WO2020045529A1

WO2020045529A1 - Novel fluorescent dye, composition for lipid droplet staining using same, and method for imaging intracellular lipid droplets using same

Info

Publication number: WO2020045529A1
Application number: PCT/JP2019/033790
Authority: WO
Inventors: 佑希立中; 公俊江副; 信之尾関; 石山　宗孝
Original assignee: 株式会社同仁化学研究所
Priority date: 2018-08-28
Filing date: 2019-08-28
Publication date: 2020-03-05
Also published as: JP2021191807A

Abstract

The present invention discloses a fluorescent dye represented by general formula (I), (II) or (III), which is specific to lipid droplets and enables lipid droplet staining with high sensitivity, while having no cytotoxicity and high intracellular retentivity, and which is applicable for kinetics observation of lipid droplets. The present invention also discloses a composition for lipid droplet staining and a method for imaging intracellular lipid droplets, each of which uses this fluorescent dye. In the formulae, each of R¹ and R² independently represents a hydrogen atom or a group that is selected from the group consisting of a cyano group, a formyl group, an aryl group, a substituted aryl group, a heteroaryl group and a substituted heteroaryl group, with at least one of R¹ and R² being a cyano group, a formyl group, an aryl group, a substituted aryl group, a heteroaryl group or a substituted heteroaryl group; R³ represents a functional group represented by general formula (i), (ii) or (iii); R¹¹ represents a functional group represented by general formula (i); and R¹² represents a nitro group, a cyano group, a sulfonyl group, a sulfoxyl group, a carbonyl group or a carboxyl group.

Description

Novel fluorescent dye, composition for staining lipid droplets using the same, and method for imaging intracellular lipid droplets

The present invention relates to a novel fluorescent dye which can be suitably used for staining lipid droplets, a composition for staining lipid droplets using the same, and a method for imaging intracellular lipid droplets.

Fat droplets (also called fat globules, oil droplets, etc.) are intracellular organelles that store neutral fats such as triglycerides inside and are surrounded by a monolayer of phospholipids. Fat droplets were mainly present in fat cells, and energy storage was considered to be the main role. Recent studies have shown that lipid droplets are widely present in cells other than fat cells.

脂肪 The fat droplets of fat cells are as large as 10 to 100 μm, and store a large amount of fat for a long time. However, the fat droplets of non-adipocytes are much smaller than 1 μm and temporarily store only a small amount of fat. Cells take up fatty acids from the blood and use them as an energy source, while re-synthesizing some fatty acids into triglycerides and storing them. This is the small fat droplet found in non-fat cells.

脂肪 As described above, conventionally, fat droplets are storage organelles of neutral lipids, and have been regarded simply as fat storage. However, recent studies have revealed that many proteins exist on the surface of lipid droplets and play an important role in regulating lipid metabolism in the body. Fat accumulation and decomposition are strictly regulated, and its breakdown is said to lead to various diseases such as obesity and diabetes. Recent studies have revealed that lipid droplets play an indispensable role not only in energy storage but also in film formation, intracellular signal transmission, and the like, and are being recognized as important intracellular organelles. (See Non-Patent Document 1) It has also been suggested that intracellular phenomena such as autophagy (see Non-Patent Document 2) and cell senescence (see Non-Patent Document 3) are associated with lipid droplets. It is hoped that the mechanism of fusion and decomposition will be elucidated in detail.

蛍光 Therefore, fluorescent dyes with high lipid droplet specificity, for elucidating the mechanism of formation, growth, fusion and decomposition of lipid droplets in detail, have attracted great interest in recent years. Since lipid droplets are expected to contribute to the diagnosis and treatment of diseases related to neutral lipid droplets such as triacylglycerol and cholesterol ester, the dynamics of lipid droplets in living cells and individuals can be observed. Molecular probes are essential. Lipid droplets form a functionally and morphologically diverse subpopulation, and no single marker has ever existed for staining all lipid droplets. One of the methods for visualizing lipid droplets is to stain a core portion (lipid ester) of the lipid droplets with a molecule having strong lipophilicity. Conventionally, imaging methods using fluorescent dyes such as Nile Red and BODYPY 493/503 have been used to observe the dynamics of lipid droplets in living cells and individuals (for example, Patent Document 1, Non-patent Documents 4 and 5). reference).

