KR20220127142A - Reporter and their applications - Google Patents

Abstract

The present invention relates to a fluorescent reporter capable of labeling nucleic acids including DNA and simultaneously exhibiting luminescent properties at an excited energy level, and to various applications thereof. The fluorescent reporter for labeling nucleic acids according to the present invention is represented by chemical formula 1 or 2.

Description

REPORTER AND THEIR APPLICATIONS

The present invention relates to a fluorescent reporter capable of labeling nucleic acids including DNA and exhibiting luminescent properties at an excited energy level, and various uses thereof.

In the field of biotechnology, fluorescent dyes are used as a means of visualizing biological phenomena at the cellular level in vivo and in vitro, or to detect biopsies and disease sites. .

Although there are biomolecules that emit light by themselves, such as green fluorescent protein (GFP), in general, biomolecules such as biological tissues, cells, and lower levels are dyed with fluorescent dyes, or biomolecules such as proteins and nucleic acids are dyed with fluorescent dyes. After labeling, it is accompanied by optical equipment that can detect fluorescence signals, and imaged data by various techniques are being obtained.

Mainly used optical analysis equipment is a fluorescence microscope, confocal microscope, flow cytometry, microarray, quantitative PCR system for cell observation. ), nucleic acid and protein separation and analysis, electrophoresis device, real-time in vivo imaging system, etc. In addition to research equipment, immunoassay technique, PCR analysis, and statistical technology are grafted. Nucleic acid and protein diagnostic kits (or biochips)-based in vitro diagnosis equipment and operating tables and endoscopic equipment for image-guided surgery are known for diagnosis and treatment, and new applications are continuously being developed. Fields and equipment with higher levels of resolution and data processing capabilities are being developed.

In order to apply fluorescent dyes to the bio field, in general, when present in a medium in which most biomolecules are present, that is, an aqueous solution, photo bleaching and quenching are small, and the molecular extinction coefficient and It is desirable to have high quantum efficiency and to be stable under various pH conditions.

Various fluorescent dyes have been used in various research fields, but fluorescent dyes that satisfy all of the above conditions are rare in the bio field, and representative structures currently used include coumarins, cyanine, and bodipy. , fluoresceins, rhodamines, pyrenes, carbopyrronin, oxazine, xanthenes, thioxanthene and acridines, etc. There is this. Among them, rhodamine derivatives and polymethine-based cyanine derivatives are predominant.

In particular, the cyanine-based fluorescent dye is connected to both ends of polymethine by a heterocyclic ring containing nitrogen, and by controlling the length of polymethine, various fluorescence can be realized over the entire range of visible and near infrared rays from 450 nm to 800 nm. In addition, there is an optical characteristic that generally has a very high extinction coefficient.

As a background art related to the present invention, there is Republic of Korea Patent Publication No. 10-2017-0026245 (published on March 8, 2017), which describes a perylene-based compound, a manufacturing method thereof, and a fluorescent dye including the same. .

An object of the present invention is to provide a novel reporter as a compound that can be widely used to observe the identification of biomolecules in the field of optical imaging.

Another object of the present invention is to provide an oligonucleotide for nucleic acid detection, a composition for nucleic acid detection, a support for nucleic acid detection, and a nucleic acid detection method comprising the novel reporter as various uses of the novel reporter as defined herein. .

According to one aspect of the present invention for solving the above technical problem, a reporter represented by the following Chemical Formula 1 or Chemical Formula 2 is provided.

[Formula 1]

[Formula 2]

here,

Ar ₁ is a substituted or unsubstituted C ₆ -C ₂₀ aryl or C ₆ -C ₂₀ heteroaryl containing at least one hetero atom,

Z ₁ is NR ₅ R ₆ or OR ₇ ,

Z ₂ is NR ₈ or O,

X is O or S;

Y is CR ₉ R ₁₀ , NR ₁₁ , O or S;

