KR20170101360A - Compound based cyanine, labeling dye, kit and contrast medium composition for biomolecule comprising the same - Google Patents

Abstract

The present invention relates to a cyanine-based compound represented by chemical formula 1, a pigment for labeling biomolecules containing the same, a kit, and a contrast medium composition. More specifically, provided is a novel cyanine-based compound capable of maintaining photo stability for a long period in aqueous solutions.

Description

TECHNICAL FIELD The present invention relates to a cyanine-based compound, a biomolecule labeling dye, a kit, and a contrast agent composition containing the same. BACKGROUND ART [0002]

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

Fluorescent dyes have been used as a means of visualizing biologic phenomena at the cellular level in vivo, in vivo, and in vitro, or for examining biostimulation and diseased sites. Green fluorescence protein (GFP) and other biomolecules which emit self-fluorescence are also present. Generally, however, biomolecules such as biotissues such as biological tissues, cells and sub-stages are dyed with fluorescent dyes, fluorescent dyes are added to biomolecules such as proteins and nucleic acids After labeling, we have obtained imaging data with various techniques, accompanied by optical equipment capable of detecting fluorescence signals.

Optical analysis equipment mainly used is a fluorescence microscope, a confocal microscope, a flow cytometry, a microarray, a quantitative PCR system In addition to equipment for research purposes, such as nucleic acid and protein separation, electrophoresis for analysis, and in vivo imaging system, immune assay, PCR analysis and statistical techniques are applied. There have been known in vitro diagnostic apparatuses based on nucleic acid and protein diagnostic kits (or biochips) and diagnostic and therapeutic apparatuses such as operating tables and endoscopic apparatuses for image-guided surgery, Equipment with higher resolution and data processing capabilities are being developed.

In order to apply fluorescent dyes to the biotechnology field, it is generally required that when most biomolecules are present in the medium in which they exist, bleaching and quenching phenomena are small and molecular extinction coefficient and quantum efficiency are large and stable under various pH conditions.

Various fluorescent dyes have been used in various research fields. However, fluorescent dyes satisfying all the above conditions in the bio field are rare, and typical structures currently used include coumarins, cyanine, bodipy, , Fluoresceins, rhodamines, pyrenes, carbopyronin, oxazine, xanthenes, thioxanthene, and acridines, and the like. . Among them, rhodamine derivatives and polymethine cyanine derivatives are predominant. In particular, the cyanine-based fluorescent dye is connected to both ends of the polymethine by a heterocyclic ring containing nitrogen, and by adjusting the length of the polymethine, a structural advantage that can realize various fluorescence from 450 nm to 800 nm in all visible and near- In addition, there is generally an optical characteristic with a very high extinction coefficient. However, as the length of the polymethine increases, the photo stability of the cyanine dye decreases.

Accordingly, there is a continuing need for the development of cyanine dyes capable of maintaining optical stability for a long time in an aqueous solution.

The present invention aims to provide a novel cyanine compound which can be widely used for observing the identification of biomolecules in the field of optical imaging, and a dye, kit and contrast agent for biomolecule marking comprising the same.

It is another object of the present invention to provide a novel cyanine compound capable of maintaining optical stability for a long time in an aqueous solution.

According to one aspect of the present invention, there is provided a cyanine compound represented by the following formula (1).

[Chemical Formula 1]

here,

Ar ₁ and Ar ₂ are each independently substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl, and Ar ₁ And 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 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 Substituted or unsubstituted aralkyl, substituted or unsubstituted aralkyl, quaternary ammonium, phosphoric acid, phosphate, ketone, sulfonate, (-COR ₆ ), aldehyde, ester (-COOR ₆ ), acyl chloride, sulfonic acid, sulfonate and -LX functional groups.

