CN110183482B

CN110183482B - Near-infrared fluorescent probe for monitoring pH of lysosome and preparation method and application thereof

Info

Publication number: CN110183482B
Application number: CN201910567239.XA
Authority: CN
Inventors: 冯素玲; 王盈盈; 毛国江; 高广琦
Original assignee: Henan Normal University
Current assignee: Henan Normal University
Priority date: 2019-06-27
Filing date: 2019-06-27
Publication date: 2022-03-01
Anticipated expiration: 2039-06-27
Also published as: CN110183482A

Abstract

The patent discloses a near-infrared fluorescent probe for monitoring lysosome pH, and a preparation method and application thereof, and belongs to the technical field of analytical chemistry. The technical scheme provided by the invention has the key points that: a near-infrared fluorescent probe for monitoring lysosome pH has the following structural formula:

the invention also specifically discloses a preparation method of the near-infrared fluorescent probe for monitoring the pH of the lysosome and application of the near-infrared fluorescent probe in selective detection of the pH in a water environment and a biological cell system. The near-infrared fluorescent probe has the advantages of emission in a near-infrared region, good selectivity, good light stability, good reversibility, excellent lysosome targeting capability and the like.

Description

Near-infrared fluorescent probe for monitoring pH of lysosome and preparation method and application thereof

Technical Field

The invention relates to a near-infrared fluorescent probe for monitoring lysosome pH, a preparation method and application thereof, and belongs to the technical field of analytical chemistry.

Background

Lysosomes, which are important acidic (pH of about 3.8 to 5.5) organelles in eukaryotic cells, are the sites of breakdown of biological macromolecules such as proteins, nucleic acids, polysaccharides, etc., and are called "digestive organs" in cells. Lysosomes play important roles in many life processes, such as endocytosis, cell growth and apoptosis, autophagy, ion metabolism, oxidative stress, and the like. Abnormal fluctuations in lysosomal pH can cause lysosomal dysfunction and are closely associated with diseases such as lysosomal storage diseases, cancer, and the like. Therefore, it is of great interest to develop analytical techniques that enable real-time monitoring of lysosomal pH.

Fluorescent molecular probes have become an indispensable tool in the field of biological imaging due to the advantages of simple operation, high response speed, high selectivity, high sensitivity, high spatial and temporal resolution and the like. In recent years, researchers have successively developed a number of fluorescent molecular probes for monitoring lysosomal pH. But these probesThe excitation and emission wavelengths of most of the probes are short (lambda)_exλ_em) The method has certain limitations in biological imaging, and has the problems of large light damage to the biological sample, small tissue penetration depth, large background fluorescence interference and the like. The near-infrared fluorescent probe (near-infrared fluorescent probe) has the advantages of small light damage to a biological sample, large tissue penetration depth, small background fluorescence interference and the like, and becomes a research hotspot in the field of fluorescent molecular probes. Based on this, researchers developed some pH fluorescent probes based on traditional near-infrared cyanine dyes (cyanine dyes). However, the pH near-infrared probe designed based on the near-infrared cyanine dye has poor light stability, and it is difficult to obtain an accurate and stable fluorescence signal in long-time and multiple imaging. The silicon rhodamine dye is a near infrared dye with excellent light stability newly reported in recent years, the light stability of the silicon rhodamine dye is far better than that of cyanine dye, and the silicon rhodamine dye is widely applied to the design of near infrared fluorescent probes for various analysis and detection objects. In addition, the silicon rhodamine is similar to common rhodamine dyes, is easy to spiro-cyclize, and facilitates construction of fluorescent probes for different detection objects.

Disclosure of Invention

Aiming at the problems and the current situation of the existing near-infrared fluorescent probe for monitoring the pH value of the lysosome, the invention provides the near-infrared fluorescent probe for monitoring the pH value of the lysosome, wherein the near-infrared fluorescent probe utilizes a morpholine group as a lysosome targeting positioning group and is used for imaging the pH value in a cell lysosome, and the probe has the characteristics of near-infrared emission, good selectivity, good light stability, good reversibility, excellent lysosome targeting capability and the like.

