JP6958781B2 - Fluorescent compound or salt thereof, ionic compound detection agent and ionic compound detection method - Google Patents

Description

The present invention relates to a fluorescent compound or a salt thereof, a detection agent for an ionic compound, and a method for detecting an ionic compound.

For example, analysis of various biomolecules in clinical tests and development of new drugs, quantitative / semi-quantitative analysis of ionic compounds in environmental water such as rivers, tap water, industrial water, etc. There is a demand for highly sensitive microanalysis.

Fluorescence analysis is used as a means for highly sensitive microanalysis of a specific compound, and a fluorescent compound for that purpose has also been devised.

For example, a ruthenium-bipyridyl polyaza compound or the like has been proposed as a fluorescent compound for analyzing a phosphate ion (phosphate group) that plays an important role in a living body such as control of information transmission in a signal transduction system. (See, for example, Patent Document 1 and Non-Patent Document 1).

In addition, various compounds containing a phenylboronic acid structure have been devised as fluorescent compounds for detecting and measuring glucose and other sugars, which are known as diagnostic markers for diabetes (for example, patents). See Documents 2-4).

^{In addition, NAD +} / NADH and NADP ⁺ / NADPH, which function as coenzymes in many redox reactions in the living body, are used for the purpose of investigating the product and enzyme activity of the redox reaction, for example, fluorescence of NADH. May be measured (see, for example, Patent Document 5).

Most of the conventional fluorescent compounds that detect and measure a specific compound as exemplified above are based on the fact that the fluorescence intensity gradually changes according to the concentration of the substance to be detected in the solution, and the fluorescence intensity is low. It is known that there is no fluorescence in the concentration range, and on the other hand, when the concentration is high, aggregates may be formed and the light may not be emitted (quenched).

On the other hand, recently, a phenomenon called agglutination-induced emission (AIE) in which fluorescence increases with aggregation has been found (see, for example, Non-Patent Documents 2 and 3). It is expected that this AIE can be used for a new type of fluorescence analysis that has never been seen before, but there are very few examples of fluorescent compounds showing AIE.

Japanese Patent No. 4138331 Japanese Patent No. 2799837 Japanese Patent No. 2883824 Japanese Patent No. 2889476 Japanese Unexamined Patent Publication No. 2-265500

Beer P. D. and Gale P. A., Anion Recognition and Sensing: The State of the Art and Future Perspectives., Angew. Chem. Int. Ed. Engl. 40 (3), 486-516, 2001. Hong Y., et al., Aggregation-induced emission., Chem. Soc. Rev. 40 (11), 5361-5388, 2011. Wu J., et al., New sensing mechanisms for design of fluorescent chemosensors emerging in recent years., Chem. Soc. Rev., 40 (7), 3483-3495, 2011.

Therefore, an object of the present invention is to provide a new fluorescent compound exhibiting aggregation-induced luminescence (AIE). It is also an object of the present invention to provide a detection agent for an ionic compound.

［式（１）中、Ｒ^１はそれぞれ独立に、水素原子又はシアノ基、ハロゲン原子若しくはフェニル基を表し、Ｒ^２はそれぞれ独立に、存在しないか又はハロゲン原子、炭素数１〜３のアルキル基、アミノ基若しくは炭素数１〜３のアルコキシ基を表し、Ｒ^３はそれぞれ独立に、存在しないか又は−Ｏ−、−Ｓ−、−ＮＨ−、−ＣＯ−若しくは−ＣＯＮＨ−で中断されていてもよい炭素数１〜２０のアルキレン基を表し、Ｘはそれぞれ独立に、存在しないか又はカチオン性基若しくはアニオン性基を表し、Ｒ^４はそれぞれ独立に、存在しないか又は−Ｏ−、−Ｓ−、−ＮＨ−、−ＣＯ−、−ＣＯＮＨ−で中断されていてもよい炭素数１〜２０のアルキル基若しくは炭素数１〜２０のアルコール基若しくは−Ｎ（Ｒ^５）_２（ここで、Ｒ^５はそれぞれ独立に、水素原子又は−Ｏ−、−Ｓ−、−ＮＨ−、−ＣＯ−、−ＣＯＮＨ−で中断されていてもよい炭素数１〜２０のアルキル基を表す。）を表す。ｐはそれぞれ独立に０〜４の整数を表し、ｑはそれぞれ独立に１〜５の整数を表し、少なくとも１つのＸはカチオン性基又はアニオン性基である。]
［２］下記式（Ｐ１）で表される、［１］に記載の発蛍光性化合物又はその塩。

［式（１）中、Ｒ^１はそれぞれ独立に、水素原子又はシアノ基、ハロゲン原子若しくはフェニル基を表し、Ｒ^２はそれぞれ独立に、存在しないか又はハロゲン原子、炭素数１〜３のアルキル基、アミノ基若しくはアルコキシ基を表し、Ｒ^３はそれぞれ独立に−Ｏ−、−Ｓ−、−ＮＨ−、−ＣＯ−又は−ＣＯＮＨ−で中断されていてもよい炭素数１〜１０のアルキレン基を表し、Ｘはカチオン性基又はアニオン性基を表し、Ｒ^４はそれぞれ独立に、存在しないか又は−Ｏ−、−Ｓ−、−ＮＨ−、−ＣＯ−、−ＣＯＮＨ−で中断されていてもよい炭素数１〜２０のアルキル基、アルコール基若しくはエチレンオキシド基を表す。ｐはそれぞれ独立に０〜４の整数を表し、ｑはそれぞれ独立に１〜５の整数を表す。]
［３］［１］又は［２］に記載の発蛍光性化合物又はその塩を有効成分とする、イオン性化合物の検出剤。
［４］前記式（１）又は前記式（Ｐ１）におけるＸがカチオン性基であり、前記イオン性化合物がアニオン性化合物である、［３］に記載のイオン性化合物の検出剤。
［５］溶媒と、被検試料と、［１］又は［２］に記載の発蛍光性化合物又はその塩とを含む混合液を調製する工程と、前記混合液の蛍光を検出する工程と、を備える、被検試料中のイオン性化合物の検出方法。 The present invention is as follows.
[1] A fluorescent compound represented by the following formula (1) or a salt thereof.

[In formula (1), R ¹ independently represents a hydrogen atom or a cyano group, a halogen atom or a phenyl group, and R ² is independently absent or an alkyl group having a halogen atom and 1 to 3 carbon atoms. , Amino group or alkoxy group having 1-3 carbon atoms, and R ³ is independently absent or interrupted by -O-, -S-, -NH-, -CO- or -CONH-. represents an even alkylene group having a carbon number of 1 to 20, X is independently, represent an absent or a cationic group or an anionic group, R ⁴ are each independently absent or -O -, - S -, -NH-, -CO-, -CONH- may be interrupted by an alkyl group having 1 to 20 carbon atoms or an alcohol group having 1 to 20 carbon atoms or -N (R ⁵ ) ₂ (where R ^{Each of 5} independently represents an alkyl group having 1 to 20 carbon atoms which may be interrupted by a hydrogen atom or -O-, -S-, -NH-, -CO-, -CONH-). p independently represents an integer of 0 to 4, q independently represents an integer of 1 to 5, and at least one X is a cationic group or an anionic group. ]
[2] The fluorescent compound or salt thereof according to [1], which is represented by the following formula (P1).

[In the formula (1), R ¹ independently represents a hydrogen atom or a cyano group, a halogen atom or a phenyl group, and R ² is independently absent or an alkyl group having a halogen atom and 1 to 3 carbon atoms. , Amino group or alkoxy group, and R ³ is an alkylene group having 1 to 10 carbon atoms which may be independently interrupted by -O-, -S-, -NH-, -CO- or -CONH-. represents, X represents a cationic group or an anionic group, each ^{R 4} is independently absent or -O -, - S -, - NH -, - CO -, - CONH- be interrupted by It represents a good alkyl group having 1 to 20 carbon atoms, an alcohol group or an ethylene oxide group. p independently represents an integer of 0 to 4, and q independently represents an integer of 1 to 5. ]
[3] An ionic compound detection agent containing the fluorescent compound according to [1] or [2] or a salt thereof as an active ingredient.
[4] The agent for detecting an ionic compound according to [3], wherein X in the formula (1) or the formula (P1) is a cationic group, and the ionic compound is an anionic compound.
[5] A step of preparing a mixed solution containing a solvent, a test sample, and the fluorescent compound according to [1] or [2] or a salt thereof, a step of detecting fluorescence of the mixed solution, and a step of detecting fluorescence of the mixed solution. A method for detecting an ionic compound in a test sample.

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide a novel fluorescent compound exhibiting aggregation-induced luminescence (AIE). In addition, an agent for detecting an ionic compound can be provided.

(A) is a photograph showing the result of Experimental Example 7. (B) is a graph showing the results of Experimental Example 7. (A) and (c) are graphs showing the results of Experimental Example 8. (B) and (d) are photographs showing the results of Experimental Example 8. (A) and (b) are graphs showing the results of Experimental Example 9. (A) and (b) are graphs showing the results of Experimental Example 10. It is a graph which shows the result of Experimental Example 11. It is a graph which shows the result of Experimental Example 11. (A) is a graph and a photograph showing the results of Experimental Example 12. (B) is a graph showing the results of Experimental Example 12. (A) is a graph and a photograph showing the results of Experimental Example 13. (B) is a graph showing the results of Experimental Example 13. (A) is a graph and a photograph showing the results of Experimental Example 14. (B) is a graph showing the results of Experimental Example 14. (A) and (b) are photographs showing the results of Experimental Example 15. It is a graph which shows the result of Experimental Example 16. (A) is a photograph showing the result of Experimental Example 17. (B) is a graph showing the results of Experimental Example 17. (A) is a photograph showing the result of Experimental Example 18. (B) is a graph showing the results of Experimental Example 18. (A) is a photograph showing the result of Experimental Example 19. (B) is a graph showing the results of Experimental Example 19. (A) and (b) are graphs showing the results of Experimental Example 20. (A) is a schematic diagram of a J-aggregate. (B) is a schematic diagram of an H-aggregate. (A) is a photograph showing the result of Experimental Example 21. (B) is a graph showing the results of Experimental Example 21. (A) is a photograph showing the result of Experimental Example 22. (B) and (c) are graphs showing the results of Experimental Example 22.

[Fluorescent compound]
In one embodiment, the present invention provides a fluorescent compound represented by the following formula (1) or a salt thereof.

