KR102846425B1 - Compound based cyanine, labeling dye, kit and contrast medium composition for biomolecule comprising the same - Google Patents

Abstract

The present invention relates to a novel cyanine compound, a dye for labeling biomolecules, a kit, and a contrast agent composition comprising the same.

Description

{COMPOUND BASED CYANINE, LABELING DYE, KIT AND CONTRAST MEDIUM COMPOSITION FOR BIOMOLECULE COMPRISING THE SAME}

The present invention relates to a novel cyanine compound, a dye for labeling biomolecules, a kit, and a contrast agent composition comprising the same.

In the field of biology, fluorescent dyes are used as a means of visualizing biological phenomena at the cellular level, both in vivo and in vitro, or for bioimaging and autopsy of diseased areas. While self-luminous biomolecules such as green fluorescent protein (GFP) exist, generally, biological tissues, cells, and even lower-level biological substances are stained with fluorescent dyes, or biomolecules such as proteins and nucleic acids are labeled with fluorescent dyes. These are then accompanied by optical equipment capable of detecting fluorescent signals, and various imaging techniques are used to obtain data.

Mainly used optical analysis equipment includes fluorescence microscopes for cell observation, confocal microscopes, flow cytometry, microarrays, quantitative PCR systems, electrophoresis systems for nucleic acid and protein separation and analysis, and real-time in vivo imaging systems. In addition to these equipment for research purposes, there are also in vitro diagnosis equipment based on nucleic acid and protein diagnostic kits (or biochips) that combine immunoassay techniques or PCR analysis and statistical techniques, and operating tables and endoscopic equipment for image-guided surgery, and equipment for diagnosis and treatment are known. New application areas and equipment with higher levels of resolution and data processing capabilities are continuously being developed.

In order to apply fluorescent dyes in the bio field, photobleaching is generally required when they are present in a medium where most biomolecules exist, i.e., an aqueous solution. It is desirable that the material has low bleaching and quenching phenomena, has high molecular extinction coefficient and quantum efficiency, and is stable under various pH conditions.

Although various fluorescent dyes have been utilized in various research fields, fluorescent dyes that satisfy all of the conditions described above in the bio field are rare. Representative structures currently in use include coumarins, cyanines, bodipy, fluoresceins, rhodamines, pyrenes, carbopyronins, oxazines, xanthenes, thioxanthenes, and acridines. Among these, rhodamine derivatives and polymethine cyanine derivatives are particularly prevalent. In particular, cyanine fluorescent dyes are linked to both ends of polymethine by a nitrogen-containing heterocycle, and not only have the structural advantage of being able to realize various fluorescence across the entire visible and near-infrared range from 450 nm to 800 nm by adjusting the length of the polymethine, but also have the optical characteristic of generally having a very high absorption coefficient. However, as the length of the polymethine increases, the problem of the photostability of the cyanine dyes deteriorating has been raised.

Therefore, there is a continuous demand for the development of cyanine dyes that can maintain photostability for long periods of time in aqueous solutions.

The present invention aims to provide a novel cyanine compound that can be widely used to observe the identification of biomolecules in the field of optical imaging, and a dye, kit, and contrast agent for labeling biomolecules containing the same.

In addition, the present invention aims to provide a novel cyanine compound that can maintain photostability for a long period of time in an aqueous solution.

According to one aspect of the present invention for solving the above-described technical problem, a cyanine compound represented by the following chemical formula 1 can be provided.

[Chemical Formula 1]

Here,

Ar ₁ and Ar ₂ are each independently a substituted or unsubstituted aryl or a substituted or unsubstituted heteroaryl, and Ar ₁ and at least one of Ar ₂ is a substituted or unsubstituted fluoranthene,

Y ₁ and Y ₂ are each independently selected from sulfur, oxygen, selenium, NR ₄ , CR ₄ R ₅ , SiR ₄ R ₅ and -CR ₄ =CR ₅ -,

R ₁ to R ₅ may each independently be selected from hydrogen, deuterium, substituted or unsubstituted C ₁ -C ₁₀ alkyl, substituted or unsubstituted C ₂ -C ₁₀ alkenyl, substituted or unsubstituted C ₂ -C ₁₀ alkynyl, substituted or unsubstituted C ₂ -C ₁₀ alkoxy, substituted or unsubstituted aryloxy, substituted or unsubstituted C ₁ -C ₁₀ haloalkyl, halogen, cyano, hydroxy, substituted or unsubstituted amino, substituted or unsubstituted amide, sulfhydryl, nitro, carboxyl, carboxylate, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted aralkyl, quaternary ammonium, phosphoric acid, phosphate, ketone (-COR ₆ ), aldehyde, ester (-COOR ₆ ), acyl chloride, sulfonic acid, sulfonate, and -LX functional group. there is.

When it is a ketone group (-COR ₆ ) or an ester group (-COOR ₆ ), R ₆ is selected from a substituted or unsubstituted C ₁ -C ₁₀ alkyl group, a substituted or unsubstituted C ₂ -C ₁₀ alkenyl group, a substituted or unsubstituted C ₂ -C ₁₀ alkynyl group, a substituted or unsubstituted C ₂ -C ₁₀ alkoxy group, a substituted or unsubstituted C ₁ -C ₁₀ haloalkyl group, and a substituted or unsubstituted C ₁ -C ₁₀ aminoalkyl group,

In the -LX functional group, L is a linker selected from 1 to 20 atoms selected from carbon, nitrogen, oxygen and sulfur, -NHCOO-, -CONH-, -CH ₂ NH-, -CH ₂ NR ₆ -, -COO-, -SO ₂ NH-, -HN-C(=NH)-NH-, -NR ₆ -, -(CH ₂ -CH ₂ -O-)p-, -CH=CH-, -C≡C-, -Ar- and -CO-Ar-NR ₆ -, R ₆ is selected from hydrogen, substituted or unsubstituted C ₁ -C ₆ alkyl, substituted or unsubstituted C ₂ -C ₁₀ alkenyl, substituted or unsubstituted C ₂ -C ₁₀ alkynyl, substituted or unsubstituted C ₂ -C ₆ alkoxy and substituted or unsubstituted C ₁ -C ₆ haloalkyl, Ar is aryl, and p is 1 to is an integer of 100, and X is carboxyl, succinimidyl ester, sulfo-succinimidyl ester, isothiocyanate, maleimide, haloacetamide, hydrazide, vinylsulphone, dichlorotriazine, phosphoramidite, alkyl halide, acyl halide, carbohydrazide, hydroxylamine, ketone, alkyne, azide, aliphatic and aromatic amine, sulfotetrafluorophenyl ester, sulfodichlorophenyl ester ester), carbonyl azide, sulfonyl chloride, sulfonyl fluoride, boronic acid, isocyanate, halogen-substituted triazine, halogen-substituted pyridine, halogen-substituted diazine, tetrafluorophenyl ester, imido ester, azidonitrophenyl, glyoxal, and aldehyde.