Ketone group (-COR ₆₎ or an ester group (-COOR ₆₎ one time, R ₆ is a substituted or unsubstituted C ₁ -C ₁₀ alkyl group, a substituted or unsubstituted C ₂ -C ₁₀ alkenyl group, a substituted or unsubstituted 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 -LX functional group, L is 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 - is a linker selected from, R ₆ is hydrogen, alkenyl 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; p is an integer from 1 to 100; , X is a carboxyl, succinimidyl ester, sulfo-succinimidyl ester, isothiocyanate, maleimide, haloacetamide, , Hydrazide, vinylsulphone, dichlorotriazine, phosphorus, The compounds of formula (I) may be selected from the group consisting of phosphoramidite, alkyl halide, acyl halide, carbohydrazide, hydroxylamine, ketone, alkyne, azide Aliphatic and aromatic amines, sulfotetrafluorophenyl esters, sulfodichlorophenyl esters, carbonyl azides, sulfonyl chlorides, sulfonyl chlorides, sulfonyl chlorides, But are not limited to, sulfonyl fluoride, boronic acid, isocyanate, halogen-substituted triazine, halogen-substituted pyridine, halogen- substituted diazine, tetrafluorophenyl ester, imido ester, azidonitrophenyl, glyoxal, and aldehyde. Is a reactive group.

In addition, when R ₁ , R ₂ , R ₃ , R ₄ , R ₅ or R ₆ are substituted, any carbon or terminal carbon in the functional group may be substituted with a sulfonic acid, sulfonate, ketone, , Acyl chloride, polyalkylene oxide, quaternary ammonium salt, ester and amide, and n is 0 or an integer of 1 to 5.

The cyanine compound represented by the formula (1) has a symmetrical or asymmetric skeleton and includes a heterocyclic ring to which fluoranthene is bonded.

The cyanine compound according to the embodiment of the present invention symmetrically or asymmetrically includes the fluoranthene-bonded heterocycle, and accordingly the fluorescence signal is generated in the relatively long wavelength band compared to the conventional cyanine dye having the same length of polymethine .

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

That is, according to various embodiments of the present invention, introduction of fluoranthene-linked heterocyclic ring enables excitation and emission in a relatively long wavelength band compared with conventional commercialized cyanine dye, It is possible to improve the light stability compared to the conventional commercialized cyanine dyes possible.

According to another aspect of the present invention, there is provided a biomolecule labeling dye comprising a cyanine compound represented by the general formula (1).

According to still another aspect of the present invention, there is provided a biomolecule marker kit comprising the cyanine compound represented by Formula (1).

According to still another aspect of the present invention, there is provided a contrast agent composition comprising a cyanine compound represented by Formula (1).

The novel cyanine compound according to the present invention contains fluoranthene in the heterocycle, and thus it is possible to absorb light having a longer wavelength as compared with a conventional cyanine dye having the same polymethine. Accordingly, a fluorescence signal similar to that of a cyanine dye having a relatively long polymethine that is commercially available and has a shorter polymethine structure can be realized, and at the same time, the optical stability can be relatively improved.

Certain terms are hereby defined for convenience in order to facilitate a better understanding of the present invention. Unless otherwise defined herein, scientific and technical terms used in the present invention may have the meanings commonly understood by one of ordinary skill in the art.

Also, unless the context clearly indicates otherwise, the singular form of the term includes plural forms thereof, and the plural forms of terms may include singular forms thereof.

New  Cyanine compound

According to an aspect of the present invention, there can be provided a cyanine-based compound represented by the following general formula (1).

[Chemical Formula 1]

In the cyanine compound represented by the formula (1), Ar ₁ and Ar ₂ are each independently substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl, and Ar ₁ And Ar < ₂ > is a substituted or unsubstituted fluoranthene.

The cyanine compound according to the embodiment of the present invention symmetrically or asymmetrically includes the fluoranthene-bonded heterocycle, and accordingly the fluorescence signal is generated in the relatively long wavelength band compared to the conventional cyanine dye having the same length of polymethine .

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

That is, according to various embodiments of the present invention, introduction of fluoranthene-linked heterocyclic ring enables excitation and emission in a relatively long wavelength band compared with conventional commercialized cyanine dye, It is possible to improve the light stability compared to the conventional commercialized cyanine dyes possible.

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 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 Substituted or unsubstituted aralkyl, substituted or unsubstituted aralkyl, quaternary ammonium, phosphoric acid, phosphate, ketone, sulfonate, (-COR ₆ ), aldehyde, ester (-COOR ₆ ), acyl chloride, sulfonic acid, sulfonate and -LX functional groups.

