KR20190117058A - Novel fluorescent compound for labelling nucleic acids and the preparation method thereof - Google Patents

Abstract

The present invention relates to a novel fluorescent compound for analyzing nucleic acids, which is very excellent in binding efficiency of biomolecules with the nucleic acids such as DNA, RNA, and the like, maintaines continuously fluorescence characteristics and efficiency, and shows excellent effects in terms of storage stability and biosafety such as temperature and moisture. In addition, the fluorescent compound of the present invention has various effects which can be dissolved in distilled water, a solvent harmless to the human body, and can be applied to a wide range of analysis without being limited to the analysis of specific cells and biological tissues.

Description

Novel fluorescent compound for labeling nucleic acids and the preparation method

The present invention improves the binding efficiency of the biomolecules with nucleic acids such as DNA and RNA when using fluorescent compounds for the analysis of cells or blood, and also has a novel stability with excellent stability to continuously maintain the fluorescence properties and efficiency. Cyanine-based fluorescent dye compound and a method for producing the same.

Since the biomaterial itself has weak or no fluorescence in the visible and near-infrared region, in the biological field, the biomaterial is used to observe biological phenomena at the cellular and subcellular level in or out of the living body or to project it in vivo to obtain optical images of contrast and diseased areas. Imaging data have been obtained through various methods that utilize fluorescent dyes or biocompatible materials labeled with fluorescent dyes together with optical equipment.

Various optical anylsis instruments used in the biotechnologies use fluorescent dyes with excitation and emission wavelengths suitable for fluorescence observation based on built-in light sources and filters as base materials or reagents. Will be chosen.

Commonly used optical analysis equipment includes fluorescence microscope, confocal microscope, flow cytometer, micro array, and quantitative polymerase chain reaction device for observing cells. In addition to research equipment such as systems, nucleic acid and protein separations, electrophoresis devices for analysis, and in vivo imaging systems, immunoassays, PCR assays, and statistical techniques Known for the diagnosis and treatment of grafted nucleic acid and protein diagnostic kits (or biochips) based in vitro diagnosis equipment and operating tables and endoscopy equipment for image-guided surgery. Applications and equipment with higher levels of resolution and data processing capabilities are being developed.

The selection of fluorescent dyes for use in the biotechnologies is important to have strong fluorescence when present in the medium in which the biomolecules are present, ie aqueous solutions and aqueous buffers, and to have excitation and fluorescence wavelengths suitable for fluorescence equipment.

In general, fluorescent dyes used for labeling biological molecules such as proteins or peptides are mostly anthranilate, 1-alkylthic isoindoles, pyrrolinones, and Bimanes, benzoxazole, benzimidazole, benzofuran, benzofurazan, naphthalenes, coumarins, cyanine, stilbenes, carbazoles ), Phenanthridine, anthracenes, bodipy, fluoresceins, eosins, rhodamines, pyrenes, chrysenes and acriri Structures such as acridines are included.

When screening fluorescent dye structures available in the biotechnologies among the many fluorophores exemplified above, fluorescence equipment and fluorescence equipment are generally used when most biomolecules are present in the medium in which they are present, ie, in aqueous and aqueous buffers. It is important to have an excitation and wavelength that fits.

The dyes that can be applied mainly in the bio field should have low photobleaching and quenching in aqueous solutions or hydrophilic conditions, have a large molecular extinction coefficient to absorb large amounts of light, and biomolecules. It should be in the visible or near infrared region of 500 nm or more away from its fluorescence range and stable under various pH conditions, but the structure of the dye which can be used for biomolecular labeling which can satisfy the above limitations is limited.

Fluorescent chromogens that meet these requirements include cyanine, rhodamine, florcein, bodiphy, coumarin, acridine, and pyrene derivatives. The reactor is introduced to allow dyes to be combined with specific substituents in the biomolecular structure. Among them, xanthane-based florcene and rhodamine, and polymethine-based cyanine derivative dye compounds are mainly commercialized.

