A long alkyl chain lysosomal fluorescent probe, and its methods, compositions, formulations, kits, and applications.

Abstract

This invention relates to a long-alkyl chain lysosomal fluorescent probe, its method, composition, formulation, kit, and application, belonging to the field of biodetection technology. Its chemical structural formulas are shown in formulas (I), (II), and (III). The pyrrolephenylquinoline salt series compounds OPQ-8C, OPQ-10C, and OPQ-12C provided by this invention have the characteristics of simple synthesis, low cost, and ultra-high photostability, making them suitable for long-term fluorescence signal monitoring based on fluorescence spectrometers and confocal fluorescence microscopes. This series of fluorescent probes maintains stable fluorescence signals under different pH environments, and its fluorescence activation mechanism is independent of acidic conditions (lysosomes). In living cells, this series of probes can specifically label lysosomes, enabling long-range real-time monitoring of dynamic changes in lysosomes. Based on the hydrophobic interaction between its long alkyl side chain and the phospholipid membrane, the probe can be positioned on the lipid membrane surface without entering the vesicle lumen.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically a long alkyl chain lysosomal fluorescent probe and its method, composition, preparation, kit and application. Background Technology

[0002] Lysosomes, as the core intracellular degradation system and signal regulation hub, maintain the activity of over 60 hydrolases by safeguarding their unique acidic microenvironment (pH 4.0-5.5), playing a central role in key biological processes such as autophagy regulation, metabolic homeostasis, developmental aging, and drug response. Lysosomes primarily achieve their complex physiological functions through dynamic morphological remodeling, spatial migration, and interactions with other organelles (such as mitochondria and endoplasmic reticulum). Therefore, precise tracing of lysosomes is crucial for studying their physiological and pathological mechanisms.

[0003] The acidic environment inside lysosomes provides ideal targets for probe design. Most current probes (such as the commercially available LysoTracker series) rely on basic amino groups in their structure for lysosomal selective localization and utilize the acidic environment of the lysosome to activate fluorescence. However, once the pH of the lysosome increases, the probe is prone to escape from the lysosome or undergo fluorescence quenching, leading to a significant decrease in imaging performance and localization stability. More seriously, the continuous accumulation of these probes within the lysosomal cavity often triggers an increase in lysosomal pH (i.e., the "alkalization effect"), resulting in the loss of their own fluorescence signal. Furthermore, to observe the dynamic changes of lysosomes over specific timescales, probes need to maintain sufficient photostability under continuous fluorescence microscopy. Unfortunately, traditional lysosomal staining probes are highly susceptible to photobleaching under strong laser excitation; this irreversible photodamage to the chromophore severely limits their application in long-term live-cell imaging. Summary of the Invention

[0004] To address the aforementioned problems in the existing technology, the present invention aims to design and provide a technical solution for a long alkyl chain lysosomal fluorescent probe, as well as a method, composition, formulation, kit, and application. This solution features simple synthesis, low cost, and ultra-high photostability, and is suitable for long-term fluorescence signal monitoring based on fluorescence spectrometers and confocal fluorescence microscopes.

[0005] The long alkyl chain lysosomal fluorescent probe is characterized by having the chemical structural formulas shown in formulas (I), (II), and (III): , The chemical names of the above fluorescent probes are as follows: (E)-1-octyl-4-(4-(1-pyrrolidine)styryl)quinoline iodide, abbreviated as OPQ-8C; (E)-1-decyl-4-(4-(1-pyrrolidine)styryl)quinoline iodide, abbreviated as OPQ-10C; (E)-1-dodecyl-4-(4-(1-pyrrolidine)styryl)quinoline iodide, abbreviated as OPQ-12C.

[0006] The probe molecules provided by this invention contain octane, decane, and dodecane alkyl chains, respectively. These long alkyl side chains can interact hydrophobically with the phospholipid membrane. Based on this interaction, the probe molecules can be stably anchored to the lipid membrane surface without penetrating the phospholipid layer into the vesicle lumen.

