A mitochondria-targeted near-infrared fluorescent probe, a preparation method thereof and application thereof in carboxylesterase detection

Abstract

The application discloses a kind of mitochondria-targeted near-infrared fluorescent probe and its preparation method and application in carboxylesterase detection, wherein the structure of the mitochondria-targeted near-infrared fluorescent probe is as follows: The near-infrared fluorescent probe SYH-SF of the application shows good selectivity and high sensitivity (detection limit is as low as 0.027 U / L) to carboxylesterase, and has good mitochondria-targeting ability (positioning coefficient Pr=0.92). Cell toxicity test shows that the probe has good biocompatibility, and confocal fluorescence microscopic imaging experiment shows that the probe has good light stability for HepG2 cells, can effectively detect carboxylesterase in mitochondria of living cells, and is suitable for confocal fluorescence imaging of intracellular endogenous carboxylesterase, evaluation of anticancer drug efficacy and differentiation between normal cells and cancer cells.

Description

Technical Field

[0001] This invention relates to a mitochondrial-targeted near-infrared fluorescent probe, its preparation method, and its application in the detection of carboxylesterases, so as to achieve near-infrared fluorescence imaging of carboxylesterases in the mitochondria of live cells. It has the advantages of selectivity, high efficiency and sensitivity, and good biocompatibility. Background Technology

[0002] As the basic unit of life, the cell is an ingenious and complex chemical machine that performs various biological activities. Various small and large biological molecules within the cell coordinate with each other to perform multiple biological functions. Enzymes are one of the most important classes of key macromolecules within the cell, playing a crucial role in various catalytic reactions essential for normal life. Carboxylesterases (CEs) are serine hydrolases widely distributed in various biological systems, capable of catalyzing the transesterification and hydrolysis of esters, thioesters, amides, and carbamates both endogenously and exogenously. As important phase I exogenous metabolic enzymes, the deficiency or abnormal regulation of CEs is directly related to human diseases such as Wolman's disease, insulin insensitivity, obesity, hyperlipidemia, atherosclerosis, hepatic steatosis, and hepatocellular carcinoma. Due to their high catalytic conversion rates and broad substrate specificity, these enzymes are widely used not only for detoxification and anti-exogenous drug treatment but also for the metabolic activation of certain prodrugs and as drug targets. Monitoring mitochondrial esterase activity is crucial not only for studying mitochondrial metabolism but also for evaluating the effectiveness of mitochondrial-targeted prodrugs. Therefore, detecting mitochondrial carboxylesterase activity is of great significance.

[0003] Currently, many fluorescent probes for the detection of carboxylesterases (CEs) have been developed due to their simple preparation, high sensitivity, high selectivity, and live organism imaging capabilities. However, due to the ubiquitous presence of esterases in mitochondria and the uncontrolled localization of fluorescent probes, there are still very few fluorescent probes developed for the accurate detection of mitochondrial esterase activity. Summary of the Invention

[0004] This invention aims to provide a mitochondrial-targeted near-infrared fluorescent probe, its preparation method, and its application in carboxylesterase detection. The technical problem to be solved is to obtain a probe through molecular design that can be used for fluorescence imaging and can identify the activity of endogenous carboxylesterases in cells. It has the advantages of selectivity, specificity, good photostability, mitochondrial targeting, and near-infrared imaging. Cytotoxicity tests show that the probe of this invention has good cell compatibility.

[0005] The mitochondrial-targeting near-infrared fluorescent probe of this invention, abbreviated as SYH-SF, uses an oxanthracene derivative as its parent material, and its structural formula is shown below:

[0006]

[0007] In this invention, the pyridine cation in the fluorescent probe SYH-SF is linked to an xanthracene derivative, forming a donor-acceptor (D-π-A) structure. This structure facilitates fluorescence emission to the near-infrared emission region and a large Stokes shift, while also enabling the probe to locate mitochondria. Upon response to carboxylesterase in living mitochondria, the SYH-SF probe exhibits enhanced fluorescence. Furthermore, the probe demonstrates high sensitivity and selectivity for CEs (carboxylesterases), specifically carboxylesterase CAS number 9016-18-6, which was used for testing.

[0008] The method for preparing the mitochondrial-targeted near-infrared fluorescent probe of the present invention includes the following steps:

[0009] Step 1: Mix 30 mL of N,N-dimethylformamide (DMF) with 10 mL of dichloromethane (DCM), add phosphorus tribromide dropwise at 0 °C, remove from ice-water bath and add cyclohexanone dropwise. After reacting for six hours, add the reaction solution dropwise to 150 mL of water, then neutralize with sodium carbonate, extract with water and DCM, and evaporate to dryness to obtain intermediate 1.

