Compound, fluorescent dye, use of compound, method for detecting RNA in sample, kit, and method for detecting infection with virus having envelope

Abstract

Provided is a compound represented by general formula (1). In formula (1): R1 is a hydrophobic group having 4 or more carbon atoms; R2 to R10 are each independently a hydrogen atom, a hydroxy group, a thiol group, a halo group, an alkyl group, an alkenyl group, an alkynyl group, an alkoxy group, an aryl group, or an amino group; and R11 is a hydrogen atom or an alkyl group.

Description

Compounds, fluorescent dyes, use of the compounds, methods for detecting RNA in a sample, kits, and methods for detecting infection with enveloped viruses

[0001] The present invention relates to compounds, fluorescent dyes, uses of the compounds, methods for detecting RNA in a sample, kits, and methods for detecting infection with an enveloped virus. This application claims priority to U.S. Provisional Application No. 63 / 560,774, filed March 4, 2024, the contents of which are incorporated herein by reference.

[0002] As the diverse biological functions of RNA become clearer, interest in intracellular RNA imaging analysis is growing. In particular, fluorescent probes (also called "fluorescent dyes") based on nucleic acid-binding small molecules are highly manipulable and extremely useful. However, most nucleic acid-binding small molecules generally exhibit higher selectivity for DNA than for RNA, making it difficult to develop RNA-selective fluorescent probes.

[0003] To the inventor's knowledge, the only commercially available RNA-selective fluorescent probe is SYTO™ RNAselect™ (Molecular Probes). This RNA-selective fluorescent probe exhibits a green fluorescence response upon binding to RNA. While this response is greater than that observed upon binding to DNA, its fluorescence wavelength lies in the green region, which is the biological background fluorescence region, and its poor photostability has been identified as a problem. Furthermore, this molecule is cell membrane permeable and stains intracellular RNA, particularly intranuclear RNA such as ribosomal RNA, making it applicable to imaging analysis, but it is unable to selectively stain nucleoli in living cells. Recently, Nucleolus Bright Green and Nucleolus Bright Red (DOJINDO) have begun to be commercially available as intranuclear RNA stains, but like SYTO RNA select, they have poor compatibility with living cells.

[0004] The inventors previously reported that a monomethine cyanine dye (BIQ) consisting of a benzo[c,d]indole ring and a quinoline ring and represented by the following structural formula has cell membrane permeability and emits red fluorescence at wavelengths (λ ｅｍ BIQ has been found to be extremely useful for live cell RNA imaging due to its significantly improved fluorescence quantum yield (φ = 657 nm) and photostability. ｆｌ ) is only 0.0085.

[0005]

[0006] Furthermore, fluorescent probes that are cell membrane permeable and have intracellular RNA selectivity have the potential to rapidly detect and visualize viruses in the environment, since many viruses in the environment contain RNA within their envelope structures. The present inventors have developed highly sensitive RNA-selective staining agents and investigated methods for detecting viruses more quickly and accurately using the RNA-selective staining agents, and have found novel compounds that are suitable as fluorescent probes that are RNA-selective and have excellent fluorescence quantum yields (Patent Document 1).

[0007] Patent No. 7029841

[0008] The compound described in Patent Document 1 would be more useful if its viable cell membrane permeability and nucleolus selectivity over cytoplasm could be improved. The object of the present invention is to provide a compound, a fluorescent dye, use of the compound, a method for detecting RNA in a sample, a kit, and a method for detecting infection with an enveloped virus, which have high viable cell membrane permeability, excellent nucleolus selectivity over cytoplasm, and exhibit high fluorescence response upon binding to RNA in the nucleolus.

[0009] One aspect of the present invention includes the following: [1] A compound represented by the following general formula (1):

[0010]

[0011] In formula (1), R １ is a hydrophobic group having 4 or more carbon atoms, and R ２ ~R １０are each independently a hydrogen atom, a hydroxy group, a thiol group, a halo group, an alkyl group, an alkenyl group, an alkynyl group, an alkoxy group, an aryl group, or an amino group; R １１ is a hydrogen atom or an alkyl group.

[0012] [2] The compound according to [1], wherein the hydrophobic group has 6 to 20 carbon atoms. [3] The compound according to [1] or [2], wherein the hydrophobic group contains an aryl group. [4] The compound according to any one of [1] to [3], wherein the hydrophobic group contains an aryl group and the hydrophobic group has 6 to 20 carbon atoms. [5] R in the general formula (1) ２ ~R １０ is a hydrogen atom, and R １１ [6] The compound according to any one of [1] to [4], wherein the hydrophobic group is -(CH ２ ) ２ C ６ H ５ , -C ６ H ５ , -C ６ H ４ -C(CH ３ ) ３ , -C ６ H ３ -(C(CH ３ ) ３ ) ２or a naphthyl group. [7] The compound according to any one of [1] to [5]. [8] The fluorescent dye according to [7], which is an RNA detection agent. [9] Use of the compound according to any one of [1] to [6] for intracellular RNA imaging.

[10] A method for detecting RNA in a sample, comprising: mixing the compound according to any one of [1] to [6] with a sample containing RNA to form a mixed sample in which the compound and the RNA in the sample are bound; and irradiating the mixed sample with light to detect RNA in the mixed sample.

[11] The method according to

[10] , wherein the sample is a living cell.

[12] A kit for detecting RNA in a sample, comprising the compound according to any one of [1] to [6], and instructions for detecting RNA in a sample.

[13] A method for detecting infection with an enveloped virus, comprising the steps of: bringing the compound according to any one of [1] to [6] and a fluorescent dye that binds to the envelope of the enveloped virus into contact with a sample suspected of containing enveloped virus particles; and irradiating the sample with light after the contacting, wherein when a fluorescent signal derived from the compound and a fluorescent signal derived from the fluorescent dye that binds to the envelope of the enveloped virus are both detected, this indicates a high possibility that the enveloped virus particles are present in the sample.

[0013] According to the above aspects, it is possible to provide a compound, a fluorescent dye, use of the compound, a method for detecting RNA in a sample, a kit, and a method for detecting infection with an enveloped virus, which have high membrane permeability in living cells, excellent selectivity for nucleoli relative to the cytoplasm, and exhibit high fluorescence response upon binding to RNA within the nucleolus.

[0014]

[0023] Figure 6 is a schematic diagram illustrating virus detection using a probe that binds to the virus particle envelope and a probe that specifically binds to the virus particle RNA.

[0024] Figure 7 is a graph showing the fluorescence intensity of each fluorescent dye for each synthetic nucleic acid in Experimental Example 3. Figure 3(A) is an RNA imaging image in Experimental Example 4, and Figure 3(B) is a graph showing the fluorescence intensity on the line in Figure 3(A). Figure 4(A) is an image showing the fluorescence of the fluorescent dye alone in Experimental Example 5, Figure 4(B) is an image showing the fluorescence of the fluorescent dye and Hoechst 33342 in Experimental Example 5, and Figure 4(C) is a graph showing the fluorescence intensity on the line in Figure 4(B). Figure 6(A) is a merged image of a differential interference contrast image (DIC) and an RNA imaging image in Experimental Example 6, and Figure 6(B) is a graph showing the fluorescence intensity on the line in Figure 6(A). 7(A) is a merged image of a differential interference contrast image (DIC) and an RNA imaging image in Experimental Example 6, and FIG. 7(B) is a graph showing the fluorescence intensity on the line in FIG. 7(A). FIG. 8(A) is a merged image of a differential interference contrast image (DIC) and an RNA imaging image in Experimental Example 6, and FIG. 8(B) is a graph showing the fluorescence intensity on the line in FIG. 8(A). FIG. 9(A) is a merged image of a differential interference contrast image (DIC) and an RNA imaging image in Experimental Example 6, and FIG. 9(B) is a graph showing the fluorescence intensity on the line in FIG. 9(A). FIG. 10(A) is a merged image of a differential interference contrast image (DIC) and an RNA imaging image in Experimental Example 6, and FIG. 10(B) is a graph showing the fluorescence intensity on the line in FIG. 10(A). Graphs showing changes in the fluorescence intensity of nucleoli over time in Experimental Example 7. 1 shows RNA imaging images of nucleoli 1 hour, 3 hours, and 5 hours after the addition of Actinomycin D in Experimental Example 8. 2 shows a graph indicating the average cross-sectional area of ​​nucleoli for each incubation time in Experimental Example 8. 3 shows the fluorescence intensity of nucleoli for each incubation time in Experimental Example 8. 4 shows RNA imaging images of nucleoli 0.5 hours after the addition of Actinomycin D in Experimental Example 8.1 shows RNA imaging images of nucleoli before the addition of Actinomycin D (control) and 3 hours after the addition in Experimental Example 8.

[0015] "First Aspect" In a first aspect of the present invention, there is provided a compound represented by the following general formula (1) (hereinafter also referred to as "compound (1)").

[0016]

[0017] In formula (1), R １ is a hydrophobic group having 4 or more carbon atoms, and R ２ ~R １０ are each independently a hydrogen atom, a hydroxy group, a thiol group, a halo group, an alkyl group, an alkenyl group, an alkynyl group, an alkoxy group, an aryl group, or an amino group; R １１ is a hydrogen atom or an alkyl group.

[0018] In this specification, R in formula (1) １ ~R １１Where a portion of the group can be substituted, the term "substituted" indicates that one or more (e.g., 1, 2, 3, 4, 5, or 6; in some embodiments, 1, 2, or 3; in other embodiments, 1 or 2) hydrogens of the group designated with "substituted" can be replaced with a group selected from the listed designated groups or with a suitable group known to one of ordinary skill in the art, provided that such replacement results in a stable compound. Suitable substituents for the substituted groups include alkyl, alkenyl, alkynyl, alkoxy, halo, haloalkyl, hydroxy, hydroxyalkyl, aryl, alkylaryl, heteroaryl, heterocycle, cycloalkyl, alkanoyl, alkoxycarbonyl, amino, alkylamino, dialkylamino, trifluoromethylthio, difluoromethyl, acetylamino, nitro, trifluoromethyl, trifluoromethoxy, carboxy, carboxyalkyl, keto, thioxo, alkylthio, alkylsulfinyl, alkylsulfonyl, arylsulfinyl, arylsulfonyl, heteroarylsulfinyl, heteroarylsulfonyl, heterocyclesulfinyl, heterocyclesulfonyl, phosphate, sulfate, hydroxylamine, hydroxyl(alkyl)amine, and cyano.

[0019] The term "halo" refers to fluoro, chloro, bromo, and iodo groups.

[0020] The term "alkyl" refers to a branched, unbranched (also called "straight chain"), or cyclic saturated hydrocarbon. In some embodiments, alkyl groups have, for example, 1 to 20 carbon atoms, often 1 to 12 carbon atoms or 1 to 6 carbon atoms. Examples of alkyl groups include, but are not limited to, methyl, ethyl, 1-propyl, 2-propyl, 1-butyl, 2-methyl-1-propyl, 2-butyl, 2-methyl-2-propyl (t-butyl), 1-pentyl, 2-pentyl, 3-pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl, 3-methyl-1-butyl, 2-methyl-1-butyl, 1-hexyl, 2-hexyl, 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 3-methyl-3-pentyl, 2-methyl-3-pentyl, 2,3-dimethyl-2-butyl, 3,3-dimethyl-2-butyl, hexyl, octyl, decyl, dodecyl, and the like. Alkyl can be unsubstituted or substituted. Substituted alkyl groups can contain one or more non-carbon and non-hydrogen atoms such as oxygen, nitrogen, sulfur, halogens, and phosphorus.

