KR20190043075A - Compound based merocyanine, labeling dye, kit and contrast medium composition for biomolecule comprising the same - Google Patents

Abstract

The present invention relates to a merocyanine-based compound, capable of representing a fluorescence signal in a visible light region of 380 nm or more, and a composition of a dye, a kit, and a contrast agent for a biomolecule marker including the merocyanine-based compound. The merocyanine-based compound of the present invention is represented by chemical formula 1. In chemical formula 1, Ar_1 is substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl, and Y_1 and Y_2 are each independently selected from sulfur, oxygen, selenium, NR_3, CR_3R_4, SiR_3R_4 and -CR_3=CR_4-.

Description

TECHNICAL FIELD The present invention relates to a biodegradable biodegradable biodegradable polymer, a biodegradable biodegradable biodegradable biodegradable biodegradable biodegradable biodegradable biodegradable biodegradable biodegradable biodegradable biodegradable biodegradable polymer,

The present invention relates to a novel merocyanine compound which exhibits a fluorescence signal in the visible light region of 380 nm or more and can be used for the detection of biomolecules, 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, but fluorescent dyes satisfying all of the above-mentioned conditions in the bio-field are quite rare. For example, coumarins, cyanine, bodipy, fluoresceins, rhodamines, pyrenes, carbopyronin, oxazine, , Xanthenes, thioxanthene, or acridines are commonly used as fluorescent dyes.

The present invention aims to provide a novel merocyanine compound having a skeleton different from that of a conventional fluorescent dye, which can be widely used for observing the identification of biomolecules in the field of optical imaging.

It is another object of the present invention to provide a biomolecule labeling dye, kit and contrast agent composition containing a novel merocyanine compound.

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

[Chemical Formula 1]

here,

Ar ₁ is substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl,

Y ₁ and Y ₂ are each independently selected from sulfur, oxygen, selenium, NR ₃ , CR ₃ R ₄ , SiR ₃ R ₄ and -CR ₃ ═CR ₄ -

Q is sulfur, oxygen or NR &_lt; a &

R ₁ is each 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 ₁ -C ₁₀ haloalkyl, Substituted or unsubstituted amide, substituted or unsubstituted amide, carboxyl, carboxylate, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, ketone (-COR ₅ ), aldehyde, ester (-COOR ₅ ), -LX functional groups and -LZ functional groups,

R _a and R ₂ to R ₄ are each independently selected from the group consisting of hydrogen, deuterium, substituted or unsubstituted C ₁ -C ₁₀ alkyl, substituted or unsubstituted C ₂ -C ₁₀ heteroalkyl including at least one heteroatom, 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, carbamate, sulfhydryl, nitro, carboxyl, carboxylate, substituted or unsubstituted Aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted aralkyl, quaternary ammonium, phosphoric acid, phosphate, ketone (-COR ₅ ), aldehyde, ester (-COOR ₅ ), acyl chloride, sulfonic acid, sulfonate , -LX functional groups and -LZ functional groups.

When R ₁ , R ₂ , R ₃ or R ₄ is a ketone group (-COR ₅ ) or an ester group (-COOR ₅ ), R ₅ is a substituted or unsubstituted C ₁ -C ₁₀ alkyl, Substituted or unsubstituted C ₂ -C ₁₀ heteroalkyl containing a heteroatom, substituted or unsubstituted C ₂ -C ₁₀ alkenyl, substituted or unsubstituted C ₂ -C ₁₀ alkynyl, substituted or unsubstituted C ₂ -C ₁₀ alkoxy, may be selected from substituted or unsubstituted C ₁ -C ₁₀ haloalkyl, substituted or unsubstituted heteroaryl and substituted or unsubstituted C ₁ -C ₁₀ amino-alkyl.

R ₉ and R ₁₀ may be selected from hydrogen, substituted or unsubstituted alkyl, and R ₉ and R ₁₀ may combine with each other to form a 4-atom, 5-atom or 6-atom hydrocarbon ring.

