JP2021191807A - Novel fluorescent dye, and composition for lipid droplet staining and method for imaging intracellular lipid droplets using the same - Google Patents

Abstract

To provide a fluorescent dye which is specific to lipid droplets and enables fluorescent staining with high sensitivity, while having no cytotoxicity but high intracellular retentivity, and which is applicable to kinetics observation, and a method for imaging intracellular lipid droplets.SOLUTION: The present invention employs a fluorescent dye represented by general formula (I) or (II). [R1 and R2 are each independently H, cyano, or the like; and R3 is substituted amino or the like.]SELECTED DRAWING: Figure 1

Description

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

Lipid droplets (also referred to as lipid globules, oil droplets, etc.) are intracellular small organs that store neutral fat such as triglyceride and are surrounded by a single layer of phospholipids. Lipid droplets are mainly present in adipocytes, and it was thought that energy storage was the main role. Recent studies have shown that lipid droplets are also widely present in cells other than adipocytes.

Lipid droplets of adipocytes are huge, 10 to 100 μm, and store a large amount of fat for a long period of time. However, the lipid droplets of non-adipocytes are much smaller, 1 μm or less, and temporarily store a very small amount of fat. Cells take up fatty acids from the blood and use them as an energy source, at which time some fatty acids are resynthesized into triglycerides and stored. Here are the small lipid droplets found on non-adipocytes.

As mentioned above, lipid droplets have traditionally been a storage organelle of triglycerides and are merely a storage of fat. However, recent studies have revealed that many proteins are present on the surface of lipid droplets and play an important role in the regulation of lipid metabolism in the body. Fat accumulation and breakdown 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 membrane formation, intracellular signal transduction, etc., and are being recognized as important intracellular organelles. (See Non-Patent Document 1) The relationship between lipid droplets and intracellular phenomena such as autophagy (see Non-Patent Document 2) and cellular senescence (see Non-Patent Document 3) has also been suggested, and the formation and growth of lipid droplets. It is hoped that the mechanism of fusion and decomposition will be elucidated in detail.

Therefore, fluorescent dyes having high lipid droplet specificity have attracted great interest in recent years for the detailed elucidation of the mechanism of lipid droplet formation, growth, fusion, and decomposition. Since lipid droplets are expected to develop new research that contributes to the diagnosis and treatment of diseases related to neutral lipid droplets such as triacylglycerol and cholesterol ester, we will observe the dynamics of lipid droplets in living cells and individuals. A capable molecular probe is indispensable. Lipid droplets form functionally and morphologically diverse subpopulations, and so far no single marker has existed to stain all lipid droplets. One of the methods for visualizing a lipid droplet is to stain the nucleus portion (lipid ester) of the lipid droplet with a molecule having a strong lipophilicity. In order to observe the dynamics of lipid droplets in living cells and individuals, imaging methods using fluorescent dyes such as Nile Red and BODYPY 493/503 have been conventionally used (for example, Patent Document 1, Non-Patent Documents 4 and 5). reference).

Japanese Patent No. 62401414

Tomohiro Yamaguchi, Showa University Pharmaceutical Magazine, Volume 1, Issue 1, 2010 Rajat Singh, Susmita Kaushik, Yongjun Wang, Youqing Xiang, Inna Novak, Masaaki Komatsu, Keiji Tanaka, Ana Maria Cuervo, Mark J. Czaja; Autophagy regulates lipid metabolism; Nature. 2009 April 30; 458 (7242): 1131-1135 Masataka Yokoyama, Sho Okada, Atsushi Nakagomi, Junji Moriya, Ippei Shimizu, Aika Nojima, Yohko Yoshida, Harumi Ichimiya, Naomi Kamimura, Yoshio Kobayashi, Shigeo Ohta, Marcus Fruttiger, Guillermina Lozano, and Tohru Minamino; Inhibition of Endothelial p53 Improves Meta Related to Dietary Obesity; Cell Reports 7, 1691-1703, June 12, 2014 Phillip Greenspan, Eugene P. Mayer, Stanley D. Fowler; Nile Red: A Selective Fluorescent Stain for Intracellular Lipid Droplets; THE JOURNAL OF CELL BIOLOGY VOLUME 100: 965-973 March 1985 Peter M. Gocze, Dale A. Freeman; Factors Underlying the Variability of Lipid Droplet Fluorescence in MA-10 Leydig Tumor Cells; Cytometry 17: 151-158 (1994)

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

BODYPY 493/503 is a bright green fluorescent dye that has a peculiar hydrophobicity and is easily distributed in hydrophobic parts such as lipid droplets, so that it is possible to detect lipid droplets. However, like Nile Red, it has intracellular background fluorescence and lacks lipid droplet detection specificity.

