A lysosome-targeted pH fluorescent probe, its preparation method and applications

Abstract

The present invention discloses a targeted lysosomal pH fluorescent probe and its preparation method and application. Using 1,4-cyclohexanedione-2,5-dimethyl dicarboxylate as the backbone, through protonation with the morpholine group, a pH fluorescent probe sensitive to acids and bases is formed. This probe can achieve high-efficiency luminescence in both solid and liquid states, has a high fluorescence quantum yield, the fluorescence intensity gradually weakens with the increase of pH, has good anti-interference ability and anti-photobleaching property, and has low cytotoxicity. Cell imaging experiments prove that this probe can effectively localize in lysosomes and can detect changes in different pH values in lysosomes and zebrafish, which has very important application value and is easy to promote.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological detection, and relates to a targeted lysosomal pH fluorescent probe, a preparation method thereof and an application thereof.

Background Art

[0002] The living system is a system with a complex environment. In this system, pH affects many aspects of cell activities, such as cell growth, autophagy, endocytosis, drug resistance, cell coupling, ion transport, etc. The intracellular pH is distributed differently in different organelles. For example, lysosomes are acidic under normal conditions, and their pH value ranges from 4.5 to 5.5. As an important organelle in eukaryotic cells, lysosomes contain a variety of hydrolases, and these enzymes have the best activity under acidic conditions and can degrade biological macromolecules such as proteins, polysaccharides, and nucleic acids. In addition to the degradation of intracellular substances by hydrolases, cytosolic acidification can also cause cell death. Acidic pH is an important biomarker of lysosomes, and even a slight abnormality in pH may cause physiological and pathological damage, thus causing some related diseases. Some studies have shown that abnormal changes in lysosomal pH can trigger neurological disorders, Alzheimer's disease, cancer, lysosomal storage diseases, etc. Therefore, monitoring the changes in lysosomal pH is crucial for further studying the physiology, cell metabolism, and pathological processes of lysosomes.

[0003] Among many pH detection methods, fluorescent molecular probes combined with laser confocal technology have the advantages of simple operation, high sensitivity, good selectivity, timely imaging, and high spatial and temporal resolution. There have been relevant studies. For example, Chinese Patent Application No. 201910687350.2 discloses a fluorescent probe for detecting lysosomal pH, a preparation method thereof and an application thereof, and provides a fluorescent probe for detecting lysosomal pH. Since the polymer chain of the probe has weakly basic dimethylamino, it is easy to aggregate in the acidic environment of lysosomes. Therefore, the probe has lysosomal targeting, and has the characteristics of high sensitivity, high specificity, strong anti-interference ability, good water solubility, etc., and has good fluorescence emission spectral characteristics (380 - 550 nm), can achieve rapid fluorescence signal response to pH changes in lysosomes and real-time visualization monitoring, and can be prepared by reacting DMAEMA, Nap-Br, PMDETA, isopropanol, and CuBr under nitrogen protection. The synthesis process is simple and easy to operate, the raw materials are cheap and easy to obtain, and the preparation cost is low.

[0004] Although a variety of pH fluorescent probes have been developed at present, most of the fluorescent probes targeting lysosomal pH have serious defects. The synthesis process of such probes is complex, the luminescence is unstable, they have a small Stokes shift, a short emission wavelength, which is not conducive to biological imaging, etc. Therefore, it is of great significance to develop new pH probes with high selectivity, high luminescence efficiency, simple synthesis, good photostability and biocompatibility, and capable of targeting lysosomes.

Summary of the Invention

[0005] Aiming at the problems in the prior art that most of the fluorescent probes targeting lysosomal pH have complex probe synthesis processes, unstable luminescence, small Stokes shifts, short emission wavelengths, and are not conducive to biological imaging, etc., the present invention provides a fluorescent probe targeting lysosomal pH, its preparation method and application, which is a fluorescent probe for detecting lysosomal pH based on the protonation process, its preparation method and application. The fluorescent probe targeting lysosomal pH is a novel fluorescent probe targeting lysosomes. This probe uses the lysosome-targeting group morpholine ring to localize in lysosomes for pH imaging of intracellular lysosomes. The probe has the advantages of simple synthesis, high sensitivity, high quantum yield, good stability, etc.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] A fluorescent probe targeting lysosomal pH, abbreviated as DMSS-AM, whose chemical structural formula is shown in formula (1):