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, a substituted or unsubstituted C ₁ -C ₆ alkyl, substituted or unsubstituted ring containing at least one heteroatom C ₂ -C ₁₀ heteroalkyl, 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 or heteroaryl; p is an integer from 1 to 100; and X is selected from the group consisting of carboxyl, succinimidyl ester, sulfo-succinimidyl ester, succinimidyl ester, isothiocyanate, maleimide, haloacetate, amide, hydrazide, vinylsulphone, dichlorotriazine, phosphoramidite, alkyl halide, acyl halide, carbohydrazide but are not limited to, carbohydrazide, hydroxylamine, ketone, alkyne, azide, aliphatic and aromatic amines, sulfotetrafluorophenyl ester, sulfodichloro A sulfonic acid group, a sulfonic acid group, a sulfonic acid group, a sulfonic acid group, a sulfonic acid group, a sulfonic acid group, a sulfonic acid group, a sulfonic acid group, a sulfonic acid group, a sulfonic acid group, a halogen-substituted triazine, a halogen-substituted pyridine, a halogen-substituted diazine, a tetrafluorophenyl ester, Is a reactive group selected from imido ester, azidonitrophenyl, glyoxal and aldehyde.

In -LZ 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, a substituted or unsubstituted C ₁ -C ₆ alkyl, substituted or unsubstituted ring containing at least one heteroatom C ₂ -C ₁₀ heteroalkyl, 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, and Z is a fluorophore capable of generating a fluorescent signal.

In one embodiment, Z is selected from the group consisting of coumarins, cyanine, bodipy, fluoresceins, rhodamines, pyrenes, carbopyronin, Oxazine, xanthenes, thioxanthene, or acridines, or may be of formula (1)

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, a sulfonic acid salt, , Heteroaryl, polyalkylene oxide, quaternary ammonium salt, ester and amide.

Here, the heteroaryl may include a salt form in which the heteroatom in the heteroaryl, such as pyridinium or quinolinium, is substituted with a specific functional group to have a (+) charge.

It is preferable that m is an integer of 1 to 3.

According to another aspect of the present invention, there can be provided a biomolecule marking dye comprising a merocyanine compound represented by the general formula (1).

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

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

The novel merocyanine compound according to the present invention can display a fluorescence signal in a visible light region of 380 nm or more in association with biomolecules. Based on this, the merocyanine compound according to the present invention can be effectively used as a biomolecule marker dye, a biomolecule marking kit, and a contrast agent composition.

FIG. 1 shows an absorption spectrum after labeling a merocyanine compound (Compound 2) according to an embodiment of the present invention with Goat Anti-Mouse IgG.
FIG. 2 is a fluorescence emission spectrum obtained by labeling a merocyanine compound (Compound 2) according to an embodiment of the present invention with Goat Anti-Mouse IgG.
FIG. 3 is a brightness spectrum after labeling the merocyanine compound (Compound 2) according to an embodiment of the present invention with Goat Anti-Mouse IgG.
4 is a brightness spectrum after labeling the merocyanine compound (Compound 7) according to another embodiment of the present invention with Goat Anti-Mouse IgG.

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.

The novel merocyanine compound

According to one aspect of the present invention, a merocyanine-based compound represented by the following formula (1) may be provided.

[Chemical Formula 1]

here,

Ar ₁ is substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl, Y ₁ and Y ₂ are each independently selected from the group consisting of sulfur, oxygen, selenium, NR ₃ , CR ₃ R ₄ , SiR ₃ R _4, and -CR ₃ = CR < ₄ > -. In one embodiment, when Y ₁ or Y ₂ is CR ₃ R ₄ , R ₃ and R ₄ may combine with each other to form a four-atom, five-atom or six-membered ring.

Also, Q can be sulfur, oxygen or NR < _a >.

R ₁ is each 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 ₁ -C ₁₀ haloalkyl, Substituted or unsubstituted amide, substituted or unsubstituted amide, carboxyl, carboxylate, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, ketone (-COR ₅ ), aldehyde, ester (-COOR ₅ ), -LX functional groups and -LZ functional groups.

