Compounds, compositions, fluorescent dye agents, fluorescent dye agent kits, and detection methods

Abstract

To provide novel pyrazinacenes (compounds) that can be used for fluorescence detection, e.g., in live cells.SOLUTION: A compound is represented by Formula (1) in the figure, where R1, R2, R3 and R4 each independently represent a group including a hydrophilic group and may be identical or different, and x and y represent 0, 1, 2 or 3 and are identical.SELECTED DRAWING: Figure 10

Description

[Technical field]

[0001] The present disclosure relates to compounds, compositions, fluorescent dye preparations, fluorescent dye preparation kits, and detection methods. [Background technology]

[0002] Fluorescent probes composed of organic molecules are used for the purpose of real-time continuous imaging (live imaging) of cells, tissues, or organs. Many fluorescent probes require irradiation with high-energy light in the ultraviolet-visible wavelength range to emit light. In addition, these fluorescent probes may be decomposed by irradiation with ultraviolet-visible light, and lose fluorescence over time. In addition, light in this wavelength range has a shallow penetration depth in living organisms, making it insufficient for three-dimensional imaging.

[0003] In order to solve such problems, development is underway of fluorescent probes that efficiently emit light using light in wavelength ranges that are highly penetrative in biological tissues, known as "biological windows" (NIR-I: 650-900 nm, NIR-II: 1000-1350 nm, NIR-III: 1550-1870 nm) (for example, Non-Patent Documents 1 and 2). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Chinese Patent Application Publication No. 104628660 (CN104628660A) [Non-patent literature]

[0005] [Non-Patent Document 1] Current Opinion in Chemical Biology Volume 14, Issue 1, February 2010, Pages 64-70 [Non-Patent Document 2] Nanoscale Horizons volume 1, Issue 3, January 2016, Pages 168-184 [Non-Patent Document 3] Journal of the American Chemical Society Volume 141, Issue 50, December, Pages 19570-19574 [Non-Patent Document 4] The Journal of Organic Chemistry Volume 74, Issue 23, November 2009, Pages 8914-8923 Summary of the Invention [Problem to be solved by the invention]

[0006] Pyrazinacenes are acene-based heterocyclic compounds in which 1,4-pyrazine units are linearly condensed, and have been attracting attention in recent years as a group of compounds whose electronic absorption and fluorescence emission wavelengths can be adjusted by the pyrazine units (e.g., Non-Patent Documents 3 and 4). Some of these compounds are known to emit fluorescence in the wavelength region of the "biological window." However, pyrazinacenes have low solubility in aqueous solvents and are highly prone to aggregation due to their molecular structure, making it difficult to introduce them into living cells in a light-emitting state (dispersed state), and it was thought that they would be difficult to use as fluorescent dyes for three-dimensional imaging, etc.

[0007] The novel pyrazinacenes (compounds) of the present disclosure can be used for fluorescent detection of living cells, etc. The present disclosure also includes compositions, fluorescent dyes, and fluorescent dye kits that include the compounds. The present disclosure also includes detection methods that use the compounds, etc. [Means for solving the problem]

[0008] A first embodiment of the compound of the present disclosure is a compound represented by formula 1 below. [ka] In formula 1, R 1 , R 2 , R 3 , and R 4 each independently represents a group containing a hydrophilic group and may be the same or different, and x and y represent 0, 1, 2, or 3, and x and y are the same. The contents of the invention are defined by the claims. Effect of the Invention

[0009] The novel pyrazinacenes (compounds) of the present disclosure can be used for fluorescent detection of live cells and the like. [Brief description of the drawings]

[0010] [Figure 1] 1H-NMR spectrum of TEG8N14 in d8-tetrahydrofuran / trifluoroacetic acid. [Diagram 2] 13C-NMR spectrum of TEG8N14 in d8-tetrahydrofuran / trifluoroacetic acid. [Diagram 3] Electrospray ionization (-) mass spectrum of TEG8N14. [Figure 4A] This is the absorption spectrum of TEG8N14 measured in deionized water. [Figure 4B] This is the absorption spectrum of TEG8N14-HPβCD measured in deionized water. [Figure 4C] 1 is an absorption spectrum of TEG8N14-CTAB measured in deionized water. [Diagram 5] Excitation spectra of TEG8N14, TEG8N14-HPβCD, and TEG8N14-CTAB measured in deionized water. [Figure 6] Emission spectra of TEG8N14, TEG8N14-HPβCD, and TEG8N14-CTAB measured in deionized water. [Figure 7] Emission spectra of TEG8N14, TEG8N14-HPβCD, and TEG8N14-CTAB measured in deionized water. [Figure 8A] This is the absorption spectrum of TEG8N14-HPβCD measured in sodium phosphate buffer (0.1 M) adjusted to pH 3. [Figure 8B] This is the absorption spectrum of TEG8N14-HPβCD measured in sodium phosphate buffer (0.1 M) adjusted to pH 4. [Figure 8C] This is the absorption spectrum of TEG8N14-HPβCD measured in sodium phosphate buffer (0.1 M) adjusted to pH 5. [Figure 8D] This is the absorption spectrum of TEG8N14-HPβCD measured in sodium phosphate buffer (0.1 M) adjusted to pH 6. [Figure 8E] This is the absorption spectrum of TEG8N14-HPβCD measured in sodium phosphate buffer (0.1 M) adjusted to pH 6.6. [Figure 8F] This is the absorption spectrum of TEG8N14-HPβCD measured in sodium phosphate buffer (0.1 M) adjusted to pH 7. [Figure 8G] This is the absorption spectrum of TEG8N14-HPβCD measured in sodium phosphate buffer (0.1 M) adjusted to pH 8. [Figure 8H] This is the absorption spectrum of TEG8N14-HPβCD measured in sodium phosphate buffer (0.1 M) adjusted to pH 9. [Figure 8J] This is the absorption spectrum of TEG8N14-HPβCD measured in sodium phosphate buffer (0.1 M) adjusted to pH 10. [Figure 9] This is the excitation spectrum of TEG8N14-HPβCD measured in sodium phosphate buffer (0.1 M). [Figure 10]Fluorescence spectrum of TEG8N14-HPβCD measured in sodium phosphate buffer (0.1 M) at an excitation wavelength of 675 nm. [Figure 11] Fluorescence spectrum of TEG8N14-HPβCD measured in sodium phosphate buffer (0.1 M) at an excitation wavelength of 743 nm. [Figure 12A] FIG. 1 shows the results of cell viability of HepG2 cells treated with different concentrations of TEG8N14 for 24 hours. [Figure 12B] FIG. 13 shows the results of cell viability of HepG2 cells treated with different concentrations of TEG8N14-HPβCD for 24 hours. [Figure 12C] FIG. 13 shows the results of cell viability of HepG2 cells treated with different concentrations of TEG8N14-CTAB for 24 hours. [Figure 13A] 13 shows confocal microscopy images of HepG2 cells stained with TEG8N14, TEG8N14-HPβCD, and TEG8N14-CTAB. [Figure 13B] Emission spectra of TEG8N14, TEG8N14-HPβCD, and TEG8N14-CTAB in the intracellular organelles of HepG2. The excitation wavelength is 675 nm. [Figure 13C] Emission spectra of TEG8N14, TEG8N14-HPβCD, and TEG8N14-CTAB in the intracellular organelles of HepG2. The excitation wavelength is 743 nm. [Figure 14] Results of staining various cell types with TEG8N14 and Hoechst 33342. [Figure 15A] Confocal fluorescence microscopy images of TEG8N14 in HepG2 cells excited with a laser wavelength of 637 nm. The images are taken at the start of time-lapse imaging, with the observation area enclosed by the dashed line. [Figure 15B]These are confocal fluorescence microscopy images of TEG8N14 in HepG2 cells excited with a laser wavelength of 637 nm, and images of the same field of view of each observation area taken 0.5 hours, 6 hours, 12 hours, and 24 hours later. [Figure 16] Confocal microscopy images of HepG2 cells stained with lysotracker green and TEG8N14-HPβCD. [Figure 17] Multicolor confocal microscopy images of HepG2 cells using TEG8N14-HPβCD, mitotracker green, and Hoechst 33342. [Figure 18A] This figure shows the results of UV-vis titration of TEG8N14 and HPβCD in deionized water (Ratio of N14:HPβCD=1:2731). [Figure 18B] This figure shows the results of UV-vis titration of TEG8N14 and HPβCD in deionized water (Ratio of N14:HPβCD=1:5461). [Figure 18C] This figure shows the results of UV-vis titration of TEG8N14 and HPβCD in deionized water (Ratio of N14:HPβCD=1:11000). [Figure 18D] This figure shows the results of UV-vis titration of TEG8N14 and HPβCD in deionized water (Ratio of N14:HPβCD=1:21923). [Figure 18E] This figure shows the results of UV-vis titration of TEG8N14 and HPβCD in deionized water (Ratio of N14:HPβCD=1:87692). [Figure 18F] This figure shows the results of UV-vis titration of TEG8N14 and HPβCD in deionized water (Ratio of N14:HPβCD=1:175307). [Figure 19A] FIG. 1 shows the UV-Vis absorbance when TEG8N14 and an excess amount of α-cyclodextrin are added to deionized water. [Figure 19B]FIG. 1 shows the UV-visible absorbance when TEG8N14 and an excess amount of β-cyclodextrin are added to ethanol-water (3:7). [Figure 19C] FIG. 1 is a graph showing the UV-Vis absorbance when TEG8N14 and an excess amount of gamma cyclodextrin are added to deionized water. [Figure 19D] FIG. 1 shows the absorbance when TEG8N14 and an excess amount of bovine serum albumin (BSA) are added to deionized water. [Figure 20A] Low magnification confocal microscopy images of HepG2 incubated with TEG8N14 and TEG8N14-HPβCD. [Figure 20B] FIG. 20B is a diagram showing the signal intensity along the arrow line in the fluorescent image of TEG8N14 in FIG. 20A. [Figure 20C] FIG. 20B is a diagram showing the signal intensity along the arrow line in the fluorescent image of TEG8N14-HPβCD in FIG. 20A. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] The following description is based on non-limiting and representative embodiments. Furthermore, a numerical range expressed using "~" means a range including the numerical values ​​before and after "~" as the lower and upper limits.

