Nitrosyl peroxide response probe as well as preparation method and application thereof

Abstract

The invention discloses a nitrosyl peroxide response probe as well as a preparation method and application thereof, and belongs to the technical field of biochemistry. The structural formula of the nitrosyl peroxide response probe is shown as a formula I, and R is hydrogen, a C1-C10 alkyl chain or an aromatic group. On the basis of replacing a rhodamine dye skeleton with phosphorus atoms, the ultrasensitive nitrosyl peroxide response probe is obtained through reasonable reduction reaction regulation and control design, destroy of a conjugated structure of molecules and formation of a non-conjugated precursor molecule with zero background fluorescence, and can be applied to nitrosyl peroxide imaging in cells and living bodies. And high-throughput screening of natural products with potential antioxidant activity is carried out.

Description

Technical Field

[0001] The invention belongs to the technical field of biochemistry, and in particular relates to a peroxide nitrosyl response probe and a preparation method and application thereof. Background Art

[0002] So far, nitrosyl peroxide (ONOO - There are many detection methods for ONOO, such as fluorescent probes, electrochemical analysis and absorption spectroscopy. Fluorescent probes have obvious advantages such as high temporal and spatial resolution, non-invasiveness and high sensitivity. However, these ONOO - The responsive fluorescent probes almost always rely on photoinduced electron transfer (PET) or intramolecular charge transfer (ICT) mechanisms to achieve quenching of the fluorescence signal. These fluorescence quenching mechanisms cannot completely avoid eliminating the intrinsic fluorescence of the probe, resulting in significantly lower sensitivity, limited response amplitude, and high detection limit of the probe. Therefore, in the process of tumors, inflammation, Alzheimer's disease, hepatorenal syndrome and other diseases, ONOO - Dynamic monitoring is difficult and elusive. Summary of the invention

[0003] In view of the above-mentioned prior art, the present invention provides a peroxide nitrosyl responsive probe and a preparation method and application thereof, so as to solve the problems of high probe intrinsic fluorescence and low sensitivity of the existing peroxide nitrosyl responsive fluorescent probe.

[0004] In order to achieve the above object, the technical solution adopted by the present invention is: to provide a peroxide nitrosyl responsive probe, the structural formula of the peroxide nitrosyl responsive probe is shown in Formula I,

[0005]

[0006] Wherein, R is hydrogen, a C1-C10 alkyl chain or an aromatic group.

[0007] Based on the above technical solution, the present invention can also be improved as follows.

[0008] Furthermore, R is hydrogen.

[0009] The beneficial effect of the above further technical scheme adopted by the present invention is: based on the replacement of the rhodamine dye skeleton with phosphorus atoms, through reasonable reduction reaction regulation and design, the conjugated structure of the molecule is destroyed to form a non-conjugated precursor molecule with zero "background" fluorescence, thereby obtaining an ultra-sensitive peroxide nitrosyl response probe.

[0010] Furthermore, the preparation method of the nitrosyl peroxide response probe comprises the following steps:

[0011] (1) dissolving m-bromoaniline, 1,4-dibromobutane and a weak base in acetonitrile and reacting at 20 to 25° C. for 10 to 12 hours to obtain a first intermediate;

[0012] (2) adding the first intermediate to a mixed solution of phosphorus oxychloride and dimethylformamide, reacting at 0-4° C. for 1-2 hours, and then heating to 60-80° C. for 12-14 hours to obtain a second intermediate;

[0013] (3) dissolving the second intermediate and the reducing agent in the first organic solvent, reacting at 20 to 25° C. for 8 to 10 hours to obtain a third intermediate;

[0014] (4) dissolving the first intermediate and the third intermediate in a second organic solvent, adding boron trifluoride etherate, and reacting at 20 to 25° C. for 5 to 8 hours to obtain a fourth intermediate;

[0015] (5) dissolving the fourth intermediate in the third organic solvent, cooling to -85 to -70°C, adding a strong base, and reacting for 1 to 2 hours; adding dichlorophenylphosphine, and reacting at 20 to 25°C for 10 to 14 hours; adding hydrogen peroxide, and reacting at 0 to 4°C for 10 to 14 hours to obtain a fifth intermediate;

