gel column chromatography using a mixed solvent of methanol:dichloromethane (1:5) as the mobile phase to obtain the pure intermediate compound 3-(2-(methylamino)ethyl)amino)-7H-benzo[d]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one (150 mg, 50% yield).

[0061] The results of nuclear magnetic resonance hydrogen spectrum, carbon spectrum and mass spectrometry are as follows:

[0062] 1 H NMR (400MHz, DMSO-d6) δ8.73 (dd, J=37.1, 7.8Hz, 1H), 8.65-8.56 (m, 1H), 8.49-8.36 (m, 2H), 7.86-7.76 (m, 1H), 7.74-7.67 (m, 1H) , 7.49-7.36 (m, 2H), 6.85 (dd, J=8.6, 4.5Hz, 1H), 3.51 (t, J=6.3Hz, 3H), 3.18 (s, 1H), 2.88 (t, J=6.4Hz, 2H), 2.40 (d, J=5.1Hz, 3H).

[0063] 13C NMR (101MHz, DMSO-d6) δ160.27(s),152.29(s),149.76(s),143.90(s),135.49(s),131.99(s),128.78(s),127.32(s),126.78(s),125 .36(s),125.05(s),124.60(s),120.98(s),119.97(s),119.81(s),115.77(s),108.21(s),104.82(s),49.65(s),42.85(s),36.09(s).

[0064] MS(ESI): m / z cacld for([M+H + ] + )C 21 H 19 N4O 343.16; found 343.18.

[0065] It can be seen that 3-(2-(methylamino)ethyl)amino)-7H-benzo[d]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one is a formula 3 (see Figure 1 ) shown in the compound.

[0066] (3) The compound represented by Formula 3 (66 mg, 0.19 mmol), 2-methyl-2-(2,4,5-trimethyl-3,6-dioxocyclohexyl-1,4-dien-1-yl)propionic acid (55 mg, 0.21 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) (77 mg, 0.38 mmol) and 1-hydroxybenzotriazole (HOBT) (54 mg, 0.38 mmol) were placed in a 25 mL round-bottom flask, and then 0.4 mL of dry triethylamine (Et3N) and 4 mL of dry dimethylformamide (DMF) were added in sequence. The reaction solution was reacted at room temperature under nitrogen protection for 24 h. After the reaction was completed, 20 mL of ice water was added to the reaction solution, and the mixture was extracted three times with dichloromethane (20 mL each time). The organic phases were combined. The mixture was then dried with 20 g of anhydrous sodium sulfate, and the solvent was subsequently removed by reduced pressure distillation to obtain a red oily crude product. The crude product was purified by silica gel column chromatography (dichloromethane: methanol = 10: 1) to obtain a pure hydrogenation-responsive coenzyme NAD(P)H fluorescent probe molecule (see Formula 1) as a red powder (61.40 mg, yield 56.3%).

[0067] The results of nuclear magnetic resonance hydrogen spectrum, carbon spectrum and mass spectrometry are as follows:

[0068] 1H NMR (400MHz, CDCl3) δ8.78 (dd, J=19.1, 6.7Hz, 1H), 8.67-8.48 (m, 2H), 8.31-7.91 (m, 2H), 7.90-7.74 (m, 1H), 7.61-7.30 (m, 4H), 6.55 ( t, J=9.3Hz, 1H), 3.81-3.69 (m, 2H), 3.50-3.39 (m, 2H), 3.12 (d, J=22.4Hz, 5H), 2.21 (d, J=4.7Hz, 3H), 1.80-1.63 (m, 6H), 1.47 (s, 6H).

[0069] 13 C NMR (101MHz, CDCl3) δ190.98(s), 187.61(s), 175.39(s), 160.89(s), 153.28(s), 150.98(s), 149.81(s), 143.83(s), 142.6 1(s), 138.67(s), 138.00(s), 135.27(s), 132.09(s), 128.70(s), 127.22(s), 125.12(s), 125.04(s), 124.66(s), 124.55(s) , 120.58(s), 120.55(s), 119.54(s), 115.97(s), 109.99(s), 103.41(s), 47.74(s), 4 7.61(s), 44.00(s), 38.18(s), 36.91(s), 29.32(s), 14.52(s), 12.53(s), 12.10(s).