In particular, dye compounds with cyanine chromophores are generally excellent in optical and pH stability, have a narrow absorption and emission wavelength range, and have a fluorescence region of 500 to 800 nm, in addition to the advantages of being easy to synthesize compounds of various absorption / excitation wavelengths. Since it does not overlap with its own fluorescent region of the biomolecule, it is easy to analyze. Although it varies slightly depending on the solvent and solubility characteristics, it has many advantages such as high molar extinction coefficient and is widely used for biological applications.

However, the conventional dye compounds as described above are not only unstable in terms of storage stability, but also exhibit a problem in that the fluorescent property is rapidly degraded with storage stability over time after being labeled on the biomolecule. Therefore, in order to overcome this problem and to apply industrially usefully, it is important to develop a novel fluorescent dye having excellent optical and pH stability, but having a narrow absorption / luminescence wavelength range and a high molar absorption coefficient in a specific wavelength range.

The present invention is to provide a novel fluorescent compound mainly used for the analysis using a fluorescent compound in the bio field, the fluorescent compound of the present invention is a problem of the conventional fluorescent compound stability and fluorescence efficiency rapidly decreases over time Can solve the problem.

Cyanine-based fluorescent compounds used for cell analysis or nucleic acid analysis (typically Sybr Green) are fluorescent compounds mainly used for coloring nucleic acids in molecular biology, and are selectively absorbed into cellular tissues by binding to proteins in serum. In the case of irradiating NIR or UV, fluorescence is emitted to enable the analysis of the degree of transfer of a target in the tissue through an optical microscope. The fluorescent compound may preferentially bind to double stranded DNA and to a lesser extent single stranded DNA.

The Sybr Green compound may be used for visualizing DNA in gel electrophoresis and the like, and may be used for flow cytometry and fluorescence microscopy. However, these compounds are known to be insignificant in cells but may cause mutations.

Therefore, the present invention is to provide a fluorescent compound having more stability and high fluorescence efficiency for the analysis of nucleic acids and the like, the novel cyanine-based fluorescent compound provided in the present invention was able to solve the above problems.

The first problem to be solved by the present invention is to provide a novel cyanine-based fluorescent compound.

The second problem to be solved by the present invention is to provide a contrast agent composition for analyzing nucleic acids, such as DNA, RNA, including the novel cyanine-based fluorescent compound.

The third problem to be solved by the present invention is to provide a manufacturing method for producing the novel fluorescent compound.

Prior art literature

Patent Literature

(Patent Document 1) Korean Patent Publication No. 10-2017-0105662

In order to solve the above problems, the present invention provides a novel fluorescent compound represented by the following [Formula 1].

[Formula 1]

In Chemical Formula 1

X is oxygen or sulfur,

R is a substituent consisting of the following formula (2) or (3).

[Formula 2]

[Formula 3]

In Formulas 2 and 3, m and n are integers of 1 to 10, R ₂ is methyl, amine (NH ₂ ), methyl substituted amine (N (CH ₃ ) ₂ ), carboxylic acid (CO ₂ H), Sulfonic acid (SO ₃ H) or sodium sulfonate salt (-SO ₃ Na).

The novel fluorescent compound according to the present invention can continuously maintain fluorescence stability and fluorescence efficiency even after a long time after being labeled on a biomolecule such as a protein or an antibody, and also has excellent biosafety.

The novel fluorescent compound of the present invention showed low absorption wavelength and low fluorescence wavelength intensity, and had a pH. 7 shows high absorption wavelength intensity, high fluorescence wavelength intensity, and also shows the difference in absorption wavelength location. It was. Changes in the pH of the body are deeply related to phenomena such as cell proliferation, apoptosis, and muscle contraction. Therefore, by measuring the pH change, studies on cellular internalization pathways as well as cancer cells can be conducted. And it can be said that the fluorescent compound of the present invention has a great advantage in that abnormal pH changes can be observed even in diseases such as Alzheimer's disease.

The fluorescent compound represented by the above [Formula 1] according to the present invention has a structure in which an alkylcarboxyamino cyanuric chloride substituent is substituted with a cyanine chromophore, showing a high fluorescence intensity at a low concentration while having a narrow wavelength range as compared with a conventional cyanine dye. , No cytotoxicity. The compound represented by the above [Formula 1] according to the present invention effectively absorbs the wavelength in the narrow region of 500 to 700 nm, and exhibits significantly improved fluorescence intensity in the narrow wavelength region of 600 to 800 nm, thereby self-fluorescence of the biological molecule. The composition that does not overlap with the region and includes it as an active ingredient may be usefully applied as a contrast agent for imaging in vivo.

