Lysosome targeted carbon monoxide fluorescent probe, preparation method and application

Abstract

The invention provides a lysosome targeted carbon monoxide fluorescent probe, a preparation method and application, the fluorescent probe is shown as a structural formula (I), the lysosome targeted carbon monoxide fluorescent probe provided by the invention takes naphthalimide as a fluorophore and morpholine as a lysosome targeted group; a benzothiadiazole group is used as a fluorescence quenching group and a CO specific 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, and fluorescent response signals in cells are weak are solved. According to the present invention, the synthesis and the purification are simple, the yield is high, the fluorescence is significantly enhanced by 10 times after the response with CO for 30 min, and the detection limit is as low as 0.90 [mu] M; the fluorescent probe has low cytotoxicity, can accurately position the lysosome, and quantitatively detects exogenous CO and endogenous CO in the lysosome.

Description

Technical Field

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

[0002] CO is regarded as a "hidden killer" because its high affinity with hemoglobin can cause hypoxia poisoning in organisms. However, recent studies have shown that CO is an endogenous gaseous signaling molecule in mammals, which is produced by the catalytic decomposition of heme by heme oxygenase (HO) and participates in various physiological processes, such as vasodilation, neurotransmission and immune response, etc., and plays a key role in regulating immune responses, inflammation and tissue damage control. In addition, CO is also closely related to the occurrence and development of various diseases (such as tumors, diabetes, Alzheimer's disease, etc.). Therefore, accurately quantifying CO molecules in organisms is of great significance for understanding their physiological and pathological functions.

[0003] CO fluorescent probes have become a powerful tool for detecting CO at the cellular level due to their advantages such as high sensitivity, good selectivity, simple operation, and in-situ detection. As one of the important organelles in cells, lysosomes have functions such as dissolving and decomposing substances inside and outside cells, maintaining cell homeostasis, participating in the processing of cell metabolic wastes and recycling nutrients. Abnormal lysosome function leads to disorders of intracellular substance metabolism, and then triggers various diseases, such as infections, inflammation and tumors, etc. Lysosome CO fluorescent probes can be specifically used for CO fluorescence imaging in lysosomes in cells to help study the role of CO in lysosomes in physiological and pathological processes.

[0004] In recent years, researchers have developed a variety of lysosome-targeted CO fluorescent probes, and most of them still have the following defects: lack of subcellular organelle localization ability; weak fluorescence response signal; complex preparation process of fluorescent probes and low yield. Summary of the Invention

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

[0006] To solve the above technical problems, one of the purposes of the present invention is to provide a lysosome-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 a lysosome-targeted carbon monoxide fluorescent probe, including the following steps: S1: React 4-bromo-1,8-naphthalic anhydride with morpholine in ethylene glycol monomethyl ether. After the reaction, a precipitate is formed. Filter by suction and collect the filter cake, then dry it in a vacuum drying oven to obtain the compound shown in Structural Formula (Ⅱ): ; S2: Add the compound with the structure shown in Structural Formula (Ⅱ) to an ethanol solution of propargylamine for reaction. After the reaction, a precipitate is formed. Filter and collect the filter cake, and purify it by silica gel column chromatography to obtain the compound shown in Structural Formula (Ⅲ): ; S3: Add tris(triphenylphosphine)ruthenium(I) chloride ligand to ultradry dichloromethane. Under nitrogen protection, react at room temperature for 1 h, then add benzothiadiazole. After the reaction solution turns from yellow to red, add the compound with the structure shown in Structural Formula (Ⅲ). After the reaction, add anhydrous methanol to the reaction solution to form a precipitate. Filter and collect the filter cake to obtain a lysosome-targeted carbon monoxide fluorescent probe.

[0008] Preferably, the reaction conditions for S1 are: reaction temperature is 100 °C, and the stirring reaction time is 12 h.

[0009] Preferably, the molar ratio of 4-bromo-1,8-naphthalic anhydride to morpholine in S1 is 7.0 - 7.5:10.5 - 11.0.

[0010] Preferably, the reaction conditions for S2 are: reaction temperature is 78 °C, and the stirring and reflux time is 12 h.

[0011] Preferably, the molar ratio of the compound with the structure shown in Structural Formula (Ⅱ) to propargylamine in S2 is 0.8 - 1:1.8 - 2.0.

[0012] Preferably, the molar ratio of tris(triphenylphosphine)ruthenium(I) chloride ligand, benzothiadiazole, and the compound with the structure shown in Structural Formula (Ⅲ) is 1:1:1.

