Endoplasmic reticulum targeting carbon monoxide fluorescent probe, preparation method and application

Abstract

The invention provides an endoplasmic reticulum targeting type carbon monoxide (CO) fluorescent probe, a preparation method and application, the fluorescent probe is shown as a structural formula (I), the endoplasmic reticulum targeting type carbon monoxide fluorescent probe takes easily modified naphthalimide as a fluorescent body, and takes methyl sulfonamide as an endoplasmic reticulum targeting group, and the structural formula (I) is shown in the description. Benzothiadiazole is used as a fluorescence quenching group and a CO recognition response site, and specific detection of CO is realized through a ruthenium ligand exchange reaction. The technical problems that an existing carbon monoxide fluorescent probe is complex in preparation and purification process, low in detection response speed, weak in intracellular fluorescent response signal and the like are solved. According to the present invention, the synthesis and the purification are simple, the yield is high, the CO response speed is rapid, the response platform can be achieved within 15 min, and the detection limit is as low as 44.7 nM; the fluorescent probe has good biocompatibility, and can be used for targeted enrichment in the endoplasmic reticulum and quantitative detection of exogenous CO and endogenous CO in the endoplasmic reticulum.

Description

Technical Field

[0001] The present invention relates to the technical field of biochemical materials, and particularly relates to an endoplasmic reticulum-targeted carbon monoxide fluorescent probe, a preparation method and an application thereof. Background Art

[0002] CO is one of the most important gaseous neurotransmitter molecules in the human body (such as NO, H2S, CO), and is closely related to many physiological and pathological processes, including anti-inflammatory response, neurotransmission, vascular smooth muscle and vasodilation. Scientific research shows that endogenous CO can be produced by the catalytic decomposition of heme by heme oxygenase in vivo. Abnormal CO levels can lead to diseases such as hypertension, heart disease, and pathological inflammation. Therefore, in order to deeply explore the specific mechanism of action of CO in vivo, it is crucial to accurately quantify the CO level in vivo.

[0003] The endoplasmic reticulum (ER), as an important sub-organelle in eukaryotic cells, is involved in various physiological activities such as protein synthesis, lipid metabolism, and calcium storage. The endoplasmic reticulum (ER) is the organelle with the largest single-layer area in eukaryotic cells. Most of the heme oxygenase that produces endogenous CO in living cells is distributed in the endoplasmic reticulum (ER), and CO plays an important role in regulating endoplasmic reticulum stress. Related research shows that endogenous CO prevents apoptosis triggered by endoplasmic reticulum (ER) stress through the protein kinase R-like endoplasmic reticulum (ER) kinase signaling pathway. However, the specific mechanism by which CO maintains endoplasmic reticulum (ER) homeostasis remains unclear. Therefore, precisely visualizing CO in the endoplasmic reticulum (ER) is of great significance for clarifying the specific regulatory function of CO and developing treatment methods for related diseases.

[0004] Compared with other analytical detection methods, the fluorescence detection method has the characteristics of high sensitivity, simple operation method, and fluorescence imaging visualization detection, and has been widely concerned in the field of bioanalysis. In the past few years, a variety of CO fluorescent probes have been reported. For example, a CO fluorescent probe based on the Pd 0 -mediated Tsuji-Trost reaction, which requires the addition of palladium salt during detection and is a two-component fluorescent probe, which increases the factors that may be interfered during the response process of the fluorescent probe to CO. In addition, the key role of CO in biological signal transduction has prompted researchers to pay more attention to the detection of CO at the subcellular level, and CO fluorescent probes with organelle targeting ability have been successively reported. However, most of them are mitochondrial-targeted CO fluorescent probes. At present, there is little attention to ER-targeted CO probes. Summary of the Invention

[0005] Based on this, the purpose of the present invention is to provide an endoplasmic reticulum-targeted carbon monoxide fluorescent probe, a preparation method and an application thereof, so as to solve the technical problems such as the complex preparation and purification process of the existing carbon monoxide fluorescent probe, slow detection response speed, weak intracellular fluorescence response signal, and mostly whole-cell level detection.

[0006] To solve the above technical problems, one of the objectives of the present invention is to provide an endoplasmic reticulum-targeted carbon monoxide fluorescent probe, and this fluorescent probe is shown as the structural formula (Ⅰ): .

[0007] Based on the same inventive concept, the present invention also provides a preparation method of an endoplasmic reticulum-targeted carbon monoxide fluorescent probe, including the following steps: S1: Dissolve 4-bromo-1,8-naphthalic anhydride and propargylamine in absolute ethanol for reaction. After the reaction is completed, a precipitate is precipitated, filtered and the filter cake is collected, and then placed in a vacuum drying oven for drying to obtain a compound shown as the structural formula (Ⅱ): ; S2: Dissolve the compound with the structure shown in the structural formula (Ⅱ), p-toluenesulfonamide and anhydrous potassium carbonate in N,N-dimethylformamide for reaction, and the reaction product is purified to obtain a compound shown as the structural formula (Ⅲ): ; S3: Add tris(triphenylphosphine)ruthenium carbonyl chloride ligand to ultradry dichloromethane, react at room temperature for 1 h under nitrogen protection, add benzothiadiazole, and after the reaction solution turns from yellow to red, add the compound with the structure shown in the structural formula (Ⅲ). After the reaction is completed, add absolute methanol to the reaction solution, precipitate, filter and collect the filter cake to obtain the endoplasmic reticulum-targeted carbon monoxide fluorescent probe RE-NS.

