A single-excitation ratio fluorescent probe Mito-XS and its application in hypochlorous acid detection

Abstract

The application discloses a single-excitation ratio fluorescent probe Mito-XS and application thereof in hypochlorous acid detection, wherein the structure of the ratio fluorescent probe responding to hypochlorous acid is as follows: the single-excitation ratio fluorescent probe Mito-XS shows good selectivity and sensitive fluorescent signal to hypochlorous acid. The coumarin skeleton in the probe enables the probe to be anchored in mitochondria and not be affected by the change of the mitochondrial membrane potential, and realizes real-time visualization of the mitochondrial morphology. Cell toxicity test shows that the probe has good biocompatibility, and confocal fluorescence microscopic imaging experiment shows that the probe has good light stability in HepG2 cells, a fast reaction speed (60s), and a low detection limit of 18nM, and can sensitively detect hypochlorous acid, and is suitable for confocal fluorescence imaging of exogenous and endogenous hypochlorous acid in cells.

Description

Technical Field

[0001] This invention relates to a single-excitation ratio fluorescent probe Mito-XS and its application in hypochlorous acid detection, to achieve fluorescence imaging of hypochlorous acid in cells, which has the advantages of short response time, single selectivity and good biocompatibility. Background Technology

[0002] Redox processes encompass the fundamental processes of almost all life functions, from bioenergy to metabolism. Mitochondria are the primary site of reactive oxygen species (ROS) production and the cellular energy metabolism center, playing a regulatory role in maintaining normal physiological functions, including metabolism, signal transduction, and redox reactions. Hypochlorous acid (HClO), as a ROS, is produced by the reaction of H₂O₂ and Cl₂. - HClO is produced under the action of myeloperoxidase (MPO). HClO plays multiple roles in living systems: on the one hand, it plays a crucial role in immune defense against invading pathogens; on the other hand, abnormal accumulation of HClO may lead to the development or exacerbation of various inflammation-related diseases, such as liver damage and cancer. Therefore, it is important to develop a highly sensitive and selective method for measuring HClO in vivo and in the environment.

[0003] In recent years, many methods for detecting ClO have been developed. - While fluorescent probes exist, most of them only exhibit an on-signal fluorescence in a single fluorescent channel, and their performance is affected by the type of instrument used, the intracellular microenvironment, the local concentration of the probe, and photobleaching. In contrast, ratiometric fluorescent probes can simultaneously modulate two emission signals, possessing self-calibration capabilities, which can greatly eliminate the aforementioned interferences and make the response signal more stable; at the same time, single-excitation ratiometric fluorescent probes can avoid signal errors caused by intensity fluctuations of different excitation sources. Therefore, developing a ClO - A single-excitation ratiometric fluorescent probe with a response is urgently needed.

[0004] To address the above issues, we rationally designed and developed a single-excitation ratiometric fluorescent probe, Mito-XS, which offers rapid response, high selectivity, and sensitivity for hypochlorous acid detection, and is suitable for analyzing mitochondrial dynamics and morphology. Compared to traditional mitochondrial anchoring methods (introducing long alkyl chains / nucleophilic addition groups such as aldehydes or benzyl chloride), we innovatively introduced a coumarin group as a mitochondrial anchoring group into Mito-X (hypochlorous acid responsive unit), enabling the probe to anchor in mitochondria and allowing for long-term monitoring of hypochlorous acid levels. Simultaneously, it serves as an internal standard for hypochlorous acid ratiometric detection and provides a stable fluorescence signal for mitochondrial dynamics and morphological analysis. (The last sentence appears to be incomplete and possibly refers to a different probe, "ClO," which is not directly related to the preceding text.) -Following the reaction, the thioanisole was partially oxidized to a sulfoxide structure, resulting in a decrease in fluorescence intensity. Based on its rational structural design, the probe Mito-XS was successfully used for real-time visualization of hypochlorous acid levels and mitochondrial dynamics and morphology during lipopolysaccharide (LPS)-induced oxidative stress. Summary of the Invention

[0005] The present invention aims to provide a single-excitation ratio fluorescent probe Mito-XS and its application in hypochlorous acid detection. The technical problem to be solved is to obtain a fluorescent probe molecule that can anchor mitochondrial dual-channel fluorescence imaging and can identify exogenous and endogenous hypochlorous acid in cells through molecular design. It has the advantages of selective specificity, good photostability and dual-channel imaging. Cytotoxicity test shows that the probe of the present invention has good cell compatibility.

