KR101621930B1 - Quencher dye for laveling biomolecule and preparation method thereof - Google Patents

Abstract

상기 [화학식 1]에서, R₁, R₂, R₃, R₄, R₅, R₆ 및 n은 각각 명세서에 정의한 바와 같다.The present invention relates to a quenching dye represented by the following formula (1) for a biomolecule marker and a method for producing the same.
[Chemical Formula 1]

R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ and n are as defined in the specification, respectively.

Description

[0001] The present invention relates to quencher dyes for biomolecule marking,

The present invention relates to a quenching dye capable of labeling various biomolecules, and more particularly to a quenching dye exhibiting strong absorption intensity in the visible and near infrared wavelength regions and exhibiting high stability under excellent solubility and water-soluble conditions, And to a process for producing the same.

A quencher is a molecule that can quench the fluorescence of a fluorescent molecule, usually a dye that has the ability to absorb light. Mechanisms of extinction phenomena are known to occur through aggregation of dyes such as fluorescence resonance energy transfer (FRET), photo-induced electron transfer and H-dimer formation.

When a quenching dye is selected for controlling or eliminating the fluorescence of the fluorescent dye, it is most important that the range of the absorption wavelength of the quenching dye covers (overlaps) a substantial part or all of the wavelength region of the fluorescent light represented by the fluorescent dye. In addition, the length between the fluorescent dye and the extinction dye is important in obtaining the effect of quenching. For example, in the case of DNA, the number of bases and the number of amino acids in the case of peptide / protein are considered. Fluorescent and extinction dyes may also be used to control the length of the linker that is labeled.

For quench dyes, which are mainly used commercially in the biotechnology field, there are quenching dyes that are paired by fluorescent dyes such as FITC (ex / em, 490/520) -BHQ-1, TRITC (ex / em, 547/572 ) -BHQ-2, Cy5.5 (ex / em, 670/690) -BHQ-3, etc. are used. In addition, fluorescent dyes generally can not emit light but can absorb only a dye structure. However, a combination of fluorescent-fluorescent dyes using FRET phenomenon is also widely utilized. Such combined fluorescence-extinction and fluorescence-fluorescent dyes are capable of imparting a kind of on / off function of fluorescence since the original fluorescence is restored or strengthened when the distance between the fluorescence-extinction and fluorescence-fluorescence dyes is distant from each other or biomolecules are separated from each other. , It is widely used in designing biosensors or activation probes capable of responding to biomarkers such as specific proteins / enzymes.

Although all the dyes can be quenching dyes if they can cover the fluorescence or the entire fluorescence spectrum of the highest absorption wavelength of the fluorescent dyes to be used, the present inventors have found that the dyes for fiber dyeing Dyes. In the case of modern fiber dyes, azo dye and anthraquinone are the most commonly used chromophores. In the case of the BHQ quenching dyes most commonly used in the biotechnology, Belongs to the azo chromophore dye family. Anthraquinone dyes are commonly used to produce blue and green colors, and absorption wavelengths are known to be greater than 550 nm.

Fluorescent or extinction dyes used in the biotechnology field are limited to only limited FDA-approved dyes such as indocyanine green or methylene blue when used alone, and in general, reactors capable of binding to the substituents of biomolecules are introduced . Although various reactors have been known, they have been verified for a long time by researchers with a high degree of substituent selectivity, reaction rate, yield, reproducibility and stability. In recent years, . For example, succinimidyl ester and isothiocyanate are most frequently used as the reactors for binding with amine groups of protein molecules, and are most widely used as a reactor for binding with thiol groups of protein molecules Is maleimide, and dichlorotriazine is mainly selected as a reactor for binding with a hydroxy group of a protein molecule. However, most of the above-mentioned reactors are difficult to maintain long-term reaction and storage stability under water-soluble conditions or in a substitution reaction. In the case of quenching dyes, optical properties are known to remain almost constant within a few nm range when there is no change in the light-absorbing chromophore structure.

The first problem to be solved by the present invention is to provide a quenching dye having a novel anthraquinone-based structure that can be widely used for observing identification of biomolecules in the field of optical molecular imaging, and a method for producing the same.

In order to attain the above object, the present invention provides an anthraquinone compound represented by the following formula 1 and a process for producing the same.

