KR101880502B1 - Compound based merocyanine, labeling dye, kit and contrast medium composition for biomolecule comprising the same - Google Patents

Abstract

The present invention relates to a novel merocyanine compound capable of labeling biomolecules by intercalating biomolecules, a biomolecule labeling dye, a kit and a contrast agent composition containing the same.

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 capable of labeling biomolecules by intercalating biomolecules, a biomolecule labeling dye, a kit and a contrast agent composition containing the same.

Fluorescent dyes have been used for the detection of a variety of biological samples involving nucleic acids and nucleic acids, including DNA and RNA. Accordingly, studies on fluorescent nucleic acid dyes that form a complex that exhibits excellent fluorescence due to specific binding or intercalation of nucleic acid have been actively conducted.

Dyes specifically bound or intercalated into nucleic acids can be used in pure solutions, cell extracts, electrophoresis gels, microarray chips, live or fixed cells, dead cells and environmental samples, and the presence of DNA and RNA in various samples And the amount of water. In particular, such fluorescent dyes can be used for the detection of nucleic acids through polymerase chain reaction (PCR), which is a representative method used in genome research and medical diagnosis.

In this case, since the quantitative PCR which is proportional to the amount of the sample nucleic acid can not be performed in the case of the general end-point PCR, the fluorescence value which changes in real time in each amplification cycle is measured and the quantitative analysis of the quantity of the nucleic acid is performed. real-time PCR or qPCR).

Such qPCR is measured quantitatively by measuring the fluorescence signal from the PCR product. For the measurement of the fluorescence signal, a detection method using a fluorescent probe and a detection method using an intercalating fluorescent dye are mainly known.

A detection method using a fluorescent probe uses a probe (for example, an oligonucleotide) labeled with a complex of a fluorescent dye and a quencher dye. When an oligonucleotide labeled with a complex of a fluorescent dye and a quencher dye is hybridized with the target sequence, the fluorescence signal is generated as the fluorescent dye is cleaved and separated from the complex.

The detection method using the above-described fluorescent probe has an advantage of high selectivity and specificity with respect to the target sequence, but it is disadvantageous in that the design is complicated and expensive because it is used in a large amount.

Detection methods using intercalating fluorescent dyes are based on DNA-binding fluorescent dyes referred to as fluorescent nucleic acid dyes or stains. Fluorescent nucleic acid dyes are advantageous because they are relatively simple molecules and therefore are relatively easy to design and manufacture.

However, since several criteria must be met in order to be used in qPCR, generally known fluorescence nucleic acid dyes can not be used for qPCR.

For example, fluorescent nucleic acid dyes used in qPCR should have sufficient stability during storage and PCR and should be resistant to the pH range of the buffer used for PCR.

In addition, when the nucleic acid is not present, the fluorescent nucleic acid dye should generate no fluorescence signal or generate only a very weak fluorescence signal, and generate a relatively strong fluorescence signal in the presence of nucleic acid.

The most commonly used dyes for labeling conventional nucleic acids are ethidium bromide.

[Bromide ethidium]

Brominated ethidium is used to stain electrophoresis gels (post-staining), and is added before the electrophoresis gel preparation to be stained with electrophoresis (pre-staining) do. In addition, since bromide ethidium generates UV light of 400 nm or less and has an emission spectrum of about 620 nm when bound to DNA, there is an advantage that the presence and position of DNA can be visually confirmed easily.

Despite the advantages of the above-mentioned brominated ethidium bromide ethidium is considered to be a mutagen and carcinogen, thus requiring considerable caution in use. In particular, it has been reported that the bromide ethidium inhibits DNA synthesis by interfering with the synthesis of DNA and RNA, thereby causing mutation.

As a result, SYBR® green Ⅰ was mainly used as a substitute fluorescent dye showing non-oil toxicity. However, since SYBR green I inhibits the PCR process, increasing the concentration of SYBR green I simply limits the maximum fluorescence signal.

In other words, the intensity of the fluorescence signal increases in proportion to the concentration of SYBR® green Ⅰ until the baseline concentration of SYBR® green Ⅰ begins to significantly inhibit the PCR process, DNA amplification is reduced with an additional increase in concentration. As a result, the intensity of the observed fluorescence signal is decreased or the threshold cycle for observing the fluorescence signal of a predetermined intensity or more is increased.

It is also known that SYBR® green Ⅰ is unstable under certain chemical conditions and therefore degrades considerably within a few days in the buffer.
A prior art related to such an intercalating fluorescent dye is Korean Patent Publication No. 10-2006-0044599.

Under the above technical background, the present invention aims to provide a merocyanine compound as an intercalating fluorescent dye suitable for use in qPCR using an intercalating fluorescent dye.

It is also an object of the present invention to provide a non-genotoxic optional merocyanine compound unlike the brominated ethidium.

It is another object of the present invention to provide a merocyanine compound capable of increasing the intensity of a fluorescence signal in proportion to the concentration of nucleic acid without increasing the number of threshold cycles.

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

It is another object of the present invention to provide a method for determining and quantifying the presence or absence of nucleic acid or cell viability using the merocyanine compound.

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

[Chemical Formula 1]

here,

Ar is a substituted or unsubstituted aromatic ring,

Y ₁ and Y ₂ are each independently selected from sulfur, oxygen, selenium, NR ₈ and -CR ₈ ═CR ₉ -

R ₁ to R ₉ 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 amides, carbamates, sulfhydryl, nitro, carboxyl, carboxylic acid salt, a substituted or unsubstituted aryl, substituted Or unsubstituted heteroaryl, substituted or unsubstituted aralkyl, quaternary ammonium, phosphoric acid, phosphate, ketone (-COR ₁₀ ), aldehyde, ester (-COOR ₁₀ ), acyl chloride, sulfonic acid, sulfonate, polyalkyl Lt; RTI ID = 0.0 > -LZ < / RTI &

When R _a is a ketone group (-COR ₁₀ ) or an ester group (-COOR ₁₀ ), R ₁₀ is substituted or unsubstituted C ₁ -C ₁₀ alkyl, at least Substituted or unsubstituted C ₂ -C ₁₀ heteroalkyl containing one heteroatom, substituted or unsubstituted C ₂ -C ₁₀ alkenyl, substituted or unsubstituted C ₂ -C ₁₀ alkynyl, substituted or unsubstituted Substituted C ₂ -C ₁₀ alkoxy, substituted or unsubstituted C ₁ -C ₁₀ haloalkyl, and substituted or unsubstituted C ₁ -C ₁₀ aminoalkyl,

R _b (Where b is an integer selected from 1 to 10), 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 sulfonate, a ketone, an aldehyde, a carboxylic acid, a carboxylic acid salt, a phosphoric acid, A polyalkylene oxide, a quaternary ammonium salt, an ester, and an amide,

m is an integer of 1 to 3,

L is a linker comprising 3 to 150 non-hydrogen atoms,

Z is a fluorescent moiety capable of generating a fluorescent signal, or has a structure represented by the formula (1)

The structure represented by the formula (1) has one or two -LZ functional groups.

Here, as a fluorescent moiety capable of generating a fluorescent signal, Z is a fluorescent moiety such as phenanthridium, coumarins, cyanine, bodipy, fluoresceins, rhodamines, May be selected from pyrenes, carbopyronin, oxazine, xanthenes, thioxanthene, and acridine.

According to one embodiment of the present invention, the -L-Z functional group may be represented by the following formula (2).

(2)

Wherein L ₁ and L ₂ are each independently a C ₁ -C ₁₂ polymethylene unit optionally containing at least one heteroatom selected from nitrogen, oxygen and sulfur, or at least one heteroatom selected from nitrogen, oxygen and sulfur And A ¹ to A ¹⁰ are each independently a chain alkyl or branched alkyl optionally containing at least one heteroatom selected from nitrogen, oxygen and sulfur, or from nitrogen, oxygen and sulfur, Each of x1 to x9 is independently 0 or an integer of 1 to 20, and y1 to y9 each independently represent 0 or 1 to 20 carbon atoms, It is an integer.

Also, Z may be selected from the group consisting of phenanthridium, coumarins, cyanine, bodipy, fluoresceins, rhodamines, pyrenes, carbopyronin ), Oxazine, xanthenes, thioxanthene, and acridine, or may have a fluorophore structure other than that.

According to another variant, one of A ¹ to A ¹⁰ may be represented by the following formula (3).

(3)

Wherein R ₁₁ is carbon, nitrogen or aryl optionally containing at least one heteroatom selected from nitrogen, oxygen and sulfur, and R ₁₂ is represented by the following formula (4).

[Chemical Formula 4]

_{^{- [A 11 - (CH 2}} ) x11 -] y11 [A 12 - (CH 2) x12 -] y12 [A 13 - (CH 2) x13 -] y13 -A 14 -L 3 -Z

Wherein L ₃ is a C ₁ -C ₁₂ polymethylene unit optionally comprising at least one heteroatom selected from nitrogen, oxygen and sulfur, or optionally containing at least one heteroatom selected from nitrogen, oxygen and sulfur Aryl, A ¹¹ to A ¹⁴ are each independently a chain alkyl or branched alkyl optionally containing at least one heteroatom selected from nitrogen, oxygen and sulfur, or at least one heteroatom selected from nitrogen, oxygen and sulfur And x 11 to x 13 are each independently 0 or an integer of 1 to 20, and y 11 to y 13 are each independently 0 or an integer of 1 to 20.

According to another aspect of the present invention, there can be provided a biomolecule labeling dye containing a merocyanine compound.

According to still another aspect of the present invention, there is provided a biomolecule marking kit comprising a merocyanine compound.

Further, according to another aspect of the present invention, a contrast agent composition comprising a merocyanine compound may be provided.

Further, according to another aspect of the present invention, an electrophoresis kit for determining the presence or absence of nucleic acid in a sample may be provided.

