阅读说明：本技术 一类近红外溶酶体荧光指示剂及应用 (Near-infrared lysosome fluorescence indicator and application ) 是由 张新富 李香丽 肖义 于 2021-07-27 设计创作，主要内容包括：一类近红外溶酶体荧光指示剂及应用,属于精细化工领域。该近红外溶酶体荧光指示剂独创性的将亲水性小分子基团引入至亲脂性BODIPY母体结构中,成功得到一系列最大发射波长在600-750 nm之间连续可调的两亲性近红外BODIPY衍生物。该设计使染料分子在水溶液中因为聚集而无荧光,在酸性脂类环境中因为解聚集而恢复荧光。同时,该类化合物易于透过细胞膜,快速靶向溶酶体,且仅在进入细胞之后发出荧光,相较其他溶酶体靶向荧光染料,具有免洗及背景荧光弱的优势。另外,该化合物使用低至纳米级浓度即可准确的标记溶酶体中,并能够用于长时间的荧光成像研究,对溶酶体的运动和形态变化进行长时间实时跟踪。(A near-infrared lysosome fluorescence indicator and application thereof belong to the field of fine chemical engineering. According to the near-infrared lysosome fluorescence indicator, a hydrophilic small molecular group is originally introduced into a parent structure of the lipophilic BODIPY, so that a series of amphiphilic near-infrared BODIPY derivatives with continuously adjustable maximum emission wavelength of 600-750nm are successfully obtained. The design enables the dye molecules to be non-fluorescent due to aggregation in aqueous solution and to recover fluorescence due to deaggregation in acidic lipid environment. Meanwhile, the compounds easily penetrate cell membranes, quickly target lysosomes, emit fluorescence only after entering cells, and have the advantages of no washing and weak background fluorescence compared with other lysosome targeted fluorescent dyes. In addition, the compound can be accurately marked in lysosomes by using the concentration as low as a nanometer level, and can be used for long-time fluorescence imaging research to track the motion and morphological change of the lysosomes in real time for a long time.)

1. A near-infrared lysosome fluorescence indicator is characterized by having the following structural general formula:

wherein R is₁，R₂Each independently is H, Cl, Br or I;

R₃＝H、CH₃、CF₃、C₃H₆COOCH₃、C₂H₄COOC₂H₅、C₂H₅、 or

X₂H or

Each Y is independently

2. The use of a class of near-infrared lysosomal fluorescence indicators according to claim 1, wherein the fluorescence indicator is used in the manufacture of a lysosome-targeted drug.

3. The use of a class of near-infrared lysosomal fluorescence indicators according to claim 1, wherein the fluorescence indicators are used for detection tracking of lysosomes.

4. The use of a class of near-infrared lysosomal fluorescence indicators according to claim 1, wherein the fluorescence indicators are used to track the location of bacterial infection, inflammation in vivo.

Technical Field

The invention relates to a near-infrared lysosome fluorescence indicator and application thereof, belonging to the field of fine chemical engineering.

Background

Lysosomes play an important role in various vital activities of cells, such as substance metabolism, cell membrane circulation, and apoptosis. Lysosomes are highly dynamic in nature and can constantly change their morphology and spatial distribution. Lysosomes are visually tracked, and active species, specific microenvironment and key physiological processes of the lysosomes are detected, so that understanding of molecular mechanisms of the lysosomes participating in life activities is facilitated, and important guiding significance is provided for treatment of diseases. To realize the above function, a fluorescent probe having excellent luminescent properties, high lysosome specificity, and little interference with the physiological function of lysosomes is required. In recent years, more lysosome-targeted fluorescent indicators are reported successively, but most of fluorescent dyes have emission peaks in the visible light range (400-650nm), wherein the fluorescent indicators in the ultraviolet region are overlapped with the autofluorescence in cells, which is not favorable for accurate imaging; most of recently reported lysosome probes and commercial lysosome indicators are concentrated in a visible region, the spectral overlapping degree among the probes is high, and the multichannel fluorescence imaging of two colors and more colors is difficult to meet. The near-infrared fluorophore has long absorption and emission wavelengths, biocompatibility is superior to that of a visible region fluorophore, imaging depth is greater than that of the visible region fluorophore, and an additional imaging waveband can be provided to match with other dyes to realize multicolor fluorescence imaging.