In addition, when R ₁ , R ₂ , R ₃ , R ₄ , R ₅ or R ₆ is substituted, any carbon or terminal carbon in the functional group may be substituted with at least one selected from sulfonic acid, sulfonate, ketone, aldehyde, carboxylic acid, carboxylate, phosphoric acid, phosphate, acyl chloride, polyalkylene oxide, quaternary ammonium salt, ester and amide, and n is 0 or an integer from 1 to 5.

The cyanine compound represented by chemical formula 1 has a symmetrical or asymmetrical skeleton and includes a heterocycle to which fluoranthene is bonded.

The cyanine compound according to an embodiment of the present invention can generate a fluorescence signal in a relatively long wavelength range compared to a conventional cyanine dye having a polymethine of the same length by symmetrically or asymmetrically including a heterocycle to which fluoranthene is bonded.

For example, a cyanine compound according to one embodiment of the present invention having a polymethine of the same length as a commercially available Cy3 dye can generate a fluorescence signal in a wavelength range similar to a commercially available Cy5 dye.

That is, according to various embodiments of the present invention, by introducing a heterocycle to which fluoranthene is bonded, excitation and emission are possible in a relatively long wavelength range compared to conventional commercialized cyanine dyes, and thus, it is possible to improve photostability compared to conventional commercialized cyanine dyes that are capable of excitation and emission in a similar wavelength range.

In addition, according to another aspect of the present invention, a dye for labeling a biomolecule comprising a cyanine compound represented by chemical formula 1 can be provided.

In addition, according to another aspect of the present invention, a biomolecule labeling kit comprising a cyanine compound represented by chemical formula 1 can be provided.

In addition, according to another aspect of the present invention, a contrast agent composition comprising a cyanine compound represented by chemical formula 1 can be provided.

The novel cyanine compound according to the present invention contains fluoranthene in the heterocycle, enabling absorption of longer wavelength light compared to conventional cyanine dyes with the same polymethine moiety. Accordingly, with a shorter polymethine structure, it is possible to realize a fluorescence signal similar to that of conventional commercially available cyanine dyes with relatively longer polymethine moieties, while at the same time exhibiting relatively improved photostability.

To facilitate understanding of the present invention, certain terms are defined herein for convenience. Unless otherwise defined herein, scientific and technical terms used herein may have the meanings commonly understood by those of ordinary skill in the relevant technical field.

Additionally, unless the context specifically states otherwise, a singular term includes its plural form, and a plural term may also include its singular form.

Novel cyanine compounds

According to one aspect of the present invention, a cyanine compound represented by the following chemical formula 1 can be provided.

[Chemical Formula 1]

In the cyanine compound represented by chemical formula 1, Ar ₁ and Ar ₂ are each independently a substituted or unsubstituted aryl or a substituted or unsubstituted heteroaryl, and Ar ₁ and at least one of Ar ₂ has a symmetrical or asymmetrical skeleton which is a substituted or unsubstituted fluoranthene.

The cyanine compound according to an embodiment of the present invention can generate a fluorescence signal in a relatively long wavelength range compared to a conventional cyanine dye having a polymethine of the same length by symmetrically or asymmetrically including a heterocycle to which fluoranthene is bonded.

For example, a cyanine compound according to one embodiment of the present invention having a polymethine of the same length as a commercially available Cy3 dye can generate a fluorescence signal in a wavelength range similar to a commercially available Cy5 dye.

That is, according to various embodiments of the present invention, by introducing a heterocycle to which fluoranthene is bonded, excitation and emission are possible in a relatively long wavelength range compared to conventional commercialized cyanine dyes, and thus, it is possible to improve photostability compared to conventional commercialized cyanine dyes that are capable of excitation and emission in a similar wavelength range.

Y ₁ and Y ₂ are each independently selected from sulfur, oxygen, selenium, NR ₄ , CR ₄ R ₅ , SiR ₄ R ₅ and -CR ₄ =CR ₅ -,

R ₁ to R ₅ may each independently be selected from hydrogen, deuterium, substituted or unsubstituted C ₁ -C ₁₀ alkyl, substituted or unsubstituted C ₂ -C ₁₀ alkenyl, substituted or unsubstituted C ₂ -C ₁₀ alkynyl, substituted or unsubstituted C ₂ -C ₁₀ alkoxy, substituted or unsubstituted aryloxy, substituted or unsubstituted C ₁ -C ₁₀ haloalkyl, halogen, cyano, hydroxy, substituted or unsubstituted amino, substituted or unsubstituted amide, sulfhydryl, nitro, carboxyl, carboxylate, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted aralkyl, quaternary ammonium, phosphoric acid, phosphate, ketone (-COR ₆ ), aldehyde, ester (-COOR ₆ ), acyl chloride, sulfonic acid, sulfonate, and -LX functional group. there is.

When Y ₁ or Y ₂ is CR ₄ R ₅ , R ₄ and R ₅ may be combined with each other to form a 4-membered, 5-membered or 6-membered hydrocarbon ring.

As used herein, the C _a -C _b functional group refers to a functional group having a to b carbon atoms. For example, the C _a -C _b alkyl group refers to a saturated aliphatic group including a straight-chain alkyl group and a branched alkyl group having a to b carbon atoms. The straight-chain or branched alkyl group has 10 or fewer carbon atoms in its main chain (e.g., straight chain of C ₁ -C ₁₀ , branched chain of C ₃ -C ₁₀ ), preferably 4 or fewer, more preferably 3 or fewer.

Specifically, alkyl can be methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, i-butyl, t-butyl, pent-1-yl, pent-2-yl, pent-3-yl, 3-methylbut-1-yl, 3-methylbut-2-yl, 2-methylbut-2-yl, 2,2,2-trimethyleth-1-yl, n-hexyl, n-heptyl and n-octyl.

Additionally, alkoxy herein refers to both an -O-(alkyl) group and an -O-(unsubstituted cycloalkyl) group, and is a straight-chain or branched hydrocarbon having one or more ether groups and 1 to 10 carbon atoms.

Specifically, it includes, but is not limited to, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, n-hexoxy, 1,2-dimethylbutoxy, cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy, etc.

Additionally, halogen herein means fluoro (-F), chloro (-Cl), bromo (-Br), or iodo (-I), and haloalkyl means alkyl substituted with the above-mentioned halogen. For example, halomethyl means methyl (-CH ₂ X, -CHX ₂ , or -CX ₃ ) in which at least one of the hydrogens of the methyl is replaced with a halogen.

In this application, aralkyl is a functional group in which aryl is substituted on the carbon of alkyl, and is a general term for -(CH ₂ ) _n Ar. Examples of aralkyl include benzyl (-CH ₂ C ₆ H ₅ ) or phenethyl (-CH ₂ CH ₂ C ₆ H ₅ ).