The present invention provides a compound labeling method comprising the step of combining the fluorescent compound represented by the above [Formula 1] and the target material.

According to the present invention, the labeling substance may be selected from fibers, biomolecules, nanoparticles or organic compounds, wherein the biomolecules are proteins, peptides, carbohydrates, sugars, fats, antibodies, proteoglycans, glycoproteins And siRNA consisting of siRNAs.

The label uses a solvent selected from the group consisting of phosphate buffer, carbonate buffer and tris buffer, an organic solvent selected from the group consisting of dimethyl sulfoxide, dimethylformamide, methanol, ethanol and acetonitrile, or water as a solvent, It is made by reacting the compound of [Formula 1] and the biomolecule, nanoparticles or organic compound at pH 5 to 12. The reaction is sufficient for 30 minutes to 48 hours at a temperature of 20 to 80 ℃.

On the other hand, in the case of biomolecules are most often dissolved in a predetermined buffer solution from the packaging unit, in order to ensure the stability of the biomolecules in many cases it requires a separate buffer or pH is not easy to adjust to the variable. The compound of [Formula 1] according to the present invention has a high stability in water-soluble conditions, not only easy to store for a long time, but also easily reacts with the protein in various buffers, reaction temperature, pH conditions, etc. to express fluorescence, for biomolecular labeling Suitable for use as

Fluorescent compounds for analyzing the nucleic acids of the present invention is very excellent in binding efficiency of the biomolecules with nucleic acids such as DNA, RNA, and at the same time to maintain the fluorescence properties and efficiency, as well as storage of temperature and moisture It also shows excellent effects in terms of stability and biosafety. In addition, the fluorescent compound of the present invention has various effects that can be dissolved in distilled water, a solvent harmless to the human body, and can be applied to a wide range of analysis without being limited to the analysis of specific cells and biological tissues.

1 shows fluorescence spectra of DNA synthesis detection using a reagent using a comparative material (compound of formula 12).
2 shows a melt curve of an experiment in which DNA synthesis was detected using a reagent using a comparative material (compound of Formula 12).
3 shows fluorescence signals and Cq values when DNA synthesis is detected using a reagent using a comparative material (compound of formula 12).
4 shows fluorescence spectra of DNA synthesis detection using reagents using compounds of Formulas 4-6.
5 shows a melt curve of an experiment in which DNA synthesis was detected using a reagent using a compound of Formulas 4 to 6. FIG.
6 shows fluorescence signals and Cq values when DNA synthesis is detected using reagents using the compounds of Formulas 4-6.
Figure 7 shows the APC wavelength analysis experiments the degree of staining of blood cells using a reagent using a comparative material and the compound of Formulas 4-6.
Figure 8 shows the change in fluorescence intensity after staining blood cells using a reagent using a comparative material and the compounds of formulas 4 to 6.
9 shows staining of cells using a reagent using a comparative material and a compound of Formulas 4 to 6. FIG.

Hereinafter, the method of preparing the fluorescent compound of the present invention and the fluorescence efficiency of the compound of the present invention will be described in detail by using an embodiment of the present invention.

The present invention provides a compound represented by the following formula (1).

[Formula 1]

In Chemical Formula 1

X is oxygen or sulfur,

R is a substituent represented by the following formula (2) or (3).

[Formula 2]

[Formula 3]

In Formulas 2 and 3, m and n are integers of 1 to 10, R ₂ is methyl, amine (NH ₂ ), methyl substituted amine (N (CH ₃ ) ₂ ), carboxylic acid (CO ₂ H), Sulfonic acid (SO ₃ H) or sodium sulfonate salt (-SO ₃ Na).

Specific compounds belonging to the present invention may be a compound represented by the following formula (4), (5) and (6).