[0012] A first embodiment of the compound of the present disclosure is a compound represented by formula 1 below. [ka]

[0013] In addition, in formula 1, R 1 , R 2 , R 3 , and R 4 each independently represents a group containing a hydrophilic group and may be the same or different, and x and y represent 0, 1, 2, or 3, and x and y are the same.

[0014] The above compound has a pyrazinacene skeleton and further has a group containing a hydrophilic group. Therefore, it has high solubility in aqueous solvents and low aggregation. Furthermore, due to the pyrazinacene skeleton, it emits light when irradiated with excitation light of a wavelength in a biological window. Since it emits light when excited by excitation light of a wavelength that is highly permeable to the inside of a living body, it is easy to use for three-dimensional imaging of living cells, etc. In addition, since the excitation light of a wavelength belonging to the biological window has lower energy than the ultraviolet-visible light that conventional fluorescent dye agents require for excitation, it is less likely to undergo photodecomposition over time and can be used for long-term continuous observation.

[0015] A second embodiment of the compound of the present disclosure is a compound in which the hydrophilic group in the compound of the first embodiment is a nonionic group.

[0016] The manner in which a compound is taken up into a living cell and / or the site at which it is taken up may be related to the presence or absence of a charge on the hydrophilic group and / or the positive or negative charge. In other words, the localization site in a living cell (the target cell or the site at which the compound is delivered) may be adjusted depending on the presence or absence of a charge on the hydrophilic group (if there is a charge, the positive or negative charge). A nonionic group can be one of the variations in that it does not have a charge.

[0017] A third embodiment of the compound of the present disclosure is a compound in which the hydrophilic group in the compound of the first embodiment is a cationic group or an anionic group.

[0018] Cationic and anionic groups are charged (carry an electric charge), and therefore can be used to adjust the manner and / or site of uptake into living cells. Furthermore, charged hydrophilic groups are likely to contribute to improved uptake into cells in an environment where a large amount of protein is present. In addition, cationic groups have a high affinity for cell membranes that have a negative (-) charge, and are more easily taken up. Additionally, anionic groups generally tend to be less toxic to living cells.

[0019] A fourth embodiment of the compound of the present disclosure is a compound in which the hydrophilic group in the compound of the first embodiment contains a (poly)oxyalkylene group. Here, the term "(poly)oxyalkylene group" refers to an oxyalkylene group or a polyoxyalkylene group.

[0020] A (poly)oxyalkylene group is one of the nonionic groups. A hydrophilic group containing a (poly)oxyalkylene group can be a nonionic group. In addition, the entire group containing a hydrophilic group can be a nonionic group. The (poly)oxyalkylene group has low toxicity to living cells. Therefore, it becomes easier to perform long-term live imaging observation using the compound. The compound of the fourth embodiment has high hydrophilicity and low toxicity at a higher level.

[0021] A fifth embodiment of the compound of the present disclosure is a compound in which the group containing a hydrophilic group in the compound of any one of the first to fourth embodiments is a substituent represented by the following formula 2: [ka]

[0022] In formula 2, Z represents the above hydrophilic group, n represents a number from 1 to 5, and * represents a bonding position.

[0023] The above compound has a pyrazinacene skeleton. The pyrazinacene skeleton is a structure similar to acene, and is a structure in which 1,4-pyrazines are linked in series. Therefore, when the group containing a hydrophilic group is a group represented by formula 2, the affinity with the skeleton is likely to be increased by the benzene ring contained in the group containing a hydrophilic group. The high affinity between the skeleton and the hydrophilic group is likely to favorably affect the ease of synthesis (price of the compound) and the resistance to aggregation. In addition, the group containing the hydrophilic group can be easily introduced using gallic acid (3,4,5-trihydroxybenzoic acid), etc. As a result, the compound can be synthesized more easily and tends to be less expensive.

[0024] A first embodiment of the composition of the present disclosure is a composition comprising a compound according to any one of the first to fifth embodiments and a surfactant.

[0025] The compounds of the first to fifth embodiments each have a group containing a hydrophilic group, and therefore have solubility in aqueous solvents. Therefore, they are easily taken up by living cells. The composition further contains a surfactant, which further improves solubility. Furthermore, aggregation between compounds is further suppressed, and as a result, even when the same amount of compound is used, stronger fluorescence is likely to be obtained compared to the compound alone.

[0026] A second embodiment of the composition of the present disclosure is a composition according to the first embodiment, in which the molar content ratio of the surfactant content to the compound content (surfactant / compound) is 1.0×10 3 That's it, 3.0 x 10 6 The composition is as follows: The content ratio is 1.0×10 3 When the content ratio is 3.0×10 or more, the solubility in the aqueous solvent is further improved, and as a result, stronger fluorescence is easily obtained. 6 When the content is equal to or less than this, the entire amount can be more easily dissolved when dissolved in a solvent, and therefore the composition tends to become homogeneous.

[0027] A third embodiment of the composition of the present disclosure is the composition of the first or second embodiment, in which the surfactant comprises cyclodextrin or a derivative thereof.

[0028] The composition tends to be less toxic to living cells. It also has better solubility in aqueous solvents. As a result, it is easy to obtain stronger fluorescence. It is also less prone to aggregation, making it easy to use as a fluorescent dye for long-term continuous observation.

[0029] A fourth embodiment of the composition of the present disclosure is the composition of the first or second embodiment, wherein the surfactant comprises a hydroxyalkylated cyclodextrin.

[0030] The composition provides stronger fluorescence, is less prone to aggregation, and has less adverse effects on living cells, making it easier to use as a fluorescent dye for long-term continuous observation.

[0031] A first embodiment of the fluorescent dye agent of the present disclosure is a fluorescent dye agent containing the compound of any one of the first to fifth embodiments, or the composition of any one of the first to fourth embodiments.

[0032] The fluorescent dye agent has high solubility in aqueous solvents and low aggregation due to the compound or composition contained therein. Furthermore, due to the pyrazinacene skeleton, it fluoresces when irradiated with excitation light of a wavelength in a biological window. Since excitation light of a wavelength with high transmittance into the living body can be used, it can be used for three-dimensional imaging of living cells, etc. In addition, excitation light of a wavelength belonging to a biological window has lower energy than the ultraviolet-visible light required by conventional fluorescent dye agents, so it is less likely to undergo photodecomposition over time and can be used for long-term continuous observation.

[0033] A first embodiment of the fluorescent dye agent kit of the present disclosure is a fluorescent dye agent kit including a first agent containing a compound of any of the first to fifth embodiments, and a second agent containing a surfactant.

[0034] The fluorescent dye kit is a kit that can be used as a fluorescent dye by mixing the first and second agents when used. Since the above kit contains the compound and the surfactant separately, the fluorescent dye can be prepared by adjusting the mass ratio of each agent according to the purpose, method of use, etc. By adjusting the mass ratio of the compound and the surfactant, the delivery site of the fluorescent dye agent in the living cell can be adjusted. In addition, the solvent used can be easily changed to an appropriate one depending on the imaging site. For example, for imaging of a specific site, better results may be obtained by dissolving the fluorescent dye agent in a hydrophilic organic solvent (e.g., alcohols). The above kit makes such use easier.

[0035] A first embodiment of the detection method of the present disclosure is a detection method comprising staining a cell, tissue, or organ with the fluorescent dye agent of the first embodiment.

[0036] The above detection method can use excitation light with a biological window wavelength derived from the fluorescent dye agent. This excitation light has high penetration into the living body, and improves the results of 3D imaging of living cells. In addition, the excitation light with a wavelength belonging to the biological window has lower energy than the ultraviolet-visible light required by conventional fluorescent dye agents, so the fluorescent dye agent is less likely to photodecompose over time, making it easy to perform continuous observation for long periods of time. Each embodiment of the compound, the composition, the fluorescent dye agent, the fluorescent dye agent kit, and the detection method will be described in detail below.

[0037] (compound) One embodiment of the compound is represented by formula 1 below: [ka]

[0038] In addition, in formula 1, R 1 , R 2 , R 3 , and R 4 R each independently represents a group containing a hydrophilic group, and may be the same or different. 1 , R 2 , R 3 , and R 4 are preferably the same. x and y represent 0, 1, 2, or 3, and x and y are the same.