[0021] The term "alkenyl" refers to a carbon-carbon sp ２ It refers to a branched or unbranched unsaturated hydrocarbon having a double bond. In some embodiments, an alkenyl group can have, for example, 2 to 10 carbon atoms, or 2 to 6 carbon atoms. In other embodiments, an alkenyl group has 2 to 4 carbon atoms. Examples of alkenyl include, but are not limited to, ethylene or vinyl, allyl, cyclopentenyl, 5-hexenyl, and the like. An alkenyl can be unsubstituted or substituted. A substituted alkenyl group can contain one or more non-carbon and non-hydrogen atoms, such as oxygen, nitrogen, sulfur, halogens, and phosphorus.

[0022] The term "alkynyl" refers to a branched or unbranched unsaturated hydrocarbon chain having a carbon-carbon, sp triple bond. In some embodiments, an alkynyl group can have, for example, 2 to 10 carbon atoms, or 2 to 6 carbon atoms. In other embodiments, an alkynyl group can have 2 to 4 carbon atoms. Examples of alkynyl groups include, but are not limited to, ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 1-octynyl, and the like. An alkynyl can be unsubstituted or substituted. Substituted alkynyl groups can contain one or more non-carbon and non-hydrogen atoms, such as oxygen, nitrogen, sulfur, halogens, and phosphorus.

[0023] The term "alkoxy" refers to -O-alkyl, where alkyl is defined herein. In some embodiments, alkoxy groups have 1 to 12 carbon atoms or 1 to 6 carbon atoms. Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, n-propoxy, iso-propoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, n-hexyloxy, 1,2-dimethylbutoxy, and the like. An alkoxy can be unsubstituted or substituted. A substituted alkoxy group can contain an oxygen bonded to a substituted alkyl group.

[0024] The term "aryl" refers to an aromatic hydrocarbon group derived by removing a hydrogen atom from a carbon atom of a parent aromatic ring. Aryl groups can have 6 to 18 carbon atoms, 6 to 14 carbon atoms, or 6 to 10 carbon atoms. Aryl groups can have a single ring (e.g., phenyl) or multiple condensed rings (fused rings) in which at least one ring is aromatic (e.g., naphthyl, dihydrophenanthrenyl, fluorenyl, or anthryl). Typical aryl groups include, but are not limited to, radicals derived from benzene, naphthalene, anthracene, biphenyl, and the like. Aryl can be unsubstituted or substituted. For example, aryl groups can be substituted with one or more substituents (as described above) to produce various substituted aryls, such as pentafluorophenyl or p-trifluoromethylphenyl.

[0025] The term "amino" refers to -NH ２ The amino group can be optionally substituted as defined for the term "substituted." For example, an amino group can be substituted with -NR ２ (wherein R is a group listed in the definition of "substituted"), for example, the group -NR ２ can include "alkylamino" (where one R is alkyl and the other R is alkyl or hydrogen) and / or "acylamino" (-N(R)C(=O)R) (where each R is independently hydrogen, alkyl, alkaryl, or aryl). An amino group can be a primary amine (-NH ２ ), a secondary amine (—NHR), or a tertiary amine.

[0026] In formula (1), R １ is a hydrophobic group having 4 or more carbon atoms. １is a hydrophobic group having 4 or more carbon atoms, the membrane permeability of living cells is enhanced, and the selectivity for nucleoli relative to the cytoplasm is improved. Examples of hydrophobic groups include, but are not limited to, alkyl groups and aryl groups. By introducing a hydrophobic group, it is preferable that the octanol / water partition coefficient of the entire compound is 0.3 or more, preferably 1.0 or more. If necessary, it can be 1.5 or more, or even 2 or more.

[0027] The number of carbon atoms in the hydrophobic group is preferably 6 to 20, more preferably 7 to 15, and even more preferably 8 to 12. When the number of carbon atoms in the hydrophobic group is within the above range, the balance between fluorescence intensity, nucleolus selectivity, and staining speed is particularly excellent. When the number of carbon atoms in the hydrophobic group is equal to or greater than the above lower limit, the hydrophobicity of the entire compound increases, and more preferable nucleolus selectivity is obtained. When the number of carbon atoms in the hydrophobic group is equal to or less than the above upper limit, more preferable fluorescence intensity is obtained.

[0028] The hydrophobic group preferably contains an aryl group. When the hydrophobic group contains an aryl group, the membrane permeability of living cells is further increased, and the selectivity for nucleoli relative to the cytoplasm is further improved. The hydrophobic group contains an aryl group, and the number of carbon atoms in the hydrophobic group is preferably 6 to 20, more preferably 7 to 15, and even more preferably 8 to 12.

[0029] The hydrophobic group is —(CH ２ ) ２ C ６ H ５ , -C ６ H ５ , -C ６ H ４ -C(CH ３ ) ３ , -C ６ H ３ -(C(CH ３ ) ３ ) ２ or a naphthyl group.

[0030] In formula (1), R ２ ~R １０ R are each independently a hydrogen atom, a hydroxy group, a thiol group, a halo, an alkyl group, an alkenyl group, an alkynyl group, an alkoxy group, an aryl group, or an amino group.２ ~R １０ are each preferably independently a hydrogen atom, a hydroxy group, a halo group, an alkyl group, an aryl group, or an amino group. ２ ~R １０ are each independently a hydrogen atom, a hydroxy group, a thiol group, a halo group, an alkyl group, an alkenyl group, an alkynyl group, an alkoxy group, an aryl group, or an amino group, and more preferably, all of them are unsubstituted. ２ ~R １０ is more preferably a hydrogen atom.

[0031] In formula (1), R １１ is a hydrogen atom or an alkyl group. １１ The number of carbon atoms in the alkyl group in R is preferably 1 to 6, more preferably 1 to 4, even more preferably 1 to 3, and particularly preferably 1 or 2. １１ is preferably unsubstituted. １１ is preferably an alkyl group, more preferably a methyl group.

[0032] In some preferred embodiments, R １ Ha-(CH ２ ) ２ C ６ H ５ , -C ６ H ５ , -C ６ H ４ -C(CH ３ ) ３ , -C ６ H ３ -(C(CH ３ ) ３ ) ２ or a naphthyl group, and R ２ ~R １０ is a hydrogen atom, and R １１ is a methyl group.

[0033] In some preferred embodiments, R １ Ha-(CH ２ ) ２ C ６ H ５ , -C ６ H ５ , or -C ６ H４ -C(CH ３ ) ３ and R ２ ~R １０ is a hydrogen atom, and R １１ is a methyl group. That is, as compound (1), compounds represented by the following structural formulas (1-1) to (1-3) are preferred. Note that the compound represented by the following structural formula (1-1) (hereinafter also referred to as "compound (1-1)") is a combination of benzo[c,d]indole and oxazolopyridine, and therefore, hereinafter, compound (1-1) will be referred to as "BIOP-C ３ Similarly, the compound represented by the following structural formula (1-2) (hereinafter also referred to as "compound (1-2)") may be referred to as "BIOP-Benzyl." The compound represented by the following structural formula (1-3) (hereinafter also referred to as "compound (1-3)") may be referred to as "BIOP-Benzyl-t-Bu." １ ~R １０ is a hydrogen atom, and R １１ A compound in which is a methyl group is sometimes called "BIOP."

[0034]

[0035] Compound (1) emits fluorescence upon binding to nucleic acids and can therefore be used as a fluorescent dye. The fluorescent dye (hereinafter also referred to as a "fluorescent probe" or "fluorescent probe") may consist solely of compound (1), or may contain components other than compound (1) as long as the effects of the present invention are not impaired. The fluorescent dye preferably consists solely of compound (1).

[0036] Furthermore, compound (1) has an excellent fluorescent response ability upon RNA binding, is more selective for RNA than DNA, and can detect RNA with high sensitivity. A fluorescent dye containing compound (1) or consisting solely of compound (1) can be applied to living cells and can selectively stain RNA-rich nucleoli at low concentrations (e.g., 1.0 μM) in a short period of time (e.g., 20 min). Because of their clear response ability, they can be suitably used for intracellular RNA imaging. That is, a fluorescent dye containing compound (1) or consisting solely of compound (1) can be suitably used as an RNA detection agent. In particular, compounds (1-1) to (1-3) have an excellent fluorescent response ability upon RNA binding and can detect RNA with higher sensitivity. A fluorescent dye containing any one or more of the compounds (1-1) to (1-3), or a fluorescent dye consisting of only any one or more of the compounds (1-1) to (1-3), is applicable to living cells, can selectively stain nucleoli, which are rich in RNA, at a low concentration (e.g., 1.0 μM) in a short time (e.g., 20 minutes), and has a clearer response performance, and therefore can be more suitably used for intracellular RNA imaging.

[0037] Compound (1) has high membrane permeability in living cells and excellent selectivity for nucleoli over cytoplasm. In particular, compounds (1-1) to (1-3) have even higher membrane permeability in living cells and excellent selectivity for nucleoli over cytoplasm.

[0038] Compound (1) may be covalently bound to other molecules such as antibodies, proteins, peptides, polypeptides, amino acids, enzymes, nucleic acids, lipids, polysaccharides, drugs, beads, solid supports (e.g., glass or plastic), etc.

[0039] Compound (1) preferably emits little or no fluorescence in the absence of nucleic acid. Fluorescence can be measured by irradiating compound (1) with light of an appropriate wavelength and monitoring the emitted fluorescence. Compound (1) preferably emits stronger fluorescence in the presence of RNA than in the presence of DNA. The fluorescence in the presence of RNA relative to the fluorescence in the presence of DNA is measured with the compound concentration constant and the RNA and DNA concentrations constant. The higher the RNA / DNA fluorescence ratio, the more suitable it is for RNA detection in the presence of DNA. The RNA / DNA fluorescence ratio is preferably 1 or more, more preferably greater than 1, even more preferably 1.2 or more, particularly preferably 1.5 or more, and most preferably 2 or more. Compound (1) meets the criteria for suitability for RNA detection.

[0040] Compound (1) may be characterized by its excitation and emission wavelengths. For example, the excitation maximum may be about 450 nm to about 650 nm. The excitation maximum between these values ​​may be about 450 nm, about 475 nm, about 500 nm, about 525 nm, about 550 nm, about 575 nm, about 600 nm, about 625 nm, about 650 nm, and any two values ​​between these. For example, the emission maximum may be about 500 nm to about 675 nm. The emission maximum between these values ​​may be about 500 nm, about 525 nm, about 550 nm, about 575 nm, about 600 nm, about 625 nm, about 650 nm, about 675 nm, and any two values ​​between these.

[0041] An example of a method for producing compound (1) will be specifically described below. Compound (1) can be produced, for example, by the following schemes 1 to 4.