Further, according to another aspect of the present invention, a method of determining the presence or absence of a nucleic acid in a sample or a method of quantifying the nucleic acid can be provided.

According to still another aspect of the present invention, a method of analyzing or quantifying the viability of cells in a sample can be provided.

The novel merocyanine compound according to the present invention can exhibit fluorescence signals in a visible light region by intercalating biomolecules and exhibits non-genetic toxicity. Based on this, the merocyanine compound Can be usefully used as biomolecule labeling dyes, biomolecule marking kits, and contrast agent compositions.

FIG. 1 shows electrophoresis results of SYBR® safe, a commercially available dye, and FIGS. 2 to 10 show electrophoresis results of merocyanine compounds according to various embodiments of the present invention.
FIG. 11 shows the absorption and emission spectrum results of SYBR® safe, a commercial dye, and FIGS. 12 and 13 show absorption and emission spectral results of merocyanine compounds according to various embodiments of the present invention.
FIG. 14 is an image showing the results of a cell permeability test of a SYBR® safe dye as a commercialized dye, and FIGS. 15 to 17 are images showing cell permeability test results of a merocyanine compound according to various embodiments of the present invention.
FIG. 18 is a graph showing the results of cell cycle analysis using propidium iodide, a commercially available cell cycle assay dye, and FIG. 19 is a graph showing the results of cell cycle analysis using Compound 6. FIG.
20 is a graph showing a melt curve of qPCR using Compound 38 as a fluorescent dye, Fig. 21 is a graph showing a melt curve of qPCR using SYBR green I as a fluorescent dye, and Fig. Is a graph showing the melt curve of qPCR using EvaGreen ^TM as a fluorescent dye.
FIGS. 23 to 25 are graphs showing results of light discoloration experiments of Compound 38, FIGS. 26 to 28 are graphs showing results of light discoloration experiments of SYBR® green I (manufactured by Invitrogen) ^TM (manufactured by Biotium), and FIG.
32 and 33 are graphs showing the amplification curves of qPCR and the melting curves of qPCR using SYBR green I and compound 38 as fluorescent dyes,
34 is a graph showing the amplification curve of qPCR according to the fluorescent dye concentration using Compound 38 as a fluorescent dye,
35 is a graph showing the amplification curve of qPCR according to the fluorescent dye concentration using SYBR green I as a fluorescent dye,
36 is a graph showing the amplification curve of qPCR according to the fluorescent dye concentration using EvaGreen ^TM as a fluorescent dye,
37 to 42 are graphs showing amplification curves and melting curves of qPCR using Compound 36, Compound 38, Compound 39 and EvaGreen ^TM as fluorescent dyes.

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 Merocyanine system  compound

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

[Chemical Formula 1]

Wherein Ar 1 is a substituted or unsubstituted aromatic ring, for example, a benzo or naphto group, Y ₁ and Y ₂ are each independently selected from the group consisting of sulfur, oxygen, selenium, NR _8, and -CR ₈ = CR ₉ -, and m is an integer of 1 to 3.

R ₁ to R ₉ 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 amides, carbamates, sulfhydryl, nitro, carboxyl, carboxylic acid salt, a substituted or unsubstituted aryl, substituted Or unsubstituted heteroaryl, substituted or unsubstituted aralkyl, quaternary ammonium, phosphoric acid, phosphate, ketone (-COR ₁₀ ), aldehyde, ester (-COOR ₁₀ ), acyl chloride, sulfonic acid, sulfonate, polyalkyl Lt; RTI ID = 0.0 > -LZ < / RTI >

R _a when an alkenyl or alkynyl imidazol (where, a is an integer selected from 1 to 9), sp ² of alkenyl-alkenyl of an sp- mixed carbon of the hybrid carbon or alkynyl group bonded directly or Al sp ² - of the alkyl bonded to the carbon of a hybrid mixed sp- or alkynyl carbon sp ³ - 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 ).

Aryl herein, unless otherwise defined, means an unsaturated aromatic ring comprising a single ring or multiple rings (preferably one to four rings) joined together or covalently bonded to each other. Non-limiting examples of aryl include phenyl, biphenyl, terphenyl, m-terphenyl, p-terphenyl, 1-naphthyl, 2- naphthyl, Anthryl, 9-anthryl, phenanthrenyl, 2-phenanthrenyl, 3-phenanthrenyl, 4-phenanthrenyl, 9-phenanthrenyl, 1-pyrenyl, And 4-pyrenyl.

As used herein, heteroaryl means a functional group in which at least one carbon atom in the aryl as defined above is replaced by a non-carbon atom such as nitrogen, oxygen or sulfur. Non-limiting examples of heteroaryl include furyl, tetrahydrofuryl, phrrolyl, pyrrolidinyl, thienyl, tetrahydrothienyl, oxazolyl, isooxa Isoxazolyl, triazolyl, thiazolyl, isothiazolyl, pyrazolyl, pyrazolidinyl, oxadiazolyl, thiadiazolyl, thiadiazolyl, Imidazolyl, imidazolinyl, pyridyl, pyridaziyl, triazinyl, piperidinyl, morpholinyl, thiophene, imidazolinyl, imidazolinyl, But are not limited to, thiomorpholinyl, pyrazinyl, piperainyl, pyrimidinyl, naphthyridinyl, benzofuranyl, benzothienyl, indolyl, , Indolizinyl, indazolyl, quinolizinyl, quinolinyl, isoquinolinyl, And examples thereof include cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, pteridinyl, quinuclidinyl, carbazoyl, acridinyl, phenazinyl, phenothizinyl, , Phenoxazinyl, purinyl, benzimidazolyl, and benzothiazolyl, and the like.

A cycloalkyl or a heterocycloalkyl containing a heteroatom herein may be understood as a cyclic structure of alkyl or heteroalkyl, respectively, unless otherwise defined.

Non-limiting examples of hydrocarbon rings include cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, and cycloheptyl. Non-limiting examples of hydrocarbon rings containing heteroatoms include 1- (1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4- Tetrahydrofuran-2-yl, tetrahydrothien-3-yl, 1-piperazinyl, 2-piperazinyl, tetrahydropyranyl, - piperazinyl, and the like.

A hydrocarbon ring containing a hydrocarbon ring or a hetero atom may also have a hydrocarbon ring, a hydrocarbon ring containing a hetero atom, an aryl or a heteroaryl, or a covalent bond.

When R _a is a ketone group (-COR ₁₀ ) or an ester group (-COOR ₁₀ ), R ₁₀ is substituted or unsubstituted C ₁ -C ₁₀ alkyl, at least Substituted or unsubstituted C ₂ -C ₁₀ heteroalkyl containing one heteroatom, substituted or unsubstituted C ₂ -C ₁₀ alkenyl, substituted or unsubstituted C ₂ -C ₁₀ alkynyl, substituted or unsubstituted Substituted C ₂ -C ₁₀ alkoxy, substituted or unsubstituted C ₁ -C ₁₀ haloalkyl, and substituted or unsubstituted C ₁ -C ₁₀ aminoalkyl.

R _b (Where b is an integer selected from 1 to 10), 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 sulfonate, a ketone, an aldehyde, a carboxylic acid, a carboxylic acid salt, a phosphoric acid, A polyalkylene oxide, a quaternary ammonium salt, an ester, and an amide.

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.

According to various variants of the present invention, R _a , where a is an integer selected from 1 to 9, may have at least one -LZ functional group.

Here, L is a linker containing 3 to 150 non-hydrogen atoms connecting the structure represented by the formula (1) and Z, which is a fluorescent group capable of generating a fluorescent signal, and specifically, 8 to 150 non- And is a linker containing a hydrogen atom.

Also, Z may be selected from the group consisting of phenanthridium, coumarins, cyanine, bodipy, fluoresceins, rhodamines, pyrenes, carbopyronin ), Oxazine, xanthenes, thioxanthene, and acridine, or may have a structure represented by Formula (1).

According to one embodiment of the present invention, the -L-Z functional group may be represented by the following formula (2).

(2)

^{_{-L 1 - [A 1 - (}} CH 2) x1 -] y1 [A 2 - (CH 2) x2 -] y2 [A 3 - (CH 2) x3 -] y3 [A 4 - (CH 2) x4 - ^{_{] y4 [A 5 - (CH}} 2) x5 -] y5 [A 6 - (CH 2) x6 -] y6 [A 7 - (CH 2) x7 -] y7 [A 8 - (CH 2) x8 -] y8 _{^{[A 9 - (CH 2)}} x9 -] y9 -A 10 -L 2 -Z

Wherein L ₁ and L ₂ are each independently a C ₁ -C ₁₂ polymethylene unit optionally containing at least one heteroatom selected from nitrogen, oxygen and sulfur, or at least one heteroatom selected from nitrogen, oxygen and sulfur And A ¹ to A ¹⁰ are each independently a chain alkyl or branched alkyl optionally containing at least one heteroatom selected from nitrogen, oxygen and sulfur, or from nitrogen, oxygen and sulfur, Each of x1 to x9 is independently 0 or an integer of 1 to 20, and y1 to y9 each independently represent 0 or 1 to 20 carbon atoms, It is an integer.

Also, Z may be selected from the group consisting of phenanthridium, coumarins, cyanine, bodipy, fluoresceins, rhodamines, pyrenes, carbopyronin ), Oxazine, xanthenes, thioxanthene, and acridine, or may have a fluorophore structure other than that.

According to another variant, one of A ¹ to A ¹⁰ may be represented by the following formula (3).

(3)

Wherein R ₁₁ is carbon, nitrogen or aryl optionally containing at least one heteroatom selected from nitrogen, oxygen and sulfur, and R ₁₂ is represented by the following formula (4).