[0039] The above compound contains a pyrazinacene skeleton, which is a group of compounds in which a plurality of 1,4-pyrazine units are linearly condensed via CC bonds. A method for synthesizing a compound containing a pyrazinacene skeleton is known. For specific examples, Non-Patent Documents 3 and 4 can be referred to. As a specific embodiment of the synthesis method, the following method can be mentioned. [ka]

[0040] In the above formula, R represents a group containing a hydrophilic group. The compound represented by formula 1 is synthesized by reacting 2,3,5,6-pyrazinetetramine with 6,7-R-substituted pyrazino[2,3-b]pyrazine-2,3-dicarbonitrile having a hydrophilic group in an aprotic polar solvent such as DMSO under basic conditions. The repeating numbers of x and y can be easily adjusted by the method described in Non-Patent Document 4. This method is more suitable when the hydrophilic group is a nonionic group. Furthermore, when the hydrophilic group contains an alkali metal salt, the corresponding alkali metal salt can be obtained by hydrolysis under strongly basic conditions using esters of carboxylic acid, phosphonic acid, and sulfonic acid.

[0041] R 1 ~R 4 Among the hydrophilic groups contained in the group represented by the formula (1), the nonionic group means a substituent that does not exhibit ionicity in water. The nonionic group is not particularly limited, but is preferably a group containing an ether structure and an ester structure, and specifically includes a group containing a (poly)oxyalkylene group and an alkoxy group (or itself).

[0042] In the (poly)oxyalkylene group, the oxyalkylene group is -OR 11 - is a divalent group represented by the formula R 11 represents an alkylene group. The number of carbon atoms in the alkylene group is not particularly limited, but is preferably 1 to 10, more preferably 1 to 8, and even more preferably 1 or 2. In a typical embodiment, the number of carbon atoms is 2 (ethylene group).

[0043] The polyoxyalkylene group is a divalent group having an oxyalkylene group as a repeating unit, -(OR 11 ) m - is expressed as R 11 The repeating number m (an integer) is not particularly limited, but is preferably 2 to 10, and more preferably 2 to 6. In addition, when referring to a "(poly)oxyalkylene group," the general formula is -(OR 11 ) m -, and m is a number equal to or greater than 1. In this case, m is preferably an integer from 1 to 10, and more preferably an integer from 1 to 6. When m is 1, the group becomes an oxyalkylene group.

[0044] The hydrophilic group containing a (poly)oxyalkylene group is, for example, 11 -(OR 11 ) m -R 12 In addition, L 11 represents a single bond or a divalent group, * represents the bonding position, R 12 represents a monovalent group. 11 The divalent group is not particularly limited, but may be -O-, -NR 13 Examples of the divalent hydrocarbon group include -, -S-, a chain-like divalent hydrocarbon group, a cyclic divalent hydrocarbon group, and a combination thereof. The chain-like or cyclic divalent hydrocarbon group is preferably an alkylene group having 1 to 18 carbon atoms, an alkenylene group having 2 to 18 carbon atoms, an alkynylene group having 2 to 18 carbon atoms, an arylene group having 5 to 18 carbon atoms, or a combination thereof, more preferably an alkylene group having 1 to 18 carbon atoms, an arylene group having 5 to 18 carbon atoms, or a combination thereof, and further preferably an arylene group having 5 to 18 carbon atoms. R 12 R represents a hydrogen atom or a monovalent group. 12 The monovalent group is not particularly limited, but examples thereof include alkyl groups having 1 to 4 carbon atoms, and a methyl group is preferable.

[0045] R 1 ~R 4 Among the hydrophilic groups contained in the group represented by the formula (I), examples of the anionic group include a carboxy group, a phosphate group, a phosphonate group, a phosphinate group, a sulfate group (-OSO3H), a sulfonic acid group, a sulfinic acid group, and salts thereof. When the anionic group is a salt, examples of the counter ion include alkali metal ions such as sodium ion, potassium ion, and lithium ion; alkaline earth metal ions such as calcium ion and magnesium ion; and ammonium ion. Among these, the hydrophilic group which is an anionic group is preferably a carboxy group, a salt of a carboxy group, a phosphonic acid group, a salt of a phosphonic acid group, a sulfonic acid group, or a salt of a sulfonic acid group.

[0046] R 1 ~R 4 Among the hydrophilic groups contained in the group represented by the formula (I), the cationic group is not particularly limited, but examples thereof include groups derived from quaternary ammonium salts, amine salts, benzalkonium salts, alkylpyridinium salts, and imidazolium salts.

[0047] In terms of achieving a more excellent effect, the group containing a hydrophilic group contained in the compound is preferably a substituent represented by the following formula 2. [ka]

[0048] In formula 2, Z represents the above-mentioned hydrophilic group, and n represents an integer of 1 to 5, more preferably 1 to 3, and further preferably 1 or 2. * represents a bonding position. Multiple Z may be the same or different, but are preferably the same.

[0049] Specific examples of the group represented by formula 2 are as follows. In the following formula, u represents the number of repetitions of the oxyethylene group and represents an integer of 0 or more, preferably 0 or 1 to 10, and more preferably 0 or 1 to 6. When u is 0, -O(CH2CH2O) u CH3 represents a methoxy group. * represents a bonding position. Multiple u's may be the same or different, but are preferably the same. M represents a counter ion. The preferred form is the same as that described for the counter ion of the anionic group.

[0050] [ka]

[0051] [ka]

[0052] [ka]

[0053] [ka]

[0054] [ka]

[0055] [ka]

[0056] [ka]

[0057] The above compound emits light by excitation light of a wavelength in the near-infrared biological window, which has cell membrane permeability. In addition, the compound has excellent solubility in water due to the presence of a hydrophilic group. The above compound can be used as a fluorescent probe in the physiological pH range. It can also be used as a ratiometric fluorescent probe. The method of use as a fluorescent probe is as described in the Examples below.

[0058] (composition) One embodiment of the composition of the present disclosure is a composition comprising the above compound and a surfactant. The surfactant is not particularly limited, and nonionic, cationic, anionic and / or amphoteric surfactants may be used. The content of the surfactant in the composition is not particularly limited, but the molar content ratio (sfct. / cmpd.) of the surfactant (sfct.) to the compound (cmpd.) in the composition is 1.0×10 2 ~1.0×10 7 , 1.0×10 3 ~3.0×10 6 , 1.0×10 3 ~2.0×10 6 , 3.0×10 3 ~2.0×10 6 , 1.0×10 4 ~2.0×10 6 , or 5.0×10 4 ~2.0×10 6 is preferred (the numerical ranges given later are more preferred). The surfactant may be used alone or in combination of two or more. When two or more surfactants are used in combination, the total content is preferably within the above range.

[0059] As the surfactant, a nonionic surfactant and / or an ionic surfactant may be used. The surfactant may be any one of the above-mentioned surfactants used alone, or a plurality of surfactants from the same category may be used in combination, or a plurality of surfactants from different categories may be used in combination. Examples of the ionic surfactant include anionic, cationic, and amphoteric surfactants. Among them, nonionic surfactants and cationic surfactants are preferred, and nonionic surfactants are more preferred.

[0060] Examples of nonionic surfactants include cyclodextrin or derivatives thereof, polyethylene glycol fatty acid esters, polyoxyethylene fatty acid glyceryl, polyoxyethylene-methylpolysiloxane copolymers, fatty acid polyoxyethylene sorbitan, polyoxyethylene alkyl ethers, maltitol hydroxy fatty alkyl ethers, alkylated polysaccharides, alkyl glucosides, sucrose fatty acid esters, polyoxyethylene hydrogenated castor oil glyceryl, polyoxyethylene sorbitol fatty acid esters, polyoxyethylene-polyoxypropylene block copolymers, tetrapolyoxyethylene-tetrapolyoxypropylene-ethylenediamine condensates, polyoxyethylene-beeswax-lanolin derivatives, alkanolamides, polyoxyethylene-propylene glycol fatty acid esters, polyoxyethylene-alkylamines, polyoxyethylene-fatty acid amides, alkylethoxydimethylamine oxides, and trioleyl phosphate.

[0061] As the nonionic surfactant, cyclodextrin or a derivative thereof is preferred. The cyclodextrin derivative means a cyclodextrin having a substituent. The substituent is not particularly limited, but a hydrocarbon group which may have a heteroatom is preferred, and among them, an alkyl group and a hydroxyalkyl group are more preferred. The number of carbon atoms in the substituent is preferably 1 to 10, more preferably 1 to 4, and even more preferably 1 to 3.

[0062] A composition containing cyclodextrin or its derivatives as a surfactant emits stronger fluorescence at the excitation wavelength of the biological window. In addition, the fluorescence is less likely to be lost over time. This is presumably because cyclodextrin or its derivatives improve the solubility of the compound in water and also reduce the likelihood of the compound agglomerating.

[0063] When the composition contains cyclodextrin or a derivative thereof (hereinafter also referred to as "cyclodextrin, etc."), the preferred range of the molar content ratio (sfct. / cmpd.) of the cyclodextrin, etc. (sfct.) to the compound (cmpd.) is 1.0×10 2 ~1.0×10 7 , 1.0×10 3 ~3.0×10 6 , 1.0×10 3 ~2.0×10 6 , 3.0×10 3 ~2.0×10 6 , 1.0×10 4 ~2.0×10 6 , or 5.0×10 4 ~2.0×10 6 (The numerical ranges given below are more preferable). The composition may contain one type of cyclodextrin, etc. alone or two or more types. When the composition contains cyclodextrin, etc., it is preferable that the total content thereof is within the above range.

