Lysosome-targeted carbon monoxide fluorescent probe, preparation method and application

Abstract

The present invention provides a lysosome-targeted carbon monoxide fluorescent probe, preparation method, and application. The fluorescent probe is shown in structural formula (I). The lysosome-targeted carbon monoxide fluorescent probe provided by the present invention uses naphthalimide as a fluorophore, morpholine as a lysosome-targeting group, a benzothiadiazole group as a fluorescence quenching group and a CO-specific recognition response site, and achieves specific detection of CO through a ruthenium ligand exchange reaction. The present invention addresses technical problems such as the complex preparation and purification processes of existing carbon monoxide fluorescent probes and weak intracellular fluorescence response signals. The present invention is simple to synthesize and purify, with high yield. After 30 minutes of response to CO, the fluorescence is significantly enhanced by 10 times, and the detection limit is as low as 0.90 μM. The fluorescent probe has low cytotoxicity, can accurately locate lysosomes, and can quantitatively detect exogenous and endogenous CO in lysosomes.

Description

Technical Field

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

[0002] CO, due to its high affinity for hemoglobin, can cause hypoxic poisoning in organisms and is considered a "hidden killer." However, recent studies have shown that CO is an endogenous gaseous signaling molecule in mammals, produced by the decomposition of ferrous heme catalyzed by heme oxygenase (HO). CO participates in numerous physiological processes, such as vasodilation, neurotransmission, and immune responses, playing a key role in regulating immune responses, inflammation, and controlling tissue damage. Furthermore, CO is closely associated with the onset and progression of numerous diseases, such as cancer, diabetes, and Alzheimer's disease. Therefore, accurate and quantitative detection of CO in vivo is crucial for understanding its physiological and pathological functions.

[0003] CO fluorescent probes have become powerful tools for detecting CO at the cellular level due to their high sensitivity, good selectivity, ease of operation, and in situ detection. Lysosomes, as important intracellular organelles, dissolve and decompose intracellular and extracellular substances, maintain cellular homeostasis, and participate in the processing of cellular metabolic waste and the recycling of nutrients. Lysosomal dysfunction leads to disorders in intracellular metabolism, which in turn triggers a variety of diseases such as infection, inflammation, and tumors. Lysosomal CO fluorescent probes can be used for targeted CO fluorescence imaging within lysosomes in cells, helping to study the role of lysosomal CO in physiological and pathological processes.

[0004] In recent years, researchers have developed a variety of CO fluorescent probes targeting lysosomes, but most of them still have the following defects: lack of subcellular organelle localization ability; weak fluorescence response signal; complex fluorescent probe preparation process 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, preparation method and application to solve technical problems such as the complex preparation and purification process of existing carbon monoxide, weak intracellular fluorescence response signal, and mostly whole-cell level detection.

[0006] In order to solve the above technical problems, one of the purposes of the present invention is to provide a lysosome-targeted carbon monoxide fluorescent probe, which is shown in structural formula (I):

[0007] .

[0008] Based on the same inventive concept, the present invention also provides a method for preparing a lysosome-targeted carbon monoxide fluorescent probe, comprising the following steps:

[0009] S1: 4-bromo-1,8-naphthalene dicarboxylic anhydride and morpholine are dissolved in ethylene glycol monomethyl ether to react. After the reaction is completed, a precipitate is precipitated, and the filter cake is collected by filtration and dried in a vacuum drying oven to obtain a compound represented by structural formula (II):

[0010] ;

[0011] S2: Add the compound having the structure represented by structural formula (II) to an ethanol solution of propargylamine to react. After the reaction is completed, a precipitate is precipitated, and the filter cake is collected by filtration and purified by silica gel column chromatography to obtain a compound represented by structural formula (III):

[0012] ;

[0013] S3: S3: Add tris(triphenylphosphine)carbonylruthenium hydrochloride ligand to ultra-dry dichloromethane, react at room temperature for 1 hour under nitrogen protection, add benzothiadiazole, and after the reaction liquid changes from yellow to red, add the compound having the structure represented by structural formula (III). After the reaction is completed, add anhydrous methanol to the reaction liquid to precipitate, and collect the filter cake by filtration to obtain a lysosome-targeted carbon monoxide fluorescent probe.

[0014] Preferably, the reaction conditions of S1 are: reaction temperature of 100° C., and stirring reaction time of 12 h.

