Lysosome targeted two-photon fluorescent probe and application thereof in beta-galactosidase detection

Abstract

The invention discloses a lysosome targeted two-photon fluorescent probe and application thereof in beta-galactosidase detection. The structure of the fluorescent probe is shown as follows: # imgabs0 #. The two-photon fluorescent probe can respond to beta-galactosidase (beta-gal) with different concentrations, and the detection limit is as low as 0.16 U / L. A cytotoxicity test shows that the probe is good in biocompatibility, a confocal fluorescence microscopic imaging experiment shows that the probe is good in cell light stability, has the capacity of monitoring endogenous and exogenous beta-gal concentration changes of cells, has lysosome targeting capacity (R2 = 0.89), and can be used for detecting the endogenous and exogenous beta-gal concentration changes of the cells. The method is suitable for confocal fluorescence imaging of beta-gal concentration change tracing in cytolysosome.

Description

Technical Field

[0001] The present invention relates to a lysosome-targeted two-photon fluorescent probe for detecting beta-galactosidase and its biological application, so as to realize the concentration detection of beta-galactosidase in solution and the fluorescent tracing of the concentration change of beta-galactosidase in the lysosome of living cells, and has the advantages of selectivity, high detection efficiency and good biocompatibility. Background Art

[0002] Lysosomes are key platforms for cells to regulate homeostasis and are widely present in various biological cells. Lysosomes are the main recycling organelles of cells. They transport inactivated macromolecules, aged organelles, and extracellular substances into the lysosomal cavity through autophagy and endocytosis. They are degraded by more than 50 acid hydrolases in the lysosomes. The resulting decomposition products are recycled to the cytoplasm through specific transporters or diffusion pathways for reuse, playing an important role in a variety of biological applications. Therefore, in-depth research on the biological characteristics and functions of lysosomes is of great significance for understanding and treating these diseases.

[0003] β-gal is a glycoside hydrolase that hydrolyzes β-galactose into monosaccharides by breaking glycosidic bonds. Abnormal β-gal in cells has been shown to be an important biomarker for cell senescence and primary ovarian cancer. High levels of β-gal activity can separate the side chains of amino polysaccharides from the core protein by hydrolyzing glycosidic bonds, leading to the decomposition of macromolecular proteoglycans and damage to the basement membrane. These evidences emphasize the importance of accurate, rapid and efficient detection and monitoring of β-gal, especially in basic biological research and early cancer diagnosis.

[0004] In biological research, a variety of methods have been developed to evaluate the activity of β-gal, including colorimetry, electrochemistry, positron emission tomography (PET), magnetic resonance imaging (MRI), etc. The fluorescence imaging method based on β-gal fluorescent probe has attracted much attention because it can exclude the interference of other signal molecules, has the advantages of specific response to β-gal, high sensitivity and real-time performance. The development of a responsive β-gal two-photon fluorescent probe will provide a powerful tool for real-time imaging of intracellular β-gal fluctuations.

[0005] In recent years, fluorescence imaging methods have been widely used to monitor biological processes, but there are still very few specific imaging of β-gal, especially fluorescent probes with two-photon function. Therefore, the development of β-gal two-photon fluorescent probes that can respond will provide a powerful tool for real-time imaging of intracellular β-gal fluctuations. Summary of the invention

[0006] The present invention aims at the deficiencies of the above-mentioned prior art and provides a lysosome-targeted two-photon fluorescent probe and its application in β-galactosidase detection. The technical problem to be solved is to obtain a molecular structure with fluorescence on-response to β-gal and lysosome targeting through molecular design, and the molecular structure is required to have the advantages of high sensitivity and resolution, selectivity and good photostability for the fluorescence response to β-gal while having the ability to target lysosomes.