[0064] In general, cyclodextrin or a derivative thereof is known to form an inclusion complex through host-guest interaction. Cyclodextrin or a derivative thereof can typically form an inclusion complex in which the guest is pierced in a skewer-like manner. However, the molar content ratio (sfct. / cmpd.) of the cyclodextrin or its derivative to the compound is 1.0×10 2 When the content ratio is more than 1.0×10, the amount of cyclodextrin or its derivative is excessive compared to the typical formulation for forming an inclusion complex. 2 In the composition containing the above, the effect is obtained not by the formation of an inclusion complex, but by the function of the cyclodextrin or its derivative as a surfactant. In other words, it is not necessary for the compound and the cyclodextrin to form an inclusion complex.

[0065] The cyclodextrin etc. may be any of α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin (or derivatives thereof). In general, the cyclodextrin etc. is often selected in consideration of the inclusion property of the host compound. However, in the above composition, the desired effect can be obtained regardless of whether α-cyclodextrin, β-cyclodextrin, or γ-cyclodextrin is used. In other words, the desired effect can be obtained regardless of the inclusion property. Compositions containing any of the cyclodextrins are also preferred because they have lower toxicity to living cells than compositions containing other surfactants.

[0066] The cyclodextrin derivative may have one or more substituents. The substituents of the cyclodextrin derivative can typically be introduced by a chemical modification method, such as by reaction with a hydroxyl group. α-cyclodextrin contains 18 hydroxyl groups, β-cyclodextrin contains 21 hydroxyl groups, and γ-cyclodextrin contains 24 hydroxyl groups. All of these may be substituted, or only a part of them may be substituted. Cyclodextrins with different degrees of substitution may also be mixed.

[0067] For example, 2-hydroxypropyl cyclodextrin, in which the hydroxyl groups are 2-hydroxypropylated, can be obtained by reacting β-cyclodextrin with propylene oxide under basic conditions. The hydroxypropyl groups are obtained by hydroxypropylating the 2-, 3-, and 6-hydroxyl groups of the glucose that constitutes the cyclodextrin. As described above, β-cyclodextrin contains 21 hydroxy groups, all of which may be hydroxypropylated, but the average degree of substitution is preferably about 4 to 9.

[0068] Examples of cyclodextrin derivatives include 2-hydroxyalkylated, methylated, acetylated, and aminated cyclodextrins. Among them, compositions containing 2-hydroxyalkylated cyclodextrins are preferred in that the solubility of the compound is improved and aggregation is suppressed, resulting in stronger fluorescence and maintaining the ability to fluoresce (the ability to emit light when irradiated with excitation light) for a longer period of time. Among them, those with an alkyl group having 1 to 5 carbon atoms are preferred, those with 1 to 4 carbon atoms are more preferred, and 2-hydroxypropyl-β-cyclodextrin is particularly preferred.

[0069] Examples of cationic surfactants include cetyltrimethylammonium bromide (cetrimonium bromide, CTAB), diallyldimethylammonium chloride, cetyltrimethylammonium chloride, stearyltrimethylammonium chloride, isostearyltrimethylammonium chloride, lauryltrimethylammonium chloride, behenyltrimethylammonium chloride, octadecyltrimethylammonium chloride, cocoyltrimethylammonium chloride, cetyltrimethylammonium bromide, stearyltrimethylammonium bromide, lauryltrimethylammonium bromide, isostearyllauryldimethylammonium chloride, dicetyldimethylammonium chloride, distearyldimethylammonium chloride, and dicocoyldimethylammonium chloride. As the cationic surfactant, cetyltrimethylammonium bromide is preferred.

[0070] Examples of anionic surfactants include N-cocoyl glutamate, coconut oil fatty acid methyl taurine salt, N-caproyl methyl taurine salt, N-lauroyl methyl taurine salt, N-myristoyl methyl taurine salt, N-palmitoyl methyl taurine salt, N-stearoyl methyl taurine salt, N-oleoyl methyl taurine salt, N-cocoyl glutamate, N-lauroyl glutamate, N-myristoyl glutamate, N-palmitoyl glutamate, N-stearoyl glutamate, N-oleoyl glutamate, lauryl phosphate, myristyl phosphate, palmityl phosphate, and stearyl phosphate. Examples of counter ions include sodium, potassium, triethanolamine, and ammonia.

[0071] Examples of amphoteric surfactants include 2-undecyl-N,N,N-(hydroxyethylcarboxymethyl)-2-imidazoline sodium, 2-cocoyl-2-imidazolinium hydroxide-1-carboxyethyloxy disodium salt, 2-heptadecyl-N-carboxymethyl-N-hydroxyethylimidazolinium betaine, lauryl dimethylaminoacetic acid betaine, alkyl betaine, amido betaine, sulfo betaine, and cocamidopropyl betaine.

[0072] The composition may contain a solvent other than the above. The solvent contained in the composition is not particularly limited, and examples thereof include water, hydrophilic organic solvents, and mixtures thereof. Examples of hydrophilic organic solvents include lower alcohols such as methanol, ethanol, propanol, isopropanol, and butanol; and non-ionic polar solvents such as dimethyl sulfoxide, dimethylformamide, acetonitrile, and tetrahydrofuran. When the composition is used as a fluorescent dye agent, these solvents can be appropriately selected according to the experimental protocol. The composition can replace conventionally used fluorescent probes, and the solvent used can be easily selected according to conventional experimental protocols.

[0073] The composition may contain other components than those described above. Examples of other components include a buffering agent. The buffering agent may be mixed with the solvent in advance. In other words, the solvent may be a buffer solution.

[0074] The composition can be produced by mixing the above-mentioned components. When the composition contains a compound, a surfactant, and a solvent, it may be heated. The temperature at that time may be appropriately selected depending on the solvent, and for example, when the solvent contains water, it may be heated to 20 to 100°C, for example.

[0075] The composition emits light when excited by near-infrared light with wavelengths in a biological window that is cell membrane permeable. In addition, since the composition contains a surfactant, the composition has improved solubility in water and inhibits compound aggregation, making it suitable for time-lapse imaging and the like, and can be used as a ratiometric fluorescent probe that can be used in a physiological pH range. The method of use will be described in the examples below.

[0076] (Fluorescent dye) One embodiment of the fluorescent dye agent of the present disclosure is a fluorescent dye agent containing the above-mentioned compound or composition. The fluorescent dye agent can be used for staining cells, tissues, or organs. It can be used in vitro or in vivo, and is suitable for three-dimensional imaging using a confocal microscope or the like because it emits light by excitation light of a wavelength in a biological window of near-infrared that has cell membrane permeability. In addition, it can be used for long-term real-time imaging and time-lapse imaging because the excitation wavelength has low toxicity to cells. It can also be used in combination with other known fluorescent dye agents. It has been experimentally confirmed that even when used in combination, it is unlikely to promote decomposition and weaken the fluorescence, or conversely, it is unlikely to affect other fluorescent dye agents and weaken the fluorescence. The results will be described later.

[0077] Fluorescent dyes have properties that make them particularly suitable for observing live cells, but can also be used to observe dead cells. The cells, tissues, organs, etc. of interest may be either animal or plant, and may be of biological origin, synthetic, or cultured. The living body is not particularly limited as long as it is a multicellular animal, and examples thereof include mammals, and among these, humans are preferred. Note that cells and tissues derived from a living body include tissues separated from a living body and tissues not separated from a living body.

[0078] Examples of cells include, but are not limited to, skin (e.g., epidermis and dermis), brain, spinal cord, esophagus, stomach, small intestine, large intestine, duodenum, rectum, liver, pancreas, gallbladder, bladder, kidney, heart, spleen, thymus, prostate, uterus, ovaries, testes, breasts, lungs, bronchi, eyes, nose, paranasal sinuses, oral cavity, pharynx, salivary glands, thyroid gland, parathyroid gland, adrenal gland, muscle, bone marrow, blood vessels, nerves, lymph nodes, peritoneum, diaphragm, and blood.

[0079] The method of using the fluorescent dye agent is the same as that of conventional fluorescent dye agents, and those skilled in the art can use it by adjusting the required concentration, pH, etc. according to the experimental protocol. Due to the properties of the above-mentioned compound, it is more suitable for use in the physiological pH range. When the composition is provided as a powder, a solvent, etc. may be added when used. Examples of the solvent to be added include the same as those exemplified as the solvent that the composition may contain.

[0080] (Fluorescent dye kit) One embodiment of the fluorescent dye agent kit of the present disclosure is a fluorescent dye agent kit including a first agent containing the compound and a second agent containing a surfactant.

[0081] The first agent may contain the compound, and may be composed of the compound alone. The first agent may contain other components that the composition may contain, such as a buffering agent. It is preferable that the first agent does not contain a surfactant. As an example, the first agent may be contained in a container. A predetermined amount of the first agent is taken out from the container and used. The first agent may be used by itself, or may be mixed with the second agent. The second agent may contain a surfactant, and may be composed of only a surfactant. The second agent may contain other components that the above-mentioned composition may contain, such as a buffering agent. The second agent does not contain the above-mentioned compound. As an example, the second agent may be contained in a container. A predetermined amount of the second agent is taken out from the container and used. The second agent may be mixed with the first agent when used.