[0008] Preferably, the reaction conditions of S1 are: the reaction temperature is 80 °C, and the stirring reaction time is 12 h.

[0009] Preferably, the molar ratio of 4-bromo-1,8-naphthalic anhydride to propargylamine in S1 is 1.7-2.0:6.0-6.5.

[0010] Preferably, the reaction conditions in S2 are: the reaction temperature is 100 °C, and the stirring and reflux condensation time is 12 h.

[0011] Preferably, the molar ratio of the compound with the structure shown in the structural formula (Ⅱ), p-toluenesulfonamide to anhydrous potassium carbonate in S2 is 1:1.8-2.0:2.0.

[0012] Preferably, the purification in S2 adopts silica gel column chromatography, and the eluent is ethyl acetate and petroleum ether, and the volume ratio of ethyl acetate to petroleum ether is 1:2.

[0013] Preferably, in the step S3, the molar ratio of tris(triphenylphosphine)ruthenium carbonyl chloride ligand, benzothiadiazole, and the compound having the structure shown in the formula (Ⅲ) is 0.15:0.16:0.15.

[0014] The present invention also provides an application of an endoplasmic reticulum-targeted carbon monoxide fluorescent probe, and the endoplasmic reticulum-targeted carbon monoxide fluorescent probe is used for detecting exogenous carbon monoxide and endogenous carbon monoxide inside and outside cells.

[0015] One or more of the above technical solutions of the present invention have the following technical effects: The endoplasmic reticulum-targeted carbon monoxide fluorescent probe provided by the present invention is simple to synthesize, has a high yield, has a fast response speed to CO, can reach the response platform in 15 minutes, and has a detection limit as low as 44.7 nM. RE-NS has good biocompatibility, can be targeted and enriched in the endoplasmic reticulum and detect exogenous and endogenous CO in the endoplasmic reticulum, providing an important tool for in-depth understanding of the function of CO in the ER. In addition, the endoplasmic reticulum-targeted carbon monoxide fluorescent probe RE-NS was successfully used to prove that the intracellular metabolism of the antihypertensive drug nifedipine can produce endogenous CO. Description of the Drawings

[0016] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the description of the embodiments will be briefly introduced below.

[0017] Figure 1 Synthesis route of the endoplasmic reticulum-targeted carbon monoxide fluorescent probe RE-NS; Figure 2 High-resolution mass spectrum of the endoplasmic reticulum-targeted carbon monoxide fluorescent probe RE-NS; Figure 3 High-resolution mass spectrum of the response product RE-CO after RE-NS detects CO; Figure 4 Fluorescence intensity ratio diagram of the reaction product of RE-NS and CO and RE-NS at 550 nm in different pH solutions; Figure 5 Figure 5 (a) shows the change of the fluorescence spectrum of RE-NS with time after reacting with CO (CORM-3 is a CO donor compound); Figure 5 (b) shows the time kinetics change of the fluorescence intensity ratio of RE-NS to the fluorescence intensity of RE-NS before reacting with CO at 550 nm; Figure 6 Figure 6 (a) shows the fluorescence spectrum diagram after reacting with different concentrations of CO; Figure 6 ​​(b) is the linear relationship between the ratio of the fluorescence intensity of RE-NS to CO response at 550 nm and the fluorescence intensity of RE-NS before response and the concentration of CO; Figure 7 Specificity analysis diagram of the endoplasmic reticulum-targeted carbon monoxide fluorescent probe RE-NS; Figure 8 、 Figure 8 (a) is the cytotoxicity diagram of the endoplasmic reticulum-targeted carbon monoxide fluorescent probe RE-NS; Figure 8 (b) is the cytotoxicity diagram of the endoplasmic reticulum-targeted carbon monoxide fluorescent probe RE-NS in response to heme; Figure 8 (c) is the cytotoxicity diagram of the endoplasmic reticulum-targeted carbon monoxide fluorescent probe RE-NS in response to CORM-3; Figure 9 、 Figure 9 (a-l) are the confocal microscopy images of the endoplasmic reticulum-targeted carbon monoxide fluorescent probe RE-NS in response to exogenous CO; Figure 9 (m) is the quantitative ratio diagram of the average fluorescence intensity of cells in the green channel; Figure 10 、 Figure 10 (a-l) are the confocal microscopy images of the endoplasmic reticulum-targeted carbon monoxide fluorescent probe RE-NS in response to endogenous CO; Figure 10 (m) is the quantitative ratio diagram of the average fluorescence intensity of cells in the green channel; Figure 11 、 Figure 11 (a-d) are the co-localization imaging diagrams of the endoplasmic reticulum-targeted carbon monoxide fluorescent probe RE-NS and the endoplasmic reticulum red dye; Figure 11 (e) is the fluorescence intensity scatter diagram; Figure 11 (f) is the fluorescence intensity distribution diagram at the same position; Figure 12 、 Figure 12 (a) is the cytotoxicity diagram of nifedipine; Figure 12 (b) is the cytotoxicity diagram of nifedipine in response to the endoplasmic reticulum-targeted carbon monoxide fluorescent probe RE-NS; Figure 13 、 Figure 13 (a-l) are the confocal imaging diagrams of the endoplasmic reticulum-targeted carbon monoxide fluorescent probe RE-NS in response to different concentrations of nifedipine; Figure 13 (m) is the quantitative diagram of the average fluorescence intensity of cells in the green channel. Specific embodiments