In addition, in order to observe the dynamics of lipid droplets, it is necessary to incubate the cells for a long period of time after introducing the fluorescent dye. However, these dyes have problems in cytotoxicity and intracellular retention, and are not suitable for long-term observation.

The present invention has been made in view of such circumstances, is specific to lipid droplets, enables highly sensitive fluorescent staining, has no cytotoxicity, has high intracellular retention, and can be applied to observing the dynamics of lipid droplets. It is an object of the present invention to provide a fluorescent dye, a composition for staining lipid droplets using the same, and a method for imaging intracellular lipid droplets.

The first aspect of the present invention according to the above object solves the above-mentioned problem by providing the fluorescent dye represented by the following general formula (I) or (II).

In the above general formulas (I) and (II), R ¹ and R ² are independently composed 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, respectively. Selected from the ^{group, at least one of R 1} and R ² is any 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 represented by the following general formula (i), a functional group represented by any one of (ii) and (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 may contain one or both of a branched and unsaturated bond, and R ⁴ and R ⁵ are direct or carbon. It may be bonded via any of an atom, an oxygen atom, a nitrogen atom and a sulfur atom to form a ring.
R ⁶ and R ⁷ are independently selected from the group consisting of hydrogen atoms, cyano groups, formyl groups, aryl groups, substituted aryl groups, heteroaryl groups and substituted heteroaryl groups, and R ⁶ and R ⁷ are of the same. At least one of them is one of a cyano group, a formyl group, an aryl group, a substituted aryl group, a heteroaryl group and 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 independently selected from the group consisting of hydrogen atoms, cyano groups, formyl groups, aryl groups, substituted aryl groups, heteroaryl groups and substituted heteroaryl groups, and R ⁹ and R ¹⁰ are of the same. At least one of them 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.

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

The second aspect of the present invention solves the above-mentioned problems by providing a composition for lipid droplet dyeing containing one or more fluorescent dyes represented by the following general formula (I) or (II). be.

In the above general formulas (I) and (II), R ¹ and R ² are independently composed 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, respectively. Selected from the ^{group, at least one of R 1} and R ² is any 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 formyl group or the following formula (i), a functional group represented by any one of (ii) and (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 may contain one or both of a branched and unsaturated bond, and R ⁴ and R ⁵ are direct or carbon. It may be bonded via any of an atom, an oxygen atom, a nitrogen atom and a sulfur atom to form a ring.
R ⁶ and R ⁷ are independently selected from the group consisting of hydrogen atoms, cyano groups, formyl groups, aryl groups, substituted aryl groups, heteroaryl groups and substituted heteroaryl groups, and R ⁶ and R ⁷ are of the same. At least one of them is one of a cyano group, a formyl group, an aryl group, a substituted aryl group, a heteroaryl group and 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 independently selected from the group consisting of hydrogen atoms, cyano groups, formyl groups, aryl groups, substituted aryl groups, heteroaryl groups and substituted heteroaryl groups, and R ⁹ and R ¹⁰ are of the same. At least one of them 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.

A third aspect of the present invention comprises the step of introducing one or more fluorescent dyes represented by the following general formula (I) or (II) into the intracellular lipid droplets.
The above problem is solved by providing a method for imaging an intracellular lipid droplet, which comprises a step of measuring the fluorescence emitted from the fluorescent dye introduced into the lipid droplet.

In the above general formulas (I) and (II), R ¹ and R ² are independently composed 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, respectively. Selected from the ^{group, at least one of R 1} and R ² is any 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 formyl group or the following formula (i), a functional group represented by any one of (ii) and (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 may contain one or both of a branched and unsaturated bond, and R ⁴ and R ⁵ are direct or carbon. It may be bonded via any of an atom, an oxygen atom, a nitrogen atom and a sulfur atom to form a ring.
R ⁶ and R ⁷ are independently selected from the group consisting of hydrogen atoms, cyano groups, formyl groups, aryl groups, substituted aryl groups, heteroaryl groups and substituted heteroaryl groups, and R ⁶ and R ⁷ are of the same. At least one of them is one of a cyano group, a formyl group, an aryl group, a substituted aryl group, a heteroaryl group and 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 independently selected from the group consisting of hydrogen atoms, cyano groups, formyl groups, aryl groups, substituted aryl groups, heteroaryl groups and substituted heteroaryl groups, and R ⁹ and R ¹⁰ are of the same. At least one of them 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.