[0016] (6) dissolving the fifth intermediate in a third organic solvent, adding tetrabutylammonium bromide, and reacting at 20 to 25° C. with oxygen for 5 to 8 hours to obtain a sixth intermediate;

[0017] (7) dissolving 2,6-dimethoxybromobenzene in a third organic solvent, cooling to -85 to -70°C, adding a strong base, and reacting for 1 to 2 hours; then adding the sixth intermediate, heating to 20 to 25°C, and reacting for 5 to 8 hours to obtain a seventh intermediate;

[0018] (8) dissolving the seventh intermediate in a third organic solvent, cooling to 0-4° C., adding a reducing agent, and reacting for 1-2 hours to obtain a peroxynitrosyl responsive probe;

[0019] Wherein, the weak base is at least one of sodium carbonate, potassium carbonate, potassium phosphate and sodium phosphate;

[0020] The strong base is one of n-butyl lithium, sec-butyl lithium and tert-butyl lithium;

[0021] The reducing agent is at least one of sodium borohydride, potassium borohydride and lithium aluminum hydride;

[0022] The first organic solvent is at least one of methanol, ethanol and dioxane;

[0023] The second organic solvent is at least one of dichloromethane and chloroform;

[0024] The third organic solvent is at least one of tetrahydrofuran and diethyl ether.

[0025] Furthermore, in step (1), the reaction temperature is 22° C. and the reaction time is 12 h;

[0026] In step (2), the reaction was carried out at 0°C for 1 h, and then heated to 70°C for 12 h;

[0027] In step (3), the reaction temperature is 22° C. and the reaction time is 8 h;

[0028] In step (4), the reaction temperature is 22° C. and the reaction time is 6 h;

[0029] In step (5), the mixture is first cooled to -78°C, and then a strong base is added and reacted for 1 hour; dichlorophenylphosphine is added and then reacted for 12 hours at 22°C; hydrogen peroxide is added and then reacted for 12 hours at 0°C;

[0030] In step (6), oxygen was bubbled in at 22° C. for 6 h;

[0031] In step (7), the mixture is first cooled to -78°C, a strong base is added and the mixture is reacted for 1 hour; after the sixth intermediate is added, the mixture is heated to 22°C and the mixture is reacted for 6 hours;

[0032] In step (8), the mixture is first cooled to 0°C, and then a reducing agent is added and the mixture is reacted for 1 hour.

[0033] Further, the weak base in step (1) is potassium carbonate;

[0034] The reducing agent in step (3) is sodium borohydride, and the first organic solvent is methanol;

[0035] The second organic solvent in step (4) is dichloromethane;

[0036] The third organic solvent in steps (5) to (8) is tetrahydrofuran;

[0037] The strong base in steps (5) and (7) is n-butyl lithium;

[0038] The reducing agent in step (8) is sodium borohydride.

[0039] The beneficial effects of the further technical scheme adopted in the present invention are: the preparation method has high yield and mild reaction conditions, and uses a phosphorus atom-substituted rhodamine derivative as the basic skeleton. By modifying the rhodamine derivative through sodium borohydride reduction, the conjugated structure of the dye molecule is broken to form a non-conjugated structure and a non-fluorescent precursor molecule, thereby obtaining a near-infrared "zero probe background fluorescence", ultra-fast, and highly selective peroxide nitrosyl response probe.

[0040] Further, the application of peroxynitrosyl responsive probes in fluorescence imaging.

[0041] Furthermore, the peroxide nitrosyl responsive probe was applied to detect peroxide nitrosyl in cells.

[0042] Furthermore, the peroxide nitrosyl responsive probe is applied to the detection of peroxide nitrosyl in vivo.

[0043] Furthermore, peroxynitrite-responsive probes were applied to screen natural products with antioxidant activity.

[0044] The beneficial effect of the above further technical solution adopted by the present invention is that the prepared peroxide nitrosyl response probe has the characteristic of "zero probe background fluorescence", and its longer emission wavelength (>740nm) can effectively avoid the interference of biological background light, and has high sensitivity, and can be used for in vitro cultured cells, peroxide nitrosyl detection and imaging in animals, and screening of natural products with potential antioxidant activity. The peroxide nitrosyl response probe has the characteristics of ultra-fast and high-sensitivity response for peroxide nitrosyl detection, which can effectively improve the accuracy of cell imaging results, and make it possible to dynamically monitor the fluctuation of peroxide nitrosyl substances in biological processes in real time.