[0007] The method for preparing a long alkyl chain lysosomal fluorescent probe is characterized by comprising the following steps: 1) 4-methylquinoline is reacted with iodoalkane (n=8, 10 or 12) by heating at 95-105℃ to produce 1-alkyl-4-methylquinoline onium salt; 2) Add 4-(1-pyrrolidine)benzaldehyde and piperidine catalyst to the product obtained in step 1), and carry out a condensation reaction in ethanol solvent at 85-95°C; 3) After the reaction is complete, the mixture is cooled and purified by column chromatography to obtain the long alkyl chain lysosomal fluorescent probe.

[0008] The reaction formula for the preparation method of this invention is shown below: .

[0009] The method for preparing a long alkyl chain lysosomal fluorescent probe is characterized in that, in step 1), the molar ratio of 4-methylquinoline to iodoalkane is 1:1.2-1.3, and the heating reaction time is 8-12 hours.

[0010] The method for preparing a long alkyl chain lysosomal fluorescent probe is characterized in that, in step 2), the molar ratio of 1-alkyl-4-methylquinoline onion salt to 4-(1-pyrrolidine)benzaldehyde is 1.1 to 1.3:1, and the condensation reaction time is 6 to 10 hours.

[0011] The composition is characterized by comprising the fluorescent probe of claim 1 or a pharmaceutically acceptable salt thereof, and optionally a solubilizer, emulsifier, filler or preservative.

[0012] The composition is characterized in that the pharmaceutically acceptable salt is one of hydrochloride, sulfate, acetate, citrate, and benzyl sulfonate.

[0013] The formulation is characterized by comprising an active ingredient and a pharmaceutical carrier, wherein the active ingredient is the fluorescent probe or the composition, and the pharmaceutical carrier comprises water or a buffer solution.

[0014] The kit is characterized in that it is a long alkyl chain lysosomal fluorescent probe kit, comprising long alkyl chain lysosomal fluorescent probes.

[0015] Application of the long alkyl chain lysosomal fluorescent probe in lysosomal fluorescence imaging.

[0016] The application of the long alkyl chain lysosomal fluorescent probe in lysosomal fluorescence imaging is characterized by specifically including: a) Applications that maintain stable fluorescence signals in pH 3–10 environments; b) Applications where the fluorescence intensity remains essentially unchanged after 7200 seconds of continuous illumination in photostability tests; c) Application of long-term dynamic real-time monitoring of lysosomes; d) Application of lipid vesicle membrane staining.

[0017] The lysosomal fluorescent probes OPQ-8C, OPQ-10C, and OPQ-12C described in this invention have ultra-high photostability and can achieve long-range real-time monitoring of dynamic changes in lysosomes.

[0018] The experimental results confirm that, firstly, in a solution environment, the fluorescence intensity of the series of fluorescent probes described in this invention remains stable even after continuous irradiation with a xenon lamp for 7200 seconds, and the fluorescence signal can be continuously monitored for a long time.

[0019] Secondly, this series of probes can effectively label lysosomes in living cells, maintaining stable fluorescence intensity under continuous laser irradiation, making them suitable for long-term real-time monitoring of lysosomal dynamic processes. This also indicates that the compounds described in this invention have broad application prospects as organic light-emitting materials and lysosomal fluorescent probes.

[0020] The beneficial effects of this invention are as follows: This invention discloses a long-alkyl-chain lysosomal fluorescent probe and its applications. Experimental results show that this series of fluorescent probes maintains stable fluorescence signals under different pH conditions, and its fluorescence activation mechanism is independent of the acidic environment (lysosome). Furthermore, this series of probes maintains continuous and stable fluorescence emission even under prolonged light exposure. In living cells, this series of probes can specifically label lysosomes, and due to its excellent photostability, enables long-range real-time monitoring of lysosomal dynamic changes. Based on the hydrophobic interaction between its long alkyl side chain and the phospholipid membrane, the probe can be localized to the lipid membrane surface without entering the vesicle lumen. Attached Figure Description