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 (8). It is preferably represented by either.

According to the present invention, the specificity of lipid droplets is high, and while strong fluorescence emission is generated in a hydrophobic environment such as lipid droplets, background emission is hardly shown in a hydrophilic environment such as cytoplasm, and fluorescence observation with high sensitivity is performed. Fluorescent dyes capable of are provided. Since the fluorescent dye of the present invention has pyrene or perylene as a fluorescent chromophore, it is relatively easy to synthesize, and a fluorescent dye having desired fluorescence characteristics (excitation spectrum, fluorescence spectrum, etc.) can be obtained by molecular design. Can be designed. 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 the lipid droplets, the dynamics of the lipid droplets in the living cells can be observed for a long period of 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 fluorescent dye, and an imaging method for intracellular lipid droplets are available. Provided.

It is a photograph showing the result of confocal microscopy of HeLa cells incubated with the compound of the present invention after treatment with oleic acid. 6 is a photograph showing the results of confocal microscopy of HeLa cells incubated with the compound of the present invention and Nile Red after treatment with oleic acid. 6 is a photograph showing the results of confocal microscopy of HeLa cells incubated with the compound of the present invention, Nile Red or BODYPY 493/503, after treatment with oleic acid. It is a photograph showing the result of confocal microscopy of HepG2 cells after 24 hours of incubation with the compound of the present invention, Nile Red or BODYPY 493/503.

The fluorescent dye according to the first embodiment of the present invention (sometimes simply abbreviated as "fluorescent dye") is represented by the following general formula (I) or (II).

In the above general formulas (I) and (II), R ¹ and R ² are independently composed 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, respectively. Selected from the ^{group, at least one of R 1} and R ² is any 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 represented by the following general formula (i), a functional group represented by any one of (ii) and (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 may contain one or both of a branched and unsaturated bond, and R ⁴ and R ⁵ are direct or carbon. It may be bonded via any of an atom, an oxygen atom, a nitrogen atom and a sulfur atom to form a ring.
R ⁶ and R ⁷ are independently selected from the group consisting of hydrogen atoms, cyano groups, formyl groups, aryl groups, substituted aryl groups, heteroaryl groups and substituted heteroaryl groups, and R ⁶ and R ⁷ are of the same. At least one of them is one of a cyano group, a formyl group, an aryl group, a substituted aryl group, a heteroaryl group and 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 independently selected from the group consisting of hydrogen atoms, cyano groups, formyl groups, aryl groups, substituted aryl groups, heteroaryl groups and substituted heteroaryl groups, and R ⁹ and R ¹⁰ are of the same. At least one of them 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.

Among the fluorescent dyes represented by the general formula (I) or (II), specific examples of preferable ones are those represented by any of the following formulas (1) to (6). Hereinafter, the "compound represented by the formula (n) (n is an integer)" may be abbreviated as "compound n".

These compounds can be synthesized using any known method using available pyrene or perylene derivatives as starting materials. For example, compound (3), compound (5) and compound (6) can be synthesized according to the scheme below.

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). In addition, compound (2) can be synthesized by the reaction of 3-bromoperylene or perylene-3-ylboronic acid with an amine using a metal catalyst.

The composition for lipid droplet staining according to the second embodiment of the present invention (hereinafter, may be abbreviated as "composition for lipid droplet staining" or "composition") is the present invention described above. The fluorescent dye according to the first embodiment of the above, that is, one or more of the fluorescent dyes represented by the above general formula (I) or (II).

Specific examples of the preferable fluorescent dye that can be used in the composition include those represented by any of the following formulas (1) to (8). Although the compound (7) and the compound (8) are known compounds, they can be preferably used in the composition.

The composition may be a solid containing one or more of the above fluorescent dyes, but may be a liquid in which one or more of the above fluorescent dyes is dissolved in a suitable solvent, solution or buffer solution. There may be. The composition for lipid droplet staining may contain 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”) is represented by the above general formula (I) or (II). It includes a step of introducing one or more fluorescent dyes into the inside of the lipid droplet in the cell and a step of measuring the fluorescence emitted from the fluorescent dye introduced into the inside of the lipid droplet.

Since the fluorescent dye used in the imaging method and its preferred specific examples are the same as in the case of the lipid droplet dyeing composition according to the second embodiment of the present invention, detailed description thereof will be omitted.

