Non-pH dependent lysosome targeted viscosity fluorescent probe and preparation method thereof

Abstract

The invention belongs to the technical field of analysis and detection, and particularly relates to a preparation method of a non-pH dependent lysosome targeted viscosity fluorescent probe, and the method comprises the following steps: reacting 8-hydroxyjulolidine-9-formaldehyde with diethyl malonate to obtain a compound 1; further reacting the compound 1 under the action of phosphorus oxychloride and N, N-dimethylformamide to obtain a compound 2; and reacting the compound 2 with benzothiazole-2-acetonitrile to generate the fluorescent probe HCB. Aiming at the problems that an existing lysosome viscosity probe has strong dependency on an acid environment and the positioning accuracy is reduced when the pH of the lysosome is changed, the non-pH-dependent probe is obtained, so that the monitoring on the viscosity change of the microenvironment of the lysosome under different acid-base conditions is realized, and the method can be used for lysosome-related viscosity imaging.

Description

Technical Field

[0001] This invention relates to the field of analytical detection technology, specifically to a pH-independent lysosomal targeted viscosity fluorescent probe and its preparation method. Background Technology

[0002] Viscosity, as a crucial parameter of the cellular microenvironment, is essential for various biological activities, such as protein folding, enzyme catalysis, and signal transduction, all of which depend on maintaining equilibrium viscosity. In other words, viscosity affects the interaction and transport of biomolecules and chemical signals within cells and subcellular structures. Abnormal viscosity changes often impair normal cellular and subcellular functions, ultimately leading to diseases such as diabetes, cardiovascular disease, and inflammation. Lysosomes, as indispensable organelles in eukaryotic cells, serve as the site of cellular digestion. Lysosomes not only act as the cell's stomach but also participate in many important physiological processes, including secretion, migration, signal transduction, apoptosis, and autophagy. Lysosomes contain a large number of acidic hydrolases and secretory proteins, functioning optimally in an acidic environment with a pH of 4.0 to 5.5, degrading and recycling damaged proteins and organelles. Abnormal lysosomal pH impairs cellular digestion, potentially leading to lysosomal storage disorders. Therefore, developing a technology to monitor lysosomal viscosity is of great significance for indicating lysosomal dysfunction.

[0003] Conventional viscometers (such as falling ball viscometers, capillary viscometers, and rotational viscometers) are not suitable for tracking viscosity changes in living cells due to their instrumental limitations. Fluorescence imaging, with its superior advantages of high sensitivity, high selectivity, and ease of operation, is widely used in in vivo visualization and sensing. Among existing related technologies, prior art CN111116539A discloses a fluorescent probe that dual-responds to lysosomal viscosity and pH in cancer cells, and prior art CN112645874A discloses a lysosomal-targeting viscosity fluorescent probe. These lysosomal viscosity detection probes are mostly pH-dependent fluorescent probes, exhibiting strong targeting to lysosomes in acidic environments. However, once the pH value increases, the probe detaches from the lysosome and cannot be accurately used to measure lysosomal viscosity.

[0004] To address the aforementioned issues, designing a pH-independent but lysosome-targeting viscosity fluorescent probe has become a pressing technical problem in this field. Summary of the Invention

[0005] To address the problem that pH-dependent lysosome-targeting fluorescent probes in the prior art are prone to leakage after lysosome damage, this application provides a non-pH-dependent lysosome-targeting viscosity fluorescent probe and its preparation method.

[0006] This application provides a pH-independent lysosomal targeting viscosity fluorescent probe, wherein the fluorescent probe is a molecular rotor compound in which a coumarin backbone and a benzothiazole backbone are conjugated by carbon-carbon double bonds, having the structure shown in the following formula HCB:

[0007] This application also provides a method for preparing the above-mentioned pH-dependent lysosomal targeting viscosity fluorescent probe, comprising the following steps: S1. Dissolve 8-hydroxyjulonidine-9-carboxaldehyde and diethyl malonate in 50-70 mL of ethanol at a mass ratio of 4.34-6.51 g: 6.40-9.60 g. Add 1-2 mL of piperidine and sonicate. Heat to 80-85 °C and reflux for 17-20 hours. Cool to room temperature and remove the solvent by rotary evaporation. Add 40-60 mL of concentrated sulfuric acid and acetic acid in a volume ratio of 1:1. Heat to 90-95 °C and stir for 16-20 hours. After cooling, pour the resulting material into ice water. Adjust the pH to weakly acidic with 40% sodium hydroxide solution. Stir and cool at 2-8 °C. Filter and dry to obtain compound 1. S2. Add 4-6 mL of N,N-dimethylformamide dropwise to 4-6 mL of phosphorus oxychloride, purge with nitrogen and heat to 60-70 °C while stirring for 30-60 min. Add this solution to a system containing 2.4-3.6 g of compound 1 and 20-25 mL of N,N-dimethylformamide. Heat to 60-70 °C and stir for 15-18 hours. After cooling, pour into ice water and adjust the pH with 40% sodium hydroxide solution until a precipitate forms. Filter, wash with water, and recrystallize by reflux with ethanol. Dry to obtain compound 2. S3. Add 0.32-0.40g of compound 2 and 0.21-0.26g of benzothiazole-2-acetonitrile to acetonitrile, heat to 75-80℃ and stir for 6-9 hours. After cooling to room temperature, a precipitate is formed, filtered and dried to obtain compound HCB.

[0008] The synthetic route of the compound HCB is as follows:

[0009] Optionally, the weak acidity of the pH adjustment in step S1 is pH = 4-6.

[0010] Optionally, 95%-100% ethanol may be used as the crystallization solvent for recrystallization in step S2.

[0011] Optionally, the amount of acetonitrile added in step S3 is 10-20 mL.

[0012] Compared with the prior art, the beneficial effects of the present invention are: the non-pH-dependent fluorescent probe maintains a constant internal charge state and membrane affinity under the entire physiological environment, and will not leak or migrate due to lysosomal damage and alkalization, ensuring that the viscosity signal always originates from the lysosome itself, and its viscosity signal is always expressed inside the lysosome, truly reflecting the lysosomal viscosity change without being excessively disturbed by pH. Attached Figure Description

[0013] Figure 1 The 1H NMR spectrum of the fluorescent probe of this invention; Figure 2 The carbon NMR spectrum of the fluorescent probe of this invention; Figure 3 This is a high-resolution mass spectrometry representation of the fluorescent probe of the present invention; Figure 4 The absorption spectra of the fluorescent probe of this invention in different solvents are shown below. Figure 5 The fluorescence spectra of the fluorescent probe of this invention in different solvents; Figure 6 This is a linear relationship graph of viscosity determination by fluorescence spectroscopy using the fluorescent probe of this invention; Figure 7 The fluorescence spectra of the fluorescent probe of this invention under different pH conditions are shown. Figure 8 The fluorescence spectrum of the coexisting cations, anions and amino acids of this invention shows the selectivity of viscosity measurement. Figure 9 This is a cellular diagram illustrating the lysosomal targeting ability of the fluorescent probe of this invention in HeLa cells. Figure 10 This is a cell diagram showing the detection of viscosity changes induced by nystatin in HeLa cells using the fluorescent probe of this invention. Figure 11 This is a cell diagram showing the targeting ability of the fluorescent probe of the present invention to lysosomes at different pH values. Detailed Implementation Example 1

[0014] S1. Weigh 4.34 g of 8-hydroxyjulonidine-9-carboxaldehyde and 6.4 g of diethyl malonate. Dissolve 1 mL of pyridine in 60 mL of anhydrous ethanol. Sonicate to obtain a homogeneous solution. Raise the temperature to 80 °C and continue stirring for 19 hours. After the reaction cools to room temperature, remove the solvent by rotary evaporation. Next, add 20 mL of concentrated sulfuric acid and 20 mL of acetic acid to the flask. Continue heating under reflux to 95 °C and stir for 19 hours. After the reaction has been allowed to stand for a period of time and cooled to room temperature, add the liquid from the flask to a beaker containing ice water. Adjust the pH of the liquid in the beaker to 5 using a 40% sodium hydroxide solution. Stir for 1 hour, cool to 4 °C over ice water, filter the precipitate, and dry to obtain compound 1.