In formula (1), R ¹ independently represents a hydrogen atom or a cyano group, a halogen atom or a phenyl group, and R ² is an independent or non-existent or halogen atom, an alkyl group having 1 to 3 carbon atoms, respectively. an amino group or an alkoxy group having 1 to 3 carbon atoms, ^{R 3} are each independently absent or -O -, - S -, - NH -, - CO- or -CONH- be interrupted by represents an alkylene group having a carbon number of 1 to 20, X is independently, represent an absent or a cationic group or an anionic group, R ⁴ are each independently absent or -O -, - S- , -NH-, -CO-, -CONH-, an alkyl group having 1 to 20 carbon atoms or an alcohol group having 1 to 20 carbon atoms or -N (R ⁵ ) ₂ (here, R ^5). Represent each independently as a hydrogen atom or an alkyl group having 1 to 20 carbon atoms which may be interrupted by a hydrogen atom or -O-, -S-, -NH-, -CO-, -CONH-). p independently represents an integer of 0 to 4, q independently represents an integer of 1 to 5, and at least one X is a cationic group or an anionic group.

In the formula (1), the ^{alkylene group represented by R 3} may have a branched chain. Further, the number of carbon atoms of the branched chain may be 1 to 10. Examples of the branched chain include a group derived from the side chain of an amino acid, and examples thereof include a methyl group and a benzyl group.

As shown in Examples described later, the fluorescent compound of this embodiment or a salt thereof exhibits aggregation-induced emission (AIE) in the presence of the ionic compound and does not fluoresce in the absence of the ionic compound. ..

In the fluorescent compound of the present embodiment or a salt thereof, when X in the above formula (1) is a cationic group, it exhibits AIE in the presence of an anionic compound and fluoresces. When X in the above formula (1) is an anionic group, it exhibits AIE in the presence of a cationic compound and emits fluorescence.

Examples of the above-mentioned cationic group include groups represented by the following formulas (2) to (4).

Further, examples of the above-mentioned anionic group include a phosphoric acid group, a carboxylic acid group, a sulfuric acid group and the like.

In the fluorescent compound of the present embodiment or a salt thereof, R ¹ represented by the above formula (1) is preferably a cyano group. Further, it is preferable that ^{R 2} represented by the above formula (1) does not exist. Further, X represented by the above formula (1) is preferably a cationic group. Further, q represented by the above formula (1) is preferably 1 to 3, and more preferably 1.

The type of salt of the fluorescent compound of the present embodiment is not particularly limited as long as the effects of the present invention can be obtained. Specifically, when X in the above formula (1) is a cationic group, for example, a salt with an inorganic acid such as sulfuric acid or nitrate, for example, a salt with a halogen atom such as chlorine, bromine or iodine, for example. Examples thereof include salts with organic acids such as acetic acid, trifluoroacetic acid, citric acid, gluconic acid, propionic acid and pantothenic acid. When X in the above formula (1) is an anionic group, for example, a salt with a metal such as an alkali metal or an alkaline earth metal, a salt with ammonia, an organic amine such as tetrabutylammonium, or an organic ammonium. And salt and the like.

[Detective agent for ionic compounds]
In one embodiment, the present invention provides a detection agent for an ionic compound containing the above-mentioned fluorescent compound or a salt thereof as an active ingredient.

As shown in Examples described later, the above-mentioned fluorescent compound or salt thereof fluoresces in the presence of the ionic compound and does not fluoresce in the absence of the ionic compound. Therefore, by using the detection agent of the present embodiment, it is possible to detect an ionic compound in a biological sample or an environment-derived sample.

Examples of the ionic compound that can be detected by the detection agent of the present embodiment include an anionic compound and a cationic compound.

Examples of the anionic compound include carboxylic acid compounds such as monocarboxylic acid, dicarboxylic acid, tricarboxylic acid and polycarboxylic acid (humic acid and the like); phosphoric acid compounds such as phosphoric acid, oligophosphate and polyphosphate; oligonucleotides. , Polynucleotide, deoxyribonucleic acid, ribonucleic acid, nicotinamide adenine dinucleotide phosphate (oxidized (NADP ⁺ ) or reduced (NADPH)), nicotinamide adenin dinucleotide (oxidized (NAD ⁺ ) or reduced (NADH)) ) And the like; anionic polysaccharides such as heparin, chondroitin sulfate, hyaluronic acid; anionic peptides, proteins and the like.

On the other hand, examples of the cationic compound include metal ions such as sodium ion and calcium ion; amine compounds such as polyamines; cationic peptides and proteins.

As will be described later in the examples, for example, by using a fluorescent compound or a salt thereof in which X represented by the above formula (1) is a cationic group, anions such as dicarboxylic acids, nucleotides, and anionic polysaccharides are used. It is possible to detect sex compounds.

For example, dicarboxylic acids are also intermediates produced in metabolic pathways such as the citric acid cycle. The dicarboxylic acid in the body, which is increased due to metabolic dysfunction, is finally excreted in the urine. Therefore, the high-concentration dicarboxylic acid group in urine serves as a marker for metabolic diseases, and a fluorescent compound (fluorescent probe, fluorescent sensor) that detects this high-concentration dicarboxylic acid group in a turn-on or switch-on manner is used as a screening test for metabolic diseases. The inspection can be simplified by using the above.

In the present specification, the turn-on type means that the fluorescent compound linearly switches from a non-fluorescent state or a state in which a negligibly weak fluorescence is emitted to a fluorescent state depending on the concentration of the compound to be detected. do. The switch-on type means that the fluorescent compound switches from a non-fluorescent state or a state in which a negligibly weak fluorescence is emitted to a fluorescent state when the concentration of the detection target compound exceeds a predetermined threshold value. Means.

[Method for detecting ionic compounds]
In one embodiment, the present invention comprises a step of preparing a mixed solution containing a solvent, a test sample, and the above-mentioned fluorinated compound or a salt thereof, and a step of detecting the fluorescence of the mixed solution. , Provide a method for detecting an ionic compound in a test sample.

In the detection method of the present embodiment, the solvent may be a liquid derived from the test sample, a liquid in which a fluorescent compound or a salt thereof is dissolved, the test sample, the fluorescent compound, or the like. It may be a liquid prepared separately from the salt of the fluorescent compound.

Moreover, it is preferable that the above-mentioned mixed solution has a pH buffering action. The pH of the solvent may be appropriately adjusted according to the intended purpose, and may be, for example, neutral, for example, acidic, or basic.

The test sample is not particularly limited, and is a biological sample such as blood, plasma, serum, urine, biological tissue, cultured cells, cultured microorganisms, cultured cells or culture medium of cultured microorganisms; water collected from a river, tap water. , Samples of industrial water and the like. Further, examples of the ionic compound to be detected include those described above.

Specifically, the fluorescence can be detected by irradiating the above-mentioned mixed solution with excitation light and detecting the generated fluorescence. The fluorescence of the mixed solution may be detected by, for example, the naked eye, or by measurement using a spectrophotometer, for example. When the fluorescence is detected with the naked eye, for example, the excitation light may be irradiated using a UV handy lamp or the like as a light source.

As will be described later in the examples, for example, the fluorescence intensity of the fluorescent compound or its salt in the presence of the test sample is compared with the fluorescence intensity of the fluorescent compound or its salt in the absence of the test sample. When the fluorescence intensity increases, it can be determined that the ionic compound is present in the test sample. In addition, the concentration of the ionic compound can be quantified by the degree of increase in fluorescence intensity.

Alternatively, as will be described later in the examples, the spectroscopic characteristics of the fluorescent compound or its salt in the presence of the test sample are the components of the fluorescent compound or its salt in the absence of the test sample. It can be determined that the ionic compound is present in the test sample when it changes from the optical characteristics. Examples of changes in spectroscopic characteristics include changes in fluorescence wavelength and changes in absorption characteristics. The change in spectroscopic characteristics may be detected by, for example, the naked eye, or by measurement using a spectrophotometer, for example.

Further, as will be described later in the examples, the type of the ionic compound in the test sample can be specified based on the change in the spectroscopic characteristics.

Next, the present invention will be described in more detail with reference to experimental examples, but the present invention is not limited to the following experimental examples.

[Experimental Example 1]
The fluorescent compound of Example 1 (hereinafter, may be referred to as “OPV-G”) was synthesized according to the following synthesis scheme 1.

(Synthesis of Compound 2)
2- (2-aminoethoxy) etherol1 (16.0 g, 152 mmol) was dissolved in dry dichloromethane (120 mL) and Boc ₂ O (33.2 g, 150 mmol) was added thereto. The reaction mixture was stirred at room temperature for 3 hours, and then the solvent was evaporated under reduced pressure. The crude product was purified by silica gel column chromatography to obtain 25.6 g of Compound 2 as a colorless oily product. Yield 82%.

^{1 1} H NMR (300 MHz, CDCl ₃ ): δ4.92 (br-s, 1H), 3.74 (m, 2H), 3.56 (m, 2H), 3.34 (m, 2H), 2. 14 (s, 1H), 1.45 (s, 9H).

(Synthesis of Compound 3)
Compound 2 (25.6 g, 125 mmol) and carbon tetrabromide (41.4 g, 125 mmol) were dissolved in dry dichloromethane (200 mL), to which triphenylphosphine (32.8 g, 125 mmol) was added. The reaction mixture was stirred at room temperature for 22 hours, and then the solvent was evaporated under reduced pressure. The crude product was purified by silica gel column chromatography to obtain 28.6 g of Compound 3 as a colorless solid. Yield 85%.

^{1 1} H NMR (300 MHz, CDCl ₃ ): δ4.91 (br-s, 1H), 3.76 (m, 2H), 3.55 (m, 2H), 3.47 (m, 2H), 3. 33 (m, 2H), 1.45 (s, 9H).

(Synthesis of Compound 4)
P-Hydroxybenzaldehyde (1.82 g, 14.9 mmol), potassium carbonate (4.12 g, 29.8 mmol) and compound 3 (4.00 g, 14.9 mmol) were suspended in dry DMF (20 mL) at 65 ° C. Was heated for 6 hours. After allowing to cool to room temperature, ethyl acetate (200 mL) was added, and the mixture was washed in the order of water and saturated brine. The organic phase was dried over anhydrous magnesium sulfate, and the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to obtain 3.97 g of Compound 4 as a colorless oily product. Yield 86%.

^{1 1} H NMR (300 MHz, CDCl ₃ ): δ9.89 (s, 1H), 7.84 (d, J = 8.9 Hz, 2H), 7.04 (d, J = 8.9 Hz, 2H), 4 .96 (br-s, 1H), 4.21 (m, 2H), 3.86 (m, 2H), 3.62 (t, J = 5.2Hz, 2H), 3.35 (q, J) = 5.2Hz, 2H), 1.44 (s, 9H).

(Synthesis of Compound 5)
Compound 4 (3.97 g, 12.8 mmol) and xylylene dicyanide (1.00 g, 6.42 mmol) were dissolved in dry ethanol (50 mL). Five drops of Bu ₄ NOW (40% in water) were added thereto, and the mixture was heated under reflux for 3 hours. The obtained precipitate was collected and washed with ethanol to obtain 4.28 g of Compound 5 as a yellow solid. Yield 90%.