[Chemical Formula 4]

_{^{- [A 11 - (CH 2}} ) x11 -] y11 [A 12 - (CH 2) x12 -] y12 [A 13 - (CH 2) x13 -] y13 -A 14 -L 3 -Z

Wherein L ₃ is a C ₁ -C ₁₂ polymethylene unit optionally comprising at least one heteroatom selected from nitrogen, oxygen and sulfur, or optionally containing at least one heteroatom selected from nitrogen, oxygen and sulfur Aryl, A ¹¹ to A ¹⁴ are each independently a chain alkyl or branched alkyl optionally containing at least one heteroatom selected from nitrogen, oxygen and sulfur, or at least one heteroatom selected from nitrogen, oxygen and sulfur And x 11 to x 13 are each independently 0 or an integer of 1 to 20, and y 11 to y 13 are each independently 0 or an integer of 1 to 20.

In addition, the merocyanine compound according to an embodiment of the present invention 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.

The merocyanine based compounds according to various embodiments 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]

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 merocyanine compounds according to various embodiments of the present invention.

In addition, if necessary, the biomolecule marker kit may further include an enzyme, a solvent (buffer solution, etc.) for intercalation in the target nucleic acid, a 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 the biomolecule marker dye according to an embodiment of the present invention, the dimeric and / or trimeric merocyanine compounds may exist in a state of being coagulated with each other due to intermolecular interaction.

At this time, the merocyanine compound in dimer and / or trimer form does not generate a fluorescence signal in the aggregated state. On the other hand, when a biomolecule (for example, nucleic acid) is present, the dimeric and / or trimeric merocyanin compounds in the aggregated state are separated from each other and are intercalated into the nucleic acid, .

That is, the presence or absence of an intercalable biomolecule serves as a quencher of a dye for biomolecule labeling according to an embodiment of the present invention.

For example, the kit for biomolecule labeling according to an embodiment of the present invention separates the nucleic acid, which is a biomolecule, by size, and then, when the merocyanine compound in the biomolecule marker dye is intercalated in the nucleic acid, And an electrophoresis kit.

Specifically, the electrophoresis kit comprises at least one compound selected from merocyanine-based compounds according to various embodiments of the present invention; At least one substance for forming a buffer, a gel matrix, a gel matrix, at least one substance for forming a surface or a surface; And an electrophoresis kit for determining the presence or absence of a nucleic acid in a sample or a sample immobilized on the surface when the nucleic acid is present in the sample.

The surface can be a solid surface, a film surface, a glass surface, a plastic surface, or a polysilicon surface. The matrix may be selected from the group consisting of alginate, collagen, peptides, fibrin, hyaluronic acid, agarose, polyhydroxyethyl methacrylate, polyvinyl alcohol, polyethylene glycol, polyethylene oxide, polyethylene glycol diacrylate, gelatin, matrigel, polylactic acid, carboxymethylcellulose, dextran, chitosan, latex and sepharose, and may be in the form of beads or membranes.

In addition, the contrast agent composition according to the above embodiments may further include a pharmaceutically acceptable carrier besides the merocyanine compound according to various embodiments of the present invention to be administered orally or parenterally.

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, the biomolecule labeling method using the biomolecule marking dye or biomolecule marking kit described above will be described.

Biomolecule labeling method using biomolecule marker dye

The method of labeling a biomolecule with a merocyanine compound according to various embodiments of the present invention can be applied to the labeling of all biomolecules capable of labeling the fluorescence of the labeled solid or semi-solid biomolecule.

By using a merocyanine compound instead of the conventional fluorescent dye, it is possible to provide a labeling method which is chemically stable with high sensitivity and excellent in operability.

According to various embodiments of the present invention, a method of labeling a biomolecule by intercalating a merocyanine compound into a target biomolecule can be implemented. It can also be used to quantify biomolecules in liquids or gels, to stain biomolecules in living cells or dead cells, and to detect and quantify biomolecules in microarrays.

Specifically, according to various embodiments of the present invention, the merocyanine compound can be used to determine the presence or absence of a nucleic acid in a sample. More specifically, when a nucleic acid is present in a sample, the nucleic acid is exposed to a merocyanine compound according to various embodiments of the present invention, and the merocyanine compound is intercalated into a nucleic acid to form a complex; And determining the fluorescence of the merocyanine-based compound or its absence, thereby determining the presence or absence of nucleic acid in the sample.

The merocyanine compound is chemically stable with high sensitivity and easily intercalated with the nucleic acid present in the sample to form a complex. Then, the formed complex is irradiated with light having a sufficient wavelength, and the presence or absence of the nucleic acid in the sample can be easily determined by the emitted fluorescence signal.

The fluorescence signal can be detected through various devices such as a plate reader, a microscope, a fluorometer, a quantum counter and a flow cell sorter, or through the naked eye.

In addition, the presence or absence of the amplified target nucleic acid can be determined using the merocyanine compound.

Specifically, there is provided a method for performing a nucleic acid amplification reaction comprising: providing a reaction mixture comprising a target nucleic acid, a reagent necessary for amplifying the target nucleic acid, and a merocyanine compound according to various embodiments of the present invention; Polymerizing the reaction mixture under conditions suitable for the formation of the amplified target nucleic acid; Illuminating the reaction mixture with light; And detecting fluorescence emission from the reaction mixture to determine the presence or absence of the amplified target nucleic acid.

The reaction mixture may contain, in addition to the target nucleic acid, amplification enzymes, primers sufficient for target nucleic acid sequence amplification, reagents such as deoxynucleoside triphosphate, and the like.

Since the merocyanine compound can be used without inhibiting the PCR reaction, it can be useful for determining the presence or absence of the amplified target nucleic acid.

In addition, the nucleic acid in the sample can be quantified using the merocyanine compound. Specifically, mixing a mixture comprising a merocyanine compound according to various embodiments of the present invention with a sample comprising a nucleic acid; Incubating the sample and the mixture for a sufficient time to cause the merocyanine compound to intercalate with the nucleic acid in the sample to form a complex and generate a fluorescence signal; And quantifying the nucleic acid in the sample by comparing fluorescence standard characteristics of the fluorescence signal to be detected with the fluorescence standard characteristic of the predetermined nucleic acid amount.

The amount of nucleic acid in the sample can be quantified relative to the fluorescence signal that is sensed based on the fluorescence standard characteristic of a particular nucleic acid amount.

A substantially primary relationship between the amount of nucleic acid and the fluorescence intensity may be used for quantification of the nucleic acid, or for cell count if a cell extract is used. In one example, the nucleic acid may be impregnated with an inert matrix such as a blot or gel, or attached to a solid surface such as a microarray chip or any other solid surface. At this time, a solution containing the merocyanine compound is applied to the surface of the nucleic acid-containing matrix, or the surface of the microarray chip or other solid surface, and incubation is performed for a sufficient time to form the dye-nucleic acid complex.

Also, according to various embodiments of the present invention, a method of intercalating a merocyanine compound into a biomolecule contained in a target cell to quantify the target cell viability and the target cell can be implemented.

Specifically, mixing a mixture comprising a merocyanine compound according to various embodiments of the present invention with a sample comprising apoptotic cells; Incubating the sample and the mixture for a sufficient time to cause the merocyanine compound to intercalate with the apoptotic cells in the sample to form a complex and generate a fluorescence signal; And quantifying the apoptotic cells in the sample by comparing fluorescence standard characteristics of the predetermined fluorescence signal with the fluorescence signal to be detected, and quantifying the viability of the cells in the sample.

According to one embodiment of the present invention, the merocyanine compound does not penetrate cells, that is, live cells, but can pass through dead cells. By using such a characteristic, it is carried out by incubating the merocyanine compound for a sufficient time to pass through the membrane of the apoptotic cell and form a complex with the nucleic acid in the membrane.

In another embodiment, a solution comprising a merocyanine compound according to various embodiments of the present invention that intercalates with apoptotic cells and a compound other than the merocyanin compound capable of intercalating with the cell, Mixing the sample comprising; Light-illuminating a sample comprising the merocyanine-based compound and other cells intercalated with the compound; And detecting fluorescence emission from the sample, wherein the fluorescence emitted from the sample intercalates with the cells in the sample together with the merocyanine compound and the other compounds, resulting in a fluorescence reaction between the cells And the emitted fluorescence differs from the fluorescence generated by the fluorescence reaction of only the merocyanine compound and may be a method of analyzing the viability of the cells in the sample.

According to one embodiment of the present invention, the merocyanine compound can intercalate with apoptotic cells. Specifically, the merocyanine compound can intercalate with the nucleic acid in the apoptotic cell by permeating the apoptotic cell.

On the other hand, compounds other than the merocyanine compound, for example, Acridine orange (AO)

Can intercalate with cells as well as dead cells as well as dead cells. In other words, Acridine orange (AO) permeates not only dead cells but also live cell membranes and can intercalate with intracellular nucleic acids.

The fluorescence signal generated from the complex formed by intercalating a nucleic acid and a compound other than the merocyanine compound and the merocyanine compound can be measured to analyze the viability of cells in the sample. Specifically, the viability of the cells can be analyzed by measuring and distinguishing the wavelength of the fluorescence signal generated by the complex of the merocyanine compound and the nucleic acid and the fluorescence signal generated by the complex of the other compound and the nucleic acid.

In addition, the present invention can be a kit for analyzing the viability of cells in a sample using the above-described specification.

Specifically, merocyanine-based compounds according to various embodiments of the present invention; And a kit for analyzing the viability of a cell in a sample in which fluorescence is detected by intercaling with the merocyanin compound when the apoptotic cell is present in the sample.

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 dye is reacted with a target nucleic acid to be labeled (that is, a merocyanine compound is intercalated in a target nucleic acid), and a single chain probe having a base sequence complementary to the target nucleic acid A fluorescent signal can be generated by preparing a nucleic acid, hybridizing the single-stranded target nucleic acid and the probe nucleic acid on the substrate, and allowing the fluorescent dye to be intercalated into the target nucleic acid.