Based on the reasons, the development of the lysosome fluorescent probe which has high spectral range division, covers the visible region to the near infrared region, has stable targeting performance and strong luminous brightness has important significance, can solve a plurality of problems of related researches on lysosomes of living cells at present and has very high practical significance.

Disclosure of Invention

The invention provides an amphiphilic near-infrared lysosome fluorescence indicator taking BODIPY as a parent body and application thereof. The compound has the following structural general formula:

wherein, RR₁，R₂Each independently is H, Cl, Br or I;

R₃＝H、CH₃、CF₃、C₃H₆COOCH₃、C₂H₄COOC₂H₅、C₂H₅、or

X₁＝X₂H or

Each Y is independently

The preparation method of the near-infrared lysosome fluorescence indicator comprises the following reaction formula and reaction steps:

i) removing water with water separator, heating compound A, B, piperidine and glacial acetic acid in organic solvent for reaction, concentrating the reaction mixture, and adding CH₂Cl₂Extracting, washing the extracted organic phase with brine, drying, concentrating, and purifying with silica gel column to obtain compound C;

ii) under the protection of argon, compound Y-N₃Adding into organic solvent, adding into solution H₂CuSO of O₄·5H₂O, and sodium ascorbate by dissolving compound C in organic solvent, adding dropwise into the above reaction solution, stirring at room temperature, concentrating the reaction mixture, and mixingCH₂Cl₂And (4) extracting, washing an extracted organic phase with brine, drying, concentrating, and purifying with a silica gel column to obtain a compound D.

Wherein R is₁、R₂、R₃、X₂And Y is as defined in the general structural formula.

The organic solvent in the step i is one or more of benzene, toluene, xylene, chlorobenzene, dichlorobenzene, dioxane, nitrogen methyl pyrrolidone, dimethyl formamide, dimethyl acetamide, dimethyl sulfoxide, hexamethyl phosphoramide, sulfolane and benzonitrile, and the reaction temperature is 90-150 ℃; the reaction temperature in the step ii is room temperature, and the solvent is one of water, dimethylformamide, dimethylacetamide, dimethyl sulfoxide, nitrogen methyl pyrrolidone and acetonitrile.

The invention has the beneficial effects that: a near-infrared lysosome fluorescence indicator and application thereof are disclosed, wherein a hydrophilic small molecular group is originally introduced into a lipophilic BODIPY parent structure by the near-infrared lysosome fluorescence indicator, so that a series of amphiphilic near-infrared BODIPY derivatives with continuously adjustable maximum emission wavelength of 600-750nm are successfully obtained. The design regulates the hydrophilicity and lipophilicity of the molecule, so that the dye molecule does not have fluorescence due to aggregation in an aqueous solution and recovers the fluorescence due to deaggregation in an acidic lipid environment. Meanwhile, the compounds easily penetrate cell membranes, quickly target lysosomes, emit fluorescence only after entering cells, and have the advantages of no washing and weak background fluorescence compared with other lysosome targeted fluorescent dyes. In addition, the compound has high molar extinction coefficient and fluorescence quantum yield, strong luminous brightness and difficult bleaching, can be accurately marked in lysosomes by using the concentration as low as a nanometer level, and can be used for long-time fluorescence imaging research to track the motion and form change of the lysosomes in real time for a long time.

Drawings

FIG. 1 shows the emission spectra of compounds D1, D2, D25 and D26 in different solvents.

Figure 2 is an image of live cell lysosomal co-localization of compounds D1, D6, D22, D25.

Figure 3 is an image of live cell lysosomal co-localization of compounds D15, D20, D46, D51.

Figure 4 is an image of live cell lysosomes under leave-on conditions of compound D25.

Figure 5 is a graphic image of live cell lysosome imaging under chloroquine-stimulated conditions of compound D25.

Detailed Description

In order to make the technical scheme of the invention more clear, the following detailed description fully describes the specific embodiment of the invention in combination with the technical scheme and the attached drawings. The invention is illustrated but not limited by the following examples in which all parts and percentages are by weight unless otherwise indicated.