[0042] (Scheme 1) First, compound (2) is used to obtain compound (3) according to J. Med. Chem., 2016, 59, 1565-1579. ４ ~R ９ is a hydrogen atom, and R １１When R is a methyl group, compound (3) is 1-methylbenzo[c,d]indol-2(1H)-one. Next, a 1,4-dioxane solution containing compound (3) and Lawesson's reagent is stirred overnight. The temperature during stirring is preferably 100 to 110°C. This solution is filtered at room temperature, and the resulting solid is washed with 1,4-dioxane to obtain compound (4). ４ ~R ９ is a hydrogen atom, and R １１ When R is a methyl group, compound (4) is 1-methylbenzo[c,d]indol-2(1H)-thione. Next, iodomethane is added to compound (4) and the mixture is heated under reflux overnight. After concentrating the solution under reduced pressure, diethyl ether is added, and the resulting solid is filtered to obtain compound (5). ４ ~R ９ is a hydrogen atom, and R １１ When is a methyl group, compound (5) is 1-methyl-2(methylthio)benzo[c,d]indole-1-ium.

[0043]

[0044] (Scheme 2) Next, compound (5) is used to obtain compound (6) and then compound (7) according to the method described in Dyes and Pigments., 1991, 15, 215-223. ４ ~R ９ is a hydrogen atom, and R １１ is a methyl group, compound (6) is 1-methyl-2(methylthio)benzo[c,d]indole-1-ium, and compound (7) is 1,2-dimethylbenzo[cd]indol-1-ium iodide.

[0045]

[0046] (Scheme 3) Separately, compound (8) is used as a starting material to obtain compound (10) via compound (9) according to Eur. J. Med. Chem., 2005, 40, 15-23. Then, compound (10) and X—CH ２ -R １(X is a halogen atom such as Cl, I, Br, etc.) is subjected to a heating reaction, and then purified to obtain compound (11). ２ , R ３ , R １０ is a hydrogen atom, compound (8) is 4-aminopyridin-3-ol, and compound (10) is 2-(methylthio)oxazolo[5,4-c]pyridine.

[0047]

[0048] (Scheme 4) Triethylamine is added to an acetonitrile solution containing compound (7) produced in Scheme 2 and compound (11) produced in Scheme 3. This solution is reacted while stirring. The reaction temperature is preferably 80 to 90°C. The reaction time is preferably 30 minutes to 1 hour. After the reaction, the solution is cooled to room temperature. Diethyl ether is then added to obtain a precipitate, which is then collected by filtration. This precipitate is purified by silica gel chromatography. The obtained crude product is dissolved in methanol, and diethyl ether is then added dropwise to obtain a precipitate, which is then collected by filtration and dried to obtain compound (1).

[0049]

[0050] "Second Aspect" In a second aspect of the present invention, there is provided a method comprising the steps of: mixing compound (1) with a sample containing RNA to form a mixed sample in which compound (1) and the RNA in the sample are bound; and irradiating the mixed sample to detect the RNA in the mixed sample.

[0051] The details of compound (1) are as explained for compound (1) of the first aspect.

[0052] The RNA-containing sample is not particularly limited and may be, for example, one or more types of cells, tissues, cell lysates, cell culture media, etc., or may be a non-biological sample. The cells may be any cells, such as bacterial cells, fungal cells, insect cells, and mammalian cells. Mammalian cells are preferably human cells. In a preferred embodiment, the cells are living cells. That is, the RNA-containing sample is preferably a living cell. The RNA-containing sample may be a solid, liquid, or suspension. The RNA-containing sample may be a biological fluid such as blood, plasma, or urine. The RNA-containing sample may be immobilized in a gel or on a membrane, bound to one or more beads, or configured in the form of an array. The RNA-containing sample may be a buffer solution or water containing a partially or fully purified nucleic acid preparation.

[0053] The step of mixing compound (1) with a sample containing RNA to form a mixed sample in which the compound and the RNA in the sample are bound may be carried out at any appropriate temperature and time. Typically, the temperature is room temperature. Examples of such temperatures include about 20°C, about 25°C, about 30°C, about 35°C, about 37°C, about 40°C, about 42°C, and any range between these two values. Temperatures above about 42°C and temperatures below about 20°C may also be applicable depending on the test sample. The time is not particularly limited and may be any time appropriate for detecting a change in fluorescence. Examples of the time length include about 10 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 60 minutes, about 90 minutes, about 120 minutes, about 180 minutes, about 240 minutes, about 300 minutes, about 360 minutes, about 420 minutes, about 480 minutes, about 540 minutes, about 600 minutes, and any range between these two values. Further extension of the time is possible depending on the sample to be tested.

[0054] The concentration of compound (1) is not particularly limited and may be any concentration at which fluorescent excitation and emission signals can be appropriately detected in the presence of RNA. An example of the concentration range is about 10 nM to 1 mM. Examples of concentrations include about 10 nM, about 100 nM, about 1 μM, about 10 μM, about 100 μM, about 1 mM, and any range between these two values.

[0055] Suitable illumination devices include portable ultraviolet lamps, mercury arc lamps, xenon lamps, lasers (such as argon and YAG lasers), and laser diodes. These illumination sources are typically optically integrated into laser scanners, fluorescence microplate readers, or standard or microfluorimeters.

[0056] The detection step may be performed by visual inspection or by the use of various measuring instruments, such as a CCD camera, a video camera, photographic film, a laser scanner device, a fluorometer, a photodiode, a quantum counter, an epifluorescence microscope, a scanning microscope, a flow cytometer, a fluorescence microplate reader, or an amplification device such as a photomultiplier tube.

[0057] The detecting step may be performed at a single time point, at multiple time points, or continuously.

[0058] "Third Aspect" In a third aspect of the present invention, there is provided a kit for detecting RNA in a sample, the kit comprising compound (1) and instructions for detecting RNA in a sample.

[0059] The details of compound (1) are as explained for compound (1) of the first aspect.

[0060] The sample is not particularly limited as long as it contains RNA. For example, it may be one or more types of cells, tissues, cell lysates, cell culture media, etc., or the sample may be a non-biological sample. The cells may be any cells, such as bacterial cells, fungal cells, insect cells, and mammalian cells. Mammalian cells are preferably human cells. In a preferred embodiment, the cells are living cells. That is, the sample is preferably a living cell. The sample may be a solid, liquid, or suspension. The sample may be a biological fluid such as blood, plasma, or urine. The sample may be immobilized in a gel or on a membrane, bound to one or more beads, or configured in the form of an array. The sample may be a buffer solution or water containing a partially or fully purified nucleic acid preparation.

[0061] In a preferred embodiment, the kit comprises a container containing compound 1. The kit may also comprise a pipette, dropper, or other sample manipulation substrate.

[0062] The kit may also include positive and / or negative control samples. Positive control samples may contain RNA and / or RNA coexisting with DNA. Negative control samples may be samples containing DNA without RNA, or may be samples containing no nucleic acid at all.

[0063] The kit may also include one or more additional dyes or stains. For example, the kit may include a total nucleic acid stain. The kit may also include a cell-permeant nucleic acid stain for distinguishing between live and dead cells.

[0064] The kit may further comprise water, buffers, buffer salts, detergents, surfactants, salts, polysaccharides, or other materials commonly used in bioassays. The kit may also comprise a solvent, such as an aqueous, non-aqueous, or aqueous / non-aqueous solvent system.

[0065] "Fourth Aspect" In a fourth aspect of the present invention, there is provided a method for detecting enveloped virus particles in a sample, the method comprising the steps of contacting a fluorescent probe consisting of compound (1) and a fluorescent probe that binds to the virus envelope with the sample suspected of containing virus particles, and irradiating the sample with light after the contact, wherein if both types of fluorescent probes exhibit a fluorescence response, this indicates a high possibility that virus particles are present in the sample.

[0066] FIG. 1 shows a schematic diagram of a method for detecting virus particles in a sample in the fourth embodiment.

[0067] A virus particle (1) has an envelope (2) as an outer shell and viral RNA (3) encapsulated in the envelope (2). Its diameter D is approximately 100 nm. Therefore, when a fluorescent dye serving as an envelope-binding probe (4) that selectively binds to the viral envelope (2) and a compound (1) serving as an RNA-binding probe (5) that selectively binds to the viral RNA (3) are brought into contact with a sample suspected of containing the virus particle (1), if the virus particle (1) is present in the sample, the envelope (2) and the fluorescent dye bind, and an increase in luminescence intensity resulting from this binding can be detected. Furthermore, the viral RNA (3) binds to the compound (1), and an increase in luminescence intensity resulting from this binding can be detected.

[0068] If no virus particles (1) are present in the sample, an increase in luminescence intensity resulting from either or both of the binding of the fluorescent dye to the envelope (2) and the binding of compound (1) to the viral RNA (3) cannot be detected.

[0069] This allows the presence or absence of virus particles in a sample to be detected or determined quickly, easily, and with high sensitivity.

[0070] Furthermore, by using two types of fluorescent dyes, compound (1) that selectively binds to the encapsulated viral RNA and a fluorescent probe that binds to the viral envelope, when both dyes bind to the virus particle, fluorescence energy transfer may occur, and by detecting the fluorescence intensity of these dyes, it may be possible to identify the type and characteristics of the virus.

[0071] The virus that constitutes the virus particle may be any virus that has RNA as its genome and also has an envelope structure, and examples thereof include, but are not limited to, coronavirus, influenza virus, herpes virus, rubella virus, hepatitis B and C virus, and AIDS virus.

[0072] The details of compound (1) are as explained for compound (1) of the first aspect.

[0073] Compound (1) can be used in combination with the following fluorescent dyes. Examples of fluorescent dyes that bind to virus envelopes include peptide fluorescent dyes that can selectively bind to the lipid bilayer membrane structure of the envelope, cell membrane staining dyes, etc. For example, compound (1) may be combined with a fluorescent dye that binds to the lipid bilayer membrane structure of the envelope, as described in Patent Document 1, to identify viruses.

[0074] The RNA-containing sample is not particularly limited and may be, for example, one or more types of cells, tissues, cell lysates, cell culture media, etc., or may be a non-biological sample. The cells may be any cells, such as bacterial cells, fungal cells, insect cells, and mammalian cells. Mammalian cells are preferably human cells. In a preferred embodiment, the cells are live cells. The RNA-containing sample may be a solid, liquid, or suspension. The RNA-containing sample may be a biological fluid, such as blood, plasma, or urine. It may also be captured in an airborne state. The RNA-containing sample may be immobilized in a gel or on a membrane, bound to one or more beads, or configured in the form of an array. The RNA-containing sample may be a buffer solution or water containing a partially or fully purified nucleic acid preparation.

[0075] The contacting step may be carried out at any appropriate temperature and time. Typically, the temperature is room temperature. Examples of such temperatures include about 20°C, about 25°C, about 30°C, about 35°C, about 37°C, about 40°C, about 42°C, and any range between these two values. Temperatures above about 42°C and temperatures below about 20°C may also be applicable depending on the test sample. The time is not particularly limited and may be any time appropriate for detecting a change in fluorescence. Examples of the time length include about 10 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 60 minutes, about 90 minutes, about 120 minutes, about 180 minutes, about 240 minutes, about 300 minutes, about 360 minutes, about 420 minutes, about 480 minutes, about 540 minutes, about 600 minutes, and any range between these two values. Further extension of the time may be possible depending on the test sample.

[0076] The medium to be contacted may be any medium. A mixture of compound (1) and a fluorescent probe that binds to the viral envelope may be directly mixed with the sample. Alternatively, for example, a medium such as a mask may be impregnated with a mixture of compound (1) and a fluorescent probe that binds to the viral envelope, and the sample suspended in the air may be captured and reacted.