[Formula 4]

[Formula 5]

[Formula 6]

Hereinafter, the embodiments of the present invention will be described in more detail, but the following examples are not intended to limit the scope of the present invention, but are described as to help understanding of the present invention.

In order to prepare the compound of the present invention, an intermediate compound represented by the following Chemical Formulas 7 to 10 should be prepared first.

Example . Preparation of Compounds of Formulas 4-6

Synthesis Example 1. Preparation of Compounds of Formulas 7 to 10 to Prepare Compounds of Formulas 4 to 6

1) Synthesis of Compound of Formula 7

2-methylbenzothiazole (1 g, 6.7 mmol, 1 eq) and iodomethane (2.38 g, 16.8 mmol, 2.5 eq) were added to 15 ml of acetonitrile and heated for 12 hours. It was refluxed. After cooling to room temperature, the reaction mixture was dried under reduced pressure to remove the organic solvent. The dried compound was washed several times with diethyl ether and dried to obtain the compound of formula 7. (1.04 g, 94.5%)

R _f = 0.5 (silica gel, MC / MeOH 9: 1 v / v)

[Formula 7]

2) Synthesis of Compound of Formula 8

Stir the compound of formula 7 (1.7 g, 10.4 mmol, 1 eq) and N, N-diphenylformamide (2.03 g, 10.4 mmol, 1 eq) with 10 ml of acetic acid Let's do it. After stirring for 4 hours at 90 ℃, cooled to room temperature. The reaction solution was lyophilized to obtain the compound of formula (8). (19 g, 100%)

R _f = 0.6 (silica gel, MC / MeOH 9: 1 v / v)

[Formula 8]

3) Synthesis of Compound of Formula 9

Heat reflux after completion of lepidine (2 g, 13.9 mmol, 1 eq) and 6-aminohexanoic acid (4.07 g, 20.9 mmol, 1.5 eq) in 20 ml of acetonitrile. Proceed. The reaction solution was dried under a reduced pressure and purified by silica gel chromatography to obtain the compound of formula (9). (2.52 g, 70.4%)

R _f = 0.4 (silica gel, MC / MeOH 9: 1 v / v)

[Formula 9]

4) Synthesis of Compound of Formula 10

Compound 8 (2.78 g, 10.4 mmol. 1 eq) and compound 9 (5.05 g, 19.6 mmol, 1.9 eq) were added to 100 ml of pyridine, stirred at 90 ° C. for 2 hours, and then cooled to room temperature. After drying under reduced pressure, the pyridine was removed, and then purified by silica gel chromatography to obtain the compound of Formula 10. (1.3 g, 31.3%)

R _f = 0.5 (silica gel, MC / MeOH 9: 1 v / v)

LC / MS, calculated C ₂₆ H ₂₆ N ₂ O ₂ S 430.56, found 431.2

[Formula 10]

Synthesis Example 2 Preparation of Compound of Formula 4

[Formula 4]

Compound of formula 10 (330 mg, 0.765 mmol, 1 eq) is completed in 30 ml of DMF. Di (N-succinimidyl) Carbonate (587 mg, 2.295 mmol, 3 eq) was added followed by N, N-diisopropylethylamine (N, N-diisopropylethylamine) (1.33 ml, 7.65 mmol, 10 eq) is added and stirred at 40 ° C. for 3 hours. After completion of the reaction, diethyl ether was added to precipitate particles, and the particles obtained by filtration were dried under reduced pressure.

The obtained compound was dried in 20 ml of DMF, and then 2- (2'-chloroethylsulfonyl) ethylamine hydrochloride (131 mg, 0.765 mmol, 1 eq) and N, N-diisopropylethylamine 666 ul, 3.83 mmol) is added and stirred at 40 ° C. for 3 hours. After the reaction was completed, diethyl ether was added to precipitate particles, and the particles obtained by filtration were dried under reduced pressure. After drying, the obtained compound was purified by silica gel chromatography to obtain the compound of formula 4 (74 mg, 17.6%).