The introduction of the fluorescent dye into the lipid droplet can be carried out by using any known method, for example, by adding the cells inoculated to the medium and incubating for a predetermined time. The fluorescence emitted from the fluorescent dye introduced into the lipid droplet can be measured by any known means, but it can be carried out by observing the fluorescence image using a confocal microscope or a fluorescence microscope. can. Since the above-mentioned fluorescent dye used in the imaging method has low cytotoxicity and high retention in the lipid droplets, it is possible to observe the lipid droplets in the living cells over time.

Next, an example carried out for confirming the action 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) having a 1- (4-methoxyphenyl) ethenyl group is as follows, respectively. According to the scheme shown, the Wittig reaction between the aldehyde derivative and triphenylphosphonium chloride (4-methoxybenzyl) was used.

Synthesis of compound (7) In a 100 mL eggplant flask, 1-pyrenecarboxyaldehyde (460 mg, 2.0 mmol), chloride (4-methoxybenzyl) triphenylphosphonium (1008 mg, 4.0 mmol), 30 mL dichloromethane, 3 mL A 50% aqueous NaOH solution was added, and the mixture was stirred overnight at room temperature. The reaction solution was extracted with dichloromethane, and the extract was distilled off by an evaporator. Purification was performed using a silica gel column and 100% chloroform as an eluent. 300 mg of slightly yellow crystals were obtained (yield 44.9%).

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-perylenecarboxyaldehyde (8) (560 mg, 2.0 mmol), triphenylphosphonium chloride (4-methoxybenzyl) (1008 mg, 4.0 mmol), 30 mL dichloromethane 3 mL of 50% NaOH aqueous solution was added, and the mixture was stirred overnight at room temperature. 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) The synthesis of compounds (5) and (6) was carried out according to the following scheme.

Synthesis of 3-bromoperylene To a 500 mL eggplant flask, perylene (1 g, 4.0 mmol) and 300 mL of dichloromethane were added, and the mixture was stirred for 1 hour to dissolve. A solution prepared by dissolving N-bromosuccinimide (1.78 g, 10.0 mmol) in 100 mL of dichloromethane was added dropwise to this solution, and the mixture was stirred overnight at room temperature. The reaction solution was concentrated with 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, 3-bromoperylene (564 mg, 1.7 mmol) and 85 mL of THF were added, and the mixture was stirred in a dry ice-acetone bath at −78 ° C. 3 mL of n-BuLi (4.8 mmol) was added dropwise to this solution, and the mixture was stirred for 1 hour. Then, 3- (N, N-dimethylamino) acrolein (2 g, 20 m mmol) was added, and the mixture was stirred overnight at room temperature. The reaction solution was distilled off by 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, 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 dissolved by stirring. To this solution was _{added 2.9 mL of TiCl 4} (2.9 mmol) and 700 μL of pyridine and refluxed overnight. After returning to room temperature, the reaction solution was extracted with dichloromethane, and the extract was distilled off by an evaporator. Purification was performed using a silica gel column and 100% chloroform as an eluent. 60 mg of black-purple crystals (yield: 23.5%) 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).

Example 2: Staining Lipid Droplets in Cells For the purpose of examining whether or not the compounds (7), (3) and (6) can specifically stain lipid droplets in living cells, lipid droplets. The staining characteristics of lipid droplets were investigated using HeLa cells that can induce lipid droplets by adding oleic acid and HepG2 cells that have a high lipid droplet content.

HeLa cells were seeded in μ-slide 8 well (Ibidi), 200 μmol / L oleic acid diluted in serum medium was added, and the cells were cultured overnight in a _{CO 2 incubator at 37 ° C.} Compound (7) and compound (3) diluted in serum medium and compound (6) of 1 μmol / L were added and incubated for 30 minutes. After that, it was observed with a confocal microscope.

HepG2 cells were seeded in μ-slide 8 well (Ibidi) and cultured overnight in a _{CO 2 incubator at 37 ° C.} Compound (7) and compound (3) diluted in serum medium and compound (6) of 1 μmol / L were added and incubated for 30 minutes. After that, it was observed with a confocal microscope.

First, an experiment was performed using HeLa cells into which lipid droplets were introduced by treatment with 200 μM oleic acid. HeLa cells in which 1 μM compound (7), compound (3) and compound (6) were 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 contrast image, and “FL” indicates a fluorescence image (the same applies hereinafter). From these results, it was confirmed that the fluorescently stained part was present in the cell.

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

In addition, imaging comparison was performed using Nile Red and BODYPY 493/503, which is also a commercially available lipid droplet indicator (see FIG. 3). For all of compound (7), compound (3) and compound (6), fluorescence images were observed for only a part of the cells, whereas when Nile Red and BODYPY 493/503 were used, the whole cytoplasm was observed. A fluorescent image was obtained. From this result, it was confirmed that when compound (7), compound (3) and compound (6) were used, a fluorescent image was specifically obtained for lipid droplets.