[0077] The concentration of compound (1) is not particularly limited and may be any concentration at which fluorescent excitation and emission signals can be appropriately detected in the presence of RNA. An example of the concentration range is about 10 nM to 1 mM. Examples of concentrations include about 10 nM, about 100 nM, about 1 μM, about 10 μM, about 100 μM, about 1 mM, and any range between these two values.

[0078] Suitable illumination devices include portable ultraviolet lamps, mercury arc lamps, xenon lamps, lasers (such as argon and YAG lasers), and laser diodes. These illumination sources are typically optically integrated into laser scanners, fluorescence microplate readers, or standard or microfluorimeters.

[0079] The detecting or determining step may be performed by visual inspection or by using various measuring instruments, such as a CCD camera, a video camera, photographic film, a laser scanner device, a fluorometer, a photodiode, a quantum counter, an epifluorescence microscope, a scanning microscope, a flow cytometer, a fluorescence microplate reader, or an amplification device such as a photomultiplier tube.

[0080] The detecting or determining step may be performed at a single time point, at multiple time points, or continuously.

[0081] When both a fluorescent signal derived from compound (1) and a fluorescent signal derived from a fluorescent probe that binds to the virus envelope are detected, it can be detected or determined that there is a high possibility that virus particles are present in the sample.

[0082] "Fifth Aspect" In the fifth aspect of the present invention, there is provided a method for detecting enveloped virus particles in a sample, the method comprising the steps of contacting a fluorescent probe consisting of compound (1) with a sample suspected of containing virus particles, and irradiating the sample with light after the contact, wherein if the fluorescent probe exhibits a fluorescence response, this indicates a high possibility that virus particles are present in the sample.

[0083] The details of each step and materials used in the fifth aspect of the present invention are the same as those explained in the fourth aspect of the present invention.

[0084] In the virus particle detection method of the fourth aspect of the present invention, a fluorescent signal derived from compound (1) and a fluorescent signal derived from a fluorescent probe bound to the envelope are detected, but the virus particle detection method of the present invention is not limited to this. As another detection technique, for example, in the fifth aspect of the present invention, a fluorescent signal derived from compound (1) is detected. This is because, when comparing RNA in virus particles with RNA in solution, virus particles are observed as bright spots with strong intensity because they are a spatially limited system. On the other hand, RNA in solution and fluorescent probes bound to the envelope glow faintly throughout the solution. This fact may be utilized to detect virus particles based on the proportion of bright spots with strong fluorescent intensity. According to the fourth aspect of the present invention, virus particles can be detected using only the fluorescent signal derived from compound (1).

[0085] "Sixth Aspect" In the sixth aspect of the present invention, there is provided a method for detecting viral infection of cells in vitro, comprising the steps of: bringing compound (1) and a fluorescent dye that binds to the viral envelope into contact with cells suspected of being infected with a virus; and irradiating the cells with light after the contact; wherein, when a fluorescent signal derived from compound (1) and a fluorescent signal derived from the fluorescent probe that binds to the viral envelope are both detected, this indicates that the cells are likely infected with a virus.

[0086] The details of compound (1) are as described for compound (1) of the first embodiment, and are the same as those of the fourth embodiment except that compound (1) and a fluorescent dye that binds to the viral envelope are brought into contact with cells suspected of being infected with a virus in vitro.

[0087] In a further embodiment of the sixth aspect of the present invention, the first threshold value is the average value of the fluorescent signal derived from compound (1) in virus-infected individuals, the median value of the fluorescent signal derived from compound (1) in virus-infected individuals, or the value of the fluorescent signal derived from compound (1) that distinguishes between virus-infected and virus-non-infected individuals. The second threshold value is the average value of the fluorescent signal derived from a fluorescent probe that binds to the virus envelope in virus-infected individuals, the median value of the fluorescent signal derived from a fluorescent probe that binds to the virus envelope in virus-infected individuals, or the value of the fluorescent signal derived from a fluorescent probe that binds to the virus envelope that distinguishes between virus-infected and virus-non-infected individuals.

[0088] When both a fluorescent signal derived from compound (1) and a fluorescent signal derived from the fluorescent probe that binds to the virus envelope are detected, and the values ​​of each signal are equal to or greater than the first threshold and the second threshold, respectively, it can be detected or determined that the virus-infected individual is highly likely to be infected with the virus.

[0089] "Seventh Aspect" In the seventh aspect of the present invention, there is provided a method for detecting viral infection of cells in vitro, comprising the steps of contacting compound (1) with cells suspected of being infected with a virus, and irradiating the cells with light after the contact, wherein detection of a fluorescent signal derived from compound (1) indicates that the cells are likely infected with a virus.

[0090] The details of each step and materials used in the seventh aspect of the present invention are the same as those explained in the sixth aspect of the present invention.

[0091] In a further embodiment of the seventh aspect of the present invention, a fluorescent signal derived from compound (1) is detected, and when the signal value is equal to or greater than a first threshold, it can be detected or determined that a virus-infected individual is likely to be infected with the virus.

[0092] In the virus particle detection method of the sixth aspect of the present invention, a fluorescent signal derived from compound (1) and a fluorescent signal derived from a fluorescent probe that binds to the envelope are detected, but the method for detecting viral infection of cells in vitro of the present invention is not limited to this. As another detection technique, for example, in the seventh aspect of the present invention, a fluorescent signal derived from compound (1) is detected. According to the seventh aspect of the present invention, viral infection of cells can be detected in vitro using only the fluorescent signal derived from compound (1).

[0093] "Eighth Aspect" In an eighth aspect of the present invention, there is provided a kit for detecting virus particles, particularly coronaviruses, in a sample, the kit comprising compound (1), a fluorescent dye that binds to the virus envelope, and instructions for detecting the virus particles.

[0094] The details of compound (1) are as explained for compound (1) of the first aspect.

[0095] In a preferred embodiment, the kit comprises a container containing compound 1. The kit may also comprise a pipette, dropper, or other sample manipulation substrate.

[0096] The kit may also include positive and / or negative control samples. Positive control samples may contain RNA and / or RNA coexisting with DNA. Negative control samples may be samples containing DNA without RNA, or may be samples containing no nucleic acid at all.

[0097] The kit may also include one or more additional dyes or stains. For example, the kit may include a total nucleic acid stain. The kit may also include a cell-permeant nucleic acid stain for distinguishing between live and dead cells.

[0098] The kit may further comprise water, buffers, buffer salts, detergents, surfactants, salts, polysaccharides, or other materials commonly used in bioassays. The kit may also comprise a solvent, such as an aqueous, non-aqueous, or aqueous / non-aqueous solvent system.

[0099] In one embodiment, when both a fluorescent signal derived from compound (1) and a fluorescent signal derived from a fluorescent probe that binds to the viral envelope are detected, the presence of a viral particle in a sample can be detected or determined.

[0100] In a further embodiment, the first threshold value is the average value of the fluorescent signal derived from compound (1) in virus-infected individuals, the median value of the fluorescent signal derived from compound (1) in virus-infected individuals, or the value of the fluorescent signal derived from compound (1) that distinguishes between virus-infected and virus-non-infected individuals. The second threshold value is the average value of the fluorescent signal derived from a fluorescent probe that binds to the virus envelope in virus-infected individuals, the median value of the fluorescent signal derived from a fluorescent probe that binds to the virus envelope in virus-infected individuals, or the value of the fluorescent signal derived from a fluorescent probe that binds to the virus envelope that distinguishes between virus-infected and virus-non-infected individuals.

[0101] When both a fluorescent signal derived from compound (1) and a fluorescent signal derived from the fluorescent probe that binds to the virus envelope are detected, and the values ​​of each signal are equal to or greater than the first threshold and the second threshold, respectively, it can be detected or determined that the virus-infected individual is highly likely to be infected with the virus.

[0102] "Ninth Aspect" In a ninth aspect of the present invention, there is provided a kit for detecting virus particles, particularly coronaviruses, in a sample, the kit comprising compound (1) and instructions for detecting the virus particles.

[0103] Details of the kit according to the ninth aspect of the present invention and the materials used therein are as described in the eighth aspect of the present invention.

[0104] In a further embodiment of the ninth aspect of the present invention, a fluorescent signal derived from compound (1) is detected, and when the signal value is equal to or greater than a first threshold, it can be detected or determined that a virus-infected individual is likely to be infected with the virus.

[0105] The kit according to the eighth aspect of the present invention includes compound (1) and a fluorescent probe that binds to the envelope, but the kit for detecting virus particles in a sample of the present invention is not limited to this. For example, a kit according to a ninth aspect of the present invention includes compound (1). The kit according to the ninth aspect of the present invention makes it possible to detect virus particles in a sample using only the fluorescent signal derived from compound (1).

[0106] The methods of the fourth to seventh aspects and the kits of the eighth and ninth aspects can be applied to the prevention and control of viral infections and the visualization of viral attachment in medical and research settings, and can also contribute to the treatment of viral infections, the development of viral infection prevention techniques, the removal of viral contamination, and the verification of viral inactivation.

[0107] The present invention can also employ the following configuration.

[0108] <1> A compound represented by the following general formula (1):

[0109]

[0110] In formula (1), R １ is a hydrophobic group having 4 or more carbon atoms, and R ２ ~R １０ are each independently a hydrogen atom, a hydroxy group, a thiol group, a halo group, an alkyl group, an alkenyl group, an alkynyl group, an alkoxy group, an aryl group, or an amino group; R １１ is a hydrogen atom or an alkyl group.

[0111] <2> The compound according to <1>, wherein the hydrophobic group has 6 to 20 carbon atoms. <3> The compound according to <1> or <2>, wherein the hydrophobic group contains an aryl group. <4> The compound according to any one of <1> to <3>, wherein the hydrophobic group contains an aryl group and the hydrophobic group has 6 to 20 carbon atoms. <5> R in the general formula (1) ２ ~R１０ is a hydrogen atom, and R １１ <6> The compound according to any one of <1> to <4>, wherein the hydrophobic group is —(CH ２ ) ２ C ６ H ５ , -C ６ H ５ , -C ６ H ４ -C(CH ３ ) ３ , -C ６ H ３ -(C(CH ３ ) ３ ) ２ <7> The compound according to any one of <1> to <6>, wherein the compound is at least one selected from the group consisting of a compound represented by the following structural formula (1-1), a compound represented by the following structural formula (1-2), and a compound represented by the following structural formula (1-3):

[0112]

[0113] <8> A fluorescent dye containing the compound according to any one of <1> to <7>. <9> The fluorescent dye according to <8>, which is an RNA detection agent. <10> Use of the compound according to any one of <1> to <7> for intracellular RNA imaging. <11> A method for detecting RNA in a sample, comprising the steps of: mixing the compound according to any one of <1> to <7> with a sample containing RNA to form a mixed sample in which the compound and the RNA in the sample are bound; and irradiating the mixed sample with light to detect RNA in the mixed sample. <12> The method according to <11>, wherein the sample is a living cell. <13> A kit for detecting RNA in a sample, comprising the compound according to any one of <1> to <7> and instructions for detecting RNA in a sample.