R _f = 0.5 (silica gel, MC / MeOH 5: 1 v / v)

LC / MS, calculated C ₃₀ H ₃₄ N ₃ O ₃ S ₂ ⁺ 548.74, found 548.2

Synthesis Example 3 Preparation of Compound of Formula 5

[Formula 5]

Compound of formula 10 (330 mg, 0.765 mmol, 1 eq) is completed in 30 ml of DMF. Di (N-succinimidyl) Carbonate (587 mg, 2.295 mmol, 3 eq) was added followed by N, N-diisopropylethylamine (N, N-diisopropylethylamine) (1.33 ml, 7.65 mmol, 10 eq) is added and stirred at 40 ° C. for 3 hours. After completion of the reaction, diethyl ether was added to precipitate particles, and the particles obtained by filtration were dried under reduced pressure.

The resulting compound was dried in 20 ml of DMF, and then dried with N, N-dimethyl-1,3-diaminopropane (N, N-Dimethyl-1,3-diaminopropane) (78 mg, 0.765 mmol, 1 eq). N, N-diisopropylethylamine 666 ul, 3.83 mmol) was added thereto, followed by stirring at 40 ° C. for 3 hours. After the reaction was completed, diethyl ether was added to precipitate particles, and the particles obtained by filtration were dried under reduced pressure. After washing several times with diethylether, the compound was obtained by drying. (117 mg, 29.7%)

R _f = 0.1 (silica gel, MC / MeOH 5: 1 v / v)

LC / MS, calcd C ₃₁ H ₃₉ N ₄ OS ⁺ 515.73, found 515.3

Synthesis Example 4 Preparation of Compound of Chemical Formula 6

[Formula 6]

Compound of formula 10 (330 mg, 0.765 mmol, 1 eq) is completed in 30 ml of DMF. Di (N-succinimidyl) Carbonate (587 mg, 2.295 mmol, 3 eq) was added followed by N, N-diisopropylethylamine (N, N-diisopropylethylamine) (1.33 ml, 7.65 mmol, 10 eq) is added and stirred at 40 ° C. for 3 hours. After completion of the reaction, diethyl ether was added to precipitate particles, and the particles obtained by filtration were dried under reduced pressure.

The resulting compound was dried in 20 ml of DMF, and octyylamine (99 mg, 0.765 mmol, 1 eq) and N, N-diisopropylethylamine 666 ul, 3.83 mmol) were added thereto and then 40 ° C. Stir for 3 hours. After the reaction was completed, diethyl ether was added to precipitate particles, and the particles obtained by filtration were dried under reduced pressure. After drying, the obtained compound was purified by silica gel chromatography to obtain a compound of formula (6). (161 mg, 38.8%)

R _f = 0.8 (silica gel, MC / MeOH 5: 1 v / v)

LC / MS, calculated C ₃₄ H ₄₄ N ₃ OS ⁺ 542.8, measured 542.3

Comparative example. Preparation of Compound Having Structure of Formula 12

<Preparation of Compound of Formula 11 to Prepare Compound of Formula 12>

Lepidine (0.757 g, 5.29 mmol, 1 eq) and 1-iodooctane (1.65 g, 6.88 mmol, 1.3 eq) were completed in 8 ml of acetonitrile and then heated to reflux. do. The reaction solution was dried under a reduced pressure, and then purified by silica gel chromatography to obtain a compound of formula 11 below. (1.9 g, 98%)

R _f = 0.6 (silica gel, MC / MeOH 9: 1 v / v)

[Formula 11]

Preparation of Compound of Formula 12

A compound of Formula 8 (130 mg, 0.487 mmol. 1 eq) and a compound of Formula 11 (187 mg, 0.487 mmol, 1 eq) were added to 100 ml of pyridine, stirred at 90 ° C. for 2 hours, and then cooled to room temperature. do. After drying under reduced pressure, pyridine was removed, and purified by silica gel chromatography to obtain a compound represented by Chemical Formula 12 below. (51 mg, 24.4%)

R _f = 0.6 (silica gel, MC / MeOH 9: 1 v / v)

LC / MS, calcd C ₂₈ H ₃₃ N ₂ S ⁺ 429.64, found 429.3

[Formula 12]

Experimental Example 1 Preparation of Reagents Using Compounds of Formulas 4 to 6

(1) Preparation of reagents for blood analysis

Dissolve 2 mg of compound of formula 4 in 3 mL of methanol and add 96.9 mL of ethylene glycol. Reagents are prepared by applying the compounds of Formulas 5 and 6 in the same manner, and a comparative reagent is prepared by applying the compounds of Formula 12 (comparative substance) as the comparative substance.