[0006] The structure of the single-excitation ratio fluorescent probe Mito-XS of this invention is shown below:

[0007]

[0008] The preparation method of the single-excitation ratio fluorescent probe Mito-XS of the present invention includes the following steps:

[0009] Step 1: Dissolve 1.78g of 7-chloroquineidine and 1.6g of iodomethane in 20mL of acetonitrile, react overnight at room temperature, evaporate the solvent, and slurry with ethyl acetate to obtain intermediate 1;

[0010] Step 2: Add 1.93g of 4-diethylaminosalicylic acid, 1.44g of Mischel acid, piperidine (2 drops) and 0.28g of K2CO3 to distilled water (5mL), then stir the mixture vigorously at room temperature for 24 hours. Acidify the resulting solution with hydrochloric acid, filter and collect the precipitate, wash it several times with water to obtain intermediate 2.

[0011] Step 3: Dissolve 2.63g of intermediate 2 in dichloromethane, add 2.8g of N-Boc piperazine, 3.8g of EDCI and 1.6g of DMAP at room temperature, then reflux for 7h. After the solvent is evaporated, separate by column chromatography. Dissolve the obtained solid in CH2Cl2 / CF3COOH solution (10mL, V / V = 1:1), stir at room temperature for 2h, concentrate under reduced pressure, and separate by column chromatography to obtain intermediate 3;

[0012] Step 4: Dissolve 1.5g of intermediate 1 and 1.8g of intermediate 3 in isopropanol, stir at 120℃ for 6 hours to obtain intermediate 4;

[0013] Step 5: Dissolve 300 mg of intermediate 4 and 180 mg of 4-methylthiobenzaldehyde in isopropanol, add three drops of piperidine, stir at 120 °C for 2 h, concentrate under reduced pressure, and separate by column chromatography to obtain the target product Mito-XS.

[0014] In step 3, the eluent used during column chromatography is methanol:dichloromethane = 1:50, v / v.

[0015] In step 5, the eluent used during column chromatography is methanol:dichloromethane = 1:20, v / v.

[0016] The synthesis route is shown below:

[0017]

[0018] The present invention relates to the application of the single excitation ratio fluorescent probe Mito-XS in the detection of hypochlorous acid.

[0019] A detection reagent was prepared using the single-excitation ratio fluorescent probe Mito-XS, which enables dual-channel imaging detection of endogenous and exogenous hypochlorous acid in cellular mitochondria.

[0020] The single-excitation ratiometric fluorescent probe Mito-XS of this invention exhibits excellent selectivity and sensitive fluorescence signal for hypochlorous acid. Its coumarin backbone allows the probe to anchor in mitochondria unaffected by changes in mitochondrial membrane potential, enabling real-time visualization of mitochondrial morphology. Cytotoxicity tests show good biocompatibility, and confocal fluorescence microscopy experiments demonstrate good photostability in HepG2 cells, a fast reaction rate (60 s), and a detection limit as low as 18 nM. It can sensitively detect hypochlorous acid and is suitable for confocal fluorescence imaging of both exogenous and endogenous hypochlorous acid in cells.