[0045] The beneficial effects of the present invention are: using attractive phosphorus-substituted rhodamine as a fluorescent skeleton to construct hydrogenated phosphorus-substituted rhodamine, and forming non-fluorescent hydrogenated phosphorus rhodamine by destroying the conjugated structure of the rhodamine dye, thereby solving the current ONOO - The problem of high probe intrinsic fluorescence in responsive fluorescent probes. The final peroxide nitrosyl responsive probe can achieve sensitive imaging of zero probe "background" fluorescence while responding to peroxide nitrosyl ultra-fast, highly sensitive, and specifically, with high recognition accuracy. Since the peroxide nitrosyl responsive probe has a long emission wavelength and high fluorescence brightness, it is basically not affected by the imaging interference caused by the spontaneous fluorescence of biological tissues during biological imaging, which makes it also have the special performance of high signal-to-noise ratio in vivo imaging, and can effectively detect endogenous ONOO - Accumulation in living cells and in mice with hepatorenal syndrome. In addition, due to the high sensitivity of the probe, high-throughput screening of natural products with potential antioxidant activity is possible, providing a useful tool for the discovery of new drugs and understanding the chemical regulation mechanism of natural products to alleviate oxidative damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 A synthetic route for the peroxynitrosyl responsive probe;

[0047] Figure 2 is the UV absorption spectrum of the peroxynitrosyl responsive probe in response to peroxynitrosyl in PBS solution;

[0048] Figure 3is the fluorescence spectrum of the peroxynitrosyl responsive probe in response to peroxynitrosyl in PBS solution;

[0049] Figure 4 is the fitting curve of the fluorescence intensity of peroxynitrosyl response at 733 mm and the concentration of peroxynitrosyl response probe;

[0050] Figure 5 The results of MTS cytotoxicity test using a peroxide nitrosyl responsive probe;

[0051] Figure 6 The imaging results of peroxide nitrosyl response probe on endogenous peroxide nitrosyl in cells; a is the result of laser confocal detection, and b is the relationship between fluorescence intensity and stimulant concentration;

[0052] Figure 7 The results of imaging endogenous peroxide nitrosyl in mice using a peroxide nitrosyl responsive probe;

[0053] Figure 8 The results of high-throughput screening of natural antioxidants using peroxynitrosyl responsive probes; a is the operation process of high-throughput screening, and b is the fluorescence detection result;

[0054] Fig. 9 The structural formulas of 23 natural products are shown below. DETAILED DESCRIPTION

[0055] The specific implementation modes of the present invention are described in detail below with reference to the embodiments.

[0056] In the examples of the present invention, m-bromoaniline, n-butyl lithium, phosphorus oxychloride, various solvents, catalysts, alkalis and other reagents were purchased from Aladdin Technology Co., Ltd., cell lines were purchased from ATCC (American Type Culture Collection), 10% fetal bovine serum (FBS) was purchased from Hyclone, and culture medium was purchased from Gibco, USA.

[0057] Embodiment: A peroxide nitrosyl responsive probe, the synthesis route of which is as follows Figure 1 As shown, the preparation method is as follows:

[0058] (1) Synthesize the first intermediate 1, the synthesis route is as follows:

[0059]

[0060] m-Bromoaniline (1.0 mmol), 1,4-dibromobutane (1.5 mmol) and potassium carbonate (3.0 mmol) were dissolved in acetonitrile (MeCN, 5 mL), mixed and stirred at 22 °C for 12 h, and then 100 mL of water was added. The organic layer was separated and the aqueous layer was extracted with ethyl acetate (30 mL x 3). The organic extract was washed with brine and purified by Na 2 SO 4 After the solvent was distilled off under reduced pressure, the product was purified by 200-300 mesh silica gel column chromatography and eluted with a mixed solution of petroleum ether and ethyl acetate (the volume ratio of the two was 20:1) to obtain the first intermediate 1.

[0061] The first intermediate 1 is a light yellow liquid with a yield of 68%. Its hydrogen spectrum data are as follows:

[0062] 1 H NMR (400 MHz, CDCl 3 )δ8.11-8.08(s,1H),4.22-4.16(t,2H),1.95-1.85(m,2H),0.94-0.88(t,3H).