R ₁ To R ₁₁ are each independently hydrogen, deuterium, substituted or unsubstituted C ₁ -C ₁₀ alkyl, substituted or unsubstituted C ₁ -C ₁₀ heteroalkyl including at least one hetero atom, substituted or unsubstituted substituted C ₂ -C ₁₀ alkenyl, substituted or unsubstituted C ₂ -C ₁₀ alkynyl, substituted or unsubstituted C ₃ -C ₂₀ cycloalkyl, substituted or unsubstituted C ₃ -C ₂₀ cycloalkenyl, substituted or unsubstituted C ₂ -C ₂₀ heterocycloalkyl, hydroxy, oxido (—O ^— ), substituted or unsubstituted C ₁ -C ₁₀ alkoxy, substituted or unsubstituted C ₃ -C ₂₀ cycloalkyloxy, substituted or unsubstituted C ₅ -C ₂₀ aryloxy, substituted or unsubstituted C ₂ -C ₂₀ heteroaryloxy, substituted or unsubstituted C ₅ -C ₂₀ aryl, substituted or unsubstituted C ₂ -C ₂₀ hetero aryl, substituted or unsubstituted C ₅ -C ₂₀ aralkyl, substituted or unsubstituted C ₁ -C ₁₀ alkylthio, substituted or unsubstituted C ₅ -C ₂₀ arylthio, substituted or unsubstituted C ₃ -C ₂₀ cycloalkylthio, substituted or unsubstituted C ₂ -C ₂₀ heteroarylthio, substituted or unsubstituted acylamino, acyloxy, substituted or unsubstituted phosphino, carboxylate (-CO ₂ ^- ), trifluoro Methylsulfonyl (-SO ₂ CF ₃ ), substituted or unsubstituted ammonium, nitro, sulfonic acid (-SO ₃ H), sulfonate, substituted sulfonyl, substituted sulfonic acid ester, substituted or unsubstituted sulfone Amide, substituted thioketone, trihalomethyl (-CF ₃ , -CCl ₃ , -CBr ₃ , -CI ₃ ), haloformyl (-COCl, -COBr, -COI), formyl (-CHO) , acyl, carboxyl, substituted ester, substituted or unsubstituted aminocarbonyl, nitro, nitroso (-N=O), fluoro (-F), chloro (-Cl), bromo (-Br ), iodo (-I), substituted or unsubstituted germanium, substituted or unsubstituted boron, substituted or unsubstituted aluminum, substituted or unsubstituted silyl, substituted or unsubstituted amide, carbamate, carboxylate, substituted or unsubstituted phosphine, substituted or unsubstituted phosphoric acid, phosphate, phosphonic acid, phosphonate, nite It is a functional group selected from reyl, hydrazine, acetal, ketal, polyalkylene oxide and -L ₁ -R ₁₂ , or two adjacent functional groups may combine with each other to form a ring.

In this case, at least one of R ₁ to R ₁₁ may be -L ₁ -R ₁₂ .

L ₁ is a single bond, substituted or unsubstituted C ₁ -C ₁₀ alkyl, substituted or unsubstituted C ₁ -C ₁₀ heteroalkyl containing at least one hetero atom, substituted or unsubstituted C ₂ -C ₁₀ alkenyl, substituted or unsubstituted C ₂ -C ₁₀ alkynyl, substituted or unsubstituted C ₃ -C ₂₀ cycloalkyl, substituted or unsubstituted C ₃ -C ₂₀ cycloalkenyl and substituted or unsubstituted C ₂ -C ₂₀ It includes at least one selected from heterocycloalkyl, and R ₁₂ is a deoxyribonucleoside represented by Formula 3 below.

[Formula 3]

here,

* represents the position at which the deoxyribonucleoside is bonded to L ₁ ,

B is a nucleobase,

R ₁₃ is selected from hydrogen, deuterium, P(OR ₁₅ )(N(R ₁₆ R ₁₇ ) and -L ₂ -R ₁₈ ,

R ₁₄ is an alcohol protecting group, hydroxy or P(OR ₁₅ )(N(R ₁₆ R ₁₇ ), or a support or nucleic acid to which a reporter represented by Formula 1 or Formula 2 is bound,

R ₁₅ to R ₁₇ are each independently selected from hydrogen, deuterium, substituted or unsubstituted C ₁ -C ₁₀ alkyl and substituted or unsubstituted C ₁ -C ₁₀ heteroalkyl containing at least one hetero atom,

L ₂ is a single bond or is selected from internucleotide phosphodiester bonds, substituted or unsubstituted C ₁ -C ₁₀ alkyl and substituted or unsubstituted C ₁ -C ₁₀ heteroalkyl containing at least one hetero atom, ,

R ₁₈ is hydroxy or P(OR ₁₅ )(N(R ₁₆ R ₁₇ ), or a support or nucleic acid to which a reporter represented by Formula 1 or Formula 2 is bound.

According to another aspect of the present invention, a reporter represented by the following Chemical Formula 5 is provided.

[Formula 5]

here,

R ₂₁ and R ₂₂ are each independently hydrogen, deuterium, substituted or unsubstituted C ₁ -C ₁₀ alkyl, substituted or unsubstituted C ₁ -C ₁₀ heteroalkyl containing at least one hetero atom, substituted or unsubstituted substituted C ₂ -C ₁₀ alkenyl, substituted or unsubstituted C ₂ -C ₁₀ alkynyl, substituted or unsubstituted C ₃ -C ₂₀ cycloalkyl, substituted or unsubstituted C ₃ -C ₂₀ cycloalkenyl, substituted or unsubstituted C ₂ -C ₂₀ heterocycloalkyl and substituted or unsubstituted C ₅ -C ₂₀ aryl and substituted or unsubstituted C ₂ -C ₂₀ heteroaryl,