[0082] The first agent and / or the second agent may contain a solvent. The solvent that the first agent and the second agent may contain is the same as that described as the solvent that the composition may contain. The first agent and / or the second agent may contain a buffering agent.

[0083] The above-mentioned composition is prepared by mixing the first agent and the second agent at any ratio. Since the fluorescent dye agent kit contains the first agent and the second agent independently, they can be mixed at any ratio to prepare a composition. If the first agent and the second agent do not contain a solvent, any solvent can be selected and used according to the experimental protocol. The composition obtained by mixing the first agent and the second agent can replace a conventional fluorescent dye agent, and the method of use is the same as that of the conventional fluorescent dye agent. Therefore, the selection of the type of solvent and the adjustment of the amount can be performed arbitrarily and easily by a person skilled in the art.

[0084] (Detection method) A first embodiment of the detection method of the present disclosure is a detection method comprising staining a cell, tissue, or organ with the above-mentioned fluorescent dye agent.

[0085] The detection method may further include observing the stained cells by fluorescence imaging. Also, the detection method may include observing the stained cells by fluorescence spectroscopy. In the observation by fluorescence imaging and fluorescence spectrum, it is preferable that the observation target (cells, etc.) is irradiated with excitation light having a wavelength in the biological window (particularly NIR-I: 650 to 900 nm). The excitation wavelength is more preferably 650 to 800 nm. The detection wavelength is not particularly limited, but is preferably 700 to 900 nm.

[0086] The detection method may further include observing the stained cells by 3D fluorescence imaging (three-dimensional fluorescence imaging). The fluorescence imaging and the fluorescence spectrum measurement may be performed continuously or intermittently for a long period of time.

[0087] The concentration of the compound during staining is not particularly limited. For example, the concentration of the compound in the staining solution is preferably 0.1 μg / mL to 100 mg / mL. The temperature of the staining solution is not particularly limited, and is preferably 0 to 100° C., and more preferably 10 to 50° C. The staining time is not particularly limited, and is preferably 1 minute to 48 hours. However, when observing continuously or intermittently for a long period of time, the observation time is not limited to the above. The pH range used is not particularly limited, but preferred ranges are pH 3 to 10, pH 5 to 10, pH 6 to 10, pH 6.6 to 10, or pH 7 to 10 (the numerical ranges listed below are more preferred). EXAMPLES

[0088] (TEG8N 14 Synthesis of Pyrazine-2,3,5,6-tetramine was prepared by the method described in [Patent Document 1]. 6,7-substituted-pyrazino[2,3b]pyrazine-2,3-dicarbonitrile derivatives were prepared by condensation of the corresponding benzyl derivatives with 5,6-diaminopyrazine-2,3-dicarbonitrile [Non-Patent Document 4].

[0089] [ka]

[0090] Freshly prepared 2,3,5,6-pyrazinetetramine (20 mg, 1.43 × 10 -4 A mixture of 6,7-bis(3,4-bis(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)phenyl)pyrazino[2,3-b]pyrazine-2,3-dicarbonitrile, 418 mg, 4.25 × 10 mol, superanhydrous dimethyl sulfoxide (1.5 mL), and freshly ground potassium carbonate (200 mg) was heated at 122 °C for 10 min. The reaction mixture was flushed with air and then ...methoxyethoxy)ethoxy)ethoxy)phenyl)pyrazino[2,3-b]pyrazine-2,3-dicarbonitrile, 418 mg, 4.25 × 10 mol, superanhydrous dimethyl sulfoxide (1.5 mL), and freshly ground potassium carbonate (200 mg) was heated at 122 °C for 10 min. -4 mol) was added to the reaction mixture, followed by additional potassium carbonate (2.16 g).

[0091] The reaction mixture was again flushed briefly with air and stirred at 122 °C for 1 h. After cooling to room temperature, DMSO was removed under reduced pressure to give a solid residue. The solid residue was dissolved by shaking in a mixture of chloroform (100 mL) and deionized water (25-30 mL). Ammonium chloride (2.5 g) was added and after further shaking, the aqueous layer was separated and extracted with chloroform (20 mL). The organic fractions were combined and dried over sodium sulfate. After filtration, the solvent was removed under reduced pressure. The crude product was first purified by column chromatography [silica gel, eluted with chloroform:methanol:triethylamine, 100:0:0 to 92:6:2]. The compound was further purified by preparative gel permeation chromatography (Biobeads SX-3, chloroform elution) and then repeated column chromatography [silica gel, eluted with chloroform:methanol:triethylamine 100:0:0 to 96:2:2] until deemed sufficiently pure. Final purification was achieved by preparative TLC [silica gel, eluted with chloroform:methanol 88:12]. Yield: 30 mg (11%).

[0092] The obtained TEG8N 14 teeth, 1 H-NMR, 13 The structure was analyzed by C-NMR and mass spectrometry (electrospray ionization). 14 of 1 H-NMR spectrum, Figure 2, shows TEG8N in d8-tetrahydrofuran / trifluoroacetic acid. 14 of 13 C-NMR spectrum, Figure 3, of TEG8N 14 The electrospray ionization (-) mass spectrum of the compound shown in FIG. 3 is the peak at m / z=1994.9, which corresponds to [TEG8N 14 -H] - was estimated to be the peak.

[0093] (TEG8N 14 -Preparation of HPβCD 2-Hydroxypropyl-β-cyclodextrin (1.3 g) was dissolved in deionized water (3 mL) and stirred at 60° C. for 1 hour. After cooling to room temperature, the solution was filtered using a microporous membrane (0.22 μm) to remove aggregates and obtain a solution. 14 (2.6 μg) was added to the solution (1 mL) and stored at -30°C overnight. The product was freeze-dried for 3 days and sterilized. 14 -HPβCD.

[0094] (TEG8N 14 Preparation of -CTAB Freshly prepared cetrimonium bromide (2mg, cationic surfactant), TEG8N 14 (2.6 μg) and deionized water (1 mL) were reacted for 5 minutes, freeze-dried for 3 days, and sterilized. 14 -CTAB.

[0095] (UV-Visible Absorption and Fluorescence Spectra) UV-visible spectra were measured on a Shimadzu UV-visible spectrophotometer. Fluorescence spectroscopic studies were performed on a JASCO V-570 UV / Vis / NIR spectrophotometer.

[0096] (cell culture) HepG2 (RCB1648; Riken BRC, Japan), SK-Mel-28 (JCRB1376; JCRB Cell Bank, Japan), HeLa (JCRB9086; JCRB Cell Bank, Japan), and MG63 (IFO50108; JCRB Cell Bank, Japan) were cultured at 37°C in a 5% CO2 / 95% air incubator.

[0097] The medium used was Eagle's minimum essential medium (MEM, Gibco, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Biowest, South America) as the basal medium, and 1% antibiotic / antibacterial agent (AA; penicillin, streptomycin, amphotericin B, Sigma Aldrich, USA) as the complete medium (CM). The medium was refreshed every 2 days.

[0098] Cell viability assay TEG8N 14 Cell viability after treatment was measured by Cell Counting Kit-8 (CCK-8, Dojindo Laboratories, Kumamoto, Japan) assay. HepG2 cells (5 × 10 4 The cells were seeded in a 24-well plate (cells / well) and cultured at 37°C under 5% CO2 / 95% air for 24 hours. After washing, the medium was diluted with various concentrations of TEG8N 14 The medium was replaced with MEM (without phenol red) containing CCK-8 (0, 10, 20, 40, 80 μg / mL). After 2 hours of culture, the cells were washed and a CCK-8 solution in MEM was added to each well. The absorbance at 450 nm of each well was measured using a microplate reader (Infinite 200PRO, Tecan, Männedorf, Switzerland). The cell viability was calculated using the following formula:

[0099] Cell viability (%)=(A sample -A blank ) / (A control -A blank ) x 100 Here, A sample A represents the average absorbance obtained from wells at a particular reagent concentration. control A indicates the average absorbance value measured from wells containing cells only. blank indicates the average absorbance value obtained from wells containing medium only. TEG8N 14 -HPβCD (0, 11, 22, 44, 88 mg / mL) and TEG8N 14 For -CTAB (0, 33, 67, 133, 267 μg / mL), incubation was performed for 0.5 h, and the same procedure was repeated to check cell viability.

[0100] (Measurement of fluorescence intensity) To confirm the fluorescence intensity of intracellular organelles, quantitative measurement of fluorescence was also performed using a microplate reader (Infinite 200PRO, Tecan, Männedorf, Switzerland). HepG2 cells (1 × 10 4 Cells (cells / well) were seeded into 96-well solid black flat-bottom polystyrene tissue culture-treated microplates and incubated for an additional 24 hours in CM at 37°C and 5% CO2 / 95% air. The cells were then washed twice with MEM and the medium was replaced with TEG8N 14 (40μg / mL, 2 hours), TEG8N 14 -HPβCD (44 mg / mL, 0.5 hours) or TEG8N 14 The medium was replaced with MEM (without phenol red) containing -CTAB (33 μg / mL, 0.5 h). After incubation at 37 °C under 5% CO2, the cells were washed and incubated in MEM (without phenol red) and the fluorescence spectrum was recorded. To avoid autofluorescence background from the cells themselves, a positive control containing only cells and medium was also prepared.