As the probe nucleic acid immobilized on the substrate in this labeling method, when the expression of a gene is examined, a library of cDNA such as cDNA, a library of genomes, or all genomes are prepared by amplification by the PCR method using a template 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.

PCR  method

In the PCR method, the probe complementary to the base sequence of the target nucleic acid to be labeled 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 double-stranded nucleic acid sequence formed by the primer consisting of the target nucleic acid and the oligonucleotide is recognized by the enzyme, Extension reaction is carried out from the 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.

In the label, 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 DNA base sequence, 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 labeling methods include a molecular beacon method, a TaqMan-PCR method, and a cycling probe method for confirming the presence of a nucleic acid having a specific sequence.

Other labeling methods

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.

Hereinafter, specific embodiments of the present invention will be described. However, the embodiments described below are only intended to illustrate or explain the present invention, and thus the present invention should not be limited thereto.

Manufacturing example

Manufacturing example  1. Synthesis of Compound 1

(1) Synthesis of intermediate 1 and intermediate 2

2- (methylthio) benzothiazole (11.115 g, 0.0614 mol) and acetonitrile (110 mL) were added to a 250 mL 1-neck reactor, and the mixture was stirred at room temperature for 5 minutes. Then, oxolane, i.e., 2-iodoethyl-1,3-dioxolane (21 g, 0.0921 mol) was added to the reactor, and the mixture was stirred under reflux for 40 hours, cooled, concentrated and then subjected to column purification to obtain Intermediate 1 mol, 85%). ^{1 H-NMR (400 MHz,} CDCl 3) δ 7.45-7.48 (m, 1H), 7.38-7.43 (m, 1H), 7.26-7.32 (m, 2H), 5.00 (t, J = 4.0 Hz, 1H) , 4.54-4.58 (m, 2H), 3.89-4.02 (m, 4H), 2.18-2.23 (m, 2H)

Subsequently, Intermediate 1 (2.37 g, 0.01 mol) and acetonitrile (30 mL) were added to a 100 mL 1-neck reactor, and the mixture was stirred at room temperature for 5 minutes. To the reactor was added methyl iodide (MeI) (1.26 g, 0.03 mol), and the mixture was stirred under reflux for 12 hours, cooled, concentrated, and ethyl acetate (50 mL) was added to precipitate a solid. The precipitated solid was filtered and vacuum dried to give Intermediate 2 (3 g, 0.00733 mol, 73%). ^{1 H-NMR (400 MHz,} CDCl 3) δ 8.51 (d, J = 8.0Hz, 1H), 7.92 (d, J = 8.4Hz, 1H), 7.76 (t, J = 8.4Hz, 1H), 7.64 ( 2H), 3.84-4.00 (m, 2H), 3.21 (s, 3H), 4.78 (t, J = 2.38 - 2.42 (m, 2H)

(2) Synthesis of intermediate 3

4-methyl-5-quinolinol (2 g, 0.0125 mol), ethyl 6-iodohexanoate (10 g, 0.0375 mol) and dimethylformamide (4 mL) were placed in a 50 mL single- For 12 hours, then cooled, concentrated and column-purified to give Intermediate 3 (3.5 g, 0.00828 mol, 67%). ^{1 H-NMR (400 MHz,} DMSO-d 6) δ 9.19 (d, J = 6.4Hz, 1H), 7.952 (t, J = 8.0Hz, 1H), 7.73-7.77 (m, 2H), 7.22 (d J = 7.6 Hz, 2H), 4.85 (t, J = 6.8 Hz, 2H), 4.01 (q, J = 7.6 Hz, 2H), 3.09 ), 1.89-1.93 (m, 2H), 1.55-1.61 (m, 4H), 1.12 (t, J = 6.8 Hz,

(3) Synthesis of intermediate 4

Intermediate 2 (1.55 g, 3.814 mmol), Intermediate 3 (1.64 g, 3.814 mmol) and dichloromethane (30 mL) were added to a 100 mL 1-necked reactor, and the mixture was stirred at room temperature for 5 minutes. Then, triethylamine (1.15 g, 11.442 mmol) was added to the reactor, followed by stirring at room temperature for 12 hours, followed by concentration and column purification to obtain Intermediate 4 (1.5 g, 2.263 mmol, 59%).

(4) Synthesis of intermediate 5

Intermediate 4 (1.5 g, 2.263 mmol) and chloroform (45 mL) were added to a 250 mL 1-necked reactor and stirred at room temperature for 5 minutes. 50% aqueous sulfuric acid solution (9 mL) was added to the reactor and stirred for 12 hours. Subsequently, water (10 mL) was added to the reactor and the mixture was extracted with dichloromethane (2 x 50 mL). The organic layer was concentrated and column-purified to obtain Intermediate 5 (0.4 g, 0.666 mmol, 29%). ^{1 H-NMR (400 MHz,} MeOD) δ 8.35 (d, J = 7.2Hz, 1H), 7.91 (d, J = 8.0Hz, 1H), 7.82 (t, J = 8.0Hz, 1H), 7.62-7.65 (m, 2H), 7.54 (d, J = 8.8 Hz, 1H), 7.42-7.44 2H), 4.13-4.20 (m, 1H), 2.87-2.92 (m, 1H), 2.52-2.56 (m, 2H), 1.46-1. 52 (m, 2H), 1.46-1. 52 (m, 2H)

(5) Synthesis of Compound 1

After stirring for 5 minutes at room temperature, TSTU (106 mg, 0.35 mmol), triethylamine (50 mg, 0.35 mmol) and dimethylformamide mu] l, 0.35 mmol) was added thereto, followed by stirring at room temperature for 15 hours. Then, triethylamine (50 μl, 0.35 mmol) and 2,2'-oxybisethylamine (13 μl, 0.119 mmol) were added thereto. The mixture was stirred at room temperature for 3 days and poured into ethyl acetate (40 mL) A solid was precipitated. The precipitated solid was filtered and the column was purified to obtain Compound 1 (110 mg, 0.0906 mmol, 26%). ^{1 H-NMR (400 MHz,} MeOD) δ 8.25 (d, J = 6.8Hz, 2H), 7.77-7.80 (m, 2H), 7.68-7.73 (m, 2H), 7.51-7.54 (m, 4H), 2H), 7.21-7.35 (m, 2H), 7.16-7.20 (m, 2H), 7.01 (d, J = 7.6Hz, 2H) 2H), 4.60-4.63 (m, 2H), 4.32-4.43 (m, 4H), 4.00-4.09 (m, 2H), 3.44-3.47 (M, 2H), 2.21 (t, J = 7.2 Hz, 4H), 1.85-1.92 (m, 4H), 1.61-1.69 (m, 4H), 1.39-1.45 m, 4H)

Manufacturing example  2. Synthesis of compounds 2 to 4

In the example of synthesizing Compound 1 from Intermediate 5 of Preparation Example 1, 4,7,10-trioxa-1,13-tridecanediamine, 1,2-bis (2 -Aminoethoxy) ethane, 1,11-diamino-3,6,9-trioxandecane, Compound 2, Compound 3 and Compound 4 were synthesized.

Compound ^{2 - 1 H-NMR (400} MHz, MeOD) δ 8.25 (d, J = 7.2Hz, 2H), 7.77 (t, J = 8.0Hz, 2H), 7.69-7.73 (m, 2H), 7.50-7.55 (m, 4H), 7.41 (t, J = 8.4 Hz, 2H), 7.30-7.32 (m, 2H), 7.17-7.20 2H), 3.50-3.56 (m, 4H), 3.48-3.50 (m, 4H), 4.04-4.10 (m, 2H) J = 6.8 Hz, 4H), 2.79-2.83 (m, 2H), 2.37-2.44 (m, 2H), 2.16 (t, J = 6H), 1.85-1.92 (m, 4H), 1.60-1.70 (m, 8H), 1.37-1.42 (m, 4H)

Compound ^{3 - 1 H-NMR (400} MHz, MeOD) δ 8.26 (d, J = 8.0Hz, 2H), 7.77-7.79 (m, 2H), 7.69-7.72 (m, 2H), 7.52-7.54 (m, 2H), 7.17-7.20 (m, 2H), 7.02 (d, J = 7.6Hz, 2H), 5.05-5.12 2H), 4.60-4.64 (m, 2H), 4.33-4.43 (m, 4H), 4.01-4.10 (m, 2H), 3.54-3.57 ), 3.32 (t, J = 6.4 Hz, 4H), 2.80-2.83 (m, 2H), 2.40-2.43 (m, 2H), 2.21 (t, J = 7.2Hz, 4H), 1.86-1.92 4H), 1.63-1.68 (m, 4H), 1.42-1.46 (m, 4H)

Compound ^{4 - 1 H-NMR (400} MHz, MeOD) δ 8.27 (d, J = 7.2Hz, 2H), 7.77-7.80 (m, 2H), 7.69-7.73 (m, 2H), 7.52-7.54 (m, 2H), 7.18-7.20 (m, 2H), 7.02 (m, J = 7.6Hz, 2H), 5.06-5.11 2H), 4.60-4.64 (m, 2H), 4.35-4.44 (m, 4H), 4.05-4.08 (m, 2H), 3.54-3.58 ), 3.32 (t, J = 4.8 Hz, 4H), 2.80-2.84 (m, 2H), 2.39-2.43 4H), 1.63-1.40 (m, 4H), 1.40-1.46 (m, 4H)

Manufacturing example  3. Synthesis of Compound 5

Production Example 3 was synthesized in the same manner as in Production Example 1 except that 5-chloro-2-methylthiobenzothiazole was used instead of 2-methylthiobenzothiazole in the production of Intermediate 1 of Production Example 1 . ^{1 H-NMR (400 MHz,} MeOD) δ 8.34 (d, J = 6.4Hz, 2H), 7.71-7.77 (m, 4H), 7.58 (m, 2H) 7.46 (t, J = 8.4Hz, 2H), 2H), 7.05 (d, J = 7.6 Hz, 2H), 5.07 (m, 2H), 4.55-4.58 2H), 2.42 (m, 2H), 2.41-2.22 (m, 2H), 4.41 (m, 4H), 400-4.03 m, 4H), 1.90 (m, 4H), 1.65 (m, 4H), 1.41

Manufacturing example  4. Synthesis of Compound 6 to Compound 8

Bis (2-aminoethoxy) ethane, 1,11-diamino-3,6-dihydroxybenzoate was used instead of 2,2'-oxybisethylamine in the synthesis example 1 of Intermediate 5 of Production Example 3, 9-trioxandecane, 4,7,10-trioxa-1,13-tridecanediamine, Compound 6, Compound 7 and Compound 8 were synthesized.