[0008]

[0009] The preparation method of the above-mentioned fluorescent probe targeting lysosomal pH includes the following steps:

[0010] 1) According to the following ratio, add 2 g (0.00876 mol) of dimethyl 1,4-cyclohexanedione-2,5-dicarboxylate (DMSS), 2.3 g (0.01767 mol) of N-(2-aminoethyl)morpholine (AM) and 35 ml of methanol (AR analytical pure) into a two-necked flask, add 2 - 3 drops of acetic acid, connect a condensing reflux tube, evacuate and fill with nitrogen for protection, and stir and react at 78 °C for 24 hours;

[0011] 2) After the material obtained in the above step is cooled to room temperature, adjust the pH to neutral with ammonia water, filter by suction to retain the solid, extract the mixed solution with ethyl acetate, wash the organic layer with ultrapure water and saturated brine, then dry with anhydrous magnesium sulfate, filter, after rotary evaporation of the solution, the obtained solid is purified by silica gel column chromatography, and the ratio of the eluent is petroleum ether:ethyl acetate = 7:1, V:V, to obtain 230 mg (0.466 mmol, 21%) of orange solid DMSS-AM.

[0012] The mechanism of the present invention is as follows:

[0013]

[0014] Due to the presence of a strong basic morpholine group, the fluorescent probe described in the present invention can aggregate in an acidic lysosomal environment, so the probe can be localized in lysosomes. Under acidic conditions, the nitrogen atom on the nitrogen-oxygen heterocycle of morpholine is protonated, and the PET effect from morpholine to the 1,4-cyclohexanedione-2,5-dimethylcarboxylate fluorophore is inhibited, resulting in an increase in fluorescence intensity. Under basic conditions, the PET process occurs, the fluorophore is inhibited, and the fluorescence intensity decreases.

[0015] The present invention also relates to the application of the above-mentioned lysosome-targeted pH fluorescent probe in detecting the pH of lysosomes in aqueous solutions, biological cell bodies, and living animals.

[0016] Furthermore, the pH of lysosomes in living animals refers to the pH of lysosomes in the biological living zebrafish.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] 1. The lysosome-targeted pH fluorescent probe described in the present invention shows good linearity in fluorescence intensity within the lysosomal environment range, has high selectivity for pH, strong anti-interference ability, and satisfactory reversibility.

[0019] 2. The preparation method of the lysosome-targeted pH fluorescent probe described in the present invention has a simple synthesis process, cheap and easily available raw materials, low preparation cost, and is easy to promote.

[0020] 3. The application of the lysosome-targeted pH fluorescent probe described in the present invention has excellent lysosome targeting ability, good photostability, low cytotoxicity to cells, good biocompatibility, low light damage to biological samples, and can real-time monitor the abnormal changes in the pH of lysosomes in cells and zebrafish, obtaining more accurate and stable optical signals and imaging effects.

Description of the Drawings

[0021] Figure 1 is the 1H NMR spectrum of the lysosome-targeted pH fluorescent probe DMSS-AM described in the embodiment of the present invention;

[0022] Figure 2 is the fluorescence spectrum of the lysosome-targeted pH fluorescent probe DMSS-AM described in the embodiment of the present invention under different pH conditions;

[0023] Figure 3It is the linear relationship graph of the fluorescence intensity and pH of a targeted lysosomal pH fluorescent probe DMSS-AM described in the embodiments of the present invention under an excitation wavelength of 468 nm;

[0024] Figure 4 It is the fluorescence spectrogram of a targeted lysosomal pH fluorescent probe DMSS-AM described in the embodiments of the present invention after reacting with different substances under different pH conditions;

[0025] Figure 5 It is the reversible response graph of a targeted lysosomal pH fluorescent probe DMSS-AM described in the embodiments of the present invention;

[0026] Figure 6 It is the graph of the lysosomal co-localization experiment of a targeted lysosomal pH fluorescent probe DMSS-AM described in the embodiments of the present invention in Hela cells;

[0027] Figure 7 It is the graph of the intracellular photostability evaluation experiment of a targeted lysosomal pH fluorescent probe DMSS-AM described in the embodiments of the present invention;