[0013] The present invention also provides an application of the lysosome-targeted carbon monoxide fluorescent probe, and the lysosome-targeted carbon monoxide fluorescent probe is used for detecting exogenous and endogenous carbon monoxide in cells.

[0014] One or more of the above technical solutions of the present invention have the following technical effects: The lysosome-targeted carbon monoxide fluorescent probe provided by the present invention is synthesized simply with a relatively high yield. After reacting with CO for 30 min, the fluorescence is significantly enhanced by 10 times, and the detection limit is as low as 0.90 μM. At the same time, the probe has relatively low cytotoxicity, can accurately locate lysosomes and detect exogenous and endogenous CO in lysosomes, and has potential application value for studying the physiological and pathological functions of CO in cell lysosomes. In addition, the lysosome-targeted carbon monoxide fluorescent probe RL-NS was successfully used to prove that the hypertensive drug - telmisartan can metabolize to produce endogenous CO intracellularly. Description of the Drawings

[0015] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments.

[0016] Figure 1 Synthetic route of the lysosome-targeted carbon monoxide fluorescent probe RL-NS; Figure 2 High-resolution mass spectrum of the lysosome-targeted carbon monoxide fluorescent probe RL-NS; Figure 3 High-resolution mass spectrum of the response product RL-CO after RL-NS detects CO; Figure 4 Fluorescence intensity ratio diagram of the reaction product of RL-NS and CO and RL-NS at 510 nm in solutions with different pH values; Figure 5 、 Figure 5 (a) Fluorescence spectrum change of the lysosome-targeted carbon monoxide fluorescent probe RL-NS with time after reacting with 200 μM CORM-3; Figure 5 (b) Time kinetic change of the fluorescence intensity ratio of RL-NS to the fluorescence intensity of RL-NS before reacting with CO at 510 nm; Figure 6 、 Figure 6 (a) Fluorescence spectrum diagram after reacting with different concentrations of CO (CORM-3 is a CO donor compound); Figure 6 (b) Linear relationship between the fluorescence intensity ratio of RL-NS to the fluorescence intensity of RL-NS before reacting with CO at 510 nm and the CO concentration; Figure 7 Specificity analysis diagram detected by the lysosome-targeted carbon monoxide fluorescent probe RL-NS; Figure 8 、 Figure 8 (a) Cytotoxicity diagram of the lysosome-targeted carbon monoxide fluorescent probe RL-NS; Figure 8 (b) Cytotoxicity diagram of the lysosome-targeted carbon monoxide fluorescent probe RL-NS reacting with heme;Figure 8 (c) is the cytotoxicity graph of the lysosome-targeted carbon monoxide fluorescent probe RL-NS in response to CORM-3; Figure 9 、 Figure 9 (a-l) are the confocal microscopy images of the lysosome-targeted carbon monoxide fluorescent probe RL-NS in response to exogenous CO; Figure 9 (m) is the quantitative graph of the average fluorescence intensity of cells in the green channel; Figure 10 、 Figure 10 (a-l) are the confocal microscopy images of the lysosome-targeted carbon monoxide fluorescent probe RL-NS in response to endogenous CO; Figure 10 (m) is the quantitative graph of the average fluorescence intensity of cells in the green channel; Figure 11 、 Figure 11 (a-d) are the co-localization images of the lysosome-targeted carbon monoxide fluorescent probe RL-NS and Lyso-Tracker Red; Figure 11 (e) is the scatter plot of fluorescence intensity; Figure 11 (f) is the fluorescence intensity distribution graph at the same position; Figure 12 、 Figure 12 (a) is the cytotoxicity graph of telmisartan, Figure 12 (b) is the cytotoxicity graph of telmisartan in response to the lysosome-targeted carbon monoxide fluorescent probe RL-NS; Figure 13 、 Figure 13 (a-l) are the confocal images of the lysosome-targeted carbon monoxide fluorescent probe RL-NS in response to different concentrations of telmisartan; Figure 13 (m) is the quantitative graph of the average fluorescence intensity of cells in the green channel. Specific embodiments

[0017] The present invention provides a lysosome-targeted carbon monoxide fluorescent probe, and this fluorescent probe is shown as the structural formula (Ⅰ): 。