[0063] (2) Synthesize the second intermediate 2, the synthesis route is as follows:

[0064]

[0065] Under nitrogen protection, the first intermediate 1 (1 mmol) was added to phosphorus oxychloride (POCl 3 , 2g, 21mmoL, 60% dispersed in mineral oil) and dimethylformamide (DMF, 20mL) in a mixed solution, the resulting solution was stirred at 0°C for 1h, then the mixture was slowly heated to 70°C and stirred for 12h, and then water was added to quench the reaction. The organic layer was separated and the aqueous layer was extracted with dichloromethane (30mL×3). The organic extract was washed with brine and washed with Na 2 SO 4 After the solvent was distilled off under reduced pressure, the product was purified by 200-300 mesh silica gel column chromatography and eluted with a mixed solution of petroleum ether and ethyl acetate (the volume ratio of the two was 6:1) to obtain the second intermediate 2.

[0066] The second intermediate 2 is a white solid with a yield of 82%. Its hydrogen spectrum data are as follows:

[0067] 1 H NMR (400 MHz, CDCl 3)δ89.14-9.11(d,1H),8.97-8.93(d,1H),7.96-7.94(s,1H),7.64-7.58(d,1H),7.42-7.36(t,1H ),7.31-7.26(t,1H),6.81-6.78(d,1H),4.23-4.17(t,2H),2.00-1.90(m,2H),1.01-0.95(t,3H).

[0068] (3) Synthesize the third intermediate 3, the synthesis route is as follows:

[0069]

[0070] The second intermediate 2 (1 mmol) and sodium borohydride (NaBH 4 , 2mmoL) was dissolved in methanol (MeOH, 10mL) and reacted at 22°C for 8h. After the reaction was complete as monitored by thin layer chromatography, the reaction mixture was added to water (100mL) and extracted with dichloromethane. The organic layer was washed with brine and water, and Na 2 SO 4 After the solvent is removed by distillation under reduced pressure, the third intermediate 3 is obtained.

[0071] The third intermediate 3 is a white solid with a yield of 98%. Its hydrogen spectrum data are as follows:

[0072] 1 H NMR (400 MHz, CDCl 3 )δ10.12-10.09(s,1H),9.22-9.19(d,1H),9.03-8.99(d,1H),8.71-8.65(t,3H),8.14-8.09(d,2H),7.72-7.68(d,1H ),7.46-7.41(t,1H),7.32-7.27(t,1H),6.95-6.91(d,1H),4.37-4.30(t,2H),2.00-1.91(m,2H),0.94-0.88(t,3H).

[0073] (4) Synthesis of the fourth intermediate 4, the synthesis route is as follows:

[0074]

[0075] The first intermediate 1 (1 mmoL) and the third intermediate 3 (1 mmoL) were dissolved in dichloromethane (DCM, 10 mL), and a boron trifluoride ether solution (BF 3 ·OEt 2, 2mmoL) was dropped into the solution. The mixture was then stirred at 22°C for 6h. After the reaction was completed by monitoring with thin layer chromatography, the solvent was removed by rotary evaporation. The crude product was purified by 200-300 mesh silica gel column chromatography and eluted with a mixed solution of petroleum ether and ethyl acetate (the volume ratio of the two was 5:1) to obtain the fourth intermediate 4.

[0076] The fourth intermediate was a light yellow solid with a yield of 67%.

[0077] (5) Synthesizing the fifth intermediate 5, the synthetic route is as follows:

[0078]

[0079] Under nitrogen protection, the fourth intermediate 4 (10 mmol) was added to the reaction bottle, and then tetrahydrofuran (THF, 40 mL) was added to dissolve the fourth intermediate 4. After the system was cooled to -78 ° C, n-butyl lithium solution (n-BuLi, 11 mmol) was dropped into the above solution, and then the mixture was stirred at -78 ° C for about 1 hour, and dichlorophenylphosphine (PPhCl) was added. 3 , 5.5mmoL), and heated to 22°C for 12h; then hydrogen peroxide (H 2 O 2 , 5 mL), react at 0°C for 12 h, and after the reaction is completed as monitored by thin layer chromatography, 100 mL of water is added to the system, extracted with dichloromethane, and the solvent is removed by rotary evaporation to obtain the fifth intermediate 5.