The invention also provides a preparation method of the near-infrared fluorescent probe for monitoring the pH of the lysosome and application of the near-infrared fluorescent probe in selective detection of the pH in water environment and biological cell bodies.

The invention adopts the following technical scheme for solving the technical problems, and the near-infrared fluorescent probe for monitoring the pH value of the lysosome is characterized in that the structural formula of the near-infrared fluorescent probe is as follows:

the preparation method of the near-infrared fluorescent probe for monitoring the pH of the lysosome is characterized by comprising the following specific steps of:

step S1: adding 6g of 3-bromo-N, N-dimethylaniline and 60 ml of anhydrous tetrahydrofuran into a dry 250 ml round-bottom flask under the protection of argon, magnetically stirring for 5 minutes to dissolve the 3-bromo-N, N-dimethylaniline, then dropwise adding 13 ml of N-butyl lithium N-hexane solution with the molar concentration of 2.4mol/L into the reaction solution, reacting at 0 ℃ for 2 hours after dropwise adding, then dissolving 2.2 ml of dichlorodimethylsilane into 10ml of anhydrous tetrahydrofuran, dropwise adding the solution into the reaction solution, reacting to room temperature after dropwise adding, stirring overnight, adding 50ml of water to quench the reaction, extracting the reaction solution with diethyl ether, drying with anhydrous sodium sulfate, and carrying out reduced pressure spin-drying on the solvent to obtain a crude product, and purifying the crude product with a silica gel column to obtain a compound 1, wherein the structural formula of the compound is as follows:

step S2: adding 500mg of compound 1, 1260mg of 2-carboxybenzaldehyde and 37.5mg of copper bromide into a 100ml glass thick-wall pressure-resistant tube, heating and stirring at 140 ℃, reacting for 5 hours, naturally cooling to room temperature, then dissolving the reaction mixture into dichloromethane, washing for three times by using a 10% NaOH solution, recovering and spin-drying a dichloromethane phase to obtain a crude product, and purifying the crude product by using a silica gel column to obtain a compound 2, wherein the structural formula of the compound is as follows:

step S3: 443mg of compound 2, 20mL of dried 1, 2-dichloroethane and 2mL of phosphorus oxychloride were charged in a 100mL round-bottomed flask, the reaction solution in the flask was heated to 85 ℃ under reflux for 4 hours, the solvent was distilled off under reduced pressure to obtain a reaction residue, the reaction residue was dissolved in 20mL of dried acetonitrile, and 5mL of triethylamine was added theretoA solution of 650mg of 4- (2-aminoethyl) -morpholine in 10mL of acetonitrile was added dropwise, the reaction mixture was stirred at room temperature overnight, the solvent was removed under reduced pressure, and 50mL of CH was used₂Cl₂The residue was dissolved and washed 3 times with 50mL of saturated aqueous NaCl solution and anhydrous Na₂SO₄Drying, evaporating the solvent by a rotary evaporator to obtain a crude product, and purifying the crude product by using a silica gel column to obtain a compound 3, wherein the structural formula is as follows:

step S4: 105mg of compound 3 was dissolved in 5mL of dry tetrahydrofuran at room temperature, 76mg of lithium aluminum hydride was added in small portions, the mixture was stirred at room temperature for 6 hours, 5mL of methanol was added to quench the reaction, the reaction mixture was distilled under reduced pressure to remove the solvent to obtain a residue, and the residue was purified by a silica gel column to obtain the target fluorescent probe compound Lyso-NIR-pH.

The near-infrared fluorescent probe for monitoring the pH of the lysosome is applied to selective detection of the pH in a water environment and a biological cell system, wherein the detection comprises fluorescence detection and cell imaging detection in a water solution.

