Fluorescence imaging reagent for lipid droplets in tissues and cells

Abstract

The present invention addresses the problem of developing a fluorescent reagent with which lipid droplets from the scale of cultured cells to the scale of an individual can be imaged at high sensitivity, and which can be used in combination with a green fluorescent protein. The present invention provides a reagent for detecting lipid droplets, the reagent containing a compound represented by formula (I). (In the formula, m represents an integer of 3-5, n represents an integer of 3-5, and R represents a C1-5 alkyl, a C6-12 aryl, a C7-13 aralkyl, or hydrogen.)

Description

Fluorescent imaging reagents for lipid droplets in cells and tissues

[0001] The present invention relates to fluorescent imaging reagents for lipid droplets in cells and tissues.

[0002] Lipid droplets (lipid droplets) are spherical intracellular organelles composed of neutral lipids, such as triacylglycerols and cholesterol esters, surrounded by a single layer of phospholipid membrane. While found primarily in adipocytes, they are ubiquitous in all cell types. While previously thought to primarily store neutral lipids, recent research has revealed their involvement in regulating intracellular lipid metabolism. Furthermore, research into the mechanisms of lipid droplet formation, growth, and degradation is progressing, including reports on lipid droplet and autophagy. Meanwhile, excessive lipid accumulation in tissues (organs) can lead to tissue dysfunction and the development of diseases such as diabetes and atherosclerosis. Furthermore, the incidence of nonalcoholic steatohepatitis (NASH), a type of hepatitis, has rapidly increased in recent years. Left untreated, NASH can progress to liver cirrhosis and liver cancer. Therefore, elucidating the mechanisms of lipid droplet formation, growth, and degradation in cells and tissues is important not only for cell biology but also for the diagnosis and treatment of these diseases. Therefore, the development of molecular probes for highly sensitive, real-time imaging of lipid droplets in living cells and tissues is essential.

[0003] Fluorescence imaging is a simple method for imaging cells and tissues in a live state and is widely used in biological and medical research. While numerous fluorescent reagents for lipid droplet imaging have been reported at the academic level, only a few have been put to practical use. Figure 1 shows currently available commercially available lipid droplet fluorescent imaging reagents. BODIPY 493 / 503 and Nile Red are widely used by researchers. BODIPY 493 / 503 exhibit green fluorescence around 500 nm and have high lipid droplet selectivity. However, they suffer from issues such as low photostability, low lipid droplet retention, and leakage of excitation light due to a small Stokes shift (the energy difference between the absorption and fluorescence maxima). Furthermore, Nile Red distributes widely in intracellular organelles other than lipid droplets, resulting in low lipid droplet selectivity. Furthermore, its absorption and fluorescence spectra vary significantly depending on the surrounding microenvironment, making it difficult to perform multi-staining with other fluorescent reagents. To solve these problems, the LipiDye and Lipi series (Lipi-Blue, Lipi-Green, Lipi-Red, and Lipi-Deep Red; the structural formula of Lipi-Deep Red is not disclosed) were developed. Although these reagents can selectively image intracellular lipid droplets, there is no information available about their use in imaging lipid droplets in living tissues.

[0004] Regarding lipid droplet imaging in living tissues, a nitrobenzene-substituted Nile Blue derivative (MNs-NB, Figure 2) has been reported in Patent Document 1. In polar solvents, MNs-NB undergoes a photoinduced electron transfer reaction between the nitrobenzene unit and Nile Blue. On the other hand, in low-polarity solvents, the photoinduced electron transfer reaction is less likely to occur, resulting in red fluorescence. While MNs-NB is a reagent capable of imaging lipid droplets in tissues, it has drawbacks, such as a low fluorescence quantum yield (0.21 in chloroform) and a small Stokes shift.

[0005] The present inventors previously synthesized a reagent (PC6X, Figure 3) for imaging lipid droplets in tissues (adipose tissue, liver, kidney, etc.) of anesthetized small animals (Patent Document 2). PC6X has a fluorescence quantum yield of 0.8 or higher in solution and is more photostable than BODIPY493 / 503 and Lipi-Green. This enables high-sensitivity imaging of lipid droplets in cells and tissues over long periods of time. Furthermore, it can also image lipid droplets in the liver of a mouse model of fatty liver. However, due to its green fluorescence, there are challenges in its use in combination with green fluorescent protein, which is widely used in the fields of medicine and biochemistry.