[0018] The present invention also provides a preparation method of the lysosome-targeted carbon monoxide fluorescent probe, including the following steps: S1: React 4-bromo-1,8-naphthalic anhydride with morpholine in ethylene glycol monomethyl ether. After the reaction ends, a precipitate is precipitated, filtered by suction and the filter cake is collected, and then placed in a vacuum drying oven to dry, obtaining a compound shown as the structural formula (Ⅱ): ; S2: Add the compound with the structure shown in formula (Ⅱ) to an ethanol solution of propargylamine for reaction. After the reaction is completed, a precipitate is formed. Filter and collect the filter cake, and purify it by silica gel column chromatography to obtain the compound shown in formula (Ⅲ): ; S3: Add tris(triphenylphosphine)ruthenium carbonyl chloride ligand to ultradry dichloromethane. Under nitrogen protection, after reacting at room temperature for 1 h, add benzothiadiazole. After the reaction solution turns from yellow to red, add the compound with the structure shown in formula (Ⅲ). After the reaction is completed, add anhydrous methanol to the reaction solution to precipitate a solid. Filter and collect the filter cake to obtain the lysosome-targeted carbon monoxide fluorescent probe RL-NS.

[0019] In step S1 of the present invention, the reaction temperature between 4-bromo-1,8-naphthalic anhydride and morpholine is 100 °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.

[0020] In step S1 of the present invention, the molar ratio of 4-bromo-1,8-naphthalic anhydride to morpholine is 7.0 - 7.5:10.5 - 11.0, preferably 7.4:10.6.

[0021] In step S2 of the present invention, the conditions for adding the compound with the structure shown in formula (Ⅱ) to an ethanol solution of propargylamine for reaction are as follows: the reaction temperature is 78 °C, and the stirring and reflux time is 12 h. Meanwhile, the eluent used for silica gel column chromatography purification is dichloromethane and methanol, and the volume ratio of dichloromethane to methanol is 99:1.

[0022] In step S2 of the present invention, the molar ratio of the compound with the structure shown in formula (Ⅱ) to propargylamine is 0.8 - 1:1.8 - 2.0, preferably 0.9:1.9.

[0023] In step S3 of the present invention, the molar ratio of tris(triphenylphosphine)ruthenium carbonyl chloride ligand, benzothiadiazole, and the compound with the structure shown in formula (Ⅲ) is 1:1:1.

[0024] In step S3 of the present invention, there is no special limitation on the reaction time. Preferably, the reaction is monitored by TLC plate until the compound with the structure shown in formula (Ⅲ) completely disappears.

[0025] The present invention also provides the application of the lysosome-targeted carbon monoxide fluorescent probe. The lysosome-targeted carbon monoxide fluorescent probe is used for detecting exogenous and endogenous carbon monoxide in cells. The action mechanism of the lysosome-targeted carbon monoxide fluorescent probe RL-NS is as follows:

[0026] The lysosome-targeted carbon monoxide fluorescent probe RL-NS provided by the present invention uses naphthalimide as a fluorophore, morpholine as a lysosome-targeting group, and benzothiadiazole as a fluorescent quencher and a CO-specific recognition and response site. Specific detection of CO is carried out through a ligand exchange reaction. 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, and then specific quantitative detection of CO can be achieved according to the change in fluorescence intensity.

[0027] 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.

[0028] 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 meaning as the general understanding of those skilled in the art to which the present invention belongs. In case of contradiction, this specification shall prevail.

[0029] Example 1 The synthesis route of a lysosome-targeted carbon monoxide fluorescent probe RL-NS is as Figure 1 shown and includes the following steps: S1: Dissolve 4-bromo-1,8-naphthalic anhydride (2.0455 g, 7.4 mmol) and morpholine (920.3 mg, 10.6 mmol) in 20 mL of ethylene glycol monomethyl ether. Heat the reaction solution to 100 °C and react for 12 h. After the reaction, a precipitate is formed. Filter by suction and collect the filter cake. Place it in a vacuum drying oven and dry (40 °C, 12 h) to obtain a light yellow solid, which is the compound shown in structural formula (Ⅱ) (966 mg, yield: 46.2%); Characterize the obtained light yellow solid, and the specific data is as follows: 1 H NMR(600 MHz, CDCl3) δ (ppm): 8.60 (d, J =7.2 Hz, 1H), 8.55 (d, J =8.4 Hz, 1H), 8.48 (d, J =8.4 Hz, 1H), 7.75 (t, J =7.8Hz, 1H), 7.26 (d, J =7.8 Hz, 1H), 4.03 (t, J= 4.5 Hz, 4H), 3.32 (t, J = 4.5 Hz, 4H). The structural formula is C 16 H3NO4. Calculated [M+Na] + molecular weight: 306.0742. The molecular weight measured by high-resolution mass spectrometry (TOF-MS): 306.0746. According to the above characterization data, the obtained white solid is the compound with the structural formula (II).