Compared with the prior art, the invention has the following beneficial effects: (1) the synthesis of the near-infrared fluorescent probe is relatively easy, and the post-treatment process is relatively simple; (2) the near-infrared fluorescent probe realizes high-selectivity and rapid detection of pH and has the capability of resisting the interference of other substances in a living body; (3) the near-infrared fluorescent probe has near-excellent lysosome targeting capability, near-infrared emission and excellent light stability, and can be applied to imaging detection in cell lysosomes. The near-infrared fluorescent probe has the characteristics of reducing the interference of autofluorescence background in a living body, reducing the light damage to a biological sample, improving the light stability and the like, so as to obtain more accurate and stable optical signals and imaging effect. Therefore, the near-infrared fluorescent probe has wide application prospect in the field of pH imaging monitoring, and has important significance for researching the action mechanism of lysosome pH in the physiological and pathological processes of organisms.

Drawings

FIG. 1 is a graph of fluorescence spectra of the fluorescence probe compound Lyso-NIR-pH prepared in example 1 under different pH conditions;

FIG. 2 is a graph of the ultraviolet-visible absorption spectrum of the fluorescent probe compound Lyso-NIR-pH prepared in example 1 under different pH conditions;

FIG. 3 is a graph showing the change of fluorescence intensity of the Lyso-NIR-pH at an emission wavelength of 675nm with pH, which is a fluorescent probe compound prepared in example 1;

FIG. 4 is a graph of the response of the fluorescence probe compound Lyso-NIR-pH prepared in example 1 to various analytes at pH7.4 (left to right): 1. h⁺(pH＝5)；2、Na⁺；3、K⁺；4、Ca²⁺；5、Mg²⁺；6、Fe³⁺；7、Cu²⁺；8、Zn²⁺；9、Mn²⁺；10、Ni²⁺；11、Cd²⁺；12、Co²⁺；13、NH4⁺；14、Ac^-；15、CO₃ ^2-；16、SO₄ ^2-；17、F^-；18、Br^-；19、I^-；20、S₂O₃ ^-；21、NO₂ ^-；22、H₂PO^4-；23、HPO₄ ^2-；24、Glutathione；25、Arginine；26、Valine；27、Tryptophan；28、Cysteine；29、Glycine；30、Homocysteine.

FIG. 5 is a pH reversible response of the fluorescence probe compound Lyso-NIR-pH prepared in example 1.

FIG. 6 is a lysosome co-localization experiment of the fluorescent probe compound Lyso-NIR-pH prepared in example 1 in HeLa cells;

FIG. 7 is an intracellular photostability evaluation experiment of the fluorescent probe compound Lyso-NIR-pH prepared in example 1.

Fig. 8 is a graph showing fluorescence images of the fluorescence probe compound Lyso-NIR-pH prepared in example 1 at different pH values (

pH

4, 5, 6).

FIG. 9 is a graph showing fluorescence imaging of the fluorescent probe compound Lyso-NIR-pH prepared in example 1 before and after the addition of chloroquine, a drug, to HeLa cells.

FIG. 10 is a fluorescence imaging of the Lyso-NIR-pH fluorescent probe compound prepared in example 1 during drug dexamethasone-induced apoptosis.

Detailed Description

The present invention is described in further detail below with reference to examples, but it should not be construed that the scope of the above subject matter of the present invention is limited to the following examples, and that all the technologies realized based on the above subject matter of the present invention belong to the scope of the present invention.