[0080] The fifth intermediate 5 is a light yellow liquid.

[0081] (6) Synthesizing the sixth intermediate 6, the synthetic route is as follows:

[0082]

[0083] The fifth intermediate 5 (1 mmoL) was added to tetrahydrofuran (THF, 50 mL), and tetrabutylammonium bromide (TBAB, 0.05 mmoL, 16 mg) was added to the system, and then the mixture was stirred at 22°C with oxygen for about 6 h. After the reaction was completed by monitoring with thin layer chromatography, the solvent was removed by rotary evaporation, and the crude product was purified by 200-300 mesh silica gel column chromatography and eluted with a mixed solution of dichloromethane and methanol (the volume ratio of the two was 100:1) to obtain the sixth intermediate 6.

[0084] The sixth intermediate 6 is a light yellow solid with a yield of 32%.

[0085] (7) Synthesis of the seventh intermediate 7, the synthesis route is as follows:

[0086]

[0087] Add 2,6-dimethoxybromobenzene (5mmoL) to tetrahydrofuran (THF, 50mL), add n-butyl lithium (n-BuLi, 5mmoL) when the system is cooled to -78°C, and continue stirring for 1h. Then, the sixth intermediate 6 (1mmoL) is dissolved in tetrahydrofuran (THF, 40mL) and added to the system, stirred at 22°C for about 6h, then 5mL of hydrochloric acid is added to quench the reaction, extracted with dichloromethane, the organic phases are combined, the solvent is removed by rotary evaporation, and the crude product is purified by 200-300 mesh silica gel column chromatography, eluted with a mixed solution of dichloromethane and methanol (the volume ratio of the two is 10:1), to obtain the seventh intermediate 7.

[0088] The seventh intermediate 7 is a blue-green solid with a yield of 74%. Its nuclear magnetic resonance spectrum data are as follows:

[0089] 1 H NMR (400 MHz, CDCl 3 )δ8.60(s,1H),7.73(dd,J=12.5,7.4Hz,6H),7.54(t,J=6.9Hz,3H),7.47(dt,J=7.1,3.5Hz,6H),7.31(d,J=8.7Hz,1H), 6.54 (dd, J=8.8, 2.3Hz, 1H), 6.48 (d, J=2.1Hz, 1H), 5.55 (d, J=28.6Hz, 1H), 3.40 (q, J=7.1Hz, 4H), 1.20 (t, J=7.1Hz, 6H).

[0090] 13 C NMR (101 MHz, CDCl 3 )δ177.7,161.9,157.2,151.1,144.0,133.3,133.2,131.9,130.3,128.9,128.8,127.3,126.4,108.9,96.5,56.4,55.4,44.8,12.5.

[0091] HRMS(ESI)C 33 H 30 NO 3 P[M+H] + 520.2040.

[0092] (8) Synthesize peroxynitrosyl responsive probe 8. The synthesis route is as follows:

[0093]

[0094] The seventh intermediate 7 (1 mmol) was added to tetrahydrofuran (THF, 50 mL). When the system was cooled to 0°C, sodium borohydride (NaBH 4 , 3mmoL), and continued stirring for 1h. After the reaction was completed by monitoring with thin layer chromatography, the solvent was removed by rotary evaporation, and the crude product was purified by 200-300 mesh silica gel column chromatography and eluted with a mixed solution of dichloromethane and methanol (the volume ratio of the two was 60:1) to obtain peroxynitrosyl responsive probe 8.

[0095] The peroxide nitrosyl responsive probe is a light green solid with a yield of 47%. Its nuclear magnetic resonance spectrum data are as follows:

[0096] 1 H NMR (400 MHz, CDCl 3 )δ8.60(s,1H),7.73(dd,J=12.5,7.4Hz,6H),7.54(t,J=6.9Hz,3H),7.47(dt,J=7.1,3.5Hz,6H),7.31(d,J=8.7Hz,1H), 6.54 (dd, J=8.8, 2.3Hz, 1H), 6.48 (d, J=2.1Hz, 1H), 5.55 (d, J=28.6Hz, 1H), 3.40 (q, J=7.1Hz, 4H), 1.20 (t, J=7.1Hz, 6H).

