Dual-targeting near-infrared ratiometric fluorescent probe and application thereof in preparation of carboxylesterase detection reagent

Abstract

The invention discloses a double-targeting near-infrared ratiometric fluorescent probe and application thereof in preparation of a carboxylesterase detection reagent. The structure of the double-targeting near-infrared ratiometric fluorescent probe is shown as follows: # imgabs0 #. A quinoline salt part in the fluorescent probe ZZD can target mitochondria, and the ZD structure after response can be positioned on lipid droplets. The fluorescent probe ZZD has the advantages of good response capability, excellent double-organelle targeting capability and near-infrared emission in living cells. The fluorescent probe ZZD is expected to become a useful fluorescent tool for researching lipid droplet and mitochondrial related diseases in the future.

Description

Technical Field

[0001] The invention relates to a dual-targeted near-infrared ratio fluorescent probe and application thereof in preparing a carboxylesterase detection reagent. Background Art

[0002] As a member of the serine esterase family, carboxylesterase participates in many physiological processes and plays a key role in homeostasis and metabolic defense systems. At the same time, it also plays an important role in catalyzing various endogenous substrates, detoxifying clinical drugs and environmental toxins, and metabolic activation of anticancer prodrugs in vivo. Importantly, when carboxylesterase is abnormally expressed in the liver, it will be highly associated with diseases such as obesity, diabetes, alcoholic fatty liver and cancer. In addition, although the lipophilicity of anticancer drugs can be increased by esterification and the membrane permeability has been reasonably improved, the carboxylesterase activity is crucial for the release of the pharmacologically active ingredients of these ester prodrugs and can be used for cancer treatment. At present, carboxylesterase is mainly studied indirectly through the detection of related proteins. However, this method does not provide information about the level of carboxylesterase activity, and the study is more complicated, and only a few methods can study the activity of carboxylesterase in living cells. For example, chromatography is usually used in conjunction with mass spectrometry and can be used to study endogenous or overexpressed carboxylesterases. But the steps of this method are still cumbersome. Therefore, the introduction of new technologies is crucial for the detection of carboxylesterase, which will play an important role in the screening of its inhibitors and clinical serum detection.

[0003] With the vigorous development of fluorescence sensing technology, the detection technology of carboxylesterase activity has also been continuously innovated and developed, and many fluorescent probes with high sensitivity, specific selectivity and high fidelity have been developed. In particular, near-infrared (NIR) fluorescent probes have the advantages of strong tissue penetration and little tissue damage, and have broad development prospects for in vivo research. However, carboxylesterase activity detection and imaging probes based on traditional sensing platforms still face severe challenges such as biological background signal interference, excitation light scattering limitations, and low biological tissue penetration. Therefore, NIR probes based on traditional excellent fluorophores (such as xanthenes, etc.) can avoid the above problems and effectively improve the resolution and signal-to-noise ratio of carboxylesterase fluorescence detection and imaging. However, most of these probes have obvious disadvantages, such as small Stokes shift and single-channel imaging, which may lead to strong autofluorescence and poor fluorescence signal-to-noise ratio. These defects may seriously affect the detection and imaging of carboxylesterase in vitro and in vivo. Therefore, there is an urgent need for a new carboxylesterase probe that can successfully overcome these shortcomings and has the ability to distinguish the levels of carboxylesterase in different organelles. Summary of the invention

[0004] The present invention aims at the deficiencies of the above-mentioned prior art and provides a dual-targeted near-infrared ratiometric fluorescent probe and its application in the preparation of a carboxylesterase detection reagent. The present invention uses a xanthene group as a fluorescent matrix and successfully designs and synthesizes a fluorescent probe ZZD that specifically recognizes carboxylesterase. In addition, the quinoline salt part in the fluorescent probe ZZD enables it to target mitochondria, and the ZD structure after response can be localized in lipid droplets. The fluorescent probe ZZD shows good responsiveness, superior dual organelle targeting ability and the advantages of near-infrared emission in living cells. In addition, the fluorescent probe of the present invention has good responsiveness to carboxylesterase in both solution and cells.