[Chemical Formula 1]

In the above formula (1)

R _1, R _2, R _3, and R ₄ are the same or different and each is independently hydrogen, a hydroxyl group, an amine group, a nitro group, a C 1 -C 6 alkyl group, having 7 to 10 groups with each other, containing from 1 to 6 carbon atoms Alkoxy group, sulfonic acid group and sulfonic acid group,

R ₅ is selected from an alkyl group having 1 to 6 carbon atoms and an alkyl group having 7 to 10 carbon atoms,

R ₆ is selected from the group consisting of a hydroxyl group, a hydrazinyl group, NH (CH ₂ ) _p NH ₂ , N-hydroxysuccinimide group, NH- (CH ₂ ) _q -N (CO) ₂ C ₂ H ₂ , 6-hydrazino-1,3,5-triazine group, NH-A-SO ₂ CH = CH ₂ ,

A is selected from (CH ₂ ) _m , para- (C ₆ H ₄ ) and meta- (C ₆ H ₄ )

m, p and q are the same as or different from each other, each independently an integer of 1 to 10,

n is an integer of 1 to 23;

The present invention also provides a light-quenching dye composition comprising an anthraquinone compound represented by the above formula (1) as an active ingredient.

The anthraquinone compound according to the present invention has broad spectrum in the visible and near infrared wavelength regions so as to cover the whole fluorescent wavelength of commercially available visible and near infrared dyes, has a high absorption spectrum and high stability and good solubility in aqueous conditions Can easily be used to label biomolecules that are manipulated on an aqueous buffer, and thus can be widely used to observe the identification of biomolecules in the field of optical molecular imaging.

1 is a graph showing the absorption spectrum of a compound according to one embodiment of the present invention and a comparative example.
FIG. 2 is a graph showing an absorption spectrum obtained by fixing the maximum absorption wavelength intensity of a compound according to an embodiment and a comparative example of the present invention to 1; FIG.
FIG. 3 is a graph showing the fluorescence-restoring effect measured by preparing a phosphor-peptide-quencher combination using the compound of Example 2 according to the present invention and performing enzymatic degradation experiments.

The present invention relates to a light-emitting dye compound which can be used in combination with a fluorescent dye widely used for labeling biomolecules. More particularly, the present invention relates to a light-emitting dye compound having a broad absorption spectrum so as to cover visible and near infrared wavelength regions, The present inventors have completed the present invention by developing an anthraquinone compound according to the present invention after an extensive effort to produce a compound having high stability and good solubility in a water-soluble condition so as to facilitate labeling of a biomolecule to be manipulated.

Hereinafter, the present invention will be described in more detail.

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

[Chemical Formula 1]

In the above formula (1)

R _1, R _2, R _3, and R ₄ are the same or different and each is independently hydrogen, a hydroxyl group, an amine group, a nitro group, a C 1 -C 6 alkyl group, having 7 to 10 groups with each other, containing from 1 to 6 carbon atoms Alkoxy group, sulfonic acid group and sulfonic acid group,

R ₅ is selected from an alkyl group having 1 to 6 carbon atoms and an alkyl group having 7 to 10 carbon atoms,

R ₆ is selected from the group consisting of a hydroxyl group, a hydrazinyl group, NH (CH ₂ ) _p NH ₂ , N-hydroxysuccinimide group, NH- (CH ₂ ) _q -N (CO) ₂ C ₂ H ₂ , 6-hydrazino-1,3,5-triazine group, NH-A-SO ₂ CH = CH ₂ ,

A is selected from (CH ₂ ) _m , para- (C ₆ H ₄ ) and meta- (C ₆ H ₄ )

m, p and q are the same as or different from each other, each independently an integer of 1 to 10,

n is an integer of 1 to 23;

을 의미한다.NH- (CH ₂ ) _q -N (CO) ₂ C ₂ H ₂ in R ₆ is

.

According to the present invention, the compound of the formula (1) may be a compound of the following formula (4) or (5).

[Chemical Formula 4]

In the above formulas (4) and (5)

R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , A, m and n are as defined in Formula 1,

R < ₇ > is hydrogen or succinimide,

R ₈ is an amine, C1 to C10 in the alkyl amines, C1 to C10 alkyl maleate already pyridyl, 2,4-dihalo-6-amino-1,3,5-triazole oscillator, from the A-SO ₂ CH = CH ₂ Can be selected.

The compound of the above formula (1) may be specifically selected from the group consisting of the following formulas (6) to (19), but is not limited thereto.

[Chemical Formula 6]

(7)

[Chemical Formula 8]

[Chemical Formula 9]

[Chemical formula 10]

(11)

[Chemical Formula 12]

[Chemical Formula 13]

[Chemical Formula 14]

[Chemical Formula 15]

[Chemical Formula 16]

[Chemical Formula 17]

[Chemical Formula 18]

[Chemical Formula 19]

The anthraquinone compound represented by the formula (1) according to the present invention can absorb wavelengths in the range of 300 to 900 nm, which are visible light and near infrared fluorescent areas.