[0021] The detection method is as follows:

[0022] The Mito-XS of this invention was dissolved in DMSO to prepare a 2 mM stock solution. 15 μL of this stock solution was then added to 3 mL of PBS solvent with different hypochlorous acid concentrations to obtain the fluorescence and UV spectra of 10 μM Mito-XS in different test solutions. With increasing hypochlorous acid concentration, the absorbance of Mito-XS increased at 420 nm. Using 420 nm as the excitation wavelength, the fluorescence remained almost unchanged at 500 nm and decreased at 625 nm, indicating its ratiometric imaging capability. The time response of Mito-XS was tested, demonstrating its excellent short response time. Selectivity tests were conducted to eliminate interference from other substances, verifying the good selectivity of Mito-XS. Cells with added Mito-XS were monitored at different time points; the fluorescence in the red and green channels remained unchanged, indicating that Mito-XS can stably image cells. Using drugs that lower mitochondrial membrane potential and fix cells, the ability of Mito-XS to anchor to mitochondria was investigated. This study also confirmed that the control structure, Mito-X after removing the coumarin group, could not anchor to mitochondria. The ability of Mito-XS to detect hypochlorous acid in cells was investigated. Compared to the control group, the green emission of the hypochlorous acid-containing sample group remained essentially unchanged, while the red emission decreased. Adding a hypochlorous acid inhibitor restored the red emission of the sample group. The addition of phorbol myristate acetate (PMA) confirmed that the probe Mito-XS could visualize changes in hypochlorous acid levels and mitochondrial morphology. The addition of an endogenous hypochlorous acid inducer further reduced the red emission. These results indicate that Mito-XS can detect both exogenous and endogenous hypochlorous acid within cells. Due to Mito-XS's excellent anchoring ability to mitochondria, successful analysis of mitochondrial dynamics and morphology was achieved.

[0023] This invention utilizes single-excitation ratiometric fluorescence detection to achieve fluorescence imaging of hypochlorous acid in cells. Mito-XS possesses excellent characteristics such as stable fluorescence imaging capability, single selectivity, and good biocompatibility. Confocal fluorescence microscopy experiments demonstrate that Mito-XS can perform fluorescence imaging of hypochlorous acid level changes and mitochondrial morphology. Attached Figure Description

[0024] Figure 1 It is a hypothetical fluorescent probe Mito-XS responding to ClO - A schematic diagram of the mechanism.

[0025] Figure 2 Mito-XS (10 μM) was added to PBS with ClO - UV-Vis absorption spectra after (10μM, 20μM).

[0026] Figure 3(a) Mito-XS (10 μM) was added to PBS with ClO - (b) Fluorescence spectrum after (0–20 μM); (c) Fluorescence intensity ratio of Mito-XS (10 μM) (I) 625nm / I 500nm ) with different concentrations of ClO - (c) Linear relationship of (0~2μM); (d) Addition of different concentrations of ClO - (d) Time-dependent changes in the fluorescence spectrum of Mito-XS (10 μM) after (0.75 μM, 1.25 μM, 2.0 μM); (d) Fluorescence intensity ratio of Mito-XS (10 μM) to other biological species in PBS (I 625nm / I 500nm ): Blank sample, Na + K + Fe 2+ Ga 2+ Cu 2+ CO3 2- Glucose, tyrosine, serine, glycine, GSH, Hcy, Cys, S 2- ROO - ,·OH,H2O2,ONOO - ClO - .

[0027] Figure 4 It is Mito-XS (10μM) with the addition of ClO - The fluorescence intensity ratio (I) at different pH values ​​(7.0-10.0) before and after 625nm / I 500nm ).

[0028] Figure 5 The image shows the survival rate of HepG2 cells under different concentrations (0 μM, 10 μM, 20 μM, 30 μM, 40 μM) of Mito-XS.

[0029] Figure 6 This is a confocal fluorescence image of mitochondria in HepG2 cells co-stained with 10 μM Mito-XS and 0.5 μM commercial mitochondrial probe (MTDR). Subsequently, carbonyl cyanide m-chlorophenylhydrazone (CCCP) was added to lower the mitochondrial membrane potential, and cells were fixed with 4% paraformaldehyde to investigate the ability of Mito-XS to anchor mitochondria.

[0030] Figure 7This is a confocal fluorescence image of mitochondria in HepG2 cells simultaneously stained with 10 μM Mito-X and 0.5 μM commercial mitochondrial probe (MTDR). Subsequently, carbonyl cyanide m-chlorophenylhydrazone (CCCP) was added to lower the mitochondrial membrane potential, and cells were fixed with 4% paraformaldehyde to investigate the ability of Mito-X to anchor mitochondria.