Example 1

i) A1(1.27mmol) and B (1.27mmol) were stirred in 30mL of dry toluene (molecular sieve for water removal before use), 1mL of piperidine and 1mL of glacial acetic acid were added for catalysis, the mixture was heated under reflux at 150 ℃, and water generated during the reaction was removed by a water separator. The reaction was monitored by TLC until the reaction was complete. After cooling to room temperature, most of toluene was removed from the reaction solution under reduced pressure, and the resulting solution was diluted with methylene chloride, washed with water and concentrated. The crude product was purified by silica gel column chromatography (dichloromethane/petroleum ether ═ 1/3) to give C1 as a solid.

ii) under argon, Compound C1 (82. mu. mol), Y1 (164. mu. mol), CuSO₄·5H₂O (82 μmol), sodium ascorbate (164 μmol) was added to a mixed solution of toluene, ethanol and water (v/v/v-12/4/1), the reaction was stirred at room temperature and monitored by TLC until compound C1 was reacted completely. The reaction mixture was concentrated and purified by silica gel column chromatography to give solid D1.

Example 2

Synthesis of compounds D2, D3 and D4 reference example 1.

Example 3

i) A2(1.27mmol) and B (1.27mmol) were stirred in 30mL of dry toluene (molecular sieve for water removal before use), 1mL of piperidine and 1mL of glacial acetic acid were added for catalysis, the mixture was heated under reflux at 150 ℃, and water generated during the reaction was removed by a water separator. The reaction was monitored by TLC until the reaction was complete. After cooling to room temperature, most of toluene was removed from the reaction solution under reduced pressure, and the resulting solution was diluted with methylene chloride, washed with water and concentrated. The crude product was purified by silica gel column chromatography (dichloromethane/petroleum ether ═ 1/3) to give C2 as a solid.

ii) under argon, Compound C2 (82. mu. mol), Y2 (164. mu. mol), CuSO₄·5H₂O (82 μmol), sodium ascorbate (164 μmol) was added to a mixed solution of toluene, ethanol and water (v/v/v-12/4/1), the reaction was stirred at room temperature and monitored by TLC until compound C2 was reacted completely. The reaction mixture was concentrated and purified by silica gel column chromatography to give solid D5.

Example 4

Synthesis of compounds D6, D7 and D8 reference was made to example 3.

Example 5

i) A3(1.27mmol) and B (1.27mmol) were stirred in 30mL of dry toluene (molecular sieve for water removal before use), 1mL of piperidine and 1mL of glacial acetic acid were added for catalysis, the mixture was heated under reflux at 150 ℃, and water generated during the reaction was removed by a water separator. The reaction was monitored by TLC until the reaction was complete. After cooling to room temperature, most of toluene was removed from the reaction solution under reduced pressure, and the resulting solution was diluted with methylene chloride, washed with water and concentrated. The crude product was purified by silica gel column chromatography (dichloromethane/petroleum ether ═ 1/3) to give C3 as a solid.

ii) under argon, Compound C3 (82. mu. mol), Y3 (164. mu. mol), CuSO₄·5H₂O (82 μmol), sodium ascorbate (164 μmol) was added to a mixed solution of toluene, ethanol and water (v/v/v-12/4/1), the reaction was stirred at room temperature and monitored by TLC until compound C3 was reacted completely. The reaction mixture was concentrated and purified by silica gel column chromatography to give solid D9.

Example 6

Synthesis of compounds D10, D11 and D12 reference was made to example 5.

Example 7

i) A4(1.27mmol) and B (1.27mmol) were stirred in 30mL of dry toluene (molecular sieve for water removal before use), 1mL of piperidine and 1mL of glacial acetic acid were added for catalysis, the mixture was heated under reflux at 150 ℃, and water generated during the reaction was removed by a water separator. The reaction was monitored by TLC until the reaction was complete. After cooling to room temperature, most of toluene was removed from the reaction solution under reduced pressure, and the resulting solution was diluted with methylene chloride, washed with water and concentrated. The crude product was purified by silica gel column chromatography (dichloromethane/petroleum ether ═ 1/3) to give C4 as a solid.

ii) under argon, Compound C4 (82. mu. mol), Y4 (164. mu. mol), CuSO₄·5H₂O (82 μmol), sodium ascorbate (164 μmol) was added to a mixed solution of toluene, ethanol and water (v/v/v-12/4/1), the reaction was stirred at room temperature and monitored by TLC until compound C4 was reacted completely. The reaction mixture was concentrated and purified by silica gel column chromatography to give solid D13.