[0028] Figure 8 It is the fluorescence imaging graph of a targeted lysosomal pH fluorescent probe DMSS-AM described in the embodiments of the present invention under different pH values (pH = 4.5, 5.5, 6.5, 7.4) in Hela cells;

[0029] Figure 9 It is the fluorescence imaging graph of a targeted lysosomal pH fluorescent probe DMSS-AM described in the embodiments of the present invention before and after adding drugs chloroquine and ammonium chloride in Hela cells;

[0030] Figure 10 It is the fluorescence imaging graph of a targeted lysosomal pH fluorescent probe DMSS-AM described in the embodiments of the present invention under different pH values (pH = 4.5, 5.5, 6.5, 7.4) in zebrafish;

[0031] Figure 11 It is the fluorescence imaging graph of a targeted lysosomal pH fluorescent probe DMSS-AM described in the embodiments of the present invention before and after adding drug chloroquine stimulation in zebrafish.

Specific Embodiments

[0032] The following further illustrates the specific embodiments of the present invention in conjunction with the embodiments.

[0033] Example 1:

[0034] The synthesis of a targeted lysosomal pH fluorescent probe (abbreviated as fluorescent probe DMSS-AM) includes the following steps:

[0035] Dimethyl 1,4-cyclohexanedione-2,5-dicarboxylate (DMSS) (2 g, 0.00876 mol), N-(2-aminoethyl)morpholine (AM) (2.3 g, 0.01767 mol) and 35 ml of methanol (AR analytical grade) were added to a two-necked flask, 2-3 drops of acetic acid were added dropwise, a condenser reflux tube was connected, the system was evacuated and filled with nitrogen for protection, and the reaction was stirred at 78 °C for 24 hours;

[0036] After cooling to room temperature, the pH was adjusted to neutral with ammonia water, the solid was filtered and retained, the mixed solution was extracted with ethyl acetate, and the organic layer was washed with ultrapure water and saturated brine, then dried with anhydrous magnesium sulfate, filtered, and the solution was rotary evaporated. The obtained solid was purified by silica gel column chromatography, and the eluent ratio was petroleum ether:ethyl acetate = 7:1, V:V. 230 mg (0.466 mmol, 21%) of orange solid DMSS-AM was obtained.

[0037] The above-mentioned targeted lysosomal pH fluorescent probe 1 The 1H NMR spectrum is shown in Figure 1 .

[0038] Figure 1 It is the 1H NMR spectrum of the targeted lysosomal pH fluorescent probe DMSS-AM described above.

[0039] Example 2:

[0040] Determination of the fluorescence spectrum of the fluorescent probe DMSS-AM in different B-R buffer solutions:

[0041] Britton-Robinson (BR) buffer solutions (40 mM) with different pH values were prepared using boric acid, phosphoric acid, acetic acid and sodium hydroxide as the acid-base system. 100 μl of a DMSS-AM solution with a concentration of 1×10 -4 mol / L was added to 5 ml of solutions with pH 1-14 respectively. After shaking well, the fluorescence spectrum of the test solution was measured (λ ex = 468 nm, slit = 3);

[0042] Its fluorescence spectrum is as shown in Figure 2 . As can be seen from Figure 2 , as the pH increases, the fluorescence intensity of the probe DMSS-AM gradually decreases and shows a red shift phenomenon;

[0043] As shown in Figure 3 , within the pH range of 4.8 - 7.0, there is a good linear relationship between the fluorescence intensity, which can be expressed by the formula Y = 486057 - 62028*X (R 2 = 0.991), where Y is the fluorescence intensity and X is the pH value.

[0044] Figure 2 It is the fluorescence spectrogram of the target lysosomal pH fluorescent probe DMSS-AM under different pH conditions;

[0045] Figure 3 It is the linear relationship diagram of the fluorescence intensity and pH of the target lysosomal pH fluorescent probe DMSS-AM under the excitation wavelength of 468 nm.