[0031] Figure 8 This is a confocal fluorescence image of a 10 μM Mito-XS, used to investigate the optical stability of Mito-XS.

[0032] Figure 9 It is 10μM Mito-XS with the addition of ClO - (a) Confocal fluorescence imaging; (b) Bar graph of fluorescence intensity in the blue and red channels; (c) Fluorescence intensity ratio of the red and green channels, to explore the exogenous response of Mito-XS to hypochlorous acid.

[0033] Figure 10 (a) is a confocal fluorescence image of 10 μM Mito-XS with added lipopolysaccharide (LPS) and puerarin (PU); (b) is a bar chart of fluorescence intensity in the blue and red channels; (c) is the fluorescence intensity ratio of the red and green channels. This study explores the ability of Mito-XS to respond to LPS-induced endogenous hypochlorous acid production and visualizes mitochondrial morphology.

[0034] Figure 11 The images show: (a) confocal fluorescence imaging of 10 μM Mito-XS within 50 min after the addition of phorbol myristoyl acetate (PMA); (b) a bar chart of fluorescence intensity in the blue and red channels; (c) the fluorescence intensity ratio of the red and green channels; and (d) mitochondrial morphology factors and branch number. Detailed Implementation

[0035] The present invention will be further illustrated by the following examples.

[0036] Example 1: Synthesis of Mito-XS

[0037] 300 mg of compound 4 and 180 mg of 4-methylthiobenzaldehyde were dissolved in isopropanol, three drops of piperidine were added, the mixture was stirred at 120 °C for 2 h, concentrated under reduced pressure, and separated by column chromatography (methanol: dichloromethane = 1:20, V / V) to obtain the target product Mito-XS.

[0038] 1H NMR (400MHz, DMSO-d6) δ8.68(d,J=8.4Hz,1H),8.08(d,J=9.3Hz,1H),8.01(d,J=7.1Hz,2 H),7.98–7.90(m,1H),7.85–7.75(m,3H),7.68(d,J=9.3Hz,1H),7.49(d,J=8.9Hz,1H),7. 35(d,J=8.1Hz,2H),7.23(s,1H),6.73(d,J=8.9Hz,1H),6.54(s,1H),4.32(s,3H),3.74( d,J=25.9Hz,6H),3.55(s,2H),3.45(d,J=7.2Hz,4H),2.52(s,3H),1.10(t,J=6.9Hz,6H). 13 C NMR(101MHz,DMSO-d6)δ157.49,156.73,147.18,144.42,144.10,139.72,135.39,131.75,130.59,130.22,1 29.47,128.27,126.06,121.55,119.86,118.57,110.06,107.51,96.80,44.75,40.18,14.63,12.83.ESI-MS m / z:calcd.For C 37 H 41 N4O3S + {[Mito-XS-I - ] +}619.2737,found,619.2727.

[0039] Example 2: Response of Mito-XS to hypochlorous acid

[0040] Mito-XS and ClO were tested - Changes in UV-Vis absorption and fluorescence spectra before and after the response. (Adding 10 μM ClO) - Subsequently, the UV-Vis absorption of Mito-XS at 420 nm decreased significantly. Figure 2 Using 420 nm as the excitation wavelength, the Mito-XS probe exhibited emission peaks at 500 nm and 625 nm. With ClO... - With increasing concentration, the fluorescence intensity of the Mito-XS probe gradually decreased at 625 nm, while the fluorescence at 500 nm remained essentially unchanged, indicating that the probe can effectively target ClO₂. - Ratio-type detection ( Figure 3 a). For example Figure 3As shown in b, the ratio of the fluorescence intensity of Mito-XS at 625 nm to that at 500 nm (I 625nm / I 500nm ) and ClO - There is a good linear relationship between concentrations, with a correlation coefficient of R. 2 =0.996, detection limit is 18 nM. For example... Figure 3 As shown in c, the Mito-XS probe can respond rapidly to ClO within 60s. - Suitable for intracellular ClO - Rapid detection.