Example 8

Synthesis of compounds D14, D15 and D16 reference is made to example 7.

Example 9

i) A5(1.27mmol) and B (1.27mmol) were stirred in 30mL of dry toluene (molecular sieve for water removal before use), 1mL of piperidine and 1mL of glacial acetic acid were added for catalysis, the mixture was heated under reflux at 150 ℃, and water generated during the reaction was removed by a water separator. The reaction was monitored by TLC until the reaction was complete. After cooling to room temperature, most of toluene was removed from the reaction solution under reduced pressure, and the resulting solution was diluted with methylene chloride, washed with water and concentrated. The crude product was purified by silica gel column chromatography (dichloromethane/petroleum ether ═ 1/3) to give C5 as a solid.

ii) under argon, Compound C5 (82. mu. mol), Y5 (164. mu. mol), CuSO₄·5H₂O (82 μmol), sodium ascorbate (164 μmol) was added to a mixed solution of toluene, ethanol and water (v/v/v-12/4/1), the reaction was stirred at room temperature and monitored by TLC until compound C5 was reacted completely. The reaction mixture was concentrated and purified by silica gel column chromatography to give solid D17.

Example 10

Synthesis of compounds D18, D19 and D20 reference is made to example 9.

Example 11

i) A6(1.27mmol) and B (1.27mmol) were stirred in 30mL of dry toluene (molecular sieve for water removal before use), 1mL of piperidine and 1mL of glacial acetic acid were added for catalysis, the mixture was heated under reflux at 150 ℃, and water generated during the reaction was removed by a water separator. The reaction was monitored by TLC until the reaction was complete. After cooling to room temperature, most of toluene was removed from the reaction solution under reduced pressure, and the resulting solution was diluted with methylene chloride, washed with water and concentrated. The crude product was purified by silica gel column chromatography (dichloromethane/petroleum ether ═ 1/3) to give C6 as a solid.

ii) under argon, Compound C6 (82. mu. mol), Y6 (164. mu. mol), CuSO₄·5H₂O (82 μmol), sodium ascorbate (164 μmol) was added to a mixed solution of toluene, ethanol and water (v/v/v-12/4/1), the reaction was stirred at room temperature and monitored by TLC until compound C6 was reacted completely. The reaction mixture was concentrated and purified by silica gel column chromatography to give solid D21.

Example 12

Synthesis of compounds D22, D23 and D24 reference example 11.

Example 13

i) A1(1.27mmol) and B (3.81mmol) were stirred in 30mL of dry toluene (molecular sieve for water removal before use), 1mL of piperidine and 1mL of glacial acetic acid were added for catalysis, the mixture was heated under reflux at 150 ℃, and water generated during the reaction was removed by a water separator. The reaction was monitored by TLC until the reaction was complete. After cooling to room temperature, most of toluene was removed from the reaction solution under reduced pressure, and the resulting solution was diluted with methylene chloride, washed with water and concentrated. The crude product was purified by silica gel column chromatography (dichloromethane/petroleum ether ═ 1/3) to give C7 as a solid.

ii) Compound C7 (66. mu. mol), Y1 (198. mu. mol), CuSO under argon₄·5H₂O (66 μmol), sodium ascorbate (132 μmol) was added to a mixed solution of toluene, ethanol and water (v/v/v-12/4/1), the reaction was stirred at room temperature and monitored by TLC until compound C7 was reacted completely. The reaction mixture was concentrated and purified by silica gel column chromatography to give solid D25.

Example 14

Synthesis of compounds D26, D27 and D28 reference example 13.