[0015] S2. Add 4 mL of DMF dropwise to a container containing 4 mL of POCl3, heat to 60 °C under nitrogen atmosphere, and stir continuously for 30 minutes. Then add the above solution to 2.4 g of compound 1 dissolved in 20 mL of DMF, heat to 60 °C, and stir continuously for 16 hours. After the reaction is complete, pour the solution into a container of 100 mL of ice water to cool. Adjust the pH of the solution in the beaker to 5 again using 40% sodium hydroxide solution, and a precipitate will form. Filter the precipitate, wash it with water, recrystallize it under reflux with ethanol, and finally filter and dry the recrystallized solid to obtain compound 2.

[0016] S3. Dissolve 0.32 g of compound 2 and 0.21 g of benzothiazole-2-acetonitrile in 5 mL of acetonitrile, heat to 80 °C and stir for 7 hours. After the reaction cools to room temperature, a precipitate forms to give compound HCB. The characterization results are as follows: Figure 1-3 As shown; 1 HNMR (600MHz, CDCl3) δ8.84 (s, 1H), 8.42 (s, 1H), 8.09 (d, J =8.1Hz, 1H), 7.88d, J =7.9Hz, 1H), 7.52-7.51 (m, 1H), 7.4-7.40 (m, 1H), 7.06 (s, 1H), 3.39 (m, J =11.5, 5.7Hz, 4H), 2.91 (t, J =6.4Hz, 2H), 2.82-2.75 (t, 2H), 2.05-1.94 (m, 4H). 13 CNMR (151MHz, CDCl3) δ (PPM) 163.87 (s), 161.57 (s), 153.80 (s), 152.46 (s), 148 .89(s), 142.66(s), 140.85(s), 134.70(s), 127.70(s), 126.64(s), 125.66(s), 123.54 (s), 121.45 (s), 119.91 (s), 117.18 (s), 110.76 (s), 108.80 (s), 106.32 ( s), 102.14(s), 50.49(s), 50.06(s), 27.34(s), 21.12(s), 20.14(s), 19.96(s). HR-MSm / z: [M+H] + C 25 H 20 N3O2S + The theoretical value is 426.1271, and the actual value is 426.1270.

[0017] Example 2

[0018] Take seven 2 μL portions of 1 mM stock solution of the fluorescent probe HCB (solvent: DMSO) and place them in clean colorimetric tubes. Add toluene, methanol, acetonitrile, ethanol, DMSO, water, and glycerol to each tube, maintaining a total volume of 2 mL. Figure 4 and Figure 5 As can be seen, the probe HCB did not show significant changes in UV absorption and fluorescence spectra in various polar and non-polar solvents such as toluene, methanol, acetonitrile, ethanol, DMSO and water, but showed a significant fluorescence change in glycerol, indicating that HCB has a selective response to viscosity and is not sensitive to polarity changes.

[0019] Example 3

[0020] Take 2 μL of 1 mM stock solution of the fluorescent probe HCB (solvent: DMSO) and add it to glycerol-water mixed solutions of different ratios. Adjust the glycerol-water volume ratio (0:100; 10:90; 20:80; 30:70; 40:60; 50:50; 60:40; 70:30; 80:20; 90:10; 100:0) to maintain the total volume at 2 mL and the probe concentration at 2 μM, and then measure its fluorescence spectrum. The fluorescence spectrum is shown below. Figure 6 As shown, fluorescence gradually increases with increasing viscosity. The system exhibits a good linear relationship between fluorescence intensity at 628 nm and viscosity (η) in the range of 11 cp to 800 cp. (Plotting lgη as the abscissa and lgI as the ordinate...) 628 Plotting lgI on the ordinate yields the linear equation lgI between viscosity and probe fluorescence intensity. 628 =4.1291+0.6354lgη(R 2 =0.9942).