Japanese Patent No. 624014

However, Nile @ Red is a hydrophobic field probe having a low fluorescence intensity in an aqueous solution and a high fluorescence intensity in a hydrophobic environment, but has background fluorescence in a cell and a poor S / N ratio. In addition, Nile @ Red causes a large change in the absorption wavelength region due to solvatochromism (a phenomenon in which the color of a compound changes due to a change in the polarity of a solvent), which affects imaging. Furthermore, observation of multiple stains other than fat droplets is difficult, and it cannot be used in combination with other organelle-specific fluorescent dyes or with other staining methods such as immunostaining.

BODYPY 493/503 is a bright green fluorescent dye, which has a unique hydrophobic property and is easily distributed in hydrophobic parts such as fat droplets, so that fat droplets can be detected. However, similar to Nile @ Red, there is background fluorescence in the cells, and the lipid droplet detection specificity is lacking.

Furthermore, in order to observe the dynamics of lipid droplets, it is necessary to carry out long-term culture after introducing a fluorescent dye into cells. However, these dyes have problems in cytotoxicity and retention in cells, and are not suitable for long-term observation.

The present invention has been made in view of the above circumstances, is specific to lipid droplets, enables high-sensitivity fluorescent staining, has no cytotoxicity, has high intracellular retention, and can be applied to dynamic observation of lipid droplets. An object of the present invention is to provide a novel fluorescent dye, a composition for staining lipid droplets using the same, and an imaging method for intracellular lipid droplets.

The first aspect of the present invention that meets the above-mentioned object solves the above-mentioned problems by providing a fluorescent dye represented by the following general formula (I), (II) or (III).

In the above general formulas (I), (II) and (III), R ¹ and R ² each independently represent a hydrogen atom, a cyano group, a formyl group, an aryl group, a substituted aryl group, a heteroaryl group and a substituted hetero group. Selected from the group consisting of aryl groups, and at least one of R ¹ and R ² is any one of a cyano group, a formyl group, an aryl group, a substituted aryl group, a heteroaryl group, and a substituted heteroaryl group;
R ³ is a functional group represented by any of the following general formulas (i), (ii), and (iii); R ¹¹ is a functional group represented by the following general formula (i); ¹² is a nitro group, a cyano group, a sulfonyl group, a sulfoxyl group, a carbonyl group or a carboxyl group,

In the above general formulas (i), (ii) and (iii),
R ⁴ and R ⁵ are each independently a hydrocarbon group having 1 to 10 carbon atoms and optionally containing one or both of a branched and unsaturated bond, and R ⁴ and R ⁵ are directly or An atom, an oxygen atom, a nitrogen atom and a sulfur atom may be bonded to each other to form a ring,
R ⁶ and R ⁷ are each independently selected from the group consisting of a hydrogen atom, a cyano group, a formyl group, an aryl group, a substituted aryl group, a heteroaryl group and a substituted heteroaryl group, and R ⁶ and R ⁷ At least one of them is a cyano group, a formyl group, an aryl group, a substituted aryl group, a heteroaryl group or a substituted heteroaryl group,
R ⁸ is selected from the group consisting of a hydrogen atom, a cyano group, a formyl group, an aryl group, a substituted aryl group, a heteroaryl group and a substituted heteroaryl group;
R ⁹ and R ¹⁰ are each independently selected from the group consisting of a hydrogen atom, a cyano group, a formyl group, an aryl group, a substituted aryl group, a heteroaryl group and a substituted heteroaryl group, and R ⁹ and R ¹⁰ At least one of them is any of a cyano group, a formyl group, an aryl group, a substituted aryl group, a heteroaryl group, and a substituted heteroaryl group.

蛍光 The fluorescent dye according to the first aspect of the present invention is preferably represented by any of the following formulas (1) to (6) and (9).

The second aspect of the present invention solves the above-mentioned problems by providing a composition for staining lipid droplets containing one or more fluorescent dyes represented by the following general formulas (I), (II) or (III). Is the solution.