R _a and R ₂ to R ₄ are each independently hydrogen, deuterium, substituted or unsubstituted C ₁ -C ₁₀ alkyl, substituted or unsubstituted C ₂ -C ₁₀ heteroalkyl containing at least one heteroatom , 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 Substituted or unsubstituted amino, substituted or unsubstituted amide, carbamate, sulfhydryl, nitro, carboxyl, carboxylic acid salt, substituted or unsubstituted C ₁ -C ₁₀ haloalkyl, halogen, cyano, Substituted or unsubstituted aryl, substituted or unsubstituted aralkyl, quaternary ammonium, phosphoric acid, phosphate, ketone (-COR ₅ ), aldehyde, ester (-COOR ₅ ), acyl chloride, sulfonic acid, Sulfonic acid salt, -LX functional group and -LZ functional group.

When R ₁ , R ₂ , R ₃ or R ₄ is a ketone group (-COR ₅ ) or an ester group (-COOR ₅ ), R ₅ is a substituted or unsubstituted C ₁ -C ₁₀ alkyl, Substituted or unsubstituted C ₂ -C ₁₀ heteroalkyl containing a heteroatom, substituted or unsubstituted C ₂ -C ₁₀ alkenyl, substituted or unsubstituted C ₂ -C ₁₀ alkynyl, substituted or unsubstituted C ₂ -C ₁₀ alkoxy, may be selected from substituted or unsubstituted C ₁ -C ₁₀ haloalkyl, substituted or unsubstituted heteroaryl and substituted or unsubstituted C ₁ -C ₁₀ amino-alkyl.

R ₉ and R ₁₀ may be selected from hydrogen, substituted or unsubstituted alkyl, and R ₉ and R ₁₀ may combine with each other to form a 4-atom, 5-atom or 6-atom hydrocarbon ring.

R _1, R _2, R ₃ or R ₄ is alkenyl or alkynyl imidazol time, alkenyl of sp ² - mixed-carbon, or alkynyl of sp- mixed carbon is bonded directly or alkenyl of sp ² - mixed-carbon or alkynyl of the alkyl bonded to the carbonyl carbon of sp ³ hybrid sp- - it may be indirectly coupled by a hybrid form of carbon.

The C _a -C _b functional group herein means a functional group having a to b carbon atoms. For example, C _a -C _b alkyl means a saturated aliphatic group, including straight chain alkyl and branched alkyl, having a to b carbon atoms, and the like. The straight chain or branched chain alkyl has 10 or fewer (for example, a straight chain of C ₁ -C ₁₀ , 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 - is a linker selected from, R ₆ is hydrogen, a substituted or unsubstituted C ₁ -C ₆ alkyl, substituted or unsubstituted ring containing at least one heteroatom C ₂ -C ₁₀ heteroalkyl, 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 or heteroaryl, and p is an integer from 1 to 100.

Non-limiting examples of X corresponding to the reactive group include carboxyl, succinimidyl ester, sulfo-succinimidyl ester, isothiocyanate, maleimide ( maleimide, haloacetamide, hydrazide, vinylsulphone, dichlorotriazine, phosphoramidite, alkyl halide, acyl (meth) acrylate, halide, carbohydrazide, hydroxylamine, ketone, alkyne, azide, aliphatic and aromatic amines, sulfotetrafluorophenyl esters (for example, sulfotetrafluorophenyl ester, sulfodichlorophenyl ester, carbonyl azide, sulfonyl chloride, sulfonyl fluoride, boronic acid, c acid, isocyanate, halogen-substituted triazine, halogen-substituted pyridine, halogen-substituted diazine, tetrafluorophenyl ester ( tetrafluorophenyl ester, imido ester, azidonitrophenyl, glyoxal, and aldehyde.

In the -L-Z functional group, L is the same linker as L of the -L-X functional group, and Z is a fluorophore capable of generating a fluorescence signal.

In one embodiment, Z is selected from the group consisting of coumarins, cyanine, bodipy, fluoresceins, rhodamines, pyrenes, carbopyronin, Oxazine, xanthenes, thioxanthene, or acridines, or may be of formula (1)

When Z is having the structure of Formula 1, L is the linker, R ₁ of formula I for the functional group Z of -LZ, R _2, R _3, R _4, R ₅ or connected to the Ar _1, or R _1, R ₂ , R < ₃ >, R < ₄ > or R < ₅ >

Merosian-based compounds according to various embodiments of the present invention may be capable of binding to and labeling target biomolecules via the above-mentioned reactive groups.