[0005] The dual-target near-infrared ratiometric fluorescent probe of the present invention, abbreviated as ZZD, is based on a xanthene derivative, and its structural formula is as follows: .

[0006] The preparation method of the dual-targeting near-infrared ratiometric fluorescent probe of the present invention comprises the following steps: Step 1: Mix 30 ml of N,N-dimethylformamide (DMF) and 10 mL of dichloromethane (DCM), and keep the solution temperature at about 0 °C. Then add phosphorus tribromide dropwise at 0 °C, then remove the ice-water bath and add 10 mL of cyclohexanone dropwise. After six hours of reaction, add the reaction solution dropwise to 150 mL of water, adjust to neutral with sodium carbonate, extract with water and DCM, filter, and spin dry to obtain compound 1. No purification is required in this step, and the reaction product can be obtained by filtration.

[0007] Step 2: Add 9.4 g of compound 1, 5.3 g of 2-hydroxy-4-methoxybenzaldehyde and 17 g of potassium carbonate to 30 mL of DMF and react for 12 h, then extract with water and DCM, spin dry the solvent, slurry (ethyl acetate: petroleum ether = 1:1, v / v), and filter to obtain compound 2.

[0008] Step 3: Add 1 g of p-cresol acetate, 1.31 g of N-bromosuccinimide and 0.24 g of dibenzoyl peroxide to 15 mL of carbon tetrachloride, reflux at 80 °C for 8 h, filter, rotary evaporate and recrystallize from methanol to obtain compound 3.

[0009] Step 4: Dissolve 300 mg of compound 3 and 210 mg of tetramethylquinoline in 6 mL of acetonitrile, reflux at 80°C for 12 h, spin dry the solvent, and separate by column chromatography (methanol: dichloromethane = 1:20, v / v) to obtain compound 4.

[0010] Step 5: Add 200 mg of compound 2, 200 mg of compound 4 and two drops of piperidine to 5 mL of ethanol, reflux at 80°C for 12 h, spin dry the solvent, slurry with ethyl acetate, and filter to obtain probe ZZD.

[0011] The synthetic route is as follows:

[0012] Application of the dual-targeted near-infrared ratiometric fluorescent probe of the present invention in the preparation of a carboxylesterase detection reagent.

[0013] The detection reagent can detect endogenous and / or exogenous carboxylesterase in cells.

[0014] In addition, the quinolyl salt part of the fluorescent probe ZZD enables it to target mitochondria, and the responsive ZD structure can be localized to lipid droplets. The fluorescent probe ZZD shows good responsiveness, superior dual organelle targeting ability and the advantages of near-infrared emission in living cells. We expect that the fluorescent probe ZZD will become a useful fluorescent tool for studying lipid droplet and mitochondrial-related diseases in the future.

[0015]

[0016] The detection method is as follows: The fluorescent probe ZZD of the present invention was dissolved in 5 mL of DMSO to prepare a 2 mM mother solution, and 15 μL of the stock solution was added to 3 mL of DMSO / HEPES (1:9, v / v) buffer (10 mM, pH=7.4) to obtain a 10 μM test solution of ZZD. A fluorescence spectrometer was used to collect three-dimensional fluorescence signals, with the excitation wavelength set to 375 to 400 nm and a scan rate of 5 nm. The emission wavelength range was 425 to 825 nm, with a step length of 5 nm. The excitation and emission slit widths were both 3 nm. Three-dimensional fluorescence detection was performed on samples of ZZD and ZZD+carboxylesterase. The UV-vis absorption spectrum and fluorescence emission spectrum obtained in a 10 mM HEPES buffer solution (containing 10% DMSO, pH=7.4) were used to detect the spectral properties of ZZD. In the reaction system, after the addition of carboxylesterase, the absorption spectrum and fluorescence emission spectrum of ZZD both showed obvious ratio responses. As the concentration of carboxylesterase increases, the UV-visible absorption of ZZD at 600 nm gradually decreases. In contrast, the absorption in the visible region (420 nm) gradually increases ( Figure 1 a). As the concentration of carboxylesterase increases, the emission peak at 720 nm gradually decreases (λex=600 nm), while the emission peak at 510 nm (λex=420 nm) gradually increases ( Figure 1 bc). The ratio of the fluorescence intensity at 510 nm to the fluorescence intensity at 720 nm (I 510 / I 720 ) shows a good linear relationship with a correlation coefficient of R 2 =0.99,( Figure 1d). It is noteworthy that the absorption and fluorescence emission spectra of ZZD in the physiological carboxylesterase concentration range (0-0.9 U / mL) changed significantly, making the probe suitable for ratiometric fluorescence imaging. To further investigate the recognition performance and mechanism of ZZD, we performed LC-MS characterization on ZZD, ZZD+carboxylesterase, and ZD groups, respectively ( Figure 2 ), the results showed that a new peak appeared in ZZD+carboxylesterase, and the peak time and molecular weight were consistent with those of the ZD peak.

