A dual-targeting near-infrared ratio fluorescent probe and application thereof in preparation of carboxylesterase detection reagent

Abstract

The application discloses a double-targeting near-infrared ratio fluorescent probe and application thereof in preparation of a carboxylesterase detection reagent, wherein the structure of the double-targeting near-infrared ratio fluorescent probe is shown in the following formula: the quinoline salt part in the fluorescent probe ZZD can target mitochondria, and the ZD structure after response can be positioned at a lipid droplet. The fluorescent probe ZZD exhibits good response capacity, superior double-organellar targeting capacity and the advantage of near-infrared emission in living cells. It is expected that the fluorescent probe ZZD will become a useful fluorescent tool for studying lipid droplet and mitochondria related diseases in the future.

Description

Technical Field

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

[0002] Carboxylesterases, members of the serine esterase family, participate in numerous physiological processes and play a key role in homeostasis and metabolic defense systems. They also play important roles in catalyzing the degradation of various endogenous substrates, detoxifying clinical drugs and environmental toxins, and in the metabolic activation of anticancer prodrugs. Importantly, when carboxylesterases are overexpressed in the liver, they are highly associated with diseases such as obesity, diabetes, alcoholic fatty liver disease, and cancer. Furthermore, while esterification can increase the lipophilicity of anticancer drugs and improve membrane permeability, carboxylesterase activity is crucial for the release of the pharmacologically active components of these ester prodrugs, making them useful for cancer therapy. Currently, carboxylesterase activity is primarily studied indirectly through the detection of related proteins. However, this method does not provide information on carboxylesterase activity levels and is more complex to study. Furthermore, only a few methods can investigate carboxylesterase activity in living cells. For example, chromatography, often combined with mass spectrometry, can be used to study endogenous or overexpressed carboxylesterases. However, this method remains 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 rapid development of fluorescence sensing technology, carboxylesterase activity detection techniques have also undergone continuous innovation and development, resulting in the development of numerous fluorescent probes with high sensitivity, specificity, and high fidelity. Near-infrared (NIR) fluorescent probes, in particular, offer advantages such as strong tissue penetration and minimal tissue damage, holding great promise for in vivo research. However, carboxylesterase activity detection and imaging probes based on traditional sensing platforms still face significant challenges, including interference from biological background signals, limited excitation light scattering, and low tissue penetration. Therefore, NIR probes based on traditionally superior fluorophores (such as xanthenes) can overcome these challenges and effectively improve the resolution and signal-to-noise ratio of carboxylesterase fluorescence detection and imaging. However, most of these probes have significant drawbacks, such as small Stokes shifts and single-channel imaging, which can lead to strong autofluorescence and poor fluorescence signal-to-noise ratio. These limitations can severely impact the detection and imaging of carboxylesterases both in vitro and in vivo. Therefore, there is an urgent need for novel carboxylesterase probes that can successfully overcome these drawbacks and possess the ability to distinguish carboxylesterase levels in different organelles. Summary of the Invention

[0004] The present invention addresses the deficiencies of the above-mentioned prior art and provides a dual-targeted near-infrared ratiometric fluorescent probe and its use in the preparation of a carboxylesterase detection reagent. Using a xanthene group as the fluorescent precursor, the present invention successfully designed and synthesized a fluorescent probe, ZZD, that specifically recognizes carboxylesterases. Furthermore, the quinolate moiety in the fluorescent probe ZZD enables it to target mitochondria, while the ZD structure after response can localize to lipid droplets. The fluorescent probe ZZD exhibits excellent responsiveness, superior dual-organelle targeting, and the advantages of near-infrared emission in living cells. Furthermore, the fluorescent probe of the present invention exhibits good responsiveness to carboxylesterases both in solution and in cells.

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

[0006] .

[0007] The preparation method of the dual-targeting near-infrared ratiometric fluorescent probe of the present invention comprises the following steps:

[0008] Step 1: Mix 30 mL of N,N-dimethylformamide (DMF) with 10 mL of dichloromethane (DCM), maintaining the solution temperature around 0°C. Phosphorus tribromide was then added dropwise at 0°C. After removing the ice-water bath, 10 mL of cyclohexanone was added dropwise. After six hours of reaction, the reaction mixture was added dropwise to 150 mL of water, adjusted to neutrality with sodium carbonate, and extracted with water and DCM. The mixture was filtered and dried to yield Compound 1. Purification is not required in this step; the product is obtained by filtration.

[0009] Step 2: 9.4 g of compound 1, 5.3 g of 2-hydroxy-4-methoxybenzaldehyde, and 17 g of potassium carbonate were added to 30 mL of DMF and reacted for 12 h. The mixture was then extracted with water and DCM, the solvent was dried, and the mixture was slurried (ethyl acetate: petroleum ether = 1:1, v / v) and filtered to obtain compound 2.