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 merocyanine compound via the linker (L), so that the steric hindrance between the biomolecule and the merocyanine compound can be alleviated, The tag can improve the labeling rate for biomolecules such as nucleic acids, proteins, carbohydrates and the like.

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, a sulfonic acid salt, , A heteroaryl, a polyalkylene oxide, a quaternary ammonium salt, an ester and an amide, and m is an integer of 1 to 3.

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.

Furthermore, the merocyanine compound represented by the 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 merocyanine compound as an organic or inorganic anion.

Examples of the counter ion of the merocyanine compound according to an embodiment of the present invention include phosphonic acid hexafluoride ion, halogen ion, phosphonic acid ion, perchloric acid ion, periodic acid ion, antimony hexafluoride ion, An inorganic acid anion such as tartaric acid hexafluoride ion, a fluoroboric acid ion and a fluoride ion, and a thiocyanate ion such as a benzenesulfonic acid ion, a naphthalenesulfonic acid ion, a p-toluenesulfonic acid ion, an alkylsulfonic acid ion, An organic acid ion such as an ion, 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.

When Ar ₁ is substituted, any carbon in the functional group is heavy hydrogen, substituted or unsubstituted C ₁ -C ₁₀ alkyl, substituted or unsubstituted ring containing at least one heteroatom C ₂ -C ₁₀ heteroalkyl, 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, carbamate, nitro, substituted or unsubstituted sulfonamide, polyalkylene oxide, , Substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted aralkyl, quaternary ammonium, phosphoric acid, phosphate, ester (-COOR ₅ ), acyl chloride, sulfonic acid, sulfonate , -LX functional group and -LZ functional group At least one can be replaced.

Further, the substituents adjacent to each other among the functional groups substituted on Ar ₁ may combine with each other to form a 4-atom, 5-atom or 6-membered ring.

More specifically, the merocyanine compound represented by the formula (1) can be represented by the following formulas (2) to (4).

(2)

(3)

[Chemical Formula 4]

here,

R ₇ and R ₈ are each independently selected from the group consisting of hydrogen, deuterium, substituted or unsubstituted C ₁ -C ₁₀ alkyl, substituted or unsubstituted C ₂ -C ₁₀ heteroalkyl containing at least one heteroatom, a C ₂ -C ₁₀ alkenyl, substituted or unsubstituted C ₂ -C ₁₀ alkynyl, substituted or unsubstituted C ₂ -C ₁₀ alkoxy, an aryloxy, a substituted or unsubstituted substituted or unsubstituted C ₁ - C ₁₀ haloalkyl, halogen, cyano, hydroxy, substituted or unsubstituted amino, substituted or unsubstituted amide, carbamate, nitro, substituted or unsubstituted sulfonamide, polyalkylene oxide, carboxyl, carboxylic acid salt , Substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted aralkyl, quaternary ammonium, phosphoric acid, phosphate, ester (-COOR ₅ ), acyl chloride, sulfonic acid, sulfonate, -LX is selected from the functional groups and functional group -LZ, Y ₃ is a sulfur, acid , _{Se, NR 3, CR 3 R 4} , SiR 3 R 4 or -CR ₃ = CR ₄ - a.

Specific examples of the merocyanine-based compounds represented by Formulas 1 to 4 according to an embodiment of the present invention 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]

[Compound 37]

[Compound 38]

[Compound 39]

[Compound 40]

[Compound 41]

[Compound 42]

[Compound 43]

[Compound 44]

[Compound 45]

[Compound 46]

[Compound 47]

[Compound 48]

[Compound 49]

[Compound 50]

[Compound 51]

[Compound 52]

[Compound 53]

[Compound 54]

[Compound 55]

[Compound 56]

[Compound 57]

[Compound 58]

[Compound 59]

[Compound 60]

[Compound 61]

[Compound 62]

[Compound 63]

[Compound 64]

[Compound 65]