[0030] S2: The compound with the structural formula (II) (257.9 mg, 0.9 mmol) was added to an ethanol solution of propargylamine (109.5 mg, 1.9 mmol) for reaction. After the reaction, a precipitate was formed. The filter cake was collected by filtration and purified by silica gel column chromatography. The eluent was dichloromethane:methanol = 99:1 (V / V), and an orange-yellow solid was obtained, which is the compound with the structural formula (III) (262.2 mg, yield: 89.9%).

[0031] The orange-yellow solid was characterized, and the specific data are as follows: 1 1H NMR(600 MHz, CDCl3) δ (ppm): 8.64(d, J = 7.2 Hz, 1H), 8.58(d, J = 7.8 Hz, 1H), 8.45(d, J = 8.4 Hz, 1H), 7.72(t, J = 7.8 Hz, 1H), 7.25(d, J = 8.4 Hz, 1H), 4.96(d, J = 2.4 Hz, 2H), 4.02(t, J = 4.5 Hz, 4H), 3.28(t, J = 4.5Hz, 4H), 2.17(t, J = 2.1 Hz, 1H). The structural formula is C 19 H 16 N2O3. Calculated [M+Na] + molecular weight: 343.1059. The molecular weight measured by high-resolution mass spectrometry (TOF-MS): 343.1060. According to the above characterization data, the obtained orange-yellow solid is the compound with the structural formula (III).

[0032] S3: Add tris(triphenylphosphine)ruthenium(II) chloride hydride ligand (294.9 mg, 0.3 mmol) to 5 mL of ultradry dichloromethane. Under nitrogen protection, after reacting at room temperature for 1 h, add benzothiadiazole (41.0 mg, 0.3 mmol). After the reaction solution changes from yellow to red, add the compound with the structure shown in formula (Ⅲ) (100.4 mg, 0.3 mmol). Monitor the reaction progress by TLC until the compound with the structure shown in formula (Ⅲ) completely disappears. After the reaction is completed, add anhydrous methanol to the reaction solution to precipitate a solid. Filter and collect the filter cake to obtain an orange powder, namely the lysosome-targeted carbon monoxide fluorescent probe RL-NS (307.9 mg, yield: 85.8%).

[0033] Characterize the obtained orange powder, and the specific data are as follows: 1 H NMR(600 MHz, CDCl3) δ (ppm): 8.45(d, J =7.3 Hz, 1H), 8.41 (d, J =8.2 Hz, 1H), 7.88-7.84 (m, 2H), 7.68 (t, J =8.3 Hz, 2H), 7.54 (d, J = 7.0 Hz, 2H), 7.40-7.37 (m, 11H), 7.23-7.19 (m, 2H), 7.14 (t, J =7.5 Hz, 2H), 7.09 (t, J= 7.3 Hz, 6H), 7.03-7.00 (m, 10H), 5.11-5.02 (m, 1H), 4.51 (d, J =6.0 Hz, 2H), 4.04 (t, J =4.4 Hz, 4H), 3.79 (s, 1H), 3.28 (t, J =4.2 Hz, 4H). 1313C NMR (151 MHz, CDCl3) δ (ppm): 163.49, 163.11, 154.94, 154.27, 134.01, 133.98, 133.94, 133.79, 132.02, 131.92, 131.88, 131.74, 130.72, 129.80, 129.41, 129.27, 128.79, 128.50, 128.42, 127.60, 127.44, 127.41, 127.38, 126.05, 125.66, 123.88, 121.25, 118.62, 117.94, 114.76, 77.21, 77.00. The structural formula is C 56 H 47 N2O4P2Ru, and its [M-Cl-BTD] was calculated + Molecular weight: 975.2055, and the molecular weight measured by high-resolution mass spectrometry (TOF-MS) is 975.2059. According to the above characterization data, the obtained orange powder is the lysosome-targeted carbon monoxide fluorescent probe RL-NS.

[0034] Test Example 1: Response of the lysosome-targeted carbon monoxide fluorescent probe RL-NS to CO CO gas was introduced into the dichloromethane solution of the lysosome-targeted carbon monoxide fluorescent probe RL-NS to obtain the response product RL-CO, and high-resolution mass spectrometry was used to measure the substances before and after the probe reaction.