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

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

Specifically, the alkyl is selected from the group consisting of methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, i-butyl, t-butyl, pent- Methylbut-2-yl, 2,2,2-trimethylet-1-yl, n-hexyl, n-heptyl, and n - octyl.

In addition, alkoxy in the context of the present invention means both an -O- (alkyl) group and an -O- (unsubstituted cycloalkyl) group, and is a straight chain or branched hydrocarbon having at least one ether group and from 1 to 10 carbon atoms.

Specific examples include methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, n- But are not limited to, cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy, and the like.

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

Aralkyl is a generic term for - (CH ₂ ) _n Ar, _wherein aryl is a functional group substituted in the carbon of the alkyl. Examples of aralkyl include benzyl (-CH ₂ C ₆ H ₅ ) or phenethyl (-CH ₂ CH ₂ C ₆ H ₅ ).

In -LX functional group, L is 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 the group consisting of hydrogen, substituted or unsubstituted C ₁ -C ₆ alkyl, substituted or unsubstituted C ₂ -C ₁₀ alkenyl, 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, p is an integer from 1 to 100 It is an integer.

X is selected from the group consisting of carboxyl, succinimidyl ester, sulfo-succinimidyl ester, isothiocyanate, maleimide, haloacetamide, But are not limited to, hydrazide, vinylsulphone, dichlorotriazine, phosphoramidite, alkyl halide, acyl halide, carbohydrazide, , Hydroxylamine, ketone, alkyne, azide, aliphatic and aromatic amines, sulfotetrafluorophenyl ester, sulfodichlorophenyl ester (for example, sulfodichlorophenyl ester, carbonyl azide, sulfonyl chloride, sulfonyl fluoride, boronic acid, isocyanate, halogen- Halogen-substituted triazines, halogen-substituted pyridines, halogen-substituted diazines, tetrafluorophenyl esters, imido esters, , Azidonitrophenyl, glyoxal, and aldehyde. ≪ / RTI >

The cyanine-based compound according to various embodiments of the present invention may be capable of binding with a target biomolecule through the above-mentioned reactive group to be labeled.

The reactive groups described above are functional groups capable of reacting with a functional group such as an amino group, imino group, thiol group or hydroxyl group of a target biomolecule. The functional groups include an amide bond, an imide bond, a urethane bond Bond, an ester bond or a guanidine bond.

In addition, the reactive group (X) is bonded to the main skeleton of the cyanine compound via the linker (L), so that steric hindrance between the biomolecule and the cyanine compound can be alleviated. Thus, , Carbohydrates, and the like can be improved.

In addition, when R ₁ , R ₂ , R ₃ , R ₄ , R ₅ or R ₆ are substituted, any carbon or terminal carbon in the functional group may be substituted with a sulfonic acid, sulfonate, ketone, , Acyl chloride, polyalkylene oxide, quaternary ammonium salt, ester and amide, and n is 0 or an integer of 1 to 5.

Here, the polyalkylene oxide is selected from the group consisting of polyethylene glycol (PEG), polypropylene glycol (PPG), polyethylene glycol-polypropylene glycol (PEG-PPG) Methacrylamide-containing polymers and copolymers.

Here, the polyalkylene oxide may be additionally substituted as necessary within the range in which 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 one or more substituents selected from the group consisting of hydroxy, alkyl ethers (methyl ether, ethyl ether, propyl ether, etc.), carboxyl methyl ether, carboxyethyl ether, benzyl ether, Methylamine. ≪ / RTI > In one embodiment, the polyalkylene oxide may be a polyalkylene oxide (mPEG) terminated with methyl ether, wherein mPEG is represented by the formula - (CH ₂ CH ₂ O) _n CH ₃ , and ethylene glycol The size of the mPEG may vary depending on the size of n corresponding to the number of call repeating units.

The cyanine compound represented by the general formula (1) may have a structure further comprising a counter ion. The counter ion can be appropriately selected in consideration of the solubility and stability of the cyanine compound as an organic or inorganic anion.