[0041] Example 3: Probe Selectivity of Mito-XS

[0042] Further, the Mito-XS probe was used to measure various analytes, including blank samples and Na. + K + Fe 2+ Ga 2+ Cu 2+ CO3 2- Glucose, tyrosine, serine, glycine, GSH, Hcy, Cys, S 2- ROO - ,·OH,H2O2,ONOO - ClO - ( Figure 3 d). The analysis results show that Mito-XS is only effective against ClO₂. - It exhibits a significant response and a fluorescence signal ratio (I) to other analytes. 625nm / I 500nm The changes are negligible. These results demonstrate that Mito-XS affects ClO₂. - It exhibits extremely high selectivity.

[0043] Example 4: pH stability of Mito-XS

[0044] To consider whether the fluorescence of the Mito-XS probe would be interfered with by a weakly alkaline environment after targeting mitochondria, the pH stability of the probe was tested. Figure 4 Two sets of buffer solutions were prepared with pH values ​​of 7, 7.4, 8, 9, and 10. One set contained 10 μM Mito-XS, and the other set contained both 10 μM Mito-XS and ClO₂. - The fluorescence signal ratio (IL) of the two groups of test solutions was compared. 625nm / I 500nmTo verify the probe's resistance to pH interference, it can be seen that the effect of pH on the fluorescence signal before and after the probe response is negligible, indicating that Mito-XS is suitable for detecting hypochlorous acid in the slightly alkaline environment of mitochondria.

[0045] Example 5: Cytotoxicity Test

[0046] We performed dark cytotoxicity assays using the MTT (5-dimethylthiazolyl-2,5-diphenyltetrazolium bromide) method. Various concentrations (0 μM, 10 μM, 20 μM, 30 μM, 40 μM) of Mito-XS were added to HepG2 cells, and after incubation in the dark for 24 hours, the results were tested. Figure 5 As shown above, Mito-XS has low biotoxicity and can be used in biological experiments.

[0047] Example 6: The ability of the Mito-XS probe to anchor mitochondria

[0048] As a powerful tool capable of simultaneously detecting hypochlorous acid and analyzing mitochondrial dynamics and morphology, Mito-XS should possess excellent mitochondrial anchoring capabilities. Figure 6 Adding the carbonyl cyanide m-chlorophenylhydrazone (CCCP) to lower the mitochondrial membrane potential revealed no significant change in the mitochondrial colocalization coefficient, indicating that the probe could anchor in mitochondria. Furthermore, after fixing cells with 4% paraformaldehyde, a significant fluorescent signal was still observed in the cells, and the mitochondrial colocalization coefficient remained stable. These results demonstrate that the probe can anchor in mitochondria in cells unaffected by mitochondrial membrane potential, and can be used for detecting mitochondrial hypochlorous acid levels and analyzing mitochondrial morphology during oxidative stress and even in fixed cells.

[0049] Example 7: The ability of probe Mito-X to anchor mitochondria

[0050] like Figure 7 Adding the carbonyl cyanide m-chlorophenylhydrazone (CCCP) to lower the mitochondrial membrane potential resulted in a significant decrease in the mitochondrial colocalization coefficient, indicating that the control structure Mito-X, after the removal of the coumarin group, could not anchor in mitochondria. Furthermore, fixing cells with 4% paraformaldehyde resulted in a significant decrease in fluorescence signal and a reduced mitochondrial colocalization coefficient. These results demonstrate that the control structure Mito-X cannot anchor in mitochondria in cells, and the mitochondrial anchoring ability of Mito-XS is attributed to the connection of the coumarin moiety.

[0051] Example 8: Stability experiment of Mito-XS in mitochondria

[0052] Long-term real-time monitoring requires probes to have good photostability; therefore, we further investigated the photostability of Mito-XS in cells. Figure 8 The fluorescence intensity of the probe remained stable in HepG2 cells for 90 minutes, with no significant signal loss observed. This indicates that Mito-XS has good photostability and can be used for long-term biological assays.