[0070] MS(ESI): m / z cacld for([M+H + ] + )C 35 H 35 N4O4575.26; found 575.09.

[0071] It can be seen that the hydrogenation-responsive coenzyme NAD(P)H fluorescent probe molecule is the compound shown in Formula 1.

[0072] The hydrogenation-responsive coenzyme NAD(P)H fluorescent probe of formula 1 and the corresponding luminophore of formula 3 were respectively prepared into aqueous solutions with a concentration of 50 μM and fluorescence spectroscopy was performed. The results are shown in FIG. Figure 2 shown.

[0073] Depend on Figure 2It can be seen that the aqueous solution of the fluorescent probe (compound shown in Formula 1) with a concentration of 50 μM has almost no fluorescence at an emission wavelength of 510 nm, while its corresponding 50 μM fluorophore (compound shown in Formula 3) has an extremely high fluorescence emission signal at 510 nm, indicating that the probe molecule has successfully achieved the successful shutdown of the fluorescence signal of its fluorophore.

[0074] The quantum yield of the hydrogenation-responsive coenzyme NAD(P)H fluorescent probe was further measured. The dosage of the compound of Formula 1, the compound of Formula 3, and quinine sulfate in the reaction system was 50 μM. The solvent for the compound of Formula 1 and the compound of Formula 3 was PBS (pH = 7.0, 0.1 M), and the solvent for quinine sulfate was H2SO4 (0.1 M). The excitation wavelength was 445 nm, and the emission wavelength was 510 nm. It is known that the fluorescence quantum yield of a 0.1 M quinine sulfate solution is ΦST = 0.54, the refractive index of a 0.1 M sulfuric acid solution is ηX = 1.3345, and the refractive index of a 0.1 M PBS solution is ηST = 1.3338.

[0075] According to the quantum yield calculation formula (Formula 1):

[0076]

[0077] The quantum yield of the obtained fluorophore (compound shown in Formula 3) is Φ1=0.2202; the quantum yield of the hydrogenation-responsive coenzyme NAD(P)H fluorescent probe (compound shown in Formula 1) is Φ2=0.00824.

[0078] It can be seen that compared with the fluorophore compound of formula 3, the fluorescence quantum yield of the fluorescent probe is almost negligible, which provides a guarantee for the application of the fluorescent probe in the construction of fluorescent sensors.

[0079] Example 2

[0080] Example 2 of the present invention is a 2-pyridine benzamide pentamethylcyclopentadienyl organic iridium catalyst as an organic metal catalyst, combined with the hydrogenation response coenzyme NAD (P) H fluorescent probe prepared in Example 1 to construct a fluorescent sensor for fluorescence detection of coenzyme NADH. The detection mechanism is as follows: Figure 3 As shown, the method is as follows:

[0081] The structure of the 2-pyridinebenzamide pentamethylcyclopentadienyl organoiridium catalyst is:

[0082]

[0083] 2.0 mL of Tris-HCl buffer solution (0.1 M, pH = 7.4) containing the hydrogenation-responsive coenzyme NAD(P)H fluorescent probe (50 μM) and 2-pyridinebenzamide pentamethylcyclopentadienyl organoiridium catalyst (5.0 μM) shown in Formula 1 was mixed with 1.0 mL of Tris-HCl buffer solution (pH 7.4) containing different concentrations (0 μM, 50 μM, 100 μM, 150 μM, 200 μM) of NADH and incubated at 37°C for 2.0 h. The fluorescence emission spectrum intensity of each sample was then measured at 510 nm under 440 nm excitation light. The results are shown in Figure 2. Figure 4 .

[0084] Depend on Figure 4 It can be seen that when the NADH concentration is in the range of 0 to 200 μM, the higher the NADH concentration, the stronger the fluorescence intensity, indicating that the constructed fluorescence sensor has good fluorescence response performance to NADH.