[0018] The present invention provides an endoplasmic reticulum-targeted carbon monoxide fluorescent probe. The endoplasmic reticulum-targeted carbon monoxide fluorescent probe RE-NS is shown as the structural formula (I): 。

[0019] The present invention also provides a method for preparing an endoplasmic reticulum-targeted carbon monoxide fluorescent probe, which comprises the following steps: S1: 4-bromo-1,8-naphthalic anhydride and propargylamine are dissolved in absolute ethanol for reaction. After the reaction is completed, a precipitate is precipitated, filtered and the filter cake is collected, and then placed in a vacuum drying oven for drying to obtain a compound with the structure shown in formula (Ⅱ): ; S2: The compound with the structure shown in formula (Ⅱ), p-toluenesulfonamide and anhydrous potassium carbonate are dissolved in N,N-dimethylformamide for reaction. The reaction product is purified to obtain a compound with the structure shown in formula (Ⅲ): ; S3: Tris(triphenylphosphine)ruthenium(I) chloride ligand is added to ultra-dry dichloromethane. Under nitrogen protection, the reaction is carried out at room temperature for 1 h. Benzothiadiazole is added. After the reaction solution turns from yellow to red, the compound with the structure shown in formula (Ⅲ) is added. After the reaction is completed, anhydrous methanol is added to the reaction solution, a precipitate is precipitated, and the filter cake is collected by filtration to obtain the endoplasmic reticulum-targeted carbon monoxide fluorescent probe RE-NS.

[0020] In S1 of the present invention, the reaction temperature of 4-bromo-1,8-naphthalic anhydride and propargylamine is 80 °C, and the stirring reaction time is 12 h. There is no special limitation on the stirring rate, and the stirring rate well-known to those skilled in the art can be adopted.

[0021] In S1 of the present invention, the molar ratio of 4-bromo-1,8-naphthalic anhydride to propargylamine is 1.7-2.0:6.0-6.5, preferably 1.8:6.3.

[0022] In S2 of the present invention, the conditions for adding the compound with the structure shown in formula (Ⅱ) to the ethanol solution of propargylamine for reaction are: the reaction temperature is 100 °C, and the stirring and reflux time is 12 h. At the same time, the purification adopts silica gel column chromatography, and the eluent is ethyl acetate and petroleum ether, and the volume ratio of ethyl acetate to petroleum ether is 1:2.

[0023] In S2 of the present invention, the molar ratio of the compound with the structure shown in formula (Ⅱ), p-toluenesulfonamide to anhydrous potassium carbonate is 1:1.8-2.0:2.0, preferably 1:1.9:2.0.

[0024] In S3 of the present invention, the molar ratio of tris(triphenylphosphine)ruthenium(I) chloride ligand, benzothiadiazole and the compound with the structure shown in formula (Ⅲ) is 0.15:0.16:0.15.

[0025] In step S3 of the present invention, there is no special limitation on the reaction time. Preferably, the reaction is monitored by a TLC plate, and the reaction can be stopped until the compound with the structure shown in formula (Ⅲ) completely disappears.

[0026] The present invention also provides the application of an endoplasmic reticulum-targeted carbon monoxide fluorescent probe. The endoplasmic reticulum-targeted carbon monoxide fluorescent probe RE-NS is used for detecting exogenous and endogenous carbon monoxide in cells. The action mechanism of the endoplasmic reticulum-targeted carbon monoxide fluorescent probe RE-NS is as follows:

[0027] The endoplasmic reticulum-targeted carbon monoxide fluorescent probe RE-NS provided by the present invention uses naphthalimide as a fluorophore. Benzothiadiazole serves as both a fluorophore quencher and a CO-specific recognition and response site, and realizes specific targeting to the endoplasmic reticulum through a methylsulfonamide group. The endoplasmic reticulum-targeted carbon monoxide fluorescent probe RE-NS specifically detects CO through a ligand exchange reaction of a ruthenium complex. Due to the intramolecular PET effect between the fluorophore and benzothiadiazole, the fluorescence is quenched. When CO exists in the system, a displacement reaction occurs between benzothiadiazole and CO, blocking the intramolecular PET, thereby restoring the fluorescence of the fluorophore. Furthermore, specific quantitative detection of CO can be achieved according to the change in fluorescence intensity.

[0028] Next, the technical solutions of the present invention will be clearly and completely described in conjunction with the accompanying drawings of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts fall within the scope of protection of the present invention.

[0029] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as having the meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In case of any conflict, this specification shall prevail.