Examples of the counter ion of the cyanine compound according to an embodiment of the present invention include a phosphonic acid hexafluoride ion, a halogen ion, a phosphonic acid ion, a perchloric acid ion, a periodic acid ion, an antimony hexafluoride ion, An inorganic acid anion such as a fluoride ion, a fluoroboric acid ion and a fluoride ion, a thiocyanate ion, a benzenesulfonic acid ion, a naphthalenesulfonic acid ion, a p-toluenesulfonic acid ion, an alkylsulfonic acid ion, a benzenecarboxylic acid ion, An organic acid ion such as an alkylcarboxylic acid ion, a trihaloalkylcarboxylic acid ion, an alkylsulfonic acid ion, a trihaloalkylsulfonic acid ion, and a nicotinic acid ion. In addition, metal compound ions such as bisphenylditol, thiobisphenol chelate and bisdiol-alpha -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 the general formula (1), when n is 1 or more, two adjacent R ₃ may bond to each other to form a hydrocarbon ring. In this case, the hydrocarbon ring may be a 4-atom, a 5-atom or a 6-atom ring, and at least one heteroatom selected from -S-, -O-, -Se-,> NR ₄ and> SiR ₄ R _{5 may} be selected As shown in FIG.

When Ar ₁ or Ar ₂ is substituted, any carbon in the functional group may be substituted by 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, Substituted or unsubstituted amino, substituted or unsubstituted amide, sulfhydryl, nitro, carboxyl, carboxylate, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted aralkyl, And may be substituted with at least one selected from the group consisting of ammonium, phosphorous, phosphate, ketone (-COR ₆ ), aldehyde, ester (-COOR ₆ ), acyl chloride, sulfonic acid, sulfonate and -LX functional groups.

More specifically, the cyanine compound represented by the formula (1) may be represented by the following formulas (2) to (6).

(2)

(3)

[Chemical Formula 4]

[Chemical Formula 5]

[Chemical Formula 6]

Wherein 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), is selected from acyl chloride, sulfonic acid, sulfonic acid salt functional group, and -LX, Y ₃ is a 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 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 compound according to various embodiments of the present invention are determined by a polymethine structure corresponding to a chromophore and a heterocyclic ring linked with fluoranthene symmetrically or asymmetrically bonded to both ends thereof, It is to be understood that the excitation and luminescence properties of the cyanine compound intended in the present invention can be maintained regardless of the kind of the reactant introduced into the compound represented by the general formulas (1) to (6).

The target biomolecules of the cyanine compounds represented by Chemical Formulas 1 to 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, lipopolysaccharides and the like. Specific examples of carbohydrates include monosaccharides, disaccharides, and polysaccharides (e.g., dextran).

At this time, the biomolecule is 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, sulphydryl), carbonyl, hydroxyl, carboxyl, phosphate, and thiophosphate, or derivative forms thereof.

In addition, the biomolecule may be an oxy or deoxypoly nucleic acid comprising at least one selected from amino, sulphydryl, carbonyl, hydroxyl, carboxyl, phosphate and thiophosphate, or derivatives thereof.

In addition, in addition to the biomolecules, the cyanine compound represented by Chemical Formulas 1 to 6 may be a drug including at least one selected from amino, sulphydryl, carbonyl, hydroxyl, carboxyl, phosphate and thiophosphate, A receptor, an enzyme or an enzyme substrate, a cell, a cell membrane, a toxin, a microorganism or a nanobio material (such as a polystyrene microsphere).

Dyes for biomolecule marking, Kit  And contrast agent composition

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

In addition, if necessary, the biomolecule labeling kit may further include an enzyme, a solvent (buffer solution, etc.) for reacting with the target biomolecule, and other reagents.

Wherein the solvent is selected from the group consisting of a buffer selected from the group consisting of phosphate buffer, carbonate buffer and Tris buffer, an organic solvent selected from dimethylsulfoxide, dimethylformamide, dichloromethane, methanol, ethanol and acetonitrile, And it is possible to control the solubility by introducing various functional groups into the cyanine compound depending on the kind of the solvent.

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

Specific examples of the pharmaceutically acceptable carrier include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose , Water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

Hereinafter, a biomolecule detection method using the biomolecule marking dye or biomolecule marking kit described above will be described.

Detection method of biomolecules using biomolecule marker dyes

The method of labeling a biomolecule with a cyanine compound represented by any one of Chemical Formulas 1 to 6 can be applied to the detection of all biomolecules capable of detecting fluorescence of labeled solid or semi-solid biomolecules.