[0097] 13 C NMR (101 MHz, CDCl 3 )δ177.7,161.9,157.2,151.1,144.0,133.3,133.2,131.9,130.3,128.9,128.8,127.3,126.4,108.9,96.5,56.4,55.4,44.8,12.5.

[0098] HRMS(ESI)C 33 H 30 NO 3 P[M+H] + 520.2040.

[0099] Experimental Example 1: UV absorption spectrum

[0100] The peroxide nitrosyl response probe prepared in the example was prepared into a mother solution with a concentration of 10 mM using the solvent DMSO, and then the mother solution was prepared into a working solution with a concentration of 10 μM using a PBS buffer, and the ultraviolet absorption value was scanned and plotted. Subsequently, the peroxide nitrosyl solution was added to the system in small amounts, and the ultraviolet absorption was scanned again and plotted. The concentration range of peroxide nitrosyl in the system was 0 to 90 μM.

[0101] The ultraviolet absorption spectrum of the peroxynitrosyl response prepared in the embodiment is as follows: Figure 2 As shown, one absorption peak appears near λ=354nm and the other appears near λ=394nm. With the addition of nitrosyl peroxide, the probe is gradually oxidized and the absorption peak at this position gradually decreases. At the same time, the absorption peak at 712nm gradually increases.

[0102] Experimental Example 2: Fluorescence Spectroscopy

[0103] The peroxide nitrosyl response probe prepared in the example was prepared into a mother solution with a concentration of 10 mM using the solvent DMSO, and then the mother solution was prepared into a working solution with a concentration of 10 μM using a PBS buffer, and the fluorescence emission spectrum was scanned and plotted with a wavelength of 660 nm as the excitation light. The peroxide nitrosyl solution was added to the system in small amounts, and the fluorescence emission was scanned again and plotted. The concentration range of peroxide nitrosyl in the system was 0 to 90 μM.

[0104] The fluorescence spectrum of the peroxynitrosyl responsive probe prepared in the embodiment is as follows: Figure 3 As shown in the figure, the relationship between the fluorescence intensity at 733 nm and the concentration of the peroxynitrosyl response probe in the system is as follows: Figure 4 As shown, with the increase of probe concentration, the fluorescence emission peak at 733 nm gradually increased.

[0105] Experimental Example 3: MTS cytotoxicity assay

[0106] HepG2 cells in the logarithmic growth phase were seeded in 96-well culture plates, with 10,000 cells per well, and cultured in DMEM (H) medium containing 10% fetal bovine serum (FBS) and 1% double antibiotics (penicillin and streptomycin, 1000 KU / L) at 37°C and 5% CO. 2 After the cells were completely attached to the wall, the peroxide nitrosyl response probe prepared in the embodiment was added at concentrations of 3.125, 6.25, 12.5, 25 and 50 μM, respectively, with 3 replicate wells for each concentration and a blank control group. After adding the peroxide nitrosyl response probe, the cells were cultured for 24 hours, and the inhibition rate of the cells was detected by the MTS method.

[0107] Taking the cell viability of the blank control group as the standard, a bar graph was drawn at concentrations of 3.125, 6.25, 12.5, 25 and 50 μM. The MTS cytotoxicity results are shown in Figure 5 As shown, at a high concentration of 25 μM, the peroxide nitrosyl responsive probe prepared in Example has a high cytotoxicity to HepG2 cells. However, when the concentration is reduced to 6.25 μM, the cell survival rate is basically not affected.

[0108] Application Example 1: Confocal Laser Imaging of Endogenous Nitrosyl Peroxide in HepG2 Cells

[0109] HepG2 cells in the logarithmic growth phase were inoculated in a 35 mm confocal culture dish and cultured overnight. Gamma interferon (IFN-γ) was added to the culture medium at concentrations of 5 μg / mL, 10 μg / mL, and 20 μg / mL, respectively, and incubated at 37°C for 60 min. Subsequently, 0.5 μL of the probe at a concentration of 10 mM was added to the culture medium and incubated at 37°C for 30 min. The peroxynitrosyl-responsive probe was imaged under a laser confocal microscope using excitation and emission filters, where λ ex =633nm,λ em =660~760nm.