The target biomolecule of the merocyanine compound according to various embodiments of the present invention 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 a reactive group bonded to any functional group or merocyanine compound of the merocyanine compound represented by Chemical Formulas (1) to (4), such as amino, sulphydryl, A carboxyl group, a hydroxyl group, a carboxyl group, a hydroxyl group, a carboxyl group, a phosphate group, and a thiophosphate group, or a derivative 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, the merocyanine-based compounds according to various embodiments of the present invention may contain, in addition to biomolecules, drugs containing at least one selected from amino, sulphydryl, carbonyl, hydroxyl, carboxyl, phosphate, and thiophosphate, Can be used to label hormones (including receptor ligands), receptors, enzymes or enzyme substrates, cells, cell membranes, toxins, microorganisms or nano-bio materials (such as polystyrene microspheres)

Dyes, kits and contrast agent compositions for biomolecule labeling

According to another aspect of the present invention, there can be provided a biomolecule labeling dye, kit and contrast agent composition containing at least one selected from merocyanine compounds represented by the general formulas (1) to (4).

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 merocyanine compound represented by Chemical Formulas 1 to 4 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 merocyanine compound represented by the formulas (1) to (4) can be applied to the detection of all biomolecules capable of detecting the fluorescence of labeled solid or semi-solid biomolecules .

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

According to various embodiments of the present invention, a method in which a merocyanine compound is directly reacted with a target biomolecule to label a biomolecule or a target biomolecule and a probe labeled with a merocyanine compound are reacted to be labeled .

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

In addition, a method of identifying a biomolecule labeled with a merocyanine 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). In this case, the enzyme recognizes a double-stranded nucleic acid sequence formed by a primer consisting of a target nucleic acid and an oligonucleotide, An elongation reaction is performed from the recognized 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 nucleotide is synthesized by adding a terminator to an ordinary nucleotide, a terminator is introduced at a certain probability to terminate the reaction, so that 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

Production Example 1: Preparation of Compound 1

5.0 g of Nucleic acids research, 40 (14), e108, 2012 and Tetrahedron, 41 (22), 5341; 1985), 6.9 g of dimethylmalonate, 3.0 g of piperidine and 100 ml of methanol, And the mixture was concentrated under reduced pressure. Then, chloroform and 50% aqueous sulfuric acid were added thereto, and the mixture was stirred for 1 hour. Thereafter, the organic layer was extracted with methylene chloride. The organic layer was concentrated under reduced pressure and purified by silica gel column chromatography to give Compound 1 (1.5 g, MS (ESI): m / z 326.2).

Production Example 2: Preparation of Compound 2 and Compound 3

400 mg of Compound 1 and 8 mL of sulfuric acid were charged into the reactor, stirred for 48 hours, cooled to 0 ° C, neutralized with aqueous sodium hydroxide solution, and filtered. The filtrate was concentrated under reduced pressure and purified by ODS column chromatography to obtain Compound 2 (122 mg, MS (ESI): m / z 389.8) and Compound 3 (30 mg, MS (ESI): m / z 346.2).

Production Example 3: Preparation of Compound 4

5.0 g of Nucleic acids research, 40 (14), e108, 2012 and Tetrahedron, 41 (22), 5341; 1985), 6.9 g of dimethylmalonate, 3.0 g of piperidine and 100 ml of methanol, And the mixture was concentrated under reduced pressure. Then, chloroform and 50% aqueous sulfuric acid were added thereto, and the mixture was stirred for 1 hour. Thereafter, the organic layer was extracted with methylene chloride, concentrated under reduced pressure, and purified by silica gel column chromatography to obtain Compound (4) (1.5 g, MS (ESI): m / z 310.2).

Production Example 4: Preparation of Compound 5

300 mg of Compound 1, 240 mg of sodium hydroxide, 5 mL of methanol and 5 mL of water were added and stirred for 12 hours, then cooled to 0 ° C and neutralized with an aqueous hydrochloric acid solution. Thereafter, the organic layer was extracted with methylene chloride. The organic layer was concentrated under reduced pressure and purified by silica gel column chromatography to give Compound 5 (120 mg, MS (ESI): m / z 312.1).