[0046] Example 3:

[0047] Fluorescence spectra of the fluorescence probe DMSS-AM for different analytes:

[0048] Add 500 μL of the 1×10 -4 mol / L DMSS-AM mother liquor to different pH (pH = 4.0, pH = 7.4) buffer solutions, and then add 50 μL of 100 mM different metal ions (Ag + , Al 3+ , Co 2+ , Cr 3+ , Cu 2+ , Hg 2+ , La 3+ , Li + , Mg 2+ , Mn 2+ , Mo 5 + , Na + , Ni 2+ , Pb 2+ , Zn 2+ , Ba 2+ , Ca 2+ , K + ) mother liquor and make up to 5 mL volumetric flask, shake well and let stand, then measure the fluorescence emission spectrum at room temperature (λ ex = 468 nm, slit = 3);

[0049] Its fluorescence spectrogram is as Figure 4 shown. As can be Figure 4 seen, in the solution with pH 4.0 or pH 7.4, the probe DMSS-AM has specific fluorescence response to acidic pH and is hardly affected by other interfering substances, meeting the monitoring requirements of the probe in the pH environment.

[0050] Figure 4 It is the fluorescence spectrogram of the target lysosomal pH fluorescent probe DMSS-AM after reacting with different substances under different pH conditions.

[0051] Example 4:

[0052] Reversibility study of the fluorescence probe DMSS-AM:

[0053] Prepare a 200 ml solution of DMSS-AM with a concentration of 1×10 -4 mol / L, and adjust its pH using hydrochloric acid and aqueous sodium hydroxide solution; first adjust the pH to pH = 4.0 with hydrochloric acid solution, detect the fluorescence emission spectrum, and then adjust the pH to pH = 7.4 with sodium hydroxide solution. Repeat the above experimental process 6 times.

[0054] Its fluorescence spectrum is as Figure 5 shown.

[0055] Figure 5 is the reversible response spectrum of the lysosome-targeting pH fluorescent probe DMSS-AM described above;

[0056] The results show that the fluorescent probe DMSS-AM has good reversibility to pH and can be used for multiple determinations of pH.

[0057] Example 5:

[0058] Lysosome co-localization study of the fluorescent probe DMSS-AM:

[0059] To verify whether DMSS-AM modified with morpholine structure can be localized in lysosomes, the inventor incubated 20 uM of DMSS-AM with Hela cells for 1 hour, and then co-incubated with the commercial lysosome dye LysoTracker-Red-DND99 (70 nM) for 50 minutes; the emission range of DMSS-AM under 488 nm laser remained between 500 nm - 540 nm, showing as a green channel, and the emission range of LTR dye under 561 nm laser remained between 570 nm - 670 nm, showing as a red channel. The imaging results are as Figure 6 shown. Its pearson correlation coefficient reached 0.86, indicating that DMSS-AM can be targeted and localized in lysosomes.

[0060] Figure 6 is the lysosome co-localization experiment of the lysosome-targeting pH fluorescent probe DMSS-AM in Hela cells described above.

[0061] Example 6:

[0062] Photostability study of the fluorescent probe DMSS-AM:

[0063] Evaluate the photostability of the probe DMSS-AM in cells by continuously irradiating with a 488 nm laser for 16 minutes. The imaging results are as Figure 7As shown, the probe DMSS-AM still showed good fluorescence intensity and cell outline after 16 minutes of irradiation. Compared with the commercial dye LysoTracker-Red-DND99, its quenching rate was visibly slower to the naked eye. The results showed that DMSS-AM had strong anti-photobleaching and stability.

[0064] Figure 7 It is a figure of the intracellular photostability evaluation experiment of the lysosome-targeting pH fluorescent probe DMSS-AM described above.

[0065] Example 7:

[0066] Fluorescence imaging study of the fluorescence probe DMSS-AM under different pH conditions in Hela cells:

[0067] The probe DMSS-AM was incubated in Hela cells for one hour, and then PBS buffer with different pH containing 10 μM nigericin sodium was added and incubated for 50 minutes. Then, imaging was performed using a confocal microscope. The imaging results are as Figure 8 shown.

[0068] It can be seen from Figure 8 that the fluorescence signal gradually decreased with the increase of the system pH. The excitation wavelength was at 488 nm, and the green channel wavelength collection range was between 500 nm and 540 nm.

[0069] Figure 8 It is a fluorescence imaging map of the lysosome-targeting pH fluorescent probe DMSS-AM described above under different pH values (pH = 4.5, 5.5, 6.5, 7.4) in Hela cells.