[0030] Example 1 The preparation route of an endoplasmic reticulum-targeted carbon monoxide fluorescent probe is as Figure 1 shown and includes the following steps: S1: Dissolve 4-bromo-1,8-naphthalic anhydride (509.1 mg, 1.8 mmol) and propargylamine (349.6 mg, 6.3 mmol) in 10 mL of absolute ethanol, and heat the mixture to 80 °C for reaction for 12 h. After the reaction is completed, cool the mixture to room temperature; a large amount of precipitate will precipitate. Filter by suction and collect the filter cake, and place it in a vacuum drying oven for drying (40 °C, 12 h) to obtain a brownish-yellow solid, that is, the compound with the structure shown in formula (Ⅱ) (487.3 mg, yield: 86.2%); The light yellow solid obtained was characterized, and the specific data are as follows: 1 1H NMR (600 MHz, CDCl3) δ (ppm): 8.69 (d, J J = 7.2 Hz, 1H), 8.59 (d, J J = 8.4 Hz, 1H), 8.45 (d, J J = 7.8 Hz, 1H), 8.05 (d, J J = 7.8 Hz, 1H), 7.86 (t, J J = 7.8 Hz, 1H), 4.95 (s, 2H), 3.32 (s, 1H). The structural formula is C 15 H8BrNO2. Calculated [M+H] + molecular weight: 313.9817. The molecular weight measured by high-resolution mass spectrometry (TOF-MS): 313.9820. According to the above characterization data, the obtained light yellow solid is the compound with the structural formula (II).

[0031] S2: The compound with the structural formula (II) (301.9 mg, 1.0 mmol), p-toluenesulfonamide (329.8 mg, 1.9 mmol) and anhydrous potassium carbonate (276.2 mg, 2.0 mmol) were dissolved in 8 mL of N,N-dimethylformamide (DMF). The mixed solution was heated to 100 °C for reaction, and the reaction progress was monitored by TLC. After the reaction was complete, the solvent was evaporated to obtain a brownish crude product. Purification was carried out by silica gel column chromatography, and the eluent was ethyl acetate: petroleum ether = 1:2 (V / V). Finally, a light yellow solid was obtained, which is the compound with the structural formula (II) (232.9 mg, yield: 60.0%).

[0032] The light yellow solid obtained was characterized, and the specific data are as follows: 1 1H NMR (600 MHz, CDCl3) δ (ppm): 8.59 (d, J J = 7.2 Hz, 1H), 8.48 (d, J J = 8.4 Hz, 1H), 8.37 (d, J J = 8.4 Hz, 1H), 8.19 (s, 1H), 7.77 (d, J J = 7.8 Hz, 2H), 7.72 (t, J J = 7.8 Hz, 1H), 7.67 (t, J J = 6.6 Hz, 1H), 7.23 (d, J= 7.8 Hz, 2H), 4.91 (s, 2H), 3.78 (s, 1H), 2.35 (s, 3H). 13 C NMR (151 MHz, CDCl3) δ (ppm): 163.13, 162.66, 144.92, 138.51, 135.35, 132.11, 131.95, 129.99, 129.01, 127.81, 127.37, 127.17, 124.94, 122.70, 119.03, 118.76, 78.43, 77.21, 77.00, 76.79, 70.59, 54.83, 29.93, 21.54. The structural formula is C 22 H 16 N2O4S, and its [M + H] + molecular weight: 405.0904 was calculated, and the molecular weight measured by high-resolution mass spectrometry (TOF-MS): 405.0901. According to the above characterization data, the obtained pale yellow solid is the compound with the structural formula (Ⅲ).

[0033] S3: Tris(triphenylphosphine)carbonylhydrochlororuthenium ligand (143.7 mg, 0.15 mmol) was added to 5 mL of ultradry dichloromethane. Under a nitrogen atmosphere, after reacting at room temperature for 1 h, benzothiadiazole (20.4 mg, 0.16 mmol) was added. After the reaction solution turned from yellow to red, the compound with the structural formula (Ⅲ) (60.7 mg, 0.15 mmol) was added, and the reaction result was monitored by TLC. After the reaction was completed, a large amount of anhydrous methanol was added to the reaction solution, and it was cooled in a refrigerator at 4 °C for 12 h to precipitate a large amount of precipitate. After filtration, the filter cake was collected to obtain an orange-yellow solid, which was the endoplasmic reticulum-targeted carbon monoxide fluorescent probe RE-NS (140.7 mg, yield: 76.5%) without purification.

[0034] The obtained orange-yellow solid was characterized, and the specific data are as follows: 1 H NMR (600 MHz, CDCl3) δ (ppm): δ8.42 (d, J = 7.2 Hz, 1H), 8.32 (d J = 8.4 Hz, 1H), 8.27 (d, J = 8.4 Hz, 1H), 7.85 (d, J = 13.8 Hz, 3H), 7.76 (d, J = 7.8 Hz, 2H), 7.68 - 7.63 (m, 3H), 7.41 (d, J= 7.2 Hz, 2H), 7.37 - 7.34 (m, 12H), 7.18 (d, J = 7.8 Hz, 2H), 7.05 (d, J = 7.2 Hz, 6H), 6.99 (d, J = 7.5 Hz, 12H), 5.05 (d, J = 15.2 Hz, 1H), 4.47 (d, J = 6.0 Hz, 2H), 2.28 (s, 3H). 13 C NMR(151 MHz, CDCl3) δ (ppm): 163.05, 162.65, 154.28, 144.69, 137.33, 135.54, 135.50, 133.98, 133.94, 133.91, 131.94, 131.80, 131.66, 131.14, 129.91, 129.34, 128.96, 128.83, 127.49, 127.45, 127.42, 127.40, 126.99, 126.80, 125.19, 123.61, 120.29, 119.63, 77.21, 77.00, 76.79, 44.95, 21.51. Molecular formula is C 59 H 47 N2O5P2RuS, and its [M - Cl - BTD] + molecular weight: 1059.1724, and the molecular weight measured by high - resolution mass spectrometry (TOF - MS) is 1059.1726. According to the above characterization data, the obtained orange - yellow solid is the endoplasmic reticulum - targeted carbon monoxide fluorescent probe RE - NS.