[0010] Step 2: Add 9.4g of intermediate 1, 5.3g of 2-hydroxy-4-methoxybenzaldehyde, and 17g of potassium carbonate to 30ml of DMF and react for 12h. Then extract with water and DCM, evaporate to dryness, and slurry (ethyl acetate: petroleum ether = 1:1, v / v). Filter to obtain intermediate 2.

[0011] Step 3: Add 1.9 g of intermediate 2 to 20 mL of DCM, add boron tribromide dropwise at 0 °C, react at room temperature for 4 h, dilute with 25 mL of DCM, extract with 20 mL of DCM and 20 mL of water, and rotary evaporate to obtain intermediate 3.

[0012] Step 4: Add 3.0g of iodomethane and 2.0g of tetramethylpyridine to 10mL of acetonitrile, stir at room temperature for 2h, and directly filter to obtain intermediate 4.

[0013] Step 5: Add 1g of intermediate 3, 1.03g of intermediate 4 and two drops of piperidine to 10mL of ethanol, reflux at 80℃ for 12h, evaporate the solvent by rotary evaporation, slurry with ethyl acetate, and then separate by column chromatography (methanol:dichloromethane = 1:20, v / v) to obtain intermediate SYH-OH.

[0014] Step 6: Add 0.1g of intermediate SYH-OH and 0.5mL of triethylamine to 5mL of LCM, add 33mg of 2-thiophenecarboxyl chloride dropwise at 0℃, react at room temperature for 2h, evaporate the solvent, and separate by column chromatography (methanol:dichloromethane = 1:20, v / v) to obtain the target product SYH-SF.

[0015] The synthesis route is shown below:

[0016]

[0017] The application of the mitochondrial-targeted near-infrared fluorescent probe of the present invention in the preparation of carboxylesterase detection reagents.

[0018] The detection reagent can be used to evaluate endogenous and exogenous carboxylesterases and anticancer drugs in imaging cells, as well as to distinguish cancer cells from normal cells.

[0019] The detection method is as follows:

[0020] The SYH-SF 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 DMSO / PBS buffer solution (pH = 7.4, 2:8, v:v) containing different concentrations of carboxylesterase. Fluorescence and UV spectra of 10 μM SYH-SF in different test solutions were obtained. With increasing carboxylesterase content, the absorbance of SYH-SF increased at 530 nm. Using 530 nm as the excitation wavelength, fluorescence increased at 645 nm, indicating its near-infrared imaging capability. Dark cytotoxicity experiments were then performed using the MTT (5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide) method. Various concentrations of SYH-SF (0 μM, 5.0 μM, 10.0 μM, 15.0 μM, 20.0 μM) were added to live HepG2 cells, and after incubation in the dark for 24 hours, cell viability was not affected. To investigate the imaging ability of SYH-SF in cells, cells treated with SYH-SF were monitored for different time periods, and the red channel fluorescence remained unchanged. These results indicate that SYH-SF can stably image in cells. To investigate the detection ability of SYH-SF in cells, a carboxylesterase inhibitor was added to the sample group, while the control group received no inhibitor; the red emission of the sample group was weakened. These results indicate that SYH-SF can perform endogenous detection of carboxylesterase within cells. To examine the potential drug screening ability of the SYH-SF probe, the anticancer drug sorafenib was added to the sample group, while the control group received no drug; the red emission of the sample group was weakened. These results indicate that the SYH-SF probe has the potential to screen drugs based on targeting CE. To investigate the ability of SYH-SF to distinguish between cancer cells and normal cells, SYH-SF (10 μM) was co-incubated with cancer cells (HepG2, 4T1) and normal cells (3T3, MH-S), followed by confocal fluorescence imaging. The fluorescence intensity of the confocal images showed that the red emission of the cancer cell group was stronger, while that of the normal cell group was weaker. The results show that the probe SYH-SF can distinguish between cancer cells and normal cells.