^{1 1} H NMR (300 MHz, CDCl ₃ ): δ7.92 (d, J = 8.9 Hz, 4H), 7.72 (s, 4H), 7.52 (s, 2H), 7.02 (d, J) = 8.9Hz, 4H), 4.96 (s, 2H), 4.20 (m, 4H), 3.86 (m, 4H), 3.63 (t, J = 5.1Hz, 4H), 3.36 (q, J = 5.1Hz, 4H), 1.45 (s, 18H).

(Synthesis of Compound 6)
Compound 5 (3.0 g, 4.06 mmol) was suspended in dry dichloromethane (15 mL) and trifluoroacetic acid (10 mL, 130 mmol) was added thereto. After stirring the reaction mixture at room temperature for 21 hours, dry diethyl ether was added to obtain 2.99 g of Compound 6 as a yellow solid. Yield 96%.

^{1 1} H NMR (300 MHz, DMSO-d ₆ ): δ8.09 (s, 2H), 8.00 (d, J = 9.0 Hz, 4H), 7.87 (s, 4H), 7.84 (s) , 6H), 7.16 (d, J = 9.0Hz, 4H), 4.25 (m, 4H), 3.84 (m, 4H), 3.68 (t, J = 5.2Hz, 4H) ), 3.03 (t, J = 5.2Hz, 4H).

(Synthesis of Compound 7)
Compound 6 (1.53 g, 2.00 mmol) was dissolved in dry dichloromethane (25 mL) with triethylamine (0.61 mL, 4.40 mmol) and 1-H-pyrazole-1- (N, N'-bis (tert). -Butyloxycarbonyl)) carboxamide (1.86 g, 6.00 mmol) was added. The reaction mixture was stirred at room temperature for 3 days. Then, chloroform (100 mL) was added, and the mixture was washed with water and saturated brine in this order. The organic phase was dried over anhydrous magnesium sulfate, and the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to obtain 1.40 g of Compound 7 as a yellow solid. Yield 68%.

^{1 1} H NMR (300 MHz, CDCl ₃ ): δ11.49 (s, 2H), 8.69 (br-s, 2H), 7.91 (d, J = 8.9 Hz, 4H), 7.72 (s) , 4H), 7.52 (s, 2H), 7.03 (d, J = 8.9Hz, 4H), 4.22 (m, 4H), 3.89 (m, 4H), 3.70 ( m, 8H), 1.51 (s, 18H), 1.49 (s, 18H).

(Synthesis of OPV-G (Example 1))
Compound 7 (1.27 g, 1.24 mmol) was suspended in dry dichloromethane (15 mL) and trifluoroacetic acid (5.0 mL, 65.0 mmol) was added thereto. After stirring the reaction mixture at room temperature for 17 hours, dry diethyl ether was added to obtain 1.00 g of OPV-G as a yellow solid. Yield 95%.

^{1 1} H NMR (300 MHz, DMSO-d ₆ ): δ8.09 (s, 2H), 8.00 (d, J = 8.9 Hz, 4H), 7.87 (s, 4H), 7.60 (s) , 2H), 7.15 (d, J = 8.9Hz, 4H), 4.23 (m, 4H), 3.82 (m, 4H), 3.59 (t, J = 5.2Hz, 4H) ), 3.32 (t, J = 5.2Hz, 4H).
^{MS (MALDI): m / z} = 623.37 ([M-H + -2CF 3 COO -] +).
calcd (%) for C ₃₈ H ₄₀ F ₆ N ₈ O ₈ (OPV-G): C53.65, H4.74, N13.17; found: C53.51, H4.82, N13.11.

[Experimental Example 2]
The fluorescent compounds of Examples 2 to 7 were synthesized according to the following synthesis scheme 2. Hereinafter, the compounds of Examples 2 to 7 are referred to as "OPV-A" (Example 2), "OPV-ImMe" (Example 3), "OPV-ImC2OMe" (Example 4), and "OPV-ImC2OH", respectively. It may be referred to as (Example 5), "OPV-ImDEO" (Example 6), or "OPV-ImTEO" (Example 7).

(Synthesis of Compound 8)
P-Hydroxybenzaldehyde (4.69 g, 38.4 mmol) and xyllenedicyanide (3.00 g, 19.2 mmol) were dissolved in dry ethanol (50 mL). Acetic acid (3.0 mL) and piperidine (5.0 mL) were added thereto, and the mixture was heated under reflux for 20 hours. After recovering the obtained precipitate, it was washed with ethanol to obtain 5.84 g of Compound 8 as a yellow solid. Yield 83%.

(Synthesis of Compound 9)
Compound 8 (2.00 g, 5.49 mmol), potassium carbonate (3.00 g, 21.7 mmol) and 1,1'-Oxybis (2-bromoethane) (12.8 g, 55.2 mmol) in dry acetone (50 mL). It was suspended in 1 and refluxed by heating at 65 ° C. for 6 hours. After allowing to cool to room temperature, the solid matter was filtered off, and the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to obtain 1.57 g of Compound 9 as a yellow solid. Yield 43%.

^{1 1} H NMR (400 MHz, CDCl ₃ ): δ7.91 (d, J = 9.0 Hz, 4H), 7.72 (s, 4H), 7.52 (s, 2H), 7.02 (d, J) = 9.0Hz, 4H), 4.22 (m, 4H), 3.93-3.89 (m, 8H), 3.51 (t, J = 6.4Hz, 4H).

(Synthesis of OPV-A, OPV-ImMe, OPV-ImC2OMe, OPV-ImC2OH, OPV-ImDEO, OPV-ImTEO)
Compound 9 (350 mg, 0.53 mmol) was dissolved in a mixed solvent (15 mL) of acetonitrile-ethanol (2: 1, v / v), and 10 equal amounts of an amine compound or an imidazole compound was added thereto. The reaction mixture was heated to reflux for 24 hours. After allowing to cool to room temperature, the precipitated solid was collected by filtration to obtain a yellow solid. Yield 44-84%.

<< OPV-A (Example 2) >>
^{1 1} H NMR (400 MHz, DMSO-d ₆ ): δ8.09 (s, 2H), 7.99 (d, J = 9.0 Hz, 4H), 7.87 (s, 4H), 7.15 (d) , J = 9.0Hz, 4H), 5.27 (t, J = 5.0Hz, 2H), 4.25 (m, 4H), 3.94 (br-s, 4H), 3.84 (br) -T, 8H), 3.63 (m, 4H), 3.48 (m, 4H), 3.13 (s, 12H).
calcd (%) forC ₄₀ H ₅₂ Br ₂ N ₄ O ₆ (OPV-A): C56.88, H6.21, N6.63; found: C56.62, H6.18, N6.62.

<< OPV-IMMe (Example 3) >>
^{1 1} H NMR (400 MHz, DMSO-d ₆ ): δ9.09 (s, 2H), 8.09 (s, 2H), 7.99 (d, J = 9.0 Hz, 4H), 7.87 (s) , 4H), 7.75 (t, J = 1.8Hz, 2H), 7.70 (t, J = 1.8Hz, 2H), 7.12 (d, J = 9.0Hz, 4H), 4 .39 (t, J = 5.0Hz, 4H), 4.20 (m, 4H), 3.86 (t, J = 5.0Hz, 4H), 3.84 (s, 6H), 3.81 (M, 4H).
calcd (%) forC ₄₀ H ₄₂ Br ₂ N ₆ O ₄ (OPV-IMMe): C57.84, H5.10, N10.12; found: C57.94, H5.13, N10.04.

<< OPV-ImC2OMe (Example 4) >>
^{1 1} H NMR (400 MHz, DMSO-d ₆ ): δ9.14 (s, 2H), 8.09 (s, 2H), 7.99 (d, J = 9.2 Hz, 4H), 7.87 (s) , 4H), 7.77 (t, J = 1.8Hz, 2H), 7.76 (t, J = 1.8Hz, 2H), 7.12 (d, J = 9.2Hz, 4H), 4 .42 (t, J = 5.2Hz, 4H), 4.36 (t, J = 5.0Hz, 4H), 4.19 (m, 4H), 3.87 (t, J = 5.2Hz, 4H), 3.81 (m, 4H), 3.68 (t, J = 5.0Hz, 4H), 3.25 (s, 6H).
calcd (%) forC ₄₄ H ₅₀ Br ₂ N ₆ O ₆ (OPV-ImC2OMe): C57.52, H5.49, N9.15; found: C57.32, H5.44, N9.12.

<< OPV-ImC2OH (Example 5) >>
^{1 1} H NMR (400 MHz, DMSO-d ₆ ): δ9.13 (s, 2H), 8.09 (s, 2H), 7.99 (d, J = 9.2 Hz, 4H), 7.87 (s) , 4H), 7.76 (t, J = 1.8Hz, 2H), 7.75 (t, J = 1.8Hz, 2H), 7.12 (d, J = 9.2Hz, 4H), 5 .18 (t, J = 5.3Hz, 2H), 4.42 (t, J = 5.2Hz, 4H), 4.23-4.19 (m, 8H), 3.87 (t, J = 5.2Hz, 4H), 3.82 (m, 4H), 3.72 (q, J = 5.3Hz, 4H).
calcd (%) forC ₄₂ H ₄₆ Br ₂ N ₆ O ₆ (OPV-ImC2OH): C56.64, H5.21, N9.44; found: C56.66, H5.24, N9.42.

<< OPV-ImDEO (Example 6) >>
^{1 1} H NMR (400 MHz, DMSO-d ₆ ): δ9.12 (s, 2H), 8.08 (s, 2H), 7.99 (d, J = 8.8 Hz, 4H), 7.87 (s) , 4H), 7.77 (t, J = 1.8Hz, 2H), 7.75 (t, J = 1.8Hz, 2H), 7.12 (d, J = 8.8Hz, 4H), 4 .42 (t, J = 5.0Hz, 4H), 4.35 (t, J = 5.0Hz, 4H), 4.19 (m, 4H), 3.87 (t, J = 5.0Hz, 4H), 3.81 (m, 4H), 3.76 (t, J = 5.0Hz, 4H), 3.53 (m, 4H), 3.40 (m, 4H), 3.21 (s) , 6H).
calcd (%) forC ₄₈ H ₅₈ Br ₂ N ₆ O ₈ (OPV-ImDEO): C57.26, H5.81, N8.35; found: C57.22, H5.87, N8.21.