[0007] The present invention designs and synthesizes a two-photon β-gal fluorescent probe PEG-gal with biphenyl as a fluorophore. Dimethylaminobiphenyl is used as an electron donor and the quaternized quinoline unit is used as an electron acceptor with a strong positive charge to form a D-Π-A structure, thereby generating an intramolecular charge transfer (ICT) effect. Galactose is introduced into the phenolic site, and the fluorescent skeleton and galactose are connected using a benzyl group to obtain the probe PEG-gal. After the probe responds to β-gal deglycosylation, the benzyl part undergoes a "1,6-elimination" process, and the fluorophore is released and the fluorescence emission is enhanced at 523nm. The optical properties and cytotoxicity tests show that PEG-gal has high selectivity, low detection limit and good biocompatibility. This study evaluates the feasibility of PEG-gal for specific fluorescence imaging of β-gal in living cell lysosomes through the analysis of cell confocal fluorescence imaging, aiming to provide a convenient chemical tool for the visualization detection of β-gal in living cell lysosomes and the study of related diseases.

[0008] The lysosome-targeted two-photon fluorescent probe of the present invention is abbreviated as PEG-gal, and its structure is as follows:

[0009] .

[0010] The preparation method of the two-photon fluorescent probe of the present invention comprises the following steps:

[0011] Step 1: Under nitrogen protection, N,N-dimethyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborol-2-yl)aniline and 4-bromo-2-hydroxybenzaldehyde were dissolved in acetone, and potassium carbonate and potassium iodide were added and heated to reflux for reaction; after the reaction, the solvent was removed by distillation under reduced pressure, and after extraction and column chromatography separation (eluent was petroleum ether: ethyl acetate = 30:1, V / V), a yellow crystalline intermediate 1 was obtained;

[0012] Step 2: Under nitrogen protection, the intermediate 1 and p-toluenesulfonic acid triethylene glycol monomethyl ether ester were dissolved in acetone, potassium carbonate and potassium iodide were added, and the mixture was heated to reflux for reaction; after the reaction, the solvent was removed by distillation under reduced pressure, and yellow crystalline intermediate 2 was obtained after extraction and column chromatography separation (eluent was petroleum ether: ethyl acetate = 10:1, V / V);

[0013] Step 3: α-acetyl bromide galactose and p-hydroxybenzaldehyde were dissolved in a mixed solution of dichloromethane and sodium hydroxide aqueous solution, and then tetrabutylammonium bromide was added and reacted at 40°C for 5 h; after the reaction, the solvent was dried by spin drying, and the product was extracted with dichloromethane, and then the solvent was removed and separated by column chromatography (eluent: petroleum ether: ethyl acetate = 20:1, V / V) to obtain a white solid intermediate 3;

[0014] Step 4: Dissolve the intermediate 3 in tetrahydrofuran solution, add sodium borohydride in batches at 0°C, stir at room temperature for 4 hours, evaporate the solvent, extract and dry, separate by column chromatography (eluent: petroleum ether: ethyl acetate = 10:1, V / V) to obtain a white solid intermediate 4;

[0015] Step 5: Dissolve the intermediate 4 in a dichloromethane solution, add phosphorus tribromide in batches at 0°C, stir at room temperature for 4 hours, evaporate the solvent, extract and dry, separate by column chromatography (eluent: petroleum ether: ethyl acetate = 5:1, V / V) to obtain a white solid intermediate 5;

[0016] Step 6: Dissolve the intermediate 5 in acetonitrile solution, add 4-methylquinoline, stir at 80°C overnight, evaporate the solvent, extract and dry, separate by column chromatography (eluent: petroleum ether: ethyl acetate = 10:1, V / V) to obtain a pink solid intermediate 6;

[0017] Step 7: Dissolve intermediate 2 in ethanol solution, add intermediate 6, stir at 40°C for 4h, evaporate the solvent, extract and dry, separate by column chromatography (eluent: dichloromethane: methanol = 10:1, V / V) to obtain purple solid intermediate 7;

[0018] Step 8: Dissolve intermediate 7 in methanol solution, add sodium methoxide in batches with stirring, stir at room temperature for 30 min, evaporate the solvent, extract and dry, separate by column chromatography (eluent: dichloromethane: methanol = 6:1, V / V) to obtain purple solid PEG-gal.