[0021] This invention utilizes near-infrared fluorescence detection to achieve fluorescence imaging of carboxylesterases in cells. It exhibits stable fluorescence imaging capabilities, and cytotoxicity tests demonstrate good cell compatibility with SYH-SF. Confocal fluorescence microscopy experiments show that SYH-SF can locate mitochondria and perform near-infrared fluorescence imaging of carboxylesterases, demonstrating its potential for evaluating the effects of anticancer drugs. Furthermore, it can distinguish between cancer cells and normal cells. Attached Figure Description

[0022] Figure 1 (a) is the fluorescence spectrum of SYH-SF (10 μM) after adding CE (0–10 U / L) to DMSO / PBS buffer solution (pH = 7.4, 2:8, v:v); (b) is the linear relationship between the fluorescence intensity of SYH-SF (10 μM) and the concentration of CE (0–6 U / L) in DMSO / PBS buffer solution (pH = 7.4, 2:8, v:v).

[0023] Figure 2 (a) shows the fluorescence intensity of SYH-SF (10 μM) for various analytes in DMSO / PBS buffer (pH = 7.4, 2:8, v:v): 1: Gly, 2: Ser, 3: Hcy, 4: Cys, 5: GSH, 6: H2O2, 7: DMR, 8: HCO3- - 9:Na + 10: Ca 2+ 11: CO3 2- ,12:ClO - ,13:GLU,14:Mg 2+ 15:K + ,16:Cu 2+ ,17:Cl - ,18:NH4 + 19: HSO3 - ,20:Fe 3+ 21: Tyr, 22: LAP, 23: NTR, 24: AchB, 25: APN, 26: AchE, 27: CE; (b) is the fluorescence intensity of SYH-SF (10 μM) in DMSO / PBS buffer (pH = 7.4, 2:8, v:v) after the addition of the above analytes (black) and subsequently (10 U / L) CE (red).

[0024] Figure 3 (a) shows the time-dependent changes in the fluorescence spectrum of SYH-SF (10 μM) after adding CE (10 U / L) to DMSO / PBS buffer solution (pH = 7.4, 2:8, v:v); (b) shows the fluorescence intensity (IL) of SYH-SF (10 μM) after the addition of CE (10 U / L). 645nmThe relationship between CE (10 U / L) and pH is shown, with red dots and black squares representing the presence and absence of CE (10 U / L), respectively.

[0025] Figure 4 The image shows the survival rate of HepG2 cells under different concentrations (0 μM, 5 μM, 10 μM, 15 μM, 20 μM) of SYH-SF.

[0026] Figure 5 This is a confocal fluorescence image of mitochondria in HepG2 cells co-stained with SYH-SF (10 μM) and a 0.5 μM commercial mitochondrial probe (Mito Green). The aim was to investigate the mitochondrial anchoring ability of SYH-SF.

[0027] Figure 6 Here are confocal fluorescence images (a) and corresponding fluorescence intensity images (b) of SYH-SF (10 μM) to investigate the optical stability of SYH-SF.

[0028] Figure 7 The image shows a confocal fluorescence image (a) and a corresponding fluorescence intensity image (b) of SYH-SF (10 μM) in response to endogenous CE in HepG2 cells.

[0029] Figure 8 The images show confocal fluorescence imaging (a) and corresponding fluorescence intensity (b) of SYH-SF (10 μM) in HepG2 cells before and after treatment with the anticancer drug sorafenib, representing the response to CE.

[0030] Figure 9 The images show confocal fluorescence imaging (a) and corresponding fluorescence intensity (b) of SYH-SF (10 μM) in cancer cells (HepG2, 4T1) and normal cells (3T3, MH-S). The aim was to investigate whether SYH-SF could distinguish between normal and cancer cells. Detailed Implementation

[0031] The technical solution of the present invention will be further illustrated below through embodiments.

[0032] Example 1: Synthesis of SYH-SF

[0033] 0.1 g of product 5 and 0.5 mL of triethylamine were added to 5 mL of LCM, and 33 mg of 2-thiophenecarboxyl chloride was added dropwise at 0 °C. The reaction was carried out at room temperature for 2 h, the solvent was evaporated, and the product was passed through a column (methanol:dichloromethane = 1:20, v / v) to obtain the target product SYH-SF, 0.09 g, yield 72%.

[0034] 1H NMR (400MHz, DMSO-d6, ppm) δ8.61(d,J=6.9Hz,2H),8.18(d,J=15.9Hz,1H),8.13(d,J=5.0Hz,1H),8.07-8.03(m,3H),7.33(d,J=7.9Hz ,3H),7.03(d,J=8.2Hz,1H),6.78(s,1H),6.66(d,J=15.9Hz,1H),4.16(s,3H),2.62-2.53(m,2H),2.49(m,2H),1.76(q,J=6.3Hz,2H). 13 C NMR(101MHz,DMSO-d6,ppm)δ159.85,152.79,152.46,151.29,150.52,144.17,135.79,135.51,134.64,131.30,129.8 9,128.90,127.10,123.46,122.33,119.70,119.36,117.16,111.64,109.29,46.34,40.02,28.84,23.89,20.13.FT-MS m / z:[SYH-SF-I - ] + :C 26 H 22 NO3S + calcd.,428.1315; found,428.1306.