[0035] As Figure 2 and 3 shown, the calculated value of the mass spectrometry peak of RL-NS [M-Cl-BTD] + is 975.2055, and the measured value is 975.2059; the calculated value of the mass spectrometry peak of the reaction product RL-CO [M-Cl] + is 1003.2004, and the measured value is 1003.2000. The measured values are in good agreement with the theoretical values. This result not only confirms that RL-NS reacts specifically with CO, but also further supports the reaction mechanism of CO substituting benzothiadiazole.

[0036] Test Example 2: Spectral test Preparation of the RL-NS mother liquor: Accurately weigh 1.1 mg of RL-NS into a 1.5 mL centrifuge tube, add 1 mL of DMSO to dissolve it, and reserve it as a 1 mM mother liquor for use.

[0037] Solution of CORM-3 (chloro(glycinato)ruthenium(III) tricarbonyl) (a CO-releasing donor compound, 1 mole of CORM-3 releases 1 mole of CO): Accurately weigh 0.9 mg of CORM-3 into a 1.5 mL centrifuge tube, add 1 mL of DMSO to dissolve it, and prepare a 3 mM stock solution.

[0038] (1) pH stability Add 30 μL of RL-NS stock solution (1 mM), 200 μL of CORM-3 stock solution (3 mM), and 70 μL of DMSO solution to 2.7 mL of PBS / DMSO (10:1, v / v) mixed solution (pH values are 4.0, 5.0, 6.0, 7.0, 8.0, 9.0) to obtain a PBS mixed solution (containing 10% DMSO) with final concentrations of 10 μM RL-NS and 200 μM CORM-3. Let the obtained mixed solution react at room temperature for 30 min, and then measure the fluorescence intensity of the mixed solution at 510 nm. (The excitation wavelength is 400 nm, the spectral detection wavelength range is 425 - 650 nm, and the slit is set at 10 nm).

[0039] It can be seen from Figure 4 that the fluorescence intensity of the fluorescent probe RL-NS is weak and shows almost no change in fluorescence intensity in buffer solutions with pH values ranging from 4.0 to 9.0, proving its good pH stability. When RL-NS responds to CO, its fluorescence intensity significantly increases in the pH range of 4.0 - 6.0. Since RL-NS has lysosome targeting, lysosomes are acidic organelles with a pH range of 4.0 - 6.0, which is the same as the optimal response range of RL-NS. Therefore, buffer solutions with a pH of 5.0 are used in subsequent experiments. Thus, RL-NS is suitable for detecting CO within the lysosomal pH range to achieve focused targeted cell imaging applications within lysosomes.

[0040] (2) Time kinetics In 2.7 mL of PBS / DMSO (10:1, v / v) mixed solution (pH value of 5.0, simulating the physiological environment of lysosomes), 30 μL of RL-NS mother liquor (1 mM) and 270 μL of DMSO solution were added to obtain a PBS mixed solution (containing 10% DMSO) with a final concentration of 10 μM RL-NS. Then, the obtained mixed solution was irradiated with ultraviolet light (294 nm) for different times (10 min, 20 min, 30 min, 40 min, 50 min, 60 min, 70 min, 80 min, 90 min, 100 min), and the fluorescence intensity of the mixed solution at 510 nm was measured. (The excitation wavelength was 400 nm, the spectral detection wavelength range was 425 - 650 nm, and the slit was set at 10 nm).

[0041] In 2.7 mL of PBS / DMSO (10:1, v / v) mixed solution (pH value of 5.0), 30 μL of RL-NS mother liquor (1 mM), 200 μL of CORM-3 mother liquor (3 mM) and 70 μL of DMSO solution were added to obtain a PBS mixed solution (containing 10% DMSO) with a final concentration of 10 μM RL-NS and 200 μM CORM-3. The obtained mixed solution was allowed to react at room temperature for different times (5 min, 10 min, 15 min, 20 min, 30 min, 40 min, 50 min, 60 min, 70 min, 80 min, 90 min, 100 min), and the fluorescence intensity of the mixed solution at 510 nm was measured. (The excitation wavelength was 400 nm, the spectral detection wavelength range was 425 - 650 nm, and the slit was set at 10 nm).

[0042] It can be seen from Figure 5 that as the response time of RL-NS to CO increases, its fluorescence intensity at 510 nm gradually increases. The fluorescence intensity increased rapidly by about 5.5 times at 10 min, increased to 10 times at 30 min, and then the increasing trend slowed down and increased to 13 times at 60 min and basically tended to an equilibrium state. Considering the detection time cost and the requirement of fluorescence enhancement multiple for imaging, 30 min was selected as the response time for subsequent experiments. In addition, RL-NS was continuously irradiated for 60 min, and the fluorescence intensity of RL-NS at 510 nm hardly changed. The results show that RL-NS has a fast CO response speed and excellent photostability.