[0010] 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.

[0011] 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.

[0012] 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.

[0013] The synthetic route is as follows:

[0014]

[0015] The invention discloses an application of a dual-targeted near-infrared ratiometric fluorescent probe in the preparation of a carboxylesterase detection reagent.

[0016] The detection reagent can detect endogenous and / or exogenous carboxylesterase in cells.

[0017] Furthermore, the quinolyl salt moiety of the fluorescent probe ZZD enables its targeting to mitochondria, while the responsive ZD structure localizes to lipid droplets. The fluorescent probe ZZD exhibits excellent responsiveness in living cells, superior dual-organelle targeting, and the advantages of near-infrared emission. We anticipate that the fluorescent probe ZZD will become a useful fluorescent tool for future studies of lipid droplet- and mitochondrial-related diseases.

[0018]

[0019] The detection method is as follows:

[0020] The fluorescent probe ZZD of the present invention was dissolved in 5 mL of DMSO to prepare a 2 mM stock solution. 15 μL of this stock solution was added to 3 mL of 10 mM DMSO / HEPES (1:9, v / v) buffer (pH 7.4) to obtain a 10 μM test solution of ZZD. Three-dimensional fluorescence signals were collected using a fluorescence spectrometer with an excitation wavelength of 375 to 400 nm and a scan rate of 5 nm. The emission wavelength range was 425 to 825 nm with a step size of 5 nm. Both the excitation and emission slit widths were 3 nm. Three-dimensional fluorescence detection was performed on ZZD and ZZD + carboxylesterase samples. The spectral properties of ZZD were determined using UV-visible absorption and fluorescence emission spectra obtained in 10 mM HEPES buffer (containing 10% DMSO, pH 7.4). Upon addition of carboxylesterase to the reaction system, both the absorption and fluorescence emission spectra of ZZD exhibited a clear ratiometric response. As the concentration of carboxylesterase increased, the UV-visible absorption of ZZD at 600 nm gradually decreased. In contrast, the absorption in the visible region (420 nm) gradually increased ( 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 1bc). 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,( Figure 1 d). It is noteworthy that the absorption and fluorescence emission spectra of ZZD changed significantly within the physiological carboxylesterase concentration range (0-0.9 U / mL), 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 ( 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.

[0021] 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 effect of temperature change on the activity of carboxylesterase is negligible ( Figure 3 c).

[0022] The present invention discloses a dual-targeted near-infrared ratiometric fluorescent probe for detecting carboxylesterase, which has good responsiveness to carboxylesterase in both solution and cells. Compared with the existing technology, we successfully designed and synthesized the fluorescent probe ZZD, which specifically recognizes carboxylesterase, using the xanthene group as the fluorescent parent. In addition, the quinolyl salt portion in the fluorescent probe ZZD enables it to target mitochondria, and the ZD structure after response can be localized to lipid droplets. The fluorescent probe ZZD exhibits 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 future research on lipid droplet and mitochondrial-related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is the ability of ZZD to respond to carboxylesterase. (a) is the UV 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; and (d) is the linear relationship between the fluorescence intensity ratio of ZZD and the concentration of carboxylesterase.

[0024] Figure 2The probe recognition mechanism of ZZD. (a) LC-MS of probe ZZD; (b) LC-MS of ZZD + carboxylesterase; (c) LC-MS of ZD.

[0025] Figure 3 The following are the selectivity and stability tests of ZZD probes: (a) the selectivity test of ZZD probe; (b) the pH stability of ZZD probe; and (c) the temperature stability of ZZD probe.

[0026] Figure 4 This is a colocalization test of ZZD probes. (a) Colocalization image of HepG2 cells cultured with ZZD and Mito-Tracker Deep Red (MTDR); (b) Intensity distribution of Mito-Tracker Deep Red (MTDR) and ZZD within a linear ROI in the merged (R+G) image. (c) Colocalization image of HepG2 cells cultured with ZZD and Nile Red; (d) Intensity distribution of Nile Red and ZZD within a linear ROI in the merged (G+R) image.

[0027] Figure 5 These are confocal fluorescence images of ZZD probe ZZD in response to exogenous carboxylesterase in cells. (a) shows the imaging of HepG2 cells in which carboxylesterase was added; (b) shows the fluorescence intensity of the green and red channels in (a).

[0028] Figure 6 The confocal fluorescence images of ZZD probe ZZD in response to endogenous carboxylesterase in cells are shown in Figure 1. (a) is the image of HepG2 cells incubated with AEBSF; (b) is the fluorescence intensity of the green and red channels in (a).