[0019] The synthetic route is as follows:

[0020] .

[0021] The invention discloses an application of a two-photon fluorescent probe in preparing a beta-gal detection reagent.

[0022] The detection reagent is in I 523nm The fluorescence intensity at is linearly related to the concentration of β-gal.

[0023] The two-photon fluorescent probe PEG-gal of the present invention uses dimethylaminobiphenyl as an electron donor and quaternized quinoline unit as an electron acceptor with a strong positive charge to form a D-Π-A structure. After reacting with β-gal, the molecular structure changes to PEG, changing the original D-Π-A structure, turning on fluorescence, and having the ability to detect β-gal in a switch-type manner. The mechanism of its response to β-gal is shown as follows:

[0024]

[0025] The detection method is as follows:

[0026] The PEG-gal of the present invention was dissolved in DMF to prepare a 2 mM mother solution, and 15 μL of the mother solution was taken in 3 mL of a mixed solvent of PBS and DMF (PBS:DMF=9 / 1) containing different concentrations of β-gal to obtain the fluorescence spectrum and UV spectrum of 10 μM PEG-gal in different test solutions. As the concentration of β-gal increased, the absorbance of PEG-gal increased at 390 nm. New fluorescence emission appeared at 523 nm and the fluorescence intensity gradually increased. Moreover, the fluorescence of PEG-gal was enhanced by more than 7 times before and after the reaction with β-gal, and it had the ability of open-type fluorescence detection, and its fluorescence intensity had a good linear relationship with the concentration of β-gal (R 2 =0.99), PEG-gal can respond normally to β-gal in the physiological pH range, can respond well to β-gal in cells, and has good intracellular lysosome imaging (Pr=0.89).

[0027] The present invention provides a lysosome-targeted two-photon fluorescent probe for detecting β-galactosidase, which can respond to different concentrations of β-gal with fluorescence on, and the effective minimum detection limit is as low as 0.16 U / L. Cytotoxicity tests show that PEG-gal has good biocompatibility, and confocal fluorescence microscopy experiments show that PEG-gal has good photostability and has the ability to monitor changes in endogenous and exogenous β-gal concentrations in cells. In addition, PEG-gal has the ability to image lysosomes (R 2 =0.89), which is suitable for confocal fluorescence imaging of β-galactosidase concentration changes in cell lysosomes. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1The UV fluorescence titration and linear relationship of the response process of PEG-gal and β-gal at different reaction times and the reaction time test. (a) Changes in the UV absorption spectrum of the reaction of PEG-gal (10μM), PEG (10μM) and β-gal (300 U / L); (b) Changes in the fluorescence emission peak of the reaction of PEG-gal (10μM) and different concentrations of β-gal (0-1000 U / L); (c) After the reaction of PEG-gal (10μM) and different concentrations of β-gal (0-90 U / L), there is a good linear relationship with the concentration of β-gal (R 2 =0.9968), with a minimum detection limit of 0.16 U / L; (d) Reaction time of PEG-gal (10 μM) and β-gal (800 U / L) under different temperature conditions.

[0029] Figure 2 (a) Two-photon performance verification diagram after the reaction of PEG-gal and β-gal; (b) Two-photon absorption cross-section diagram at different excitation wavelengths after the reaction of PEG-gal and β-gal.

[0030] Figure 3 This is a pH stability test of PEG-gal. This test investigates the pH stability of PEG-gal (10 μM) after reacting with β-gal (800 U / L) for 30 minutes, and investigates whether PEG-gal is affected by the pH in the physiological range during the reaction with β-gal.