[0101] (confocal microscope) A confocal laser microscope system (A1RHD25, Nikon, Japan) was used to perform fluorescence imaging of the nuclei of various cultured animal cells at 37°C under 5% CO2 / 95% air conditions using a stage incubator system (Tokai Hit Co., Ltd.). Nikon Plan Apo 60x / 1.11 objective lens was used for low magnification observation, and Nikon Plan Apo 100x / 1.45 objective lens was used for high magnification observation.

[0102] First, cells (5 × 10 4 The cells were then washed twice with MEM red and the medium was replaced with TEG8N. 14 (40μg / mL, 2 hours), TEG8N 14 -HPβCD (44 mg / mL, 0.5 hours) or TEG8N 14 The medium was replaced with MEM (containing no phenol red) containing -CTAB (33 μg / mL, 0.5 h).

[0103] After incubation at 37°C under a 5% CO2 atmosphere, the cells were washed and co-stained with Hoechst 33342, mitotracker green, or lysotracker green (Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer's instructions. After washing, the cells were incubated in MEM. 14 , and TEG8N 14 Images of cells stained with -HPβCD were acquired in the red channel (λex=637nm, λem=663-738nm). Cells stained with Hoechst 33342 were excited at 405nm and emitted at 430-475nm. Cells stained with mitotracker or lysotracker green were acquired in the green channel (λex=488nm, λem=500-550nm). In addition, a positive control containing only cells was prepared to avoid autofluorescence background from the cells themselves. Images were analyzed and deconvolved using Nikon's NIS-Elements software.

[0104] (Time-lapse imaging) For time-lapse observation, a Nikon Confocal A1 (A1RHD25, Nikon, Japan) equipped with a Nikon Plan Apo 100× / 1.45 objective lens was used. The cells were incubated at 37°C under 5% CO2 / 95% air conditions using a stage incubator system (Tokai Hit Co., Ltd.). In particular, HepG2 cells were incubated in TEG8N 14 (40 μg / mL, 2 h). After washing HepG2 cells twice with MEM, the cells were incubated in MEM (phenol red-free, 1% AA). Confocal images were recorded in the red channel (λex=637 nm, λem=663-738 nm) and in bright field mode (time: 24 h, time interval: 0.5 h).

[0105] (Statistical analysis and reproducibility) Statistical data were presented as mean ± standard deviation (SD). Statistical data were further calculated by one-way analysis of variance (ANOVA) using Origin Pro software (8.0). Values ​​were considered significantly different at 95% and 99% confidence intervals. Each experiment was independently repeated at least three times with similar results.

[0106] (Optical characteristics as a fluorescent dye) Figure 4A shows the TEG8N measured in deionized water. 14 Figure 4B shows the absorption spectrum of TEG8N measured in deionized water. 14 Figure 4C shows the absorbance spectrum of TEG8N-HPβCD measured in deionized water. 14 1 is an absorption spectrum of -CTAB. Figure 5 shows the TEG8N measured in deionized water. 14 , TEG8N 14 -HPβCD and TEG8N 14 The excitation spectrum of -CTAB was observed at a fluorescence wavelength of 760 nm.

[0107] Figure 6 shows the TEG8N measured in deionized water. 14 , TEG8N 14 -HPβCD and TEG8N 14 The emission spectrum of -CTAB. The excitation wavelength (λex) in Figure 6 is 675 nm.

[0108] Figure 7 shows the TEG8N measured in deionized water. 14 , TEG8N 14 -HPβCD and TEG8N 14 The emission spectrum of -CTAB. The excitation wavelength (λex) in Figure 7 is 743 nm.

[0109] From the results in Figures 4A to 4C and Figures 5 to 7, TEG8N 14 , TEG8N 14 -HPβCD and TEG8N 14 It was confirmed that both -CTAB and TEG8N have absorbance in the NIR-I range (650-900 nm) in deionized water. 14 -HPβCD and TEG8N 14 It was confirmed that -CTAB emits light more efficiently at wavelengths of NIR-I (650 to 900 nm) in deionized water, as is evident from the fluorescence spectrum for excitation light of 675 nm or 743 nm. From the above experimental results, TEG8N 14 , TEG8N 14 -HPβCD and TEG8N 14 -CTAB has the potential to be used as a fluorescent dye in vivo. 14 -CTAB shows the strongest luminescence, while TEG8N 14 For both of these, light emission sufficient for practical use was obtained.

[0110] (Relationship between optical properties and pH) 8A to 8J show the TEG8N measured in sodium phosphate buffer (0.1 M) adjusted to pH 3, 4, 5, 6, 6.6, 7, 8, 9, and 10, respectively. 14 FIG. 9 shows the absorption spectrum of TEG8N measured in sodium phosphate buffer (0.1 M). 14 The excitation spectrum of TEG8NβCD was measured at an excitation wavelength of 675 nm in sodium phosphate buffer (0.1 M). 14 FIG. 11 shows the fluorescence spectrum of TEG8N in sodium phosphate buffer (0.1 M) at an excitation wavelength of 743 nm. 14 Fluorescence spectrum of -HPβCD.

[0111] From the results in Figures 8A to 8J and Figures 9 to 11, TEG8N 14 The absorbance and fluorescence intensity of -HPβCD were found to vary depending on the pH. In particular, the fluorescence intensity was high in the pH range of 5-10, even higher in the pH range of 6-10, even higher in the pH range of 6.6-10, and especially high in the pH range of 7-10. In terms of trends, the fluorescence intensity increased in the pH range of 3 to 8, and decreased in the pH range of 9 to 10.

[0112] From the above results, TEG8N 14 It has been confirmed that -HPβCD emits light more efficiently at wavelengths of NIR-I (650 to 900 nm) and its emission intensity changes depending on the pH, so it can be used as a fluorescent dye that can emit light specifically (depending on the surrounding pH) at specific sites in a living body, etc.

[0113] (cell viability) FIG. 12A shows the effect of different concentrations of TEG8N 14 FIG. 12B shows the results of cell viability of HepG2 cells treated with different concentrations of TEG8N for 24 hours. 14FIG. 12C shows the results of cell viability of HepG2 cells treated with TEG8N-HPβCD for 24 hours. 14 Figure 13 shows the results of cell viability of HepG2 cells treated with -CTAB for 24 hours. All results are shown as percentages of the dye-free control. Data represent the mean ± SD (standard deviation) (n=3). Significant differences: *p<0.05; **p<0.01; ***p<0.001.

[0114] The results in Figure 12A show that TEG8N 14 It was confirmed that there was no significant difference in cell viability within the range of 0 to 80 μg / mL. The results in Figure 12B show that TEG8N 14 In the case of -HPβCD, there was no significant difference in cell viability at concentrations up to 50 mg / mL. In other words, it was confirmed that cell viability was maintained at a higher level even at higher concentrations. Also, TEG8N 14 In the case of -CTAB, when the concentration was 33μg / mL or more, the cell viability was slightly decreased. Furthermore, when the concentration was 133μg / mL or more, the cell viability was further decreased. Furthermore, when the concentration was 267μg / mL or more, the cell viability was further decreased. In other words, when the concentration was less than 267μg / mL, the cell viability was higher, when the concentration was less than 133μg / mL, the cell viability was even higher, and when the concentration was less than 33μg / mL, the cell viability was particularly high. From the above results, TEG8N 14 -HPβCD and TEG8N 14 is TEG8N 14 -It has been confirmed to be less toxic than CTAB.

[0115] (Confocal microscopy) FIG. 13A shows TEG8N 14 , TEG8N 14 -HPβCD and TEG8N 14Confocal microscopy images of HepG2 cells stained with -CTAB and CTAB, respectively. Images were selected from 3-4 similar images. The scale bar is 5 μm. All specimens were also stained with Hoechst 33342. Hoechst 33342 is a commonly used nuclear stain with the characteristics of λex=352 nm, λem=461 nm.

[0116] In FIG. 13A, images are arranged in a two-dimensional matrix along horizontal rows and vertical columns. 14 , TEG8N 14 -HPβCD and TEG8N 14 Images of the same field of view stained with -CTAB are arranged. In the same row (vertical arrangement), blue channel, red channel, overlay of blue and red channels, and bright-field (BF) images are arranged.

[0117] The blue channel images are all blue in the original image (gray in the figure) and are due to Hoechst 33342. On the other hand, the red channel images are all red in the original image and are due to TEG8N 14 , TEG8N 14 -HPβCD and TEG8N 14 The red areas are represented in white (light areas) in Figure 13A.

[0118] From the second row of images in Figure 13A, TEG8N 14 , TEG8N 14 -HPβCD and TEG8N 14 It was revealed that both TEG8N and -CTAB can be used as fluorescent dyes. Furthermore, the third row of images shows that even when used in combination with Hoechst 33342, TEG8N 14 , TEG8N 14 -HPβCD and TEG8N 14 It was found that -CTAB does not cause problems such as immediate decomposition (discoloration).