[0085] Furthermore, through data processing, it was found that the logarithm of fluorescence intensity and NADH concentration showed a good linear relationship in the range of 6.25 to 200 μM: I = 3181.8 log C NADH -1697.1, R 2 =0.9869, and the detection limit was 1.0 μM.

[0086] Example 3

[0087] In Example 3 of the present invention, an organic iridium catalyst 2-pyridinebenzamide pentamethylcyclopentadiene is used as an organic metal catalyst, which is combined with the hydrogenation-responsive coenzyme NAD(P)H fluorescent probe prepared in Example 1 to construct a fluorescent sensor, and the selectivity of the hydrogenation-responsive coenzyme NAD(P)H fluorescent probe for coenzyme NADH fluorescence detection is detected as follows:

[0088] Take 20 μM of the compound of formula 1, 5.0 μM of the organic iridium catalyst 2-pyridinebenzamide pentamethylcyclopentadiene, and 3.0 v / v% methanol, then add 10 μM of reduced glutathione (GSH), oxidized glutathione (GSSG), captopril (CAP), adenine (Ade), L-cysteine ​​hydrochloride anhydrate (L-cysteine ​​HCl), glucose (Glu) or coenzyme NADH, add disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution at pH = 7.0, react at 37°C for 1.0 h, and then detect the fluorescence response of these biomolecules at 510 nm. The results are as follows. Figure 5 shown.

[0089] Depend on Figure 5It can be seen that when coenzyme NADH is used as the analyte, the fluorescence intensity of the system can reach 7000, while when other molecules are used instead of coenzyme NADH as the analyte, the fluorescence intensity is lower, indicating that the constructed sensor has good specificity for coenzyme NADH detection.

[0090] Example 4

[0091] In Example 4 of the present invention, an organic iridium catalyst 2-pyridinebenzamide pentamethylcyclopentadienyl organic iridium catalyst is used as an organic metal catalyst and combined with the hydrogenation-responsive coenzyme NAD(P)H fluorescent probe prepared in Example 1 to construct a fluorescent sensor for fluorescence imaging detection of intracellular coenzyme NADH. The method is as follows:

[0092] A human non-small cell lung cancer cell line (A459) was selected for cell passage. When the cell density reached 80%, the culture medium was removed and the cells were washed once with phosphate buffer solution PBS (0.1M, pH=7.4). A fluorescent probe (30.0 μM), an organic metal catalyst 2-pyridinebenzamide pentamethylcyclopentadiene (10.0 μM), and a coenzyme NADH (100 μM) were added to 1.5 ml of cell culture medium and incubated in a 37°C incubator for 1.0 h. The results are shown in FIG. Figure 6 .

[0093] After incubation, the cells were imaged using a confocal inverted fluorescence microscope. Figure 6 It can be seen that the cells showed obvious green fluorescence and had high lysosomal targeting.

[0094] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A hydrogenation-responsive coenzyme NAD(P)H fluorescent probe, characterized in that The structure of the hydrogenation-responsive coenzyme NAD(P)H fluorescent probe is shown in Formula 1: Formula 1.