[0035] Example 2: Response of SYH-SF to carboxylesterase

[0036] The SYH-SF of this invention was dissolved in DMSO to prepare a 2 mM stock solution. 15 μL of the SYH-SF stock solution was added to 3 mL of DMSO / PBS buffer solution (pH = 7.4, 2:8, v:v). Different concentrations of CE were added to obtain the fluorescence spectra of the probe SYH-SF at different CE concentrations. Figure 2 a). The fluorescence intensity at 645 nm versus CE concentration curve showed a good linear relationship (R0). 2 =0.98, Figure 2 (b) The detection limit was calculated to be 0.027 U / L. This indicates that the probe SYH-SF can respond sensitively to carboxylesterases.

[0037] Example 3: Probe selectivity and competitiveness of SYH-SF

[0038] The SYH-SF probe is used to measure various analytes, including 1: Gly, 2: Ser, 3: Hcy, 4: Cys, 5: GSH, 6: H2O2, 7: DMR, and 8: HCO3. - 9:Na + 10: Ca 2+ 11: CO3 2- ,12:ClO - ,13:GLU,14:Mg 2+ 15:K + ,16:Cu 2+ ,17:Cl - ,18:NH4 + 19: HSO3 - ,20:Fe 3+ 21: Tyr, 22: LAP, 23: NTR, 24: AchB, 25: APN, 26: AchE, 27: CE. Analysis results ( Figure 2 a) This indicates that SYH-SF significantly enhances the fluorescence of carboxylesterases, while the fluorescence changes of other analytes are negligible. These results demonstrate that SYH-SF exhibits high selectivity for carboxylesterases.

[0039] The SYH-SF probe is used to add various analytes when measuring CEs, including 1: Gly, 2: Ser, 3: Hcy, 4: Cys, 5: GSH, 6: H2O2, 7: DMR, and 8: HCO3. - 9:Na + 10: Ca 2+ 11: CO3 2- ,12:ClO - ,13:GLU,14:Mg 2+ 15:K + ,16:Cu 2+ ,17:Cl - ,18:NH4 + 19: HSO3 - ,20:Fe 3+ 21: Tyr, 22: LAP, 23: NTR, 24: AchB, 25: APN, 26: AchE. Analysis results ( Figure 2 (b) This indicates that when SYH-SF responds to carboxylesterases, the effect of other analytes on the fluorescence of the SYH-SF probe is negligible. These results demonstrate that SYH-SF exhibits good competitiveness against carboxylesterases.

[0040] Example 3: Time response and pH stability of the SYH-SF probe

[0041] like Figure 3 As shown in figure a, the time dependence of the fluorescence spectrum of SYH-SF (10 μM) after the addition of CE (10 U / L) is shown. The fluorescence stabilizes after 40 min, which is beneficial for the sensitive detection of carboxylesterase.

[0042] To address the potential interference of the SYH-SF probe's fluorescence in a weakly alkaline environment after targeting mitochondria, the pH stability of the probe was tested accordingly. Figure 3 (b) Using phosphoric acid and sodium hydroxide diluted with multiple times of water, buffer solutions with pH values ​​of 6.59, 6.81, 7, 7.22, 7.4, 7.6, and 7.8 were prepared. 3 mL of each of the eight PBS buffer solutions was placed in centrifuge tubes, and the probe stock solution was added to each tube. Carboxylesterase test solution was added to the PBS buffer solutions at different pH values, and the fluorescence intensity of the two test solutions was compared to verify the probe's resistance to pH interference.

[0043] Example 4: Cytotoxicity Test

[0044] We performed dark cytotoxicity assays using the MTT (5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide) method. Various concentrations (0 μM, 5.0 μM, 10.0 μM, 15.0 μM, 20.0 μM) of SYH-SF were added to live HepG2 cells, and after incubation in the dark for 24 hours, the results were tested. Figure 4 As shown above, SYH-SF has low biotoxicity and can be used in biological applications.