[0006] Patent No. 6241014 WO 2020 / 189721

[0007] As mentioned above, currently available fluorescent reagents for lipid droplet imaging are limited to cultured cells. Furthermore, the non-commercial compound MNs-NB also faces many challenges in practical application. Therefore, the objective of the present invention is to develop a fluorescent reagent that can image lipid droplets with high sensitivity at the cultured cell level and at the individual level, and that can be used in combination with green fluorescent protein. It is believed that such a fluorescent reagent could significantly contribute to the development of diagnostic and therapeutic agents for diseases resulting from excessive fat accumulation.

[0008] The present inventors have conducted extensive research to solve the above problems and have developed a blue fluorescent reagent having a coumarin skeleton. They have discovered that the use of this reagent enables selective fluorescent imaging of lipid droplets in cells and tissues, and have completed the present invention. The gist of the present invention is as follows.

[0009] [1] A reagent for detecting lipid droplets, comprising a compound represented by the following general formula (I):

[0010]

[0011] (wherein m represents an integer of 3 to 5, n represents an integer of 3 to 5, and R represents alkyl having 1 to 5 carbon atoms, aryl having 6 to 12 carbon atoms, aralkyl having 7 to 13 carbon atoms, or hydrogen.) [2] The detection reagent according to [1], wherein R represents alkyl having 1 to 4 carbon atoms or phenyl. [3] The detection reagent according to [1] or [2], wherein m and n are 3. [4] The detection reagent according to any of [1] to [3], for detecting lipid droplets in a biological sample. [5] The detection reagent according to [4], wherein the biological sample is a cell or tissue. [6] The detection reagent according to any of [1] to [3], for detecting lipid droplets in a living individual. [7] A method for detecting lipid droplets, comprising the step of administering the detection reagent according to any of [1] to [6] to a biological sample or a living individual (in one embodiment, excluding humans). [8] Use of a compound represented by general formula (I) above in detecting lipid droplets. [9] Use of the compound represented by the above general formula (I) in the production of a reagent for detecting lipid droplets.

[10] A compound represented by the following general formula (I)':

[0012]

[0013] (wherein m represents an integer of 3 to 5, n represents an integer of 3 to 5, and R' represents alkyl having 1 to 5 carbon atoms, aryl having 6 to 12 carbon atoms, or aralkyl having 7 to 13 carbon atoms.)

[11] The compound according to

[10] , wherein R' represents alkyl having 1 to 4 carbon atoms or phenyl.

[12] The compound according to

[10] or

[11] , wherein m and n are 3.

[0014] According to the present invention, it is possible to provide a blue fluorescent reagent that enables selective fluorescent imaging of lipid droplets in cells and tissues.

[0015] Figure 1 shows the structural formula of a commercially available lipid droplet imaging reagent. Figure 2 shows the structural formula of a Nile Blue derivative (MNs-NB). Figure 3 shows the structural formula of the lipid droplet imaging reagent described in Patent Document 2 (PC6X) and the comparative example (C6-OBu). Figure 4 shows the structural formula of the lipid droplet imaging reagent described in the examples of the present invention. Figure 5 shows the absorption and fluorescence spectra of the compounds of the present invention (DBC30 series). Figure 6 shows fluorescent imaging images (photographs) obtained by adding the DBC30 series and a commercially available lipid droplet imaging reagent to HeLa cells. Figure 7 shows the results of an evaluation of the intracellular photostability of DBC30, C6-OBu, and a commercially available lipid droplet imaging reagent. Figure 8 shows co-stained fluorescent imaging images (photographs) obtained by DBC30 and a commercially available lipid droplet imaging reagent (BODIPY493 / 503). Figure 9 shows co-stained fluorescent imaging images (photographs) using DBC30-Bu and a commercially available lipid droplet imaging reagent (BODIPY493 / 503). Figure 10 shows co-stained fluorescent imaging images (photographs) using DBC30-Ph and a commercially available lipid droplet imaging reagent (BODIPY493 / 503). Figure 11 shows multicolor imaging images (photographs) using DBC30 and commercially available lipid droplet imaging reagents (Mito Tracker Green, Lyso Tracker Red). Figure 12 shows fluorescence intensity imaging images (intensity) and fluorescence lifetime imaging (FLIM) images (photographs) of adipose tissue and lipid droplets in healthy mice and liver steatosis model mice administered DBC30. Figure 13 shows fluorescence imaging images (photographs) of subcutaneous adipose tissue, abdominal adipose tissue, skeletal muscle, and kidneys in DBC30-administered mice.