Compound ^{6 - 1 H-NMR (400} MHz, MeOD) δ 8.33-8.36 (m, 2H), 7.74-7.78 (m, 4H), 7.62-7.64 (m, 2H), 7.46-7.51 (m, 2H), 2H), 4.30-7.34 (m, 2H), 7.25 (t, J = 7.6Hz, 2H), 7.05-7.07 2H), 2.41 (m, 4H), 3.47-3.48 (m, 4H) 2H), 2.17-2.20 (m, 4H), 1.89-1.92 (m, 4H), 1.62-1.66 (m, 4H), 1.40-1.42

Compound ^{8 - 1 H-NMR (400} MHz, MeOD) δ 8.36-8.38 (m, 2H), 7.75-7.82 (m, 4H), 7.62-7.65 (m, 2H), 7.49-7.54 (m, 2H), 2H), 4.60-4.63 (m, 2H), 4.44-7.34 (m, 2H), 7.27 (t, J = 7.2 Hz, 2H), 7.08-7.10 4.50 (m, 4H), 3.45 (t, J = 6.0 Hz, 4H), 3.20 (m, 2H), 3.50-3.59 2H), 2.19 (t, J = 6.4 Hz, 4H), 1.90 - 1.97 (m, 4H), 1.63 - 1.72 (m, 8 H), 1.40 - 1.65 (m, 4 H)

The absorption spectra (λ _abs), a light emitting spectrum (λ _em), to measure the molar absorption coefficient (ε) and quantum efficiency of SYBR® safe Table 1 are commercially available compounds (1) to the compound 8 obtained in Preparation Example 1-4 Respectively.

Compounds 1 to 8 obtained in Preparation Examples 1 to 4 exhibited similar molar extinction coefficients to SYBR® safe, and particularly excellent quantum efficiency.

Manufacturing example  5. Synthesis of Compound 9 to Compound 20

In the example of synthesizing Compound 1 from Intermediate 5 of Preparation Example 1, 1,6-diaminoheptane, 1,8-diaminooctane, 1,10-dianodecane Compound 9 to Compound 13 were synthesized using 1,1-diaminododecane, 1,12-diaminododecane, and 3,3-diamino-N-methyl dipropylamine.

Compound 14 was synthesized by using an amine bridge represented by the following formula instead of 2,2'-oxybisethylamine in an example of synthesizing Compound 1 from Intermediate 5 of Preparation Example 1.

Instead of 2,2'-oxybisethylamine, piperazine, 2,6-diaminopyridine, p -phenylamine and 1,4-diaminonaphthalene were used in the synthesis of Compound 1 from Intermediate 5 of Preparation Example 1 Compound 15 to Compound 18 were synthesized.

Compound 19 was synthesized by using an amine bridge represented by the following formula instead of 2,2'-oxybisethylamine in Synthesis Example 1 from Intermediate 5 of Preparation Example 1.

Compound 20 was synthesized by using an amine bridge represented by the following formula instead of 2,2'-oxybisethylamine in an example of synthesizing Compound 1 from Intermediate 5 of Preparation Example 1.

Manufacturing example  6. Synthesis of Compound 36

(1) Synthesis of intermediate 1 and intermediate 2

(52.6 g, 0.290 mol), potassium iodide (64.3 g, 0.387 mol) and acetonitrile (320 mL) were added to a 1 L 1-liter reactor equipped with a stirrer 50 < [deg.] ≫ C for 1 hour. Then, 2-methylthio benzoxazole (32.0 g, 0.193 mol) was added to the reactor and stirred for 20 hours under reflux. After cooling, the mixture was concentrated and subjected to column purification to obtain Intermediate 1 (30.2 g, 0.120 mol, 62%). ^{1 H-NMR (400 MHz,} CDCl 3) δ 7.40-7.28 (m, 4H), 4.93 (t, J = 4.4Hz, 1H), 4.33 (t, J = 6.8Hz, 2H), 4.10-3.90 (m , 2H), 3.84-3.78 (m, 2H), 2.22-2.00 (m, 2H)

Intermediate 1 (1.0 g, 3.979 mmol), methyl tosylate (MeOTs) (0.9 mL, 5.968 mmol) and dimethylformamide (2 mL) were then added to a 50 mL 1-necked reactor at 120 ° C for 1 hour After cooling, ethyl acetate was added to the reactor, followed by stirring at room temperature. The supernatant was then discarded and vacuum dried to give intermediate 2 (0.9 g, 2.057 mmol, 52%). ^{1 H-NMR (300 MHz,} MeOD) δ 7.95-7.89 (m, 2H), 7.73-7.66 (m, 4H), 7.22 (d, J = 7.8Hz, 2H), 4.96 (t, J = 3.6Hz, 2H), 3.93-3.77 (m, 4H), 3.19 (s, 3H), 2.44-2. 35 (m, 5H)

(2) Synthesis of intermediate 3

4-methyl-5-quinolinol (2 g, 0.0125 mol), ethyl 6-iodohexanoate (10 g, 0.0375 mol) and dimethylformamide (4 mL) were placed in a 50 mL single- For 12 hours, then cooled, concentrated and purified by column to give intermediate 3 (3.6 g, 0.00828 mol, 67%). ^{1 H-NMR (400 MHz,} DMSO-d 6) δ 9.19 (d, J = 6.4Hz, 1H), 7.952 (t, J = 8.0Hz, 1H), 7.73-7.77 (m, 2H), 7.22 (d J = 7.6 Hz, 2H), 4.85 (t, J = 6.8 Hz, 2H), 4.01 (q, J = 7.6 Hz, 2H), 3.09 ), 1.89-1.93 (m, 2H), 1.55-1.61 (m, 4H), 1.12 (t, J = 6.8 Hz,

(3) Synthesis of intermediate 4

Intermediate 2 (1.87 g, 4.292 mmol), Intermediate 3 (1.45 g, 4.292 mmol) and dichloromethane (20 mL) were added to a 100 mL 1-necked reactor, and the mixture was stirred at room temperature for 5 minutes. Then, triethylamine (TEA) (0.868 g, 8.584 mmol) was added to the reactor, followed by stirring at room temperature for 12 hours, followed by concentration and column purification to obtain Intermediate 4.

(4) Synthesis of intermediate 5

Intermediate 4 (8.6 g, 0.0133 mmol), 50% aqueous sulfuric acid solution (50 mL) and chloroform (250 mL) were added to a 500 mL 1-necked reactor and stirred at room temperature. Subsequently, water (10 mL) was added to the reactor, and the mixture was extracted with dichloromethane (2 x 50 mL). The organic layer was concentrated and column-purified to obtain Intermediate 5 (0.37 g, 0.665 mmol, 5%). ^{1 H-NMR (400 MHz,} MeOD) δ 8.25 (d, J = 6.8Hz, 1H), 7.80 (t, J = 8.4Hz, 1H), 7.69 (d, J = 7.6Hz, 2H), 7.53-7.47 (m, 3H), 7.44-7.40 (m 1H), 7.08 (d, J = 8.0 Hz, 1H), 5.31-5.27 (m, 1H), 4.57-4.50 (m, 2H), 4.47-4.40 J = 7.2 Hz, 2H), 1.99-1.92 (m, 2H), 2.31-2.27 (m, 1.71-1.64 (m, 2H), 1.51-1. 43 (m, 2H)

(5) Synthesis of Compound 36

After adding intermediate 5 (123 mg, 0.220 mmol), TBTU (106 mg, 0.330 mmol) and dimethylformamide (2 mL) to a 250 mL 1-neck reactor and stirring at room temperature for 5 minutes, triethylamine mu] l, 0.660 mmol) and 2,2'-oxybisethylamine (23 [mu] l, 0.220 mmol) were added thereto, followed by stirring at room temperature for 12 hours. Subsequently, the reaction solution was poured into ethyl acetate (40 mL) to precipitate a solid. The precipitated solid was filtered and the column was purified to obtain Compound 36 (30 mg, 0.0906 mmol, 11%). ^{1 H-NMR (400 MHz,} MeOD) δ 8.21 (d, J = 7.2Hz, 2H), 7.77-7.72 (m, 2H), 7.66 (d, J = 8.0Hz, 2H), 7.60 (t, J = 2H), 7.44-7.37 (m, 2H), 7.06-7.03 (m, 2H), 5.24-2.18 2H), 2.52-2.39 (m, 4H), 4.44-4.37 (m, 4H), 4.14-4.07 2H), 2.23 (t, J = 7.2 Hz, 4H), 1.94-1.91 (m, 4H), 1.70-1.65 (m, 4H), 1.49-1.

Manufacturing example  7. Synthesis of Compound 37 to Compound 39

Bis (2-aminoethoxy) ethane, 1,11-diamino-3,6-dihydroxybenzoate was used in place of 2,2'-oxybisethylamine in the synthesis of Compound 1 from Intermediate 5 of Preparation Example 6, Compound 37, Compound 38 and Compound 39 were synthesized using 9-trioxandecane, 4,7,10-trioxa-1,13-tridecanediamine.