[0043] Sensitivity test In a 2.7 mL PBS / DMSO (10:1, v / v) mixed solution (pH = 5.0), 30 μL of the RL-NS mother liquor (1 mM) and 270 μL of the DMSO solution were added to obtain a PBS mixed solution (containing 10% DMSO) with a final concentration of 10 μM RL-NS. Then, CORM-3 solutions with different concentrations (concentration range: 0 - 200 μM) were added respectively. After reacting at room temperature for 30 min, the fluorescence emission spectra of the solutions obtained after adding different concentrations of CORM-3 were measured at 510 nm (excitation wavelength: 400 nm, spectral detection wavelength range: 425 - 650 nm, slit setting: 10 nm). The fluorescence enhancement factor of the fluorescent probe was plotted against the corresponding detected CO concentrations (0 - 100 μM) to obtain a linear relationship graph, and the detection limit of the fluorescent probe was calculated by 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 RL-NS has no obvious fluorescence. When it responds to CO, the fluorescence is significantly enhanced, and the fluorescence signal intensity increases with the increase of the CO concentration. When the concentration of CO is between 0 - 100 μM, there is a good linear relationship between the fluorescence intensity ratio of RL-NS at 510 nm and the concentration of CO. The linear fitting equation is y = 0.06x + 0.88, and the correlation coefficient is as high as 0.9986. Based on the linear fitting results, the detection limit of the probe RL-NS for CO was calculated to be 0.90 μM. The experimental results show that RL-NS has a high signal-to-noise ratio for CO.

[0044] Selectivity test In a 2.7 mL PBS / DMSO (10:1, v / v) mixed solution (pH 5.0), 30 µM of the RL-NS stock solution (1 mM) and 270 µM of the DMSO solution were added to obtain a PBS mixed solution with a final concentration of 10 µM RL-NS (containing 10% DMSO). Competitive interfering molecules were added, and the final concentration of CO in the final fluorescence probe sample was 200 µM (20-fold equivalents of the fluorescence probe), and the concentrations of sodium sulfite, sodium sulfide, sodium thiosulfate, hydrogen peroxide, tert-butyl hydroperoxide, and sodium hypochlorite were 400 µM (40-fold equivalents of the fluorescence 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 were 1 mM (100-fold equivalents of the fluorescence probe), and the final concentration of glutathione GSH in the test solution was 10 mM (1000-fold equivalents of the fluorescence probe). The resulting mixed solution was allowed to react at room temperature for 30 min, and then the fluorescence intensity of the mixed solution at 510 nm was measured. (The excitation wavelength was 400 nm, the spectral detection wavelength range was 425 nm - 650 nm, and the slit was set at 10 nm).

[0045] It can be seen from Figure 7 that other interfering substances did not cause significant changes in the fluorescence intensity of RL-NS at 510 nm, which was almost the same as the fluorescence signal of the fluorescence probe itself. When RL-NS reacted with CO (200 μM), the fluorescence signal increased significantly by about 9.5 times. The results showed that RL-NS had good selectivity for CO and could achieve selective detection of CO under acidic physiological conditions mimicking lysosomes.

[0046] Test Example 3: Cytotoxicity Test (1) Cytotoxicity of RL-NS The effect of the fluorescence probe RL-NS on cell viability was determined by the MTT assay. RAW 264.7 cells were seeded in 96-well plates, 100 μL per well, with approximately 1.0×10 4 cells per well on average, and cultured in a humidified incubator containing 5% carbon dioxide at 37°C for 24 h.

[0047] a: Different concentrations of RL-NS (0, 10, 20, 30, 40, 50 μM) were added and co-incubated with RAW 264.7 cells at 37°C for 24 h. Then, 20 μL of MTT solution (0.1 mg / mL) was added to each well, and the cells were incubated at 37°C for 45 min. Finally, the old medium was aspirated and 200 μL of DMSO was added, and the absorbance value of each well at 490 nm was recorded using a microplate reader.

[0048] b: Incubate the cells with a culture medium containing hemin at a final concentration of 100 μM for 30 min, 3 h, and 6 h. Then, gently aspirate the medium and wash the cells with PBS. Treat the cells with 10 μM RL-NS for 1 h, then add 20 μL of MTT solution and co-incubate at 37°C for 45 min. After incubation, aspirate the old medium, add 200 μL of DMSO to each well, and measure the absorbance value of each well at 490 nm using a microplate reader.