[0016] The present invention will be described below. <Reagent for detecting lipid droplets> One aspect of the present invention relates to a reagent for detecting lipid droplets (hereinafter, sometimes referred to as "reagent for detecting lipid droplets of the present invention"), which comprises a compound represented by the following general formula (I). Here, lipid droplets refer to spherical droplets containing lipids, for example, contained within cells. The compound represented by general formula (I) is a compound having the following structure:

[0017]

[0018] In general formula (I), m represents an integer of 3 to 5. From the viewpoints of photophysical properties, synthesis, etc., m is preferably 3. In general formula (I), n represents an integer of 3 to 5. From the viewpoints of photophysical properties, synthesis, etc., n is preferably 3.

[0019] In general formula (I), R is an alkyl having 1 to 5 carbon atoms, an aryl having 6 to 12 carbon atoms, an aralkyl having 7 to 13 carbon atoms, or hydrogen. The alkyl having 1 to 5 carbon atoms may be a linear alkyl, a branched alkyl, or a cycloalkyl, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, s-butyl, t-butyl, and pentyl. The aryl having 6 to 12 carbon atoms may be a substituted or unsubstituted aryl, such as phenyl, tolyl, xylyl, and naphthyl. The aralkyl having 7 to 13 carbon atoms may be a substituted or unsubstituted aralkyl, such as benzyl and phenethyl. The substituents include, but are not limited to, alkyl having 1 to 4 carbon atoms, alkoxy, and halogen. From the viewpoints of photophysical properties and synthesis, R is preferably alkyl having 1 to 4 carbon atoms or phenyl.

[0020] Specific examples of the compound represented by the above general formula (I) include the compounds listed below, but the present invention is not limited to these.

[0021]

[0022] The photophysical properties of the fluorescence of the compound represented by general formula (I), such as the absorption and fluorescence maximum wavelengths, the fluorescence quantum yield (Φ f ) and fluorescence lifetime (τ f ) can be measured by known measurement methods. For example, the absorption / fluorescence maximum wavelength and fluorescence quantum yield can be measured using a luminescence quantum yield measurement device or the like, with a sample prepared by dissolving the compound represented by general formula (I) in a solvent or the like. The fluorescence lifetime can be measured by measuring the fluorescence lifetime (τ ｆ ) can be measured.

[0023] Fluorescence quantum yield (Φ f ) can be changed depending on the structure of the compound, the type of solvent, etc., and is not particularly limited, but is, for example, 0.5 or more, 0.7 or more, 0.8 or more, or 0.9 or more. ｆ ) can be changed depending on the structure of the compound, the type of solvent, etc., and is not particularly limited, but is, for example, 2.0 ns (nanoseconds) or more, 2.2 ns or more, 2.4 ns or more, or 2.5 ns or more.

[0024] The maximum excitation wavelength of the compound represented by general formula (I) in a solvent can be changed depending on the structure of the compound, the type of solvent, etc., and is not particularly limited, but is, for example, 390 nm to 460 nm. The maximum fluorescence wavelength in the solvent can also be appropriately set, but is, for example, 460 nm to 500 nm.

[0025] <<Method for Producing Compounds>> The compounds represented by general formula (I) can be produced according to the description in the Examples below and known organic synthesis methods.

[0026] <Reagent> The lipid droplet detection reagent of the present invention includes a compound having the above structure. This structure provides excellent photophysical properties in fluorescence (blue fluorescence, fluorescence quantum yield, fluorescence lifetime, Stokes shift, etc.). In particular, since it has the above-mentioned excellent photophysical properties in various solvents, it is useful as a lipid droplet detection reagent not only in cells but also in living individuals. In addition, the above structure provides excellent lipid droplet selectivity and intracellular retention. Therefore, it can be used as a highly specific lipid droplet detection reagent.

[0027] The lipid droplet detection reagent of the present invention may consist solely of the compound represented by general formula (I), or may further contain solvents, additives, and compounds other than the compounds used in the present invention as a lipid droplet detection reagent, as long as the effects of the present invention are not hindered.