In the general formulas (I), (II) and (III), R ¹ and R ² each independently represent a hydrogen atom, a cyano group, a formyl group, an aryl group, a substituted aryl group, a heteroaryl group, and a substituted heteroaryl Selected from the group consisting of groups, and at least one of R ¹ and R ² is any one of a cyano group, a formyl group, an aryl group, a substituted aryl group, a heteroaryl group, and a substituted heteroaryl group;
R ³ is a formyl group or a functional group represented by any of the following general formulas (i), (ii) and (iii), and R ¹¹ is a functional group represented by the following general formula (i) R ¹² is any one of a nitro group, a cyano group, a sulfonyl group, a sulfoxyl group, a carbonyl group and a carboxyl group;

In a third aspect of the present invention, a step of introducing one or a plurality of fluorescent dyes represented by the following general formulas (I), (II) or (III) into lipid droplets in cells:
The object is achieved by providing a method for imaging intracellular lipid droplets, which comprises a step of measuring fluorescence emitted from the fluorescent dye introduced into the lipid droplets.

In the above general formulas (I), (II) and (III), R ¹ and R ² each independently represent a hydrogen atom, a cyano group, a formyl group, an aryl group, a substituted aryl group, a heteroaryl group and a substituted hetero group. Selected from the group consisting of aryl groups, and at least one of R ¹ and R ² is any one of a cyano group, a formyl group, an aryl group, a substituted aryl group, a heteroaryl group, and a substituted heteroaryl group;
R ³ is a formyl group or a functional group represented by any of the following general formulas (i), (ii) and (iii), and R ¹¹ is a functional group represented by the following general formula (i) R ¹² is any one of a nitro group, a cyano group, a sulfonyl group, a sulfoxyl group, a carbonyl group and a carboxyl group;

In the composition for staining lipid droplets according to the second aspect of the present invention and the method for imaging intracellular lipid droplets according to the third aspect of the present invention, the fluorescent dye is represented by the following formulas (1) to (9). It is preferable to be represented by any of them.

According to the present invention, lipid droplet specificity is high, and strong fluorescence is emitted in a hydrophobic environment such as a lipid droplet, but little background emission is exhibited in a hydrophilic environment such as a cytoplasm and fluorescence observation with high sensitivity Is provided. Since the fluorescent dye of the present invention has naphthalene, pyrene or perylene as a fluorescent chromophore, it is relatively easy to synthesize, and has a desired fluorescent property (excitation spectrum, fluorescence spectrum, etc.) by molecular design. Design dyes. This makes it possible not only to observe clear lipid droplets in cells but also to realize multiple staining for studying the relationship with other organelles by combining fluorescent dyes having different wavelengths. Furthermore, since the fluorescent dye of the present invention has low cytotoxicity and high retention in lipid droplets, the dynamics of lipid droplets in living cells can be observed for a long time. As described above, according to the present invention, a fluorescent dye as a useful tool for analyzing the biological function of lipid droplets in living cells, a composition for staining lipid droplets using the same, and an imaging method for intracellular lipid droplets are disclosed. Provided.

It is a photograph which shows the result of the confocal microscope observation of the HeLa cell incubated with the compound of this invention after oleic acid treatment. It is a photograph which shows the result of the confocal microscope observation of the HeLa cell incubated with the compound of this invention and Nile @ Red after oleic acid treatment. It is a photograph which shows the result of the confocal microscope observation of the HeLa cell incubated with the compound of this invention, Nile @ Red or BODYPY @ 493/503 after oleic acid treatment. It is the photograph which shows the result of the confocal microscope observation of HepG2 cell after incubating with the compound of this invention, Nile @ Red or BODYPY @ 493/503, and after 24 hours. FIG. 9 is a view showing a measurement result of flow cytometry in Example 3. 10 is a histogram showing the relationship between each cell group and the fluorescence intensity obtained from the results of flow cytometry in Example 3.

The fluorescent dye according to the first embodiment of the present invention (may be simply referred to as “fluorescent dye”) is represented by the following general formula (I), (II) or (III). .