[0021] Example 4

[0022] Two μL of the fluorescent probe HCB stock solution was added to 2 mL of glycerol-phosphate buffer solutions with glycerol concentrations of 0%, 60%, and 90% (v / v), respectively, for analysis. The signal stability of the probe at different pH levels and its reliability in response to viscosity were evaluated by varying the pH of the buffer solutions (2.0–11.0). Figure 7 It can be seen that the fluorescence emission of HCB probe did not fluctuate significantly in the pH range of 2.0-11.0, regardless of the viscosity of the probe at 0%, 60% or 90%. However, within the same pH range, the fluorescence intensity increased monotonically as the viscosity of the system increased from 0% to 90%, showing a viscosity-dominated response characteristic that is not sensitive to pH changes.

[0023] Example 5

[0024] Take 2 μL of the stock solution of the fluorescent probe HCB and add it to 2 mL of deionized water. Then add 500 μM of various anions, cations, and amino acids respectively. For the comparative experiment, take 2 μL of the stock solution of the fluorescent probe and add it to 2 mL of glycerol. Then detect it on a fluorometer. Figure 8 The response of the HCB probe to viscosity and other anions, cations and amino acids was found to be much higher for viscosity than for other ions.

[0025] Example 6

[0026] The probe of this invention was applied to the targeting of lysosomes by fluorescent probes in HeLa cells. The specific steps were as follows: HeLa cells were co-cultured for 30 minutes at 37°C with 10 μM of the fluorescent probe HCB and 1.5 μM of the lysosome green targeting reagent Lyso Tracker Green. Afterwards, the HeLa cells were washed three times with PBS buffer (pH=7.4), and the co-localization results were observed under a confocal laser scanning microscope. Furthermore, the co-localization experiment of probe HCB with mitochondria was compared with the above-described lysosome co-localization experiment. Using the same method, HeLa cells were co-incubated for 30 minutes at 37°C with 10 μM of the fluorescent probe and 1.5 μM of the mitochondrial deep red targeting reagent Mito Tracker Deep Red, and the experimental results were observed under a confocal laser scanning microscope. Figure 9 As can be seen, the probe HCB has a Pearson colocalization coefficient of up to 0.86 with commercially available lysosomes, while the Pearson colocalization coefficient with mitochondria is only 0.07, indicating that the probe has a good targeting effect on lysosomes.

[0027] Example 7

[0028] To detect the ability of the HCB probe to detect viscosity in living cells and its targeting ability to lysosomes during viscosity changes, the following experiments were conducted. First, HeLa cells were co-cultured with 5 μM HCB for 10 minutes, and the cells were observed using a laser confocal microscope. Then, 15 μM nystatin, a viscosity initiator, was added and the cells were cultured for 1 hour and 1.5 hours, showing a gradual increase in cell fluorescence intensity. To further demonstrate that the viscosity detected by HCB more accurately reflects lysosomal viscosity (i.e., HCB still has good targeting ability to lysosomes during viscosity changes), 5 μM HCB and 1.5 μM Lyso Tracker Green were simultaneously added to HeLa cells and co-cultured for 10 minutes, followed by laser confocal imaging. Then, 15 μM nystatin was added, and the intracellular viscosity changes and probe-lysosomal co-localization were observed after 0.5 hours, 1 hour, 1.5 hours, and 2 hours. Figure 10The results showed that the HCB probe can be used to monitor the continuous increase in viscosity caused by nystatin. During the viscosity increase, the colocalization coefficient between HCB and lysosomes remained at a high level, indicating that HCB still has good targeting ability to lysosomes during viscosity changes.

[0029] Example 8

[0030] Prepare three trays of HeLa cells. After washing the HeLa cells three times with phosphate buffer at different pH values ​​(5.0, 7.4, 9.0), co-culture the probe HCB and HeLa cells for 10 minutes, then add Lyso Tracker Green (1.5 μM) and co-culture for another 30 minutes. Finally, perform laser confocal imaging on the cells. Figure 11 The results show that the HCB probe can be used to detect lysosomal viscosity while avoiding pH interference, providing a powerful tool for studying viscosity changes when lysosomes are functioning normally and when damaged.