[0110] The results are as follows Figure 6 As shown in the figure, the peroxide nitrosyl response probe can well recognize endogenous peroxide nitrosyl and light up the cells. At the same time, as the concentration of the stimulator (IFN-γ) increases, the probe signal increases accordingly, based on which endogenous peroxide nitrosyl imaging of cells can be achieved.

[0111] Application Example 2: In vivo imaging of liver peroxide nitrosyl in a mouse liver injury model

[0112] A mouse drug-induced liver injury model was constructed, and D-galactosamine (100 mg / kg, prepared with normal saline, injection volume of 100 μL) was injected into the mouse peritoneum. After 12 hours, the model was successfully established, and the mice were randomly divided into an experimental group and a control group. The peroxide nitrosyl response probe was dissolved in DMSO, and then prepared into a working solution with a concentration of 2 mg / mL with normal saline. The final working solution contained 25% DMSO. 100 μL of the working solution was injected into the control group mice and the experimental group mice respectively through the tail vein of the mouse, and then the changes in the fluorescence signal in the liver were monitored by a small animal in vivo imager.

[0113] The results are as follows Figure 7 As shown, in the liver area of ​​mice, the fluorescence signal of mice with liver damage was significantly higher than that of mice in the normal control group, indicating that the probe molecule has the ability to specifically identify nitrosyl peroxide in small animals with high sensitivity (detection limit of 18.9nM) and high signal-to-noise ratio.

[0114] Application Example 3: High-throughput screening of natural antioxidants

[0115] The peroxide nitrosyl response probe was used to evaluate 23 natural products with different chemical structures (such as polyphenols, quinones, alkaloids and sesquiterpenes). Each natural product was prepared into a mother solution with a concentration of 10mM using DMSO. HepG2 cells in the logarithmic growth phase were inoculated in a 24-well plate and cultured overnight. 10μL of each natural product mother solution was added to each well of the 24-well plate and incubated in a 37℃ constant temperature incubator for 12h. The probe was then added and incubated for 1h. The 24-well plate was placed on a high-content imager to collect cell fluorescence images and statistically analyze the results.

[0116] The operation process and fluorescence results of high-throughput screening are as follows Figure 8 As shown, Figure 8 The structural formulas of the 23 natural products corresponding to 2 to 24 in b are as follows Fig. 9 As shown, there are two natural products (luteolin and quercetin, corresponding to Figure 8 16 and 20 in b) can significantly reduce the fluorescence intensity, indicating that endogenous ONOO - Down-regulated. Both compounds have structural features of polyphenol derivatives and have potential antioxidant capacity. This shows that the probe has high sensitivity and successfully screened natural products with potential antioxidant activity, which can provide a useful tool for the discovery of new drugs to reduce oxidative damage and for understanding the chemical regulation mechanism of natural products.

[0117] In summary, the present invention uses the rhodamine skeleton substituted with phosphorus atoms as the basis, breaks the conjugated structure of the dye molecule through a reduction reaction, and forms a non-conjugated structure and non-fluorescent form of the probe molecule. Since the peroxide nitrosyl response probe is a non-conjugated structure, it can achieve the regulation of "zero probe background fluorescence". While quickly identifying peroxide nitrosyl, it also has very high sensitivity and selectivity, which is beneficial to the dynamic monitoring of peroxide nitrosyl in animals. In addition, since the probe has the characteristics of near-infrared emission, it has low biological background fluorescence and high imaging signal-to-noise ratio. The preparation method of the present invention has high yield and mild reaction conditions, and the prepared probe is sensitive and highly selective.

[0118] Although the specific implementation of the present invention is described in detail in conjunction with the embodiments, it should not be understood as limiting the scope of protection of this patent. Within the scope described in the claims, various modifications and variations that can be made by those skilled in the art without creative work still fall within the scope of protection of this patent.

Claims

1. A nitrosyl peroxide responsive probe, characterized in that: The structural formula of the peroxide nitrosyl responsive probe is shown in Formula I, Wherein, R is hydrogen, a C1-C10 alkyl chain or an aromatic group.