Example 1

Synthesis of fluorescent probe compound Lyso-NIR-pH

(1) Synthesis of Compound 1

Adding 6g of 3-bromo-N, N-dimethylaniline and 60 ml of anhydrous tetrahydrofuran into a dry 250 ml round-bottom flask under the protection of argon, magnetically stirring for 5 minutes to dissolve the 3-bromo-N, N-dimethylaniline, then dropwise adding 13 ml of N-butyl lithium N-hexane solution with the molar concentration of 2.4mol/L into the reaction solution, reacting at 0 ℃ for 2 hours after dropwise adding, dissolving 2.2 ml of dichlorodimethylsilane in 10ml of anhydrous tetrahydrofuran, then dropwise adding the solution into the reaction solution, reacting to room temperature after dropwise adding, stirring overnight, adding 50ml of water to quench the reaction, extracting the reaction solution with diethyl ether (50 ml × 2), washing the extracted diethyl ether solution with saturated NaCl aqueous solution (50 ml × 3), and adding anhydrous Na₂SO₄Drying, evaporating the solvent by using a rotary evaporator to obtain a crude product, purifying the crude product by using a silica gel column, wherein the size of a silica gel particle is 200-300 meshes, and the volume ratio of an eluent is 80:1 of petroleum ether/ethyl acetate to obtain a compound 1, a yellow oily substance, 3.35g, the yield is 75%, and the synthetic route is as follows:

(2) synthesis of Compound 2

500mg of Compound 1(1.68mmol), 1260mg of 2-carboxybenzaldehyde (8.4mmol) and 37.5mg of copper bromide (0.168mmol) were added to a 100ml glass thick-walled pressure-resistant vesselSealing the tube, placing the tube in an oil bath kettle, heating and stirring at 140 ℃ for 5 hours, naturally cooling to room temperature, dissolving the reaction mixture in 50ml of dichloromethane, washing with 10% NaOH solution (50 ml. times.3), removing unreacted acidic by-products such as 2-carboxybenzaldehyde, and collecting the dichloromethane phase with anhydrous Na₂SO₄Drying, evaporating the solvent by using a rotary evaporator to obtain a crude product, purifying the crude product by using a silica gel column, wherein the size of a silica gel particle is 200-300 meshes, and the volume ratio of an eluent is petroleum ether/ethyl acetate (2: 1), so that a compound 2, a green solid, 0.33g and 45% of yield are obtained, and the synthetic route is as follows:

(3) synthesis of Compound 3

In a 100mL round bottom flask, 443mg of Compound 2, 20mL of dry 1, 2-dichloroethane, and 2mL of phosphorus oxychloride were added. The reaction solution in the flask was heated to 85 ℃ and refluxed for 4 hours, and the solvent was distilled off under reduced pressure to obtain a reaction residue. The reaction residue is dissolved in 20mL of dry acetonitrile and 5mL of triethylamine are added, to which 10mL of a solution of 4- (2-aminoethyl) -morpholine in acetonitrile containing 650mg are added dropwise. The reaction mixture was stirred at room temperature overnight, the solvent was removed under reduced pressure, and 50mL of CH was used₂Cl₂The residue was dissolved and washed with saturated aqueous NaCl (50 ml. times.3) anhydrous Na₂SO₄Drying, and evaporating the solvent by a rotary evaporator to obtain a crude product. The crude product was purified by silica gel column with a particle size of 200-300 mesh and eluent ratio of dichloromethane/methanol 25:1 to give compound 3, a grayish green solid compound, 277.0mg, 51.3% yield. The synthetic route is as follows:

(4) synthesis of Compound 4

105mg of Compound 3 are dissolved in 5mL of dry tetrahydrofuran at room temperature, and a total of 76mg of lithium aluminum hydride are added in small portions. After stirring the reaction at room temperature for 6 hours, the reaction was quenched by adding 5mL of methanol. The reaction solution was distilled under reduced pressure to remove the solvent to obtain a residue, and the residue was purified by a silica gel column to obtain the target fluorescent probe compound Lyso-NIR-pH, 38.0mg, yield 36.2%. The synthetic route is as follows:

example 2

Determination of fluorescence spectrogram of fluorescence probe compound Lyso-NIR-pH under different pH conditions