In the above general formulas (i), (ii) and (iii),
R ⁴ and R ⁵ are each independently a hydrocarbon group having 1 to 10 carbon atoms and optionally containing one or both of a branched and unsaturated bond, and R ⁴ and R ⁵ are directly or One or more of an atom, an oxygen atom, a nitrogen atom (amino group) and a sulfur atom (thioether group, sulfinyl group or sulfonyl group) (a ring in which a nitrogen atom, an oxygen atom and a sulfur atom are formed as constituents of a functional group) May also be included within the group) to form a ring,
R ⁶ and R ⁷ are each independently selected from the group consisting of a hydrogen atom, a cyano group, a formyl group, an aryl group, a substituted aryl group, a heteroaryl group and a substituted heteroaryl group, and R ⁶ and R ⁷ At least one of them is a cyano group, a formyl group, an aryl group, a substituted aryl group, a heteroaryl group or a substituted heteroaryl group,
R ⁸ is selected from the group consisting of a hydrogen atom, a cyano group, a formyl group, an aryl group, a substituted aryl group, a heteroaryl group and a substituted heteroaryl group;
R ⁹ and R ¹⁰ are each independently selected from the group consisting of a hydrogen atom, a cyano group, a formyl group, an aryl group, a substituted aryl group, a heteroaryl group and a substituted heteroaryl group, and R ⁹ and R ¹⁰ At least one of them is any of a cyano group, a formyl group, an aryl group, a substituted aryl group, a heteroaryl group, and a substituted heteroaryl group.

Among the fluorescent dyes represented by the general formulas (I), (II) or (III), preferred specific examples are represented by any of the following formulas (1) to (6) and (9). Things. Hereinafter, the “compound represented by the formula (n) (n is an integer)” may be abbreviated as “compound n”.

Compounds (1) to (8) can be synthesized by using any available method using a commercially available pyrene or perylene derivative as a starting material. For example, compound (3), compound (5) and compound (6) can be synthesized according to the following scheme.

Compounds (1) and (4) can be synthesized from an aldehyde derivative using the same reagents and conditions as in the reaction for synthesizing compound (6) from compound (5). The compound (2) can be synthesized by reacting 3-bromoperylene or perylene-3-ylboronic acid with an amine using a metal catalyst.

Compound (9) can be synthesized, for example, by reacting 1-nitronaphthalene-4-sulfonyl chloride with Nile Blue according to the following scheme.

The composition for fat droplet dyeing according to the second embodiment of the present invention (hereinafter, may be abbreviated as “composition for fat droplet dyeing” or “composition”) is the present invention described above. , Ie, one or more of the fluorescent dyes represented by the above general formulas (I), (II) or (III).

具体 Specific examples of preferred fluorescent dyes that can be used in the composition include those represented by any of the following formulas (1) to (9). The compound (7) and the compound (8) are known compounds, but can be preferably used for the composition.

The composition may be in the form of a solid containing one or more of the above-mentioned fluorescent dyes, or in the form of a liquid in which one or more of the above-mentioned fluorescent dyes is dissolved in an appropriate solvent, solution or buffer solution. There may be. The composition for lipid droplet staining may comprise one or more other dyes that can be used for specific or non-specific staining of lipid droplets or other organelles.

The method for imaging intracellular lipid droplets according to the third embodiment of the present invention (hereinafter, may be abbreviated as “imaging method”) may be any of the above general formulas (I), (II) and (III). ) And a step of measuring the fluorescence emitted from the fluorescent dye introduced into the lipid droplet inside the cell.

蛍光 Fluorescent dyes used in the imaging method and preferred specific examples thereof are the same as in the case of the composition for staining lipid droplets according to the second embodiment of the present invention, and thus detailed description is omitted.

The introduction of the fluorescent dye into the lipid droplets can be performed using any known method. For example, the fluorescent dye can be added to cells seeded in a medium and incubated for a predetermined time. The measurement of the fluorescence emitted from the fluorescent dye introduced inside the fat droplet can be performed using any known means, but can be performed by observing a fluorescent image using a confocal microscope or a fluorescent microscope. it can. Since the above-described fluorescent dye used in the imaging method has low cytotoxicity and high retention in fat droplets, it is possible to observe fat droplets in living cells over time.

Next, an example performed to confirm the operation and effect of the present invention will be described.
Example 1 Synthesis of Fluorescent Dye [1]: Synthesis of Compounds (7) and (3) The synthesis of compounds (7) and (3) each having a 1- (4-methoxyphenyl) ethenyl group was as follows. According to the scheme shown, the reaction was carried out using a Wittig reaction between an aldehyde derivative and (4-methoxybenzyl) triphenylphosphonium chloride.

Synthesis of Compound (7) In a 100 mL eggplant flask, 1-pyrenecarboxaldehyde (460 mg, 2.0 mmol), (4-methoxybenzyl) triphenylphosphonium chloride (1,008 mg, 4.0 mmol), 30 mL of dichloromethane, and 3 mL of A 50% NaOH aqueous solution was added, and the mixture was stirred at room temperature overnight. The reaction solution was extracted with dichloromethane, and the extract was distilled off with an evaporator. Purification was performed using a silica gel column and 100% chloroform as an eluent. 300 mg of pale yellow crystals were obtained (44.9% yield).