In the -LX functional group, L is a linker selected from 1 to 20 atoms selected from carbon, nitrogen, oxygen and sulfur, -NHCOO-, -CONH-, -CH ₂ NH-, -CH ₂ NR ₆ -, -COO-, -SO ₂ NH-, -HN-C(=NH)-NH-, -NR ₆ -, -(CH ₂ -CH ₂ -O- ₎ p-, -CH=CH-, -C≡C-, -Ar- and -CO-Ar-NR ₆ -, wherein, R ₆ is selected from hydrogen, substituted or unsubstituted C _{1 -C 6} alkyl, substituted or unsubstituted C ₂ -C ₁₀ alkenyl, substituted or unsubstituted C ₂ -C 10 alkynyl, substituted or unsubstituted C 2 -C ₆ alkoxy and substituted or unsubstituted C ₁ -C ₆ haloalkyl, Ar is aryl, and p is 1 to It is an integer of 100.

X is carboxyl, succinimidyl ester, sulfo-succinimidyl ester, isothiocyanate, maleimide, haloacetamide, hydrazide, vinylsulphone, dichlorotriazine, phosphoramidite, alkyl halide, acyl halide, carbohydrazide, hydroxylamine, ketone, alkyne, azide, aliphatic and aromatic amine, sulfotetrafluorophenyl ester, sulfodichlorophenyl ester, carbonyl A reactive group selected from carbonyl azide, sulfonyl chloride, sulfonyl fluoride, boronic acid, isocyanate, halogen-substituted triazine, halogen-substituted pyridine, halogen-substituted diazine, tetrafluorophenyl ester, imido ester, azidonitrophenyl, glyoxal, and aldehyde.

The cyanine compound according to various embodiments of the present invention may be capable of labeling a target biomolecule by binding to it through the reactive group described above.

Here, the reactive groups described above are functional groups that can react with functional groups such as amino groups, imino groups, thiol groups or hydroxyl groups of the target biomolecule, and can form a covalent bond such as an amide bond, an imide bond, a urethane bond, an ester bond or a guanidine bond between the cyanine compound and the target biomolecule.

In addition, the reactive group (X) can alleviate steric hindrance between a biomolecule and a cyanine compound by being bound to the main skeleton of the cyanine compound via a linker (L), thereby improving the labeling rate for biomolecules such as nucleic acids, proteins, and carbohydrates having complex structures.

In addition, when R ₁ , R ₂ , R ₃ , R ₄ , R ₅ or R ₆ is substituted, any carbon or terminal carbon in the functional group may be substituted with at least one selected from sulfonic acid, sulfonate, ketone, aldehyde, carboxylic acid, carboxylate, phosphoric acid, phosphate, acyl chloride, polyalkylene oxide, quaternary ammonium salt, ester and amide, and n is 0 or an integer from 1 to 5.

Here, the polyalkylene oxide includes polyethylene glycol (PEG), polypropylene glycol (PPG), polyethylene glycol-polypropylene glycol (PEG-PPG) copolymers, and N-substituted methacrylamide-containing polymers and copolymers as water-soluble polymer functional groups.

Here, the polyalkylene oxide may be further substituted as needed within the range where the properties of the polymer are maintained. For example, the substitution may be a chemical bond to increase or decrease the chemical or biological stability of the polymer. As a specific example, any carbon or terminal carbon in the polyalkylene oxide may be substituted with hydroxy, alkyl ether (methyl ether, ethyl ether, propyl ether, etc.), carboxylmethyl ether, carboxyethyl ether, benzyl ether, dibenzylmethylene ether, or dimethylamine. In one embodiment, the polyalkylene oxide may be a polyalkylene oxide terminated with methyl ether (mPEG), wherein mPEG is represented by the chemical formula -(CH ₂ CH ₂ O) _n CH ₃ , and the size of mPEG may vary depending on the size of n corresponding to the number of ethylene glycol repeating units.

In addition, the cyanine compound represented by Chemical Formula 1 may have a structure further including a counter ion. The counter ion may be an organic or inorganic anion and may be appropriately selected in consideration of the solubility and stability of the cyanine compound.

Examples of counter ions of the cyanine compound according to one embodiment of the present invention include inorganic acid anions such as phosphoric acid hexafluoride ion, halogen ion, phosphoric acid ion, perchlorate ion, periodate ion, antimony hexafluoride ion, tartaric acid hexafluoride ion, fluoroboric acid ion, and tetrafluoride ion; and organic acid ions such as thiocyanate ion, benzenesulfonic acid ion, naphthalenesulfonic acid ion, p-toluenesulfonic acid ion, alkylsulfonic acid ion, benzenecarboxylic acid ion, alkylcarboxylic acid ion, trihaloalkylcarboxylic acid ion, alkylsulfonic acid ion, trihaloalkylsulfonic acid ion, and nicotinic acid ion. In addition, metal compound ions such as bisphenylditol, thiobisphenol chelate, and bisdiol-α-diketone, metal ions such as sodium and potassium, and quaternary ammonium salts can also be selected as counter ions.

In the cyanine compound represented by chemical formula 1, when n is 1 or more, two adjacent R ₃ can combine with each other to form a hydrocarbon ring. At this time, the hydrocarbon ring can be a 4-membered, 5-membered, or 6-membered ring, and can optionally include at least one heteroatom selected from -S-, -O-, -Se-, >NR ₄ , and >SiR ₄ R ₅ .

In addition, when Ar ₁ or Ar ₂ is substituted, any carbon in the functional group is deuterium, substituted or unsubstituted C ₁ -C ₁₀ alkyl, substituted or unsubstituted C ₂ -C ₁₀ alkenyl, substituted or unsubstituted C ₂ -C ₁₀ alkynyl, substituted or unsubstituted C ₂ -C ₁₀ alkoxy, substituted or unsubstituted aryloxy, substituted or unsubstituted C ₁ -C ₁₀ haloalkyl, halogen, cyano, hydroxy, substituted or unsubstituted amino, substituted or unsubstituted amide, sulfhydryl, nitro, carboxyl, carboxylate, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted aralkyl, quaternary ammonium, phosphoric acid, phosphate, ketone (-COR ₆ ), aldehyde, ester (-COOR ₆ ), acyl chloride, sulfonic acid, sulfonate, and -Can be substituted with at least one selected from the LX functional group.

More specifically, the cyanine compound represented by Chemical Formula 1 can be represented by Chemical Formulas 2 to 6 below.

[Chemical Formula 2]

[Chemical Formula 3]

[Chemical Formula 4]

[Chemical Formula 5]

[Chemical Formula 6]

Here, R ₁₁ to R ₂₆ are each independently hydrogen, deuterium, substituted or unsubstituted C ₁ -C ₁₀ alkyl, substituted or unsubstituted C ₂ -C ₁₀ alkenyl, substituted or unsubstituted C ₂ -C ₁₀ alkynyl, substituted or unsubstituted C ₂ -C ₁₀ alkoxy, substituted or unsubstituted aryloxy, substituted or unsubstituted C ₁ -C ₁₀ haloalkyl, halogen, cyano, hydroxy, substituted or unsubstituted amino, substituted or unsubstituted amide, sulfhydryl, nitro, carboxyl, carboxylate, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted aralkyl, quaternary ammonium, phosphoric acid, phosphate, ketone (-COR ₆ ), aldehyde, ester (-COOR ₆ ), acyl chloride, sulfonic acid, sulfonate, and -LX is selected from functional groups, wherein Y ₃ is sulfur, oxygen, selenium, NR ₄ , CR ₄ R ₅ , SiR ₄ R ₅ or -CR ₄ =CR ₅ -.