2. The peroxide nitrosyl responsive probe according to claim 1, characterized in that: The R is hydrogen.

3. The method for preparing the nitrosyl peroxide responsive probe according to claim 1 or 2, characterized in that: The following steps are involved: (1) dissolving m-bromoaniline, 1,4-dibromobutane and a weak base in acetonitrile and reacting at 20 to 25° C. for 10 to 12 hours to obtain a first intermediate; (2) adding the first intermediate to a mixed solution of phosphorus oxychloride and dimethylformamide, reacting at 0-4° C. for 1-2 hours, and then heating to 60-80° C. for 12-14 hours to obtain a second intermediate; (3) dissolving the second intermediate and the reducing agent in a first organic solvent, and reacting at 20 to 25° C. for 8 to 10 hours to obtain a third intermediate; (4) dissolving the first intermediate and the third intermediate in a second organic solvent, adding boron trifluoride etherate, and reacting at 20 to 25° C. for 5 to 8 hours to obtain a fourth intermediate; (5) dissolving the fourth intermediate in a third organic solvent, cooling to -85 to -70°C, adding a strong base, and reacting for 1 to 2 hours; adding dichlorophenylphosphine, and reacting at 20 to 25°C for 10 to 14 hours; adding hydrogen peroxide, and reacting at 0 to 4°C for 10 to 14 hours to obtain a fifth intermediate; (6) dissolving the fifth intermediate in a third organic solvent, adding tetrabutylammonium bromide, and reacting at 20 to 25° C. with oxygen for 5 to 8 hours to obtain a sixth intermediate; (7) dissolving 2,6-dimethoxybromobenzene in a third organic solvent, cooling to -85 to -70°C, adding a strong base, and reacting for 1 to 2 hours; then adding the sixth intermediate, heating to 20 to 25°C, and reacting for 5 to 8 hours to obtain a seventh intermediate; (8) dissolving the seventh intermediate in a third organic solvent, cooling to 0-4° C., adding a reducing agent, and reacting for 1-2 hours to obtain a peroxynitrosyl responsive probe; Wherein, the weak base is at least one of sodium carbonate, potassium carbonate, potassium phosphate and sodium phosphate; The strong base is one of n-butyl lithium, sec-butyl lithium and tert-butyl lithium; The reducing agent is at least one of sodium borohydride, potassium borohydride and lithium aluminum hydride; The first organic solvent is at least one of methanol, ethanol and dioxane; The second organic solvent is at least one of dichloromethane and chloroform; The third organic solvent is at least one of tetrahydrofuran and diethyl ether.

4. The method for preparing the nitrosyl peroxide responsive probe according to claim 3, characterized in that: In the step (1), the reaction temperature is 22° C. and the reaction time is 12 h; In step (2), the reaction was carried out at 0°C for 1 hour, and then heated to 70°C for 12 hours; In step (3), the reaction temperature is 22° C. and the reaction time is 8 h; In step (4), the reaction temperature is 22° C. and the reaction time is 6 h; In the step (5), the mixture is first cooled to -78°C, a strong base is added and the mixture is reacted for 1 hour; dichlorophenylphosphine is added and the mixture is reacted for 12 hours at 22°C; hydrogen peroxide is added and the mixture is reacted for 12 hours at 0°C; In the step (6), oxygen is added to react at 22° C. for 6 hours; In the step (7), the mixture is first cooled to -78°C, a strong base is added and the mixture is reacted for 1 hour; after the sixth intermediate is added, the mixture is heated to 22°C and the mixture is reacted for 6 hours; In the step (8), the mixture is first cooled to 0° C., and then a reducing agent is added and the mixture is reacted for 1 hour.

5. The method for preparing the nitrosyl peroxide responsive probe according to claim 3, characterized in that: The weak base in step (1) is potassium carbonate; The reducing agent in step (3) is sodium borohydride, and the first organic solvent is methanol; The second organic solvent in step (4) is dichloromethane; The third organic solvent in steps (5) to (8) is tetrahydrofuran; The strong base in steps (5) and (7) is n-butyl lithium; The reducing agent in step (8) is sodium borohydride.

6. Use of the peroxynitrosyl responsive probe according to claim 1 or 2 in fluorescence imaging.

7. The use according to claim 6, characterized in that: The peroxide nitrosyl response probe is used for detecting peroxide nitrosyl in cells.

8. The use according to claim 6, characterized in that: The peroxide nitrosyl response probe is used for detecting peroxide nitrosyl in a living body.

9. The use according to claim 6, characterized in that: The peroxynitrosyl responsive probe is used to screen natural products with antioxidant activity.

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