Compound ^{37 - 1 H-NMR (400} MHz, MeOD) δ 8.20 (d, J = 7.2, 2H), 7.76-7.70 (m, 2H), 7.65 (d, J = 8.0Hz, 2H), 7.61-7.57 ( 2H), 7.48-7.42 (m, 6H), 7.39-7.35 (m, 2H), 7.04-7.01 2H), 3.57 (s, 4H), 3.51-3.48 (m, 4H), 3.36-3.30 (m, 4H), 2.83-2.80 ), 2.48-2.40 (m, 2H), 2.25-2.21 (m, 4H), 1.93-1.89 (m, 4H)

Compound ^{38 - 1 H-NMR (400} MHz, MeOD) δ 8.20 (d, J = 7.2Hz, 2H), 7.77-7.72 (m, 2H), 7.66 (d, J = 8.0Hz, 2H), 7.62-7.59 (m, 2H), 7.47-7.44 (m, 6H), 7.41-7.36 (m, 2H), 7.03 (d, J = 8.0Hz, 2H), 5.25-5.19 2H), 4.44-4.39 (m, 4H), 4.13-4.07 (m, 2H), 3.59-3.58 (m, 8H), 3.50-3.48 2H), 2.21 (t, J = 7.2 Hz, 4H), 1.94-1.89 (m, 4H), 1.69-1.65 (m, 4H), 1.44-1.43 (m, 4H)

Compound ^{39 - 1 H-NMR (400} MHz, MeOD) δ 8.22-8.21 (m, 2H), 7.78-7.72 (m, 2H), 7.67 (d, J = 8.0Hz, 2H), 7.63-7.60 (m, 2H), 7.51-7.44 (m, 6H), 7.41-7.37 (m, 2H), 7.05-7.03 4H), 3.48-3.45 (m, 4H), 3.23-3.20 (m, 4H), 3.50-3.53 (m, ), 2.85-2.82 (m, 2H), 2.50-2.42 (m, 2H), 2.20 (t, J = 7.2 Hz, 4H), 1.97-1.89 (m, 4H), 1.73-1.68 1.46-1.40 (m, 4H)

The absorption spectra (λ _abs ), the emission spectrum (λ _em ), the molar extinction coefficient (ε) and the quantum efficiency of the compounds 36 to 39 obtained in Production Examples 6 and 7 and commercially available SYBR®safe were measured, Respectively.

The compounds 36 to 39 obtained in Preparation Examples 6 and 7 exhibited similar molar extinction coefficients to those of SYBR®safe, and particularly excellent in terms of quantum efficiency.

Experimental Example

Experimental Example  1. Nucleic Acid Labeling Experiment

To compare the nucleic acid labeling properties of SYBR® safe, a commercially available dye, Compound 1 to Compound 8, Compound 38 prepared according to Preparation Example, 1% agarose gel (40 ml 1X TAE buffer + 0.4 g agarose) mu] l each to prepare nine agarose gels.

[SYBR® safe]

The prepared nine agarose gels were completely immersed in 1X TAE buffer, and then 10 μl, 5 μl, 2.5 μl, 1 μl, and 0.5 μl of DNA samples were loaded into 5 wells, respectively. Electrophoresis was performed for 30 minutes at a power of 100V. The electrophoresis results are shown in FIGS. 1 and 2 to 10. FIG.

2 to 10 show electrophoresis results of the compounds 1 to 8 and 38, respectively.

Referring to FIGS. 1 to 10, when the merocyanine compound according to various embodiments of the present invention is used as a dye in comparison with SYBR® safe, it can be confirmed that the background signal is uniform. safe, and the readability of the labeling results was improved.

In addition, in the case of SYBR® safe, although the overall band brightness is dark despite UV irradiation for 0.5 second, referring to FIG. 6 to FIG. 10, even though UV is irradiated for a shorter time than SYBR® safe, Can be observed.

FIG. 11 shows the absorption and emission spectrum results of SYBR® safe, which is a commercially available dye, and FIGS. 12 and 13 show absorption and emission spectral results of compound 3 and compound 6, respectively.

SYBR® safe, Compound 3, and Compound 6 are both intercalated into DNA, and the absorption wavelength is shifted to a longer wavelength and the fluorescence intensity is increased. However, the fluorescence intensities Was significantly improved over the fluorescence intensity of SYBR® safe.

(Λ _abs ), luminescence spectrum (λ _em ) and quantum efficiency measured in the presence of commercially available SYBR® safe DNA and the compounds 1 to 8 and 36 to 39 were measured and are shown in Table 3 below.

division λ _abs (nm) ? _em (nm) Quantum efficiency SYBR®safe 503 535 0.021 Compound 1 517 541 1.00 Compound 2 517 541 0.93 Compound 3 517 540 0.86 Compound 4 517 539 0.88 Compound 5 517 537 0.86 Compound 6 517 538 0.73 Compound 7 517 538 0.71 Compound 8 517 538 0.80 Compound 36 492 511 0.67 Compound 37 492 512 0.64 Compound 38 492 512 0.76 Compound 39 492 513 0.70

Compounds 1 to 8 and 36 to 39 show fluorescence signals in the wavelength range similar to that of commercially available SYBR® safe in the presence of DNA, while they show significantly higher quantum efficiency than SYBR® safe.

Experimental Example  2. Cell permeability experiment

In order to confirm the cell permeability of SYBR® safe and compounds 1, 4 and 6 according to one embodiment of the present invention, heliocytosis was used.

Hela cells were grown in MEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin (10,000 units penicillin and 10,000 ug / mL streptomycin, Invitrogen) under a humidified atmosphere of CO2 And cultured at 37 ° C. Hela cells were washed with medium 3, and 1 μM of SYBR® safe and compound 6 were treated with Hela Cells and cultured at 37 ° C. for 30 minutes. After washing with media No. 3 as above, images were observed with a microscope. The results are shown in Figs. 14 to 17.

FIG. 14 is an image showing the results of a cell permeability test of SYBR ^® safe, which is a commercial dye, and FIG. 15 is an image showing the results of cell permeability test of Compound 1, FIG. 16, and Compound 6.

Referring to FIG. 14, SYBR® safe shows cell permeability. Referring to Figures 15-17, it can be seen that compounds according to various embodiments of the invention do not penetrate cells.

Experimental Example  3. Cell cycle analysis

After treating the cells with a busy ~ 1x10 ⁶ 5x10 ⁵ per well in 6-well plates at a vehicle and TNFα (tumor necrosis factor alpha), as then transferred to FACS tubes 1ml PBS (4 ℃) recovered by trypsin-EDTA And washed. Then, 300 μl of PBS (4 ° C) was added to the tube, and the cells were allowed to float.

After vortexing the suspended cells, 700 μl of 100% Et-OH was added dropwise and the cells were fixed at 4 ° C for 1 hour. Cells were fixed with Et-OH in PBS (phosphate-buffered saline) and then suspended in 1 ml of PBS were added 10 μl of Compound 6 (2 μM) or PI (propidium iodide) (50 mg / ml) and 1 μl of RNase at a concentration of 1 mg / ml, And analyzed by flow cytometry.

In other words, gating nuclei using FSC-A and SSC-A, and adjusting the FITC-A and FITC-W plots to voltage, distinguish singlets. The results of the discrimination of G0 / G1, S and G2 / M groups are shown in FIGS. 5A and 5B. In FIG. 5A and FIG. .

FIG. 18 is a graph showing the results of cell cycle analysis using propidium iodide, a commercially available cell cycle assay dye, and FIG. 19 is a graph showing the results of cell cycle analysis using Compound 6. FIG.

Comparing FIG. 18 and FIG. 19, it can be seen that the results of the cell cycle analysis of FIG. 19 are clearly distinguished from each other in comparison with FIG. 18, and the visibility of signals is improved.

Experimental Example  4. Return mutation  Experiment (Ames test)

For the purpose of evaluating the genotoxicity of Compound 6 according to one embodiment of the present invention, the Ames test using a bacterial strain was performed.

The growth of the test strain was monitored by the compound 6, EtBr and SYBR® safe treatment in the test system treated with the S9 mixture, which is a metabolic enzyme, in the mouse strain Tryptophan (Salmonella typhimurium TA98 strain), which is a cysteine freeze-preserved histidine Respectively.

The test strain in the deep freezer was inoculated into a sterile liquid medium (2.5% Oxoid Nutrient Broth No. 2) and cultured in a shaking incubator (37 ° C, 200 rpm) for 10 hours. Minimal Glucose Agarplates were prepared by dispensing 20 ml each of 1.5% Bacto agar (Difco), Vogel-Bonner medium E and 2% glucose in a Petri dish (90 × 15 mm). Top agar was prepared with 0.6% agar and 0.5% NaCl, and histidine-biotin was added to the top agar for Salmonellatyphimurium strain to a concentration of 0.05 mM.

In a heating block preheated to high temperature sterilized top agar at 45 ° C, the blots were dispensed in sterile tubes (12 x 75 mm) in a volume of 2 ml, and then 0.1 ml of compound 6, EtBr and SYBR® safe and S9 mixture, ml, shake for 2 to 3 seconds with vortex-mixer, pour into a minimal glucose agar plate, tilt in various directions, and then harden. When the top agar hardens, the plate is turned upside down and cultured at 37 ° C for 48 hours.

The treatment concentrations (μg / plate) of Compound 6, EtBr and SYBR® safe and the number of colonies counted per plate are as follows.

compound Concentration (μg / plate) Number of colonies per plate EtBr 0 19 ± 4 40 3051 ± 181 Compound 6 0 20 ± 4 40 57 ± 3

As can be seen from the results in Table 4, the number of colonies did not significantly increase in S9 metabolic TA98 strain when compared with the number of EtBr colonies known to exhibit genotoxicity. That is, Compound 6 can sufficiently replace EtBr as a non-oil toxic dye.

compound Concentration (μg / plate) Number of colonies per plate SYBR®safe 0 24 ± 2 1.6 81 ± 7 Compound 6 0 20 ± 4 8 24 ± 6

As can be seen from the results in Table 5, the SYBR® safe dye currently available has a greater number of colonies than compound 6 even at a lower concentration (1.6 μg / plate) than the compound 6 treatment concentration (8 μg / plate).