[0021] Figure 1 The fluorescence intensity diagrams of 5 μM OPQ-8C, OPQ-10C and OPQ-12C at 700 nm in PBS buffer solutions with different pH values ​​are shown. Excitation wavelength: 561 nm. Figure 2 The photostability test results for 5 μM OPQ-8C, OPQ-10C, and OPQ-12C in aqueous solution are shown. Excitation wavelength: 561 nm; Emission wavelength: 700 nm. Figure 3 The fluorescence intensity ratio of 5 μM OPQ-8C, OPQ-10C and OPQ-12C in glycerol-water systems of different viscosities at 700 nm is shown. Excitation wavelength: 561 nm. Figure 4 Confocal fluorescence images, fluorescence colocalization scatter plots, and fluorescence intensity-distance distribution maps based on white lines in the merged images of live HeLa cells co-stained with OPQ-8C / OPQ-10C / OPQ-12C and Lyso-Tracker Green; OPQ series probes, excitation wavelength: 561 nm, fluorescence acquisition band: 620-700 nm; Lyso-Tracker Green, excitation wavelength: 488 nm, fluorescence acquisition band: 500-560 nm; scale bar: 10 μm; Figure 5 To evaluate the photostability of the OPQ series probes under confocal fluorescence microscopy, live HeLa cells were stained with 5µM OPQ-8C, OPQ-10C, and OPQ-12C for 10 min, respectively. The stained cells were then continuously imaged (50 images in total), and the fluorescence intensity of all images was quantitatively analyzed. Excitation wavelength: 561nm; fluorescence acquisition band: 620-700nm; scale bar = 10μm. Figure 6 The image shows fluorescence images of mixed lipid vesicles stained with 5μM probes OPQ-8C, OPQ-10C, and OPQ-12C. Excitation wavelength: 561nm, fluorescence acquisition band: 620-700nm. Detailed Implementation

[0022] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention are within the scope of protection of the present invention.

[0023] Example 1: Synthesis of probes OPQ-8C, OPQ-10C and OPQ-12C 4-Methylquinoline (0.172 g, 1.2 mmol) and iodoalkane (n=8 / 10 / 12) (1.5 mmol) were added sequentially to a round-bottom flask, and the reaction mixture was stirred at 100 °C for 10 h. The progress of the reaction was monitored using a silica gel plate for thin-layer chromatography. After the reactants were completely consumed, 4-(1-pyrrolidine)benzaldehyde (0.175 g, 1 mmol) and 200 μL piperidine were added to the mixture using ethanol (8 mL) as solvent, and the mixture was stirred at 95 °C for another 8 h. After cooling to room temperature, the mixture was purified by column chromatography (CH2Cl2 / MeOH, 100:1, v / v) to give a purple-black solid (OPQ-8C, 0.39 g, 72%; OPQ-10C, 0.32 g, 56%; OPQ-12C, 0.28 g, 47%).