^{1 H NMR (400 MHz, CDCl} 3): δ = 3.81 (3H, s), 6.98 (2H, d, J = 7.6 Hz), 7.31 (1H, 7.30, d, J = 16.0 Hz), 7.63 (2H, d, J = 7.6 Hz), 7.98-8.19 (8H, m), 8.31 (1H, d, J = 8.0 Hz), 8.50 (1H, d, J = 9.2 Hz).

Synthesis of Compound (3) In a 100 mL eggplant flask, 3-perylenecarboxaldehyde (8) (560 mg, 2.0 mmol), (4-methoxybenzyl) triphenylphosphonium chloride (1,008 mg, 4.0 mmol), 30 mL of dichloromethane Then, 3 mL of a 50% aqueous NaOH solution was added, and the mixture was stirred at room temperature overnight. The precipitated crystals were filtered to obtain 198 mg of orange crystals (yield 25.7%).

^{1 H NMR (400 MHz, CDCl} 3): δ = 3.87 (3H, s), 6.95 (2H, d, J = 7.2 Hz), 7.15 (1H, d, J = 16.4 Hz), 7.47-7.57 (5H, m), 7.67-7.72 (3H, m), 7.75 (1H, J = 7.6 Hz), 8.08 (1H, d, J = 8.8 Hz), 8.19-8.25 (4H, m).

[2]: Synthesis of Compounds (5) and (6) Compounds (5) and (6) were synthesized according to the following scheme.

Synthesis of 3-bromoperylene Perylene (1 g, 4.0 mmol) and 300 mL of dichloromethane were added to a 500 mL eggplant flask, and stirred for 1 hour to dissolve. To this solution, a solution of N-bromosuccinimide (1.78 g, 10.0 mmol) dissolved in 100 mL of dichloromethane was added dropwise, and the mixture was stirred at room temperature overnight. The reaction solution was concentrated by an evaporator to obtain yellow crystals. The crystals were washed with 100 mL of methanol to obtain 1.6 g of yellow crystals (yield: 99%).

¹ H NMR (400 MHz, CDCl ₃ ): δ = 7.48-7.52 (2H, m), 7.59 (1H, t, J = 15.6 Hz), 7.67-7.73 (2H, m), 7.77 (1H, d, J = 7.6 Hz), 8.01 (1H, d, J = 8.4 Hz), 8.09 (1H, J = 9.2 Hz), 8.17-8.26 (3H, m).

Synthesis of Compound (5) To a 200 mL eggplant flask were added 3-bromoperylene (564 mg, 1.7 mmol) and 85 mL of THF, and the mixture was stirred in a dry ice-acetone bath at -78 ° C. To this solution, 3 mL of n-BuLi (4.8 mmol) was added dropwise and stirred for 1 hour. Thereafter, 3- (N, N-dimethylamino) acrolein (2 g, 20 mmol) was added, and the mixture was stirred overnight at room temperature. The reaction solution was distilled off with an evaporator. Purification was performed using a silica gel column and 100% chloroform as an eluent. 220 mg (yield: 42.2%) of slightly yellow crystals were obtained.

^{1 H NMR (400 MHz, CDCl} 3): δ = 6.87 (1H, dd, J = 16.0, 8.8 Hz), 7.53 (2H, t, J = 7.4 Hz), 7.62 (1H, t, J = 4.0 Hz) , 7.76 (2H, dd, J = 8.8 Hz), 7.84 (1H, d, J = 8.8 Hz), 8.05 (1H, d, J = 8.4 Hz), 8.22-8.31 (5H, m), 9.86 (1H, d, J = 8.4 Hz).

Synthesis of Compound (6) Compound (5) (220 mg, 0.7 mmol), malononitrile (190 mg, 2.9 mmol), and 55 mL of dichloroethane were added to a 300-mL eggplant flask and stirred to dissolve. To this solution, 2.9 mL of TiCl ₄ (2.9 mmol) and 700 μL of pyridine were added, and the mixture was refluxed overnight. After returning to room temperature, the reaction solution was extracted with dichloromethane, and the extract was distilled off with an evaporator. Purification was performed using a silica gel column and 100% chloroform as an eluent. 60 mg (yield: 23.5%) of black purple crystals were obtained.