[0049] c: Incubate the cells with different concentrations of CORM-3 (50, 100, 200 μM) for 30 min, then culture the cells with a culture medium containing 10 μM RL-NS for 1 h, then add 20 μL of MTT solution and co-incubate with the cells at 37°C for 45 min. Finally, add 200 μL of DMSO and measure the absorbance value of each well at 490 nm using a microplate reader.

[0050] It can be seen from Figure 8 that after incubating RAW 264.7 cells with different concentrations (0 - 20 μM) of RL-NS for 24 h, the cell viability still remains above 80%. In addition, after co-incubating the cells with the products resulting from the response of RL-NS to endogenous and exogenous CO, the cell viability is about 90%, indicating that RL-NS has the advantage of low cytotoxicity and can be used for the detection of CO in biological samples.

[0051] Test Example 4: Cell Imaging (1) Exogenous CO Imaging The exogenous CO fluorescence imaging of RAW 264.7 cells is divided into 4 groups. First, seed the cells in a 6-well plate containing glass slides (about 5.0×10 4 cells per well) and culture at 37°C for 24 h. Divide the cells into four groups for the exogenous CO imaging experiment. Among them, the cells in the first group are not treated with anything; the cells in the second group are only treated with RL-NS (10 μM) for 35 min; the cells in the third group are treated with 100 μM CORM-3 and 10 μM RL-NS for 35 min; the cells in the fourth group are treated with 200 μM CORM-3 and 10 μM RL-NS for 35 min. After 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).

[0052] It can be seen from Figure 9It can be seen that when the cells were treated only with RL-NS, there was almost no fluorescence signal in the green channel. Further, fluorescence signals were observed in the green channel in the cells treated with 100 μM and 200 μM CORM-3, and the fluorescence signals increased with the increase in the concentration of CORM-3. 200 μM CORM-3 could increase the fluorescence intensity of the cells by about 3.3 times. Therefore, RL-NS has the ability to quantitatively track exogenous CO in cells.

[0053] (2)Intracellular 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 was added and incubated with RAW 264.7 cells at 37 °C for 0, 2, 4, and 6 h, respectively. Then, the culture medium containing 10 μM RL-NS was added for culture (35 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 the cells were washed 3 times with PBS buffer solution and observed under a laser confocal microscope (Nikon A1R), and bright-field and dark-field images were taken (excitation wavelength: 488 nm, emission wavelength: 500 - 550 nm).

[0054] It can be seen from Figure 10 that RAW 264.7 cells treated only with the fluorescent probe RL-NS (10 μM) for 35 min had almost no fluorescence. After incubation in the culture medium containing hemin (100 μM) for 2, 4, and 6 h, obvious green fluorescence was shown, and the fluorescence intensity increased with the prolongation of the incubation time. Co-incubation with hemin for 6 h could increase the fluorescence intensity of the cells by about 2.6 times. The results indicate that RL-NS can detect endogenous CO at the cellular level.

[0055] (3)Cell co-localization imaging 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 incubated with a culture medium containing 10 μM RL-NS. After 35 min of incubation, a commercially available lysosomal dye was added and incubated at 37 °C for 40 min. Finally, the stained cells were washed three times with PBS buffer and observed under a laser confocal microscope (Nikon A1R), and bright-field and dark-field photographs were taken (excitation wavelengths: 488 nm, 638 nm; emission wavelengths: 500 - 550 nm, 663 - 738 nm).

[0056] It can be seen from Figure 11 that the fluorescence of cells co-stained with the commercial lysosomal dye and the probe RL-NS overlapped well in the green channel and the red channel. After calculation, the Pearson correlation coefficient between RL-NS and the dye reached 0.93, and the fluorescence intensity signals of green and red at the same position changed consistently. The results showed that RL-NS has the property of targeting cell lysosomes and can be used to detect CO in lysosomes.

[0057] Test Example 5: Telmisartan Metabolism (1)Cytotoxicity of Telmisartan The biocompatibility of telmisartan at concentrations of 50 - 200 μM was evaluated by CCK-8 cell proliferation assay. First, the RAW 264.7 cell suspension was inoculated into 96-well plates, 100 μL per well, with an average of about 1.0×10 4 cells per well, and cultured in a humidified incubator containing 5% carbon dioxide at 37 °C for 24 h.