Considering that the staining concentration of the gel is usually 1 to 3 μg / ml (3 to 8 μg / plate) upon electrophoresis, within this concentration range, Compound 6 may exhibit lower genotoxicity than SYBR® safe.

Experimental Example  5. qPCR  Experiment

(1) qPCR is a 20 μl reaction solution containing 10 μl of 2X Real-Time PCR Master Mix (Cellsafe), Hela cDNA of various concentrations, 1 μl of 10 pmol forward primer, 1 μl of 10 pmol reverse primer and 1 μl of compound 38 . The fragment in the cDNA was amplified using a primer (GAPDH). Cycles performed at 95 ° C for 5 minutes, at 95 ° C for 10 seconds, at 60 ° C for 20 seconds, and at 72 ° C for 15 seconds were repeated 40 times and fluorescence was measured at 72 ° C. The results of qPCR are shown in Table 6 below.

division cDNA content 10 ng 1 ng 100 pg 10 pg 1 pg Compound 38 Number of cycles (Ct) 19.649 23.267 26.852 29.901 33.256 Tm (占 폚) 83.982 83.982 83.982 83.982 83.982

Here, the threshold value cycle number Ct generally means the number of cycles at which the fluorescence signal reaches a certain threshold value. For example, in the qPCR amplification graph, it means the number of cycles at which the fluorescence signal starts to cross the reference line .

When qPCR was performed using the compound 38 as a fluorescent dye, the amplification efficiency was 97.44%, confirming that the amplification efficiency of qPCR did not decrease even when the compound 38 was contained in the reaction solution.

In addition, referring to Table 6, it can be confirmed that the sensitivity of the compound 38 to the qPCR reaction is high in view of the fact that a PCR product is generated even when a very low concentration of cDNA sample such as 1 pg is used.

(2) qPCR consists of 10 μl of TOPreal ^™ qPCR 2X PreMIX (TagMan Probe, Enzynomics), Hela cDNA of various concentrations, 1 μl of 0.5 μM forward primer, 1 μl of 0.5 μM reverse primer and SYBR® green I Was performed using CFX96 in a 20 [mu] l reaction solution containing compound 38 (1X). Hela cDNA fragments were amplified using 0.5 μM forward primer 5'-GTATGACAACAGCCTCAAGAT-3 'and 0.5 μM reverse primer 5'-AGTCCTTCCACGATACCAAA-3'. Cycles performed at 95 ° C for 10 minutes, at 95 ° C for 10 seconds, at 60 ° C for 15 seconds, and at 72 ° C for 15 seconds were repeated 40 times and fluorescence was measured at 72 ° C. The qPCR was repeated three times in total, and the qPCR results are shown in Figs. 32 and 33 and Table 7 below.

division cDNA content Number of cycles (Ct) End point RFU Tm (占 폚) SYBR®green I 1 ng 24.70 6209.44 82.50 0.1 ng 28.47 5458.15 82.50 0.01 ng 31.97 4736.22 82.50 0.001 ng 35.66 2818.84 82.50 0.0001 ng 36.99 1250.30 82.50 Compound 38 1 ng 24.45 31885.43 83.17 0.1 ng 28.12 24568.08 83.00 0.01 ng 31.42 16545.25 83.00 0.001 ng 35.63 5033.85 83.00 0.0001 ng 36.88 1818.80 83.25

When qPCR was performed using the compound 38 as a fluorescent dye, it was confirmed that the Ct value was about 0.5 smaller than that of SYBR green I, and the RFU value was about 5 times or more.

(3) qPCR was prepared by mixing 10 μl of 2X Real-Time PCR Master Mix (DQ372, Biophotics), 2 μl of Hg DNA (25 ng / μl), 1 μl of Primer (HSP98) (Compound 38, SYBR® green I (Invitrogen) or EvaGreen ^™ (Biotium)) using ABI 7500 FAST. Cycles performed at 95 ° C for 15 minutes, at 95 ° C for 10 seconds, at 60 ° C for 10 seconds, and at 72 ° C for 30 seconds were repeated 40 times and fluorescence was measured at 72 ° C. The qPCR was repeated three times in total, and the qPCR results are shown in Figs. 34 to 36 and Table 8 below.

Dyed dye Fluorescent dye concentration 1X 0.5X 0.25X Compound 38 Number of cycles (Ct) 23.17 23.61 24.53 Fluorescence intensity (Rn) 37.83 21.42 11.18 Tm (占 폚) 79.04 79.2 79.04 SYBR®green I Number of cycles (Ct) N / D 25.61 24.93 Fluorescence intensity (Rn) 0.22 8.04 4.01 Tm (占 폚) 89.3 80.75 80.13 EvaGreen ^TM Number of cycles (Ct) 25.29 25.8 26.82 Fluorescence intensity (Rn) 17.25 9.58 4.59 Tm (占 폚) 79.04 79.04 78.89

When qPCR was performed using the compound 38 as a fluorescent dye, it was confirmed that the Ct value was smaller than that in the case of using SYBR green I or EvaGreen ^TM at the same dilution rate. In addition, the fluorescence intensity is about twice as high as that of using the same dilution of SYBR® green Ⅰ or EvaGreen ^™ .

Also, from the results in Table 8, it can be seen that Compound 38 at a higher concentration than that of SYBR green I can be used without inhibiting qPCR reaction.

20 is a graph showing a melt curve of qPCR using Compound 38 as a fluorescent dye, Fig. 21 is a graph showing a melt curve of qPCR using SYBR green I as a fluorescent dye, and Fig. Is a graph showing the melt curve of qPCR using EvaGreen ^TM as a fluorescent dye.

Referring to FIGS. 20-22, qPCR using Compound 38 and EvaGreen ^™ shows a single specific peak, while qPCR using SYBR® green I shows an extra primer-dimer peak have.

(4) qPCR was prepared by mixing 10 μl of 2X Real-Time PCR Master Mix (Cellsafe), fluorescent dyes (Compound 36, Compound 38, Compound 39, SYBR®green I (Invitrogen) or EvaGreen ^™ Biotium)), 4 μl of Hela cDNA (0.5 ng / μl) and 1 μl of primer (GAPDH). Cycles performed at 95 ° C for 10 minutes, 95 ° C for 10 seconds, and 60 ° C for 1 minute were repeated 46 times and fluorescence was measured at 60 ° C step. The qPCR results are shown in Figs. 37 to 42 and Table 9 below.

Dyed dye Fluorescent dye concentration 2X 1X 0.5X Compound 36 Number of cycles (Ct) 39.66 30.55 24.32 End point RFU 14645 21485 11100 Tm (占 폚) 78.3 84.0 84.0 Compound 38 Number of cycles (Ct) 26.2 24.59 25.40 End point RFU 27677 15460 7212 Tm (占 폚) 84.0 83.5 83.5 Compound 39 Number of cycles (Ct) 26.87 24.69 25.31 End point RFU 24717 13320 6176 Tm (占 폚) 84.0 83.5 83.5 EvaGreen ^TM Number of cycles (Ct) 27.48 27.09 30.80 End point RFU 10223 5941 2487 Tm (占 폚) 83.5 83.3 83.0

When Compound 38 and Compound 39 were used at a relatively high concentration (2X, 1X) of fluorescent dye concentration, it was confirmed that the Ct value was low and the fluorescence intensity was high as compared with the case of using EvaGreen ^TM having the same dilution factor. In the case of a relatively low concentration (0.5X) of fluorescent dye concentration, the compound 36 was also found to have a low Ct value and a high fluorescence intensity as compared with SYBR green I or EvaGreen ^TM at the same dilution rate.

Experimental Example  6. Thermal Stability Experiment

In order to confirm the stability of the fluorescent dye in the PCR reaction, 20 μl of PCR reaction buffer containing 1 μl of compound 38 and 1 μl of EvaGreen ^™ (manufactured by Biotium) was prepared. Each PCR reaction buffer was heated to 95 ° C. and absorbance was measured according to the incubation time at 95 ° C.

As shown in the following Table 10, it can be seen that the absorbance variation according to the standing time at 95 ° C is smaller than that of EvaGreen ^TM . That is, the high temperature stability of Compound 38 will be greater than EvaGreen ^TM .

division Time left (hr) 0 0.5 One 2 3 Absorbance Compound 38 0.127 0.149 0.162 0.163 0.178 EvaGreen ^TM 0.095 0.125 0.135 0.167 0.190

Experimental Example  7. Light fade ( 광도 ) Experiment

Real-time PCR was performed in a 20 μl reaction solution containing only 5 μl of fluorescent dyes (Compound 38, SYBR® green I (Invitrogen) or EvaGreen ^™ (Biotium)) at various concentrations (dilution times). A cycle of 30 seconds at 25 DEG C and 30 seconds at 25 DEG C was repeated 60 times and fluorescence intensity was measured for each cycle (1 minute) to observe light discoloration of fluorescent dyes at various concentrations (dilution multiples).

The light discoloration test results of compound 38 as measured by the above method are shown in Figs. 23 to 25, and the results of light discoloration test of SYBR green I (Invitrogen) are shown in Figs. 26 to 28 and EvaGreen ^TM (manufactured by Biotium) The results of the light discoloration test are shown in Figs. 29 to 31.

FIG. 26 shows the change in fluorescence intensity measured with SYBR green I at a dilution factor of 0.5X, FIG. 27, and 0.25X, and FIG. 28 shows a change in fluorescence intensity with a dilution factor of 0.125X. In SYBR green I, the half- It is only time.