Example 15

i) A7(1.27mmol) and B (3.81mmol) were stirred in 30mL of dry toluene (molecular sieve for water removal before use), 1mL of piperidine and 1mL of glacial acetic acid were added for catalysis, the mixture was heated under reflux at 150 ℃, and water generated during the reaction was removed by a water separator. The reaction was monitored by TLC until the reaction was complete. After cooling to room temperature, most of toluene was removed from the reaction solution under reduced pressure, and the resulting solution was diluted with methylene chloride, washed with water and concentrated. The crude product was purified by silica gel column chromatography (dichloromethane/petroleum ether ═ 1/3) to give C8 as a solid.

ii) Compound C8 (66. mu. mol), Y2 (198. mu. mol), CuSO under argon₄·5H₂O (66 μmol), sodium ascorbate (132 μmol) was added to a mixed solution of toluene, ethanol and water (v/v/v-12/4/1), the reaction was stirred at room temperature and monitored by TLC until compound C8 was reacted completely. The reaction mixture was concentrated and purified by silica gel column chromatography to give solid D29.

Example 16

Synthesis of compounds D30, D31 and D32 reference example 15.

Example 17

i) A8(1.27mmol) and B (3.81mmol) were stirred in 30mL of dry toluene (molecular sieve for water removal before use), 1mL of piperidine and 1mL of glacial acetic acid were added for catalysis, the mixture was heated under reflux at 150 ℃, and water generated during the reaction was removed by a water separator. The reaction was monitored by TLC until the reaction was complete. After cooling to room temperature, most of toluene was removed from the reaction solution under reduced pressure, and the resulting solution was diluted with methylene chloride, washed with water and concentrated. The crude product was purified by silica gel column chromatography (dichloromethane/petroleum ether ═ 1/3) to give C9 as a solid.

ii) Compound C9 (66. mu. mol), Y3 (198. mu. mol), CuSO under argon₄·5H₂O (66 μmol), sodium ascorbate (132 μmol) was added to a mixed solution of toluene, ethanol and water (v/v/v-12/4/1), the reaction was stirred at room temperature and monitored by TLC until compound C9 was reacted completely. The reaction mixture was concentrated and purified by silica gel column chromatography to give solid D33.

Example 18

Synthesis of compounds D34, D35 and D36 reference is made to example 17.

Example 19

i) A9(1.27mmol) and B (3.81mmol) were stirred in 30mL of dry toluene (molecular sieve for water removal before use), 1mL of piperidine and 1mL of glacial acetic acid were added for catalysis, the mixture was heated under reflux at 150 ℃, and water generated during the reaction was removed by a water separator. The reaction was monitored by TLC until the reaction was complete. After cooling to room temperature, most of toluene was removed from the reaction solution under reduced pressure, and the resulting solution was diluted with methylene chloride, washed with water and concentrated. The crude product was purified by silica gel column chromatography (dichloromethane/petroleum ether ═ 1/3) to give C10 as a solid.

ii) Compound C10 (66. mu. mol), Y7 (198. mu. mol), CuSO under argon₄·5H₂O (66. mu. mol), sodium ascorbate (132. mu. mol) added to toluene, ethanol and waterThe solution was mixed (v/v/v-12/4/1), the reaction was stirred at room temperature and monitored by TLC until compound C10 was reacted to completion. The reaction mixture was concentrated and purified by silica gel column chromatography to give solid D37.

Example 20

Synthesis of compounds D38, D39 and D40 reference example 19.

Example 21

i) A10(1.27mmol) and B (3.81mmol) were stirred in 30mL of dry toluene (molecular sieve for water removal before use), 1mL of piperidine and 1mL of glacial acetic acid were added for catalysis, the mixture was heated under reflux at 150 ℃, and water generated during the reaction was removed by a water separator. The reaction was monitored by TLC until the reaction was complete. After cooling to room temperature, most of toluene was removed from the reaction solution under reduced pressure, and the resulting solution was diluted with methylene chloride, washed with water and concentrated. The crude product was purified by silica gel column chromatography (dichloromethane/petroleum ether ═ 1/3) to give C11 as a solid.

ii) Compound C11 (66. mu. mol), Y5 (198. mu. mol), CuSO under argon₄·5H₂O (66 μmol), sodium ascorbate (132 μmol) was added to a mixed solution of toluene, ethanol and water (v/v/v-12/4/1), the reaction was stirred at room temperature and monitored by TLC until compound C11 was reacted completely. The reaction mixture was concentrated and purified by silica gel column chromatography to give solid D41.