¹ H NMR (400 MHz, CDCl ₃ ): δ = 7.41 (1H, t, J = 13.0), 7.61 (3H, m), 7.76 (3H, m), 7.90 (1H, t, J = 10.0 Hz), 7.98 (1H, d, J = 8.0 Hz), 8.06 (1H, d, J = 14.8 Hz), 8.23-8.32 (4H, m).

Synthesis of Compound (9) Compound (9) was synthesized by stirring Nile Blue and 1-nitronaphthalene-4-sulfonyl chloride in DMF in the presence of diisopropylethylamine.

Example 2: Staining of lipid droplets in cells (1)
In order to examine whether the compound (7), the compound (3), and the compound (6) can specifically stain lipid droplets in living cells, the lipid droplets are small, but fat is added by adding oleic acid. The staining characteristics of lipid droplets were examined using HeLa cells capable of inducing droplets and HepG2 cells having a high lipid droplet content.

HeLa cells were seeded in μ-slide 8 well (Ibidi), 200 μmol / L oleic acid diluted with a serum medium was added, and the cells were cultured overnight at 37 ° C. in a CO ₂ incubator. 0.1 μmol / L of the compound (7) and the compound (3) diluted with a serum medium were added, and 1 μmol / L of the compound (6) was added, and the mixture was incubated for 30 minutes. Thereafter, observation was performed with a confocal microscope.

HepG2 cells were seeded in μ-slide 8 well (Ibidi) and cultured overnight at 37 ° C. in a CO ₂ incubator. 0.1 μmol / L of the compound (7) and the compound (3) diluted with a serum medium were added, and 1 μmol / L of the compound (6) was added, and the mixture was incubated for 30 minutes. Thereafter, observation was performed with a confocal microscope.

First, an experiment was performed using HeLa cells into which lipid droplets were introduced by treatment with 200 μmol / L oleic acid. HeLa cells in which 1 μmol / L of compound (7), compound (3) and compound (6) were each incubated at 37 ° C. for 15 minutes were evaluated using a confocal microscope. The results are shown in FIG. In FIG. 1, "DIC" indicates a differential interference image, and "FL" indicates a fluorescent image (the same applies hereinafter). From these results, it was confirmed that a fluorescently stained portion was present in the cells.

At the same time, the fluorescence observed from a co-staining experiment with Nile Red (100 nmol / L), a commercially available lipid droplet indicator, confirmed that compound (3) was localized in the lipid droplets (see FIG. 2). .

イメージング In addition, imaging comparison was performed using Nile Red and BODYPY 493/503, which is also a commercially available fat droplet indicator (see FIG. 3). In each of the compound (7), the compound (3) and the compound (6), a fluorescent image was observed only in a part of the cells, whereas when Nile Red and BODYPY $ 493/503 were used, the whole cytoplasm was A fluorescent image was obtained. From these results, it was confirmed that when the compound (7), the compound (3) and the compound (6) were used, a fluorescent image could be specifically obtained for fat droplets.

Furthermore, using HepG2 cells containing a large amount of lipid droplets, the cells were observed 24 hours after staining of the lipid droplets. FIG. 4 shows the results. Both Nile @ Red and BODYPY @ 493/503 have low intracellular retention properties, and after 24 hours from the staining, the dye leaks out of the cells and a fluorescent image specific to lipid droplets cannot be observed. ) And compound (3), the fluorescence image specific to the lipid droplets is maintained even after 24 hours from the staining, the retention in the cell is high, and the lipid droplets are observed for a long period of time. It was confirmed that it was possible.

Example 3: Staining of lipid droplets in cells (2)
The flow cytometer can detect the fluorescence intensity of each individual cell simply, quickly and with high sensitivity, and can quantify the characteristics of the cell population. When analyzing lipid droplets using a flow cytometer, a higher lipid droplet specificity is required because the detected fluorescent signal depends on the amount of fluorescent dye labeled on the cells. In flow cytometer analysis, cells sometimes cause autofluorescence in a short wavelength region. Excitation by a 488 nm laser caused by a constituent material of the cell causes slight emission even without fluorescent staining, and false positives may be detected. On the longer wavelength side, auto-fluorescence interference is reduced, and a fluorescent dye that emits fluorescence exceeding 600 nm is required. Using the compound (9), evaluation of the amount of intracellular lipid droplets by a flow cytometer was examined.