[0114] <14> A method for detecting infection with an enveloped virus, comprising the steps of: contacting a sample suspected of containing enveloped virus particles with compound (1) according to any one of <1> to <7> and a fluorescent dye that binds to the envelope of the enveloped virus; and irradiating the sample with light after the contact, wherein detection of both a fluorescent signal derived from compound (1) and a fluorescent signal derived from the fluorescent dye that binds to the envelope of the enveloped virus indicates a high possibility that the enveloped virus particles are present in the sample. <15> A method for detecting infection with an enveloped virus, comprising the steps of: contacting a sample suspected of containing virus particles with compound (1) according to any one of <1> to <7>; and irradiating the sample with light after the contact, wherein detection of a fluorescent signal derived from compound (1) indicates a high possibility that the sample contains coronavirus particles. <16> A method for detecting viral infection of cells in vitro, comprising the steps of: bringing compound (1) according to any one of <1> to <7> and a fluorescent dye that binds to the viral envelope into contact with cells suspected of being infected with a virus; and irradiating the cells with light after the contact, wherein detection of both a fluorescent signal derived from compound (1) and a fluorescent signal derived from the fluorescent probe that binds to the viral envelope indicates a high possibility that the cells are infected with a virus. <17> A method for detecting viral infection of cells in vitro, comprising the steps of: bringing compound (1) according to any one of <1> to <7> into contact with cells suspected of being infected with a virus; and irradiating the cells with light after the contact, wherein detection of a fluorescent signal derived from compound (1) indicates a high possibility that the cells are infected with a virus.<18> A kit for detecting infection with an enveloped virus, comprising: compound (1) according to any one of <1> to <7>; a fluorescent dye that binds to the coronavirus envelope; and instructions for detecting viral infection of cells. <19> A kit for detecting infection with an enveloped virus, comprising compound (1) according to any one of <1> to <7>; and instructions for detecting viral infection of cells.

[0115] The disclosures of all patent applications and publications cited herein are hereby incorporated by reference in their entirety.

[0116] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the examples described below.

[0117] Experimental Example 1 Synthesis of Compounds (Fluorescent Dyes) Examples 1-1 to 1-3 are working examples, and Examples 1-4 to 1-7 are comparative examples.

[0118] Example 1-1: Synthesis of BIOP-benzyl-t-Bu (Production of BIOP-benzyl-t-Bu) The synthesis scheme of BIOP-benzyl-t-Bu, which is a compound of formula (1-3), is as follows.

[0119]

[0120] As shown in Scheme 1-1 below, 1-methyl-2-(methylthio)benzo[cd]indol-1-ium iodide (compound (5-1)) was obtained via compound (3-1) and compound (4-1) according to J. Med. Chem., 2016, 59, 1565-1579, using enzo[c,d]indole-2(1H)-one (compound (2-1)) and iodomethane as starting materials.

[0121]

[0122] Next, as shown in Scheme 2-1, using compound (5-1) as a starting material, 1,2-dimethylbenzo[c,d]indole-1-im iodide (compound (7-1)) was obtained via compound (6-1) according to Dyes Pigm., 1991, 15, 215-223.

[0123]

[0124] As shown in the following scheme 3-1, separately, 2-(methylthio)oxazolo[5,4-c]pyridine (compound (10-1)) was obtained via compound (9-1) according to Eur. J. Med. Chem., 2005, 40, 15-23. using 4-aminopyridin-3-ol (compound (8-1)) as a starting material. Then, compound (10-1) and X—CH ２ -R １ As the methyl group, 1-(tert-butyl)-4-(chloromethyl)benzene was subjected to a heating reaction, followed by purification to obtain 5-(4-(tert-butyl)benzyl)-2-(methylthio)oxazolo[5,4-c]prydine-5-ium iodide (compound 11-1).

[0125]

[0126] As shown in the following scheme 4-1, compound (7-1) and compound (11-1) were reacted by heating in the presence of triethylamine, and then purified to obtain compound (1-3), BIOP-Bneyzl-tBu.

[0127]

[0128] (Identification of BIOP-Benzyl-t-Bu) BIOP-Benzyl-t-Bu was identified using nuclear magnetic resonance (NMR) and electrospray ionization mass spectrometry (ESI-MS).

[0129] <<Analysis results by nuclear magnetic resonance>> ・Device used: Bruker, product name "Avance III 500" ・Magnetic field strength: 500 MHz, Spectral width: 3089 Hz ・ １ H-NMR spectrum １H-NMR (500MHz, DMSO-d ６ , ppm): δ9.86 (d, J=7.3Hz, 1H), 9.62 (s, 1H), 8.92 (d, J=7.0Hz, 1H), 8.27 (d, J=7.9Hz, 1H), 8.20 (d, J=6.7Hz, 1H), 7.94 (t, J=7. 6Hz, 1H), 7.72 (d, J = 8.2Hz, 1H), 7.66 (t, J = 7.8Hz, 1H), 7.50-7.45 (m, 5H), 6.34 (s, 1H), 5.76 (s, 2H), 3.78 (s, 3H), 1.26 (s, 9H). １ The H-NMR spectrum is predicted from the structural formula of compound (1). １ The position of H and the integral ratio are consistent with the measured values.

[0130] <<Analysis results by electrospray ionization mass spectrometry>> ・Apparatus used: JEOL, product name "JMS-T100CS" ・ESI-MS spectrum Calculated value of BIOP-Benzyl-t-Bu (Calcd([M ＋ ]): 446.22, actual value (found([M ＋ ])): 446.04.

[0131] From the above nuclear magnetic resonance and electrospray ionization mass spectrometry, the structure of BIOP-Benzyl-t-Bu was identified.

[0132] (Results of measuring the properties of BIOP-Benzyl-t-Bu) <<Fluorescence measurement>> ・Device used: JASCO Corporation, product name "FP-6500" To calculate the fluorescence quantum yield, an ethanol solution of Rhodamine 6G (fluorescence quantum yield φ ｆｌ = 0.95, Photochem. Photobiol., 2002, 75, 327.) was used as a reference.

[0133] <<Fluorescence Microscope Measurement>> Equipment used: GE Healthcare, product name "Deltavision Elite microscopy system" A TRITC filter was used to detect the fluorescent signal of BIOP-Benzyl-t-Bu.

[0134] BIOP-Benzyl-t-Bu was almost non-fluorescent in its free state, but showed a clear light-up response upon addition of total RNA derived from E. coli (excitation wavelength λex: 530 nm, fluorescence wavelength λem: 576.0 nm). The fluorescence quantum convergence φ of BIOP-Benzyl-t-Bu in the RNA-bound state was ｆｌ is calculated to be 0.11, and the quantum convergence φ of this BIOP-Benzyl-t-Bu ｆｌ The quantum convergence φ of a monomethine cyanine dye (BIQ) in which benzo[c,d]indole and quinoline are bonded is 0.0085, which is nearly 13 times the value.

[0135] <Example 1-2: BIOP-C ３ Synthesis of -Benzyl (BIOP-C ３ Preparation of BIOP-C, a compound of formula (1-1) ３ The synthesis scheme of -Benzyl is as follows:

[0136]

[0137] Compound (7-1) was obtained in the same manner as in Example 1-1. ２ -R １ 2-(methylthio)-5-(3-phenylpropyl)oxazolo[5,4-c]pyridin-5-ium iodide (compound 11-2) was obtained in the same manner as in Scheme 3-1 of Example 1-1, except that (3-bromopropyl)benzene was used as the aryl group. Next, as shown in Scheme 4-2 below, compound (7-1) and compound (11-2) were reacted by heating in the presence of triethylamine, and then purified to obtain compound (1-1), BIOP-C ３ -Bneyzl was obtained.

[0138]

[0139] (BIOP-C ３ -Identification of Benzyl) BIOP-C ３ Identification of BIOP-Benzyl-t-Bu was performed using nuclear magnetic resonance (NMR) and electrospray ionization mass spectrometry (ESI-MS), similar to that of BIOP-Benzyl-t-Bu. １H-NMR spectrum １ H-NMR (500MHz, DMSO-d ６ , ppm): δ9.87 (d, J = 7.3Hz, 1H), 9.48 (s, 1H), 8.83 (d, J = 6.7Hz, 1H), 8.27 (d, J = 7.9Hz, 1H), 8.19 (d, J=6.7Hz, 1H), 7.95 (t, J=7.6Hz, 1H), 7.72 (d, J=8.2Hz, 1H), 7.67 (t, J=7.8Hz, 1H), 7.46 (d, J=7.0Hz, 1H), 7.28 (t, J=7.5Hz, 2H), 7.22 (d, J=7.0Hz, 2H), 7.18 (t, J=7.2Hz, 1H), 6.36 (s, 1H), 4.63 (t, J = 7.2Hz, 2H), 3.78 (s, 3H), 2.67 (t, J = 7.8Hz, 2H), 2.33-2.27 (m, 2H). ・ESI-MS spectrum BIOP-C ３ -Benzyl calculated value (Calcd([M ＋ ]): 418.19, actual value (found([M ＋ ])): 418.04. From the above nuclear magnetic resonance and electrospray ionization mass spectrometry, BIOP-C ３ The structure of -Benzyl was identified.

[0140] (BIOP-C ３ Measurement results of BIOP-Benzyl characteristics) BIOP-C was measured using the same equipment and under the same conditions as BIOP-Benzyl-t-Bu. ３ Fluorescence measurement and fluorescence microscope measurement were also performed for BIOP-C. ３ Although β-benzyl is almost non-fluorescent by itself, it showed a clear light-up response upon addition of total RNA derived from E. coli (excitation wavelength λex: 530.0 nm, emission wavelength λem: 572.0 nm). ３ Fluorescence quantum convergence φ in the RNA-bound state of -Benzyl ｆｌ was calculated to be 0.23.

[0141] Example 1-3: Synthesis of BIOP-benzyl (Production of BIOP-benzyl) The synthesis scheme of BIOP-benzyl, which is a compound of formula (1-2), is as follows.

[0142]

[0143] Compound (7-1) was obtained in the same manner as in Example 1-1. ２ -R １ 5-benzyl-2-(methylthio)oxazolo[5,4-c]pyridin-5-ium iodide (compound 11-3) was obtained in the same manner as in Scheme 3-1 of Example 1-1, except that (bromomethyl)benzene was used as the aryl group. Next, as shown in Scheme 4-3 below, compound (7-1) and compound (11-3) were reacted by heating in the presence of triethylamine, and then purified to obtain compound (1-2), BIOP-Bneyzl.

[0144]

[0145] (Identification of BIOP-Benzyl) BIOP-Benzyl was identified using nuclear magnetic resonance (NMR) and electrospray ionization mass spectrometry (ESI-MS), similar to BIOP-Benzyl-t-Bu. １ H-NMR spectrum １ H-NMR (500MHz, DMSO-d ６ , ppm): δ9.87 (d, J = 7.3Hz, 1H), 9.64 (s, 1H), 8.93 (d, J = 6.7Hz, 1H ), 8.27 (d, J = 7.9Hz, 1H), 8.22 (d, J = 6.7Hz, 1H), 7.95 (t, J = 7.8Hz, 1H), 7.73 (d, J = 8.2Hz, 1H), 7.66 (t, J = 7.8Hz, 1H), 7.55 (d, J = 6.7H z, 2H), 7.47-7.41 (m, 5H), 6.35 (s, 1H), 5.81 (s, 2H), 3.78 (s, 3H). ・ESI-MS spectrum Calculated value of BIOP-Benzyl (Calcd([M ＋ ]): 390.16, actual value (found([M ＋ ])): 390.01. From the above nuclear magnetic resonance and electrospray ionization mass spectrometry, the structure of BIOP-Benzyl was identified.