[0015] Preferably, the molar ratio of 4-bromo-1,8-naphthalene dicarboxylic anhydride to morpholine in S1 is 7.0-7.5:10.5-11.0.

[0016] Preferably, the reaction conditions of S2 are: reaction temperature of 78° C., stirring and condensation reflux time of 12 h.

[0017] Preferably, the molar ratio of the compound having the structure represented by structural formula (II) to propargylamine in S2 is 0.8-1:1.8-2.0.

[0018] Preferably, the molar ratio of the tris(triphenylphosphine)carbonylruthenium hydrochloride ligand, benzothiadiazole and the compound having the structure represented by structural formula (III) is 1:1:1.

[0019] The present invention also provides an application of a lysosome-targeted carbon monoxide fluorescent probe, wherein the lysosome-targeted carbon monoxide fluorescent probe is used to detect exogenous carbon monoxide and endogenous carbon monoxide in cells.

[0020] The above one or more technical solutions of the present invention have the following technical effects:

[0021] The lysosome-targeted carbon monoxide fluorescent probe provided by the present invention is simple to synthesize and has a high yield. After 30 minutes of reaction with CO, the fluorescence is significantly enhanced 10-fold, with a detection limit as low as 0.90 μM. Furthermore, the probe has minimal cytotoxicity and can accurately locate lysosomes and detect exogenous and endogenous CO in lysosomes, showing potential applications in studying the physiological and pathological functions of CO in cellular lysosomes. Furthermore, the lysosome-targeted carbon monoxide fluorescent probe RL-NS was successfully used to demonstrate that the hypertensive drug telmisartan can be metabolized within cells to produce endogenous CO. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments.

[0023] Figure 1 , the synthetic route of lysosome-targeted carbon monoxide fluorescent probe RL-NS;

[0024] Figure 2 , high-resolution mass spectrometry of the lysosome-targeted carbon monoxide fluorescent probe RL-NS;

[0025] Figure 3 , high-resolution mass spectrum of the response product RL-CO after RL-NS detects CO;

[0026] Figure 4 , the fluorescence intensity ratio of the reaction products of RL-NS and CO and RL-NS at 510 nm in different pH solutions;

[0027] Figure 5 、 Figure 5 (a) The fluorescence spectrum of the lysosome-targeted carbon monoxide fluorescent probe RL-NS changes with time after responding to 200 μM CORM-3; Figure 5 (b) The time-dependent dynamics of the ratio of the fluorescence intensity of RL-NS after the response to CO to the fluorescence intensity of RL-NS before the response at 510 nm.

[0028] Figure 6 、 Figure 6 (a) Fluorescence spectra of the response to the addition of different concentrations of CO (CORM-3 is a CO donor compound); Figure 6 (b) The linear relationship between the ratio of the fluorescence intensity of RL-NS in response to CO and the fluorescence intensity of RL-NS before response and CO concentration at 510 nm;

[0029] Figure 7 , specificity analysis diagram of lysosome-targeted carbon monoxide fluorescent probe RL-NS detection;

[0030] Figure 8 、 Figure 8 (a) is the cytotoxicity diagram of the lysosome-targeted carbon monoxide fluorescent probe RL-NS; Figure 8 (b) is a cytotoxicity diagram of the lysosome-targeted carbon monoxide fluorescent probe RL-NS in response to heme; Figure 8 (c) is a cytotoxicity graph of the lysosome-targeted carbon monoxide fluorescent probe RL-NS in response to CORM-3;

[0031] Figure 9 、 Figure 9 (a1) are confocal microscopic 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 mean fluorescence intensity of cells in the green channel;

[0032] Figure 10 、 Figure 10 (a1) are confocal microscopic 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 mean fluorescence intensity of cells in the green channel;

[0033] Figure 11 、 Figure 11 (ad) are co-localization images of the lysosome-targeted carbon monoxide fluorescent probe RL-NS and Lyso-Tracker Red; Figure 11 (e) is a scatter plot of fluorescence intensity; Figure 11 (f) is the fluorescence intensity distribution diagram at the same position;

[0034] Figure 12 、 Figure 12 (a) is the cytotoxicity diagram of telmisartan, Figure 12 (b) is a cytotoxicity graph of telmisartan in response to the lysosome-targeted carbon monoxide fluorescent probe RL-NS;

[0035] Figure 13 、 Figure 13 (a1) is the confocal imaging of the response of the lysosome-targeted carbon monoxide fluorescent probe RL-NS to different concentrations of telmisartan; Figure 13 (m) is the quantitative graph of the mean fluorescence intensity of cells in the green channel. DETAILED DESCRIPTION

[0036] The present invention provides a lysosome-targeted carbon monoxide fluorescent probe, which is shown in structural formula (I):

[0037] .