[0017] In addition, we also studied the anti-interference ability of ZZD to other common species in physiological environments. Figure 3 As shown in a, ZZD exhibits ultra-high selectivity for carboxylesterase, and the effects of other species are basically negligible. In addition, the fluorescence of ZZD is basically unaffected at pH values ​​of 7.50-8.25 ( Figure 3 b) and the change in temperature has little effect on the activity of carboxylesterase ( Figure 3 c).

[0018] The present invention discloses a dual-targeted near-infrared ratiometric fluorescent probe for detecting carboxylesterase, which has good response to carboxylesterase in both solution and cells. Compared with the prior art, we successfully designed and synthesized the fluorescent probe ZZD that specifically recognizes carboxylesterase using the xanthene group as the fluorescent matrix. In addition, the quinoline salt part in the fluorescent probe ZZD enables it to target mitochondria, and the ZD structure after response can be localized in lipid droplets. The fluorescent probe ZZD shows good response ability, superior dual organelle targeting ability and the advantages of near-infrared emission in living cells. We expect that the fluorescent probe ZZD will become a useful fluorescent tool for studying lipid droplet and mitochondrial related diseases in the future. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is the ability of ZZD to respond to carboxylesterase. Where: (a) is the ultraviolet absorption spectrum of ZZD; (b) is the fluorescence emission spectrum of ZZD at 510 nm; (c) is the fluorescence emission spectrum of ZZD at 720 nm; (d) is the linear relationship between the fluorescence intensity ratio of ZZD and the concentration of carboxylesterase.

[0020] Figure 2 This is the probe recognition mechanism of ZZD. Among them: (a) is the LC-MS of probe ZZD; (b) is the LC-MS of ZZD+carboxylesterase; (c) is the LC-MS of ZD.

[0021] Figure 3It is a test of the selectivity and stability of the ZZD probe. Among them: (a) is the selectivity experiment of the probe ZZD; (b) is the pH stability of the probe ZZD; (c) is the temperature stability of the probe ZZD.

[0022] Figure 4 This is a probe co-localization test of ZZD. Among them: (a) is the co-localization image of HepG2 cells cultured with ZZD and Mito-Tracker Deep Red (MTDR); (b) is the intensity distribution of Mito-Tracker Deep Red (MTDR) and ZZD in the linear ROI in the merged (R+G) image. (c) is the co-localization image of HepG2 cells cultured with ZZD and Nile Red; (d) is the intensity distribution of Nile Red and ZZD in the linear ROI area in the merged (G+R) image.

[0023] Figure 5 This is a confocal fluorescence imaging diagram of the probe ZZD responding to exogenous carboxylesterase in cells. Wherein: (a) is the imaging of HepG2 cells with carboxylesterase added; (b) is the fluorescence intensity of the green channel and the red channel in (a).

[0024] Figure 6 The confocal fluorescence imaging diagram of the response of ZZD probe to endogenous carboxylesterase in cells, wherein: (a) is the imaging of HepG2 cells with AEBSF added; (b) is the fluorescence intensity of the green channel and the red channel in (a).

[0025] Figure 7 This is the confocal fluorescence imaging of lipid droplets and mitochondria in HepG2 cells under starvation state using probe ZZD. Among them: (a) is the imaging of lipid droplets and mitochondria in HepG2 cells under starvation state; (b) is the size and number of lipid droplets in (a); (c) is the average branch length of mitochondria in (a); (d) is the fluorescence intensity in (a). DETAILED DESCRIPTION

[0026] The present invention will be further described below by way of examples.