The measurement of fluorescence spectra was determined in 40mM Britton-Robinson buffer solution (containing 1% DMSO). Lyso-NIR-pH fluorescent probes were dissolved in dimethyl sulfoxide (DMSO) to make 500 μ M stock solutions. The test solution Lyso-NIR-pH (5.0 μ M) was prepared from a dilution of the 500 μ M stock solution described above. The different pH was obtained by adding a trace of an equal concentration of HCl or NaOH. The test solution was stored at room temperature for 30min, and the fluorescence spectrum was measured at an excitation wavelength of 620 nm. The fluorescence emission spectrum range is 640-800 nm, the excitation gap is 3nm, and the emission gap is 3 nm. Fluorescence spectrum As shown in FIG. 1, when the pH of BR buffer was greater than 7.4, the probe Lyso-NIR-pH was almost non-fluorescent because it was a stable non-fluorescent spiro structure. When the pH was decreased from 7.4 to 3, a significantly enhanced near infrared fluorescence signal occurred at 675nm due to H⁺The induced helical loop opened and fluorescence intensity increased 1400-fold. The fluorometric instrument used was a Perkin Elmer LS55 spectrofluorometer.

Example 3

Determination of ultraviolet-visible absorption spectrogram of fluorescence probe compound Lyso-NIR-pH under different pH conditions

FIG. 2 is a diagram showing an ultraviolet-visible absorption spectrum of a probe Lyso-NIR-pH (5. mu.M) at pH7.4 to pH 3. As can be seen from FIG. 2, a gradually increasing absorption peak was observed at 655nm as the pH was decreased. This indicates that the probe Lyso-NIR-pH responds to pH with its spiro structure in proton H⁺Is opened and sucked under the action ofThe yield is increased. The Instrument for measuring the ultraviolet-visible absorption spectrum was a TU-1900 type ultraviolet-visible spectrophotometer (Beijing Purkinje General Instrument Co., Ltd.).

Example 4

pH titration curve of Lyso-NIR-pH of fluorescent probe compound

FIG. 3 is a pH titration curve of the probe Lyso-NIR-pH. As can be seen from FIG. 3, the fluorescence intensity of the probe at 675nm gradually increased as the pH decreased (pH7.4 to pH 3). We plot the fluorescence intensity of the probe with pH according to Henderson-Hasselbalch equation to obtain FIG. 3, and further determine the pK of the probe Lyso-NIR-pH versus pH_aIt was 4.63. Lysosomes generally have a pH of 3.8-5.5, whereas the probe Lyso-NIR-pK of the pH_aJust within this range facilitates fluorescence monitoring imaging of lysosomal pH.

Example 5

Selective investigation of the fluorescent Probe Compound Lyso-NIR-pH

Selectivity is an important indicator for evaluating the performance of fluorescent probes. As shown in fig. 4, the fluorescence intensity of Lyso-NIR-pH (5 μ M) at pH7.4 shows that 20 times equivalent of a common cation (Na) was added⁺、K⁺、Ca²⁺、Mg²⁺、Fe³⁺、Cu²⁺、Zn²⁺、Al³⁺、Mn²⁺、Ni²⁺、Cd²⁺、Co²⁺、NH₄ ⁺) After that, there was no significant increase in fluorescence. In addition, when 20 times equivalent of common anion (Ac) was added separately^-、CO₃ ^2-、SO₄ ^2-、F^-、I^-、S₂O₃ ^-、NO^2-、Cl^-、Br^-、H₂PO^4-、HPO₄ ^2-) The increase in fluorescence intensity of Lyso-NIR-pH was also negligible. The fluorescence interference of various amino acids (valine, tryptophan, cysteine, glycine and homocysteine) and glutathione on the Lyso-NIR-pH value can be ignored in 200 times of equivalent. These results indicate that Lyso-NIR-pH has a specific fluorescence response to acidic pH, other analytes do not significantly interfere with the probe, and the requirement for p in practical biological samples can be metH monitoring requirements.

Example 6

Reversible investigation of the fluorescence Probe Compound Lyso-NIR-pH

Subsequently, we examined the reversibility of Lyso-NIR-pH (fig. 5). As can be seen from the figure, the fluorescence intensity value of the buffer system with the probe Lyso-NIR-pH (5 mu M) can be maintained above 90% of the initial value after three cycles of switching of the pH (pH7 to pH 3.5), and the song result shows that the probe Lyso-NIR-pH has better reversibility to the pH and can be used for multiple measurements of the pH.