[0058] a: Different concentrations of telmisartan (0, 50, 100, 150, 200 μM) were added and co-incubated with RAW 264.7 cells at 37 °C for 2 h. Then, 10 μL of CCK-8 solution (5.0 mg / mL) was added to each well, and the cells were incubated at 37 °C for 1 h. Finally, the absorbance value of each well at 450 nm was recorded using a microplate reader.

[0059] b: The cells were incubated with culture media containing different concentrations of telmisartan (0, 50, 100, 150, 200 μM) for 2 h. Then, the culture medium was gently aspirated and the cells were washed with PBS. A culture medium containing RL-NS at a final concentration of 10 μM was added and incubated for 1 h. Then, 10 μL of CCK-8 solution was added to each well and co-incubated with the cells at 37 °C for 1 h. Subsequently, the absorbance value of each well at 450 nm was measured using a microplate reader.

[0060] It can be seen fromFigure 12 It can be seen that after co-incubating telmisartan at different concentrations (50 - 200 μM) with RAW 264.7 cells, the cell viability still remained above 80%. In addition, after co-incubating 10 μM RL-NS in cells with telmisartan at different concentrations (50 - 200 μM), the cell viability was all above 80%, indicating that both telmisartan and the probe response product of telmisartan have good biocompatibility.

[0061] (2) Cell imaging of telmisartan This experiment was divided into four groups. Cells were seeded in a 6-well plate with 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 culture media containing telmisartan with final concentrations of 50 μM, 100 μM, and 200 μM were added, and co-incubated with RAW 264.7 cells at 37°C for 2 h. Then, a culture medium containing 10 μM RL-NS was added and cultured at 37°C for 35 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).

[0062] It can be Figure 13 seen that as the concentration of telmisartan gradually increased, the fluorescence of cells in the green channel gradually enhanced. When cells were treated with 50 μM telmisartan, the fluorescence intensity was 1.6 times stronger than that of the control group; when the telmisartan concentration was increased to 200 μM, the cell fluorescence intensity increased to 2.2 times that of the control group. The results indicate that the hypertension drug telmisartan can produce endogenous CO in cells.

Claims

1. A lysosome-targeted carbon monoxide fluorescent probe, characterized in that, The fluorescent probe is shown in the structural formula (Ⅰ): 。 2. A preparation method of the lysosome-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 morpholine are dissolved in ethylene glycol monomethyl ether for reaction. After the reaction is completed, a precipitate is formed, filtered and the filter cake is collected, and then dried in a vacuum drying oven to obtain a compound shown in the structural formula (Ⅱ); ； S2: The compound with the structure shown in the structural formula (Ⅱ) is added to an ethanol solution of propargylamine for reaction. After the reaction is completed, a precipitate is formed, filtered and the filter cake is collected, and then purified by silica gel column chromatography to obtain a compound shown in the structural formula (Ⅲ); ； S3: Tris(triphenylphosphine)ruthenium(I) chloride ligand is added to ultra-dry dichloromethane. Under nitrogen protection, after reacting 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 the structural formula (Ⅲ) is added. After the reaction is completed, anhydrous methanol is added to the reaction solution, a precipitate is formed, filtered and the filter cake is collected to obtain the lysosome-targeted carbon monoxide fluorescent probe RL-NS.

3. The preparation method of the lysosome-targeted carbon monoxide fluorescent probe according to claim 2, characterized in that: The reaction conditions of S1 are: the reaction temperature is 100 °C, and the stirring reaction time is 12 h.

4. The preparation method of the lysosome-targeted carbon monoxide fluorescent probe according to claim 2, wherein: In S1, the molar ratio of 4-bromo-1,8-naphthalic anhydride to morpholine is 7.0 - 7.5:10.5 - 11.

0.

5. The preparation method of the lysosome-targeted carbon monoxide fluorescent probe according to claim 2, wherein: The reaction conditions of S2 are: the reaction temperature is 78 °C, and the stirring and reflux condensation time is 12 h.

6. The preparation method of the lysosome-targeted carbon monoxide fluorescent probe according to claim 2, characterized in that: In S2, the molar ratio of the compound with the structure shown in the structural formula (Ⅱ) to propargylamine is 0.8 - 1:1.8 - 2.

0.

7. The preparation method of the lysosome-targeted carbon monoxide fluorescent probe according to claim 2, characterized in that: The molar ratio of tris(triphenylphosphine)ruthenium(I) chloride ligand, benzothiadiazole and the compound with the structure shown in the structural formula (Ⅲ) is 1:1:

1.

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

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