The anthraquinone compound represented by the formula (1) according to the present invention may be labeled on a biomolecule containing at least one of an amine group, a hydroxyl group and a thiol group,

The biomolecule may be any one selected from the group consisting of proteins, peptides, carbohydrates, sugars, fats, antibodies, proteoglycans, glycoproteins and siRNA.

The biomolecule may be a biomolecule that is not physically or chemically modified, but is not limited thereto, and may be a biomolecule modified physically or chemically. The physically or chemically modified biomolecule may be, for example, a biomolecule labeled with a compound exhibiting fluorescence.

In the method using the anthraquinone compound represented by the above formula (1) according to the present invention, the anthraquinone compound may be labeled on a biomolecule to be utilized in biomolecule research using absorbed light. Alternatively, Labeled with a biomolecule labeled with a compound to control or quench the fluorescence.

As a method for labeling an anthraquinone compound represented by the above formula (1) on a biomolecule, a buffer selected from the group consisting of phosphate buffer solution, carbonate buffer solution and Tris buffer solution, dimethylsulfoxide, dimethylformamide, methanol, ethanol and Acetonitrile or water, and reacting the biomolecule with the compound of formula (I) at a pH of 5 to 12. The reaction may be carried out at a temperature of 20 to 80 DEG C for 30 minutes to 48 hours.

In the case of biomolecules, most of the biomolecules are dissolved in a predetermined buffer from the package unit. In order to secure the stability of the biomolecule, a separate buffer or pH is often required. The compound of formula (I) according to the present invention easily absorbs visible light and near-infrared light by easily reacting with proteins in various buffer solutions, reaction temperatures, pH conditions and the like, and is suitable for use as biomolecule markers.

The present invention provides a process for producing an anthraquinone compound represented by the above formula (1).

The anthraquinone compound represented by the formula (1) according to the present invention may be prepared by the steps of: converting a compound represented by the following formula (2) into a diazonium salt; And reacting a compound represented by the following formula (3).

[Chemical Formula 2] < EMI ID =

R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ and n are the same as defined in the above formula (1).

The method for producing the diazonium salt is not particularly limited as long as the method for producing the diazonium salt is not limited. For example, the compound of the formula 2 may be prepared by adding hydrochloric acid to the hydrochloride salt and then adding sodium nitrite to the hydrochloride salt. (Hereinafter, referred to as a sieving method), the compound of formula 2 may be prepared by adding sodium nitrite to an excessive amount, followed by addition of an excess of hydrochloric acid (hereinafter referred to as a reverse diazo method).

The method for preparing the compound of formula (1) may be performed by dissolving or adding hydrochloric acid salt in an amount of 3 to 50 equivalents of concentrated hydrochloric acid per equivalent of the compound of formula (2) 1 to 10 equivalents of sodium nitrite may be added in an acidic condition at 20 to 5 ° C and pH 1 to 5.5 to form a diazonium salt. The compound of the formula 3 may be added to an aqueous (neutral to alkaline) solution Or an organic solvent to react with a diazonium salt,

Alternatively, the compound of formula (2) is dissolved in an organic solvent to lower the temperature to -20 to 5 占 폚, 1 to 10 equivalents of sodium nitrite is added to 1 equivalent of the compound of formula (2), and then concentrated hydrochloric acid 3 ≪ / RTI > to 50 equivalents.

The organic solvent may be at least one selected from the group consisting of 1,2-dichloroethane, dichloromethane, chloroform, acetonitrile, dioxane, tetrahydrofuran, N, N-dimethylformamide, methanol and ethanol.

The optimum coupling pH at the time of coupling of the above formulas (2) and (3) may vary depending on the functional groups R ₁ , R ₂ , R ₃ , R ₄ , R ₅ and R ₆ , 10, and the reaction time may be 1 to 24 hours, but the present invention is not limited thereto, and the reaction is shown in the following reaction formula (1).

[Reaction Scheme 1]

The step of reacting the compound of formula (2) with the compound of formula (3) to produce the compound of formula (1) may be carried out in 1-step, but not limited thereto, and may proceed in 2-step or more.