[0028] <Lipid Droplet Detection Method> One aspect of the present invention relates to a lipid droplet detection method (hereinafter sometimes referred to as the "lipid droplet detection method of the present invention"), which includes a step of administering a lipid droplet detection reagent of the present invention to a biological sample or a biological individual (in one embodiment, excluding humans). Another aspect of the present invention relates to a lipid droplet detection method, which involves administering a solution containing a lipid droplet detection reagent of the present invention and a solubilizing agent to a biological sample or a biological individual (in one embodiment, excluding humans). The compound represented by general formula (I) used as the lipid droplet detection reagent may be poorly water-soluble. In this case, the compound represented by general formula (I) may be dissolved in an organic solvent in which the compound represented by general formula (I) is soluble, and the solution prepared by mixing with an aqueous solution containing a solubilizing agent can be administered to a biological sample or a biological individual (in one embodiment, excluding humans). The solubilizing agent is not limited as long as it can impart water solubility to the compound represented by general formula (I) and is biocompatible. For example, biocompatible proteins such as albumin, gelatin, and casein are preferred. One or more solubilizing agents can be used in combination. The solubilizing agent can be used in an aqueous solution at, for example, 1 to 30% by mass, preferably 5 to 20% by mass, and more preferably 7.5 to 10% by mass. The compound represented by general formula (I) can be appropriately adjusted, but can be used in an organic solvent or in a solution prepared by mixing an organic solvent in which the compound represented by general formula (I) is soluble with an aqueous solution containing the solubilizing agent at, for example, 0.01 to 50 mM, preferably 0.1 to 5 mM, and more preferably 0.5 to 1 mM. The lipid droplet detection method of the present invention can further include a step of detecting the lipid droplet detection reagent of the present invention. Detection of the lipid droplet detection reagent can be carried out based on known detection methods for fluorescent reagents.

[0029] The lipid droplet detection reagent of the present invention can be used, for example, as a detection reagent for detecting lipid droplets in a biological sample. The biological sample is not limited to, but is, for example, a cell or isolated tissue. In addition, the lipid droplet detection reagent of the present invention can be applied to and detected in a living body, and can be used as a detection reagent for detecting lipid droplets in cells, tissues, etc. in a living body.

[0030] The lipid droplet detection reagent of the present invention can specifically detect lipid droplets present within cells. Therefore, it is useful as a lipid droplet detection reagent in cells. Detection of lipid droplets present within cells can be performed, for example, as follows. The lipid droplet detection reagent of the present invention is added to cells that contain or are expected to contain lipid droplets. The fluorescent signal of the lipid droplet detection reagent of the present invention is then observed using a fluorescence microscope or the like to detect lipid droplets within the cells. The amount of the lipid droplet detection reagent of the present invention added to cells can be varied appropriately depending on the cells used and the proportion of lipid droplets, but it can be added to cells at a final concentration of, for example, 0.01 to 100 μM, preferably 0.1 to 10 μM. When the lipid droplet detection reagent of the present invention is dissolved in a solvent and then added to cells, the solvent is not limited, and examples of the solvent that can be used include organic solvents such as n-hexane, dibutyl ether, ethyl acetate, acetonitrile, and dimethyl sulfoxide. Cells to which the lipid droplet detection reagent of the present invention can be added are not particularly limited as long as they contain or are expected to contain lipid droplets, and examples include 3T3-L1 cells and isolated adipocytes. Alternatively, cells containing no lipid droplets or cells containing a low amount of lipid droplets may be used in which lipid droplets have been artificially formed. Examples of cells containing no lipid droplets or cells containing a low amount of lipid droplets include HeLa cells, UEET-12 cells, and NIH3T3 cells. Examples of methods for forming lipid droplets include methods that induce lipid droplets by adding oleic acid to cells.