[0029] Figure 7 Confocal fluorescence images of lipid droplets and mitochondria in starved HepG2 cells using probe ZZD. (a) Images of lipid droplets and mitochondria in starved HepG2 cells; (b) lipid droplet size and number in (a); (c) average mitochondrial branch length in (a); and (d) fluorescence intensity in (a). DETAILED DESCRIPTION

[0030] The present invention will be further described below by way of examples.

[0031] Example 1: Synthesis of ZZD

[0032] 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 and refluxed at 80°C for 12 h. The solvent was spin-dried, slurried with ethyl acetate, and filtered to obtain probe ZZD.

[0033] 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.

[0034] Example 2: Spectral Test of ZZD

[0035] Spectral analysis was carried out in a 10 mM HEPES buffer solution (containing 10% DMSO, pH = 7.4) at room temperature. The concentration of the probe ZZD for ultraviolet-visible light and fluorescence measurement was 10 μM. The stock solutions of metal ions, anions, ROS, amino acids and enzymes were all dissolved in 20 mM ultrapure water. The stock solution of the enzyme was stored at 37°C to ensure the activity of the enzyme. All stock solutions were prepared as needed. In the cuvette, the probe ZZD, analyte and PBS were added in turn, and after the mixture was stable, ultraviolet and fluorescence titration tests were carried out. First, we tested the ultraviolet-visible absorption spectrum changes of ZZD after a series of carboxylic esterase responses. With the increase of carboxylic esterase concentration, the ultraviolet-visible absorption of ZZD at 600 nm gradually decreased. On the contrary, the absorption in the visible region (420 nm) gradually increased Figure 1 a). As shown in Figure 1 b and 1 c, as expected, with the increase of carboxylic esterase 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 that the ratio of fluorescence intensity at 510 nm to fluorescence intensity at 720 nm (I 510 / I 720 ) shows a good linear relationship, with a correlation coefficient of R 2 =0.99.

[0036] Example 3: Probe recognition mechanism of ZZD

[0037] LC-MS characterization was carried out on ZZD, ZZD+carboxylic esterase and ZD three groups respectively by LC-MS Figure 2 ), and the results showed that new peaks appeared in ZZD+carboxylic esterase, and the peak time and molecular weight were consistent with those of ZD.

[0038] Example 4: Probe selectivity and stability test of ZZD

[0039] The intracellular environment is relatively complex, and there are various anions and cations, biological thiols, amino acids and enzymes. In order to ensure that ZZD can accurately detect carboxylic esterase, we carried out an exclusion experiment to exclude whether various anions and cations, biological thiols, amino acids and enzymes in the cell would interfere with ZZD before it entered the cell for imaging. The fluorescent probe ZZD was used to measure various analytes, including 1. Mg 2+ ; 2. Na + ; 3. Cl - ; 4. SO4 2- ; 5. glycine; 6. cysteine; 7. homocysteine; 8. glutathione; 9. ClO -; 10. H2O2; 11. Bovine serum albumin; 12. Glutamyltransferase; 13. Acetylcholinesterase; 14. Leucine aminopeptidase; 15. Nitroreductase; 16. Carboxylesterase. Analysis results ( Figure 3 a) ZZD exhibits a significant ratiometric fluorescence reduction effect on carboxylesterase, while the fluorescence changes for various analytes, including anions and cations, biothiols, amino acids, and enzymes, are negligible. These results demonstrate that analytes such as anions and cations, biothiols, amino acids, and enzymes have no effect on the fluorescent probe ZZD, which exhibits exceptional selectivity for carboxylesterase.

[0040] At the same time, the pH stability of the probe was tested ( Figure 3 b). We tested the probe in 3 mL of DMSO / HEPES (1:9, v / v) buffer solutions with pH values ​​ranging from 6.5 to 8.25. The fluorescent probe ZZD showed no significant changes between pH 7.50 and 8.25. In contrast, the fluorescent probe ZZD showed a significant, sudden increase in response at pH 6.50 to 7.00. This is likely due to the protonation of the nitrogen atom on the quinoline group after the fluorescent probe's response, while there was no significant change between pH 7.50 and 8.25. The results indicate that the fluorescent probe maintains good stability in a weakly alkaline environment of pH 7.50 to 8.25, both before and after its response. Furthermore, mitochondria are also in a weakly alkaline environment, making our fluorescent probe well suited for the detection of carboxylesterase in cells.

[0041] To explore the temperature sensitivity of carboxylesterase, we incubated the carboxylesterase at 30, 35, 37, 40, and 45°C for 10 minutes, and then added the test solution containing the probe 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 showed a slight increase. Similarly, when the temperature rose from 37°C to 45°C, the fluorescence intensity ratio of the fluorescent probe ZZD also showed a slight increase. These results indicate that temperature fluctuations have negligible effects on carboxylesterase activity.