[0024] OPQ-8C: 1 H NMR (DMSO- d 6 , 400 MHZ): δ (ppm) 9.11 (d, J = 6.7 Hz, 1H), 9.02 (dd, J = 8.9, 1.5 Hz, 1H), 8.42 (d, J = 8.9 Hz, 1H), 8.32 (d, J = 6.9Hz, 1H), 8.24-8.14 (m, 2H), 8.02-7.91 (m, 2H), 7.87 (d, J = 9.0 Hz, 2H), 6.68(d, J = 9.0 Hz, 2H), 4.85 (t, J = 7.5 Hz, 2H), 3.44-3.35 (m, 4H), 2.00 (p, J = 3.4 Hz, 4H), 1.92 (p, J = 7.7 Hz, 2H), 1.41-1.20 (m, 10H), 0.87-0.82 (m, 3H). 13 C NMR (DMSO- d 6, 100 MHz) δ (ppm): 153.78, 150.37, 146.35, 145.83,138.30, 135.15, 132.19, 128.82, 127.16, 126.52, 123.24, 119.28, 114.27,112.95, 112.57, 56.29, 47.93, 31.62, 29.72, 29.01, 28.95, 26.32, 25.42,22.51, 14.42. HRMS (m / z): [M] + calculated for C 29 H 37 N2 + , 413.2952; found,413.2953. OPQ-10C: 1 H NMR (DMSO- d 6 , 400 MHZ): δ (ppm) 9.10 (d, J = 6.9 Hz, 1H),9.02 (dd, J = 8.9, 1.4 Hz, 1H), 8.41 (d, J = 8.9 Hz, 1H), 8.32 (d, J = 6.9Hz, 1H), 8.22-8.16 (m, 2H), 8.00-7.93 (m, 2H), 7.87 (d, J = 9.0 Hz, 2H), 6.68(d, J = 9.0 Hz, 2H), 4.84 (t, J = 7.4 Hz, 2H), 3.42-3.37 (m, 4H), 2.00 (p, J = 3.5 Hz, 4H), 1.91 (t, J = 7.5 Hz, 2H), 1.37-1.21 (m, 14H), 0.86-0.82 (m,3H). 13 C NMR (DMSO- d 6, 100 MHz) δ (ppm): 153.81, 150.39, 146.39, 145.84,138.32, 135.15, 132.20, 128.82, 127.16, 126.53, 123.24, 119.29, 114.26,112.96, 112.59, 56.30, 47.93, 31.74, 29.69, 29.32, 29.12, 28.97, 26.29,25.43, 22.56, 14.44. HRMS (m / z): [M] + calculated for C 31 H 41 N2 + , 441.3265;found, 441.3270. OPQ-12C: 1 H NMR (DMSO- d 6 , 400 MHZ): δ (ppm) 9.10 (d, J = 6.9 Hz, 1H),9.02 (dd, J = 8.9, 1.4 Hz, 1H), 8.41 (d, J = 8.7 Hz, 1H), 8.32 (d, J = 6.9Hz, 1H), 8.22-8.15 (m, 2H), 8.01-7.92 (m, 2H), 7.87 (d, J = 9.0 Hz, 2H), 6.68(d, J = 9.0 Hz, 2H), 4.84 (t, J = 7.4 Hz, 2H), 3.43-3.37 (m, 4H), 2.00 (p, J = 3.4 Hz, 4H), 1.90 (q, J = 8.2 Hz, 2H), 1.38-1.20 (m, 18H), 0.87-0.81 (m,3H). 13 C NMR (DMSO- d 6, 100 MHz) δ (ppm): 153.78, 150.36, 146.36, 145.84,138.30, 135.14, 132.20, 128.81, 127.17, 126.51, 123.25, 119.27, HRMS (m / z): [M] + Calculated for C 33 H 45 N2 + ,469.3578; found, 469.3581. In Example 1 above, in step 1), the heating temperature is 95°C or 105°C, the molar ratio of 4-methylquinoline to iodoalkane is 1:1.2 or 1:1.3, and the heating reaction time is 8 or 12 hours; in step 2), the condensation reaction temperature is 85°C or 95°C, the molar ratio of 1-alkyl-4-methylquinoline onium salt to 4-(1-pyrrolidine)benzaldehyde is 1.1:1 or 1.3:1, and the condensation reaction time is 6 or 10 hours; other steps are the same as in Example 1, and the probe described in this invention can also be prepared.

[0025] Example 2: Stability testing of probes OPQ-8C, OPQ-10C, and OPQ-12C under different pH conditions The pH of the PBS buffer was adjusted to different ranges from 3 to 10 using HCl or NaOH. Subsequently, test solutions containing 5 μM OPQ-8C / OPQ-10C / OPQ-12C were prepared from these buffers at different pH values, and the fluorescence intensity of each probe at 700 nm was measured as a function of pH using a fluorescence spectrometer. The results showed that the fluorescence intensities of OPQ-8C, OPQ-10C, and OPQ-12C remained stable without significant fluctuations when the solution pH changed from 3 to 10, indicating that the fluorescence of the probes was not dependent on acidic activation and was insensitive to pH changes.