<< OPV-ImTEO (Example 7) >>
^{1 1} H NMR (400 MHz, DMSO-d ₆ ): δ9.13 (s, 2H), 8.08 (s, 2H), 7.99 (d, J = 9.0 Hz, 4H), 7.87 (s) , 4H), 7.77 (t, J = 1.8Hz, 2H), 7.76 (t, J = 1.8Hz, 2H), 7.12 (d, J = 9.0Hz, 4H), 4 .39 (t, J = 5.0Hz, 4H), 3.77 (t, J = 5.0Hz, 4H), 3.55-3.53 (m, 4H), 3.49-3.45 ( m, 8H), 3.42-3.40 (m, 4H), 3.23 (s, 6H).

[Experimental Example 3]
According to the following synthesis scheme 3, the fluorescent compounds of Examples 8 to 10 were synthesized using the compounds 10a, 10b and 10c as starting materials. In Scheme 3, "*" represents a bond. Hereinafter, the compounds of Examples 8 to 10 may be referred to as "G2" (Example 8), "G4" (Example 9), and "G6" (Example 10), respectively. The procedure for synthesizing each compound was the same as that for Synthesis Scheme 1 described above. More specifically, G2 (Example 8) was obtained from compounds 10a via 11a, 12a, and 13a. In addition, G4 (Example 9) was obtained from compound 10b via 11b, 12b, and 13b. Further, G6 (Example 8) was obtained from the compound 10c via 11c, 12c and 13c.

<< G2 (Example 8) >>
^{1 1} H NMR (400 MHz, DMSO-d ₆ ): δ8.08 (s, 2H), 7.99 (d, J = 9.2 Hz, 4H), 7.86 (s, 4H), 7.63 (t) , J = 5.4Hz, 2H), 7.14 (d, J = 9.2Hz, 4H), 7.13 (br-s, 8H), 4.13 (t, J = 6.2Hz, 4H) , 3.30 (q, J = 6.2Hz, 4H), 1.97 (quint, J = 6.2Hz, 4H).
^{MS (MALDI): m / z} = 563.1 [M-H + -2CF 3 COO -] +.
calcd (%) for C ₃₆ H ₃₆ F ₆ N ₈ O ₆ (G2): C54.68, H4.59, N14.17; found: C54.30, H4.55, N14.07.

<< G4 (Example 9) >>
^{1 1} H NMR (400 MHz, DMSO-d ₆ ): δ8.07 (s, 2H), 7.86 (s, 4H), 7.78 (t, J = 5.4 Hz, 2H), 7.77 (t) , J = 5.4Hz, 2H), 7.73 (d, J = 2.0Hz, 2H), 7.61 (dd, J = 8.8, 2.0Hz, 2H), 7.21 (br- s, 16H), 7.20 (d, J = 8.8Hz, 2H), 4.13 (t, J = 6.4Hz, 4H), 4.10 (t, J = 6.4Hz, 4H), 3.28 (m, 8H), 2.00 (quint, J = 6.4Hz, 4H), 1.98 (quint, J = 6.4Hz, 4H).
^{MS (MALDI): m / z} = 793.0 [M-3H + -4CF 3 COO -] +.
_{^{HRMS (ESI): m / z}} calcd for [M-CF 3 COO -] +: 1135.4155; found: 1135.4157.

<< G6 (Example 10) >>
^{1 1} H NMR (400 MHz, DMSO-d ₆ ): δ8.09 (s, 2H), 7.88 (s, 4H), 7.86 (t, J = 5.6 Hz, 4H), 7.75 (t) , J = 5.6Hz, 2H), 7.40 (s, 4H), 7.25 (br-s, 24H), 4.09 (t, J = 6.4Hz, 8H), 4.05 (t) , J = 6.4Hz, 4H), 3.35 (q, J = 6.4Hz, 4H), 3.30 (q, J = 6.4Hz, 8H), 2.01 (quint, J = 6. 4Hz, 8H), 1.88 (quint, J = 6.4Hz, 4H).
^{MS (MALDI): m / z} = 1023.2 [M-5H + -6CF 3 COO -] +.
_{^{HRMS (ESI): m / z}} calcd for [M-CF 3 COO -] +: 1593.5503; found: 1593.5510.

[Experimental Example 4]
The fluorescent compound of Example 11 was synthesized according to the following synthesis scheme 4. In Scheme 4, "*" represents a bond. Hereinafter, the compound of Example 11 may be referred to as "DEO-OPV-CN-G".

(Synthesis of Compound 14)
Aniline (1.61 g, 17.3 mmol), potassium carbonate (9.56 g, 69.2 mmol), potassium iodide (1.43 g, 8.65 mmol) and 1-bromo-2- (2-methoxyethoxy) ethane (9). .50 g, 51.9 mmol) was suspended in dry DMF (5.0 mL) and heated at 80 ° C. for 24 hours. After allowing to cool to room temperature, ethyl acetate (200 mL) was added, and the mixture was washed in the order of water and saturated brine. The organic phase was dried over anhydrous magnesium sulfate, and the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to obtain 4.27 g of Compound 14 as a colorless oily product. Yield 83%.

^{1 1} H NMR (400 MHz, CDCl ₃ ): δ7.20 (dd, J = 8.4,7.6 Hz, 2H), 6.71 (d, J = 8.4 Hz, 2H), 6.66 (t, J = 7.6Hz, 1H), 3.63-3.519 (m, 16H), 3.39 (s, 6H).

(Synthesis of Compound 15)
Compound 14 (4.26 g, 14.3 mmol) was dissolved in dry DMF (5.0 mL) and phosphorus oxytrichloride (2.0 mL, 21.5 mmol) was added thereto. The reaction mixture was heated at 60 ° C. for 3 hours. Then, water (100 mL) and ethyl acetate (200 mL) were added, and the organic phase was washed with water and saturated brine in this order. The organic phase was dried over anhydrous magnesium sulfate, and the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to obtain 3.48 g of Compound 15 as a yellow oily product. Yield 75%.

^{1 1} H NMR (400 MHz, CDCl ₃ ): δ9.73 (s, 1H), 7.71 (d, J = 9.2 Hz, 2H), 6.76 (d, J = 9.2 Hz, 2H), 3 .68 (m, 8H), 3.61 (m, 4H), 3.52 (m, 4H), 3.38 (s, 6H).

(Synthesis of Compound 16)
Diethyl (4-Cyanobenzyl) phosphonate (3.24 g, 12.8 mmol), sodium hydride (60% in oil, 642 mg, 16.1 mmol) was suspended in dry THF (15 mL), wherein compound 15 (3. 47 g, 10.7 mmol) was added. The reaction mixture was stirred at room temperature for 4 hours. After distilling off the solvent under reduced pressure, water was added and the reaction product was extracted with chloroform. The organic phase was washed with water and saturated brine in that order. The organic phase was dried over anhydrous magnesium sulfate, and the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to obtain 3.64 g of compound 16 as a yellow solid. Yield 80%.

^{1 1} H NMR (400 MHz, CDCl ₃ ): δ7.58 (d, J = 8.4 Hz, 2H), 7.51 (d, J = 8.4 Hz, 2H), 7.38 (d, J = 8. 8Hz, 2H), 7.12 (d, J = 16Hz, 1H), 6.85 (d, J = 16Hz, 1H), 6.71 (d, J = 8.8Hz, 2H), 3.68- 3.52 (m, 16H), 3.39 (s, 6H).
¹³ C NMR (100 MHz, CDCl ₃ ): δ148.1, 142.8, 132.4, 132.3, 128.3, 126.1, 124.2, 121.9, 119.3, 111.7, 109.1, 71.9, 70.6, 68.3, 59.1, 50.9.

(Synthesis of Compound 17)
Compound 16 (3.64 g, 8.57 mmol) was dissolved in dry THF (10 mL) and DIBAL (1.0 M THF solution, 17.1 mL) was added to it. The reaction mixture was stirred at room temperature for 18 hours, and then a 10% aqueous acetic acid solution was added to stop the reaction. The reaction product was extracted with chloroform, and the organic phase was washed with water and saturated brine in that order. The organic phase was dried over anhydrous magnesium sulfate, and the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to obtain 1.65 g of Compound 17 as an orange solid. Yield 45%.

^{1 1} H NMR (400 MHz, CDCl ₃ ): δ9.96 (s, 1H), 7.83 (d, J = 8.4 Hz, 2H), 7.60 (d, J = 8.4 Hz, 2H), 7 .41 (d, J = 9.0Hz, 2H), 7.19 (d, J = 16Hz, 1H), 6.92 (d, J = 16Hz, 1H), 6.72 (d, J = 9. 0 Hz, 2H), 3.68-3.53 (m, 16H), 3.39 (s, 6H).
¹³ C NMR (100 MHz, CDCl ₃ ): δ191.6, 148.1, 144.5, 134.4, 132.3, 130.2, 128.3, 126.2, 124.5, 122.5, 111.7, 71.9, 70.6, 68.3, 59.1, 50.9.

(Synthesis of Compound 18)
Compound 17 (800 mg, 1.87 mmol) and compound 21 (600 mg, 1.87 mmol) were dissolved in dry ethanol (10 mL). Three drops of Bu ₄ NOW (40% in water) were added thereto, and the mixture was heated under reflux for 12 hours. The obtained precipitate was collected and washed with ethanol to obtain 1.11 g of compound 18 as an orange solid. Yield 81%.

^{1 1} H NMR (400 MHz, CDCl ₃ ): δ7.86 (d, J = 8.4 Hz, 2H), 7.61 (d, J = 8.8 Hz, 2H), 7.54 (d, J = 8. 4Hz, 2H), 7.40 (d, J = 8.8Hz, 2H), 7.39 (s, 1H), 7.12 (d, J = 16Hz, 1H), 6.99 (d, J = 8.8Hz, 2H), 6.91 (d, J = 16Hz, 1H), 6.72 (d, J = 8.8Hz, 2H), 4.97 (br-s, 1H), 4.17 ( m, 2H), 3.85 (m, 2H), 3.68-3.53 (m, 18H), 3.39 (s, 6H), 3.36 (m, 2H), 1.45 (s) , 9H).
¹³ C NMR (100 MHz, CDCl ₃ ): δ159.3, 155.9, 147.7, 140.3, 139.9, 132.0, 130.5, 129.5, 128.1, 127.5. 127.1, 126.2, 124.9, 123.0, 118.5, 115.0, 111.7, 109.4, 79.3, 71.9, 70.6, 70.4, 69. 3,68,3,67,4,59.1,50.9,40.3,28.4.
MS (MALDI): m / z = 752.71 ([M + Na] ⁺ ).

(Synthesis of Compound 19)
Compound 18 (980 mg, 1.34 mmol) was suspended in dry dichloromethane (3.0 mL) and trifluoroacetic acid (2.0 mL, 26.1 mmol) was added thereto. After stirring the reaction mixture at room temperature for 6 hours, dry diethyl ether was added to obtain 904 mg of compound 19 as an orange solid. Yield 91%.