[0031] The lipid droplet detection reagent of the present invention can specifically detect lipid droplets in tissues, as well as lipid droplets and adipose tissue in living organisms (living organisms). Therefore, it is useful as a reagent for detecting lipid droplets in tissues and lipid droplets and adipose tissue in vivo. Detection of lipid droplets present in tissue can be performed, for example, as follows: The lipid droplet detection reagent of the present invention is added to tissue containing or expected to contain lipid droplets. Lipid droplets contained in the tissue can then be detected by observing the fluorescent signal of the lipid droplet detection reagent of the present invention using a fluorescence microscope or the like. The amount of the lipid droplet detection reagent of the present invention added to tissue can be varied as appropriate depending on the tissue used and the proportion of lipid droplets, but it can be added to tissue at a final concentration of, for example, 0.01 to 100 μM, preferably 0.1 to 10 μM. When the lipid droplet detection reagent of the present invention is dissolved in a solvent and then added to tissue, the solvent can be, but is not limited to, organic solvents such as n-hexane, dibutyl ether, ethyl acetate, acetonitrile, and dimethyl sulfoxide. Furthermore, it can also be administered in combination with a biocompatible liquid. Alternatively, as described above, a solution prepared by mixing an organic solvent containing the lipid droplet detection reagent of the present invention with an aqueous solution containing a solubilizer can be added to tissue. Tissues that can be detected by the lipid droplet detection reagent of the present invention include, but are not limited to, subcutaneous fat, visceral fat, and ectopic fat (e.g., fat accumulated in organs such as muscle, liver, heart, pancreas, and kidney).

[0032] Detection of lipid droplets present in a living individual can be performed, for example, as follows. The lipid droplet detection reagent of the present invention is administered to a living individual. Then, the fluorescent signal of the lipid droplet detection reagent of the present invention is observed using a bioimaging technique such as a confocal microscope, allowing adipose tissue to be detected in the living individual without immobilizing the living individual. Examples of administration forms for the lipid droplet detection reagent of the present invention include intravenous administration, subcutaneous administration, and intramuscular administration. The dosage of the lipid droplet detection reagent of the present invention varies depending on the subject and administration form, but can be administered in the range of, for example, 0.01 to 1.0 μmol / kg body weight, preferably 0.1 to 0.5 μmol / kg body weight. When the lipid droplet detection reagent of the present invention is dissolved in a solvent and then administered to a living individual, the solvent can be, but is not limited to, organic solvents such as n-hexane, dibutyl ether, ethyl acetate, acetonitrile, and dimethyl sulfoxide. Furthermore, it can also be administered in combination with a biocompatible liquid. As described above, a solution prepared by mixing an organic solvent containing the lipid droplet detection reagent of the present invention with an aqueous solution containing a solubilizer can be added to a living organism. The living organism to be administered is not particularly limited, and examples thereof include vertebrates and invertebrates, including mammals (mouse, human, pig, dog, rabbit, human, etc.).

[0033] <Compound of the Present Invention> The compound represented by the following general formula (I)' is a novel compound synthesized by the present invention. That is, one aspect of the present invention relates to a compound represented by the following general formula (I)' (hereinafter, sometimes referred to as "compound of the present invention"). The compound represented by general formula (I)' is a compound having the following structure:

[0034]

[0035] In general formula (I)', m represents an integer of 3 to 5. From the viewpoints of photophysical properties, synthesis, etc., m is preferably 3. In general formula (I)', n represents an integer of 3 to 5. From the viewpoints of photophysical properties, synthesis, etc., n is preferably 3.

[0036] In general formula (I)', R' is alkyl having 1 to 5 carbon atoms, aryl having 6 to 12 carbon atoms, or aralkyl having 7 to 13 carbon atoms. Specific examples of alkyl, aryl, and aralkyl in R' are the same as those described above in the section <Reagents for detecting lipid droplets>. From the viewpoints of photophysical properties, synthesis, etc., R' is preferably alkyl having 1 to 4 carbon atoms or phenyl.

[0037] The present invention will be specifically described below with reference to examples, but these are merely examples of the present invention and the scope of the present invention is not limited to these examples.

[0038] <Synthesis Example> Compounds DBC30, DBC30-Bu, and DBC30-Ph were synthesized as follows.

[0039] DBC30: 7-Dibutylamino-2-oxo-2H-1-benzopyran-3-carbaldehyde (150 mg, 0.5 mmol) and N-methyl-1,2-benzenediamine (61 mg, 0.5 mmol) were added to 2-ethoxyethanol (15 mL) and stirred at 120 °C for 24 h. The solvent was removed under reduced pressure, and the resulting crude product was purified using a flash autopurifier (Isolera Spektra, Biotage) (silica gel column, developing solvent: n-hexane:ethyl acetate (1:1, v / v)) to give DBC30 (yield: 83 mg, 41%).