[0119] Figures 13B and 13C show TEG8N in intracellular organelles of HepG2. 14 , TEG8N 14 -HPβCD, TEG8N 14 13B shows the emission spectrum of -CTAB. The excitation wavelength in FIG. 13B is 675 nm, and the excitation wavelength in FIG. 13C is 743 nm. The results of this study were verified by confocal microscopy images and confirmed that the observed fluorescence was not due to autofluorescence. It was also confirmed that there was no spectral shift in the dye spectrum after introduction into cells. For use as a fluorescent dye, it is necessary that the complex formed between the fluorescent dye and the organelle does not cause a shift in absorbance or fluorescence, and this characteristic was confirmed.

[0120] From Figures 13B and 13C, it was confirmed that fluorescence could be detected even when two different excitation wavelengths (675 nm and 743 nm) were used. Since the selection range of excitation wavelengths within the NIR-I range is wide and various hardware can be used, it has excellent properties as a fluorescent dye agent. This is because confocal microscopes may have limitations on the excitation wavelengths that can be used.

[0121] Figure 14 shows the TEG8N 14 and Hoechst 33342 staining of various cell types. Scale bar is 5 μm. In FIG. 14, images are arranged in a two-dimensional matrix along horizontal rows and vertical columns. In the same row (horizontally lined up), HepG2, SK-Mel-28, and MG63 are compared with TEG8N. 14 Images of the same field stained with and Hoechst 33342 are collocated. In addition, the blue channel, the red channel, their overlap, and a bright-field image (BF) are arranged in the same row (vertical alignment).

[0122] The blue channel images are all blue in the original images and are due to Hoechst 33342. The blue parts are shown as grey in Fig. 14. On the other hand, the red channel images are all red in the original images and are due to TEG8N 14 The red parts are shown in white (bright parts) in Figure 14. From the results in Figure 14, TEG8N 14 It has become clear that TEG8N can be used as a fluorescent dye for various cells. 14 -HPβCD and TEG8N 14 For -CTAB, the results are similar.

[0123] (Time-lapse imaging) 15A and 15B show the TEG8N in HepG2 cells excited with a laser wavelength of 637 nm. 14 The confocal fluorescence microscopy images of TEG8N are shown in Fig. 15A. Fig. 15A is an image at the start of the experiment, and the observation areas are enclosed by dashed lines. Fig. 15B is an image of the same field of view of each observation area 0.5 h, 6 h, 12 h, and 24 h later. The white arrows in each image indicate TEG8N 14 The above shows the nuclear image stained with TEG8N. 14 It was confirmed that the dynamics of cell nuclei could be observed by live imaging.

[0124] This is TEG8N 14 This clearly indicates that the excitation wavelength is in the NIR-I range and that even long-term irradiation with excitation light causes low toxicity to the observed cells. It was also confirmed that photobleaching is unlikely to occur because the dye emits light with excitation light in the NIR-I range. Photobleaching refers to the process in which a dye is decomposed and its fluorescence disappears due to irradiation with excitation light, and is thought to occur particularly easily in dyes that require excitation light with shorter wavelengths. Although not shown in the figure, the fact that there is little cell damage due to long-term irradiation with excitation light is similar to that of TEG8N, which has a similar fluorescent wavelength. 14 -HPβCD and TEG8N 14For -CTAB, the results are similar.

[0125] (Confirmation of localization) Figure 16 shows the results of lysotracker green and TEG8N. 14 16(a) to (d) are confocal microscopy images of HepG2 cells stained with -HPβCD. All of the images in Fig. 16(a) to (d) show images of the same field of view. Fig. 16(a) shows the image by Lysotracker Green (green channel), Fig. 16(b) shows the image by TEG8N 14 Figure 16(c) shows the superimposed image of the two, and Figure 16(d) shows the bright-field image. The scale bar is 2 μm.

[0126] In Fig. 16(a), the gray areas are green in the original image. In Fig. 16(b), the white areas (bright areas) are red in the original image. Lysotracker Green is known as a common lysosomal marker dye.

[0127] The Pearson correlation coefficient plot of the red and green channels detected from the overlapped images was R = 0.76, indicating a positive correlation. 14 It was confirmed that -HPβCD has high localization to lysosomes.

[0128] (Multicolor confocal microscopy) Figure 17 shows the TEG8N 14 -Multicolor confocal microscopy images of HepG2 cells using HPβCD, mitotracker green, and Hoechst 33342. Images were selected from 2 to 3 similar images. The scale bar is 20 μm. Mitotracker green is a common green fluorescent mitochondrial stain, and Hoechst 33342 is a common blue fluorescent dye for nuclear staining. These were used for costaining.

[0129] Figures 17(a) to (d) all show images of the same field of view. Figure 17(a) shows the TEG8N 14 Figure 17(b) is an image stained with -HPβCD (red channel), Figure 17(b) is an image stained with mitotracker green (green channel), Figure 17(c) is an image stained with Hoechst 33342 (blue channel), Figure 17(d) is a superimposed image of Figures 17(a) to (c), and Figure 17(e) is a bright field image.

[0130] In Fig. 17(a), the white (bright) parts are red in the original image, in Fig. 17(b), the bright parts are green in the original image, and in Fig. 17(c), the grey parts are blue in the original image. In Figures 17(a) to (c), different areas were stained, and the stained areas were also confirmed in the superimposed image in Figure 17(d). 14 It was found that -HPβCD is not easily decomposed when used in combination with other fluorescent dyes, and can also be used for co-staining.

[0131] (Results: Effect of amount of HPβCD added) 18A-18F show the results of the analysis of TEG8N in deionized water. 14 The results of UV-vis titration of TEG8N and HPβCD are shown in the figures. 14 The ratio of N14 to HPβCD is as shown in the table below. The "Concentration" column in the table below indicates the concentration in the composition. The "Ratio of N14:HPβCD" column indicates the ratio of TEG8H to HPβCD. 14 and HPβCD. The letters A to F correspond to the letters in each of Figures 18A to 18F. That is, A (1:2731) corresponds to the spectrum in Figure 18A. [Table 1]

[0132] From the results of Figures 18A to 18F, it was confirmed that the peak in the 700 to 800 nm region becomes larger depending on the amount of HPβCD added, and the peak position also shifts to the longer wavelength side (this tendency was also observed for "1:43846" not shown). This is because the addition of HPβCD increased the 14 This indicates that the dissolution of TEG8N has progressed. 14 is an amphiprotic molecule that can react with both acids and bases, and the shift in the absorption peak can be considered to represent the solubility in the solvent.

[0133] Specifically, TEG8N 14 The molar content ratio of HPβCD to the content of TEG8N (HPβCD / TEG8N 14 ) is 1.0 × 10 3 ~3.0×10 6 It was confirmed that a more excellent effect can be obtained when the upper limit is set to 2.0×10 or more. That is, when the upper limit is set to 2.0×10 or more, the peak in the 700-800 nm region becomes larger, and when the upper limit is set to 2.0×10 or less, HPβCD is easily dissolved, and the entire composition including the solvent tends to become more uniform. 6 It is more preferable that the ratio is less than or equal to the above.

[0134] From the same viewpoint as above, the content ratio is 3.0×10 3 ~2.0×10 6 is more preferable, and 1.0×10 4 ~2.0×10 6 More preferably, 5.0×10 4 ~2.0×10 6 is particularly preferred. In addition, the above experimental results show that in a composition containing a solvent, TEG8N 14 It was speculated that it was unlikely that HPβCD formed a one-to-one (or one-to-many) inclusion compound with HPβCD. It was confirmed that it was unlikely that the composition of the present disclosure exerted its function by forming an inclusion compound with HPβCD, and it was highly likely that the composition exerted its function by a different effect (effect as a surfactant).

[0135] (Effect of surfactant type) 19A to 19D show TEG8N 14 FIG. 19A shows the relationship between the type of surfactant added to the solution and the UV-vis (ultraviolet-visible) absorbance. 14 FIG. 19B shows the absorbance when TEG8N was added to ethanol-water (3:7) and an excess amount of α-cyclodextrin was added. 14 FIG. 19C shows the UV-visible absorbance when TEG8N was added to deionized water and an excess amount of β-cyclodextrin was added. 14 FIG. 19D shows the UV-visible absorbance when TEG8N was added to deionized water and an excess amount of gamma cyclodextrin was added. 14 1 is a graph showing absorbance when an excess amount of bovine serum albumin (BSA) is added.

[0136] As mentioned above, TEG8N 14 is an amphiprotic molecule that can react with both acids and bases, and the shift in the absorption peak can be considered to represent the solubility in the solvent. When β-cyclodextrin was used, the peak at 700-800 nm was larger than when α-cyclodextrin, γ-cyclodextrin, or BSA were used, so it was confirmed that β-cyclodextrin or its derivatives (HBβCD is a derivative of β-cyclodextrin) further increased the solubility.

[0137] On the other hand, even when α-cyclodextrin, γ-cyclodextrin, and BSA were used, a peak or shoulder was observed at 700 to 800 nm, and it was confirmed that the solubility was improved. In other words, it was confirmed that they are suitable as surfactants contained in the composition. Among them, α-cyclodextrin and γ-cyclodextrin showed higher solubility than BSA.