Production Example 5: Preparation of Compound 6

5.0 g of Nucleic acids research, 40 (14), e108; 2012 and Tetrahedron, 41 (22), 5341; 1985), 6.9 g of dimethylmalonate, 3.0 g of piperidine, And the mixture was concentrated under reduced pressure. Then, chloroform and 50% aqueous sulfuric acid were added thereto, and the mixture was stirred for 1 hour. Thereafter, the organic layer was extracted with methylene chloride, concentrated under reduced pressure, and purified by silica gel column chromatography to obtain Compound 6 (1.5 g, MS (ESI): m / z 376.2).

Production Example 6: Preparation of Compound 7 and Compound 8

400 mg of Compound 6 and 8 mL of sulfuric acid were charged into the reactor, stirred for 48 hours, cooled to 0 ° C, neutralized with aqueous sodium hydroxide solution, and filtered. The filtrate was concentrated under reduced pressure and purified by ODS column chromatography to obtain Compound 7 (122 mg, MS (ESI): m / z 439.7) and Compound 8 (30 mg, MS (ESI): m / z 395.8).

Preparation Example 7: Preparation of Compound 65

(Dyes and Pigments (2014), 101, 1-8, Nucleic acids research, 40 (14), e108; 2012 and Tetrahedron, 41 (22), 5341; 1985) in place of Intermediate 1 in Production Example 1 Compound 65 was prepared (0.4 g, MS (ESI): m / z 442.2) in the same manner as in Production Example 1.

Experimental Example 1

The absorption spectra, emission spectra, molar extinction coefficients and quantum efficiencies of the respective compounds obtained in Production Examples 1 to 7 were measured and shown in Table 1 below.

division menstruum λ _ex (nm) ? _em (nm) ε (Lmol ^-1 cm ^-1 ) φ Compound 1 DMSO 456 479 63,000 0.900 Compound 2 PBS 451 487 44,000 0.796 H ₂ O 453 489 45,000 0.718 DMSO 468 500 30,000 0.768 MeOH 463 493 50,000 0.625 CH ₂ Cl ₂ 429 495 20,000 0.169 Compound 3 PBS 423 492 28,000 0.759 H ₂ O 423 492 28,000 0.764 DMSO 416 470 27,000 0.645 MeOH 410 484 27,000 0.667 CH ₂ Cl ₂ 403 464 22,000 0.227 Compound 4 DMSO 492 516 47,000 0.910 Compound 5 PBS 454 483 49,000 0.633 H ₂ O 452 483 50,000 0.062 DMSO 466 490 49,000 0.913 EtOH 462 485 51,000 0.412 Compound 6 DMSO 474 503 53,000 0.928 Compound 7 H ₂ O 470 502 45,000 0.649 DMSO 486 517 43,000 0.788 EtOH 482 511 47,000 0.587 Compound 8 DMSO 442 480 32,000 0.767 Compound 9 DMSO 492 516 47,000 0.910 Compound 16 DMSO 494 519 47,000 0.935 PBS 492 516 51,000 0.417 H ₂ O 492 516 48,000 0.405 EtOH 490 516 47,000 0.872 CH ₂ Cl ₂ 488 515 49,000 0.137 Compound 60 DMSO 476 513 42,000 - H ₂ O 476 523 38,000 - EtOH 474 519 42,000 - CH ₂ Cl ₂ 470 511 41,000 - Compound 61 DMSO 480 525 47,000 - PBS 468 518 46,000 - H ₂ O 468 516 46,000 - EtOH 480 525 49,000 - CH ₂ Cl ₂ 484 522 46,000 - Compound 62 DMSO 492 516 46,000 0.958 PBS 486 520 29,000 0.193 H ₂ O 468 520 26,000 0.194 EtOH 486 516 47,000 0.820 CH ₂ Cl ₂ 486 511 42,000 0.944 Compound 63 DMSO 416 470 15,000 - PBS 434 507 17,000 - H ₂ O 432 506 17,000 - EtOH 416 485 15,000 - CH ₂ Cl ₂ 414 466 15,000 - Compound 64 DMSO 412 472 26,000 0.84 PBS 423 503 27,000 0.95 H ₂ O 422 500 26,000 0.91 EtOH 408 483 28,000 0.78 CH ₂ Cl ₂ 414 468 22,000 0.01

As shown in Table 1, the merocyanine-based compounds according to various embodiments of the present invention are soluble in various solvents and exhibit high extinction coefficient and quantum efficiency, and can emit fluorescence signals in the visible light wavelength band of 380 nm have.