[0035] Test Example 1: Response of the endoplasmic reticulum - targeted carbon monoxide fluorescent probe RE - NS to CO CO gas was introduced into the dichloromethane solution of the endoplasmic reticulum - targeted carbon monoxide fluorescent probe RE - NS to generate the response product RE - CO, and high - resolution mass spectrometry was performed on RE - NS and RE - CO.

[0036] As Figure 2 and 3 shown, the calculated value of the mass spectrometry peak of RE - NS for [M - Cl - BTD] + is 1059.1724, and the measured value is 1059.1726; the calculated value of the mass spectrometry peak of the reaction product RE - CO for [M - Cl] +The theoretical value is 1087.1674, and the measured value is 1087.1677. The measured value is in good agreement with the theoretical value. This result not only confirms the specific reaction between RE-NS and CO, but also further supports the reaction mechanism of CO substituting benzothiadiazole.

[0037] Test Example 2: Spectral Test Preparation of RE-NS mother liquor: Take 28 μL of DOTAP chloroform solution (1,2-dioleoyl-3-trimethylammonium propane, 25 mg / mL) and 100 μL of RE-NS (1 mM chloroform solution) and mix them well in a glass bottle. Evaporate the solvent with a nitrogen stream. The obtained lipid film is dried under vacuum at 40 °C for 12 h to remove residual chloroform. Hydrate the lipid film with 1 mL of ultrapure water and sonicate for 5 min to obtain a clear liposome solution of 100 μM RE-NS for use as the mother liquor.

[0038] CORM-3 (tricarbonylchloro(glycinato)ruthenium) (a CO-generating donor compound, 1 mole of CORM-3 releases 1 mole of CO) solution: Accurately weigh 0.9 mg of CORM-3 into a 1.5 mL centrifuge tube, add 1.5 mL of ultrapure water to dissolve it, and prepare a 2 mM mother liquor.

[0039] (1) pH Stability Add 100 μL of RE-NS mother liquor (100 μM) and 100 μL of CORM-3 mother liquor (2 mM) to 1.8 mL of PBS buffer solution (pH values are 4.0, 5.0, 6.0, 7.0, 8.0, 9.0) to obtain a PBS mixed solution with a final concentration of 5 μM RE-NS and 100 μM CORM-3. Let the above mixed solution react at room temperature for 15 min, and then measure the fluorescence intensity of the mixed solution at 550 nm (excitation wavelength is 450 nm, spectral detection wavelength range is 480 nm - 700 nm, slit setting is 5 nm).

[0040] The pH stability of the fluorescent probe is crucial for the accuracy of the detection results. As Figure 4 can be seen, RE-NS maintains a stable low fluorescence background within the pH range of 4.0 - 9.0, indicating that the fluorescent probe itself has good pH stability. When RE-NS responds to CO, within the pH range of 4.0 - 8.0, the fluorescence intensity increases significantly by 9.5 - 10.5 times, and the increase multiple does not show obvious fluctuations with the change of pH. Even under the conditions of weak acidity (pH = 4.0) to weak alkalinity (pH = 8.0), RE-NS can still maintain excellent CO response performance. The results show that RE-NS has high pH stability, laying a good foundation for its biomedical applications such as cell imaging.

[0041] (2)Time dynamics Add 100 μL of the RE-NS stock solution (100 µM) to 1.9 mL of PBS buffer solution (pH = 7.4, simulating the physiological environment) to obtain a PBS mixed solution with a final concentration of 5 μM RE-NS. After irradiating the above mixed solution with ultraviolet light (294 nm) for different times (10 min, 20 min, 30 min, 40 min, 50 min, 60 min), measure the fluorescence intensity of the mixed solution at 550 nm (the excitation wavelength is 450 nm, the spectral detection wavelength range is 480 nm - 700 nm, and the slit is set at 5 nm).

[0042] Add 100 μL of the RE-NS stock solution (100 µM) and 100 μL of the CORM-3 stock solution (2 mM) to 1.8 mL of PBS buffer solution (pH = 7.4) to obtain a PBS mixed solution with a final concentration of 5 μM RE-NS and 100 μM CORM-3. After allowing the above mixed solution to react at room temperature for different times (2 min, 4 min, 5 min, 6 min, 8 min, 10 min, 20 min, 30 min, 40 min, 50 min, 60 min), measure the fluorescence intensity of the mixed solution at 550 nm (the excitation wavelength is 450 nm, the spectral detection wavelength range is 480 nm - 700 nm, and the slit is set at 5 nm).

[0043] As Figure 5 can be seen, as the reaction time between RE-NS and CO increases, the fluorescence intensity at 550 nm increases significantly, reaching more than 9 times the initial value within 10 min and basically reaching a stable equilibrium state after 15 min. Therefore, 15 min is selected as the response time for subsequent experiments. After continuously irradiating RE-NS with a 294 nm ultraviolet lamp for 60 min, the fluorescence intensity of RE-NS at 550 nm hardly changes. The results show that the RE-NS probe not only has a rapid CO response ability but also exhibits excellent photostability, providing an important guarantee for its application in cell imaging.