[0042] Example 5: ZZD probe co-localization test

[0043] To determine whether the fluorescent probe ZZD can simultaneously target both mitochondria and lipid droplets, we performed mitochondrial colocalization experiments using the commercial mitochondrial dye Mito-Tracker Deep Red (MTDR) and probe ZZD, as well as co-incubation with the commercial lipid droplet dye Nile Red. The colocalization coefficients were 0.89 and 0.93, respectively. These results demonstrate that the fluorescent probe ZZD can simultaneously target lipid droplets and mitochondria after entering cells, without crosstalk between the two. Analysis of mitochondrial-lipid droplet dual localization morphology facilitates the study of interactions between the two.

[0044] Example 6: Response of probe ZZD to exogenous carboxylesterase

[0045] The ability of ZZD to respond to carboxylesterase has been clearly verified in 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 incubating the two groups of experiments 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 control group, cells clearly showed strong fluorescence in the red channel and weak fluorescence in the green channel. Next, the experimental group enhanced the intracellular response of the probe ZZD, and significant changes were observed in the corresponding fluorescence images of the red and green channels. Compared with the control group, the red channel fluorescence intensity decreased, while the green channel fluorescence intensity increased, confirming the response of ZZD to exogenous carboxylesterase in the cells.

[0046] Example 7: Response of probe ZZD to endogenous carboxylesterase

[0047] We also conducted related research on endogenous carboxylesterase in cells. The control group only added probe ZZD, while the experimental group first added serine protease inhibitor (AEBSF) for incubation, then added probe ZZD for incubation, washed away the excess fluorescent probe ZZD with PBS, and then added new culture medium. Figure 6 As shown, cells in the control group clearly show strong fluorescence in the red channel and weak fluorescence in the green channel. Next, the experimental group weakened the intracellular response of the probe ZZD, and significant changes were observed in the corresponding fluorescence images of the red and green channels. Compared to the control group, the red channel fluorescence intensity increased, while the green channel fluorescence intensity decreased, confirming the response of ZZD to exogenous carboxylesterase in the cells.

[0048] Example 8: Dynamic changes in organelles during starvation

[0049] Cells undergo autophagy in a starved environment, however, autophagy process consumes the lipid droplets that provide energy. Cells shift their metabolism from glucose metabolism to mitochondrial fatty acid (FA) oxidation. The FAs released from lipid droplets by lipolysis or lipophagy are oxidized by mitochondria as substrates for beta oxidation and citric acid cycle, which requires the lipid droplets to be close to mitochondria to trigger. However, the energy transport kinetics mechanism from lipid droplets to mitochondria has not been extensively studied. Therefore, visualizing the kinetic interaction between lipid droplets and mitochondria during starvation is crucial for understanding lipid metabolism and energy homeostasis. We treated HepG2 cells with 0 h, 3 h, 6 h starvation, respectively, to study the dynamic changes of lipid droplets and mitochondria during starvation induction. As shown in FIG. 1A, the fluorescent probe ZZD was labeled in green and red channels to mark lipid droplets and mitochondria in HepG2 cells, respectively. After starvation treatment, the green fluorescence (lipid droplets) intensity in cells decreased significantly, indicating that lipid droplets were significantly consumed during starvation induction. Unlike this, the red channel intensity of mitochondria did not change significantly during starvation, which is mainly because lipid droplets provide energy for starved cells, and mitochondria obtain energy. This also proves that the close contact of lipid droplets with mitochondria helps efficient energy transfer from lipid droplets to mitochondria. Figure 7 As shown in FIG. 1A, the fluorescent probe ZZD was labeled in green and red channels to mark lipid droplets and mitochondria in HepG2 cells, respectively. After starvation treatment, the green fluorescence (lipid droplets) intensity in cells decreased significantly, indicating that lipid droplets were significantly consumed during starvation induction. Unlike this, the red channel intensity of mitochondria did not change significantly during starvation, which is mainly because lipid droplets provide energy for starved cells, and mitochondria obtain energy. This also proves that the close contact of lipid droplets with mitochondria helps efficient energy transfer from lipid droplets to mitochondria.

Claims

1. A dual-targeted near-infrared ratiometric fluorescent probe, abbreviated as ZZD, characterized in that Its structural formula is shown below: 。 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. The ice-water bath was then removed and cyclohexanone was added dropwise. After reacting for six hours, the reaction solution was added dropwise to water, and then adjusted to neutral with sodium carbonate. The mixture was 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, followed by extraction with water and DCM. The solvent was dried, the mixture was 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 and 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 portion of 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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