[0038] The present invention also provides a method for preparing a lysosome-targeted carbon monoxide fluorescent probe, comprising the following steps:

[0039] S1: 4-bromo-1,8-naphthalene dicarboxylic anhydride and morpholine are dissolved in ethylene glycol monomethyl ether to react. After the reaction is completed, a precipitate is precipitated, and the filter cake is collected by filtration and dried in a vacuum drying oven to obtain a compound represented by structural formula (II):

[0040] ;

[0041] S2: Add the compound having the structure represented by structural formula (II) to an ethanol solution of propargylamine to react. After the reaction is completed, a precipitate is precipitated, and the filter cake is collected by filtration and purified by silica gel column chromatography to obtain a compound represented by structural formula (III):

[0042] ;

[0043] S3: Add tris(triphenylphosphine)carbonylruthenium hydrochloride ligand to ultra-dry dichloromethane, react at room temperature for 1 hour under nitrogen protection, then add benzothiadiazole. After the reaction liquid changes from yellow to red, add the compound having the structure represented by structural formula (III). After the reaction is completed, add anhydrous methanol to the reaction liquid to precipitate, and collect the filter cake by filtration to obtain the lysosome-targeted carbon monoxide fluorescent probe RL-NS.

[0044] In S1 of the present invention, the reaction temperature of 4-bromo-1,8-naphthalene dicarboxylic anhydride and morpholine is 100° C., and the stirring reaction time is 12 hours. There is no particular limitation on the stirring rate, and any stirring rate known to those skilled in the art can be used.

[0045] In S1 of the present invention, the molar ratio of 4-bromo-1,8-naphthalene dicarboxylic anhydride to morpholine is 7.0-7.5:10.5-11.0, preferably 7.4:10.6.

[0046] The compound represented by structural formula (II) in S2 of the present invention is added to an ethanol solution of propargylamine for reaction at a temperature of 78°C and with stirring and reflux for 12 hours. Furthermore, silica gel column chromatography purification employs eluents of dichloromethane and methanol, with a volume ratio of dichloromethane to methanol of 99:1.

[0047] In S2 of the present invention, the molar ratio of the compound having the structure represented by structural formula (II) to propargylamine is 0.8-1:1.8-2.0, preferably 0.9:1.9.

[0048] In S3 of the present invention, the molar ratio of the tris(triphenylphosphine)carbonylruthenium hydrochloride ligand, benzothiadiazole and the compound having the structure represented by structural formula (III) is 1:1:1.

[0049] There is no particular limitation on the reaction time in S3 of the present invention. The reaction is preferably monitored by TLC plate and the reaction is allowed to proceed until the compound having the structure represented by structural formula (III) completely disappears.

[0050] The present invention also provides the use of a lysosome-targeted carbon monoxide fluorescent probe, which is used to detect both exogenous and endogenous carbon monoxide in cells. The mechanism of action of the lysosome-targeted carbon monoxide fluorescent probe RL-NS is as follows:

[0051]

[0052] The lysosome-targeted carbon monoxide fluorescent probe RL-NS provided by the present invention uses naphthalimide as the fluorophore, morpholine as the lysosome-targeting group, and benzothiadiazole as the fluorophore quencher and CO-specific recognition response site. CO is specifically detected through a ligand exchange reaction. Due to the intramolecular PET interaction between the fluorophore and benzothiadiazole, fluorescence is quenched. When CO is present in the system, benzothiadiazole undergoes a displacement reaction with CO, blocking the intramolecular PET and restoring fluorescence of the fluorophore. Consequently, specific quantitative detection of CO can be achieved based on changes in fluorescence intensity.

[0053] The technical solution of the present invention will be described clearly and completely below in conjunction with the accompanying drawings of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0054] Throughout this specification, unless otherwise specified, the terms used herein should be understood as having the same meaning as commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In the event of any conflict, the present specification shall take precedence.