[0044] (3)Sensitivity Add 100 μL of the RE-NS mother liquor (100 µM) to 1.9 mL of PBS buffer solution (pH = 7.4) to obtain a PBS mixed solution with a final concentration of 5 μM RE-NS. Then, add CORM-3 solutions with different concentrations (concentration range: 0 - 100 μM). After reacting at room temperature for 15 min, measure the fluorescence intensity of the above mixed solution at 550 nm (excitation wavelength: 450 nm, spectral detection wavelength range: 480 nm - 700 nm, slit setting: 5 nm). Plot the fluorescence enhancement factor of the fluorescent probe against the corresponding detected CO concentrations (0 - 100 μM) to obtain a linear relationship graph, and calculate the detection limit of the fluorescent probe through 3δ / k. (In the formula, k is the slope of the linear fitting, and δ is the standard deviation of the fluorescence intensity values of 10 blank samples). It can be seen from Figure 6 that in the initial state, due to the fluorescence quenching effect of benzothiadiazole, RE-NS hardly shows fluorescence; when it responds to CO, benzothiadiazole is replaced by CO, resulting in a weakened quenching effect and a significant increase in fluorescence intensity. As the CO concentration increases, the fluorescence intensity shows a concentration-dependent increase, and the maximum enhancement factor can reach 10 times. When the CO concentration is between 0 - 100 μM, there is a good linear relationship between the fluorescence intensity ratio of RE-NS at 550 nm and the CO concentration. The linear regression equation is y = 1.0x + 0.72, and the correlation coefficient is 0.9942. The detection limit of RE-NS for CO is calculated to be 44.7 nM. The results show that the RE-NS probe has excellent sensitivity and a wide linear detection range for CO.

[0045] (4) Selectivity The selectivity, i.e., anti-interference ability, of the fluorescent probe is the key to ensuring the detection accuracy. Add 100 μL of the RE-NS mother liquor (100 µM) to 1.9 mL of PBS buffer solution (pH = 7.4) to obtain a test solution with a final concentration of 5 μM RE-NS. Add various competitive interfering molecules. The final concentration of CO in the final fluorescent probe sample is 100 μM (20-fold equivalent of the fluorescent probe), and the concentrations of sodium sulfite, sodium sulfide, sodium thiosulfate, hydrogen peroxide, tert-butyl hydroperoxide, and sodium hypochlorite are 400 μM (80-fold equivalent of the fluorescent probe). The final concentrations of copper sulfate, zinc chloride, magnesium chloride, iron chloride, lysine Lys, leucine Leu, tyrosine Tyr, and sodium citrate SSC in the test solution are 1 mM (200-fold equivalent of the fluorescent probe), and the final concentration of glutathione GSH in the test solution is 10 mM (2000-fold equivalent of the fluorescent probe). After the above test solution is placed at room temperature for 15 min for reaction, measure the fluorescence intensity of the test solution at 550 nm (the excitation wavelength is 450 nm, the spectral detection wavelength range is 480 nm - 700 nm, and the slit is set at 5 nm).

[0046] It can be seen from Figure 7 that the fluorescence intensity change of RE-NS at 550 nm was not caused by other interfering analytes, which was almost the same as the fluorescence signal intensity of the fluorescent probe itself. When RE-NS reacted with CO (100 μM), an obvious enhancement phenomenon of the fluorescence signal occurred. The results show that RE-NS has excellent selectivity for CO and can achieve selective detection of CO under physiological conditions.

[0047] Test Example 3: Cytotoxicity Test (1) Cytotoxicity of RE-NS The effect of the fluorescent probe RE-NS on cell viability was determined by the CCK-8 detection method. Seed RAW 264.7 cells in a 96-well plate, 100 μL per well, with approximately 1.0×10 4 cells per well on average, and culture them in a humidified incubator containing 5% carbon dioxide at 37°C for 24 h.

[0048] a: Add different concentrations of RE-NS (0, 10, 20, 30, 40, 50 μM) respectively, and co-incubate with RAW 264.7 cells at 37°C for 24 h. Then add 10 μL of CCK-8 solution (5.0 mg / mL) to each well, and incubate the cells at 37°C for 1 h. Finally, use a microplate reader to record the absorbance value of each well at 450 nm.

[0049] b: After co-incubating RAW 264.7 cells with a heme solution at a final concentration of 100 μM for 1, 2, and 4 h at 37°C, gently aspirate the culture medium and wash the cells with PBS. Add fresh culture medium and treat the cells with 5 μM RE-NS for 1 h. Then add 10 μL of CCK-8 solution to each well and co-incubate with the cells for 1 h. Subsequently, use a microplate reader to measure the absorbance value of each well at 450 nm.

[0050] c: After incubating the cells with different concentrations of CORM-3 (50, 100, 200 μM) for 30 min, culture the cells with a culture solution containing 5 μM RE-NS for 1 h, then add 10 μL of CCK-8 solution and co-incubate with the cells at 37°C for 1 h. Then use a microplate reader to measure the absorbance value of each well at 450 nm.

[0051] It can be seen from Figure 8 that when RAW 264.7 cells were co-incubated with different concentrations (0 - 50 μM) of RE-NS for 24 h, the cell viability remained above 80%. When the products after the reaction of RE-NS with endogenous or exogenous CO were co-incubated with the cells, the cell viability was about 90%. The experimental results fully demonstrated that both RE-NS and its reaction products have good biocompatibility and low cytotoxicity and can be used for CO detection in biological samples.