For example, the above-mentioned formula (1) may be represented by the following formula (4) or (5)

[Chemical Formula 4]

In the above Chemical Formula 4 or Chemical Formula 5,

R ₁ , R ₂ , R ₃ , R ₄ , R _5, and n are each as defined for Formula 1,

R < ₇ > is hydrogen or succinimide,

R ₈ is an amine, C1 to C10 in the alkyl amines, C1 to C10 alkyl maleate already pyridyl, 2,4-dihalo-6-amino-1,3,5-triazole oscillator, from the A-SO ₂ CH = CH ₂ Selected,

A is selected from (CH ₂ ) _m , para- (C ₆ H ₄ ) and meta- (C ₆ H ₄ )

m is an integer of 1 to 10;

The compound wherein R ₇ is succinimide in the above formula 4 is obtained by reacting a compound wherein R ₇ is hydrogen in the above formula 4 with N, N'-disuccinimidyl carbonate (DSC) or N, N ', N', N'-tetramethyluronium hexafluorophosphate (O- (benzotriazol- '-tetramethyluroniumhexafluorophosphate, HBTU) in an organic solvent,

The [Formula 5] R ₈ in the A-SO ₂ CH = CH ₂ The compound is the [formula 4] R ₇ are those wherein the vinyl succinimide sulfonyl alkyl amine _{(NH 2 -A-SO 2 CH} = CH ₂ , A is as defined for the above formula (1)) or a vinylsulfonylalkylamine hydrochloride in an organic solvent,

The [Formula 5] R ₈ in the amine, C1 to C10 alkyl _{amine, A-SO 2 CH = CH} 2, C1 to C10 alkyl maleate-6-amino-1,3 as already pyridyl and 2,4-dihalo , 5-triazine group can be prepared by reacting the compound of formula (4) with an amine compound or an amine compound protected with a protecting group,

The amine compound may be at least one selected from the group consisting of hydrazine, alkyldiamine (NH ₂ (CH ₂ ) _p NH ₂ , p is an integer of 1 to 10), vinylsulfonylalkylamine (NH ₂ -A-SO ₂ CH═CH ₂ , (NH ₂ (CH ₂ ) _q -N (CO) ₂ C ₂ H ₂ , q is an integer of 1 to 10) and 2,4-dihalo-6- Hydrazinyl-l, 3,5-triazine.

Specific examples of the amine compound include hydrazine, ethylenediamine, N- (2-aminoethyl) maleimide and 4,6-dichloro-2-hydrazinyl-1,3,5-triazine. The protected amine compound may be, but is not limited to, tert-buthyl carbazate.

The compound of formula (4) may be a compound wherein R ₇ is hydrogen in the formula (4), but preferably R ₇ is succinimide.

The organic solvent used in the reaction may be the same or different and each independently selected from the group consisting of 1,2-dichloroethane, dichloromethane, chloroform, acetonitrile, dioxane, tetrahydrofuran, N, N-dimethylformamide, methanol, ethanol May be at least one selected from the group consisting of

The compound of Formula 2 may be prepared by dispersing bromine-amine acid in an aqueous solution using a dispersing agent such as Dowell, adding a benzene derivative containing an amine group and a copper catalyst, and conducting a condensation reaction at 15 to 80 ° C .

The present invention provides a light-quenching dye composition comprising an anthraquinone compound represented by the above formula (1) as an active ingredient.

The anthraquinone compound of the formula (1) according to the present invention can absorb wavelengths in the range of 300 to 900 nm which are visible and near-infrared wavelengths, and using the composition containing it as an effective component, the fluorescence represented by the biomolecule disappears Or restoration function, and thus can be usefully applied to biomolecule imaging.

The present invention provides a biomolecule marking method comprising the step of binding a biomolecule with an anthraquinone compound represented by the above formula (1).

The biomolecule includes at least one functional group selected from the group consisting of an amine group, a hydroxyl group and a thiol group. The label includes a vinylsulfone group, an amine group or a halogen group present in the compound of formula (1) A hydroxyl group or a thiol group.

The biomolecule may be any one or more selected from the group consisting of proteins, peptides, carbohydrates, sugars, fats, antibodies, proteoglycans, glycoproteins and siRNA.

The biomolecule may be a biomolecule that is not physically or chemically modified, but is not limited thereto, and may be a biomolecule modified physically or chemically. The physically or chemically modified biomolecule may be, for example, a biomolecule labeled with a compound exhibiting fluorescence.

The compound exhibiting fluorescence is preferably a compound exhibiting fluorescence in the range of 300 to 900 nm.