[0027] Example 1: Synthesis of ZZD Compound 2 (200 mg, 0.8 mmol), compound 4 (200 mg, 0.5 mmol) and two drops of piperidine were added to 5 mL of ethanol, refluxed at 80 °C for 12 h, the solvent was spin-dried, slurried with ethyl acetate, and filtered to obtain probe ZZD.

[0028] 1H NMR (400 MHz, DMSO-d6) δ 9.21 (d, J = 6.8 Hz, 1H), 8.81 (d, J =8.6 Hz, 1H), 8.57 (d, J = 15.0 Hz, 1H), 8.25 (d, J = 8.9 Hz, 1H), 8.04 (t, J= 8.1 Hz, 1H), 7.81 (t, J = 7.8 Hz, 1H), 7.40 (d, J = 8.3 Hz, 2H), 7.33 (d, J= 15.1 Hz, 1H), 7.19 – 7.14 (m, 2H), 7.12 (d, J = 2.5 Hz, 1H), 6.91 (s, 1H),6.76 (dd, J = 8.5, 2.5 Hz, 1H), 6.15 (s, 2H), 3.85 (s, 3H), 2.70 (t, J = 6.1Hz, 2H), 2.58 (t, J = 6.1 Hz, 2H), 2.24 (s, 3H), 1.86 – 1.70 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δ 175.19, 169.69, 161.76, 155.29, 154.19, 153.47, 150.93,146.59, 138.94, 138.45, 135.15, 132.83, 128.90, 127.54, 126.56, 123.15,122.93, 115.53, 114.58, 113.28, 111.81, 101.16, 58.08, 56.96, 29.90, 24.44,21.35, 20.77. FT-MS m / z: [M-Br] +: C34H30NO4 + calcd., 516.2169; found, 516.2181.

[0029] Example 2: Spectral test of ZZD Spectral analysis was performed in 10 mM HEPES buffer solution (containing 10% DMSO, pH=7.4) at room temperature. The concentration of probe ZZD used for UV-visible and fluorescence measurements was 10 μM. The mother solutions of metal ions, anions, ROS, amino acids, and enzymes were dissolved in 20 mM ultrapure water. The mother solutions of enzymes were stored at 37°C to ensure the activity of the enzymes. All mother solutions were prepared as needed. Probe ZZD, analyte, and PBS were added to the cuvette in sequence, and UV and fluorescence titration tests were performed after the mixture stabilized. First, we tested the changes in the UV-visible absorption spectra of ZZD after a series of carboxylesterase responses. As the concentration of carboxylesterase increased, the UV-visible absorption of ZZD at 600 nm gradually decreased. In contrast, the absorption in the visible light region (420 nm) gradually increased ( Figure 1 a). Figure 1 As shown in Figure 1b and 1c, as expected, with the increase of carboxylesterase concentration, the emission peak at 720 nm gradually decreased (λex = 600 nm), while the emission peak at 510 nm (λex = 420 nm) gradually increased. Figure 1 d shows the ratio of the fluorescence intensity at 510 nm to the fluorescence intensity at 720 nm (I 510 / I 720 ) showed a good linear relationship, with a correlation coefficient of R 2 =0.99.

[0030] Example 3: Probe recognition mechanism of ZZD The three groups of ZZD, ZZD+carboxylesterase and ZD were characterized by LC-MS. Figure 2 ), the results showed that a new peak appeared in ZZD+carboxylesterase, and the peak time and molecular weight were consistent with those of the ZD peak.