Experimental Example 2

Goat Anti-Mouse IgG was diluted with 0.1 M sodium bicarbonate buffer to prepare 1 mg / ml protein solution, and 125 μl of protein solution was added to each of 8 tubes. Dye solution (5 mg / ml) containing Compound 2 prepared according to Preparation Example 2 was placed in 1, 2, 3, 5, 7, 9, 11 and 13 μl of the prepared tube and immediately vortexed. Then, the reaction solution was added to the Amicon centrifuge filter after shaking the locker for 30 minutes at room temperature. Filtered with PBS (14000 rpm, 10 min, 5 times) until free dye was removed with Centrifuge.

After centrifugation, the filtrate was collected (1000 rpm, 2 min), diluted with 1 ml of PBS, and the fluorescence was measured by UV, PL and microplate reader.

The ratio of the dye to the protein was determined using the following equation.

A _dye : Absorbance at the maximum absorption wavelength of the dye after labeling

A ₂₈₀ : absorbance at ₂₈₀ nm

E _prot : molar extinction coefficient of protein at 280 nm (210,000 for IgG)

E _dye : molar extinction coefficient of dye at maximum absorption wavelength

X: absorbance at 280 nm / absorbance at maximum absorption wavelength of Compound 2

Referring to FIG. 1 to FIG. 3 showing the spectra after labeling compound 2 with Goat Anti-Mouse IgG, spectral patterns after labeling can be confirmed, and as the D / P ratio increases from 1 to 5, , Respectively.

Experimental Example 3

Goat Anti-Mouse IgG was diluted with 0.1 M sodium bicarbonate buffer to prepare 1 mg / ml protein solution, and 125 μl of protein solution was added to each of 7 tubes. Dye solution (5 mg / ml) containing Compound 7 prepared according to Preparation Example 6 was placed in 1, 2, 3, 4, 5, 6, and 7 μl of prepared tubes and immediately vortexed. Then, the reaction solution was added to the Amicon centrifuge filter after shaking the locker for 30 minutes at room temperature. Filtered with PBS (14000 rpm, 10 min, 5 times) until free dye was removed with Centrifuge.

After centrifugation, the filtrate was collected (1000 rpm, 2 min), diluted with 1 ml of PBS, and the fluorescence was measured with a microplate reader.

Referring to FIG. 4 showing the brightness spectrum after labeling Compound 7 with Goat Anti-Mouse IgG, it can be confirmed that the maximum brightness is obtained when the D / P ratio is 6.

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 (9)

A merocyanine compound represented by the following formula (1): < EMI ID =
[Chemical Formula 1]