Example 7

Lysosome co-localization experiment of fluorescence probe compound Lyso-NIR-pH

To examine the ability of the Lyso-NIR-pH probe to target lysosomes in cells, we performed co-localization experiments using the probe Lyso-NIR-pH (5 μ M) in contrast to the commercial lysosomal dye LysoTracker Green DND-26(500nM) and the mitochondrial dye Mito Tracker Green FM (1 μ M). First, we performed lysosomal co-localization experiments on HeLa cells. As shown in FIGS. 6a-6d, emission of Green fluorescence (510-550nm) was observed in the Green channel (FIG. 6a), which is the emission of the LysoSensor Green DND-26, and emission of red fluorescence (650-720nm) was observed in the red channel (FIG. 6b), which is the emission of red light from the probe Lyso-NIR-pH, and the co-localization coefficients of the two dyes were 0.90 by software treatment (FIG. 6 c). At the same time, the linear distribution of probe fluorescence with that of the commercial lysosomal dye showed a clear synchronicity (fig. 6 d). This suggests that the probe Lyso-NIR-pH is mainly accumulated in lysosomes and can be localized to lysosomes. Subsequently, we performed co-localization experiments of the probe with commercial mitochondrial dyes (6e-6 h). As shown, there was no significant overlap of the probe with the commercial mitochondrial localization agent fluorescence (FIG. 6g) and the linear profile was not synchronized (FIG. 6h), and the co-localization coefficient between the two was only 0.43 from FIG. 6g by software processing, which further confirms that Lyso-NIR-pH is lysosomal targeting, not mitochondrial targeting.

Example 8

Photostability investigation of the fluorescent Probe Compound Lyso-NIR-pH

By reaction with a conventional near-infrared dye Cy5-N₃By contrast, we examined the photostability of the fluorescent probe compound Lyso-NIR-pH (fig. 7). As shown, the fluorescence intensity of the fluorescent probe compound Lyso-NIR-pH was maintained at 88% of the initial value after 50 continuous excitation imaging, while the conventional near infrared dye Cy5-N was used₃The fluorescence intensity of (2) showed a significant decrease (10% of the initial value). The result shows that the fluorescence probe compound Lyso-NIR-pH has better light stability, can meet the requirement of in vivo long-time imaging, and obtains a stable fluorescence signal. The excitation wavelength was 635nm and the power was 20%.

Example 9

Imaging study of lysosome pH values of cells affected by fluorescence probe compound Lyso-NIR-pH to different pH buffer solutions

In HeLa cells, the fluorescence imaging of the probe Lyso-NIR-pH on changes in the pH of the lysosomes was examined. The specific operation steps are as follows: a549 cells and HeLa cells were incubated with Lyso-NIR-pH (5. mu.M) in PBS buffer (pH7.4) at 37 ℃ for 30min and then in a medium containing 10. mu.M Nigericin (a H)^+/K⁺Ionophore, to homogenize the pH inside and outside the cells) at different pH values (4.0, 5.0 and 6.0) in buffer at 37 ℃ for 30 min. Then, the image was obtained by confocal microscopy, and the image was obtained as shown in FIG. 8. As can be seen, the fluorescence signal gradually decreases with increasing pH of the system. The excitation wavelength is 635nm, and the red channel wavelength collection range is 650-720 nm.

Example 10

Imaging of lysosome pH changes induced by the drug chloroquine with the fluorescent probe compound Lyso-NIR-pH

The fluorescent probe compound Lyso-NIR-pH (5 μ M) was applied for monitoring of alkaline drug chloroquine-induced lysosomal pH changes (fig. 9). As shown in the figure, when chloroquine (100 mu M) is added within 150 seconds, the fluorescence intensity of cells is obviously reduced, and the result shows that the basic drug chloroquine induces the increase of the pH value of lysosome, so that the probe is in a spiral ring structure, the fluorescence is obviously reduced, and the capability of the probe for monitoring the pH value in the lysosome in real time is also shown.