Specific examples of the compounds represented by the chemical formulas 1 to 6 are as follows.

[Compound 1]

[Compound 2]

[Compound 3]

[Compound 4]

[Compound 5]

[Compound 6]

[Compound 7]

[Compound 8]

[Compound 9]

[Compound 10]

[Compound 11]

[Compound 12]

[Compound 13]

[Compound 14]

[Compound 15]

[Compound 16]

[Compound 17]

[Compound 18]

[Compound 19]

[Compound 20]

[Compound 21]

[Compound 22]

[Compound 23]

[Compound 24]

[Compound 25]

[Compound 26]

[Compound 27]

[Compound 28]

[Compound 29]

[Compound 30]

[Compound 31]

[Compound 32]

[Compound 33]

[Compound 34]

[Compound 35]

[Compound 36]

The excitation and luminescence properties of the cyanine compounds according to various embodiments of the present invention are determined by a polymethine structure corresponding to a chromophore and a heterocycle having fluoranthene bonded symmetrically or asymmetrically to both ends thereof. It should be understood that the excitation and luminescence properties of the cyanine compounds intended in the present invention can be maintained regardless of the type of reactive group introduced into the compounds represented by Chemical Formulas 1 to 6.

The biomolecule that is the target of the cyanine compound represented by Chemical Formula 1 to Chemical Formula 6 disclosed herein may be at least one selected from antibodies, lipids, proteins, peptides, carbohydrates, and nucleic acids (including nucleotides).

Specific examples of lipids include fatty acids, phospholipids, and lipopolysaccharides, while specific examples of carbohydrates include monosaccharides, disaccharides, and polysaccharides (e.g., dextrans).

At this time, the biomolecule may include at least one selected from amino, sulfhydryl, carbonyl, hydroxyl, carboxyl, phosphate, and thiophosphate as a functional group for reacting with any functional group of the cyanine compound represented by Chemical Formulas 1 to 6 or a reactive group bonded to the cyanine compound represented by Chemical Formulas 1 to 6, or may have a derivative form thereof.

Additionally, the biomolecule may be an oxy or deoxy polynucleic acid comprising at least one selected from amino, sulfhydryl, carbonyl, hydroxyl, carboxyl, phosphate and thiophosphate or having a derivative form thereof.

In addition, in addition to biomolecules, the cyanine compounds represented by Chemical Formulas 1 to 6 can be used to label drugs, hormones (including receptor ligands), receptors, enzymes or enzyme substrates, cells, cell membranes, toxins, microorganisms or nanobiomaterials (such as polystyrene microspheres) containing at least one selected from amino, sulfhydryl, carbonyl, hydroxyl, carboxyl, phosphate and thiophosphate.

Dyes, kits and contrast agent compositions for biomolecule labeling

According to another aspect of the present invention, a dye, kit and contrast agent composition for biomolecule labeling can be provided, the dye, kit and contrast agent composition including at least one selected from the cyanine compounds represented by Chemical Formulas 1 to 6.

Additionally, if necessary, the biomolecule labeling kit may further include enzymes, solvents (buffers, etc.), and other reagents for reaction with target biomolecules.

Here, a buffer selected from the group consisting of a phosphate buffer, a carbonate buffer, and a Tris buffer, an organic solvent selected from dimethyl sulfoxide, dimethylformamide, dichloromethane, methanol, ethanol, and acetonitrile, or water can be used as a solvent, and it is possible to control the solubility by introducing various functional groups to the cyanine compound depending on the type of solvent.

In addition, the contrast agent composition according to the above embodiment may further include a pharmaceutically acceptable carrier in addition to the cyanine compound represented by Chemical Formula 1 to Chemical Formula 6 for oral or parenteral administration.

Specific examples of pharmaceutically acceptable carriers include lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil.

Below, a method for detecting a biomolecule using the biomolecule labeling dye or biomolecule labeling kit described above will be described.

Method for detecting biomolecules using biomolecule labeling dyes

The method of labeling a biomolecule with a cyanine compound represented by Chemical Formula 1 to Chemical Formula 6 can be applied to the detection of all biomolecules by a detection method that measures the fluorescence of the labeled solid or semi-solid state biomolecule.

By using cyanine compounds instead of conventional fluorescent dyes, a highly sensitive, chemically stable, and highly operable detection method can be provided.

According to various embodiments of the present invention, a method of labeling a biomolecule by directly reacting a cyanine-based compound with the target biomolecule or a method of labeling by reacting a target biomolecule with a probe labeled with a cyanine-based compound can be implemented.

Additionally, a method for detecting biomolecules using target-specific interactions can be implemented by introducing an appropriate reactive group into a cyanine compound depending on the type of target biomolecule.

In addition, a method for identifying biomolecules labeled with cyanine compounds through electrophoresis may be implemented.

DNA microarray method

The DNA microarray method labels the target nucleic acid to be detected by reacting it with a dye, prepares a single-chain probe nucleic acid having a complementary base sequence to the target nucleic acid, and hybridizes the single-chain denatured target nucleic acid and the probe nucleic acid on a substrate to measure the fluorescence of the target nucleic acid.

In the present detection method, as the probe nucleic acid fixed to the substrate, when investigating the expression of a gene, a library of cDNA such as cDNA, a library of genomes, or a nucleic acid prepared by amplification by PCR using the entire genome as a template can be used.

In addition, when investigating genetic mutations, etc., various oligonucleotides that correspond to mutations, etc. can be synthesized and used based on a known sequence that serves as a standard.

The method for fixing probe nucleic acids to a substrate can be selected according to the type of nucleic acid and the type of substrate. For example, a method can be used to electrostatically bind DNA to a cation-treated substrate, such as polylysine, by utilizing the charge of the DNA.

Meanwhile, a target nucleic acid labeled with a labeling dye is prepared by mixing and reacting a single-stranded target nucleic acid with a labeling dye. The reaction temperature is preferably room temperature to 60°C, and the reaction time is preferably 2 to 48 hours.

Next, the labeled target nucleic acid is spotted onto the substrate and hybridized.

Hybridization is preferably performed at room temperature to 70°C for 2 to 48 hours. Through hybridization, a target nucleic acid with a complementary base sequence to the probe nucleic acid selectively binds to the probe nucleic acid. The substrate is then washed and dried at room temperature.

Next, the fluorescence intensity of the dry substrate surface is measured using a fluorescence laser scanner. The level of gene expression can be monitored based on the fluorescence intensity.