[0055] Example 1

[0056] The synthetic route of a lysosome-targeted carbon monoxide fluorescent probe RL-NS is as follows: Figure 1 As shown, the following steps are included:

[0057] S1: 4-Bromo-1,8-naphthalene dicarboxylic anhydride (2.0455 g, 7.4 mmol) and morpholine (920.3 mg, 10.6 mmol) were dissolved in 20 mL of ethylene glycol monomethyl ether. The reaction solution was heated to 100°C for 12 h. After the reaction, a precipitate was separated and filtered, and the filter cake was collected and dried in a vacuum drying oven (40°C, 12 h) to obtain a light yellow solid, i.e., the compound represented by structural formula (II) (966 mg, yield: 46.2%).

[0058] The obtained light yellow solid was characterized, and the specific data are 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 =7.8 Hz, 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, its [M+Na] is calculated + The molecular weight is 306.0742, and the molecular weight measured by high-resolution mass spectrometry (TOF-MS) is 306.0746. According to the above characterization data, the obtained white solid is a compound represented by structural formula (II).

[0059] S2: The compound having the structure represented by structural formula (II) (257.9 mg, 0.9 mmol) was added to an ethanol solution of propargylamine (109.5 mg, 1.9 mmol) to carry out a reaction. After the reaction, a precipitate was precipitated, and the filter cake was collected by filtration and purified by silica gel column chromatography with an eluent of dichloromethane:methanol = 99:1 (V / V) to obtain an orange-yellow solid, i.e., the compound having structural formula (III) (262.2 mg, yield: 89.9%).

[0060] The obtained orange-yellow solid was characterized, and the specific data are as follows: 1 H 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, its [M+Na] + The molecular weight is 343.1059, and the molecular weight measured by high-resolution mass spectrometry (TOF-MS) is 343.1060. Based on the above characterization data, it can be seen that the obtained orange-yellow solid is a compound represented by structural formula (III).

[0061] S3: Tris(triphenylphosphine)carbonylruthenium hydrochloride ligand (294.9 mg, 0.3 mmol) was added to 5 mL of ultra-dry dichloromethane. Under nitrogen, the reaction was allowed to react at room temperature for 1 h. Benzothiadiazole (41.0 mg, 0.3 mmol) was then added. After the reaction solution turned from yellow to red, the compound represented by structural formula (III) (100.4 mg, 0.3 mmol) was added. The reaction progress was monitored by TLC until the compound represented by structural formula (III) completely disappeared. After the reaction was completed, anhydrous methanol was added to the reaction solution to precipitate the precipitate. The filter cake was collected by filtration to obtain an orange powder, namely, the lysosome-targeted carbon monoxide fluorescent probe RL-NS (307.9 mg, 85.8% yield).

[0062] The obtained orange powder was characterized, 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). 13C 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, its [M-Cl-BTD] + The molecular weight is 975.2055, and the molecular weight measured by high-resolution mass spectrometry (TOF-MS) is 975.2059. Based on the above characterization data, the obtained orange powder is the lysosome-targeted carbon monoxide fluorescent probe RL-NS.

[0063] Test Example 1: Lysosome-targeted carbon monoxide fluorescent probe RL-NS responds to CO test

[0064] CO gas was introduced into a dichloromethane solution of the lysosome-targeted carbon monoxide fluorescent probe RL-NS to obtain the response product RL-CO, and the substances before and after the probe reaction were measured by high-resolution mass spectrometry.

[0065] like Figure 2 and 3 As shown, the calculated mass spectrum peak value of RL-NS [M-Cl-BTD] + The calculated mass spectrum peak value of the reaction product RL-CO is [M-Cl] + The measured value is 1003.2004, and the theoretical value is 1003.2000. The measured value is highly consistent with the theoretical value. This result not only confirms the specific reaction of RL-NS with CO, but also further supports the reaction mechanism of CO replacing benzothiadiazole.

[0066] Test Example 2: Spectrum Test

[0067] Preparation of RL-NS stock solution: 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 for use as a 1 mM stock solution.

[0068] CORM-3 (tricarbonylchloro(glycinyl)ruthenium) (a CO donor compound, 1 mol of CO released to produce 1 mol of CO) solution: Accurately weigh 0.9 mg of CORM-3 into a 1.5 mL centrifuge tube and dissolve it in 1 mL of DMSO to prepare a 3 mM stock solution.