[0026] See results Figure 1 The fluorescence intensity of 5 μM OPQ-8C, OPQ-10C, and OPQ-12C at 700 nm in PBS buffer solutions with different pH values. Excitation wavelength: 561 nm.

[0027] Example 3: Photostability Test of Probes OPQ-8C, OPQ-10C and OPQ-12C in Solution Using a xenon lamp in a fluorescence spectrometer, 5 μM aqueous solutions of OPQ-8C, OPQ-10C, and OPQ-12C were continuously irradiated for 7200 seconds, and the fluorescence intensity changes at 700 nm were monitored. The results showed that the fluorescence emission intensity of the three probes remained stable throughout the irradiation process, with only minor fluctuations compared to the initial values, confirming the excellent photostability of the OPQ series probes in solution.

[0028] See results Figure 2 Photostability tests of 5μM OPQ-8C, OPQ-10C, and OPQ-12C in aqueous solution. Excitation wavelength: 561nm; Emission wavelength: 700nm.

[0029] Example 4: Response test of probes OPQ-8C, OPQ-10C and OPQ-12C to viscosity Glycerol and water, with their significantly different viscosities, were selected as solvents to construct glycerol-water mixtures with varying ratios. In these systems, the fluorescence intensity of OPQ-8C, OPQ-10C, and OPQ-12C significantly increased with increasing glycerol content (i.e., viscosity), consistent with the presence of rotatable units in their molecular structures. Quantitative analysis showed that when the solution viscosity increased to pure glycerol (100%), the fluorescence intensity of the three probes increased by approximately 35-fold, 43-fold, and 47-fold, respectively, fully demonstrating the highly sensitive fluorescence response of the OPQ series probes to changes in environmental viscosity.

[0030] See results Figure 3 Fluorescence intensity ratios of 5 μM OPQ-8C, OPQ-10C, and OPQ-12C at 700 nm in glycerol-water systems of different viscosities. Excitation wavelength: 561 nm.

[0031] Example 5: HeLa cell culture HeLa cells were cultured in high-glucose medium (H-DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin. They were passaged every 2-3 days in a 37°C, 5% CO2, saturated humidity incubator. Once the cells reached the logarithmic growth phase, they were cultured on slides: ① Cell slides were immersed in anhydrous ethanol for 30 min, dried under an alcohol lamp, and placed in disposable 35mm culture dishes for later use; ② Confluent cells in 100 mL cell culture flasks were washed three times with PBS, digested with 1 mL of 0.25% trypsin for 2-3 minutes, the trypsin was carefully poured off, fresh culture medium was added, and the cells were repositioned and counted. The cell density was controlled by the amount of culture medium added, aiming for a final cell concentration of 1 × 10⁻⁶ cells / mL. 5 Then, the cells are seeded into the culture dish containing the cell spreads mentioned above, and placed in a 5% CO2 incubator at 37°C to allow the cell spreads to grow, thus obtaining the cells for the experiment.

[0032] Example 6: Counterstaining experiments with OPQ-8C / OPQ-10C / OPQ-12C and Lyso-Tracker Green First, probes OPQ-8C, OPQ-10C, and OPQ-12C were dissolved in DMSO to prepare a 5 mM stock solution. After cells adhered to the confocal culture dish, they were stained with 5 μM OPQ-8C / OPQ-10C / OPQ-12C and incubated at 37°C for 10 min. The culture medium was then discarded, and the cells were washed three times with PBS to remove unbound dye. Next, the cells were stained with 0.2 μM Lyso-Tracker Green for 10 min. After washing three times with PBS, the cell growth side of the slide was placed face down in the culture dish, and the localization of the probes in the cells was observed using a laser scanning confocal fluorescence microscope. The results showed that the fluorescence distribution areas of OPQ-8C, OPQ-10C, and OPQ-12C highly overlapped with those of LTG within the cells. The fluorescence colocalization scatter plot shows a linear distribution extending from the origin to the upper right corner, indicating a high degree of overlap between the red and green channel signals, with Pearson colocalization coefficients of 0.89, 0.87, and 0.84, respectively. Furthermore, the distance-dependent fluorescence intensity distribution of the OPQ series probes and Lyso-Tracker Green is highly synchronized. Therefore, this confirms that the probes OPQ-8C, OPQ-10C, and OPQ-12C described in this invention can specifically label lysosomes.