On the other hand, the label may be a buffer selected from the group consisting of phosphate buffer, carbonate buffer and Tris buffer as a solvent, an organic solvent selected from the group consisting of dimethylsulfoxide, dimethylformamide, methanol, ethanol and acetonitrile, And reacting the compound of formula (I) with the biomolecule at a pH of 5 to 12. The reaction may be carried out at a temperature of 20 to 80 DEG C for 30 minutes to 48 hours.

In the case of biomolecules, most of the biomolecules are dissolved in a predetermined buffer from the package unit. In order to secure the stability of the biomolecule, a separate buffer or pH is often required. The compound of formula (I) according to the present invention has high stability under water-soluble conditions and is easy to store for a long time. In addition, since the compound easily reacts with proteins in various buffer solutions, reaction temperatures and pH conditions to produce fluorescence, . ≪ / RTI >

Hereinafter, the present invention will be described in more detail by way of examples and comparative experiments. However, the examples are for illustrative purposes only and are not intended to limit the scope of the invention.

Example

The experimental apparatus, analysis equipment and reagents used in the examples and comparative experiments will be described.

LC / MS was measured by electrospray ionization (ESI) using an LC / MSD SL from Agilent Technology. The absorbance and absorption at the maximum wavelength (λ _abs ) of the synthesized dyes were measured with a Lambda 45 from Perkin Elmer, and the emission value (λ _fl ) at the emission wavelength and maximum emission wavelength was measured using a Perkin Elmer LS-55 .

Column chromatography for the separation and purification of organic compounds used kieselgel 60 (230-400 mesh) from Merck as silicagel in the normal phase and Silica gel 60GF254 (0.25 g / ml) for thin layer chromatography (TLC) mm, Merck) was used as a glass plate, and ultraviolet rays of 254 nm and 365 nm were used for compound identification on TLC. In the case of reverse phase, the glass plate coated with silica gel 60 RP-18 F254S (0.25 mm, Merck) was used as the TLC. In the case of column chromatography, Fraction Collector, a medium pressure liquid chromatography (MPLC) A reversed phase column Lichroprep RP-18 (40 to 63 [mu] m, Merck) was connected to R-660.

The reagents were mainly Aldrich and TCI. Solvents requiring purification were purified and used according to known methods and all reactions were carried out in a nitrogen stream unless otherwise noted.

Manufacturing example

Manufacturing example  1: Preparation of a compound of the formula (2-1)

After 10 g (24.7 mmol, 1 eq) of sodium bromoaminate was dispersed in 100 mL of distilled water, 6.3 g of sodium carbonate and 1.3 g of sodium sulfite were added and the mixture was stirred at 35 DEG C for 30 minutes. Then, 2,5-diaminobenzenesulfonic acid 5.2 g (24.7 mmol, 1 eq) was added thereto, and the mixture was further stirred for 30 minutes. Then, 0.1 g of copper chloride was added thereto and further stirred for 30 minutes, followed by filtration, and the filtrate was distilled under reduced pressure. The obtained compound was purified using a reversed phase column to obtain a compound represented by the formula (2-1). (5.23 g, 43.3%).

Rf = 0.45 (RP-18C, acetonitrile: distilled water = 1: 3 v / v)

Manufacturing example  2

Production Example 2.1: Preparation of Formula (3-1)

6.87 g (64 mmol, 1 eq) of N-methylaniline was stirred with 100 mL of 1,2-dichlorobenzene for 30 minutes. To the mixture was added 15 g (77 mmol, 1.2 eq) of 6-bromohexanoic acid and refluxed at 140 占 폚 for 12 hours. After completion of the reaction, 500 mL of hexane was added, stirred for 10 minutes, dried under reduced pressure, and purified by silica gel column chromatography to obtain Compound 3-1. (7.48 g, 52.8%).

Rf = 0.49 (dichloromethane: nucleic acid: methanol = 10: 1: 1 v / v)

LC / MS: 221.1 (Note, the theoretical C ₁₃ H ₁₉ NO ₂ 221.3)

Production Example 2.2: Preparation of Formula 3-2

Compound 3-2 was obtained according to the method of Production Example 2.1 except that 10 g (45 mmol, 1 eq) of 8-bromooctanoic acid was used instead of 15 g (77 mmol, 1.2 eq) of 6-bromohexanoic acid . (3.55 g, 31.6%).