[0045] Example 5: Co-localization of probes SYH-SF

[0046] After SYH-SF staining, filamentous mitochondria were clearly visible, comparable to those stained with Mito Green. Furthermore, the fluorescence distribution of SYH-SF showed good overlap with that of Mito Green, with a localization coefficient of 0.92. Figure 5 ).

[0047] Example 6: Stability experiment of SYH-SF in mitochondria

[0048] The photostability of SYH-SF in cells is crucial because long-term monitoring is often required when studying cellular physiological processes such as apoptosis. Figure 6 In photostability experiments in HepG2 cells, the fluorescence intensity remained stable within 120 minutes, with no significant signal loss observed. This indicates that SYH-SF exhibits good photostability and can be further tested over extended periods.

[0049] Example 7: Imaging of intracellular carboxylesterases by SYH-SF

[0050] The control group was cultured with SYH-SF for 0.5 h, followed by confocal fluorescence microscopy analysis. Figure 7 As shown, the fluorescence emitted by the cells can be seen in the red channel. The experimental group was treated with AEBSF for 3 hours to inhibit intracellular CEs activity. Figure 7 After incubation with the probe for 0.5 h, the fluorescence intensity of the red channel in the confocal image significantly decreased. This confirms that as CE activity is inhibited, the SYH-SF response also weakens.

[0051] Example 8: SYH-SF for cell imaging evaluation of the anticancer drug sorafenib

[0052] The control group was cultured with SYH-SF for 0.5 h, followed by confocal fluorescence microscopy analysis. Figure 8 As shown, the fluorescence emitted by the cells can be seen in the red channel. The experimental group was treated with the anticancer drug sorafenib for 3 hours, followed by incubation with a probe for 0.5 hours. Figure 8 The fluorescence intensity of the red channel in the confocal image was significantly reduced. This confirms that with the addition of sorafenib, CE activity was inhibited, and the SYH-SF response weakened accordingly. This suggests that SYH-SF has the potential for drug evaluation and screening.

[0053] Example 9: SYH-SF for cell imaging to distinguish cancer cells from normal cells

[0054] HepG2, 4T1, 3T3, and MH-S cells were cultured for 30 minutes using the probe SYH-SF, followed by confocal fluorescence imaging. Results were obtained as follows: Figure 9 The series of images shown indicate that the fluorescence intensity of the red channel of the probe SYH-SF is enhanced in cancer cells HepG2 and 4T1, while the fluorescence intensity of the red channel is weakened in normal cells 3T3 and MH-S, demonstrating that the probe can distinguish between cancer cells and normal cells.

Claims

1. A mitochondrial-targeting near-infrared fluorescent probe, characterized in that... Its structural formula is shown below: 。 2. The method for preparing the mitochondrial-targeted near-infrared fluorescent probe according to claim 1, characterized in that... Includes the following steps: Step 1: Mix N,N-dimethylformamide with dichloromethane, add phosphorus tribromide dropwise at 0°C, remove from ice-water bath and add cyclohexanone dropwise. After reacting for six hours, add the reaction solution dropwise to water, then neutralize with sodium carbonate, extract with water and DCM, and evaporate to dryness to obtain intermediate 1. Step 2: Add intermediate 1, 2-hydroxy-4-methoxybenzaldehyde and potassium carbonate to DMF and react for 12 h. Then extract with water and DCM, evaporate to dryness, pulp, and filter to obtain intermediate 2. Step 3: Add intermediate 2 to DCM, add boron tribromide dropwise at 0℃, react at room temperature for 4 h, dilute DCM, extract with DCM and water, and rotary evaporate to obtain intermediate 3; Step 4: Add iodomethane and tetramethylpyridine to acetonitrile, stir at room temperature for 2 h, and directly filter to obtain intermediate 4; Step 5: Add intermediates 3 and 4 and piperidine to ethanol, reflux at 80°C for 12 h, evaporate the solvent by rotary evaporation, slurry with ethyl acetate, and then separate by column chromatography to obtain intermediate SYH-OH; Step 6: Add intermediate SYH-OH and triethylamine to DCM, add 2-thiophenecarboxyl chloride dropwise at 0℃, react at room temperature for 2 h, evaporate the solvent, and separate the target product SYH-SF by column chromatography; The synthesis route is shown below: 。 3. The application of the mitochondrial-targeted near-infrared fluorescent probe of claim 1 in the preparation of carboxylesterase detection reagents.

4. The application according to claim 3, characterized in that: The detection reagent can image endogenous and exogenous carboxylesterases in cells, evaluate anticancer drugs, and distinguish cancer cells from normal cells.

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