[0069] (1) pH stability

[0070] To 2.7 mL of a PBS / DMSO (10:1, v / v) mixture (pH 4.0, 5.0, 6.0, 7.0, 8.0, or 9.0), 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 obtain a final concentration of 10 μM RL-NS and 200 μM CORM-3 in PBS (containing 10% DMSO). The resulting mixture was allowed to react at room temperature for 30 minutes. The fluorescence intensity of the mixture was then measured at 510 nm (excitation wavelength: 400 nm, detection wavelength range: 425-650 nm, slit setting: 10 nm).

[0071] Depend on Figure 4 The fluorescent probe RL-NS exhibited weak fluorescence intensity and little fluorescence intensity change in buffer solutions with a pH range of 4.0-9.0, demonstrating its excellent pH stability. When RL-NS reacted with CO, its fluorescence intensity significantly increased within the pH range of 4.0-6.0. Because RL-NS targets lysosomes, which are acidic organelles with a pH range of 4.0-6.0, this coincides with the optimal response range of RL-NS. Therefore, subsequent experiments were conducted using a buffer solution with a pH of 5.0. Therefore, RL-NS is suitable for detecting CO within the lysosomal pH range, enabling focused intralysosomal imaging applications.

[0072] (2) Time dynamics

[0073] To 2.7 mL of a PBS / DMSO (10:1, v / v) mixture (pH 5.0, simulating the physiological environment of lysosomes), 30 μL of RL-NS stock solution (1 mM) and 270 μL of DMSO were added to obtain a final concentration of 10 μM RL-NS in PBS (containing 10% DMSO). The resulting mixture was then irradiated with UV light (294 nm) for various times (10 min, 20 min, 30 min, 40 min, 50 min, 60 min, 70 min, 80 min, 90 min, and 100 min). The fluorescence intensity of the mixture was measured at 510 nm (excitation wavelength: 400 nm, detection wavelength range: 425–650 nm, slit setting: 10 nm).

[0074] To 2.7 mL of a PBS / DMSO (10:1, v / v) mixture (pH 5.0), 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 obtain a final concentration of 10 μM RL-NS and 200 μM CORM-3 in PBS (containing 10% DMSO). The resulting mixture was incubated at room temperature for various times (5 min, 10 min, 15 min, 20 min, 30 min, 40 min, 50 min, 60 min, 70 min, 80 min, 90 min, and 100 min). The fluorescence intensity of the mixture was measured at 510 nm (excitation wavelength: 400 nm, detection wavelength range: 425-650 nm, slit setting: 10 nm).

[0075] Depend on Figure 5 As the response time of RL-NS to CO increases, its fluorescence intensity at 510 nm gradually increases. At 10 minutes, the fluorescence intensity rapidly increases by approximately 5.5 times, reaching a 10-fold increase at 30 minutes. The enhancement trend then slows, reaching a 13-fold increase at 60 minutes, essentially reaching equilibrium. Considering the detection time cost and the fluorescence enhancement factor required for imaging, a 30-minute response time was selected for subsequent experiments. Furthermore, the fluorescence intensity of RL-NS at 510 nm remained virtually unchanged after continuous irradiation for up to 60 minutes. These results demonstrate that RL-NS exhibits a rapid CO response and excellent photostability.

[0076] Sensitivity test

[0077] To 2.7 mL of a PBS / DMSO (10:1, v / v) mixture (pH 5.0) was added 30 μL of the RL-NS stock solution (1 mM) and 270 μL of DMSO solution to obtain a final 10 μM RL-NS solution in PBS (containing 10% DMSO). Then, various concentrations of CORM-3 solutions (ranging from 0 to 200 μM) were added. After incubation at room temperature for 30 minutes, the fluorescence emission spectra of the resulting solutions were measured at 510 nm (excitation wavelength 400 nm, detection wavelength range 425-650 nm, slit setting 10 nm). The fluorescence enhancement factor of the fluorescent probe was plotted against the corresponding CO concentrations (0-100 μM). A linear relationship was obtained, and the detection limit of the fluorescent probe was calculated using 3δ / k (where k is the slope of the linear fit and δ is the standard deviation of the fluorescence intensity values of 10 blank samples).