[0052] Test Example 4: Cell Imaging (1) Exogenous CO Imaging The exogenous CO fluorescence imaging of RAW 264.7 cells was divided into 4 groups. First, seed the cells in a 6-well plate containing glass slides (2 mL per well, approximately 5.0×10 4 cells) and culture at 37°C for 24 h. Divide the cells into four groups for exogenous CO imaging experiments. The cells in the first group were not treated with anything; the cells in the second group were treated with RE-NS (5 μM) for 30 min; the cells in the third group were treated with 50 μM CORM-3 and 5 μM RE-NS for 30 min; the cells in the fourth group were treated with 100 μM CORM-3 and 5 μM RE-NS for 30 min. After the incubation, aspirate the old culture medium, add 1 mL of 4% paraformaldehyde to fix the cells for 3 min, then wash 3 times with PBS buffer solution, observe under a laser confocal microscope (Nikon A1R), and take pictures of the bright field and dark field (excitation wavelength: 488 nm, emission wavelength: 500 - 550 nm).

[0053] It can be seen from Figure 9It can be seen that when the cells were treated only with RE-NS, there was almost no fluorescence signal in the green channel. After treating the cells with 50 μM and 100 μM CORM-3 respectively, a significant fluorescence enhancement was observed in the green channel, and the fluorescence signal intensity showed an obvious concentration dependence. The fluorescence intensity of the 100 μM CORM-3 treatment group was enhanced by 2.7 times compared with the control group. The results proved that RE-NS has the ability to quantitatively detect and track exogenous CO in living cells.

[0054] (2)Endogenous CO imaging In this experiment, hemin was used to stimulate the cells to produce endogenous CO. The cells were seeded in a 6-well plate containing glass slides (about 5.0×10 4 cells per well) and cultured at 37 °C for 24 h. Then the old culture medium was aspirated, and the culture medium containing 100 μM hemin at the final concentration was added. The cells were incubated with RAW 264.7 cells at 37 °C for 1, 2, and 4 h respectively, and then the culture medium containing 5 μM RE-NS was added for culture (30 min). After the incubation, the old culture medium was aspirated, 1 mL of 4% paraformaldehyde was added to fix the cells for 3 min, and then the cells were washed 3 times with PBS buffer solution. Observation was carried out under a laser confocal microscope (Nikon A1R), and bright-field and dark-field photographs were taken for analysis (excitation wavelength: 488 nm, emission wavelength: 500 - 550 nm).

[0055] It can be seen from Figure 10 that the control group cells treated only with 5 μM RE-NS for 30 min showed almost no fluorescence signal. In contrast, significant green fluorescence signals were observed in the cells treated with 100 μM hemin for 1, 2, and 4 h, and the fluorescence intensity increased with the prolongation of the hemin treatment time. The fluorescence intensity of the cells treated with hemin for 4 h was enhanced by about 3 times compared with the control group. The results showed that RE-NS has the ability to quantitatively detect endogenous CO in cells, providing a powerful tool for studying endogenous CO in cells.

[0056] (3)Cell co-localization imaging The RAW 264.7 cells were seeded at 2×10 4Cells were inoculated at a density of [density value] in 15-mm glass-bottom cell culture dishes and cultured at 37 °C for 24 h. Then, the cells were incubated with a culture medium containing CORM-3 at a final concentration of 100 μM for 30 min. Subsequently, the cells were washed three times with PBS buffer solution, and a culture medium containing 5 μM RE-NS was added. After incubating for 30 min, a commercially available endoplasmic reticulum red dye was added and incubated at 37 °C for 30 min. Finally, the stained cells were washed three times with PBS buffer solution, observed under a laser confocal microscope (Nikon A1R), and photographed in bright field and dark field (excitation wavelengths: 488 nm, 638 nm; emission wavelengths: 500 - 550 nm, 663 - 738 nm).

[0057] It can be seen from Figure 11 that the fluorescence of the cells stained with the commercially available endoplasmic reticulum red dye overlapped well in the green channel and the red channel. After calculation, the Pearson correlation coefficient between RE-NS and the dye reached 0.88, and the fluorescence intensity signals of green and red at the same position changed consistently. The results showed that RE-NS has the property of targeting the endoplasmic reticulum of cells and can be used to detect CO in the endoplasmic reticulum.

[0058] Test Example 5: Nifedipine Metabolism (1) Nifedipine cytotoxicity The cytotoxicity of nifedipine was detected by CCK-8 cell proliferation assay. First, the RAW 264.7 cell suspension was inoculated in 96-well plates, 100 μL per well, with approximately [cell number] cells per well on average, and cultured in a humidified incubator containing 5% carbon dioxide at 37 °C for 24 h. 4 (1) Add different concentrations of nifedipine (0, 100, 200, 300, 400, 500 μM) and co-incubate with RAW 264.7 cells at 37 °C for 2 h. Then, add 10 μL of CCK-8 solution (5.0 mg / mL) to each well and incubate at 37 °C for 1 h. Finally, use a microplate reader to record the absorbance value of each well at 450 nm.

[0059] (2) Incubate the cells with different concentrations of nifedipine (100 - 500 μM) solution for 2 h. Then, gently aspirate the culture medium and wash the cells with PBS. Add a culture medium containing 5 μM RE-NS and incubate for 1 h. Then, add 10 μL of CCK-8 solution to each well and co-incubate with the cells at 37 °C for 1 h. Subsequently, use a microplate reader to measure the absorbance value of each well at 450 nm.