Rf = 0.55 (dichloromethane: nucleic acid: methanol = 10: 1: 1 v / v)

LC / MS: 249.99 (see the theoretical value C ₁₅ H ₂₃ NO ₂ 249.35)

Example

Example  1: Preparation of compound of formula (6)

3 g (6.13 mmol, 1 eq) of the compound of Preparation Example 1 was dissolved in a mixed solution of 100 ml of water and 50 ml of methanol, cooled to 10 ° C or lower, and 550 mg (7.97 mmol, 1.3 eq) of sodium nitrite Was slowly added to the cooled solution. Then, 6.4 mL (61.3 mmol, 10 eq) of concentrated hydrochloric acid was added and reacted for 1 hour. A solution of 1.36 g (6.13 mmol, 1 eq) of the compound of Preparation 2.1 dissolved in 50 mL of methanol was added thereto. The mixture was further stirred, adjusted to pH 6 by adding an aqueous solution of sodium carbonate, and stirred until the next day. After completion of the reaction, the compound obtained by distillation under reduced pressure was purified using a reversed phase column to prepare a compound of formula (6). (853 mg, 19.3%).

Rf = 0.45 (RP-18C, acetonitrile: distilled water = 1: 3 v / v)

LC / MS = 719.6 (see the theoretical value C ₃₃ H ₂₉ N ₅ O ₁₀ S ₂ ^{2 -} 719.14)

? _abs (PBS): 481 nm, 626 nm

Example  2: Preparation of compound of formula (7)

400 mg (0.55 mmol, 1 eq) of the compound of Example 1 was dissolved in 50 mL of DMF, the temperature was raised to 40 DEG C, and 0.96 mL (5.54 mmol, 10 eq) of N, N-diisopropyladilyamine was added. 426 mg (1.66 mmol, 3 eq) of N, N'-disuccinimidyl carbonate was dissolved in 1 mL of DMF, added to the reaction mixture, and further stirred for 1 hour. After completion of the reaction, ethyl acetate was added to form a precipitate, followed by filtration and drying to obtain the desired compound.

Example  3: Preparation of compound of formula 8

After the compound of Example 2 was dissolved in 40 mL of DMF, 0.96 mL of N, N-diisopropylidiylamine and 115 mg (0.55 mmol, 1 eq) of 2- (2'-chloroethylsulfonyl) And the mixture was stirred at 40 占 폚 (24 hours or longer). After completion of the reaction, ethyl acetate was added to the reaction mixture to form a precipitate, followed by filtration and drying, followed by purification using a reverse phase column to obtain the desired compound. (97 mg, 21.0%).

R _f = 0.4 (RP-18C, acetonitrile: distilled water = 1: 4 v / v)

LC / MS = 838.93 (see the theoretical value C ₃₇ H ₃₈ N ₆ O ₁₁ S ₃ 838.93)

λ _abs (PBS): 481 nm, 626 nm

Example  4: Preparation of compound of formula 11

The procedure of Example 3 was repeated except for using N- (2-aminoethyl) maleimide instead of 2- (2'-chloroethylsulfonyl) ethylamine hydrochloride to obtain the desired compound .

Rf = 0.45 (RP-C18, acetonitrile: distilled water = 3: 7 v / v)

LC / MS = 841.5 (see, theoretical value C ₃₉ H ₃₅ N ₇ O ₁₁ S ₂ ^{2 -} 841.18)

? _abs (PBS): 481 nm, 626 nm

Example  5: Preparation of the compound of formula (13)

Compound (13) was prepared by the method of Example 1, except that the compound of Preparation 2.2 was used in place of the compound of Preparation 2.1. (570 mg, 14.9%).

Rf = 0.25 (RP-18C, acetonitrile: distilled water = 1: 3 v / v)

LC / MS = 747.7 (see the theoretical value C ₃₅ H ₃₃ N ₅ O ₁₀ S ₂ ^{2 -} 747.17)

? _abs (PBS): 472 nm, 624 nm

Example  6: Preparation of compound of formula 14

The objective compound was prepared by the method of Example 2 except that the compound of Example 5 was used instead of the compound of Example 1.

Example  7: Preparation of compound of formula (15)

The objective compound was prepared by the method of Example 3 except that the compound of Example 6 was used instead of the compound of Example 2. (35 mg, 18.1%).

Rf = 0.5 (RP-18C, acetonitrile: distilled water = 1: 2 v / v)

LC / MS = 865.0 (see the theoretical value C ₃₉ H ₄₁ N ₆ O ₁₁ S ^{3 -} 865.20)

? _abs (PBS): 472 nm, 624 nm

Example  8: Preparation of the compound of formula (16)

Except that the compound of Example 6 was used in place of the compound of Example 2 and tert-butyl carbazate was used instead of 2- (2'-chloroethylsulfonyl) ethylamine hydrochloride. 3, the desired compound was prepared.