FIGS. 23 and 29 show changes in fluorescence intensity measured when Compound 38 or EvaGreen ^™ was used at a dilution factor of 1X, FIGS. 24 and 30 were 0.5X, and FIGS. 25 and 31 were 0.25X. If used, the same concentration of EvaGreen ^TM And a half-life similar to or higher than that of < RTI ID = 0.0 >

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 (16)

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

here,
Ar is phenyl or naphthyl,
Y ₁ and Y ₂ are each independently sulfur or oxygen,
R ₁ to R ₆ are each independently hydrogen, deuterium, C ₁ -C ₁₀ alkyl, at least one containing a hetero atom C ₂ -C ₁₀ heteroalkyl, C ₂ -C ₁₀ alkenyl, C ₂ -C ₁₀ alkynyl carbonyl, C ₂ -C ₁₀ alkoxy, aryloxy, C ₁ -C ₁₀ haloalkyl, halogen, hydroxy, amino, amide, sulfhydryl, aralkyl, 4-nitro, carboxyl, carboxylic acid salt, aryl, heteroaryl, Phosphoric acid, phosphate, ester (-COOR ₈ ), sulfonic acid, sulfonate, polyalkylene oxide and -LZ functional groups,
R ₇ is independently selected from the group consisting of hydrogen, deuterium, C ₁ -C ₁₀ alkyl, C ₂ -C ₁₀ heteroalkyl containing at least one heteroatom, C ₂ -C ₁₀ alkenyl, C ₂ -C ₁₀ alkynyl, C ₂ -C ₁₀ alkoxy, aryloxy, C ₁ -C ₁₀ haloalkyl, halogen, hydroxy, amino, amide, sulfhydryl, nitro, carboxyl, carboxylic acid salt, aryl, heteroaryl, aralkyl, quaternary ammonium, Phosphoric acid, phosphate, ester (-COOR ₈ ), sulfonic acid, sulfonic acid salt and polyalkylene oxide,
When R ₂ , R ₃ , R ₄ , R ₅ , R ₆ or R ₇ is an ester group (-COOR ₈ ), R ₈ is a substituted or unsubstituted C ₁ -C ₁₀ alkyl, containing at least one heteroatom Substituted or unsubstituted 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 alkyl Alkoxy, substituted or unsubstituted C ₁ -C ₁₀ haloalkyl and substituted or unsubstituted C ₁ -C ₁₀ aminoalkyl,
When R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R ₇ and R ₈ are substituted, any carbon or terminal carbon in the functional group may be substituted with a sulfonic acid, sulfonate, ketone, aldehyde, And may be substituted with at least one member selected from phosphoric acid, phosphate, acyl chloride, polyalkylene oxide, quaternary ammonium salt, ester and amide,
m is 2,
L is a linker consisting of 3 to 150 non-hydrogen atoms and containing at least one selected from nitrogen, oxygen, carbonyl, phenyl, naphthyl, pyridine, pyrrole, piperazine, triazine,
Z is selected from the group consisting of phenanthridium, coumarins, cyanine, bodipy, fluoresceins, rhodamines, pyrenes, carbopyronin, Oxazine, xanthenes, thioxanthene and acridine, or has a structure represented by the formula (1)
The structure represented by the formula (1) has one or two-LZ functional groups.
The method according to claim 1,
When L comprises at least one nitrogen, said nitrogen is a tertiary amino or quaternary ammonium,
Merocyanine compound.
The method according to claim 1,
-LZ functional group is a merocyanine-based compound represented by the following formula (2): < EMI ID =
(2)
^{_{-L 1 - [A 1 - (}} CH 2) x1 -] y1 [A 2 - (CH 2) x2 -] y2 [A 3 - (CH 2) x3 -] y3 [A 4 - (CH 2) x4 - ^{_{] y4 [A 5 - (CH}} 2) x5 -] y5 [A 6 - (CH 2) x6 -] y6 [A 7 - (CH 2) x7 -] y7 [A 8 - (CH 2) x8 -] y8 _{^{[A 9 - (CH 2)}} x9 -] y9 -A 10 -L 2 -Z
here,
L ₁ and L ₂ are each independently a C ₁ -C ₁₂ polymethylene unit optionally containing at least one heteroatom selected from nitrogen, oxygen and sulfur, or at least one heteroatom selected from nitrogen, oxygen and sulfur, Optionally containing aryl,
A ¹ to A ¹⁰ each independently represents a chained alkyl or branched alkyl optionally containing at least one hetero atom selected from nitrogen, oxygen and sulfur, or at least one heteroatom selected from nitrogen, oxygen and sulfur, Or a 5-membered ring or 6-membered ring,
x1 to x9 each independently represents 0 or an integer of 1 to 20,
y1 to y9 each independently represents 0 or an integer of 1 to 20,
Z is selected from the group consisting of phenanthridium, coumarins, cyanine, bodipy, fluoresceins, rhodamines, pyrenes, carbopyronin, Oxazine, xanthenes, thioxanthene, and acridine. The term " oxazine "
The method of claim 3,
One of A ¹ to A ¹⁰ is a merocyanine-based compound represented by the following formula (3)
(3)

here,
R < ₁₁ > is aryl optionally containing at least one heteroatom selected from carbon, nitrogen or nitrogen, oxygen and sulfur,
R ₁₂ is represented by the following formula (4)
[Chemical Formula 4]
_{^{- [A 11 - (CH 2}} ) x11 -] y11 [A 12 - (CH 2) x12 -] y12 [A 13 - (CH 2) x13 -] y13 -A 14 -L 3 -Z
here,
L ₃ is a C ₁ -C ₁₂ polymethylene unit optionally containing at least one heteroatom selected from nitrogen, oxygen and sulfur or is aryl optionally containing at least one heteroatom selected from nitrogen, oxygen and sulfur ,
A ¹¹ to A ¹⁴ each independently represents a chained alkyl or branched alkyl optionally containing at least one heteroatom selected from nitrogen, oxygen and sulfur, or at least one heteroatom selected from nitrogen, oxygen and sulfur, Or a 5-membered ring or 6-membered ring,
x 11 to x 13 are each independently 0 or an integer of 1 to 20,
y11 to y13 each independently represents 0 or an integer of 1 to 20;
A biomolecule marking dye comprising the merocyanine compound according to any one of claims 1 to 4.
6. The method of claim 5,
The merocyanine compound is a compound that is intercalated in a nucleic acid,
Dye for biomolecule marking.
The method according to claim 6,
The biomolecule is at least one nucleic acid selected from single-stranded RNA, double-stranded RNA, single-stranded DNA and double-stranded DNA
Dye for biomolecule marking.
A biomolecule labeling dye according to claim 5; Or a biomolecule labeling dye according to claim 5 or a buffer solution.
A merocyanine based compound according to any one of claims 1 to 4;
At least one substance for forming a buffer, a gel matrix, a gel matrix, at least one substance for forming a surface or a surface; And
Instructions for use;
When the nucleic acid is present in the sample, the matrix or the surface
An electrophoresis kit for determining the presence or absence of nucleic acids in a sample.
When the nucleic acid is present in the sample,
Exposing the nucleic acid to the merocyanine compound according to any one of claims 1 to 4 so that the merocyanine compound is intercalated in the nucleic acid to form a complex; And
Determining the presence or absence of the nucleic acid in the sample comprising determining fluorescence or absence of the merocyanine based compound.
A method for performing a nucleic acid amplification reaction,
Providing a reaction mixture comprising a target nucleic acid, a reagent necessary for amplifying the target nucleic acid, and a merocyanine compound according to any one of claims 1 to 4;
Polymerizing the reaction mixture under conditions suitable for the formation of the amplified target nucleic acid;
Illuminating the reaction mixture with light; And
And detecting fluorescence emission from the reaction mixture
And determining the presence or absence of the amplified target nucleic acid.
Mixing a mixture comprising a merocyanine compound according to any one of claims 1 to 4 with a sample comprising a nucleic acid;
Incubating the sample and the mixture for a sufficient time to cause the merocyanine compound to intercalate with the nucleic acid in the sample to form a complex and generate a fluorescence signal; And
And comparing the fluorescence standard characteristic of the fluorescence signal to be measured with a predetermined nucleic acid amount to quantify the nucleic acid in the sample
A method for quantifying a nucleic acid in a sample.
Mixing a mixture comprising a merocyanine compound according to any one of claims 1 to 4 with a sample comprising apoptotic cells;
Incubating the sample and the mixture for a sufficient time to cause the merocyanine compound to intercalate with the apoptotic cells in the sample to form a complex and generate a fluorescence signal; And
Comparing the fluorescence signal detected with the fluorescence standard characteristic of the determined dead cell volume to quantify the dead cell in the sample
A method for quantifying the viability of a cell in a sample.
Comprising an aqueous solution and a cell comprising a merocyanin compound according to any one of claims 1 to 4 and a compound other than the merocyanin compound capable of intercalating with cells intercalating with apoptotic cells Mixing a sample to be analyzed;
Light-illuminating a sample comprising the merocyanine-based compound and other cells intercalated with the compound; And
Detecting fluorescence emission from the sample,
The fluorescence emitted from the sample is generated by fluorescence reaction between cells of the merocyanine compound and other compounds intercalated with the cells in the sample,
The emitted fluorescence is different from the fluorescence generated by the fluorescence reaction of the merocyanine compound only
A method for analyzing the viability of a cell in a sample.
A merocyanine based compound according to any one of claims 1 to 4; And
Includes user manual,
When the apoptotic cell is present in the sample, fluorescence is detected by intercaling with the merocyanine compound
A kit for analyzing the viability of cells in a sample.
A contrast agent composition comprising a merocyanine compound according to any one of claims 1 to 4.

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