[0078] Depend on Figure 6 RL-NS exhibits no significant fluorescence. However, upon exposure to CO, its fluorescence increases significantly, and the intensity of the fluorescence signal increases with increasing CO concentration. Within the CO concentration range of 0-100 μM, the fluorescence intensity ratio of RL-NS at 510 nm exhibits a good linear relationship with the CO concentration. The linear fit equation is y = 0.06x + 0.88, with a correlation coefficient of 0.9986. Based on the linear fit results, the detection limit of the probe RL-NS for CO was calculated to be 0.90 μM. These experimental results demonstrate that RL-NS exhibits a high signal-to-noise ratio for CO.

[0079] Optional testing

[0080] 30 µM RL-NS stock solution (1 mM) and 270 µM DMSO solution were added to 2.7 mL of PBS / DMSO (10:1, v / v) mixed solution (pH 5.0) to obtain a PBS mixed solution (containing 10% DMSO) with a final concentration of 10 µM RL-NS. Competing interfering molecules were added. The final CO concentration in the fluorescent probe sample was 200 µM (20 equivalents of the fluorescent probe), the concentrations of sodium sulfite, sodium sulfide, sodium thiosulfate, hydrogen peroxide, tert-butyl hydroperoxide, and sodium hypochlorite were 400 µM (40 equivalents of the fluorescent probe), the final concentrations of copper sulfate, zinc chloride, magnesium chloride, ferric chloride, lysine Lys, leucine Leu, tyrosine Tyr, and sodium citrate SSC in the test solution were 1 mM (100 equivalents of the fluorescent probe), and the final concentration of glutathione GSH in the test solution was 10 mM (1000 equivalents of the fluorescent probe). The resulting mixed solution was allowed to react at room temperature for 30 minutes, and then the fluorescence intensity of the mixed solution at 510 nm was measured (excitation wavelength was 400 nm, spectral detection wavelength range was 425 nm-650 nm, and the slit was set to 10 nm).

[0081] Depend on Figure 7 As can be seen, other interfering substances did not cause a significant change in the fluorescence intensity of RL-NS at 510 nm, maintaining a fluorescence signal that was nearly identical to that of the fluorescent probe itself. When RL-NS reacted with CO (200 μM), the fluorescence signal was significantly enhanced by approximately 9.5-fold. These results demonstrate that RL-NS has excellent selectivity for CO and can selectively detect CO under acidic physiological conditions that mimic lysosomes.

[0082] Test Example 3: Cytotoxicity Test

[0083] (1) RL-NS cytotoxicity

[0084] The effect of fluorescent probe RL-NS on cell viability was determined by MTT assay. RAW 264.7 cells were seeded in 96-well plates, with 100 μL per well and an average of approximately 1.0×10 4 The cells were cultured in a humidified incubator containing 5% carbon dioxide at 37°C for 24 h.

[0085] a: RL-NS at various concentrations (0, 10, 20, 30, 40, and 50 μM) was added and 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. The absorbance of each well at 490 nm was recorded using a microplate reader.

[0086] b: Cells were incubated with culture medium containing a final concentration of 100 μM hemin for 30 minutes, 3 hours, and 6 hours. The medium was then aspirated and the cells washed with PBS. Cells were treated with 10 μM RL-NS for 1 hour, followed by the addition of 20 μL of MTT solution and incubation at 37°C for 45 minutes. Following incubation, the medium was aspirated, and 200 μL of DMSO was added to each well. The absorbance of each well was measured at 490 nm using a microplate reader.

[0087] c: After incubating cells with various concentrations of CORM-3 (50, 100, and 200 μM) for 30 minutes, the cells were incubated with culture medium containing 10 μM RL-NS for 1 hour. Then, 20 μL of MTT solution was added and incubated with the cells at 37°C for 45 minutes. Finally, 200 μL of DMSO was added, and the absorbance of each well was measured at 490 nm using a microplate reader.

[0088] Depend on Figure 8 The results show that after incubating RAW 264.7 cells with different concentrations of RL-NS (0-20 μM) for 24 hours, the cell viability remained above 80%. In addition, after co-incubation with cells containing RL-NS and products of endogenous and exogenous CO response, the cell viability was around 90%, indicating that RL-NS has the advantage of low cytotoxicity and can be used for CO detection in biological samples.