Example  9: Preparation of compound of formula (17)

The objective compound was prepared by the same procedure as in Example 3 except that the compound of Example 6 was used instead of the compound of Example 2 and ethylenediamine was used instead of 2- (2'-chloroethylsulfonyl) ethylamine hydrochloride .

Example  10: Preparation of compound of formula 18

Example 3 was repeated except that the compound of Example 6 was used instead of the compound of Example 2 and N- (2-aminoethyl) maleimide was used in place of 2- (2'-chloroethylsulfonyl) ethylamine hydrochloride. The desired compound was prepared.

Example  11: Preparation of compound of formula 19

Except that the compound of Example 8 was used in place of the compound of Example 2 and cyanuric chloride was used instead of 2- (2'-chloroethylsulfonyl) ethylamine hydrochloride, the objective compound was prepared Respectively.

Test Example  1: Absorption spectrum comparison experiment

In order to confirm the absorption intensity and spectrum of the anthraquinone compound according to the present invention, the compounds of Examples 1 and 6 and the commercially available BHQ-3 as a comparative example were purchased and the absorption spectrum comparison experiment was conducted. The compounds of Example 1 and Example 6 of the present invention were dissolved in distilled water and BHQ-3 was dissolved in a small amount of DMSO according to the manufacturer's instructions to prepare a stock solution. The absorption spectrum was measured by diluting with distilled water, and it is shown in Fig.

BHQ-3 was not solely dissolved in water, and when dissolved in DMSO and diluted with water, it remained dissolved.

As shown in Fig. 1, the compounds of Examples 1 and 6 exhibited absorption spectra in the range of 300 to 800 nm, which is broader than that of BHQ-3. In addition, BHQ-3 had a wavelength band exhibiting a negative (-) absorption value in the vicinity of 750 to 800 nm, but the compounds of Examples 1 and 6 did not show a negative absorption value.

2 is a graph in which the measured absorption spectrum is recalculated by fixing the intensity at the maximum absorption wavelength to 1. The compounds of Examples 1 and 6 exhibited absorption spectra in the range of 300 to 800 nm, while the comparative examples showed spectra in the range of 300 to 750 nm, indicating that the anthraquinone compounds according to the present invention absorb Can be confirmed. Further, it absorbs more wavelengths of 650 nm or more, confirming that the anthraquinone compound according to the present invention is effective for absorption of near-infrared fluorescence.

Test Example  2: Fluorescent Extinction  Effect measurement

Near infrared fluorescent dyes such as Cy5.5 NHS ester are labeled at the N-terminus of NH2-GPLGVRGKBB-COOH, which is a peptide substrate selectively degraded to MMP-2 protease by a combination of a known phosphor-peptide-quencher, Peptide-quencher derivative (Cy5.5-peptide-BHQ-3) in which the amine of BHQ-3 is labeled. When MMP-2 protease is added to the derivative, the peptide is decomposed and the near-infrared fluorescence that has been quenched by the quenching dye is restored.

In order to confirm the quenching effect of the anthraquinone compound according to the present invention, the peptide substrate was labeled with FNR-675 NHS ester (BioActs), which is a fluorescent dye showing the same fluorescence as Cy5.5, and the compound of Example 2 was bound FNR-675) -peptide- (compound of formula 7).

An enzyme degradation experiment was performed by adding the above (FNR-675) -peptide- (compound of formula (7)) with MMP-2 protease.

 50 μL of TCNB buffer (0.1 M Tris, 5 mM calcium chloride, 200 mM NaCl, 0.1% Brij), 50 μL of MMP-2 enzyme and 1 μL of 100 mM p-aminophenyl mercuric acid were mixed After 10 μl aliquots were diluted with 90 μl TCNB buffer, MMP-2 enzyme was activated by reacting at 37 ° C for 1 hour. (FNR-675) -peptide- (Formula 7) was prepared with TCNB buffer at a concentration of 1 mg / 2 mL, and 40 μL aliquot was diluted with 3,960 μL of TNCB buffer to prepare a peptide substrate solution. The MMP-2 enzyme-activating solution was added to the peptide substrate solution, and the degree of fluorescence recovery with time was measured while maintaining the temperature at 37 ° C, which is shown in FIG. The fluorescence intensity was measured at an excitation wavelength of 675 nm and a fluorescent wavelength of 698 nm.

As shown in FIG. 3, the fluorescence was restored over time, and after 300 minutes, the fluorescence was restored 15 times or more.