[0053] Example 9: Imaging of intracellular hypochlorous acid and visualization of mitochondrial morphology using Mito-XS

[0054] HepG2 cells were co-incubated with Mito-XS for 0.5 hours, followed by confocal fluorescence microscopy analysis. Figure 9 As shown, bright fluorescence signals were observed in both the green and red channels. Next, exogenous ClO₂ was added to the experimental group cells. - Subsequently, the fluorescence intensity of the green channel remained essentially unchanged, while the fluorescence intensity of the red channel significantly decreased. This confirms that Mito-XS can achieve exogenous ClO₂ in cells. - The response.

[0055] Cells were treated with lipopolysaccharide (LPS) for 12 hours to induce the production of endogenous ClO. - ( Figure 10 The sample was then co-incubated with the probe for 0.5 h for confocal imaging. The results showed that the fluorescence intensity of the green channel remained essentially unchanged, while the red channel fluorescence weakened. Upon addition of the antioxidant puerarin (PU), the red channel fluorescence recovered, confirming that Mito-XS can effectively target endogenous ClO2 in cells. - The response was positive. Simultaneously, the mitochondrial morphology changed from filamentous to blurred dots and then back to filamentous, indicating that Mito-XS is capable of visualizing mitochondrial morphology.

[0056] Example 10: Imaging of hypochlorous acid and visualization of mitochondrial morphology during PMA-induced oxidative stress

[0057] like Figure 11 Upon addition of phorbol myristate acetate (PMA), the red channel fluorescence gradually weakened, indicating an increase in hypochlorous acid content in cells under PMA stimulation. Subsequently, thanks to the stable fluorescence signal of coumarin, analysis of mitochondrial morphology via the green fluorescence channel revealed that with increasing PMA stimulation time, the number of mitochondrial branches gradually increased, mitochondria gradually contracted inward, and mitochondrial morphology factors gradually decreased. This may be due to increased mitochondrial division caused by mitochondrial damage resulting from increased hypochlorous acid content. This demonstrates that the Mito-XS probe is a powerful tool capable of simultaneously visualizing changes in hypochlorous acid levels and mitochondrial morphology and their interactions.

Claims

1. A single-excitation ratiometric fluorescent probe, Mito-XS, characterized in that... Its structural formula is: 。 2. The method for preparing the ratiometric fluorescent probe Mito-XS according to claim 1, characterized in that... Includes the following steps: Step 1: Dissolve 7-chloroquineidine and iodomethane in acetonitrile, react at room temperature, evaporate the solvent, and slurry with ethyl acetate to obtain intermediate 1; Step 2: Add 4-diethylaminosalicylic acid, Mischel acid, piperidine and K2CO3 to distilled water, then stir the mixture at room temperature for 24 hours. Acidify the resulting solution with hydrochloric acid, filter and collect the precipitate, wash with water to obtain intermediate 2. Step 3: Dissolve intermediate 2 in dichloromethane, add N-Boc piperazine, EDCI and DMAP at room temperature, reflux for 7 h, evaporate the solvent and separate by column chromatography, dissolve the obtained solid in CH2Cl2 / CF3COOH solution, stir at room temperature for 2 h, concentrate under reduced pressure, and separate by column chromatography to obtain intermediate 3; Step 4: Dissolve intermediate 1 and intermediate 3 in isopropanol and stir at 120°C for 6 h to obtain intermediate 4; Step 5: Dissolve intermediate 4,4-methylthiobenzaldehyde in isopropanol, add piperidine dropwise, stir at 120°C for 2 h, concentrate under reduced pressure, and separate by column chromatography to obtain the target product Mito-XS; The synthesis route is shown below: 。 3. The application of the ratiometric fluorescent probe Mito-XS according to claim 1 in the detection of hypochlorous acid, wherein the application is for non-disease diagnosis and / or treatment purposes.

4. The application according to claim 3, characterized in that: A detection reagent was prepared using the ratiometric fluorescent probe Mito-XS, which can anchor mitochondria and enable real-time visualization of mitochondrial morphology.

5. The application according to claim 3, characterized in that: A detection reagent was prepared using the ratiometric fluorescent probe Mito-XS, which enables dual-channel imaging detection of endogenous and exogenous hypochlorous acid in mitochondria under single excitation.

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