[0031] Example 4: Probe selectivity and stability test of ZZD The intracellular environment is relatively complex and contains a variety of anions and cations, biothiols, amino acids, and enzymes. To ensure that ZZD can accurately detect carboxylesterase, we conducted exclusion experiments to determine whether the various anions and cations, biothiols, amino acids, and enzymes in the cell would cause interference before ZZD entered the cell for imaging. The fluorescent probe ZZD was used to measure a variety of analytes, including 1. Mg 2+ ; 2. Na + ; 3. Cl - ; 4. SO 4 2- ; 5. Glycine; 6. Cysteine; 7. Homocysteine; 8. Glutathione; 9. ClO - 10. H 2 O 2; 11. Bovine serum albumin; 12. Glutamyl transferase; 13. Acetylcholinesterase; 14. Leucine aminopeptidase; 15. Nitroreductase; 16. Carboxylesterase. Analysis results ( Figure 3 a) ZZD has a significant ratio fluorescence attenuation effect on carboxylesterase, while the fluorescence changes of various analytes such as anions and cations, biothiols, amino acids and enzymes are negligible. These results prove that various analytes such as anions and cations, biothiols, amino acids and enzymes have no effect on the fluorescent probe ZZD, and the fluorescent probe ZZD shows super high selectivity for carboxylesterase.

[0032] At the same time, the pH stability of the probe was tested ( Figure 3 b). We tested the probes in 3 mL buffer solutions of DMSO / HEPES (1:9, v / v) at pH=6.5-8.25. The fluorescent probe ZZD showed no significant changes between pH=7.50-8.25. On the contrary, the fluorescent probe ZZD showed a sudden increase in response effect at pH=6.50-7.00 after the response. This may be due to the protonation of the nitrogen atom on the quinoline group after the fluorescent probe responded, while there was no significant change between pH=7.50-8.25. The results show that the fluorescent probe has good stability in the weak alkaline environment of pH=7.50-8.25 before and after the response. At the same time, the mitochondria of the cells are also in a weak alkaline environment. Our fluorescent probe can be well used for the detection of carboxylesterase in cells.

[0033] To explore the temperature sensitivity of carboxylesterase, we incubated carboxylesterase at 30, 35, 37, 40, and 45°C for 10 min and then added the probe-containing test solution to quantify the activity ( Figure 3 c). When the temperature dropped from 37°C to 30°C, the dual-channel fluorescence intensity ratio of the fluorescent probe ZZD increased slightly. At the same time, when the temperature increased from 37°C to 45°C, the fluorescence intensity ratio of the fluorescent probe ZZD also increased slightly. These results indicate that the effect of temperature change on the activity of carboxylesterase is negligible.

[0034] Example 5: ZZD probe co-localization test In order to determine whether the fluorescent probe ZZD can simultaneously target two organelles, mitochondria and lipid droplets, we used the mitochondrial commercial dye Mito-Tracker Deep Red (MTDR) and probe ZZD to perform mitochondrial colocalization experiments and co-incubated with the lipid droplet commercial dye Nile Red, with colocalization coefficients of 0.89 and 0.93, respectively. These results together indicate that the fluorescent probe ZZD can simultaneously target lipid droplets and mitochondria after entering the cell, without crosstalk between them, and the analysis of mitochondrial-lipid droplet dual localization morphology is helpful to study the interaction between the two.

[0035] Example 6: Response of probe ZZD to exogenous carboxylesterase The ability of ZZD to respond to carboxylesterase has been clearly verified in the solution, and the corresponding study was then carried out in cells. The control group only added probe ZZD, and the experimental group added probe ZZD and carboxylesterase. After the two groups of experiments were incubated together for 30 minutes, the excess fluorescent probe ZZD and carboxylesterase were washed away with PBS, and new culture medium was added. Figure 5 As shown, in the cells of the Control group, strong fluorescence in the red channel and weak fluorescence in the green channel can be clearly seen. Then, the experimental group enhanced the response of the probe ZZD in the cells, and it can be clearly seen that the corresponding fluorescence images in the red channel and green channel have changed significantly. Compared with the Control group, the fluorescence intensity of the red channel is weakened, while the fluorescence intensity of the green fluorescence channel is enhanced, confirming the response of ZZD to exogenous carboxylesterase in cells.