HeLa cells into which lipid droplets were introduced by treatment with 200 μmol / L oleic acid (indicated by “OA” in FIGS. 5 and 6) and 5 μmol / L TriacsinΔC (acyl-CoA synthetase inhibitor) were added (FIG. 5). , 6).) HeLa cells with reduced lipid droplets were prepared, and 0.5 μmol / L of compound (9) was incubated at 37 ° C. for 2 hours. Evaluation was performed using a meter. FIG. 5 shows the results. From the results of FIG. 5, it was confirmed that the change in the amount of intracellular lipid droplets can be read out as a change in the fluorescence intensity by the flow cytometer.

において Comparing the histograms obtained in FIG. 6 between HeLa cells treated with oleic acid (OL) and untreated HeLa cells (Control), a significant difference in fluorescence intensity was observed. From the results, it was revealed that the compound (9) specifically stains lipid droplets and is a practical reagent in evaluating an appropriate amount of intracellular fat using a flow cytometer.

Comparing the histograms obtained with untreated HeLa cells (Control) and those obtained with HeLa cells (TC) that inhibited lipid droplet formation, it was confirmed that the intracellular fluorescence signal was attenuated. This result indicates that compound (9) is also useful for searching for a lipid droplet inhibitor.

The present invention allows various embodiments and modifications without departing from the broad spirit and scope of the present invention. Further, the above-described embodiment is for describing the present invention, and does not limit the scope of the present invention. That is, the scope of the present invention is shown not by the embodiment but by the claims. Various modifications made within the scope of the claims and equivalents thereof are considered to be within the scope of the present invention.

This application is based on Japanese Patent Application No. 2018-158925 filed on August 28, 2018 and includes the specification, claims, drawings, and abstract. The disclosure in the above-mentioned Japanese Patent Application is incorporated herein by reference in its entirety.

Claims

A fluorescent dye represented by the following general formula (I), (II) or (III).

In the above general formulas (I), (II) and (III), R 1 and R 2 each independently represent a hydrogen atom, a cyano group, a formyl group, an aryl group, a substituted aryl group, a heteroaryl group and a substituted hetero group. Selected from the group consisting of aryl groups, and at least one of R 1 and R 2 is any one of a cyano group, a formyl group, an aryl group, a substituted aryl group, a heteroaryl group, and a substituted heteroaryl group;
R 3 is a functional group represented by any of the following general formulas (i), (ii), and (iii); R 11 is a functional group represented by the following general formula (i); 12 is a nitro group, a cyano group, a sulfonyl group, a sulfoxyl group, a carbonyl group or a carboxyl group,

In the above general formulas (i), (ii) and (iii),
R 4 and R 5 are each independently a hydrocarbon group having 1 to 10 carbon atoms and optionally containing one or both of a branched and unsaturated bond, and R 4 and R 5 are directly or An atom, an oxygen atom, a nitrogen atom and a sulfur atom may be bonded to each other to form a ring,
R 6 and R 7 are each independently selected from the group consisting of a hydrogen atom, a cyano group, a formyl group, an aryl group, a substituted aryl group, a heteroaryl group and a substituted heteroaryl group, and R 6 and R 7 At least one of them is a cyano group, a formyl group, an aryl group, a substituted aryl group, a heteroaryl group or a substituted heteroaryl group,
R 8 is selected from the group consisting of a hydrogen atom, a cyano group, a formyl group, an aryl group, a substituted aryl group, a heteroaryl group and a substituted heteroaryl group;
R 9 and R 10 are each independently selected from the group consisting of a hydrogen atom, a cyano group, a formyl group, an aryl group, a substituted aryl group, a heteroaryl group and a substituted heteroaryl group, and R 9 and R 10 At least one of them is any of a cyano group, a formyl group, an aryl group, a substituted aryl group, a heteroaryl group, and a substituted heteroaryl group.
The fluorescent dye according to claim 1, wherein the fluorescent dye is represented by any one of the following formulas (1) to (6) and (9).
A composition for staining lipid droplets, comprising one or more fluorescent dyes represented by the following general formulas (I), (II) or (III).