Meanwhile, although the above hybridization has been described with respect to a method of fixing a probe nucleic acid to a substrate, a method of fixing a target nucleic acid labeled with a dye in advance to a substrate and spotting the probe nucleic acid on the substrate may also be used.

PCR method

The PCR method labels a probe complementary to the base sequence of the target nucleic acid to be detected with a dye, reacts the target nucleic acid with the probe before or after amplification of the target nucleic acid, and measures the fluorescence of the target nucleic acid.

Specifically, the elongation reaction of the target nucleic acid is carried out by an enzyme (DNA polymerase, RNA polymerase), and at this time, the enzyme recognizes the double-stranded nucleic acid sequence formed by the target nucleic acid and the primer composed of oligonucleotides, and the elongation reaction is carried out from this recognized position, amplifying only the target gene region.

When an enzyme synthesizes, the synthetic reaction takes place using nucleotides (dNTP, NTP) as raw materials.

At this time, if a nucleotide having a dye is mixed with a normal nucleotide (dNTP, NTP) at an arbitrary ratio, a nucleic acid with a dye introduced at that ratio can be synthesized.

Additionally, by introducing nucleotides having amino groups at an arbitrary ratio by PCR and then combining a labeling dye, a nucleic acid with a labeling dye introduced can be synthesized.

When an enzyme synthesizes, the synthesis reaction takes place using nucleotides as raw materials. If the OH of the 3' nucleotide is changed to H, the nucleic acid elongation reaction no longer takes place and the reaction ends at that point.

This nucleotide, ddNTP (dideoxy nucleotide triphospate), is called a terminator.

When synthesizing nucleic acids by mixing terminators into regular nucleotides, the terminators are introduced with a certain probability and the reaction is terminated, so nucleic acids of various lengths are synthesized.

When this is separated by size using gel electrophoresis, the DNA is arranged in order of length. Here, if each type of base of the terminator is labeled with a different labeling dye, a tendency dependent on each base is observed at the end point (3' end) of the synthetic reaction, and by reading the fluorescence information starting from the labeling dye labeled on the terminator, the base sequence information of the target nucleic acid can be obtained.

Additionally, a primer labeled with a pre-labeled dye instead of a terminator can be used to hybridize with the target nucleic acid.

PNA (peptide nucleic acid) can also be used as a probe. PNA replaces the pentose-phosphate backbone of nucleic acids with a polyamide backbone made up of glycine units. Its three-dimensional structure closely resembles that of nucleic acids, and it binds very specifically and strongly to nucleic acids with complementary base sequences. This allows it to be used not only in conventional DNA analysis methods such as ISH (in-situ hybridization), but also as a reagent for telomere research by applying it to telomere PNA probes.

Detection can be performed, for example, by contacting double-stranded DNA with PNA, which has a base sequence complementary to all or part of the base sequence of the DNA and is labeled with a labeling dye, to hybridize, heating the mixture to produce single-stranded DNA, slowly cooling the mixture to room temperature to prepare a PNA-DNA complex, and measuring its fluorescence.

The above example describes a method of amplifying a target nucleic acid by PCR and measuring the fluorescence of the product. However, in this method, it is necessary to confirm the size of the product by electrophoresis and then measure the fluorescence intensity to investigate the amount of the amplified product.

To this end, the amount of product can be measured in real time using a probe designed to generate fluorescence by utilizing the energy transfer of a fluorescent dye and hybridizing it to the product of the PCR method.

For example, labeled DNA for donors and acceptors can be used. Specific detection methods include molecular beacon methods, TaqMan-PCR, and cycling probe methods, which identify the presence of specific nucleic acid sequences.

Additionally, when proteins are the target of detection, dyes are used to detect proteins after electrophoresis.

Typically, a method is used to stain proteins by penetrating a dye, such as Coomassie Brilliant Blue (CBB), into the gel after electrophoresis and then irradiating it with UV light to cause it to glow.

However, while the conventional method using dyes is simple, its sensitivity is low, at around 100 ng, making it unsuitable for detecting trace amounts of protein. Furthermore, because the dyes must penetrate through the gel, the staining process requires a significant amount of time.

In this regard, in the present invention, proteins are labeled by separating proteins by size through electrophoresis and then binding a labeling dye to the separated proteins.

The labeling dye of the present invention possesses a reactive group, reacts rapidly and quantitatively with proteins, and exhibits high sensitivity, making it suitable for detecting trace amounts of proteins. Furthermore, size-separated proteins can be identified by mass spectrometry.

Here, any of the simple proteins such as albumin, globulin, glutelin, histone, protamine, and collagen, and complex proteins such as nucleoprotein, glycoprotein, riboprotein, phosphoprotein, and metalloprotein can be detected.

For example, three types of labeling dyes are used to correspond to the staining dyes of phosphoprotein, glycoprotein, and total protein, and in a protein sample separated by two-dimensional electrophoresis, phosphoprotein, glycoprotein, and total protein can be stained.

Additionally, since proteins can be identified by mass spectrometry such as TOF-Mass, it can be applied to the diagnosis and treatment of diseases such as cancer or viral infections that produce specific proteins.

Collagen is a protein that constitutes the connective tissue of animals and has a unique fibrous structure. It is composed of three polypeptide chains, and these peptide chains assemble to form three-stranded chains. Collagen is generally a protein with very low immunogenicity and is widely used in fields such as food, cosmetics, and pharmaceuticals.

Aptamers can also be used as probes. Aptamers are composed of oligonucleotides and can assume various characteristic three-dimensional structures depending on their base sequence. Therefore, they can bind to any biomolecule, including proteins, through their three-dimensional structures. This property can be exploited by binding aptamers labeled with a dye to a specific protein, allowing the subject to be indirectly detected from changes in fluorescence resulting from structural changes in the protein upon binding to the subject.

Other detection methods

In addition, the labeling dye of the present invention can also be used in a method for detecting biomolecules using specific binding.

That is, in detecting a subject composed of a biomolecule or a subject modified by a modifier, one of the binding substances that specifically bind to the subject or the binding substances that specifically bind to the modifier can be labeled with a labeling dye, and fluorescence from the labeled binding substance can be measured.

Here, the combination of the subject or the modifier and the binding material may use antigen-antibody, hapten-antihapten antibody, biotin-avidin, Tag-anti Tag antibody, lectin-glycoprotein or hormone-receptor.

Specifically, a binding substance such as an antibody labeled with a labeling dye is reacted with an antigen present in a substrate, solution, bead, or antibody, so that a specific antigen can be detected through antigen-specific interaction of the antibody.

Antigens can include proteins, polysaccharides, nucleic acids, and peptides. In addition to antigens, haptens such as low-molecular-weight molecules such as FITC or dinitrophenyl groups can also be used. At this time, combinations of antigens (or haptens) and antibodies include GFP and anti-GFP antibodies, and FITC and anti-FITC antibodies.