By using a cyanine compound instead of a conventional fluorescent dye, a detection method that is chemically stable with high sensitivity and excellent in operability can be provided.

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

In addition, a biomolecule detection method using a target-specific interaction may be implemented by introducing an appropriate reactive group into the cyanine-based compound depending on the type of the target biomolecule.

In addition, a method of identifying a biomolecule labeled with a cyanine-based compound through electrophoresis may be implemented.

DNA Microarray method

In the DNA microarray method, a single-stranded probe nucleic acid having a base sequence complementary to a target nucleic acid is prepared by reacting a target nucleic acid to be detected with a dye, and a target nucleic acid and a probe nucleic acid, And the fluorescence of the target nucleic acid is measured.

As the probe nucleic acid immobilized on the substrate in this detection method, when the expression of a gene is examined, a library of cDNA such as cDNA, a library of the genome, or all the genomes are prepared as templates (amplified type) and amplified by the PCR method Can be used.

In the case of examining a mutation or the like of a gene, it is possible to use a synthesized oligonucleotide corresponding to a mutation based on a known sequence which becomes a standard.

The method of immobilizing the probe nucleic acid on the substrate can be appropriately selected depending on the kind of the nucleic acid and the type of the substrate. For example, a method of electrostatic bonding to a substrate surface-treated with a cation such as polylysine using the charge of DNA may be used.

On the other hand, a target nucleic acid labeled with a labeling dye is prepared by mixing and reacting a target nucleic acid denatured with a single chain and a labeling dye. The reaction temperature is preferably from room temperature to 60 ° C and the reaction time is from 2 hours to 48 hours.

The labeled target nucleic acid is then spotted onto the substrate and hybridized.

The hybridization is preferably carried out at room temperature to 70 ° C and for 2 to 48 hours. By hybridization, a target nucleic acid having a base sequence complementary to the probe nucleic acid selectively binds to the probe nucleic acid. Thereafter, the substrate is cleaned and dried at room temperature.

Subsequently, the fluorescent intensity of the dried substrate surface is measured by a fluorescent laser scanner method. The level of gene expression can be monitored by fluorescence intensity.

In the meantime, the hybridization has been described as a method of immobilizing a probe nucleic acid on a substrate. Alternatively, a method of immobilizing a target nucleic acid labeled with a dye in advance on a substrate and dropping the probe nucleic acid on the substrate may be used.

PCR  method

In the PCR method, the probe complementary to the base sequence of the target nucleic acid to be detected is labeled with a dye, and the target nucleic acid is reacted with the probe before or after the amplification of the target nucleic acid, and fluorescence of the target nucleic acid is measured.

Specifically, the elongation reaction of the target nucleic acid is carried out by an enzyme (DNA polymerase, RNA polymerase). At this time, the double-stranded nucleic acid sequence formed by the primer consisting of the target nucleic acid and the oligonucleotide is recognized by the enzyme, An elongation reaction takes place from one position, and only the desired gene region is amplified.

When the enzyme is synthesized, the synthesis reaction is carried out using the nucleotide (dNTP, NTP) as a raw material.

At this time, when a nucleotide having a dye in an ordinary nucleotide (dNTP, NTP) is mixed at an arbitrary ratio, a nucleic acid into which the dye has been introduced can be synthesized.

In addition, a nucleotide having an amino group at an arbitrary ratio may be introduced by PCR, followed by binding of a labeling dye to synthesize a nucleic acid into which a labeling dye has been introduced.

When an enzyme is synthesized, a synthesis reaction is carried out using a nucleotide as a raw material. If the 3 'OH of the nucleotide at this time is changed to H, the extension reaction of the nucleic acid is no longer carried out. do.

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

When a nucleic acid is synthesized by adding a terminator to an ordinary nucleotide, a terminator is introduced at a certain probability to terminate the reaction. Thus, nucleic acids of various lengths are synthesized.

When it is size-separated by gel electrophoresis, the DNA is lined up in order of length. Here, if each kind of the terminator is labeled with another labeling dye, the tendency to depend on each base is observed at the end point (3 'end) of the synthesis reaction, and fluorescence information The base sequence information of the target nucleic acid can be obtained.