Example 11

Imaging of apoptosis-induced lysosomal pH changes by the fluorescent probe compound Lyso-NIR-pH

Finally, probe Lyso-NIR-pH (5 μ M) was used for monitoring lysosomal pH changes during apoptosis (fig. 10). During apoptosis, lysosomal pH can undergo proton leakage, leading to an increase in pH. As shown in the figure, when 2 mu M dexamethasone (drug for inducing apoptosis) is added, the cell morphology is obviously changed within 2 hours, which indicates that the cell really undergoes the apoptosis process. At the same time, the fluorescence of the cells also gradually decreases. The results show that the probe Lyso-NIR-pH successfully realizes the monitoring of the lysosome pH rising process in the apoptosis process.

The foregoing embodiments illustrate the principles, principal features and advantages of the invention, and it will be understood by those skilled in the art that the invention is not limited to the foregoing embodiments, which are merely illustrative of the principles of the invention, and that various changes and modifications may be made therein without departing from the scope of the principles of the invention.

Claims

1. A near-infrared fluorescent probe for monitoring lysosome pH is characterized in that the structural formula of the near-infrared fluorescent probe is as follows:

2. the method for preparing the near-infrared fluorescent probe for monitoring the pH of the lysosome according to claim 1, which is characterized by comprising the following specific steps:

step S1: adding 3g of bromine-N, N-dimethylaniline and 60 ml of anhydrous ether into a dry 250 ml round-bottom flask under the protection of argon at-78 ℃, magnetically stirring for 5 minutes to dissolve the mixture, then dropwise adding 13 ml of N-butyl lithium N-hexane solution with the molar concentration of 2.4mol/L into the reaction solution, reacting at 0 ℃ for 2 hours after dropwise adding, then dissolving 2.2 ml of dichlorodimethylsilane into 10ml of anhydrous ether, dropwise adding the mixture into the reaction solution, reacting to room temperature after dropwise adding, stirring overnight, adding 50ml of water to quench the reaction, extracting the reaction solution with ether, drying the anhydrous sodium sulfate, and performing reduced pressure spin-drying on the solvent to obtain a crude product, and purifying the crude product by using a silica gel column to obtain a compound 1, wherein the structural formula is as follows:

step S3: in a 100mL round-bottom flask, 443mg of compound 2, 20mL of dried 1, 2-dichloroethane and 2mL of phosphorus oxychloride were added, the reaction solution in the flask was heated to 85 ℃ under reflux for 4 hours, the solvent was distilled off under reduced pressure to obtain a reaction residue, the reaction residue was dissolved in 20mL of dried acetonitrile and 5mL of triethylamine was added, then 10mL of an acetonitrile solution containing 650mg of 4- (2-aminoethyl) -morpholine was further added dropwise thereto, the reaction solution was stirred at room temperature overnight, the solvent was removed under reduced pressure, and then 50mL of CH was used₂Cl₂The residue was dissolved and washed 3 times with 50mL of saturated aqueous NaCl solution and anhydrous Na₂SO₄Drying, evaporating the solvent by a rotary evaporator to obtain a crude product, and purifying the crude product by a silica gel column to obtain a gray-green solid compound 3, wherein the structural formula is as follows:

step S4: dissolving 105mg of compound 3 in 5mL of dry tetrahydrofuran at room temperature, adding 76mg of lithium aluminum hydride in multiple times, stirring at room temperature for reaction for 3 hours, adding 5mL of methanol to quench the reaction, removing the solvent from the reaction solution by using a rotary evaporator to obtain a residue, and purifying the residue by using a silica gel column to obtain the target fluorescent probe compound Lyso-NIR-pH.

3. The use of the near-infrared fluorescent probe for monitoring lysosomal pH of claim 1 for selective detection of pH in aqueous environments, biological cell systems, wherein detection includes fluorescence detection in aqueous solution and cellular imaging detection.