[0040] 1 H NMR (400 MHz, CDCl3, TMS): δ8.18(1H, s), 7.73-7.79(1H, d), 7.26-7.42(3H, m), 6.56-6.63(1H, d), 6.50(1H, s), 3.80(3H, s), 3.31-3.41(4H, t), 1.57-1.67(3H, m), 1.32-1.44(4H, m), 0.94-1.02(6H, t) ESI-MS (m / z) of DBC30: calcd for C 25 H 29 N3O2[M+H] + : 403.23, found: 404.3

[0041]

[0042] <DBC30-Bu> 7-Dibutylamino-2-oxo-2H-1-benzopyran-3-carbaldehyde (151 mg, 0.5 mmol) and N-butyl-1,2-benzenediamine (93 mg, 0.5 mmol) were added to 2-ethoxyethanol (15 mL) and stirred at 120 °C for 24 h. The solvent was removed under reduced pressure, and the resulting crude product was purified using a flash autopurifier (Isolera Spektra, Biotage) (silica gel column, developing solvent: n-hexane:ethyl acetate (1:1, v / v)) to give DBC30-Bu (yield: 47 mg, 20%).

[0043] 1 H NMR (400 MHz, CDCl3, TMS): δ8.13(1H, s), 7.73-7.80(1H, d), 7.38-7.45 (1H, m), 7.31-7.38 (1H, m), 6.55-6.63 (1H, d), 6.50 (1H, s), 4.20-4.30 (2H, t), 3.28-3. 43 (4H, t), 1.72-1.86 (2H, m), 1.56-1.66 (4H, m), 1.32-1.45 (4H, m), 1.16-1.30 (2H, m), 0.94-1.02 (6H, t), 0.80-0.89 (3H, t) ESI-MS (m / z) of DBC30-Bu: calcd for C 28 H 35 N3O2[M+H] + : 445.27, found: 446.4

[0044]

[0045] DBC30-Ph: 7-Dibutylamino-2-oxo-2H-1-benzopyran-3-carbaldehyde (150 mg, 0.5 mmol) and N-phenyl-1,2-benzenediamine (270 mg, 1.6 mmol) were added to 2-ethoxyethanol (15 mL) and stirred at 120 °C for 24 h. The solvent was removed under reduced pressure, and the resulting crude product was purified using a flash autopurifier (Isolera Spektra, Biotage) (silica gel column, developing solvent: n-hexane:ethyl acetate (1:1, v / v)) to give DBC30-Ph (yield: 83 mg, 41%).

[0046] 1 H NMR (400 MHz, CDCl3, TMS): δ8.13 (1H, s), 7.76-7.93 (1H, d), 7.27-7.50 (6H, m), 6.48-6.61 (1H, d), 6.36 (1H, s), 3.20-3.42 (4H, t), 1.49-1.65 (4H, m), 1.27-1.45 (4H, m), 0.89-1.02 (6H, t) ESI-MS (m / z) of DBC30-Ph: calcd for C 30 H 31 N3O2[M+H] + : 465.24, found: 466.3

[0047]

[0048] <Measurement method> (Measurement of maximum absorption wavelength, maximum fluorescence wavelength, and fluorescence quantum yield) The maximum absorption wavelength (λ ) of the above compound in each solvent was measured using a luminescence quantum yield measurement device (C9920-01; manufactured by Hamamatsu Photonics). abs / nm), fluorescence maximum wavelength (λ flu / nm) and fluorescence quantum yield (Φ f The absorption spectrum was measured using an ultraviolet-visible spectrophotometer (Ubest-550; manufactured by JASCO Corporation), and the fluorescence emission spectrum was measured using a fluorescence spectrophotometer (F-7000; manufactured by Hitachi).

[0049] (Measurement of Fluorescence Lifetime) The fluorescence lifetime (τ) of the above compounds in each solvent was measured using a compact fluorescence lifetime measurement device (Quantaurus-Tau; manufactured by Hamamatsu Photonics). f ) was measured.

[0050] The fluorescence yield, i.e., the fluorescence quantum yield (Φ f ) represents the ratio of photons emitted as fluorescence to photons absorbed by a substance. Therefore, the higher the fluorescence yield, the better the luminescence efficiency and the stronger the luminescence intensity. f ) has a value specific to the molecule.