In the above general formulas (I), (II) and (III), R 1 and R 2 each independently represent a hydrogen atom, a cyano group, a formyl group, an aryl group, a substituted aryl group, a heteroaryl group and a substituted hetero group. Selected from the group consisting of aryl groups, and at least one of R 1 and R 2 is any one of a cyano group, a formyl group, an aryl group, a substituted aryl group, a heteroaryl group, and a substituted heteroaryl group;
R 3 is a formyl group or a functional group represented by any of the following general formulas (i), (ii) and (iii), and R 11 is a functional group represented by the following general formula (i) R 12 is any one of a nitro group, a cyano group, a sulfonyl group, a sulfoxyl group, a carbonyl group and a carboxyl group;

In the above general formulas (i), (ii) and (iii),
R 4 and R 5 are each independently a hydrocarbon group having 1 to 10 carbon atoms and optionally containing one or both of a branched and unsaturated bond, and R 4 and R 5 are directly or An atom, an oxygen atom, a nitrogen atom and a sulfur atom may be bonded to each other to form a ring,
R 6 and R 7 are each independently selected from the group consisting of a hydrogen atom, a cyano group, a formyl group, an aryl group, a substituted aryl group, a heteroaryl group and a substituted heteroaryl group, and R 6 and R 7 At least one of them is a cyano group, a formyl group, an aryl group, a substituted aryl group, a heteroaryl group or a substituted heteroaryl group,
R 8 is selected from the group consisting of a hydrogen atom, a cyano group, a formyl group, an aryl group, a substituted aryl group, a heteroaryl group and a substituted heteroaryl group;
R 9 and R 10 are each independently selected from the group consisting of a hydrogen atom, a cyano group, a formyl group, an aryl group, a substituted aryl group, a heteroaryl group and a substituted heteroaryl group, and R 9 and R 10 At least one of them is any of a cyano group, a formyl group, an aryl group, a substituted aryl group, a heteroaryl group, and a substituted heteroaryl group.
4. The composition for staining lipid droplets according to claim 3, wherein the fluorescent dye is represented by any of the following formulas (1) to (9).
A step of introducing one or a plurality of fluorescent dyes represented by the following general formulas (I), (II) or (III) into lipid droplets in cells;
Measuring the fluorescence emitted from the fluorescent dye introduced into the inside of the lipid droplet.

In the general formulas (I), (II) and (III), R 1 and R 2 each independently represent a hydrogen atom, a cyano group, a formyl group, an aryl group, a substituted aryl group, a heteroaryl group, and a substituted heteroaryl Selected from the group consisting of groups, and at least one of R 1 and R 2 is any one of a cyano group, a formyl group, an aryl group, a substituted aryl group, a heteroaryl group, and a substituted heteroaryl group;
R 3 is a formyl group or a functional group represented by any of the following general formulas (i), (ii) and (iii), and R 11 is a functional group represented by the following general formula (i) R 12 is any one of a nitro group, a cyano group, a sulfonyl group, a sulfoxyl group, a carbonyl group and a carboxyl group;

In the above general formulas (i), (ii) and (iii),
R 4 and R 5 are each independently a hydrocarbon group having 1 to 10 carbon atoms and optionally containing one or both of a branched and unsaturated bond, and R 4 and R 5 are directly or An atom, an oxygen atom, a nitrogen atom and a sulfur atom may be bonded to each other to form a ring,
R 6 and R 7 are each independently selected from the group consisting of a hydrogen atom, a cyano group, a formyl group, an aryl group, a substituted aryl group, a heteroaryl group and a substituted heteroaryl group, and R 6 and R 7 At least one of them is a cyano group, a formyl group, an aryl group, a substituted aryl group, a heteroaryl group or a substituted heteroaryl group,
R 8 is selected from the group consisting of a hydrogen atom, a cyano group, a formyl group, an aryl group, a substituted aryl group, a heteroaryl group and a substituted heteroaryl group;
R 9 and R 10 are each independently selected from the group consisting of a hydrogen atom, a cyano group, a formyl group, an aryl group, a substituted aryl group, a heteroaryl group and a substituted heteroaryl group, and R 9 and R 10 At least one of them is any of a cyano group, a formyl group, an aryl group, a substituted aryl group, a heteroaryl group, and a substituted heteroaryl group.
The imaging method for intracellular lipid droplets according to claim 5, wherein the fluorescent dye is represented by any of the following formulas (1) to (9).