[0033] See results Figure 4 Confocal fluorescence images, fluorescence colocalization scatter plots, and fluorescence intensity-distance distribution maps based on the white lines in the merged images of viable HeLa cells stained with OPQ-8C / OPQ-10C / OPQ-12C and Lyso-Tracker Green are presented. Figure ① shows the fluorescence image of the OPQ series probes (excitation wavelength: 561 nm; fluorescence acquisition band: 620-700 nm); Figure ② shows the fluorescence image of Lyso-Tracker Green (excitation wavelength: 488 nm; fluorescence acquisition band: 500-560 nm); Figure ③ is the merged image of ① and ②; Figure ④ shows the fluorescence colocalization scatter plot and colocalization coefficients based on Figure ③; Figure ⑤ shows the fluorescence intensity-distance distribution map along the white lines shown in Figure ③. Scale bar = 10 μm.

[0034] Example 7: Photostability assay of HeLa cells stained with OPQ series probes Three cell-inoculated slides were washed three times with PBS and stained with 5 μM OPQ-8C, OPQ-10C, and OPQ-12C, respectively, and incubated at 37°C for 10 min. The stained cell slides were then removed and washed three times with PBS to remove unbound dye. The slides were placed cell-side down in glass-bottomed culture dishes, and a series of images were continuously captured under laser irradiation of the same intensity. The results showed that even after capturing 50 images, OPQ-8C, OPQ-10C, and OPQ-12C maintained high fluorescence intensity. Quantitative fluorescence intensity analysis of the 50 consecutive images indicated that the fluorescence signal of the probes within the lysosomes remained stable under continuous laser irradiation, with no obvious photobleaching observed. Therefore, the OPQ series probes are confirmed to have ultra-high photostability and are suitable for studying dynamic lysosomal processes requiring long-term imaging.

[0035] See results Figure 5 Images show the photostability evaluation of the OPQ series probes during confocal fluorescence microscopy imaging. Live HeLa cells were stained for 10 min with 5 µM OPQ-8C, OPQ-10C, and OPQ-12C, respectively. Subsequently, the stained cells were continuously imaged under the same laser intensity (a total of 50 images were acquired), and the photostability of the probes was evaluated by quantitatively analyzing the fluorescence intensity of all images. Excitation wavelength: 561 nm; fluorescence acquisition band: 620-700 nm; scale bar = 10 μm.

[0036] Example 8: Staining mixed lipid vesicles with OPQ series probes 1. First, prepare mixed lipid vesicles: 1) Prepare a 6 mL mixture of chloroform and ethanol (volume ratio 2:1); 2) Weigh 18 mg of DOPC, DPPC and CL, and 2 mg of DOPG and DPPG respectively, and place them in five 5 mL glass bottles to prepare stock solutions of 18 mg / mL and 2 mg / mL respectively. 3) Mix each bottle thoroughly by shaking; 4) Transfer 25 μL of LDOPC, DPPC, DOPG and DPPG stock solutions, and 13 μL of CL stock solution to the same glass bottle and mix thoroughly by shaking; 5) Perform film formation under nitrogen protection: Connect the nitrogen outlet to the pipette tip, slightly tilt the glass bottle containing the mixed solution to spread the solution evenly along the inner wall of the bottle, and slowly rotate the bottle until the solvent evaporates completely; 6) After film formation, place the opened glass bottle in a 500 mL round-bottom flask, connect the oil pump, and evacuate for 30 minutes; 7) Remove the glass bottle, fill it with 0.1 M sucrose solution, tighten the cap, and place it in a 55 ℃ oven for 24 hours; 8) Finally, the formation of a white cloud-like substance in the bottle indicates that the mixed lipid vesicles have been successfully prepared. 2. Staining the prepared mixed lipid vesicles using OPQ series probes: 1) Add 1 μL of 5 mM probe stock solution to 200 μL of 0.1 M glucose solution, mix well, and obtain the probe staining solution; 2) Add 10 μL of a white, cloudy suspension (i.e., the prepared mixed lipid vesicles) to the above probe staining solution, mix gently, and complete the staining. The results showed that the OPQ series probes were only enriched on the vesicle surface, and their fluorescence signal was not detected in the vesicle cavity, indicating that the hydrophobic interaction between the long alkyl chain of the probe and the vesicle phospholipid membrane effectively anchored it to the membrane.