[0138] (Low magnification confocal microscope image) FIG. 20A shows TEG8N 14 , and TEG8N 14Low magnification confocal microscopy images of HepG2 incubated with -HPβCD. Six images are arranged in a 2 × 3 matrix. The first row (horizontal row) shows TEG8N 14 In the second row, the sample was TEG8N. 14 Images of the same field of view of a sample using -HPβCD are arranged. The first row (vertical) shows bright-field images, the second row shows fluorescent images (originally in red), and the third row shows an image in which the bright-field and fluorescent images are superimposed. The scale bar indicates 20 μm.

[0139] FIG. 20B shows the TEG8N of FIG. 14 FIG. 20C is a diagram showing the signal intensity along the arrow line in the fluorescent image of TEG8N in FIG. 14 The figure shows the signal intensity along the arrow line in the fluorescent image of -HPβCD. From the above results, it was confirmed that a uniform dye distribution was obtained even at low magnification and that the fluorescence was not autofluorescence.

[0140] (Consideration) Conventional fluorescent dyes have the following five major problems: First, to realize 3D imaging of live cells, there are few fluorescent dyes that can be used with excitation light in the near-infrared biological window with cell membrane permeability. Most conventional fluorescent dyes require excitation light of 650 nm or less, which results in a shallow penetration depth and false positives, limiting imaging to 2D.

[0141] Second, a major challenge in imaging cell organelles is the lack of suitable ratiometric fluorescent probes with pKa suitable for imaging in the physiological pH range.

[0142] Third, there is a lack of fluorescent probes with high photostability, low toxicity, and long retention time within organelles that can be used for real-time imaging of live cells. Most fluorescent dyes have poor intracellular retention time and photostability, limiting their application in long-term imaging, and can be degraded or bleached upon repeated exposure to excitation light in the UV-visible wavelength range. Low-energy near-infrared light, especially light with wavelengths of 650-900 nm, has lower phototoxicity to living cells than ultraviolet-visible light and is therefore a promising tool for real-time imaging of living cells.

[0143] Fourth, conventional fluorescent dyes have low solubility or do not emit in the required spectral range. Fifth, the fluorescent dyes aggregate and no longer emit light.

[0144] As shown in the examples, the compounds and compositions of the present disclosure solve all of the above problems in the following ways. First, the compounds absorb and emit in the NIR-I biologically transparent range and have low toxicity, making them suitable for live 3D imaging of cells.

[0145] Second, the compounds are amphiprotic (react with acids and bases and selectively react at low and high pH), and therefore have pKa suitable for imaging applications and respond in the physiological pH range, making them suitable as ratiometric fluorescent probes.

[0146] Third, the above compounds and compositions have properties (low toxicity, long intracellular residence time, high photostability) that make them suitable for use in combination with NIR light for long-term 3D imaging of living cells.

[0147] Fourth, the linear fused pyrazine unit that constitutes the above compounds has suitable properties as efficient NIR emitting materials for fluorescence imaging in three spatial axes, high water solubility, more sensitive detection of pH in the NIR region, and reliable monitoring of multiple signaling activities in live cells for long-term imaging.

[0148] Fifth, the composition acts as an effective, low-toxicity carrier system, reducing aggregation of the fluorescent dye agent and delivering the fluorescent dye agent to the desired location.

[0149] The compounds and compositions containing them are shown to be a new class of highly efficient NIR emitting materials for continuous real-time imaging of live cells. These fluorescent dyes can be applied not only for imaging but also for more accurate and sensitive detection of pH in the near-infrared biological range.

[0150] Previous ratiometric fluorescent probes for pH imaging, such as BCECF and SNARF-1, have neutral pKa values ​​and are susceptible to photobleaching, which can limit their effectiveness and accuracy in long-term imaging studies. Meanwhile, the compounds mentioned above are based on extended pyrazinacenes and are specifically designed to be highly efficient and stable, allowing for more reliable monitoring of multiple signaling activities in living cells over long periods of time. This increased sensitivity and stability will enable researchers to gain a deeper understanding of the complex molecular interactions and reactions involved in signaling cascades.

[0151] Another important technical benefit of the development of extended pyrazinacene-based materials for fluorescence imaging is the ability to image in three spatial axes (xyz), providing a more comprehensive understanding of the spatial organization of signaling pathways in live cells. Using these compounds and compositions containing them, researchers can create more detailed and precise signaling maps that visualize the precise coordination of the molecular components involved in these pathways. This increased resolution allows researchers to better understand how different molecules and enzymes interact in space and time, providing important insights into the complex processes that govern cellular function and behavior.

[0152] Finally, the development of extended pyrazinacene-based materials for continuous real-time imaging of living cells has important technical implications for a wide range of research fields, including cell biology, neuroscience, and cancer biology. These materials enable researchers to gain a better understanding of the cellular mechanisms underlying a wide range of biological processes, from the control of gene expression to the propagation of action potentials within cells. A better understanding of the fundamental mechanisms of cellular function will have important implications for the development of novel therapeutics and interventions for a variety of diseases and disorders. Ultimately, the technical impact of the development of these compounds and compositions is far-reaching, providing researchers with powerful new tools to investigate the complex and fascinating world of living cells, and is transformative in enabling 3D imaging due to its deep penetrating capabilities.

[0153] The economic impact of this development is also noteworthy. The ability to use these new materials to continuously image live cells in real time with high sensitivity and precision will have a major impact on the field of biological research. Providing a more efficient and reliable way to monitor multiple signaling activities in live cells over time will enable researchers to better understand biological processes and develop new treatments for diseases. This, in turn, will have a positive impact on the healthcare industry, leading to improved health outcomes for patients and potentially reducing medical costs in the long run. Furthermore, the development of these new materials may also have economic implications for the materials science industry. The ability to construct highly efficient near-infrared emitting materials based on extended pyrazinacenes could lead to the development of new materials with unique optical properties and applications beyond fluorescence imaging. These materials could be used in a variety of hospitals and industries, creating new opportunities for innovation and economic growth.

[0154] The above compounds and compositions, which are a new class of highly efficient near-infrared emitting materials based on extended pyrazinacenes developed for intracellular imaging, offer several advantages over existing fluorescent dyes. First, extended pyrazinacenes have improved fluorescence properties compared to existing fluorescent dyes. The highly conjugated aromatic system of pyrazinacenes improves fluorescence properties and provides brighter and more stable fluorescent signals, enabling long-term imaging within specific organelles. In addition, the use of near-infrared wavelengths reduces interference from cellular autofluorescence, making the fluorescent signal easier to distinguish from background noise in cells and culture media.

[0155] Second, pyrazinacenes are more sensitive to pH changes in the intracellular environment than existing fluorescent dyes. (5-8) This is because the presence of multiple pyrazine units in their structure allows for greater interaction with the surrounding environment. This sensitivity to pH changes is crucial for studying many cellular processes, including a wide range of diseases, enzyme activity, and cell signaling.

[0156] Third, extended pyrazinacene has a longer retention time in live cells compared to existing fluorescent dyes. One of the main challenges in intracellular imaging is photobleaching, where dyes lose fluorescence after repeated excitation with light. This leads to loss of signal over time, making it difficult to obtain reliable long-term imaging data. However, pyrazinacene has been shown to retain its fluorescence even after multiple excitations. This allows for more accurate and reliable imaging data to be obtained over a longer period of time.

[0157] In summary, the use of extended pyrazinacenes for intracellular imaging offers significant advantages compared to existing fluorescent dyes, including improved fluorescence properties in the NIR region, increased sensitivity to pH changes, and improved photostability and retention time in live cells. Furthermore, pyrazinacene-based probes can be used for multiplex imaging to simultaneously monitor different cellular processes using multiple probes, which can provide a more comprehensive understanding of the complex signaling networks within cells.

Claims

1. A compound represented by formula 1. 【Chemistry 1】 (In formula 1, R １ , R ２ , R ３ , and R ４ each independently represents a group containing a hydrophilic group, and may be the same or different; x and y each represent 0, 1, 2, or 3, and x and y are the same.

2. The compound according to claim 1 , wherein the hydrophilic group is a nonionic group.

3. The compound according to claim 1 , wherein the hydrophilic group is a cationic group or an anionic group.

4. The compound of claim 1 , wherein the hydrophilic group comprises a (poly)oxyalkylene group.

5. The compound according to any one of claims 1 to 4, wherein the group containing a hydrophilic group is a group represented by the following formula 2: 【Chemistry 2】 (In formula 2, Z represents the hydrophilic group, n represents a number from 1 to 5, and * represents a bonding position.)

6. A composition comprising the compound according to any one of claims 1 to 5 and a surfactant.

7. The molar ratio of the content of the surfactant to the content of the compound is 1.0×10 ３ That's it, 3.0 x 10 ６ 7. The composition of claim 6, wherein:

8. 8. The composition of claim 6 or 7, wherein the surfactant comprises a cyclodextrin or a derivative thereof.

9. 8. The composition of claim 6 or 7, wherein the surfactant comprises a hydroxyalkylated cyclodextrin.

10. A fluorescent dye agent comprising the compound according to any one of claims 1 to 5 or the composition according to any one of claims 6 to 9.

11. 6. A fluorescent dye agent kit comprising a first agent containing the compound according to claim 1 and a second agent containing a surfactant.

12. A detection method comprising staining a cell, tissue, or organ with the fluorescent dye agent according to claim 10.

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