[0031] Figure 4 This is a selective test of PEG-gal. The fluorescence intensity changes after PEG-gal (10 μM) reacted with common metal ions (100 µM), reactive oxygen species (100 µM), enzymes (1000 U / L), and β-gal (800 U / L) were investigated.

[0032] Figure 5 This is a study on the co-localization of PEG-gal in lysosomal imaging. PEG-gal and 1μg / mL lysosomal deep red commercial dye (Lyso-Tracker Deep Red) were co-incubated in cells and then confocal imaging was performed to explore the lysosomal imaging ability of PEG-gal.

[0033] Figure 6 This is a test in which PEG-gal is used to detect the β-gal content in different cells (4T1, 3T3, A549, HepG2, HeLa). PEG-gal (10 μM) was incubated with different cells for 30 minutes and then confocal imaging was performed to explore the ability of PEG-gal to detect β-gal in different cells.

[0034] Figure 7 This is a test of using PEG-gal to detect endogenous β-gal. (a) 4T1 cells were induced with 50 μM doxorubicin (Dox), and then 50 μM D-glucose was added to inhibit β-gal activity. PEG-gal (10 μM) was added and incubated for 30 minutes before confocal imaging. (b) is an analysis of the fluorescence intensity of the two channels in (a) to explore the ability of PEG-gal to monitor β-gal produced in cells. DETAILED DESCRIPTION

[0035] The present invention will be further described below by way of examples.

[0036] Example 1: Synthesis of PEG-gal

[0037] Intermediate 2 (116 mg, 0.3 mmol) and intermediate 6 (116 mg, 0.2 mmol) were dissolved in ethanol solution, two drops of piperidine solution were added, stirred at 40°C for 4 h, the solvent was spun off, extracted and dried, and separated by column chromatography (eluent: dichloromethane: methanol = 10: 1, V / V) to obtain purple solid intermediate 7. Intermediate 7 (47 mg, 0.05 mmol) was dissolved in methanol solution, sodium methoxide (27 mg, 0.5 mmol) was added in batches under stirring, stirred at room temperature for 30 min, the solvent was evaporated, extracted and dried, and separated by column chromatography (eluent: dichloromethane: methanol = 6: 1) to obtain purple solid PEG-gal.

[0038] 1H NMR (400 MHz, Methanol-d4) δ 9.06 (d, J = 6.6 Hz, 1H), 8.64 (d, J =8.6 Hz, 1H), 8.26 -8.16 (m, 3H), 8.09 - 7.99 (m, 2H), 7.88 (t, J = 7.7 Hz,1H), 7.66 (d, J = 8.2 Hz, 1H), 7.49 - 7.42 (m, 2H), 7.33 - 7.25 (m, 2H), 7.14(dd, J = 9.1, 2.4 Hz, 3H), 7.06 (d, J = 1.7 Hz, 1H), 6.73 - 6.68 (m, 2H),5.94 (s, 2H), 4.87 (d, J = 7.7 Hz, 1H), 4.26 (dd, J = 5.7, 3.0 Hz, 2H), 4.02- 3.97 (m, 2H), 3.90 (d, J = 3.3 Hz, 1H), 3.82 - 3.62 (m, 10H), 3.61 - 3.49(m, 4H), 3.40 - 3.34 (m, 2H), 3.17 (s, 3H), 2.95 (s, 6H), 2.90 - 2.84 (m,1H). 13 C NMR (151 MHz, Methanol-d4) δ 160.25, 159.64, 156.12, 152.13, 147.68,146.33, 141.23, 139.51, 136.21, 132.86, 130.41, 130.07, 128.53, 128.41,127.64, 123.14, 120.25, 120.08, 119.39, 118.54, 116.46, 113.63, 110.00,102.59, 76.97, 74.75, 72.83, 72.16, 71.59, 71.57, 71.28, 70.84, 70.17, 69.10,62.41, 60.50, 59.04, 40.55.