[0070] Example 8:

[0071] Fluorescence imaging study of the fluorescence probe DMSS-AM before and after adding the drugs chloroquine and ammonium chloride in Hela cells:

[0072] The probe DMSS-AM was incubated in Hela cells at 37 °C for 1 hour, and then 100 μM chloroquine and 10 mM ammonium chloride were added respectively and incubated for 1 hour. After the incubation, 4% paraformaldehyde was added to fix for 15 minutes, and then fluorescence imaging was performed. The imaging results are as Figure 9 shown.

[0073] The results showed that after adding chloroquine and ammonium chloride, the pH of lysosomes in cells increased and the fluorescence intensity decreased, proving that the probe can be used to detect the pH in lysosomes.

[0074] Figure 9 It is a fluorescence imaging map of the lysosome-targeting pH fluorescent probe DMSS-AM before and after adding the drugs chloroquine and ammonium chloride in Hela cells.

[0075] Example 9:

[0076] Fluorescence imaging study of the fluorescent probe DMSS-AM in zebrafish under different pH conditions:

[0077] Under a 28 °C environment, zebrafish eggs were incubated until they reached the larval stage. They were incubated for one hour in zebrafish culture medium with different pH values (pH = 4.5, pH = 5.5, pH = 6.5, pH = 7.4) containing DMSS-AM, a small amount of anesthetic was added, and then they were placed in a confocal dish for fluorescence imaging. Fluorescence signals were collected at 500 - 540 nm (green channel). The imaging results are as Figure 10 shown.

[0078] The results showed that as the pH increased, the fluorescence signal gradually decreased, and the fluorescence signal was the strongest at pH 4.5. In addition, the zebrafish were stimulated with 300 μM chloroquine for 1 hour and then fluorescence imaging was performed. The imaging results are as Figure 11 shown. After being stimulated with the drug chloroquine, the fluorescence signal weakened, indicating that chloroquine induced an increase in the pH of lysosomes. Both can prove that the probe DMSS-AM can be used to monitor the dynamic pH changes in vivo.

[0079] Figure 10 is the fluorescence imaging diagram of the lysosome-targeted pH fluorescent probe DMSS-AM in zebrafish under different pH values (pH = 4.5, 5.5, 6.5, 7.4);

[0080] Figure 11 is the fluorescence imaging diagram before and after the lysosome-targeted pH fluorescent probe DMSS-AM in zebrafish was stimulated with the drug chloroquine.

[0081] The above description is a detailed description of the preferred and feasible embodiments of the present invention. However, the embodiments are not intended to limit the scope of the patent application of the present invention. Any equivalent changes or modifications made under the technical spirit disclosed by the present invention shall fall within the scope of the patent covered by the present invention.

Claims

1. A lysosomal pH fluorescent probe, characterized in that: It is referred to as DMSS-AM, and its chemical structure is shown in formula (1):

2. The method for preparing a lysosomal pH fluorescent probe according to claim 1, characterized in that: The following steps are involved: 1) Add 2 g of dimethyl 1,4-cyclohexanedione-2,5-dicarboxylate, 2.3 g of N-(2-aminoethyl)morpholine, and 35 ml of methanol to a two-necked flask in the following proportions. Add 2-3 drops of acetic acid dropwise. Connect a condenser reflux tube, evacuate, fill with nitrogen, and stir at 78°C for 24 hours. 2) After the material obtained in the previous step was cooled to room temperature, the pH was adjusted to neutral with aqueous ammonia, the solid was retained by filtration, the mixed solution was extracted with ethyl acetate, and the organic layer was washed with ultrapure water and saturated brine, then dried over anhydrous magnesium sulfate, filtered, and the solution was rotary evaporated. The resulting solid was purified by silica gel column chromatography with an eluent ratio of petroleum ether: ethyl acetate = 7:1, V:V, to obtain 230 mg of DMSS-AM as an orange solid.

3. The use of a lysosomal pH-targeted fluorescent probe according to claim 1, characterized in that: The targeted lysosomal pH fluorescent probe is used to detect the pH of lysosomes in aqueous solutions, biological cells, and living animals.

4. The use of a lysosomal pH fluorescent probe according to claim 3, characterized in that: The lysosome pH in living animals refers to the lysosome pH in living zebrafish.

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