Labeled antigens can be used in various measurement methods such as immunostaining, ELISA, Western blotting, and flow cytometry.

Furthermore, the labeling dye of the present invention can be used to observe intracellular signaling phenomena. Various enzymes are involved in internal signaling and the resulting cellular responses. A representative signaling phenomenon is known to involve the activation of specific protein kinases, which induce protein phosphorylation and initiate signaling.

The binding and hydrolysis of nucleotides (e.g., ATP or ADP) play a crucial role in their activity, and by introducing a labeling dye into a nucleotide derivative, intracellular signaling phenomena can be observed with high sensitivity.

Additionally, the labeling dye of the present invention can be used to observe gene expression phenomena using RNA interference (RNAi).

RNAi is a method of suppressing expression by degrading the mRNA of a target gene by introducing double-stranded RNA (dsRNA) into cells. It is possible to observe the RNAi phenomenon by labeling the designed dsRNA with a labeling dye.

In addition, the labeling dye of the present invention can be used as a dye for confirming the transcription level of a target nucleic acid or the expression level of a target protein by including a reactive group capable of labeling a target nucleic acid or a target protein in a tissue or cell.

Manufacturing example

Manufacturing Example 1: Preparation of Compound 1

Intermediate 1-1 (2.0 g), 1,3-propanesultone (2.2 g), and sulfolane (20 mL) were added to a reactor and stirred at 140°C for 36 hours. The reaction mixture was cooled to room temperature, isopropyl alcohol was added, and the mixture was filtered to obtain 2.3 g of Intermediate 1-2.

2.3 g of intermediate 1-2, 3.4 g of diphenylformamidine, and 69 mL of acetic acid were added to a reactor and stirred under reflux for 18 hours. After the reaction solution was cooled to room temperature, EA was added and filtered to obtain 2.5 g of intermediate 1-3.

0.3 g of intermediate 1-3, 0.2 g of intermediate 1-4 (Tetrahedron letters, 48(33), 5825; 2007), 0.2 g of triethylamine, 0.2 g of acetic anhydride, and 9 mL of N,N-dimethylformamide were added to a reactor and stirred at room temperature for 4 hours. Then, ethyl acetate was added to the reaction solution, filtered, and separated by ODS column chromatography to obtain 30 mg of compound 1 (MS (ESI): m/z 953.2).

Manufacturing Example 2: Preparation of Compound 2

Compound 2 was prepared by reacting intermediate 1-3 and intermediate 1-5 (Bioorganic and medicinal chemistry letters, 22(2), 1242; 2012) in the same manner as in Manufacturing Example 1 (MS (ESI): m/z 874.2).

Manufacturing Example 3: Preparation of Compound 3

Compound 3 was prepared by reacting intermediates 1-6 and 1-7 in the same manner as in Manufacturing Example 1 (MS (ESI): m/z 605.3).

Experimental example

For each compound obtained in the above manufacturing examples 1 to 3, the absorption spectrum, emission spectrum, molar extinction coefficient, and quantum efficiency were measured and are shown in Table 1 below.

division menstruum λ _ex (nm) λ _em (nm) ε(Lmol ^-1 cm ^-1 ) φ Compound 1 PBS 603 627 84,000 0.060 Compound 2 PBS 597 621 84,000 0.100 Compound 3 DMSO 657 676 109,000 0.250

As described in Table 1 above, Compound 1, Compound 2 and Compound 3 synthesized according to Preparation Examples 1 to 3 contain a heterocycle to which fluoranthene is bonded, either symmetrically or asymmetrically.

At this time, it can be confirmed that the wavelength at which excitation and emission occur is similar to that of the commercially available Cy5 dye, although the length of the polymethine corresponding to the chromophore (chromogen) in compounds 1, 2, and 3 is the same as that of the commercially available Cy3 dye.

That is, according to various embodiments of the present invention, since excitation and emission are possible in a relatively long wavelength range compared to a cyanine compound having a simple indole structure by introducing a heterocycle to which fluoranthene is bonded, there is no need to cause structural instability by increasing the length of polymethine for excitation and emission in a long wavelength range.

In the above examples, the wavelength ranges where excitation and emission occur were confirmed only for compounds 1, 2, and 3, but it is also quite possible to predict that the cyanine compounds represented by chemical formulas 1 to 6 will exhibit fluorescence characteristics in a longer wavelength range than the excitation and emission wavelength ranges expected from the length of polymethine, as a heterocyclic structure to which fluoranthene is bonded is introduced.

Above, one embodiment of the present invention has been described, but a person having ordinary skill in the art will be able to modify and change the present invention in various ways by adding, changing, deleting or adding components, etc., within the scope that does not depart from the spirit of the present invention described in the claims, and this will also be considered to be included within the scope of the rights of the present invention.

Claims (8)

A cyanine compound represented by the following chemical formula 1:
[Chemical Formula 1]