[0039] Example 2: Optical titration, linear relationship and response time of PEG-gal in response to β-gal in solvent

[0040] To investigate the optical titration of PEG-gal in solvent in response to β-gal, UV absorption and fluorescence titrations were performed using β-gal (0 - 1000 U / L). Figure 1 (a) It can be seen that as the concentration of β-gal increases, the ultraviolet absorption of PEG-gal at 480 nm gradually disappears, and the ultraviolet absorption at 400 nm gradually increases. Figure 1 (b) It can be seen that with the addition of β-gal, PEG-gal reached the titration endpoint at 800 (U / L). Figure 1 (c) It can be seen that the fluorescence signal of PEG-gal shows a good linear relationship in the detection of β-gal (0-90 U / L), and the linear correlation coefficient R 2 =0.99, and the minimum detection limit is 0.16 U / L. Figure 1 (d) Monitor the reaction time of PEG-gal in response to β-gal (800 U / L) in solvent, it can be seen that the reaction between PEG-gal and β-gal is basically completed at 30 minutes. This shows that PEG-gal and β-gal can emit good fluorescence signals after reaction, and β-gal can be accurately monitored in solvent.

[0041] Example 3: Two-photon performance of PEG-gal in response to β-gal in solvent

[0042] In order to investigate the two-photon performance of PEG-gal in response to β-gal (800 U / L) in solvent. Figure 2 (a), according to the two-photon verification formula Log(I out )=k*Log(I in )+b calculated, the slope is 2, proving that the probe PEG-gal has two-photon performance after responding to β-gal. Figure 2 (b) This shows that the two-photon absorption cross section of PEG-gal in response to β-gal reaches 52 GM at 840 nm.

[0043] Example 4: pH stability of PEG-gal in solvent in response to β-gal

[0044] To investigate the pH stability of PEG-gal in response to β-gal (800 U / L) in solvent. Figure 3 , comparing the fluorescence changes of PEG-gal and β-gal in different pH solvents after 30 minutes of reaction, which shows that PEG-gal can respond to β-gal within the pH of the physiological environment.

[0045] Example 5: Selective response experiment of PEG-gal in response to β-gal in solvent

[0046] To investigate the specificity of PEG-gal in response to β-gal (800 U / L) in solvent, PEG-gal was used in solvent with common metal ions (1-8: Blank, Fe 2+ , Cu 2+ , K + ,Mg 2+ , Na + , Ca 2+ , Ni 2+ , 100 µM ), reactive oxygen species (9-19:HPO4 2- ,CO3 2- , H2O2, HClO, GSH, Cys, Hcy, ONOO - , H2S, SO3 2- , SO4 2- , 100 µM), enzymes (20-24: carboxylesterase, sulfatase, nitroreductase, tyrosinase, acetylcholinesterase, 1000 U / L), etc. Figure 4 This indicates that PEG-gal is not affected by other metal ions, enzymes, and other reactive oxygen species, but has a good response ability only to β-gal, and the fluorescence intensity after response is much higher than that of other detection substances.

[0047] Example 6: Lysosomal imaging of PEG-gal

[0048] In order to explore the ability of PEG-gal to image lysosomes in cells, we used Lyso-Tracker Deep Red to perform co-localization studies with PEG-gal in cells. Figure 5 As shown, the green channel of PEG-gal (λ em =520±20 nm; λ ex = 440 nm) and the red channel of Lyso-Tracker Deep Red (λ em = 668 ± 20 nm; λ ex = 647 nm) overlapped well, and the Pearson colocalization coefficient of PEG-gal and Lyso-Tracker Deep Red was calculated to be 0.89. These results indicate that PEG-gal has the ability to track lysosomes.

[0049] Example 7: Response of PEG-gal to β-gal in different cells

[0050] It is very important to know whether PEG-gal can be used to detect β-gal in cells, because it reflects whether PEG-gal can be used to monitor the concentration changes of β-gal in cells. Compared with other cells, the expression of β-gal in 4T1 (breast tumor cell line) cells is higher. Figure 6 After adding PEG-gal to the cells and incubating them, the fluorescence intensity of the 523nm channel in 4T1 cells was much higher than that in other cells.