^{1 1} H NMR (400 MHz, DMSO-d ₆ ): δ7.90 (d, J = 8.4 Hz, 2H), 7.88 (s, 1H), 7.73 (br-s, 3H), 7.71 (D, J = 8.8Hz, 2H), 7.67 (d, J = 8.4Hz, 2H), 7.44 (d, J = 8.8Hz, 2H), 7.28 (d, J = 16Hz, 1H), 7.09 (d, J = 8.8Hz, 2H), 7.01 (d, J = 16Hz, 1H), 6.72 (d, J = 8.8Hz, 2H), 4. 21 (m, 2H), 3.83 (m, 2H), 3.67 (t, J = 5.2Hz, 2H), 3.56-3.43 (m, 16H), 3.25 (s, 6H), 3.02 (t, J = 5.2Hz, 2H).
¹³ C NMR (100 MHz, DMSO-d ₆ ): δ159.1, 158.1 (q, ² J = 31 Hz, CF ₃ COO), 147.7, 140.2, 140.1, 131.9, 130. 7, 129.4, 128.2, 127.1, 126.6, 126.1, 124.1, 122.3, 118.4, 117.3 (q, ¹ _{J = 298 Hz, CF 3} COO), 115.1, 111.5, 108.1, 71.3, 69.7, 68.8, 67.9, 67.2, 66.8, 58.1, 50.2, 38.6.
MS (MALDI): m / z = 630.63 ([M-CF ₃ COO] ⁺ ).

(Synthesis of Compound 20)
Compound 19 (800 mg, 1.08 mmol) was dissolved in dry dichloromethane (10 mL) with Et ₃ N (0.23 mL, 1.62 mmol) and 1-H-pyrazole-1- (N, N-bis (tert-). Butyloxy-carbonyl)) carboxamide (503 mg, 1.62 mmol) was added. The reaction mixture was stirred at room temperature for 20 hours. Then, chloroform (100 mL) was added, and the mixture was washed with water and saturated brine in this order. The organic phase was dried over anhydrous magnesium sulfate, and the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to obtain 745 mg of compound 20 as an orange solid. Yield 79%.

^{1 1} H NMR (400 MHz, CDCl ₃ ): δ11.5 (s, 1H), 8.68 (br-t, 1H), 7.85 (d, J = 8.6 Hz, 2H), 7.59 (d) , J = 8.8Hz, 2H), 7.53 (d, J = 8.6Hz, 2H), 7.40 (d, J = 8.8Hz, 2H), 7.39 (s, 1H), 7 .12 (d, J = 16Hz, 1H), 6.99 (d, J = 8.8Hz, 2H), 6.90 (d, J = 16Hz, 1H), 6.72 (d, J = 8. 8Hz, 2H), 4.19 (m, 2H), 3.87 (m, 2H), 3.70-3.53 (m, 20H), 3.39 (s, 6H), 1.51 (s) , 9H), 1.48 (s, 9H).
¹³ C NMR (100 MHz, CDCl ₃ ): δ163.5, 159.4, 156.3, 153.0, 147.7, 140.2, 139.8, 132.0, 130.5, 129.5 128.1, 127.4, 127.1, 126.2, 124.9, 123.1, 118.5, 115.1, 111.7, 109.5, 83.0, 79.2, 71. 9,70.6,69.5,69,3,68,4,67,5,59.1,50.9,40.5,28.3,28.0.
MS (MALDI): m / z = 694.65 ([(DEO-OPV-CN-G) -CF ₃ COOH + Na] ⁺ ).

(Synthesis of DEO-OPV-CN-G (Example 11))
Compound 20 (700 mg, 0.80 mmol) was suspended in dry dichloromethane (3.0 mL) and trifluoroacetic acid (2.0 mL, 26.1 mmol) was added thereto. After stirring the reaction mixture at room temperature for 4 hours, dry diethyl ether was added to obtain 381 mg of DEO-OPV-CN-G as a red solid. Yield 61%.

^{1 1} H NMR (400 MHz, DMSO-d ₆ ) δ7.90 (d, J = 8.6 Hz, 2H), 7.87 (s, 1H), 7.70 (d, J = 9.2 Hz, 2H), 7.66 (d, J = 8.6Hz, 2H), 7.51 (t, J = 5.4Hz, 1H), 7.44 (d, J = 9.2Hz, 2H), 7.28 (d) , J = 16Hz, 1H), 7.10 (br-s, 4H), 7.09 (d, J = 9.2Hz, 2H), 7.01 (d, J = 16Hz, 1H), 6.72 (D, J = 9.2Hz, 2H), 4.18 (m, 2H), 3.81 (m, 2H), 3.60-3.43 (m, 18H), 3.33 (t, J) = 5.4Hz, 2H), 3.25 (s, 6H).
¹³ C NMR (100 MHz, DMSO-d ₆ ) δ159.1, 158.6 (q, ² J = 32 Hz, CF ₃ COO), 157.1, 147.7, 140.2, 140.1, 131.9 , 130.7, 129.4, 128.1, 127.1, 126.6, 126.1, 124.1, 122.3, 118.4, 116.7 (q, ¹ J = 296Hz, CF ₃₎ COO), 115.1, 111.5, 108.1, 71.3, 69.7, 68.8, 68.6, 67.9, 67.3, 58.1, 50.3, 40.8 ..
MS (MALDI): m / z = 672.66 ([M-CF ₃ COO] ⁺ ).

[Experimental Example 5]
The fluorescent compounds of Examples 12 to 15 represented by the following formula (5) were synthesized according to the same synthesis procedure as the synthesis scheme 4 described above.

In formula (5), ^{the compound in which R 5} is -CH ₃ is the compound of Example 12 (hereinafter, may be referred to as "Me-OPV-CN-G"), and R ⁵ is -n-C ₄ what is H ₉ are the compound of example 13 (hereinafter sometimes referred to as _"C 4 -OPV-CN-G".), those ^{wherein R 5} is a group represented by the following formula (6) The compound of Example 14 (hereinafter, may be referred to as “C ₄ ^* -OPV-CN-G”), and ^{the compound in which R 5} is −n—C ₁₂ H ₂₅ is the compound of Example 15 (hereinafter, it may be referred to as “C 4 * -OPV-CN-G”). hereinafter referred to as _"C 12 -OPV-CN-G".).

［式（６）中、「＊」は結合手を表し、「†」は不斉炭素原子を表す。］

[In equation (6), "*" represents a bond and "†" represents an asymmetric carbon atom. ]

<< Me-OPV-CN-G (Example 12) >>
^{1 1} H NMR (400 MHz, DMSO-d ₆ ): δ7.91 (d, J = 8.6 Hz, 2H), 7.87 (s, 1H), 7.70 (d, J = 9.0 Hz, 2H) , 7.67 (d, J = 8.6Hz, 2H), 7.52 (t, J = 5.4Hz, 1H), 7.47 (d, J = 8.8Hz, 2H), 7.30 ( d, J = 16Hz, 1H), 7.10 (br-s, 4H), 7.09 (d, J = 9.0Hz, 2H), 7.03 (d, J = 16Hz, 1H), 6. 74 (d, J = 8.8Hz, 2H), 4.19 (m, 2H), 3.81 (m, 2H), 3.59 (m, 2H), 3.33 (q, J = 5. 4Hz, 2H), 2.95 (s, 6H).
¹³ C NMR (100 MHz, DMSO-d ₆ ): δ159.1, 158.7 (q, ² J = 31 Hz, CF ₃ COO), 157.1, 150.2, 140.2, 140.1, 131. 9, 130.8, 129.4, 127.9, 127.1, 126.5, 126.1, 124.5, 122.5, 118.4, 117.1 (q, ^{1 J = 298 Hz, CF} ₃ COO), 115.1, 112.2, 108.2, 68.8, 68.6, 67.3, 40.8 (One peak is overlapped into solvent peak at ca. 40 ppm).
MS (MALDI): m / z = 496.48 ([M-CF ₃ COO] ⁺ ).

<< C _4- OPV-CN-G (Example 13) >>
¹ H NMR (400 MHz, DMSO-d ₆ ): δ7.90 (d, J = 8.4 Hz, 2H), 7.85 (s, 1H), 7.70 (d, J = 9.0 Hz, 2H) , 7.65 (d, J = 8.4Hz, 2H), 7.49 (t, J = 5.6Hz, 1H), 7.42 (d, J = 8.6Hz, 2H), 7.25 ( d, J = 16Hz, 1H), 7.10 (br-s, 4H), 7.08 (d, J = 9.0Hz, 2H), 6.97 (d, J = 16Hz, 1H), 6. 66 (d, J = 8.6Hz, 2H), 4.19 (m, 2H), 3.81 (m, 2H), 3.60 (t, J = 5.6Hz, 2H), 3.33 ( q, J = 5.6Hz, 2H), 3.30 (t, J = 7.6Hz, 4H), 1.52 (quint, J = 7.6Hz, 4H), 1.33 (sext, J = 7) .6Hz, 4H), 0.93 (t, J = 7.6Hz, 6H).
¹³ C NMR (100 MHz, DMSO-d ₆ ): δ159.0, 158.3 (q, ² J = 32 Hz, CF ₃ COO), 157.1, 147.7, 140.13, 140.11, 131. 7, 130.8, 129.3, 128.1, 127.0, 126.6, 126.0, 123.7, 122.0, 118.3, 116.8 (q, ^{1 J = 297Hz, CF} ₃ COO), 115.1, 111.6, 108.1, 68.7, 68.6, 67.3, 49.9, 40.8, 29.0, 19.6, 13.7.
MS (MALDI): m / z = 580.57 ([M-CF ₃ COO] ⁺ ).

<< C ₄ ^* -OPV-CN-G (Example 14) >>
^{1 1} H NMR (400 MHz, DMSO-d ₆ ): δ7.90 (d, J = 8.6 Hz, 2H), 7.87 (s, 1H), 7.70 (d, J = 8.6 Hz, 2H) , 7.65 (d, J = 8.6Hz, 2H), 7.55 (br-s, 1H), 7.42 (d, J = 9.0Hz, 2H), 7.26 (d, J = 16Hz, 1H), 7.10 (br-s, 4H), 7.09 (d, J = 8.6Hz, 2H), 6.98 (d, J = 16Hz, 1H), 6.68 (d, J = 9.0Hz, 2H), 4.19 (m, 2H), 3.81 (m, 2H), 3.59 (m, 2H), 3.40-3.29 (m, 4H), 3 .07 (dd, J = 15Hz, 8.0Hz, 2H), 1.80 (m, 2H), 1.40 (m, 2H), 1.10 (m, 2H), 0.88 (t, J) = 7.6Hz, 6H), 0.84 (d, J = 6.4Hz, 6H).
¹³ C NMR (100 MHz, DMSO-d ₆ ): δ159.0, 158.4 (q, ² J = 31 Hz, CF ₃ COO), 157.1, 148.0, 140.12140.11, 131.7, 130.7, 129.3, 127.9, 127.0, 126.5, 125.9, 123.5, 122.0, 118.3, 117.1 (q, ¹ _{J = 298 Hz, CF 3} COO ), 115.1, 112.2, 108.1, 68.7, 68.6, 67.3, 57.6, 40.8, 32.4, 26.4, 16.7, 11.2.
MS (MALDI): m / z = 608.62 ([M-CF ₃ COO] ⁺ ).