[0051] (Fluorescence Intensity Imaging and Fluorescence Lifetime Imaging) Fluorescence intensity imaging and fluorescence lifetime imaging were obtained using a confocal laser microscope (IX-73; Olympus) equipped with a confocal scanner (DCS-120; Becker & Hickl).

[0052] Example 1 The compounds of the present invention synthesized in the above synthesis example (Figure 4, DBC30, DBC30-Bu, and DBC30-Ph) have an 8-dibutylaminocoumarin skeleton. Figure 5 shows the absorption and fluorescence spectra of DBC30, DBC30-Bu, and DBC30-Ph in dibutyl ether and acetonitrile. Table 1 lists their photophysical parameters. The absorption maximum wavelength was observed at 401-412 nm, and the fluorescence maximum wavelength was observed at 467-484 nm, exhibiting blue fluorescence. Each maximum wavelength shifts to longer wavelengths as the polarity of the solvent increases. The fluorescence quantum yield is 0.9 or higher in all solvents.

[0053]

[0054] Example 2: A comparative experiment was conducted on the performance of DBC30, DBC30-Bu, DBC30-Ph, and a commercially available blue imaging reagent (Lipi-Blue) for imaging lipid droplets in cultured cells. The items were luminescence intensity and photostability.

[0055] Figure 6 shows fluorescence images obtained by adding each fluorescent reagent to a final concentration of 500 nM to HeLa cells cultured for 48 hours in the presence of 400 μM oleic acid, and then irradiating them with the excitation light required for imaging (DBC30, DBC30-Bu, DBC30-Ph, Lipi-Blue: 430 nm) after 60 minutes of culture, and observing them using a confocal laser scanning microscope.It can be seen that the fluorescence intensity of HeLa cells added with DBC30, DBC30-Bu, or DBC30-Ph is greater than that of HeLa cells added with Lipi-Blue.

[0056] Photostability is an important factor for long-term measurements such as tracking the formation, fusion, and disassembly of lipid droplets. HeLa cells were cultured for 48 hours in the presence of 400 μM oleic acid, and each fluorescent reagent was added to a final concentration of 100 nM (DBC30, C6-OBu, BODIPY493 / 503) or 2 μM (Lipi-Blue). After 30 minutes of incubation, the cells were exposed to the excitation light required for imaging (DBC30, C6-OBu, Lipi-Blue: 430 nm, BODIPY493 / 503: 488 nm), and images were captured using a confocal laser scanning microscope. Photostability was evaluated by performing 500 scans, with 50 scans per set. Figure 7 shows the fluorescence intensity ratio versus scan number. Compared to conventional reagents such as Lipi-Blue, DBC30 exhibited superior photostability.

[0057] Example 3: To clarify the lipid droplet selectivity of DBC30, DBC30-Bu, and DBC30-Ph, we performed co-staining experiments with the commercially available green fluorescent lipid droplet reagent BODIPY493 / 503 (Figures 8-10). Each fluorescent reagent was added to HeLa cells cultured for 48 hours in the presence of 400 μM oleic acid to a final concentration of 100 nM. After 30 minutes of incubation, the cells were irradiated with the excitation light required for imaging and observed. The excitation wavelength for DBC30, DBC30-Bu, and DBC30-Ph was 430 nm, with observation wavelengths of 460-500 nm. The excitation wavelength for BODIPY493 / 503 was 488 nm, with observation wavelengths of 510-560 nm. The blue (pseudocolor) colors of DBC30, DBC30-Bu, and DBC30-Ph and the green (pseudocolor) colors of BODIPY493 / 503 were observed at the same location within the cells, indicating that DBC30, DBC30-Bu, and DBC30-Ph have lipid droplet selectivity.

[0058] Example 4: Because DBC30 fluoresces in the blue wavelength region, we investigated co-staining with intracellular organelle-selective reagents that exhibit green or red fluorescence. The green fluorescent reagent used was Mito Tracker Green (excitation wavelength: 488 nm, observation wavelength: 510-560 nm), which exhibits mitochondrial accumulation, and the red fluorescent reagent used was Lyso Tracker Red (excitation wavelength: 570 nm, observation wavelength: >590 nm), which exhibits lysosomal accumulation. As shown in Figure 11, each fluorescent reagent was distributed to different intracellular organelles (blue: lipid droplets, green: mitochondria, red: lysosomes), allowing us to obtain information about their shape and location. This allows for multicolor imaging and can be used in combination with green fluorescent protein.