[0037] See results Figure 6 Fluorescence images of mixed lipid vesicles stained with 5 μM probes OPQ-8C, OPQ-10C, and OPQ-12C. Excitation wavelength: 561 nm; fluorescence acquisition band: 620-700 nm.

[0038] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A long alkyl chain lysosomal fluorescent probe, characterized in that, Its chemical structural formulas are shown in formulas (I), (II) and (III): 。 2. The method for preparing a long alkyl chain lysosomal fluorescent probe according to claim 1, characterized in that... Includes the following steps: 1) 4-methylquinoline is reacted with iodoalkane (n=8, 10 or 12) by heating at 95-105℃ to produce 1-alkyl-4-methylquinoline onium salt; 2) Add 4-(1-pyrrolidine)benzaldehyde and piperidine catalyst to the product obtained in step 1), and carry out a condensation reaction in ethanol solvent at 85-95°C; 3) After the reaction is complete, the mixture is cooled and purified by column chromatography to obtain the long alkyl chain lysosomal fluorescent probe.

3. The method for preparing a long alkyl chain lysosomal fluorescent probe according to claim 2, characterized in that... In step 1), the molar ratio of 4-methylquinoline to iodoalkane is 1:1.2-1.3, and the heating reaction time is 8-12 hours.

4. The method for preparing a long alkyl chain lysosomal fluorescent probe according to claim 2, characterized in that... In step 2), the molar ratio of 1-alkyl-4-methylquinoline onion salt to 4-(1-pyrrolidine)benzaldehyde is 1.1 to 1.3:1, and the condensation reaction time is 6 to 10 hours.

5. A composition, characterized in that... It includes the fluorescent probe of claim 1 or a pharmaceutically acceptable salt thereof, and optionally a solubilizer, emulsifier, filler or preservative.

6. The composition according to claim 5, characterized in that: The pharmaceutically acceptable salt is one of the following: hydrochloride, sulfate, acetate, citrate, and benzyl sulfonate.

7. A formulation, characterized in that... It includes an active ingredient and a pharmaceutical carrier, wherein the active ingredient is the fluorescent probe of claim 1 or the composition of claim 5, and the pharmaceutical carrier includes water or a buffer solution.

8. A reagent kit, characterized in that, The kit is a long alkyl chain lysosomal fluorescent probe kit, comprising the long alkyl chain lysosomal fluorescent probe as described in claim 1.

9. The application of the long alkyl chain lysosomal fluorescent probe as described in claim 1 in lysosomal fluorescence imaging.

10. The application of the long alkyl chain lysosomal fluorescent probe as described in claim 9 in lysosomal fluorescence imaging, characterized in that... Specifically, it includes: a) Applications that maintain stable fluorescence signals in pH 3–10 environments; b) Applications where the fluorescence intensity remains essentially unchanged after 7200 seconds of continuous illumination in photostability tests; c) Application of long-term dynamic real-time monitoring of lysosomes; d) Application of lipid vesicle membrane staining.

Images