Example 22

Synthesis of compounds D42, D43 and D44 reference example 21.

Example 23

i) A11(1.27mmol) and B (3.81mmol) were stirred in 30mL of dry toluene (molecular sieve for water removal before use), 1mL of piperidine and 1mL of glacial acetic acid were added for catalysis, the mixture was heated under reflux at 150 ℃, and water generated during the reaction was removed by a water separator. The reaction was monitored by TLC until the reaction was complete. After cooling to room temperature, most of toluene was removed from the reaction solution under reduced pressure, and the resulting solution was diluted with methylene chloride, washed with water and concentrated. The crude product was purified by silica gel column chromatography (dichloromethane/petroleum ether ═ 1/3) to give C12 as a solid.

ii) Compound C12 (66. mu. mol), Y6 (198. mu. mol), CuSO under argon₄·5H₂O (66 μmol), sodium ascorbate (132 μmol) was added to a mixed solution of toluene, ethanol and water (v/v/v-12/4/1), the reaction was stirred at room temperature and monitored by TLC until compound C12 was reacted completely. The reaction mixture was concentrated and purified by silica gel column chromatography to give solid D45.

Example 24

Synthesis of compounds D46, D47 and D48 reference example 23.

Example 25

i) A12(1.27mmol) and B (3.81mmol) were stirred in 30mL of dry toluene (molecular sieve for water removal before use), 1mL of piperidine and 1mL of glacial acetic acid were added for catalysis, the mixture was heated under reflux at 150 ℃, and water generated during the reaction was removed by a water separator. The reaction was monitored by TLC until the reaction was complete. After cooling to room temperature, most of toluene was removed from the reaction solution under reduced pressure, and the resulting solution was diluted with methylene chloride, washed with water and concentrated. The crude product was purified by silica gel column chromatography (dichloromethane/petroleum ether ═ 1/3) to give C13 as a solid.

ii) Compound C13 (66. mu. mol), Y8 (198. mu. mol), CuSO under argon₄·5H₂O (66 μmol), sodium ascorbate (132 μmol) was added to a mixed solution of toluene, ethanol and water (v/v/v-12/4/1), the reaction was stirred at room temperature and monitored by TLC until compound C13 was reacted completely. The reaction mixture was concentrated and purified by silica gel column chromatography to give solid D49.

Example 26

Synthesis of compounds D50, D51 and D52 reference is made to example 25.

Example 27

Compounds D1, D2, D25, D26 were subjected to property studies. D1, D2, D25, D26 were dissolved in DMSO to obtain a 1mM stock solution. The stock solution was further diluted with acetonitrile to 1 μ M of the test solution and 1 μ L of trifluoroacetic acid (0.1% by volume) was added. These test solutions were tested for absorption, emission spectra, quantum yield and molar extinction coefficient. The absorption spectrum was measured by an Agilent 8453 uv-vis spectrophotometer and the fluorescence spectrum was measured by an Agilent spectrometer. Table 1 shows the maximum absorption wavelengths (. lamda.) of the compounds D1, D2, D25 and D26 in acetonitrile_ab) Maximum emission wavelength (λ)_em) Molar extinction coefficient (. epsilon.) and fluorescence quantum yield (. phi.)¹). As can be seen from the data in the table, the maximum emission wavelength of the compounds D1, D2, D25 and D26 is between 600 and 750nm, and the compounds have higher molar extinction coefficient and fluorescence quantum yield.

Table 1 Spectrum Properties in D1, D2, D25, D26 acetonitrile

Example 28

Compounds D1, D2, D25, D26 were subjected to property studies. D1, D2, D25, D26 were dissolved in DMSO to obtain a 1mM stock solution. Stock solution was mixed with 2mM CTAB (pH 3), ddH₂O (pH 7) was further diluted to 1. mu.M test solution. These test solutions were subjected to absorption and emission spectrum tests. The spectral properties were measured as in example 13.