[0146] (Results of measuring the properties of BIOP-Benzyl) Fluorescence and fluorescence microscopy measurements were also carried out on BIOP-Benzyl using the same equipment and under the same conditions as for BIOP-Benzyl-t-Bu. BIOP-Benzyl is almost non-fluorescent on its own, but showed a clear light-up response upon the addition of total RNA derived from E. coli (excitation wavelength λex: 530.0 nm, emission wavelength λem: 574.0 nm). The fluorescence quantum convergence φ of BIOP-Benzyl in the RNA-bound state was also ｆｌ was calculated to be 0.25.

[0147] Example 1-4: Synthesis of BIOP-nBu (Production of BIOP-nBu) The synthesis scheme of BIOP-nBu, which is a compound of formula (1'-4), is as follows.

[0148]

[0149] Compound (7-1) was obtained in the same manner as in Example 1-1. ２ -R １ 5-butyl-2-(methylthio)oxazolo[5,4-c]pyridin-5-ium iodide (compound 11-4) was obtained in the same manner as in Scheme 3-1 of Example 1-1, except that 1-bromobutane was used as the methyl group. Next, as shown in Scheme 4-4 below, compound (7-1) and compound (11-4) were reacted by heating in the presence of triethylamine, and then purified to obtain compound (1'-4), BIOP-nBu.

[0150]

[0151] (Identification of BIOP-nBu) BIOP-nBu was identified using nuclear magnetic resonance (NMR) and electrospray ionization mass spectrometry (ESI-MS), similar to the case of BIOP-Benzyl-t-Bu. １ H-NMR spectrum １ H-NMR (500MHz, DMSO-d ６, ppm): δ9.87 (d, J=7.3Hz, 1H), 9.49 (d, J=3.4Hz, 1H), 8.83 (d, J=6.7Hz, 1H), 8.2 7 (d, J = 8.2Hz, 1H), 8.21 (d, J = 6.7Hz, 1H), 7.95 (t, J = 7.8Hz, 1H), 7.72 (d, J = 8.2Hz , 1H), 7.66 (t, J = 7.8Hz, 1H), 7.46 (d, J = 7.0Hz, 1H), 6.35 (s, 1H), 4.57 (t, J = 7.3H z, 2H), 3.78 (s, 3H), 1.91-1.97 (m, 2H), 1.30-1.36 (m, 2H), 0.94 (t, J = 7.5Hz, 3H). ・ESI-MS spectrum Calculated value of BIOP-nBu (Calcd([M ＋ ]): 356.1757, actual value (found([M ＋ ])):356.1946. From the above nuclear magnetic resonance and electrospray ionization mass spectrometry, the structure of BIOP-nBu was identified.

[0152] (Results of Characteristic Measurement of BIOP-nBu) Fluorescence measurement and fluorescence microscope measurement were also performed on BIOP-nBu using the same equipment and measurement conditions as for BIOP-Benzyl-t-Bu. BIOP-nBu is almost non-fluorescent when used alone, but showed a clear light-up response upon addition of total RNA derived from E. coli (excitation wavelength λex: 530 nm, emission wavelength λem: 570 nm). Furthermore, the fluorescence quantum convergence φ of BIOP-nBu in the RNA-bound state was ｆｌ was calculated to be 0.37.

[0153] Example 1-5: Synthesis of BIOP-sulfone (Production of BIOP-sulfone) The synthesis scheme of BIOP-sulfone, which is a compound of formula (1'-5), is as follows.

[0154]

[0155] Compound (7-1) was obtained in the same manner as in Example 1-1. ２ -R １3-(2-(methylthio)oxazolo[5,4-c]pyridin-5-ium-5-yl)propane-1-sulfonate, sodium salt iodide (compound 11-5) was obtained in the same manner as in Scheme 3-1 of Example 1-1, except that sodium 3-bromopropane-1-sulfonate was used as the base. Next, as shown in Scheme 4-5 below, compound (7-1) and compound (11-5) were reacted by heating in the presence of triethylamine, and then purified to obtain compound (1'-5), BIOP-Sulfone.

[0156]

[0157] (Identification of BIOP-Sulfone) BIOP-Sulfone was identified using nuclear magnetic resonance (NMR) and electrospray ionization mass spectrometry (ESI-MS), similar to BIOP-Benzyl-t-Bu. １ H-NMR spectrum １ H-NMR (500MHz, DMSO-d ６ , ppm): δ9.88 (d, J=7.3Hz, 1H), 9.51 (s, 1H), 8.83 (d, J=6.1Hz, 1H), 8. 26 (d, J=7.9Hz, 1H), 8.20 (d, J=6.7Hz, 1H), 7.96 (t, J=7.6Hz, 1H), 7.7 2 (d, J=8.5Hz, 1H), 7.67 (d, J=7.0Hz, 1H), 7.45 (d, J=7.3Hz, 1H), 6.37 (s, 1H), 5.76 (s, 2H), 3.78 (s, 3H), 2.64 (s, 2H), 2.14 (t, J=7.2Hz, 2H). ESI-MS spectrum Calculated value of BIOP-Benzyl (Calcd([M ＋ ]): 44.10, actual value (found([M ＋ ])): 444.23. From the above nuclear magnetic resonance and electrospray ionization mass spectrometry, the structure of BIOP-Sulfone was identified.

[0158] (Results of Characteristic Measurement of BIOP-Sulfone) Fluorescence measurement and fluorescence microscope measurement were also carried out for BIOP-Sulfone using the same equipment and measurement conditions as for BIOP-Benzyl-t-Bu. BIOP-Sulfone is almost non-fluorescent on its own, but showed a clear light-up response upon addition of total RNA derived from Escherichia coli (excitation wavelength λex: 530 nm, emission wavelength λem: 573.0 nm). Furthermore, the fluorescence quantum convergence φ of BIOP-Sulfone in the RNA-bound state was ｆｌ was calculated to be 0.28.

[0159] <Example 1-6: BIOP-C ３ Synthesis of —COOH> BIOP-C, a compound of formula (1′-6) ３ The synthesis scheme of —COOH is as follows:

[0160]

[0161] Compound (7-1) was obtained in the same manner as in Example 1-1. ２ -R １ 5-(3-carboxypropyl)-2-(methylthio)oxazolo[5,4-c]pyridin-5-ium iodide (compound 11-6) was obtained in the same manner as in Scheme 3-1 of Example 1-1, except that tert-butyl 4-bromobutanoate was used as the ester. Next, as shown in Scheme 4-6 below, compound (7-1) and compound (11-6) were reacted by heating in the presence of triethylamine, and then purified to obtain compound (1'-6), BIOP-C ３ -COOH was obtained.

[0162]

[0163] (BIOP-C ３ Identification of -COOH) BIOP-C ３ The —COOH group was identified using nuclear magnetic resonance (NMR) and electrospray ionization mass spectrometry (ESI-MS), as with BIOP-Benzyl-t-Bu. １ H-NMR spectrum １ H-NMR (500MHz, DMSO-d ６, ppm): δ 9.85-9.93 (1H), 9.52-9.58 (1H), 8.81-8.89 (1H), 8.24-8.32 (1H), 8.17-8.24 (1H), 7.92-8.04 (1H), 7.69-7.77 (1H), 7.63-7.69 (1H), 7.43-7.52 (1H), 4.56-4.66 (2H), 3.75-3.79 (2H), 2.11-2.23 (4H). ESI-MS spectrum Calculated value of BIOP-Benzyl (Calcd([M ＋ ]): 386.15, actual value (found([M ＋ ])): 386.00. From the above nuclear magnetic resonance and electrospray ionization mass spectrometry, BIOP-C ３ The structure of -COOH was identified.

[0164] (BIOP-C ３ Measurement results of -COOH properties) BIOP-C was measured using the same equipment and under the same conditions as BIOP-Benzyl-t-Bu. ３ Fluorescence measurement and fluorescence microscope measurement were also performed on —COOH. ３ -COOH is almost non-fluorescent by itself, but showed a clear light-up response upon addition of total RNA derived from E. coli (excitation wavelength λex: 530.0 nm, emission wavelength λem: 571.0 nm). ３ Fluorescence quantum convergence φ in the RNA-bound state of -COOH ｆｌ was calculated to be 0.31.

[0165] Example 1-7: Synthesis of BIOP (Production of BIOP) BIOP, a compound of formula (1'-7), was obtained according to ACS Omega, 2022, 7, 27, 23744-23748.

[0166]

[0167] (Identification of BIOP) BIOP was identified using nuclear magnetic resonance (NMR) and electrospray ionization mass spectrometry (ESI-MS), similar to BIOP-Benzyl-t-Bu. １ H-NMR spectrum １ H-NMR (500MHz, DMSO-d ６, ppm): δ 9.88 (d, J = 7.3 Hz, 1H), 9.41 (s, 1H), 8.74 (d, J = 6.7 Hz, 1H), 8.26 (d, J = 8.2 Hz, 1H), 8.19 (d, J = 6.7 Hz, 1H), 7.95 (t, J = 7.6 Hz, 1H), 7.72 (d, J = 8.2 Hz, 1H), 7.66 (t, J = 7.6 Hz, 1H), 7.45 (d, J = 7.3 Hz, 1H), 6.35 (s, 1H), 4.33 (s, 3H), 3.77 (s, 3H). ESI-MS spectrum Calculated value of BIOP (Calcd([M ＋ ]): 314.1288, actual value (found([M ＋ ])):314.1744. From the above nuclear magnetic resonance and electrospray ionization mass spectrometry, the structure of BIOP was identified.

[0168] (Results of BIOP Characteristic Measurement) BIOP was also measured by fluorescence and fluorescence microscope using the same equipment and measurement conditions as BIOP-Benzyl-t-Bu. BIOP is almost non-fluorescent when used alone, but showed a clear light-up response upon addition of total RNA derived from E. coli (excitation wavelength λex: 530 nm, emission wavelength λem: 570 nm). The fluorescence quantum convergence φ of BIOP in the RNA-bound state was also measured. ｆｌ was calculated to be 0.52.

[0169] The compounds synthesized in Experimental Example 1, namely, BIOP-Benzyl-t-Bu as compound (1-3), BIOP-C as compound (1-1), ３ -Benzyl, BIOP-Benzyl which is the compound (1-2), BIOP-nBu which is the compound (1'-4), BIOP-Sulfone which is the compound (1'-5), and BIOP-C which is the compound (1'-6). ３ Using —COOH and BIOP, which is the compound (1′-7), as fluorescent dyes, the following Experimental Examples 2 to 8 were carried out.

[0170] [Experimental Example 2: Fluorescence response to biologically derived nucleic acids] Examples 2-1 to 2-3 are working examples, and Examples 2-4 to 2-7 are comparative examples. <Example 2-1> (Materials and Methods) Calf thymus DNA (Sigma-Aldrich) or Escherichia coli total RNA (Thermo Fisher Scientific) was used as the sample. The sample was mixed with BIOP-Benzyl-t-Bu as a fluorescent dye, and the fluorescence spectrum was measured in PBS buffer when irradiated with 530 nm light as excitation light. The BIOP-Benzyl-t-Bu concentration was 1.0 μM, and the nucleic acid concentration was 1000 μM (M = mol / L per nucleotide, pH 7.0 at 25°C).

[0171] <Examples 2-2 to 2-7> BIOP-C instead of BIOP-Benzyl-t-Bu ３ -Benzyl, BIOP-Benzyl, BIOP, BIOP-nBu, BIOP-Sulfone, BIOP-C ３ The same procedure as in Example 2-1 was carried out except that --COOH was used as the fluorescent dye, and these were designated as Examples 2-2 to 2-7, respectively.