In addition, a primer labeled with a labeling dye may be used in advance to hybridize with the target nucleic acid instead of the terminator.

PNA (peptide nucleic acid) may also be used as a probe. PNA is a substitution of pentane / phosphate skeleton, which is a basic skeleton structure of nucleic acid, with a polyamide skeleton of glycine unit. It has a three-dimensional structure that resembles that of a nucleic acid and is highly specific for a nucleic acid having a complementary base sequence Combine strongly. Thus, it can be used as a reagent for telomere studies by applying it to a telomere (telomere) PNA probe as well as a conventional DNA analysis method such as an in-situ hybridization (ISH) method.

For the detection, for example, double-stranded DNA is hybridized by contacting it with PNA labeled with a labeling dye having a base sequence complementary to all or a part of the base sequence of DNA, and the mixture is heated to produce single-stranded DNA , And the mixture is slowly cooled to room temperature to prepare a PNA-DNA complex, and the fluorescence thereof can be measured.

In this example, the method of amplifying the target nucleic acid by the PCR method to measure the fluorescence of the product has been described. However, in this method, the size of the product is confirmed by electrophoresis, and then the fluorescence intensity is measured, Needs to be.

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

For example, DNA labeled donor and acceptor can be used. Specific detection methods include a molecular beacon method, a TaqMan-PCR method, and a cycling probe method in which the presence of a nucleic acid having a specific sequence is confirmed.

In addition, when a protein is to be detected, a staining dye is used for detection of the protein after electrophoresis.

In general, a method is used in which a staining dye such as CBB (Coomassie Brilliant Blue) is infiltrated into the gel after electrophoresis to stain the protein and irradiate with UV light to emit light.

However, the method using a conventional dye is simple, but the sensitivity is as low as 100 ng and is not suitable for the detection of trace amounts of protein. In addition, since the dye penetrates the dye through the gel, there is also a problem that it takes much time to dye.

On the other hand, in the present invention, the protein is size-separated by electrophoresis, and the protein is labeled by binding a labeling dye to the separated protein.

The labeling dye of the present invention has a reactive group, reacts rapidly with the protein quantitatively, and is highly sensitive, so that it is suitable for the detection of a trace amount of protein. In addition, the size-separated proteins can be identified by mass analysis.

Here, the protein may be any of complex proteins such as simple proteins such as albumin, globulin, glutelin, histone, protamine and collagen, nuclear protein, glycoprotein, riboprotein, phosphorous protein and metal protein .

For example, in a protein sample separated by two-dimensional electrophoresis using three kinds of labeling dyes corresponding to phosphorylation protein, glycoprotein and total protein dye, phosphorus protein, glycoprotein and total protein were stained can do.

In addition, since the proteins can be identified by mass analysis such as TOF-Mass, the present invention can be applied to diagnosis and treatment of diseases such as infections caused by cancer or viruses that produce special proteins.

Collagen is a protein that constitutes the connective tissue of an animal and has a unique fibrous structure. That is, it is composed of a triple polypeptide chain, and the peptide chains are gathered to form a triple chain. Collagen is generally a very low immunogenicity protein and is widely used in the fields of foods, cosmetics, pharmaceuticals and the like.

In addition, an aptamer may be used for the probe. Since the aptamer is composed of an oligonucleic acid and can have various stereostructures depending on the base sequence, it can bind to all biomolecules including proteins through the steric structure. By using this property, it is possible to indirectly detect the subject from the fluorescence change due to the structural change of the protein due to binding with the subject, by binding the platemar labeled with the labeling dye to the specific protein.

Other detection methods

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

That is, in the detection of the subject, which is made of a biomolecule or modified by a modifier, one of the binding substances specifically binding to the subject or the binding substance specifically binding to the modifying substance is used for labeling Labeled with a dye, and the fluorescence from the labeled binding substance can be measured.

Here, the antigen-antibody, the hapten-anti-integrin antibody, the biotin-avidin, the Tag-anti Tag antibody, the lectin-glycoprotein or the hormone-receptor can be used for the combination of the test substance or the modulating substance and the binding substance.