(2) Preparation of reagents for real-time PCR analysis

1 mM of reagent is prepared by dissolving 548.74 mg of a compound of Formula 4 in 1 mL of dimethyl sulfoxide. 1 mM reagent was prepared by dissolving 515.74 mg of the compound of Formula 5, 542.8 mg of the compound of Formula 6, and 429 mg of the comparative compound, respectively, in 1 mL of dimethyl sulfoxide.

Experimental Example 2 Fluorescence Analysis of Compounds of Formulas 4 to 6 by Real-Time PCR

The comparative material and the compounds of Formulas 4, 5 and 6 were compared and verified by real-time PCR through the intercalating method, which is a method using a double stranded DNA binding dye. CDNA extracted from HeLa cell was used, and primers were used as b-actin (Forward 5'-ATCTGGCACCACACCTTCTA-3 ', Reverse 5'-CGTCATACTCCTGCTTGCTG-3'). Real-time PCR reactions were performed using the CFX96 touch real-time PCR detection system (BIO-RAD). The PCR mixture comprises 2 uL of forward primer, 2 uL of reverse primer, 10 uL of PCR master mix (TAKARA), a comparative substance and 1 uL of each compound (Formula 4, Formula 5, Formula 6) and 1 uL of template cDNA. A total of 20 uL of reaction solution. PCR conditions were as follows: (a) Pre-denaturing step 1 minute at 95 ° C (b) 30 cycles 1 cycle consisting of 15 seconds at 95 ° C, 30 seconds at 55 ° C and 1 minute at 72 ° C And detects a fluorescence wavelength of 650 nm at a cycle of 1 minute at 72 ° C.

Comparative material confirmed a result that can not detect DNA synthesis. As shown in FIGS. 1 and 2 in the comparative material, no DNA synthesis fluorescence signal was observed, and as shown in FIG. 3, the Cq value was not confirmed at all.

On the other hand, the compound of Formula 4 and the compound of Formula 5 can be seen to fluoresce in combination with DNA when DNA amplification, as shown in FIG. It was also confirmed that the Cq value was also detected (FIG. 6). In the case of the compound of Formula 6, the fluorescence did not show superior fluorescence as the compounds of Formulas 4 and 5, but exhibited an appropriate degree of fluorescence. As can be seen in Figure 5 (melt curve) the compound of the formula 4 to 6 showed the same results as the above results.

Experimental Example 3 Analysis of Nucleic Acid Staining Dye by Blood Cell Staining Reagent

FACS analysis

Analysis was performed using Fluorescence Activated Cell Sorting (hereinafter referred to as FACS) to verify blood cell staining reagents prepared using comparative substances and compounds of Formulas 4, 5 and 6. Samples were prepared by lysing erythrocytes using Sysmex viscosity control reagent E-check (high concentration) using sysmex-4DL and removing the supernatant after centrifugation to prepare samples using the remaining white blood cells. The prepared samples were treated with blood cell staining reagents made of the respective compounds, FACS analysis was performed, and white blood cells and comparative substances that were not treated with blood cell staining reagents were used as controls. Thermo fisher's Attune NxT instrument was used and measured under conditions of FSC 180, SSC 320, BL1 180, RL1 150, RL2 165, YL1 200, and analyzed at APC wavelength. As shown in FIG. 7, all of the blood cell staining reagents prepared with the compounds of Chemical Formulas 4 to 6 were stained. It was confirmed that the comparative material was also slightly stained, but when the change in the fluorescence intensity was confirmed after treating the blood cell staining reagent, the intensity of the fluorescence intensity of the dyeing reagent using the compound of formulas 4 to 6 was much stronger than that of the comparative material. It was confirmed that (Fig. 8).