Here,
Ar ₁ and Ar ₂ are each independently a substituted or unsubstituted phenyl, a substituted or unsubstituted naphthyl, a substituted or unsubstituted fluorene, a substituted or unsubstituted fluoranthene, a substituted or unsubstituted carbazole, a substituted or unsubstituted dibenzofuran, or a substituted or unsubstituted dibenzothiophene, wherein Ar ₁ and at least one of Ar ₂ is a substituted or unsubstituted fluoranthene,
When Ar ₁ or Ar ₂ is substituted, any carbon in the functional group is deuterium, C ₁ -C ₁₀ alkyl substituted or unsubstituted with at least one substituent, C 2 -C ₁₀ alkenyl substituted or unsubstituted with at least one substituent, C ₂ -C ₁₀ alkynyl substituted or unsubstituted with at least one substituent, C _{2 -C 10} alkoxy substituted or unsubstituted with at least one substituent, C ₆ -C ₂₄ aryloxy substituted or unsubstituted with at least one substituent, C ₁ -C ₁₀ haloalkyl substituted or unsubstituted with at least one substituent, halogen, cyano, hydroxy, amino substituted or unsubstituted with at least one substituent, amide substituted or unsubstituted with at least one substituent, sulfhydryl, nitro, carboxyl, carboxylate, C ₆ -C ₂₄ aryl substituted or unsubstituted with at least one substituent, C ₅ -C ₂₃ heteroaryl, aralkyl substituted or unsubstituted with at least one substituent, quaternary ammonium, phosphoric acid, phosphate, ketone (-COR ₆ ), aldehyde, ester (-COOR ₆ ), acyl chloride, sulfonic acid, sulfonate and -LX functional group,
Y ₁ and Y ₂ are each independently selected from sulfur, oxygen and CR ₄ R ₅ ,
R ₁ and R ₂ are each independently selected from hydrogen, deuterium, substituted or unsubstituted C ₁ -C ₁₀ alkyl, substituted or unsubstituted C ₂ -C ₁₀ alkenyl, substituted or unsubstituted C ₂ -C ₁₀ alkynyl, substituted or unsubstituted C ₂ -C ₁₀ alkoxy, substituted or unsubstituted aryloxy, substituted or unsubstituted C ₁ -C ₁₀ haloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted aralkyl, and -LX functional group,
R ₃ is hydrogen, deuterium, substituted or unsubstituted C ₁ -C ₁₀ alkyl, substituted or unsubstituted C ₂ -C ₁₀ alkenyl, substituted or unsubstituted C ₂ -C ₁₀ alkynyl, substituted or unsubstituted C ₂ -C ₁₀ alkoxy, substituted or unsubstituted aryloxy, substituted or unsubstituted C ₁ -C ₁₀ haloalkyl, halogen, substituted or unsubstituted amino, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted aralkyl or -LX functional group,
R ₄ and R ₅ are each independently selected from hydrogen, deuterium, substituted or unsubstituted C ₁ -C ₁₀ alkyl, substituted or unsubstituted C ₂ -C ₁₀ alkenyl, substituted or unsubstituted C ₂ -C ₁₀ alkynyl, substituted or unsubstituted C ₂ -C ₁₀ alkoxy, substituted or unsubstituted aryloxy, substituted or unsubstituted C ₁ -C ₁₀ haloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted aralkyl and -LX functional group,
In the -LX functional group, L is a linker selected from 1 to 20 atoms selected from carbon, nitrogen, oxygen and sulfur, -NHCOO-, -CONH-, -CH ₂ NH-, -CH ₂ NR ₆ -, -COO-, -SO ₂ NH-, -HN-C(=NH)-NH-, -NR ₆ -, -(CH ₂ -CH ₂ -O-)p-, -CH=CH-, -C≡C-, -Ar- and -CO-Ar-NR ₆ -, and R ₆ is hydrogen, C ₁ -C ₆ alkyl substituted or unsubstituted with at least one substituent, C ₂ -C ₁₀ alkenyl substituted or unsubstituted with at least one substituent, C ₂ -C ₁₀ alkynyl substituted or unsubstituted with at least one substituent, C ₂ -C ₆ alkoxy substituted or unsubstituted with at least one substituent and C ₁ -C ₆ substituted or unsubstituted with at least one substituent. is selected from haloalkyl, Ar is aryl, p is an integer from 1 to 100, X is carboxyl, succinimidyl ester, sulfo-succinimidyl ester, isothiocyanate, maleimide, haloacetamide, hydrazide, vinylsulphone, dichlorotriazine, phosphoramidite, alkyl halide, acyl halide, carbohydrazide, hydroxylamine, ketone, alkyne, azide, aliphatic and aromatic amine, sulfotetrafluorophenyl ester ester), sulfodichlorophenyl ester, carbonyl azide, sulfonyl chloride, sulfonyl fluoride, boronic acid, isocyanate, halogen-substituted triazine, halogen-substituted pyridine, halogen-substituted diazine, tetrafluorophenyl ester, imido ester, azidonitrophenyl, glyoxal, and aldehyde.
n is 0 or an integer from 1 to 5,
The above aryl is a single ring or an unsaturated aromatic ring containing 2 to 4 rings connected to each other by fusion or covalent bonds,
The above heteroaryl is an unsaturated aromatic ring in which one or more carbon atoms in the aryl are replaced with a heteroatom,
The above heteroatom is selected from non-carbon atoms including nitrogen (N), oxygen (O) and sulfur (S),
The above substituents are selected from sulfonic acid, sulfonate, ketone, aldehyde, carboxylic acid, carboxylate, phosphoric acid, phosphate, acyl chloride, hydroxy, polyalkylene oxide, quaternary ammonium salt, _C6 - _C24 aryl, ester and amide, wherein the polyalkylene oxide is terminated with methyl ether and contains ethylene glycol as a repeating unit.
A cyanine compound represented by the following chemical formula 1:
[Chemical Formula 1]