Furthermore, using HepG2 cells having a high lipid droplet content, the cells were observed 24 hours after staining of the lipid droplets. The results are shown in FIG. Both Nile Red and BODYPY 493/503 have low intracellular retention, and 24 hours after staining, the dye leaks from the cells and a lipid droplet-specific fluorescent image cannot be observed, whereas the compound (7) ) And the compound (3), the fluorescent image specific to the lipid droplet is maintained even after 24 hours have passed from the staining, the intracellular retention is high, and the long-term observation of the lipid droplet is observed. It was confirmed that it is possible.

Claims (6)

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

In the above general formulas (I) and (II), R ¹ and R ² are independently composed 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, respectively. Selected from the ^{group, at least one of R 1} and R ² is any 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 represented by the following general formula (i), a functional group represented by any one of (ii) and (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 may contain one or both of a branched and unsaturated bond, and R ⁴ and R ⁵ are direct or carbon. It may be bonded via any of an atom, an oxygen atom, a nitrogen atom and a sulfur atom to form a ring.
R ⁶ and R ⁷ are independently selected from the group consisting of hydrogen atoms, cyano groups, formyl groups, aryl groups, substituted aryl groups, heteroaryl groups and substituted heteroaryl groups, and R ⁶ and R ⁷ are of the same. At least one of them is one of a cyano group, a formyl group, an aryl group, a substituted aryl group, a heteroaryl group and 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 independently selected from the group consisting of hydrogen atoms, cyano groups, formyl groups, aryl groups, substituted aryl groups, heteroaryl groups and substituted heteroaryl groups, and R ⁹ and R ¹⁰ are of the same. At least one of them 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. The fluorescent dye according to claim 1, wherein the fluorescent dye is represented by any of the following formulas (1) to (6).

A composition for lipid droplet staining containing one or more fluorescent dyes represented by the following general formula (I) or (II).

In the above general formulas (I) and (II), R ¹ and R ² are independently composed 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, respectively. Selected from the ^{group, at least one of R 1} and R ² is any 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 formyl group or the following formula (i), a functional group represented by any one of (ii) and (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 may contain one or both of a branched and unsaturated bond, and R ⁴ and R ⁵ are direct or carbon. It may be bonded via any of an atom, an oxygen atom, a nitrogen atom and a sulfur atom to form a ring.
R ⁶ and R ⁷ are independently selected from the group consisting of hydrogen atoms, cyano groups, formyl groups, aryl groups, substituted aryl groups, heteroaryl groups and substituted heteroaryl groups, and R ⁶ and R ⁷ are of the same. 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.
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 independently selected from the group consisting of hydrogen atoms, cyano groups, formyl groups, aryl groups, substituted aryl groups, heteroaryl groups and substituted heteroaryl groups, and R ⁹ and R ¹⁰ are of the same. At least one of them 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. The composition for lipid droplet dyeing according to claim 3, wherein the fluorescent dye is represented by any of the following formulas (1) to (8).

The step of introducing one or more fluorescent dyes represented by the following general formula (I) or (II) into the intracellular lipid droplets, and
A method for imaging an intracellular lipid droplet, which comprises a step of measuring the fluorescence emitted from the fluorescent dye introduced into the lipid droplet.

In the above general formulas (I) and (II), R ¹ and R ² are independently composed 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, respectively. Selected from the ^{group, at least one of R 1} and R ² is any 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 formyl group or the following formula (i), a functional group represented by any one of (ii) and (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 may contain one or both of a branched and unsaturated bond, and R ⁴ and R ⁵ are direct or carbon. It may be bonded via any of an atom, an oxygen atom, a nitrogen atom and a sulfur atom to form a ring.
R ⁶ and R ⁷ are independently selected from the group consisting of hydrogen atoms, cyano groups, formyl groups, aryl groups, substituted aryl groups, heteroaryl groups and substituted heteroaryl groups, and R ⁶ and R ⁷ are of the same. At least one of them is one of a cyano group, a formyl group, an aryl group, a substituted aryl group, a heteroaryl group and 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 independently selected from the group consisting of hydrogen atoms, cyano groups, formyl groups, aryl groups, substituted aryl groups, heteroaryl groups and substituted heteroaryl groups, and R ⁹ and R ¹⁰ are of the same. At least one of them 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. The method for imaging intracellular lipid droplets according to claim 5, wherein the fluorescent dye is represented by any of the following formulas (1) to (8).

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