As shown in FIG. 1, a, b, c, D are CTAB (2mM), ddH at pH 3 of D1, D2, D25, D26(1 μ M), respectively₂Emission spectrum in O (pH 7). CTAB (cetyl trimethyl ammonium bromide) is a cationic surfactant, the critical micelle concentration (cmc) of the CTAB is 0.9mM, and the CTAB exists in a micelle form in a 2mM CTAAB (pH 3) aqueous solution, thereby not only having the function of solubilizing dye molecules, but also simulating the acidic microenvironment of lysosomes. As can be seen from the figure, in 2mM CTAB at pH 3, the four compounds all exhibited stable emission in a lysosome-like microenvironment, while in H at pH 7₂No fluorescence exists in the O control group, so that the four compounds have no fluorescence characteristic outside cells, only show stable and strong fluorescence in the cells, and can be directly subjected to confocal imaging by preliminarily judging without washing by PBS (phosphate buffer solution) and can be used for lysosome fluorescence labeling.

Example 29

The spectral properties of the compounds D6, D15, D20, D22, D46, D51 are given in the form of the table below, under the same test conditions as in example 14 and example 15. As can be seen from the data in Table 2, the compounds D6, D15, D20, D22, D46 and D51 have the maximum emission wavelength between 600 and 750nm, and also have higher molar extinction coefficient and fluorescence quantum yield. In 2mM CTAB at pH 3, six compounds all exhibited stable emission in a lysosome-like microenvironment, while at pH 7H₂No fluorescence exists in O, so that the compound can be directly subjected to confocal imaging without PBS (phosphate buffer solution) cleaning, and can be used for lysosome fluorescence labeling.

TABLE 2 spectral properties of D6, D15, D20, D22, D46, D51

Example 30

Fluorescence imaging experiments were performed on compounds D1, D6, D22, D25, and RAW 264.7 cells at 37 deg.C with 5% CO₂After 24 hours incubation in the cell incubator, confocal imaging was performed by incubating for 5 minutes with 1 μ M staining for D1 and D6, for 60 minutes with 1 μ M staining for D22 and D25, and for 5 minutes with 1 μ M staining for DND-26 green lysosomal probe, washed three times with PBS.

As shown in fig. 2, the first row is a confocal imaging picture of DND-26, the second row is a confocal imaging picture of D1, D6, D22 and D25 from left to right, and the specific targeting of compounds D1, D6, D22 and D25 to lysosome can be clearly observed through the confocal imaging picture.

Example 31

Fluorescence imaging experiments were performed on compounds D15, D20, D46, D51, and RAW 264.7 cells at 37 deg.C with 5% CO₂After 24 hours incubation in the cell incubator, confocal imaging was performed by 1 μ M staining for D15 and D20 for 5 minutes, 1 μ M staining for D46 and D51 for 60 minutes, 1 μ M staining for DND-26 green lysosomal probe for 5 minutes, three washes with PBS.

Fig. 3 is a cell imaging co-localization map of compounds D15, D20, D46, D51, as shown in fig. 3, it can be clearly observed from the co-focused imaging picture that compounds D15, D20, D46, D51 specifically target lysosomes.

Example 32

Fluorescence imaging experiments were performed on compound D25 using RAW 264.7 cells at 37 ℃ with 5% CO₂After 24 hours incubation in the cell incubator, confocal imaging was performed directly using 100nM D25 stain for 60 minutes. As shown in fig. 4, it can be clearly observed from confocal imaging pictures that compound D25 can still be used for lysosomal imaging studies under the wash-free condition.

Example 33

Fluorescence imaging experiments were performed on compound D25 using RAW 264.7 cells at 37 ℃ with 5% CO₂Incubation in cell incubator for 24 hoursAfter incubation for 60 min with 1 μ M D25 staining, followed by incubation for 5min with 1 μ MDND-26 green lysosomal probe staining, three washes with PBS, and 20 μ M chloroquine addition for confocal imaging. Chloroquine can cause protons to escape from the lysosome, serving to raise the pH of the lysosome.

As shown in FIG. 5, under the condition of co-staining with DND-26, after addition of 20. mu.M chloroquine, the fluorescence intensity of DND-26 decreased significantly within 5min, while the fluorescence of compound D25 changed only slightly. It can be seen that the fluorescence intensity of compound D25 is stable and independent of lysosomal pH changes, indicating the location and morphology of lysosomes correctly.