<< C _12- OPV-CN-G (Example 15) >>
^{1 1} H NMR (400 MHz, DMSO-d ₆ ): δ7.90 (d, J = 8.8 Hz, 2H), 7.87 (s, 1H), 7.70 (d, J = 9.0 Hz, 2H) , 7.65 (d, J = 8.8Hz, 2H), 7.50 (t, J = 5.6Hz, 1H), 7.42 (d, J = 8.8Hz, 2H), 7.26 ( d, J = 16Hz, 1H), 7.10 (br-s, 4H), 7.09 (d, J = 9.0Hz, 2H), 6.97 (d, J = 16Hz, 1H), 6. 64 (d, J = 8.8Hz, 2H), 4.19 (m, 2H), 3.81 (m, 2H), 3.59 (t, J = 5.6Hz, 2H), 3.33 ( q, J = 5.6Hz, 2H), 3.29 (t, J = 6.8Hz, 4H), 1.51 (br-quint, 4H), 1.30-1.24 (m, 36H), 0.85 (t, J = 6.8Hz, 6H).
¹³ C NMR (100 MHz, DMSO-d ₆ ): δ159.0, 158.8 (q, ² J = 32 Hz, CF ₃ COO), 157.1, 147.6, 139.9, 139.8, 131. ^{8,130.5,129.4,128.0,126.9,126.5,125.9,123.7,121.9,118.3,116.8 (q, 1 J = 296Hz} , CF ₃ COO), 115.0, 111.3, 108.0, 68.7, 68.6, 67.2, 50.2, 40.8, 31.4, 29.24, 29.19, 29. 0,28.9,26.9,26.5,22.2,13.8. (Two carbon peaks of dodecyl group were overlapped.)
MS (MALDI): m / z = 804.94 ([M-CF ₃ COO] ⁺ ).

[Experimental Example 6]
The fluorescent compound of Example 16 was synthesized according to the following synthesis scheme 5. Hereinafter, the compound of Example 16 may be referred to as “OPV-CN-G”.

(Synthesis of Compound 21)
4-Hydroxybenzyl cyanide (2.48 g, 18.6 mmol), potassium carbonate (5.14 g, 37.2 mmol) and compound 3 (5.00 g, 18.6 mmol) were suspended in dry DMF (20 mL). It was heated at 65 ° C. for 5 hours. After allowing to cool to room temperature, ethyl acetate (200 mL) was added, and the mixture was washed in the order of water and saturated brine. The organic phase was dried over anhydrous magnesium sulfate, and the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to obtain 4.25 g of Compound 21 as a colorless solid. Yield 71%.

^{1 1} H NMR (400 MHz, CDCl ₃ ): δ7.23 (d, J = 8.8 Hz, 2H), 6.93 (d, J = 8.8 Hz, 2H), 4.95 (br-s, 1H) , 4.12 (m, 2H), 3.82 (m, 2H), 3.61 (t, J = 5.6Hz, 2H), 3.35 (q, J = 5.6Hz, 2H), 1 .44 (s, 9H).
¹³ C NMR (100 MHz, CDCl ₃ ): δ158.4, 155.9, 129.0, 122.1, 118.1, 115.2, 79.2, 70.4, 69.3, 67.4 40.2, 28.4, 22.8.
MS (MALDI): m / z = 342.71 ([M + Na] ⁺ ).

(Synthesis of Compound 22)
Compound 21 (2.00 g, 6.24 mmol) and terephthalaldehyde (419 mg, 3.12 mmol) were dissolved in dry ethanol (20 mL). Three drops of Bu ₄ NOW (40% in water) were added thereto, and the mixture was heated under reflux for 3 hours. The obtained precipitate was collected and washed with ethanol to obtain 1.03 g of Compound 22 as a yellow solid. Yield 45%.

^{1 1} H NMR (400 MHz, CDCl ₃ ): δ7.96 (s, 4H), 7.64 (d, J = 8.8 Hz, 4H), 7.44 (s, 2H), 7.01 (d, J) = 8.8Hz, 4H), 4.95 (br-s, 2H), 4.18 (m, 4H), 3.85 (m, 4H), 3.63 (t, J = 5.6Hz, 4H) ), 3.36 (q, J = 5.6Hz, 4H), 1.45 (s, 18H).
¹³ C NMR (100 MHz, CDCl ₃ ): δ159.8, 155.9, 138.6, 135.3, 129.5, 127.4, 126.9, 117.9, 115.1, 112.2. 79.3, 70.5, 69.3, 67.5, 40.3, 28.4.
MS (MALDI): m / z = 760.92 ([M + Na] ⁺ ).

(Synthesis of Compound 23)
Compound 22 (900 mg, 1.22 mmol) was suspended in dry dichloromethane (5.0 mL) and trifluoroacetic acid (3.0 mL, 39.2 mmol) was added thereto. After stirring the reaction mixture at room temperature for 7 hours, dry diethyl ether was added to obtain 876 mg of compound 23 as a yellow solid. Yield 94%.
^{1 1} H NMR (400 MHz, DMSO-d ₆ ): δ8.04 (s, 4H), 7.99 (s, 2H), 7.79 (br-s, 6H), 7.75 (d, J = 8) 8.8Hz, 4H), 7.12 (d, J = 8.8Hz, 4H), 4.22 (m, 4H), 3.83 (m, 4H), 3.68 (t, J = 5.6Hz) , 4H), 3.02 (t, J = 5.6Hz, 4H).
¹³ C NMR (100 MHz, DMSO-d ₆ ): δ159.5, 158.1 (q, ² J = 32 Hz, CF ₃ COO), 139.4, 135.4, 129.3, 127.4, 126. _{^{1,117.9,117.3 (q, 1 J = 298Hz}} , CF 3 COO), 115.2,110.9,68.8,67.3,66.9,38.6.
MS (MALDI): m / z = 538.82 ([MH-2CF ₃ COO] ⁺ ).

(Synthesis of Compound 24)
Compound 23 (782 mg, 1.02 mmol) was dissolved in dry dichloromethane (10 mL) with Et ₃ N (0.43 mL, 3.06 mmol) and 1-H-pyrazole-1- (N, N'-bis (tert). -Butyloxy-carbonyl)) carboxamide (950 mg, 3.06 mmol) was added. The reaction mixture was stirred at room temperature for 20 hours. Then, chloroform (100 mL) was added, and the mixture was washed with water and saturated brine in this order. The organic phase was dried over anhydrous magnesium sulfate, and the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to obtain 479 mg of compound 24 as a yellow solid. Yield 46%.

^{1 1} H NMR (400 MHz, CDCl ₃ ): δ11.48 (s, 2H), 8.67 (br-s, 2H), 7.96 (s, 4H), 7.63 (d, J = 8.8 Hz) , 4H), 7.43 (s, 2H), 7.01 (d, J = 8.8Hz, 4H), 4.21 (m, 4H), 3.88 (m, 4H), 3.70 ( m, 8H), 1.51 (s, 18H), 1.48 (s, 18H).
¹³ C NMR (100 MHz, CDCl ₃ ): δ163.5, 159.9, 156.3, 153.0, 138.5, 135.4, 129.5, 127.4, 126.9, 117.9, 115.2, 112.2, 83.0, 79.3, 69.6, 69.2, 67.6, 40.5, 28.3, 28.0.
MS (MALDI): m / z = 644.88 ([(OPV-CN-G) + Na-2CF ₃ COOH] ⁺ ).

(Synthesis of OPV-CN-G (Example 16))
Compound 24 (400 mg, 0.39 mmol) was suspended in dry dichloromethane (5.0 mL) and trifluoroacetic acid (3.0 mL, 39.2 mmol) was added thereto. After stirring the reaction mixture at room temperature for 10 hours, dry diethyl ether was added to obtain 300 mg of OPV-CN-G as a yellow solid. Yield 90%.

^{1 1} H NMR (400 MHz, DMSO-d ₆ ): δ8.04 (s, 4H), 7.99 (s, 2H), 7.75 (d, J = 8.8 Hz, 4H), 7.57 (br) -S, 2H), 7.13 (br-s, 8H), 7.11 (d, J = 8.8Hz, 4H), 4.20 (m, 4H), 3.81 (m, 4H), 3.59 (t, J = 5.6Hz, 4H), 3.33 (t, J = 5.6Hz, 4H).
¹³ C NMR (100 MHz, DMSO-d ₆ ): δ159.5, 158.7 (q, ² J = 31 Hz, CF ₃ COO), 157.4, 139.4, 135.4, 129.3, 127. 4,126.1, 117.9, 117.1 (q, ¹ J = 297 Hz, CF ₃ COO), 115.2, 110.9, 68.8, 68.6, 67.4, 40.8.
MS (MALDI): m / z = 622.94 ([MH-2CF ₃ COO] ⁺ ).

[Experimental Example 7]
The fluorescence characteristics of the fluorescent compound (OPV-G) of Example 1 with respect to the dicarboxylic acid were examined.

L-tartaric acid or meso-tartaric acid was added to OPV-G as a dicarboxylic acid, and the fluorescence was measured. The measurement conditions were as follows.

(Measurement condition)
Concentration of OPV-G: 10 μM, in HEPES buffer (10 mM, pH 7.4).
Concentrations of L-tartaric acid and meso-tartaric acid: 5.0 mM.
Excitation wavelength λ _ex : 388 nm
Measuring device: Perkin-Elmer LS55
Measurement temperature: 25 ° C
Measuring cell: 1 mm quartz cell

The measurement results are shown in FIG. As shown in FIG. 1 (a), it was observed with the naked eye that the fluorescent compound of Example 1 fluoresces against tartaric acid. Further, as shown in FIG. 1 (b), despite the same measurement conditions, the fluorescence intensity when L-tartaric acid is added gives 2.5 times stronger fluorescence intensity than that when meso-tartaric acid is added. Was recognized.