[0031] Furthermore, the performance comparison of this application with three existing non-pH-dependent fluorescent probes is shown in the table below:

[0032] Comparative Example a is the probe IVDI disclosed by Rui Yang's team, published in the journal *Analytica Chimica Acta*, titled "Exquisite visualization of mitophagy and monitoring the increase of lysosomal micro-viscosity in mitophagy with an unusual pH-independent lysosomal rotor"; Comparative Example b is probe compound 4 disclosed by Rui Zhu's team, published in the journal *Tetrahedron Letters*, titled "Synthesis of a new coumarin dye for pH independent staining of lysosomes"; Comparative Example c is the probe Lyso-NCO disclosed by Wenjuan Wang's team, published in the journal *Journal of Materials Chemistry B*, titled "pH-Independent two-photon fluorescent lysotrackers for real-time monitoring autophagy"; Comparative Example d is HCB from this application. The comparison shows that the first probe relies on viscosity to target lysosomes, but other organelles may have high viscosity, resulting in inaccurate targeting; the second probe reacts with enzymes in lysosomes, and emits fluorescence after the reaction, achieving targeting of lysosomes, but it cannot be used to detect viscosity; the third probe utilizes specific targeting groups of lysosomes, but the emission wavelength is short and does not respond to viscosity; the fluorescent probe of this application utilizes specific targeting groups of lysosomes to target lysosomes and responds to viscosity, with a longer emission wavelength, which can effectively avoid interference from biological background fluorescence.

[0033] The above embodiments are merely illustrative of the technical solutions and feasibility of the present invention and are not intended to limit the present invention. Any equivalent substitutions, improvements, or modifications made by those skilled in the art to the present invention without departing from the spirit and principles disclosed herein shall fall within the protection scope defined by the claims of the present invention.

Claims

1. A pH-independent lysosomal targeting viscosity fluorescent probe, characterized in that, The fluorescent probe is a molecular rotor compound in which the coumarin skeleton and the benzothiazole skeleton are conjugated by carbon-carbon double bonds, and has the structure shown in the formula HCB: ； HCB.

2. A method for preparing a non-pH-dependent lysosomal targeting viscosity fluorescent probe as described in claim 1, characterized in that, Includes the following steps: S1. Dissolve 8-hydroxyjulonidine-9-carboxaldehyde and diethyl malonate in 50-70 mL of ethanol at a mass ratio of 4.34-6.51 g: 6.40-9.60 g. Add 1-2 mL of piperidine and sonicate. Heat to 80-85 °C and reflux for 17-20 hours. Cool to room temperature and remove the solvent by rotary evaporation. Add 40-60 mL of concentrated sulfuric acid and acetic acid in a volume ratio of 1:

1. Heat to 90-95 °C and stir for 16-20 hours. After cooling, pour the resulting material into ice water. Adjust the pH to weakly acidic with 40% sodium hydroxide solution. Stir and cool at 2-8 °C. Filter and dry to obtain compound 1. S2. Add 4-6 mL of N,N-dimethylformamide dropwise to 4-6 mL of phosphorus oxychloride, purge with nitrogen and heat to 60-70 °C while stirring for 30-60 min. Add this solution to a system containing 2.4-3.6 g of compound 1 and 20-25 mL of N,N-dimethylformamide. Heat to 60-70 °C and stir for 15-18 hours. After cooling, pour into ice water and adjust the pH with 40% sodium hydroxide solution until a precipitate forms. Filter, wash with water, and recrystallize by reflux with ethanol. Dry to obtain compound 2. S3. Add 0.32-0.40g of compound 2 and 0.21-0.26g of benzothiazole-2-acetonitrile to acetonitrile, heat to 75-80℃ and stir for 6-9 hours. After cooling to room temperature, a precipitate is formed, filtered and dried to obtain compound HCB.

3. The preparation method according to claim 2, characterized in that, The weak acidity of pH adjustment mentioned in step S1 is pH = 4-6.

4. The preparation method according to claim 2, characterized in that, In step S2, recrystallization uses 95%-100% ethanol as the crystallization solvent.

5. The preparation method according to claim 2, characterized in that, In step S3, the amount of acetonitrile added is 10-20 mL.

Images