here,
Ar ₁ is substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl,
Y ₁ and Y ₂ are each independently selected from sulfur, oxygen, selenium, NR ₃ , CR ₃ R ₄ , SiR ₃ R ₄ and -CR ₃ ═CR ₄ -
Q is sulfur, oxygen or NR &_lt; a &
R ₁ is each 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 ₁ -C ₁₀ haloalkyl, Substituted or unsubstituted amide, substituted or unsubstituted amide, carboxyl, carboxylate, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, ketone (-COR ₅ ), aldehyde, ester (-COOR ₅ ), -LX functional groups and -LZ functional groups,
R _a and R ₂ to R ₄ are each independently selected from the group consisting of hydrogen, deuterium, substituted or unsubstituted C ₁ -C ₁₀ alkyl, substituted or unsubstituted C ₂ -C ₁₀ heteroalkyl including at least one heteroatom, 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, carbamate, sulfhydryl, nitro, carboxyl, carboxylate, substituted or unsubstituted Aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted aralkyl, quaternary ammonium, phosphoric acid, phosphate, ketone (-COR ₅ ), aldehyde, ester (-COOR ₅ ), acyl chloride, sulfonic acid, sulfonate , -LX functional groups and -LZ functional groups,
When R ₁ , R ₂ , R ₃ or R ₄ is a ketone group (-COR ₅ ) or an ester group (-COOR ₅ ), R ₅ is a substituted or unsubstituted C ₁ -C ₁₀ alkyl, at least one heteroatom substituted or unsubstituted, including a C ₂ -C ₁₀ heteroalkyl, substituted or unsubstituted C ₂ -C ₁₀ alkenyl, substituted or unsubstituted C ₂ -C ₁₀ alkynyl, substituted or unsubstituted C ₂ - C ₁₀ alkoxy, is selected from substituted or unsubstituted C ₁ -C ₁₀ haloalkyl, substituted or unsubstituted heteroaryl and substituted or unsubstituted C ₁ -C ₁₀ aminoalkyl,
R ₉ and R ₁₀ are selected from hydrogen, substituted or unsubstituted alkyl,
In the -LX and -LZ functional groups,
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 -, 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 heteroalkyl containing at least one heteroatom, 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 or heteroaryl, p is an integer from 1 to 100,
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, which is a reactive group,
Z is a fluorophore capable of generating a fluorescence signal,
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 a sulfonic acid, a sulfonic acid salt, a carboxylic acid, a carboxylic acid salt, a phosphoric acid, Aryl, polyalkylene oxide, quaternary ammonium salt, ester and amide,
m is an integer of 1 to 3;
The method according to claim 1,
When Ar ₁ is substituted, any carbon in the functional group is heavy hydrogen, substituted or unsubstituted C ₁ -C ₁₀ alkyl, substituted or unsubstituted ring containing at least one heteroatom C ₂ -C ₁₀ heteroalkyl, 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, carbamate, nitro, substituted or unsubstituted sulfonamide, polyalkylene oxide, , Substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted aralkyl, quaternary ammonium, phosphoric acid, phosphate, ester (-COOR ₅ ), acyl chloride, sulfonic acid, sulfonate , -LX functional group and -LZ functional group The at least one substitution,
Merocyanine compound.
The method according to claim 1,
Z may be selected from the group consisting of coumarins, cyanine, bodipy, fluoresceins, rhodamines, pyrenes, carbopyronin, oxazine, xanthenes, thioxanthene, acridines and a structure represented by the formula (1)
Merocyanine compound,
The method according to claim 1,
R ₉ and R ₁₀ are bonded to each other to form a 4-atom, 5-atom or 6-atom hydrocarbon ring,
Merocyanine compound.
The method according to claim 1,
Wherein the merocyanine compound is at least one selected from the group consisting of compounds represented by the following formulas:
Merocyanine compound:
(2)

(3)

[Chemical Formula 4]

here,
R ₇ and R ₈ are each independently selected from the group consisting of hydrogen, deuterium, substituted or unsubstituted C ₁ -C ₁₀ alkyl, substituted or unsubstituted C ₂ -C ₁₀ heteroalkyl containing at least one heteroatom, a C ₂ -C ₁₀ alkenyl, substituted or unsubstituted C ₂ -C ₁₀ alkynyl, substituted or unsubstituted C ₂ -C ₁₀ alkoxy, an aryloxy, a substituted or unsubstituted substituted or unsubstituted C ₁ - C ₁₀ haloalkyl, halogen, cyano, hydroxy, substituted or unsubstituted amino, substituted or unsubstituted amide, carbamate, nitro, substituted or unsubstituted sulfonamide, polyalkylene oxide, carboxyl, carboxylic acid salt , Substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted aralkyl, quaternary ammonium, phosphoric acid, phosphate, ester (-COOR ₅ ), acyl chloride, sulfonic acid, sulfonate, -LX Functional groups and -LZ functional groups,
Y ₃ is sulfur, oxygen, selenium, NR ₃ , CR ₃ R ₄ , SiR ₃ R ₄ or -CR ₃ = CR ₄ -.
A biomolecule marking dye comprising the merocyanine compound according to any one of claims 1 to 5.
The method according to claim 6,
Wherein the biomolecule is at least one selected from antibodies, lipids, proteins, peptides, carbohydrates and nucleic acids,
Dye for biomolecule marking.
A biomolecule marking kit comprising the biomolecule marking dye according to claim 7.
6. A contrast agent composition comprising a merocyanine compound according to any one of claims 1 to 5.

Images