[Experimental Example 8]
The properties of the fluorescent compound (OPV-G) of Example 1 were further investigated. When L-tartaric acid was added to OPV-G and the ultraviolet-visible absorption spectrum was measured, the maximum absorption peak at 370 nm decreased and a new absorption peak was observed at 425 nm (see FIG. 2 (a)). This suggests the formation of J-aggregates. By observing the aqueous dispersion with a fluorescence microscope, the formation of fibrous aggregates was observed (see FIG. 2 (b)). On the other hand, with respect to the addition of meso-tartaric acid, the maximum absorption peak at 370 nm was greatly reduced and the wavelength was shifted to 342 nm by a short wavelength (see FIG. 2 (c)). This suggests that an H-aggregate is formed. By observing the aqueous dispersion with a fluorescence microscope, the formation of particulate aggregates was observed (see FIG. 2 (d)).

Here, the J-aggregate and the H-aggregate will be described. FIG. 16A is a schematic diagram showing an example of a J-aggregate. In FIG. 16A, OPV-G (20) forms a J-aggregate 100 by the interaction between the cationic group 21 of OPV-G (20) and the negative charge 11 of hyaluronic acid 10, which will be described later. Shows how it is fluorescing. Further, FIG. 16B is a schematic diagram showing an example of an H-aggregate. In FIG. 16B, OPV-G (20) forms an H-aggregate 200 due to the interaction between the cationic group 21 of OPV-G (20) and the negative charge 31 of heparin 30, which will be described later. Shows how it is fluorescent.

As shown in FIGS. 16A and 16B, in the J-aggregate, the fluorescent compounds are piled up while sliding in the long axis direction of the molecule (slip-stacked fashion). On the other hand, in the H-aggregate, the fluorescent compounds are densely stacked in the direction perpendicular to the long axis direction of the molecule.

[Experimental Example 9]
The properties of the fluorescent compound (OPV-G) of Example 1 were further investigated. A fluorescence titration test was carried out by sequentially adding L-tartaric acid or meso-tartaric acid as a dicarboxylic acid to OPV-G (concentration: 10 μM). The measurement conditions were the same as in Experimental Example 7.

The results are shown in FIG. In the figure, F ₀ represents the fluorescence intensity in the absence of the dicarboxylic acid, F represents the fluorescence intensity in the presence of the dicarboxylic acid, and ΔF = FF ₀ . Therefore, ΔF / F ₀ means the degree of change in fluorescence intensity.

As shown in FIG. 3A, with respect to the addition of L-tartaric acid, fluorescence (λ _em : 518 nm) was observed from 250 μM with respect to the increase in concentration, showing a maximum of 45-fold increase in fluorescence. .. On the other hand, with respect to the addition of meso-tartaric acid, fluorescence (λ _em : 518 nm) was observed from 50 μM, and a maximum of 18-fold increase in fluorescence was observed. FIG. 3 (b) is an enlargement of the low concentration region of FIG. 3 (a). From this result, it was confirmed that OPV-G imparted a fluorescence response depending on the three-dimensional structure (L-form, meso-form) of tartaric acid.

[Experimental Example 10]
The properties of the fluorescent compound (OPV-G) of Example 1 were further investigated. A fluorescence titration test was carried out by sequentially adding fumaric acid or maleic acid as dicarboxylic acids to OPV-G (concentration: 10 μM). The measurement conditions were the same as in Experimental Example 7.

The results are shown in FIG. As shown in FIG. 4 (a), with respect to the addition of fumaric acid, fluorescence (λ _em : 520 nm) was observed from 200 μM with respect to the increase in concentration, showing a maximum of 54-fold increase in fluorescence. On the other hand, with respect to the addition of maleic acid, fluorescence (λ _em : 514 nm) was observed from 100 μM, and a maximum of 20-fold increase in fluorescence was observed. FIG. 4B is an enlargement of the low concentration region of FIG. 4A. From this result, it was confirmed that OPV-G provided a fluorescence response depending on the positional isomer structure (trans-form, cis-form) of fumaric acid and maleic acid.

[Experimental Example 11]
The properties of the fluorescent compound (OPV-G) of Example 1 were further investigated. A fluorescence titration test was carried out by sequentially adding oxalic acid, malonic acid, succinic acid, glutaric acid or adipic acid as dicarboxylic acids to OPV-G (concentration: 10 μM). The measurement conditions were the same as in Experimental Example 7.

The results are shown in FIGS. 5 and 6. As shown in FIG. 5, when acetic acid was sequentially added as a monocarboxylic acid, no fluorescence was observed. On the other hand, with respect to the addition of the dicarboxylic acid, fluorescence (λ _em : 515 nm) was observed with respect to the increase in concentration. As shown in FIG. 6, it is recognized that the concentration at which the fluorescence intensity is observed (concentration threshold value) and the fluorescence intensity are peculiar to the dicarboxylic acid structure.

As shown in the results of Experimental Examples 6 to 8, it was revealed that when the fluorescent compound of Example 1 was used, the dicarboxylic acid could be detected in a turn-on or switch-on manner at a constant dicarboxylic acid concentration. Furthermore, it was clarified that the dicarboxylic acid can be identified by converting and amplifying the chemical structure information of the dicarboxylic acid into the threshold value of the fluorescence response, the fluorescence intensity, and the fluorescence wavelength.

[Experimental Example 12]
The properties of the fluorescent compound (OPV-G) of Example 1 were further investigated. A fluorescence titration test was carried out by sequentially adding polyacrylic acid (average molecular weight 5000) as a polycarboxylic acid to OPV-G (concentration: 10 μM). The measurement conditions were the same as in Experimental Example 7.

The results are shown in FIG. As shown in FIG. 7 (a), with respect to the addition of polyacrylic acid, fluorescence (λ _em : 520 nm) was observed in a turn-on or switch-on manner with respect to the increase in concentration. Further, as shown in FIG. 7 (b), the fluorescence intensity was observed from 0.1 ppm and reached saturation at 1.3 ppm.

As described above, it was clarified that the polyacrylic acid concentration can be measured from the fluorescence intensity in the concentration range of 0.1 to 1.3 ppm by using the fluorescent compound of Example 1.

[Experimental Example 13]
The characteristics of the fluorescent compound (OPV-IMMe) of Example 3 were examined. A fluorescence titration test was carried out by sequentially adding polyacrylic acid (average molecular weight 5000) as a polycarboxylic acid to OPV-IMMe (concentration: 20 μM). The measurement conditions were the same as in Experimental Example 7.

The results are shown in FIG. As shown in FIG. 8 (a), with respect to the addition of polyacrylic acid, fluorescence (λ _em : 515 nm) was observed in a turn-on or switch-on manner with respect to the increase in concentration. Further, as shown in FIG. 8 (b), the fluorescence intensity was observed from 0.1 ppm and reached saturation at 4.0 ppm.

As described above, it was clarified that the polyacrylic acid concentration can be measured from the fluorescence intensity in the concentration range of 0.1 to 4.0 ppm by using the fluorescent compound of Example 3.

[Experimental Example 14]
The characteristics of the fluorescent compound (OPV-ImDEO) of Example 6 were examined. A fluorescence titration test was carried out by sequentially adding polyacrylic acid (average molecular weight 5000) as a polycarboxylic acid to OPV-ImDEO (concentration: 60 μM). The measurement conditions were the same as in Experimental Example 7.

The results are shown in FIG. As shown in FIG. 9A, with respect to the addition of polyacrylic acid, fluorescence (λ _em : 513 nm) was observed in a turn-on or switch-on manner with respect to the increase in concentration. Further, as shown in FIG. 9B, the fluorescence intensity was observed from 0.1 ppm and reached saturation at 15 ppm.

As described above, it was clarified that the polyacrylic acid concentration can be measured from the fluorescence intensity in the concentration range of 0.1 to 15 ppm by using the fluorescent compound of Example 6.

[Experimental Example 15]
The fluorescence characteristics of the fluorescent compound (OPV-G) of Example 1 and the fluorescent compound (G2) of Example 8 were examined for nucleotides. Specifically, ATP and NADPH were added as nucleotides to OPV-G and G2, and the fluorescence was measured. The chemical formulas of ATP and NADPH are shown below.

The measurement conditions were the same as in Experimental Example 7. However, EtOH / HEPES buffer (10 mM, pH 7.4) (1: 9 v / v) was used as the solvent. The measurement results are shown in FIGS. 10 (a) and 10 (b). When ATP or NADPH was added to the fluorescent compound, OPV-G showed a similar fluorescence response to both ATP and NADPH, as shown in FIG. 10 (a). On the other hand, as shown in FIG. 10B, in G2, a clear difference in fluorescence intensity was observed between ATP and NADPH.

As can be inferred from the results of Experimental Examples 7 and 8 described above, G2 forms a J-aggregate with ATP and exhibits strong fluorescence, whereas NADPH forms an H-aggregate with NADPH. It was considered to show weak fluorescence intensity.

From the results of Experimental Example 15, it was clarified that OPV-G can detect nucleotides non-specifically, and G2 can selectively detect ATP from among nucleotides.

[Experimental Example 16]
The influence of the salt concentration on the fluorescence of the fluorescent compounds of Example 8 (G2) and Example 10 (G6) was examined. More specifically, sodium chloride (NaCl) was sequentially added as a salt to G2 and G6 to carry out a fluorescence titration test. The measurement conditions were the same as in Experimental Example 7.

The measurement results are shown in FIG. As shown in FIG. 11, G2 was found to fluoresce with the addition of sodium chloride. On the other hand, in G6, fluorescence due to the addition of sodium chloride was not observed.

As shown in the results of Experimental Example 16, G2 self-associates and fluoresces in the presence of salt concentration, whereas G6 does not fluoresce even under physiological salt concentration (about 150 mM). That is, it became clear that they did not meet themselves.

[Experimental Example 17]
The characteristics of the fluorescent compound (G6) of Example 10 were further investigated. Specifically, under physiological salt concentration conditions (NaCl: 125 mM, KCl: 5 mM, CaCl ₂ : 1 mM, MgCl ₂ : 0.5 mM), AMP, ADP or ATP is added as a nucleotide to G6 for fluorescence. A titration test was performed. The measurement conditions were the same as in Experimental Example 7.

The measurement results are shown in FIGS. 12 (a) and 12 (b). As shown in FIG. 12 (a), it was observed with the naked eye that G6 selectively fluoresces with respect to ATP. Further, as shown in FIG. 12 (b), it was revealed that G6 selectively exhibits a fluorescence response to ATP among nucleotides.

As shown in the results of Experimental Examples 16 to 17, when G6 is used, ATP can be selectively detected in a turn-on or switch-on manner under physiological salt concentration conditions, and the concentration of ATP can be measured from the fluorescence intensity. Became clear. Therefore, it can be said that G6 is an ATP detection agent.