[0172] (Results) The fluorescence intensity at 575.0 nm and RNA selectivity of each fluorescent dye are shown in Table 1. In Table 1, "Molecular Weight" indicates the molecular weight of the fluorescent dye, and "ClogP ｏｗ " indicates the octanol / water partition coefficient, "Intensity at 575.0 nm" indicates the fluorescence intensity at 575.0 nm when calf thymus DNA or E. coli total RNA was used as a sample, and "RNA / DNA" indicates the ratio of the fluorescence intensity at 575.0 nm when E. coli total RNA was used as a sample to the fluorescence intensity at 575.0 nm when calf thymus DNA was used as a sample, i.e., RNA selectivity.

[0173] As shown in Table 1, the fluorescence intensity was high regardless of the hydrophilicity and hydrophobicity of the fluorescent dye as indicated by ClogPow. It was also found that RNA selectivity was not correlated with the hydrophilicity and hydrophobicity of the fluorescent dye. As the molecular weight of the fluorescent dye increased, the fluorescence intensity and RNA selectivity decreased. In other words, it was shown that the molecular weight of the fluorescent dye has a negative correlation with the fluorescence intensity and RNA selectivity. These results suggest that the responsiveness and selectivity to nucleic acids are dominated by the three-dimensional structure of the molecule.

[0174]

[0175] In addition, each fluorescent dye alone was added to a PBS buffer solution, and the fluorescence intensity at 575.0 nm (Probe only) was measured when irradiated with 530 nm light as excitation light. The other conditions were the same as those for the measurement described above. The results are shown in Table 2. In Table 2, F / F ０ indicates the ratio of the fluorescence intensity when E. coli total RNA was used as a sample to the fluorescence intensity of the probe only.

[0176]

[0177] The fluorescence intensity ratio increased when a hydrophobic group with four or more carbon atoms was introduced, and when a sulfonic acid group was introduced. A clear increase in fluorescence intensity was observed for all fluorescent dyes when E. coli total RNA was included. In other words, the BIOP substituents did not affect the fluorescence response mechanism or the binding mode between the fluorescent dye and RNA. This indicates that the interaction between RNA and the fluorescent dye is not the cause of the improved nucleolar selectivity of the fluorescent dye.

[0178] [Experimental Example 3: Fluorescence response to synthetic nucleic acids] Examples 3-1 to 3-3 are working examples, and Examples 3-4 to 3-7 are comparative examples. <Example 3-1> (Materials and Methods) Chemically synthesized DNA (Nihon Gene Research Institute) was prepared using G １３ , C １３ , A １３ and T １３ was prepared. １３ and C １３ double-stranded DNA or A １３ and T １３The double-stranded DNA was obtained by annealing in PBS buffer at 95°C. Chemically synthesized RNA (Gene Design) was prepared using G １３ , C １３ , A １３ and U １３ was prepared. １３ and C １３ double-stranded RNA or A １３ and U １３ The double-stranded RNA was obtained by annealing in PBS buffer at 95°C. Single-stranded or double-stranded DNA and single-stranded or double-stranded RNA (hereinafter sometimes collectively referred to as synthetic nucleic acids) were each mixed with BIOP-Benzyl-t-Bu in PBS buffer to prepare a mixture. The fluorescent dye concentration was 1.0 μM, and the concentration of each synthetic nucleic acid was 20 μM. Each mixture was irradiated with excitation light at a wavelength of 530.0 nm, and the fluorescence intensity at 575.0 nm was measured.

[0179] <Examples 3-2 to 3-7> BIOP-C instead of BIOP-Benzyl-t-Bu ３ -Benzyl, BIOP-Benzyl, BIOP, BIOP-nBu, BIOP-Sulfone, BIOP-C ３ The same procedure as in Example 3-1 was carried out except that --COOH was used as the fluorescent dye, and these were designated as Examples 3-2 to 3-7, respectively.

[0180] (Results) Figure 2 shows the fluorescence intensity of each fluorescent dye for each synthetic nucleic acid. For all fluorescent dyes, the fluorescence intensity was greater for double-stranded nucleic acids than for single-stranded nucleic acids. This suggests that the nucleic acids and fluorescent dyes bind by insertion of the fluorescent dye between base pairs (i.e., intercalation). It also suggests that there is no difference in the binding mode between DNA and RNA. Furthermore, the fluorescence intensity was greater for single-stranded guanine in DNA and RNA. This suggests binding to a guanine quadruplex.

[0181] [Experimental Example 4: Detection of RNA in Cells] Examples 4-1 to 4-3 are examples, and Examples 4-4 to 4-6 are comparative examples. Example 4-1 (Materials and Methods) Five thousand MCF-7 cells were cultured in a 5% CO atmosphere. ２The cells were incubated in RPMI 1640 medium containing 10% FCS at 37°C. The medium was then replaced with the above medium containing 1 μM BIOP-Benzyl-t-Bu, and the cells were incubated in 5% CO ２ The cells were incubated at 37°C for 20 minutes. After washing twice with HBSS(+) buffer, the cells were observed under a fluorescence microscope in HBSS(+) buffer. The cells were photographed using a TRITC filter set (Ex: 545 / 27; Em: 597 / 45) with an exposure time of 0.1 seconds and a transmittance of 10%.

[0182] <Examples 4-2 to 4-6> BIOP-C instead of BIOP-Benzyl-t-Bu ３ -Benzyl, BIOP-Benzyl, BIOP-nBu, BIOP-Sulfone, BIOP-C ３ The same procedure as in Example 4-1 was carried out except that --COOH was used as the fluorescent dye, and these were designated as Examples 4-2 to 4-6, respectively.

[0183] (Results) Figure 3(A) shows RNA imaging images of Examples 4-1 to 4-4, and Figure 3(B) shows the fluorescence intensity on the line in Figure 3(A) (the intensity along the direction of the arrow in Figure 3(A) is shown from left to right in Figure 3(B)). As shown in Figures 3(A) and 3(B), when BIOP-Benzyl-t-Bu of Example 4-1 was applied to RNA imaging using live MCF-7 cells, it was confirmed that BIOP-Benzyl-t-Bu can selectively stain nucleoli rich in RNA even at a low concentration (1.0 μM) and for a short period of time (20 minutes). Similarly, BIOP-C of Examples 4-2 to 4-4 ３ It was confirmed that BIOP-Benzyl, BIOP-Benzyl, and BIOP-nBu could selectively stain nucleoli even at low concentrations (1.0 μM) and for a short time (20 minutes). Furthermore, it was found that there were differences in fluorescence intensity and RNA selectivity between the fluorescent dyes.

[0184] On the other hand, BIOP-Sulfone or BIOP-C, which are Examples 4-5 to 4-6, ３When -COOH was applied to RNA imaging using live MCF-7 cells, it was not possible to stain the live cells (not shown). This indicates that highly hydrophilic fluorescent dyes are not applicable to live cell imaging. This is thought to be due to the inability of highly hydrophilic fluorescent dyes to penetrate cell membranes, specifically lipid bilayer membranes.

[0185] Experimental Example 5: Evaluation of nucleolus selectivity in living cells Examples 5-1 to 5-3 are examples, and Examples 5-4 to 5-5 are comparative examples. Example 5-1 (Materials and Methods) MCF-7 cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and 2% penicillin / streptomycin at 37°C and 5% CO ２ The cells were cultured in an incubator. For fluorescence imaging, MCF-7 cells were seeded in Coverglass Chamber 8-well (Iwaki) at a density of 5,000 cells / well and cultured for 24 hours. They were then incubated for 20 minutes in medium (400 μL / well) containing the DNA staining dye Hoechst 33342 (176 nM). After washing twice with HBSS(+) buffer, the cells were incubated for 20 minutes in medium (400 μL / well) containing BIOP-Benzyl-t-Bu (1 μM). After washing twice with HBSS(+) buffer, the cells were imaged in HBSS(+) buffer using a Deltavision Elite microscopy system. The following filter sets were used for imaging: DAPI filter set for Hoechst 33342 (Ex: 390 / 18; Em: 435 / 48); TRITC filter set for fluorescent dyes (Ex: 545 / 27; Em: 597 / 45).

[0186] The fluorescence intensity and organelle selectivity of stained cells were calculated using Fiji-Image J according to the following method. An ROI was created in an area containing only the cells to be analyzed, and an image was extracted. Next, a certain threshold was set for the fluorescence intensity of the image, and the area emitting fluorescence above the threshold was designated as the entire cell, and the average fluorescence intensity in that area was calculated. The same procedure was performed on the nuclear and nucleolus regions, and the average fluorescence intensity in the entire nuclear region (excluding nucleoli) and the average fluorescence intensity in the nucleolus region were calculated. This procedure was performed on nine cells, and selectivity was evaluated based on the fluorescence intensity in each region and its ratio.

[0187] The "fluorescence intensity on the white line" was calculated using a function on the Deltavision Elite microscopy system.

[0188] <Examples 5-2 to 5-5> BIOP-C instead of BIOP-Benzyl-t-Bu ３ The same procedures as in Example 5-1 were carried out except that BIOP-Benzyl, BIOP-Benzyl, BIOP and BIOP-nBu were used as the fluorescent dyes, and these were designated as Examples 5-2 to 5-5, respectively.

[0189] (Results) Figure 4(A) is an image showing the fluorescence of the fluorescent dye alone, Figure 4(B) is an image showing the fluorescence of the fluorescent dye and Hoechst 33342, and Figure 4(C) shows the fluorescence intensity on the white line in Figure 4(B) (in Examples 5-1 to 5-3 and 5-5, the intensity along the direction of the arrow in Figure 4(B) is shown from left to right in Figure 4(C), and in Example 5-4, the intensity along the direction of the arrow in Figure 4(B) is shown from left to right in Figure 4(C). In Figure 4(C), the solid line shows the intensity of the fluorescent dye, and the dashed line shows the fluorescence intensity of Hoechst 33342.

[0190] 5 is a graph showing the fluorescence intensity in the cytoplasm, nucleus, and nucleolus in Examples 5-1 to 5-5. The solid line represents the ratio of the fluorescence intensity in the nucleolus to the fluorescence intensity in the cytoplasm, and the dashed line represents the fluorescence intensity in the nucleolus to the fluorescence intensity in the nucleus.

[0191] As shown in Figure 5, the ratio of nucleolar fluorescence intensity to cytoplasmic fluorescence intensity was higher in Examples 5-1 to 5-3, in which a substituent R having 4 or more carbon atoms was introduced into BIOP, compared to Example 5-4, which was BIOP, and Example 5-5, in which a substituent R having 3 carbon atoms was introduced into BIOP. In Example 5-1, the ratio of nucleolar fluorescence intensity to cytoplasmic fluorescence intensity increased 1.7 times compared to Example 5-5. From the above, it can be said that the fluorescent dyes used in Examples 5-1 to 5-3 had improved nucleolar selectivity relative to cytoplasm. Possible reasons for the improved nucleolar selectivity compared to BIOP include a change in the interaction between RNA and each fluorescent dye or a change in the staining process by the fluorescent dye in live cells.