[0036] Example 7: Response of probe ZZD to endogenous carboxylesterase in cells We also conducted related research on endogenous carboxylesterase in cells. The control group only added probe ZZD, and the experimental group first added serine protease inhibitor (AEBSF) for incubation, then added probe ZZD for incubation, washed off the excess fluorescent probe ZZD with PBS, and then added new culture medium. Figure 6 As shown, in the cells of the Control group, strong fluorescence in the red channel and weak fluorescence in the green channel can be clearly seen. Then, the experimental group weakened the response of the probe ZZD in the cells, and it can be clearly seen that the corresponding fluorescence images in the red channel and green channel showed obvious changes. The fluorescence intensity of the red channel was enhanced compared with the Control group, while the fluorescence intensity of the green fluorescence channel was weakened, confirming the response of ZZD to exogenous carboxylesterase in cells.

[0037] Example 8: Dynamic changes of organelles during starvation Cells undergo autophagy when they are in a starving environment, and the autophagic process consumes energy-providing lipid droplets. Cells shift their metabolism from glucose metabolism to mitochondrial fatty acid (FA) oxidation. FA released from lipid droplets through lipolysis or lipophagy are oxidized by mitochondria as substrates for β-oxidation and the citric acid cycle, a process that requires lipid droplets to be close to mitochondria to be stimulated. However, the kinetic mechanism of energy transport from lipid droplets to mitochondria has not been widely studied. Therefore, visualizing the kinetic interaction between lipid droplets and mitochondria during starvation is crucial for understanding lipid metabolism and energy homeostasis. We starved HepG2 cells for 0 h, 3 h, and 6 h, respectively, to study the dynamic changes of lipid droplets and mitochondria during starvation induction. As Figure 7 As shown, the fluorescent probe ZZD is used in HepG2 cells in two channels, green and red, to mark lipid droplets and mitochondria, respectively. After starvation treatment, the intensity of green fluorescence (lipid droplets) in cells decreased significantly, indicating that lipid droplets were significantly consumed during starvation induction. In contrast, during starvation, the intensity of the mitochondrial red channel did not change significantly, mainly because lipid droplets provide energy for cells in a starving state, while mitochondria obtain energy. This also proves that the close contact between lipid droplets and mitochondria contributes to the efficient transfer of energy from lipid droplets to mitochondria.

Claims

1. A dual-target near-infrared ratiometric fluorescent probe, abbreviated as ZZD, characterized in that Its structural formula is as follows: 。 2. The method for preparing the dual-targeting near-infrared ratiometric fluorescent probe according to claim 1, characterized in that The steps include: Step 1: In an ice-water bath, N,N-dimethylformamide and dichloromethane were mixed, and then phosphorus tribromide was added dropwise, and then cyclohexanone was added dropwise after removing the ice-water bath. After reacting for six hours, the reaction solution was added dropwise to water, and then adjusted to neutral with sodium carbonate, extracted with water and DCM, filtered, and dried to obtain compound 1; Step 2: Compound 1, 2-hydroxy-4-methoxybenzaldehyde and potassium carbonate were added to DMF for reaction, and then extracted with water and DCM, the solvent was spin-dried, slurried, and filtered to obtain compound 2; Step 3: Add p-cresol acetate, N-bromosuccinimide and dibenzoyl peroxide to carbon tetrachloride, reflux for 8 h, filter, rotary evaporate, and recrystallize from methanol to obtain compound 3; Step 4: Compound 3 and tetramethylquinoline were dissolved in acetonitrile, refluxed for 12 h, the solvent was dried, and separated by column chromatography to obtain compound 4; Step 5: Compound 2, compound 4 and piperidine were added to ethanol, refluxed for 12 h, the solvent was spin-dried, slurried with ethyl acetate, and filtered to obtain probe ZZD; The synthetic route is as follows: 。 3. Use of the dual-targeted near-infrared ratiometric fluorescent probe according to claim 1 in the preparation of a carboxylesterase detection reagent.

4. The use according to claim 3, characterized in that: The detection reagent can detect endogenous and / or exogenous carboxylesterase in cells.

5. The use according to claim 3, characterized in that: The quinolyl salt part in the fluorescent probe ZZD enables it to target mitochondria, and the ZD structure after response can be localized in lipid droplets; 。 6. The use according to claim 4, characterized in that: The ratio of the fluorescence intensity of the fluorescent probe ZZD at 510 nm to the fluorescence intensity at 720 nm is 510 / I 720 It is linearly related to the concentration of carboxylesterase.

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