[Experimental Example 18]
The fluorescence characteristics of the fluorescent compound (DEO-OPV-CN-G) of Example 11 with respect to anionic polysaccharides were examined. Specifically, a fluorescence titration test was carried out by adding heparin, chondroitin sulfate or hyaluronic acid as anionic polysaccharides to DEO-OPV-CN-G. The chemical formulas of heparin, chondroitin sulfate and hyaluronic acid are shown below.

The measurement conditions were as follows.
(Measurement condition)
Concentration of DEO-OPV-CN-G: 30 μM in HEPES buffer (10 mM, pH 7.4).
Excitation wavelength λ _ex : 400 nm
Measuring device: Perkin-Elmer LS55
Measurement temperature: 25 ° C
Measuring cell: 1 mm quartz cell

The measurement results are shown in FIGS. 13 (a) and 13 (b). As shown in FIG. 13 (a), when an anionic polysaccharide was added to DEO-OPV-CN-G, an increase in the intensity of red fluorescence was observed. Further, as shown in FIG. 13 (b), it was revealed that DEO-OPV-CN-G gives a linear fluorescence response to an increase in the concentration of anionic polysaccharides.

As shown in the results of Experimental Example 18, DEO-OPV-CN-G is used to selectively detect anionic polysaccharides, especially heparin, in a turn-on or switch-on manner at a constant anionic polysaccharide concentration. It was clarified that the concentration of anionic polysaccharides can be measured from the fluorescence intensity. Therefore, it can be said that DEO-OPV-CN-G is an anionic polysaccharide detection agent or a heparin detection agent.

[Experimental Example 19]
The fluorescence characteristics of the fluorescent compound (OPV-G) of Example 1 with respect to anionic polysaccharides were examined. Specifically, a fluorescence titration test was carried out by adding heparin, chondroitin sulfate or hyaluronic acid as anionic polysaccharides to OPV-G. The measurement conditions were the same as in Experimental Example 7.

The measurement results are shown in FIGS. 14 (a) and 14 (b). As shown in FIG. 14 (a), the addition of anionic polysaccharides to OPV-G gave the strongest fluorescence intensity to the addition of hyaluronic acid. Further, as shown in FIG. 14 (b), it was revealed that OPV-G gives a linear fluorescence response to an increase in the concentration of anionic polysaccharides.

[Experimental Example 20]
The properties of the fluorescent compound (OPV-G) of Example 1 were further investigated. Specifically, hyaluronic acid and heparin were added to OPV-G, and the ultraviolet-visible absorption spectrum was measured.

As a result, as shown in FIG. 15A, the addition of hyaluronic acid reduced the maximum absorption peak at 370 nm and a new absorption peak was observed at 430 nm. This suggests that a J-aggregate was formed. FIG. 16A shows a schematic diagram of the J-aggregate. In FIG. 16 (a), OPV-G (20) forms a J-aggregate 100 and fluoresces due to the interaction between the cationic group 21 of OPV-G (20) and the negative charge 11 of hyaluronic acid 10. doing.

On the other hand, as shown in FIG. 15B, the addition of heparin significantly reduced the maximum absorption peak at 370 nm. This suggests that an H-aggregate was formed. FIG. 16B shows a schematic diagram of the H-aggregate. In FIG. 16 (b), OPV-G (20) forms an H-aggregate 200 and fluoresces due to the interaction between the cationic group 21 of OPV-G (20) and the negative charge 31 of heparin 30. ing.

As shown in the results of Experimental Examples 19 and 20, it was revealed that OPV-G can detect anionic polysaccharides in a turn-on or switch-on manner at a constant anionic polysaccharide concentration. In addition, OPV-G can selectively detect hyaluronic acid by forming J-aggregates in the presence of hyaluronic acid, which has the lowest charge number among anionic polysaccharides, and the concentration of hyaluronic acid. It became clear that can be measured from the fluorescence intensity. Therefore, OPV-G can be said to be an anionic polysaccharide detection agent or a hyaluronic acid detection agent.

[Experimental Example 21]
The properties of the fluorescent compound (OPV-G) of Example 1 were further investigated. Specifically, heparin (final concentration 2.5 ppm), chondroitin sulfate (final concentration 5.5 ppm) or hyaluronic acid (final concentration 8.5 ppm) was added to OPV-G having a final concentration of 10 μM as an anionic polysaccharide. And the fluorescence was measured. The measurement conditions were the same as in Experimental Example 7 except that the pH of the solution was 1.5.

FIG. 17A is a photograph showing the experimental results. Further, FIG. 17B is a graph showing the results of measuring the fluorescence intensity of each sample of FIG. 17A. As a result, as shown in FIGS. 17 (a) and 17 (b), when an anionic polysaccharide was added to OPV-G under the condition of pH 1.5, the strongest fluorescence intensity was given by the addition of chondroitin sulfate.

As shown in the results of Experimental Example 21, it was clarified that OPV-G can selectively detect anionic polysaccharides, particularly chondroitin sulfate, in an acidic condition in a turn-on or switch-on manner. .. Therefore, OPV-G can be said to be an anionic polysaccharide detection agent or a chondroitin sulfate detection agent.

From the results of Experimental Examples 18 to 21, it was clarified that the selectivity for anionic polysaccharides can be controlled by appropriately adjusting the chemical structure and solution conditions of the fluorescent compound.

[Experimental Example 22]
The fluorescence characteristics of the fluorescent compound (OPV-CN-G) of Example 16 with respect to a dicarboxylic acid were examined. Specifically, L-tartaric acid or meso-tartaric acid was added as a dicarboxylic acid to OPV-CN-G, and the fluorescence was measured. The measurement conditions were as follows.

(Measurement condition)
Concentration of OPV-CN-G: 10 μM, in DMSO / HEPES buffer (10 mM, pH 7.4) (1: 2 v / v).
Concentrations of L-tartaric acid and meso-tartaric acid: 3.0 mM.
Excitation wavelength λ _ex : 365 nm (UV handy lamp)

The measurement results are shown in FIGS. 18 (a) to 18 (c). FIG. 18A is a photograph showing the experimental results. Further, FIGS. 18 (b) and 18 (c) are graphs showing the results of measuring the spectroscopic characteristics of each sample. As shown in FIG. 18 (a), it was observed with the naked eye that OPV-CN-G emits a different fluorescent color depending on the chemical structure of tartaric acid. Specifically, OPV-CN-G alone was blue, whereas it showed green fluorescence in the presence of L-tartaric acid and yellow fluorescence in the presence of meso-tartaric acid. Further, as shown in FIGS. 18 (b) and 18 (c), it was revealed that OPV-CN-G has spectroscopic characteristics corresponding to the chemical structure of the dicarboxylic acid.

As shown in the results of Experimental Example 22, it is clear that the chemical structure of the dicarboxylic acid can be spectroscopically identified by the difference in emission color with the naked eye or using a spectrophotometer by using OPV-CN-G. It became.

[Experimental Example 23]
In the above formula (1), comprising a spacer which alkylene group represented by R ³ is derived from amino acids, fluorogenic compound of Example 17 (hereinafter referred to as "OPV-CN-G ^*".) The Synthesized. The chemical formula ^{of OPV-CN-G *} is shown in the following formula (7). In the formula (7), the group represented by "‡" is a group derived from an amino acid (alanine). In addition, "†" represents an asymmetric carbon atom.

As a result of irradiating OPV-CN-G ^* with ultraviolet rays and observing the fluorescence with the naked eye, ^{it is clear that OPV-CN-G *} emits fluorescence of different wavelengths in the solid state and the state dissolved in a solvent (DMSO). It became. This result indicates that OPV-CN-G ^* fluoresces at different wavelengths depending on the change in agglutination morphology.

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide a novel fluorescent compound exhibiting aggregation-induced luminescence (AIE). In addition, an agent for detecting an ionic compound can be provided.

10 ... hyaluronic acid, 11,31 ... negative charge, 20 ... OPV-G, 21 ... cationic group, 30 ... heparin, 100 ... J-aggregate, 200 ... H-aggregate.

Claims (5)

A fluorescent compound represented by the following formula (1) or a salt thereof.

[In formula (1), R ¹ each independently represent a hydrogen atom or a cyano group, a halogen atom or phenyl group, at least one R ¹ is a cyano group, each R ² is independently, Ha androgenic atom, Represents an alkyl group having 1 to 3 carbon atoms, an amino group or an alkoxy group having 1 to 3 carbon atoms, and R ³ is independently absent or -CH ₂ − is -O-, -S-, -NH. It may be interrupted by being replaced by −, −CO− or −CONH−, where −O−, −S−, −NH−, −CO− or −CONH− are not adjacent. Represents an alkylene group having 1 to 20 carbon atoms, and X is independently absent or a cationic group or a phosphoric acid group represented by any of the following formulas (2) to (4), a carboxylic acid group and a sulfuric acid. Represents an anionic group selected from the groups

In the absence of X, R ⁴ adjacent to X is -N (R ⁵ ) ₂ (where R ⁵ is independently a hydrogen atom or -CH _2- but -O-, -S-,-. It may be interrupted by being replaced by NH-, -CO-, -CONH-, where -O-, -S-, -NH-, -CO- or -CONH- may be adjacent. Represents an alkyl group having 1 to 20 carbon atoms.)
When X is a cationic group represented by the above formula (2) or an anionic group selected from a phosphate group, a carboxylic acid group and a sulfate group, R ⁴ adjacent to the X does not exist. When X is a cationic group represented by the above formula (3) or (4), R ⁴ adjacent to X is a hydrogen atom, or -CH _2- is -O-, -S-, It may be interrupted by being replaced by −NH−, −CO−, −CONH−, where −O−, −S−, −NH−, −CO− or −CONH− are adjacent. There is no alkyl group with 1 to 20 carbon atoms or -CH ₂ CH ₂ OH or -N (R ⁵ ) ₂ (Here, R ⁵ is an independent hydrogen atom or -CH _2- is -O-,- It may be interrupted by being replaced by S-, -NH-, -CO-, -CONH-, where -O-, -S-, -NH-, -CO- or -CONH- Represents an alkyl group having 1 to 20 carbon atoms that is not adjacent to each other). p independently represents an integer of 0 to 4, q independently represents an integer of 0 to 5, at least one q is not 0, and at least one X is the cationic group or the anionic group. be. ] The fluorescent compound according to claim 1 or a salt thereof, which is represented by the following formula (9) or (10).

An ionic compound detection agent containing the fluorescent compound according to claim 1 or 2 or a salt thereof as an active ingredient. The ionic compound according to claim 3, wherein X in the formula (1) is a cationic group represented by any of the following formulas (2) to (4), and the ionic compound is an anionic compound. Detection agent.

A step of preparing a mixed solution containing a solvent, a test sample, and the fluorescent compound according to claim 1 or 2 or a salt thereof.
A method for detecting an ionic compound in a test sample, comprising a step of detecting the fluorescence of the mixed solution.

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