Specifically, a specific antigen can be detected through an antigen-specific interaction of an antibody by reacting a binding substance such as an antibody labeled with a labeling dye against an antigen present on a substrate, a solution, a bead or an antibody.

The antigen may be a protein, a polysaccharide, a nucleic acid, a peptide, or the like. In addition to the antigen, a ligand such as a low molecular weight molecule such as FITC or dinitrophenyl group may be used. In this case, the combination of antigen (or hapten) and antibody includes GFP, anti-GFP antibody, FITC and anti-FITC antibody.

The labeled antigens can be used for various measurement methods such as immuno-staining, ELISA, Western blotting, flow cytometry and the like.

Also, by using the labeling dye of the present invention, intracellular signaling phenomenon can be observed. Various enzymes and the like are involved in internal signal transduction or cell reaction. In a typical signal transduction phenomenon, it is known that a specific protein kinase is activated, thereby inducing protein phosphorylation and initiating signal transduction.

Binding and hydrolysis of nucleotides (for example, ATP or ADP) play a significant role in their activity, and intracellular signal transduction can be observed with high sensitivity by introducing a labeling dye into the nucleotide derivative.

In addition, the labeling dye of the present invention can be used for observation of gene expression phenomenon using RNA interference (RNAi).

RNAi is a method of degrading mRNA of a target gene by introducing double-stranded RNA (dsRNA) into a cell, thereby inhibiting the expression. 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 a cell.

Manufacturing example

Manufacturing example  1: Preparation of compound 1

2.0 g of Dyes and pigments (96,535; 2013), 2.2 g of 1,3-propane sultone and 20 mL of sulfolane were charged into the reactor and stirred at 140 DEG C for 36 hours. After the reaction solution was cooled to room temperature, isopropyl alcohol was added thereto 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 the reactor and refluxed with stirring for 18 hours. After the reaction solution was cooled to room temperature, EA was added thereto and filtration was conducted to obtain 2.5 g of Intermediate 1-3.

0.2 g of triethylamine, 0.2 g of acetic anhydride and 9 mL of N, N-dimethylformamide were added to a reactor, And the mixture was stirred at room temperature for 4 hours. Ethyl acetate was then added to the reaction solution, followed by filtration and separation by ODS column chromatography to obtain 30 mg of Compound 1 (MS (ESI): m / z 953.2).

Manufacturing example  2: Preparation of compound 2

(MS (ESI): m / z 874.2 (M + H)) was prepared by reacting Intermediate 1 - 3 with Intermediate 1 (Bioorganic and medicinal chemistry letters, 22 ).

Manufacturing example  3: Preparation of compound 3

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

Experimental Example

The absorption spectra, emission spectra, molar extinction coefficients and quantum efficiencies of the respective compounds obtained in Production Examples 1 to 3 were measured and are shown in Table 1 below.

division menstruum λ _ex (nm) ? _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 shown in Table 1, in the case of Compound 1, Compound 2 and Compound 3 synthesized according to Production Examples 1 to 3, the fluoranthene-containing heterocycle is symmetrically or asymmetrically contained.

At this time, although the length of the polymethine corresponding to the chromophore (color material) in the compound 1, the compound 2 and the compound 3 is the same as the length of the polymethine in the commercially available Cy3 dye, Which is similar to the Cy5 dye.

That is, according to various embodiments of the present invention, since fluoranthene-bonded heterocyclic ring is introduced, excitation and luminescence are possible in a relatively long wavelength band as compared with a cyanine compound having a simple indole structure, And there is no need to cause structural instability by increasing the length of the polymethine for luminescence.

In the above examples, the excitation and emission wavelength of the compound 1, the compound 2 and the compound 3 were confirmed, but the cyanine compound represented by the general formulas (1) to (6) was also introduced with the fluoranthene-bonded heterocyclic structure It can be expected that it will exhibit the fluorescence characteristics in the long wavelength band rather than the excitation and emission wavelength band expected from the polymethine length.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit of the invention as set forth in the appended claims. The present invention can be variously modified and changed by those skilled in the art, and it is also within the scope of the present invention.