Considering the results of Test Examples 1 and 2, the compound of Formula 1 according to the present invention is excellent in the ability to absorb wavelengths in visible and near-infrared regions, and is useful for fluorescence extinction or restoration of biomolecules It is expected to be possible.

Claims (12)

An anthraquinone compound represented by the following Chemical Formula 1 and absorbing a wavelength in a range of 300 to 900 nm:
[Chemical Formula 1]

In the above formula (1)
R ₁ is SO ₃ H,
R ₂ , R ₃ and R ₄ are hydrogen,
R ₅ is selected from an alkyl group having 1 to 6 carbon atoms and an alkyl group having 7 to 10 carbon atoms,
R ₆ is selected from the group consisting of a hydrazinyl group, NH (CH ₂ ) _p NH ₂ , N-hydroxysuccinimide group, NH- (CH ₂ ) _q -N (CO) ₂ C ₂ H ₂ , 6-hydrazino-1,3,5-triazine group, NH-A-SO ₂ CH = CH ₂ ,
A is selected from (CH ₂ ) _m , para- (C ₆ H ₄ ) and meta- (C ₆ H ₄ )
m, p and q are the same as or different from each other, each independently an integer of 1 to 10,
n is an integer of 1 to 23; The anthraquinone compound according to claim 1, wherein the compound represented by Formula 1 is represented by Formula 4 or Formula 5:
[Chemical Formula 4]

In the above formulas (4) and (5)
R ₁ is SO ₃ H,
R ₂ , R ₃ and R ₄ are hydrogen,
R ₅ is selected from an alkyl group having 1 to 6 carbon atoms and an alkyl group having 7 to 10 carbon atoms,
R < ₇ > is succinimide,
R ₈ is an amine, C1 to C10 in the alkyl amines, C1 to C10 alkyl maleate already pyridyl, 2,4-dihalo-6-amino-1,3,5-triazole oscillator, from the A-SO ₂ CH = CH ₂ Selected,
A is selected from (CH ₂ ) _m , para- (C ₆ H ₄ ) and meta- (C ₆ H ₄ )
m is an integer of 1 to 10,
n is an integer of 1 to 23; The anthraquinone compound according to claim 1, wherein the formula 1 is any one selected from the group consisting of the following formulas (7) to (12) and (14)
(7)

[Chemical Formula 8]

[Chemical Formula 9]

[Chemical formula 10]

(11)

[Chemical Formula 12]

[Chemical Formula 14]

[Chemical Formula 15]

[Chemical Formula 16]

[Chemical Formula 17]

[Chemical Formula 18]

[Chemical Formula 19]

delete The anthraquinone compound according to claim 1, which is labeled with a biomolecule comprising an amine group, a hydroxyl group or a thiol group. 6. The anthraquinone compound according to claim 5, wherein the biomolecule is at least one selected from the group consisting of proteins, peptides, carbohydrates, sugars, fats, antibodies, proteoglycans, glycoproteins and siRNA. (2) to a diazonium salt; And a step of reacting a compound represented by the following formula (3) to obtain a compound represented by the following formula (1): < EMI ID =
[Chemical Formula 2] < EMI ID =

[Chemical Formula 1]

In the above formulas (1) to (3)
R ₁ is SO ₃ H,
R ₂ , R ₃ and R ₄ are hydrogen,
R ₅ is selected from an alkyl group having 1 to 6 carbon atoms and an alkyl group having 7 to 10 carbon atoms,
R ₆ is selected from the group consisting of a hydrazinyl group, NH (CH ₂ ) _p NH ₂ , N-hydroxysuccinimide group, NH- (CH ₂ ) _q -N (CO) ₂ C ₂ H ₂ , 6-hydrazino-1,3,5-triazine group, NH-A-SO ₂ CH = CH ₂ ,
A is selected from (CH ₂ ) _m , para- (C ₆ H ₄ ) and meta- (C ₆ H ₄ )
m, p and q are the same as or different from each other, each independently an integer of 1 to 10,
n is an integer of 1 to 23; A quencher dye composition comprising the anthraquinone compound of claim 1 as an active ingredient. delete A biomolecule marking method comprising the step of binding an anthraquinone compound of claim 1 with a biomolecule of a mammal other than human or a human biomolecule,
Wherein the biomolecule comprises at least one functional group selected from an amine group, a hydroxyl group and a thiol group, and the anthraquinone compound of the first aspect is bound to the functional group 11. The labeling method according to claim 10, wherein the biomolecule is any one or more selected from the group consisting of proteins, peptides, carbohydrates, sugars, fats, antibodies, proteoglycans, glycoproteins and siRNA. delete

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