[0059] Example 5: Fluorescence imaging of intracellular lipid droplets and adipose tissue using DBC30, DBC30-Bu, and DBC30-Ph is shown. The DBC30 series has extremely low solubility in water (physiological saline), making it difficult to dissolve directly. Furthermore, when a 5 mM stock solution dissolved in dimethyl sulfoxide (DMSO) was added to physiological saline (10% by volume), DBC30, DBC30-Bu, and DBC30-Ph precipitated, making them unsuitable for administration to mice. Therefore, when a 5 mM stock solution was added to physiological saline containing 10% bovine serum albumin (10% by volume), the precipitation of DBC30, DBC30-Bu, and DBC30-Ph was suppressed. For MNs-NB administration, a DMSO stock solution was directly administered to mice. Because DMSO administration can cause shock death in mice, the administration method of the present invention is considered to be a safer method. Here, we performed fluorescence imaging experiments using a fluorescence lifetime imaging microscope (FLIM) that can capture fluorescence lifetime images in addition to fluorescence intensity images, by administering 100-200 μL (50-100 nmol) of a 500 μM solution into the tail vein of anesthetized mice.

[0060] Example 6: Fatty liver model mice are known to accumulate larger amounts of lipid droplets in the liver than normal mice. Here, we created fatty liver model mice by feeding mice (BALB / cAJcl) an ultra-high-fat, choline-deficient, methionine-reduced diet for two weeks. Figure 12 shows fluorescence intensity imaging images and fluorescence lifetime imaging (FLIM) images obtained after administering 50 nmol of DBC30 to normal mice and fatty liver model mice. Small lipid droplets were observed within hepatocytes in mice fed a normal diet. On the other hand, large lipid droplets were observed throughout the liver surface in fatty liver model mice (2 weeks), indicating lipid accumulation in the liver.

[0061] Example 7 Various adipose tissues and lipid droplets exist within an individual. These were imaged using DBC30. Figure 13 shows fluorescent images of subcutaneous adipose tissue, abdominal adipose tissue, skeletal muscle, and kidney obtained after administering DBC30 (50 nmol) to mice (BALB / cAJcl). Fluorescence derived from DBC30 was observed in areas where lipids accumulated. In particular, in the kidney, small lipid droplets distributed within tubular cells were successfully imaged, and it is expected that this image will be useful as a tool for studying the relationship between lipids in lifestyle-related diseases such as diabetes and renal dysfunction.

[0062] Based on the above results, the compounds containing DBC30, DBC30-Bu, and DBC30-Ph developed in this invention exhibit blue fluorescence and are new reagents that can be used to image intracellular lipid droplets and adipose tissue and lipid droplets in living individuals.

[0063] The present invention can be used for fluorescent imaging of adipose tissue and lipid droplets in biological samples and living individuals.

Claims

1. A reagent for detecting lipid droplets, comprising a compound represented by the following general formula (I): 【Chemistry 1】 (In the formula, m represents an integer from 3 to 5; n is an integer from 3 to 5, R is alkyl having 1 to 5 carbon atoms, aryl having 6 to 12 carbon atoms, aralkyl having 7 to 13 carbon atoms, or or hydrogen.)

2. 2. The detection reagent according to claim 1, wherein R is alkyl having 1 to 4 carbon atoms or phenyl.

3. The detection reagent according to claim 1, wherein m and n are 3.

4. The detection reagent according to claim 1 for detecting lipid droplets in a biological sample.

5. The detection reagent according to claim 4 , wherein the biological sample is a cell or tissue.

6. The detection reagent according to claim 1, for detecting lipid droplets in a living individual.

7. A step of administering the detection reagent according to any one of claims 1 to 6 to a biological sample or a living individual (excluding humans); A method for detecting lipid droplets, comprising:

8. A compound represented by the following general formula (I)'. 【Chemistry 2】 (In the formula, m represents an integer from 3 to 5; n is an integer from 3 to 5, R' is an alkyl having 1 to 5 carbon atoms, an aryl having 6 to 12 carbon atoms, or an aralkyl having 7 to 13 carbon atoms. It's a kill.)

9. 9. The compound according to claim 8, wherein R' is alkyl having 1 to 4 carbon atoms or phenyl.

10. 10. The compound according to claim 8 or 9, wherein m and n are 3.

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