Claims (8)

A cyanine compound represented by the following formula (1)
[Chemical Formula 1]

here,
Ar ₁ and Ar ₂ are each independently substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl, and Ar ₁ And 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 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 Substituted or unsubstituted aralkyl, substituted or unsubstituted aralkyl, quaternary ammonium, phosphoric acid, phosphate, ketone, sulfonate, (-COR ₆ ), aldehyde, ester (-COOR ₆ ), acyl chloride, sulfonic acid, sulfonate and -LX functional groups,
When R ₁ , R ₂ , R ₃ , R ₄ or R ₅ is a ketone group (-COR ₆ ) or an ester group (-COOR ₆ ), R ₆ is a substituted or unsubstituted C ₁ -C ₁₀ alkyl group, Or an 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 -LX functional group, L is 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 - is a linker selected from, R ₆ is hydrogen, alkenyl 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; p is an integer from 1 to 100; , X is a carboxyl, succinimidyl ester, sulfo-succinimidyl ester, isothiocyanate, maleimide, haloacetamide, , Hydrazide, vinylsulphone, dichlorotriazine, phosphorus, The compounds of formula (I) may be selected from the group consisting of phosphoramidite, alkyl halide, acyl halide, carbohydrazide, hydroxylamine, ketone, alkyne, azide Aliphatic and aromatic amines, sulfotetrafluorophenyl esters, sulfodichlorophenyl esters, carbonyl azides, sulfonyl chlorides, sulfonyl chlorides, sulfonyl chlorides, But are not limited to, sulfonyl fluoride, boronic acid, isocyanate, halogen-substituted triazine, halogen-substituted pyridine, halogen- substituted diazine, tetrafluorophenyl ester, imido ester, azidonitrophenyl, glyoxal, and aldehyde. Is a reactive group,
When R ₁ , R ₂ , R ₃ , R ₄ , R ₅ or R ₆ are substituted, any carbon or terminal carbon in the functional group may be substituted with at least one substituent selected from the group consisting of sulfonic acid, sulfonate, ketone, aldehyde, carboxylic acid, , Acyl chloride, polyalkylene oxide, quaternary ammonium salt, ester and amide,
n is 0 or an integer of 1 to 5;
The method according to claim 1,
n is 1 or more,
Two R ₃ are bonded to each other -S-, -O-, -Se-,> NR 4 , and> SiR ₄ R _5, optionally form a hydrocarbon ring including the at least one hetero atom selected from,
Cyanine compound.
The method according to claim 1,
When Ar ₁ or Ar ₂ is substituted, any carbon in the functional group can be 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 Substituted or unsubstituted amido, unsubstituted amino, substituted or unsubstituted amide, sulfhydryl, nitro, carboxyl, carboxylate, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted aralkyl, quaternary ammonium Substituted with at least one member selected from the group consisting of phosphoric acid, phosphate, ketone (-COR ₆ ), aldehyde, ester (-COOR ₆ ), acyl chloride, sulfonic acid, sulfonate and-
Cyanine compound.
The method according to claim 1,
Wherein the cyanine compound is at least one compound selected from compounds represented by the following formulas:
Cyanine compounds:
(2)

(3)

[Chemical Formula 4]

[Chemical Formula 5]

[Chemical Formula 6]

here,
R ₁₁ to R ₂₆ are independently selected from the group consisting of 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, 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 Substituted or unsubstituted aralkyl, substituted or unsubstituted aralkyl, quaternary ammonium, phosphoric acid, phosphate, ketone (-COR), substituted or unsubstituted heteroaryl, substituted or unsubstituted heteroaryl, ₆ ), aldehydes, esters (-COOR ₆ ), acyl chlorides, sulfonic acids, sulfonates and -LX functional groups,
Y ₃ is sulfur, oxygen, selenium, NR ₄ , CR ₄ R ₅ , SiR ₄ R ₅ or -CR ₄ = CR ₅ -.
A biomolecule labeling dye comprising the cyanine compound according to any one of claims 1 to 4.
6. The method of claim 5,
Wherein the biomolecule is at least one selected from antibodies, lipids, proteins, peptides, carbohydrates and nucleic acids,
Dye for biomolecule marking.
A biomolecule labeling dye according to claim 5,
Kit for biomolecule labeling.
A contrast agent composition comprising a cyanine compound according to any one of claims 1 to 4.