[0192] Experimental Example 6: Evaluation by Nuclease Treatment Examples 6-1 to 6-3 are working examples, and Examples 6-4 to 6-5 are comparative examples. Example 6-1 (Materials and Methods) The MCF-7 cells evaluated in Example 4-3 were treated with DNA enzyme (DNase) or RNA enzyme (RNase). Then, differential interference contrast (DIC) images and RNA imaging images were obtained for each cell under the same conditions as in Experimental Example 4. In addition, the "fluorescence intensity above the white line" was calculated under the same conditions as in Experimental Example 5.

[0193] <Examples 6-2 to 6-5> BIOP-C instead of BIOP-Benzyl ３ The same procedures as in Example 6-1 were carried out except that BIOP-Benzyl, BIOP-Benzyl-t-Bu, BIOP-nBu and BIOP were used as the fluorescent dye, and these were designated as Examples 6-2 to 6-5, respectively.

[0194] Figure 6(A) shows a merged image of a differential interference contrast image (DIC) and an RNA imaging image of Example 6-1 using BIOP-Benzyl, and Figure 6(B) shows the fluorescence intensity on the line in Figure 6(A) (the intensity along the direction of the arrow in Figure 6(A) is shown from left to right in Figure 6(B)). Figure 7(A) shows the fluorescence intensity of BIOP-C ３7(B) shows a merged image of a differential interference contrast image (DIC) and an RNA imaging image of Example 6-2 using BIOP-Benzyl-t-Bu, and FIG. 7(A) shows the fluorescence intensity on the line in FIG. 7(A) (the intensity along the direction of the arrow in FIG. 7(A) is shown from left to right in FIG. 7(B)). FIG. 8(A) shows a merged image of a differential interference contrast image (DIC) and an RNA imaging image of Example 6-3 using BIOP-Benzyl-t-Bu, and FIG. 8(B) shows the fluorescence intensity on the line in FIG. 8(A) (the intensity along the direction of the arrow in FIG. 8(A) is shown from left to right in FIG. 8(B)). 9(A) shows a merged image of a differential interference contrast image (DIC) and an RNA imaging image of Example 6-4 using BIOP-nBu, and FIG. 9(B) shows the fluorescence intensity on the line in FIG. 9(A) (the intensity along the direction of the arrow in FIG. 9(A) is shown from left to right in FIG. 9(B)). FIG. 10(A) shows a merged image of a differential interference contrast image (DIC) and an RNA imaging image of Example 6-5 using BIOP, and FIG. 10(B) shows the fluorescence intensity on the line in FIG. 10(A) (the intensity along the direction of the arrow in FIG. 10(A) is shown from left to right in FIG. 10(B)). In FIGS. 6 to 10, RNase indicates cells treated with RNase, DNase indicates cells treated with DNase, and Control indicates cells not treated with either RNase or DNase.

[0195] In all cases of Examples 6-1 to 6-5, the fluorescent signal intensity of the nucleoli was significantly reduced only when treated with RNase. On the other hand, when treated with DNase, there was no change in the signal in the nucleoli compared to the control. From these results, it is believed that the fluorescent response in the nucleoli is due to the binding of the fluorescent dye to RNA in the nucleoli, regardless of the BIOP substituent.

[0196] Experimental Example 7 Evaluation of staining process by time lapse Examples 7-1 to 7-3 are examples, and Examples 7-4 to 7-5 are comparative examples. Example 7-1 (Materials and Methods) MCF-7 cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and 2% penicillin / streptomycin at 37°C and 5% CO ２The cells were cultured in an incubator. MCF-7 cells were seeded at a density of 5,000 cells / well in a Coverglass Chamber 8-well (Iwaki) and cultured for 24 hours. After washing twice with HBSS(+) buffer, HBSS(+) buffer (400 μL / well) containing BIOP-Benzyl-t-Bu (0.5 μM) was added. Starting 4 minutes after addition, images were acquired at regular intervals using a Deltavision Elite microscopy system. Images were taken at intervals from 4 to 20 minutes, and at 20-minute intervals from 20 to 60 minutes.

[0197] The change in fluorescence intensity in the nucleolus region was calculated using Fiji-Image J as follows: ROIs were created based on the shape of the nucleolus 60 minutes after the addition of BIOP-Benzyl-t-Bu, and the ROIs were used to calculate the average fluorescence intensity in the nucleolus over time.

[0198] <Examples 7-2 to 7-5> BIOP-C instead of BIOP-Benzyl-t-Bu ３ The same procedures as in Example 7-1 were carried out except that BIOP-Benzyl, BIOP-Benzyl, BIOP and BIOP-nBu were used as the fluorescent dyes, and these were designated as Examples 7-2 to 7-5, respectively.

[0199] (Results) Figure 11 shows the change in fluorescence intensity in nucleoli over time in Examples 7-1 to 7-5. In Examples 7-1 and 7-2, the fluorescence intensity in nucleoli increased to approximately 1500 at 12 minutes. This suggests that increasing the hydrophobicity of the fluorescent dye reduces the time required to stain nucleoli. In other words, increasing the hydrophobicity of the fluorescent dye improves staining efficiency. It is also believed that improving the hydrophobicity of the fluorescent dye increases the amount of fluorescent dye introduced into cells. It is believed that the binding between RNA and the fluorescent dye in the cytoplasm becomes saturated, causing the fluorescent dye to accumulate in the nucleoli. As a result, it is believed that the RNA detection intensity in nucleoli is increased relative to the cytoplasm.

[0200] In Examples 7-3 and 7-5, where the rate of increase in fluorescence intensity increased over time, it was suggested that the nucleoli were stained next to the cytoplasm in the cells. BIOP-Benzyl-t-Bu in Example 7-1 was the best in terms of the fluorescence intensity of the nucleoli, the ratio of the fluorescence intensity of the nucleoli to the fluorescence intensity of the cytoplasm, and the staining rate.

[0201] Experimental Example 8: Imaging of nucleolar morphological changes (Materials and Methods) HeLa cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and 2% penicillin / streptomycin at 37°C and 5% CO ２ The cells were cultured in an incubator. HeLa cells were seeded onto glass-based dishes (Iwaki) at a density of 10,000 cells / dish and cultured for 24 hours. The above medium (1 mL / dish) containing actinomycin D (2 μg / mL) was added and incubated at 37°C for 0.5 to 5 hours. After washing twice with HBSS(+) buffer, the above medium (1 mL / dish) containing BIOP-Bneyzl-tBu (1 μM) was added and incubated at 37°C for 20 minutes. After washing twice with HBSS(+) buffer again, images of nucleoli were taken using a Deltavision Elite microscopy system before the addition of Actinomycin D (control), and 0.5, 1, 2, 3, 4, and 5 hours after the addition.

[0202] The size and fluorescence intensity of nucleoli at each incubation time were quantitatively evaluated using Fiji-Image J (n=13).

[0203] (Results) Figure 12 shows RNA imaging images of nucleoli before the addition of Actinomycin D (control, indicated as (-)con), and 1 hour (1 h), 3 hours (3 h), and 5 hours (5 h) after the addition of Actinomycin D. As shown in Figure 12, after the addition of Actinomycin D, the nucleoli of HeLa cells were observed to shrink over time.

[0204] Figure 13 is a graph showing the average cross-sectional area of ​​nucleoli for each incubation time, and Figure 14 is a graph showing the fluorescence intensity of nucleoli for each incubation time.

[0205] As shown in Figure 13, the average cross-sectional area of ​​nucleoli was reduced by 52% one hour after the addition of Actinomycin D. Morphological changes were gradual one hour and thereafter after the addition of Actinomycin D, and the average cross-sectional area of ​​nucleoli was reduced by 67% five hours later.

[0206] As shown in Figure 14, the fluorescence intensity of the nucleolus did not decrease monotonically with incubation time, but rather decreased with increasing incubation time after passing through a mountain-like change in intensity, suggesting that this may reflect changes in RNA density.

[0207] Figure 15 shows an RNA imaging image of a nucleolus 0.5 hours after the addition of Actinomycin D. As shown in Figure 15, a relatively large area of ​​weak fluorescence is present inside the nucleolus indicated by the arrow. This area is expected to be a region where RNA has decreased and fluorescence intensity has decreased.

[0208] Figure 16 shows RNA imaging images of nucleoli 3 hours after the addition of actinomycin D. As shown in Figure 16, an onion-skin-shaped fluorescent region is present around the nucleolus indicated by the arrow. If this fluorescent region indicates the presence of RNA, it suggests that the morphological changes and decay process of nucleoli can be captured. In other words, it suggests that after the addition of the fluorescent dye of the present invention, long-term observation can be used to track nucleolar shrinkage and apoptosis.

[0209] The above suggests that the dynamics of nucleoli can be evaluated from multiple angles using the fluorescent dye of the present invention by combining quantitative analysis of nucleolus cross-sectional area and fluorescence intensity using image analysis software with visual observation of morphological changes.

[0210] In conclusion, this study demonstrated that BIOP with a hydrophobic substituent functions as a highly bright fluorescent probe that can be applied to imaging nucleoli in living cells.

[0211] According to the above aspects, it is possible to provide a compound, a fluorescent dye, use of the compound, a method for detecting RNA in a sample, a kit, and a method for detecting infection with an enveloped virus that have high membrane permeability in living cells and excellent selectivity for nucleoli relative to the cytoplasm.

Claims

1. A compound represented by the following general formula (1): (In formula (1), R １ is a hydrophobic group having 4 or more carbon atoms, and R ２ ~R １０ are each independently a hydrogen atom, a hydroxy group, a thiol group, a halo group, an alkyl group, an alkenyl group, an alkynyl group, an alkoxy group, an aryl group, or an amino group; R １１ is a hydrogen atom or an alkyl group.

2. The compound according to claim 1, wherein the hydrophobic group has 6 to 20 carbon atoms.

3. The compound of claim 1, wherein the hydrophobic group comprises an aryl group.

4. The compound according to claim 1, wherein the hydrophobic group comprises an aryl group and the hydrophobic group has 6 to 20 carbon atoms.

5. R in the general formula (1) ２ ~R １０ is a hydrogen atom, and R １１ The compound according to claim 1, wherein is a methyl group.

6. The hydrophobic group is -(CH ２ ) ２ C ６ H ５ , -C ６ H ５ , -C ６ H ４ -C(CH ３ ) ３ , -C ６ H ３ -(C(CH ３ ) ３ ) ２ or a naphthyl group.

7. A fluorescent dye containing the compound according to any one of claims 1 to 6.

8. The fluorescent dye according to claim 7, which is an RNA detection agent.

9. Use of a compound according to any one of claims 1 to 6 for intracellular RNA imaging.

10. A method for detecting RNA in a sample, comprising the steps of: mixing a compound according to any one of claims 1 to 6 with a sample containing RNA to form a mixed sample in which the compound and the RNA in the sample are bound; and irradiating the mixed sample with light to detect the RNA in the mixed sample.

11. The method of claim 10, wherein the sample is a living cell.

12. A kit for detecting RNA in a sample, comprising a compound according to any one of claims 1 to 6, and instructions for detecting RNA in the sample.

13. A method for detecting infection with an enveloped virus, comprising the steps of: contacting a sample suspected of containing enveloped virus particles with the compound according to any one of claims 1 to 6 and a fluorescent dye that binds to the envelope of the enveloped virus; and irradiating the sample with light after said contacting, wherein when a fluorescent signal derived from the compound and a fluorescent signal derived from the fluorescent dye that binds to the envelope of the enveloped virus are both detected, this indicates a high possibility that enveloped virus particles are present in the sample.

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