2. A method for preparing the hydrogenation-responsive coenzyme NAD(P)H fluorescent probe according to claim 1, characterized in that: The steps include: (1) Preparation of 3-bromo-7H-benzo[d]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one: 4-Bromo-1,8-naphthalene dicarboxylic anhydride and o-phenylenediamine are dissolved in glacial acetic acid and heated under reflux for reaction. After the reaction, the solution is cooled to room temperature and the solid is collected by filtration to obtain a crude intermediate compound 3-bromo-7H-benzo[d]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one. The collected crude solid product is then transferred to a saturated aqueous solution of sodium bicarbonate and filtered. The obtained solid is rinsed with deionized water and analytical grade ethanol in sequence to obtain a pure bright yellow solid 3-bromo-7H-benzo[d]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one. (2) Preparation of 3-(2-(methylamino)ethyl)amino)-7H-benzo[d]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one: The 3-bromo-7H-benzo[d]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one obtained above is dissolved in ethylene glycol monoethyl ether solvent, and then an excess amount of the compound N-methylethane-1,2-diamine is added and refluxed. After the reaction, the reaction solution is cooled to room temperature, and then an appropriate amount of ice water is added and extracted with dichloromethane. The organic phase is collected and the organic solvent is removed by distillation under reduced pressure to obtain a crude intermediate product of 3-(2-(methylamino)ethyl)amino)-7H-benzo[d]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one. The crude product is further separated and purified by column chromatography to obtain a pure intermediate product of 3-(2-(methylamino)ethyl)amino)-7H-benzo[d]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one. (3) Preparation of hydrogenation-responsive coenzyme NAD(P)H fluorescent probe: 3-(2-(methylamino)ethyl)amino)-7H-benzo[d]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one, 2-methyl-2-(2,4,5-trimethyl-3,6-dioxocyclohexyl-1,4-dien-1-yl)propionic acid, EDCI, and HOBT were dissolved in dry Et3N solvent and DMF solvent, and reacted under light-proof and nitrogen protection conditions. After the reaction was completed, an appropriate amount of ice water was added to the reaction solution, followed by extraction with dichloromethane. The organic phase was collected and dried over anhydrous sodium sulfate, and the organic solvent was then distilled off under reduced pressure to obtain a crude hydrogenation-responsive coenzyme NAD(P)H fluorescent product. The crude product was further separated and purified by column chromatography to obtain a pure red powdery hydrogenation-responsive coenzyme NAD(P)H fluorescent probe.

3. The method for preparing the hydrogenation-responsive coenzyme NADPH fluorescent probe according to claim 2, wherein: In step (1), the molar ratio of 4-bromo-1,8-naphthalene dicarboxylic anhydride to o-phenylenediamine is 1:1-5, and the molar volume ratio of o-phenylenediamine to glacial acetic acid is 1 mmol:10-100 mL; the reaction temperature is 100-120° C., and the reaction time is 3-24 h.

4. The method for preparing the hydrogenation-responsive coenzyme NADPH fluorescent probe according to claim 2, wherein: The molar ratio of 3-bromo-7H-benzo[d]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one to N-methylethane-1, 2-diamine in step (2) is 1:1-5; the molar volume ratio of 3-bromo-7H-benzo[d]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one to ethylene glycol monoethyl ether is 1 mmol:5-20 mL; the reflux reaction time is 5-10 h, the temperature is 100-150° C., and the reflux reaction conditions are to avoid light; the conditions for chromatographic column separation are: silica gel column chromatography, and the mobile phase is methanol:dichloromethane = 1:

5.

5. The method for preparing the hydrogenation-responsive coenzyme NADPH fluorescent probe according to claim 2, wherein: In step (3), the molar ratio of 3-(2-(methylamino)ethyl)amino)-7H-benzo[d]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one, 2-methyl-2-(2,4,5-trimethyl-3,6-dioxocyclohexyl-1,4-dien-1-yl)propionic acid, Et3N, EDCI, and HOBT is 1:1~2:2~4:2~4, and the reaction time is 5~24 h; the number of dichloromethane extractions is 2~5 times; and the column chromatography purification conditions are: silica gel column chromatography, and the mobile phase is methanol:dichloromethane = 1:

10.

6. A hydrogenation-responsive coenzyme NAD(P)H fluorescence sensor, characterized in that The hydrogenation-responsive coenzyme NAD(P)H fluorescent sensor is prepared by combining the hydrogenation-responsive coenzyme NAD(P)H fluorescent probe according to claim 1 with a catalyst; The catalyst is a 2-pyridinebenzamide pentamethylcyclopentadienyl organic iridium catalyst, and its structure is: 。 7. Use of the hydrogenation-responsive coenzyme NAD(P)H fluorescence sensor according to claim 6 in the preparation of an intracellular hydrogenation-responsive coenzyme NAD(P)H fluorescence imaging analysis reagent.

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