[0089] Test Example 4: Cell Imaging

[0090] (1) Exogenous CO imaging

[0091] Exogenous CO fluorescence imaging of RAW 264.7 cells was divided into four groups. First, cells were seeded in a 6-well plate with a glass slide (approximately 5.0 × 10 cells per well). 4 The cells were cultured at 37°C for 24 hours. The cells were divided into four groups for exogenous CO imaging experiments: the first group received no treatment; the second group was treated with RL-NS (10 μM) alone for 35 minutes; the third group was treated with 100 μM CORM-3 and 10 μM RL-NS for 35 minutes; and the fourth group was treated with 200 μM CORM-3 and 10 μM RL-NS for 35 minutes. After the incubation period, the culture medium was aspirated and the cells were fixed with 1 mL of 4% paraformaldehyde for 3 minutes. The cells were then washed three times with PBS buffer and observed under a laser confocal microscope (Nikon A1R), and both bright-field and dark-field images were taken (excitation wavelength: 488 nm, emission wavelength: 500-550 nm).

[0092] Depend on Figure 9As can be seen, cells treated with RL-NS alone showed almost no fluorescence signal in the green channel. Further, cells treated with 100 μM and 200 μM CORM-3 showed fluorescence in the green channel, and this fluorescence signal increased with increasing CORM-3 concentrations. 200 μM CORM-3 increased the fluorescence intensity by approximately 3.3-fold. Therefore, RL-NS has the ability to quantitatively track exogenous CO in cells.

[0093] (2) Endogenous CO imaging in cells

[0094] In this experiment, heme was used to stimulate cells to produce endogenous CO. Cells were seeded in 6-well plates with glass slides (approximately 5.0 × 10 cells per well). 4 The culture medium was aspirated and heme culture medium was added to a final concentration of 100 μM. This was then incubated with RAW 264.7 cells at 37°C for 0, 2, 4, and 6 hours. Culture medium containing 10 μM RL-NS was then added for 35 minutes. Following incubation, the culture medium was aspirated and the cells were fixed with 1 mL of 4% paraformaldehyde for 3 minutes. The cells were then washed three times with PBS and observed under a laser confocal microscope (Nikon A1R). Brightfield and darkfield images were taken (excitation wavelength: 488 nm, emission wavelength: 500-550 nm).

[0095] Depend on Figure 10 As shown, RAW 264.7 cells treated with the fluorescent probe RL-NS (10 μM) for only 35 minutes exhibited almost no fluorescence. However, after incubation in culture medium containing heme (100 μM) for 2, 4, and 6 hours, they displayed distinct green fluorescence, which increased with incubation time. Co-incubation with heme for 6 hours increased the fluorescence intensity by approximately 2.6-fold. These results demonstrate that RL-NS can detect endogenous CO at the cellular level.

[0096] (3) Cell co-localization imaging

[0097] RAW 264.7 cells were cultured at 2 × 10 4Cells were seeded at a density of 10 μM in 15 mm glass-bottomed cell culture dishes and cultured at 37°C for 24 h. The cells were then incubated with CORM-3 culture medium at a final concentration of 100 μM for 30 min. The cells were then washed three times with PBS buffer and incubated with culture medium containing 10 μM RL-NS for 35 min. A commercially available lysosomal dye was then added and incubated at 37°C for 40 min. The stained cells were washed three times with PBS buffer and observed under a laser confocal microscope (Nikon A1R). Brightfield and darkfield images were taken (excitation wavelengths: 488 nm, 638 nm; emission wavelengths: 500-550 nm, 663-738 nm).

[0098] Depend on Figure 11 Cells co-stained with a commercial lysosomal dye and the RL-NS probe showed good fluorescence overlap in the green and red channels. The calculated Pearson correlation coefficient between RL-NS and the dye reached 0.93, and the green and red fluorescence intensity signals at the same location showed consistent changes. These results demonstrate that RL-NS has the ability to target lysosomes and can be used to detect CO in lysosomes.

[0099] Test Example 5: Telmisartan Metabolism

[0100] (1) Telmisartan cytotoxicity

[0101] The biocompatibility of telmisartan at concentrations of 50-200 μM was evaluated by CCK-8 cell proliferation assay. First, RAW 264.7 cell suspension was seeded in a 96-well plate, with 100 μL per well and an average of approximately 1.0×10 4 The cells were cultured in a humidified incubator containing 5% carbon dioxide at 37°C for 24 h.

[0102] a: Telmisartan at different concentrations (0, 50, 100, 150, and 200 μM) was added and 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 of each well at 450 nm was recorded using a microplate reader.