[0060] It can be seen from

[0061] It can be seen from Figure 12It can be seen that after incubation with nifedipine drugs at different concentrations (100 - 500 μM) for 2 h, the cell viability of RAW 264.7 cells still remained above 90%. After co-incubation of 5 μM RE-NS in cells with nifedipine at different concentrations (100 - 500 μM), the cell viability was above 80%. The experimental results indicate that both nifedipine and the response product of nifedipine and RE-NS have extremely low cytotoxicity and good biocompatibility.

[0062] (2)Cell imaging of nifedipine This experiment was divided into four groups. Cells were seeded in 6-well plates containing glass slides (2 mL per well, approximately 5.0×10 4 cells) and cultured at 37°C for 24 h. Then, the old culture medium was aspirated, and culture media containing nifedipine with final concentrations of 100 μM, 200 μM, and 300 μM were added, and incubated with RAW 264.7 cells at 37°C for 2 h. Then, a culture medium containing 5 μM RE-NS was added and cultured at 37°C for 30 min. After incubation, the old culture medium was aspirated, 1 mL of 4% paraformaldehyde was added to fix the cells for 3 min, and then washed 3 times with PBS buffer solution. Observation was carried out under a laser confocal microscope (Nikon A1R), and bright-field and dark-field photographs were taken (excitation wavelength: 488 nm, emission wavelength: 500 - 550 nm).

[0063] It can be seen from Figure 13 that the fluorescence intensity of the cell group co-incubated with nifedipine increased significantly with the increase in drug concentration. Quantitative analysis of fluorescence intensity showed that 100 μM and 300 μM nifedipine increased the cell fluorescence intensity by 2.2 times and 3.4 times, respectively. The experimental results not only confirmed that the antihypertensive drug nifedipine can metabolize to produce endogenous CO in cells, but also provided an experimental basis for the detection application potential of CO fluorescence probes in the field of drug metabolism.

Claims

1. An endoplasmic reticulum-targeted carbon monoxide fluorescent probe, characterized in that, The fluorescent probe is shown as the structural formula (Ⅰ): 。 2. A method for preparing an endoplasmic reticulum-targeted carbon monoxide fluorescent probe as described in claim 1, characterized in that, It includes the following steps: S1: 4-Bromo-1,8-naphthalic anhydride and propargylamine are dissolved in absolute ethanol for reaction. After the reaction is completed, a precipitate is formed, filtered by suction and the filter cake is collected, then placed in a vacuum drying oven to dry, obtaining a compound shown as the structural formula (Ⅱ); ； S2: The compound with the structure shown as the structural formula (Ⅱ), p-toluenesulfonamide and anhydrous potassium carbonate are dissolved in N,N-dimethylformamide for reaction. The reaction product is purified to obtain a compound shown as the structural formula (Ⅲ); ； S3: Tris(triphenylphosphine)ruthenium(I) chloride ligand is added to ultradry dichloromethane. Under nitrogen protection, the reaction is carried out at room temperature for 1 h. Then benzothiadiazole is added. After the reaction solution turns from yellow to red, the compound with the structure shown as the structural formula (Ⅲ) is added. After the reaction is completed, anhydrous methanol is added to the reaction solution to precipitate a solid, which is filtered and the filter cake is collected to obtain an endoplasmic reticulum-targeted carbon monoxide fluorescent probe RE-NS.

3. The preparation method of the endoplasmic reticulum-targeted carbon monoxide fluorescent probe according to claim 2, wherein: The reaction conditions of S1 are: the reaction temperature is 80 °C and the stirring reaction time is 12 h.

4. The preparation method of the endoplasmic reticulum-targeted carbon monoxide fluorescent probe according to claim 2, characterized in that: In S1, the molar ratio of 4-bromo-1,8-naphthalic anhydride to propargylamine is 1.7 - 2.0:6.0 - 6.

5.

5. The preparation method of the endoplasmic reticulum-targeted carbon monoxide fluorescence probe according to claim 2, wherein: The reaction conditions of S2 are: the reaction temperature is 100 °C and the stirring and reflux time is 12 h.

6. The preparation method of the endoplasmic reticulum-targeted carbon monoxide fluorescent probe according to claim 2, characterized in that: In S2, the molar ratio of the compound with the structure shown as the structural formula (Ⅱ), p-toluenesulfonamide to anhydrous potassium carbonate is 1:1.8 - 2.0:2.

0.

7. The preparation method of the endoplasmic reticulum-targeted carbon monoxide fluorescent probe according to claim 2, wherein: In S2, the purification is carried out by silica gel column chromatography, and the eluent is ethyl acetate and petroleum ether, and the volume ratio of ethyl acetate to petroleum ether is 1:

2.

8. The preparation method of the endoplasmic reticulum-targeted carbon monoxide fluorescent probe according to claim 2, wherein: In S3, the molar ratio of tris(triphenylphosphine)ruthenium(I) chloride ligand, benzothiadiazole and the compound with the structure shown as the structural formula (Ⅲ) is 0.15:0.16:0.

15.

9. Use of the endoplasmic reticulum-targeted carbon monoxide fluorescent probe according to claim 1, characterized in that: The application of the endoplasmic reticulum-targeted carbon monoxide fluorescent probe for detecting exogenous carbon monoxide and endogenous carbon monoxide in cells.

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