Here,
Ar ₁ and Ar ₂ are each independently a substituted or unsubstituted phenyl, a substituted or unsubstituted naphthyl, a substituted or unsubstituted fluorene, a substituted or unsubstituted fluoranthene, a substituted or unsubstituted carbazole, a substituted or unsubstituted dibenzofuran, or a substituted or unsubstituted dibenzothiophene, wherein Ar ₁ and at least one of Ar ₂ is a substituted or unsubstituted fluoranthene,
When Ar ₁ or Ar ₂ is substituted, any carbon in the functional group is deuterium, C ₁ -C ₁₀ alkyl substituted or unsubstituted with at least one substituent, C 2 -C ₁₀ alkenyl substituted or unsubstituted with at least one substituent, C ₂ -C ₁₀ alkynyl substituted or unsubstituted with at least one substituent, C _{2 -C 10} alkoxy substituted or unsubstituted with at least one substituent, C ₆ -C ₂₄ aryloxy substituted or unsubstituted with at least one substituent, C ₁ -C ₁₀ haloalkyl substituted or unsubstituted with at least one substituent, halogen, cyano, hydroxy, amino substituted or unsubstituted with at least one substituent, amide substituted or unsubstituted with at least one substituent, sulfhydryl, nitro, carboxyl, carboxylate, C ₆ -C ₂₄ aryl substituted or unsubstituted with at least one substituent, C ₅ -C ₂₃ heteroaryl, aralkyl substituted or unsubstituted with at least one substituent, quaternary ammonium, phosphoric acid, phosphate, ketone (-COR ₆ ), aldehyde, ester (-COOR ₆ ), acyl chloride, sulfonic acid, sulfonate and -LX functional group,
Y ₁ and Y ₂ are each independently selected from sulfur, oxygen and CR ₄ R ₅ ,
R ₁ and R ₂ are each independently selected from hydrogen, deuterium, substituted or unsubstituted C ₁ -C ₁₀ alkyl, substituted or unsubstituted C ₂ -C ₁₀ alkenyl, substituted or unsubstituted C ₂ -C ₁₀ alkynyl, substituted or unsubstituted C ₂ -C ₁₀ alkoxy, substituted or unsubstituted aryloxy, substituted or unsubstituted C ₁ -C ₁₀ haloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted aralkyl, and -LX functional group,
R ₃ is hydrogen, deuterium, substituted or unsubstituted C ₁ -C ₁₀ alkyl, substituted or unsubstituted C ₂ -C ₁₀ alkenyl, substituted or unsubstituted C ₂ -C ₁₀ alkynyl, substituted or unsubstituted C ₂ -C ₁₀ alkoxy, substituted or unsubstituted aryloxy, substituted or unsubstituted C ₁ -C ₁₀ haloalkyl, halogen, substituted or unsubstituted amino, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted aralkyl or -LX functional group,
R ₄ and R ₅ are each independently selected from hydrogen, deuterium, substituted or unsubstituted C ₁ -C ₁₀ alkyl, substituted or unsubstituted C ₂ -C ₁₀ alkenyl, substituted or unsubstituted C ₂ -C ₁₀ alkynyl, substituted or unsubstituted C ₂ -C ₁₀ alkoxy, substituted or unsubstituted aryloxy, substituted or unsubstituted C ₁ -C ₁₀ haloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted aralkyl and -LX functional group,
In the -LX functional group, L is a linker selected from 1-20 atoms selected from carbon, nitrogen, oxygen and sulfur, -NHCOO-, -CONH-, -CH ₂ NH-, -CH ₂ NR ₆ -, -COO-, -SO ₂ NH-, -HN-C(=NH)-NH-, -NR ₆ -, -(CH ₂ -CH ₂ -O-) _p -, -CH=CH-, -C≡C-, -Ar- and -CO-Ar-NR ₆ -, and R ₆ is hydrogen, C ₁ -C ₆ alkyl substituted or unsubstituted with at least one substituent, C ₂ -C ₁₀ alkenyl substituted or unsubstituted with at least one substituent, C ₂ -C ₁₀ alkynyl substituted or unsubstituted with at least one substituent, C ₂ -C ₆ alkoxy substituted or unsubstituted with at least one substituent and C ₁ -C ₆ substituted or unsubstituted with at least one substituent. is selected from haloalkyl, Ar is aryl, p is an integer from 1 to 100, X is carboxyl, succinimidyl ester, sulfo-succinimidyl ester, isothiocyanate, maleimide, haloacetamide, hydrazide, vinylsulphone, dichlorotriazine, phosphoramidite, alkyl halide, acyl halide, carbohydrazide, hydroxylamine, ketone, alkyne, azide, aliphatic and aromatic amine, sulfotetrafluorophenyl ester ester), sulfodichlorophenyl ester, carbonyl azide, sulfonyl chloride, sulfonyl fluoride, boronic acid, isocyanate, halogen-substituted triazine, halogen-substituted pyridine, halogen-substituted diazine, tetrafluorophenyl ester, imido ester, azidonitrophenyl, glyoxal, and aldehyde.
n is an integer from 1 to 5,
Two adjacent R ₃ are bonded to each other to form a 4- to 6-membered hydrocarbon ring optionally containing at least one heteroatom,
The above aryl is a single ring or an unsaturated aromatic ring containing 2 to 4 rings connected to each other by fusion or covalent bonds,
The above heteroaryl is an unsaturated aromatic ring in which one or more carbon atoms in the aryl are replaced with a heteroatom,
The above heteroatom is selected from non-carbon atoms including nitrogen (N), oxygen (O) and sulfur (S),
The above substituents are selected from sulfonic acid, sulfonate, ketone, aldehyde, carboxylic acid, carboxylate, phosphoric acid, phosphate, acyl chloride, hydroxy, polyalkylene oxide, quaternary ammonium salt, _C6 - _C24 aryl, ester and amide, wherein the polyalkylene oxide is terminated with methyl ether and contains ethylene glycol as a repeating unit.
In the second paragraph,
The ring formed by two adjacent R _3s joining together is selected from the group consisting of deuterium, C ₁ -C ₁₀ alkyl substituted or unsubstituted with at least one substituent, C ₂ -C ₁₀ alkenyl substituted or unsubstituted with at least one substituent, C ₂ -C ₁₀ alkynyl substituted or unsubstituted with at least one substituent, C ₂ -C ₁₀ alkoxy substituted or unsubstituted with at least one substituent, C ₆ -C ₂₄ aryloxy substituted or unsubstituted with at least one substituent, C ₁ -C ₁₀ haloalkyl substituted or unsubstituted with at least one substituent, halogen, cyano, hydroxy, amino substituted or unsubstituted with at least one substituent, amide substituted or unsubstituted with at least one substituent, sulfhydryl, nitro, carboxyl, carboxylate, C ₆ -C ₂₄ aryl substituted or unsubstituted with at least one substituent, C ₅ -C ₂₃ heteroaryl substituted or unsubstituted with at least one substituent, or Substituted with at least one selected from unsubstituted aralkyl, quaternary ammonium, phosphoric acid, phosphate, oxo(=O), aldehyde, ester(-COOR ₆ ), acyl chloride, sulfonic acid, sulfonate and -LX functional groups,
The above substituents are selected from sulfonic acid, sulfonate, ketone, aldehyde, carboxylic acid, carboxylate, phosphoric acid, phosphate, acyl chloride, hydroxy, polyalkylene oxide, quaternary ammonium salt, _C6 - _C24 aryl, ester and amide, wherein the polyalkylene oxide is terminated with methyl ether and contains ethylene glycol as a repeating unit.
Cyanine compounds.
In claim 1 or 2,
The above cyanine compound is at least one selected from compounds represented by the following chemical formula:
Cyanine compounds:
[Chemical Formula 2]

[Chemical Formula 3]

[Chemical Formula 4]

[Chemical Formula 5]

[Chemical Formula 6]

Here,
R ₁₁ to R ₂₆ are hydrogen, deuterium, C ₁ -C ₁₀ alkyl substituted or unsubstituted with at least one substituent, C ₂ -C ₁₀ alkenyl substituted or unsubstituted with at least one substituent, C ₂ -C ₁₀ alkynyl substituted or unsubstituted with at least one substituent, C ₂ -C ₁₀ alkoxy substituted or unsubstituted with at least one substituent, C ₆ -C ₂₄ aryloxy substituted or unsubstituted with at least one substituent, C ₁ -C ₁₀ haloalkyl substituted or unsubstituted with at least one substituent, halogen, cyano, hydroxy, amino substituted or unsubstituted with at least one substituent, amide substituted or unsubstituted with at least one substituent, sulfhydryl, nitro, carboxyl, carboxylate, C ₆ -C ₂₄ aryl substituted or unsubstituted with at least one substituent, C ₅ -C ₂₃ heteroaryl substituted or unsubstituted with at least one substituent, Selected from substituted or unsubstituted aralkyl, quaternary ammonium, phosphoric acid, phosphate, ketone (-COR ₆ ), aldehyde, ester (-COOR ₆ ), acyl chloride, sulfonic acid, sulfonate and -LX functional groups,
The above substituents are selected from sulfonic acid, sulfonate, ketone, aldehyde, carboxylic acid, carboxylate, phosphoric acid, phosphate, acyl chloride, hydroxy, polyalkylene oxide, quaternary ammonium salt, _C6 - _C24 aryl, ester and amide, wherein the polyalkylene oxide is terminated with methyl ether and contains ethylene glycol as a repeating unit,
Y ₃ is sulfur, oxygen or CR ₄ R ₅ .
A dye for labeling a biomolecule comprising a cyanine compound according to claim 1 or 2.
In paragraph 5,
The biomolecule is at least one selected from antibodies, lipids, proteins, peptides, carbohydrates and nucleic acids.
Dyes for labeling biomolecules.
Containing a dye for biomolecule labeling according to Article 5,
Kit for biomolecule labeling.
A contrast agent composition comprising a cyanine compound according to claim 1 or 2.