[0103] b: Cells were incubated with different concentrations of telmisartan (0, 50, 100, 150, and 200 μM) for 2 hours. The medium was then aspirated, the cells washed with PBS, and RL-NS culture medium was added to a final concentration of 10 μM and incubated for 1 hour. 10 μL of CCK-8 solution was then added to each well and incubated with the cells at 37°C for 1 hour. The absorbance of each well was then measured at 450 nm using a microplate reader.

[0104] Depend on Figure 12 It can be seen that after co-incubation of RAW 264.7 cells with different concentrations of telmisartan (50-200 μM), the cell viability remained above 80%. In addition, after co-incubation of 10 μM RL-NS with different concentrations of telmisartan (50-200 μM), the cell viability was above 80%, indicating that both telmisartan and the telmisartan-probe response product have good biocompatibility.

[0105] (2) Cell imaging of telmisartan

[0106] The experiment was divided into four groups. Cells were seeded in 6-well plates with glass slides (about 5.0×10 cells per well). 4 The cells were incubated at 37°C for 24 hours. The culture medium was then aspirated and telmisartan culture medium at final concentrations of 50 μM, 100 μM, and 200 μM was added. The cells were incubated with RAW 264.7 cells at 37°C for 2 hours. The cells were then incubated with culture medium containing 10 μM RL-NS at 37°C for 35 minutes. Following the incubation period, the culture medium was aspirated and the cells were fixed with 1 mL of 4% paraformaldehyde for 3 minutes. The cells were then washed three times with PBS and observed under a laser confocal microscope (Nikon A1R). Brightfield and darkfield images were taken (excitation wavelength: 488 nm, emission wavelength: 500-550 nm).

[0107] Depend on Figure 13 As the concentration of telmisartan gradually increased, the fluorescence of the cells in the green channel gradually increased. When the cells were treated with 50 μM telmisartan, their fluorescence intensity increased by 1.6 times compared to the control group. When the telmisartan concentration was increased to 200 μM, the cell fluorescence intensity increased by 2.2 times that of the control group. These results indicate that the hypertension drug telmisartan can generate 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 method for preparing a lysosome-targeted carbon monoxide fluorescent probe according to claim 1, characterized in that: The steps include: S1: dissolving 4-bromo-1,8-naphthalene dicarboxylic anhydride and morpholine in ethylene glycol monomethyl ether to react. After the reaction is complete, a precipitate is separated, filtered, and the filter cake is collected and dried in a vacuum drying oven to obtain a compound having structural formula (II). ； S2: adding the compound having the structure represented by formula (II) to an ethanol solution of propargylamine to carry out a reaction. After the reaction is completed, a precipitate is precipitated, and the filter cake is collected by filtration and purified by silica gel column chromatography to obtain a compound represented by formula (III); ； S3: Add tris(triphenylphosphine)carbonylruthenium hydrochloride ligand to ultra-dry dichloromethane, react at room temperature for 1 hour under nitrogen protection, then add benzothiadiazole. After the reaction liquid changes from yellow to red, add the compound having the structure represented by structural formula (III). After the reaction is completed, add anhydrous methanol to the reaction liquid to precipitate, and collect the filter cake by filtration to obtain the lysosome-targeted carbon monoxide fluorescent probe RL-NS.

3. The method for preparing a lysosome-targeted carbon monoxide fluorescent probe according to claim 2, wherein: The reaction conditions of S1 are: reaction temperature of 100° C., and stirring reaction time of 12 h.

4. The method for preparing a lysosome-targeted carbon monoxide fluorescent probe according to claim 2, wherein: The molar ratio of 4-bromo-1,8-naphthalene dicarboxylic anhydride to morpholine in S1 is 7.0-7.5:10.5-11.

0.

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

6. The method for preparing a lysosome-targeted carbon monoxide fluorescent probe according to claim 2, wherein: The molar ratio of the compound having the structure represented by structural formula (II) and propargylamine in S2 is 0.8-1:1.8-2.

0.

7. The method for preparing a lysosome-targeted carbon monoxide fluorescent probe according to claim 2, wherein: The molar ratio of the tris(triphenylphosphine)carbonylruthenium hydrochloride ligand, benzothiadiazole and the compound having the structure represented by structural formula (III) is 1:1:1.

Images