Experimental Example 4 Identification of Nucleic Acid Dyeing Dye through Cell Experiment

Fluorescence Microscopy Analysis with Live Cells

HeLa cell lines cultured in Dulbeco'Modified Eagle'Media (DMEM), 10% FBS, 1% Penicillin / Streptomycin medium were dispensed with 1 × 10 ⁵ HeLa cell lines in a confocal dish for fluorescence microscopy. After 24 hours after dispensing, the medium was replaced with serum-free DMEM medium, and the control material prepared by dissolving in DMSO and the compound of Formula 4, Formula 5, and Formula 6 were treated to 2.5uM, respectively, and pH 7.4 10 After washing with mM Phosphate buffered saline (hereinafter referred to as 1X PBS), the phenol free DMEM medium was replaced with fluorescence microscopy. The analysis was performed under Nikon ECLIPSE Ti-U X200, Cy5 filter. In the case of the comparative material as a control from the analysis results shown in FIG. 9, the dye is stained in the cytoplasm outside the nucleus, not the cell nucleus, whereas three compounds of the dye dyes modified by the chemical structure shown in the present invention (Formula 4, Formula 5, Formula 6) was confirmed that all are stained in the nucleus. Among them, it was confirmed that the compound of the formula (5) is best stained in the nucleus.

The present invention is not limited to the above-described embodiments, but can be variously modified and changed by those skilled in the art, and can be used in various biological and chemical fields, as defined in the appended claims. It is included in the spirit and scope of the present invention.

Claims (11)

Fluorescent compound represented by the following formula (1)
[Formula 1]

In Chemical Formula 1
X is oxygen or sulfur,
R is a substituent represented by the following formula (2) or (3).
[Formula 2]

[Formula 3]

In Formulas 2 and 3, m and n are integers of 1 to 10, R ₂ is methyl, amine (NH ₂ ), methyl substituted amine (N (CH ₃ ) ₂ ), carboxylic acid (CO ₂ H), Sulfonic acid (SO ₃ H) or sodium sulfonate salt (-SO ₃ Na). According to claim 1, wherein the compound of Formula 1 is a fluorescent compound characterized in that the compound represented by the formula (4)
[Formula 4]

According to claim 1, wherein the compound of Formula 1 is a fluorescent compound characterized in that the compound represented by the formula (5)
[Formula 5]

According to claim 1, wherein the compound of Formula 1 is a fluorescent compound characterized in that the compound represented by the formula
[Formula 6]

An intermediate compound for preparing the compound of claim 1, wherein the intermediate compound is represented by the following Chemical Formula 10

Biomolecule labeling contrast agent composition comprising a fluorescent compound represented by the formula (1)
[Formula 1]

In Chemical Formula 1
X is oxygen or sulfur,
R is a substituent represented by the following formula (2) or (3).
[Formula 2]

[Formula 3]

In Formulas 2 and 3, m and n are integers of 1 to 10, R ₂ is methyl, amine (NH ₂ ), methyl substituted amine (N (CH ₃ ) ₂ ), carboxylic acid (CO ₂ H), Sulfonic acid (SO ₃ H) or sodium sulfonate salt (-SO ₃ Na). According to claim 6, wherein the compound of Formula 1 is a biomolecule labeling contrast composition, characterized in that the compound represented by the following formula (4)
[Formula 4]

According to claim 6, wherein the compound of formula 1 is a biomolecule labeling contrast composition, characterized in that the compound represented by the formula (5)
[Formula 5]

According to claim 6, wherein the compound of formula 1 is a biomolecule labeling contrast composition, characterized in that the compound represented by the following formula (6)
[Formula 6]

Biomolecule labeling kit comprising a contrast agent composition comprising a fluorescent compound represented by Formula 1 below
[Formula 1]

In Chemical Formula 1
X is oxygen or sulfur,
R is a substituent represented by the following formula (2) or (3).
[Formula 2]

[Formula 3]

In Formulas 2 and 3, m and n are integers of 1 to 10, R ₂ is methyl, amine (NH ₂ ), methyl substituted amine (N (CH ₃ ) ₂ ), carboxylic acid (CO ₂ H), Sulfonic acid (SO ₃ H) or sodium sulfonate salt (-SO ₃ Na). The biomolecule labeling kit of claim 10, wherein the biomolecule is DNA or RNA.

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