[0051] Example 8: Endogenous β-gal test of PEG-gal in cells

[0052] In order to explore the ability of PEG-gal to monitor changes in cellular β-gal, 4T1 cells were treated with Dox ( Figure 7 ). Compared with the control group, doxorubicin (Dox) was added to the cells to treat 4T1 cells to induce senescence, increase the expression of intracellular β-gal (β-galactosidase), and the red channel fluorescence increased. After adding β-gal inhibitor D-glucose to inhibit β-gal activity, the red channel fluorescence intensity weakened. The results show that PEG-gal can monitor the changes of β-gal in this process.

Claims

1. A lysosome-targeted two-photon fluorescent probe, referred to as PEG-gal, characterized in that Its structure is as follows: 。 2. The method for preparing the two-photon fluorescent probe according to claim 1, characterized in that The steps include: Step 1: Under nitrogen protection, N,N-dimethyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborol-2-yl)aniline and 4-bromo-2-hydroxybenzaldehyde are dissolved in acetone, and potassium carbonate and potassium iodide are added and heated to reflux for reaction; after the reaction, the solvent is removed by distillation under reduced pressure, and yellow crystalline intermediate 1 is obtained after extraction and column chromatography separation; Step 2: Under nitrogen protection, the intermediate 1 and triethylene glycol monomethyl ether p-toluenesulfonate are dissolved in acetone, potassium carbonate and potassium iodide are added, and the mixture is heated to reflux for reaction; after the reaction is completed, the solvent is removed by distillation under reduced pressure, and yellow crystalline intermediate 2 is obtained after extraction and column chromatography separation; Step 3: dissolving α-acetyl bromide galactose and p-hydroxybenzaldehyde in a mixed solution of dichloromethane and sodium hydroxide aqueous solution, then adding tetrabutylammonium bromide, and reacting at 40° C.; after the reaction is completed, the solvent is dried, the product is extracted with dichloromethane, and then the solvent is removed, and column chromatography is performed to obtain a white solid intermediate 3; Step 4: Dissolve the intermediate 3 in tetrahydrofuran solution, add sodium borohydride in batches at 0°C, stir at room temperature for 4 hours, evaporate the solvent, extract and dry, separate by column chromatography to obtain a white solid intermediate 4; Step 5: Dissolve intermediate 4 in dichloromethane solution, add phosphorus tribromide in batches at 0°C, stir at room temperature for 4 hours, evaporate the solvent, extract and dry, separate by column chromatography to obtain white solid intermediate 5; Step 6: Dissolve the intermediate 5 in acetonitrile solution, add 4-methylquinoline, stir at 80°C overnight, evaporate the solvent, extract and dry, separate by column chromatography to obtain a pink solid intermediate 6; Step 7: Dissolve intermediate 2 in ethanol solution, add intermediate 6, stir at 40°C for 4h, evaporate the solvent, extract and dry, separate by column chromatography to obtain purple solid intermediate 7; Step 8: Dissolve the intermediate 7 in methanol solution, add sodium methoxide in batches under stirring, stir at room temperature for 30 min, evaporate the solvent, extract and dry, separate by column chromatography to obtain a purple solid PEG-gal; The synthetic route is as follows: 。 3. Use of the two-photon fluorescent probe according to claim 1 in the preparation of β-gal detection reagents.

4. The use according to claim 3, characterized in that: The detection reagent is in I 523nm The fluorescence intensity at is linearly related to the concentration of β-gal.

5. The use according to claim 3, characterized in that: The detection reagent targets lysosomes.

6. The use according to claim 3, characterized in that: The detection limit of the detection reagent for β-gal is 0.16 U / L.

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