R ₂₃ to R ₃₀ are each independently hydrogen, deuterium, substituted or unsubstituted C ₁ -C ₁₀ alkyl, substituted or unsubstituted C ₁ -C ₁₀ heteroalkyl containing at least one hetero atom, substituted or unsubstituted substituted C ₂ -C ₁₀ alkenyl, substituted or unsubstituted C ₂ -C ₁₀ alkynyl, substituted or unsubstituted C ₃ -C ₂₀ cycloalkyl, substituted or unsubstituted C ₃ -C ₂₀ cycloalkenyl, substituted or unsubstituted C ₂ -C ₂₀ heterocycloalkyl, hydroxy, oxido (—O ^— ), substituted or unsubstituted C ₁ -C ₁₀ alkoxy, substituted or unsubstituted C ₃ -C ₂₀ cycloalkyloxy, substituted or unsubstituted C ₅ -C ₂₀ aryloxy, substituted or unsubstituted C ₂ -C ₂₀ heteroaryloxy, substituted or unsubstituted C ₅ -C ₂₀ aryl, substituted or unsubstituted C ₂ -C ₂₀ hetero aryl, substituted or unsubstituted C ₅ -C ₂₀ aralkyl, substituted or unsubstituted C ₁ -C ₁₀ alkylthio, substituted or unsubstituted C ₅ -C ₂₀ arylthio, substituted or unsubstituted C ₃ -C ₂₀ cycloalkylthio, substituted or unsubstituted C ₂ -C ₂₀ heteroarylthio, substituted or unsubstituted acylamino, acyloxy, substituted or unsubstituted phosphino, carboxylate (-CO ₂ ^- ), trifluoro Methylsulfonyl (-SO ₂ CF ₃ ), substituted or unsubstituted ammonium, nitro, sulfonic acid (-SO ₃ H), sulfonate, substituted sulfonyl, substituted sulfonic acid ester, substituted or unsubstituted sulfone Amide, substituted thioketone, trihalomethyl (-CF ₃ , -CCl ₃ , -CBr ₃ , -CI ₃ ), haloformyl (-COCl, -COBr, -COI), formyl (-CHO) , acyl, carboxyl, substituted ester, substituted or unsubstituted aminocarbonyl, nitro, nitroso (-N=O), fluoro (-F), chloro (-Cl), bromo (-Br ), iodo (-I), substituted or unsubstituted germanium, substituted or unsubstituted boron, substituted or unsubstituted aluminum, substituted or unsubstituted silyl, substituted or unsubstituted substituted or unsubstituted amides, carbamates, carboxylates, substituted or unsubstituted phosphines, substituted or unsubstituted phosphoric acids, phosphates, phosphonic acids, phosphonates, It is a functional group selected from nitrile, hydrazine, acetal, ketal, polyalkylene oxide and -L ₁ -R ₁₂ , or two adjacent functional groups may combine with each other to form a ring.

In addition, R ₃₁ to R ₃₄ are each independently hydrogen, deuterium, substituted or unsubstituted C ₁ -C ₁₀ alkyl, substituted or unsubstituted C ₁ -C ₁₀ heteroalkyl containing at least one hetero atom, and -L It is selected from ₁ -R ₁₂ , and at least one of R ₃₁ to R ₃₄ may be -L ₁ -R ₁₂ .

L ₁ is a single bond, substituted or unsubstituted C ₁ -C ₁₀ alkyl, substituted or unsubstituted C ₁ -C ₁₀ heteroalkyl containing at least one hetero atom, substituted or unsubstituted C ₂ -C ₁₀ alkenyl, substituted or unsubstituted C ₂ -C ₁₀ alkynyl, substituted or unsubstituted C ₃ -C ₂₀ cycloalkyl, substituted or unsubstituted C ₃ -C ₂₀ cycloalkenyl and substituted or unsubstituted C ₂ -C ₂₀ It includes at least one selected from heterocycloalkyl, and R ₁₂ is a deoxyribonucleoside represented by Formula 3 below.

[Formula 3]

here,

* represents the position at which the deoxyribonucleoside is bonded to L ₁ ,

B is a nucleobase,

R ₁₃ is selected from hydrogen, deuterium, P(OR ₁₅ )(N(R ₁₆ R ₁₇ ) and -L ₂ -R ₁₈ ,

R ₁₄ is an alcohol protecting group, hydroxy or P(OR ₁₅ )(N(R ₁₆ R ₁₇ ), or a support or nucleic acid to which a reporter represented by Formula 1 or Formula 2 is bound,

R ₁₅ to R ₁₇ are each independently selected from hydrogen, deuterium, substituted or unsubstituted C ₁ -C ₁₀ alkyl and substituted or unsubstituted C ₁ -C ₁₀ heteroalkyl containing at least one hetero atom,

L ₂ is a single bond or is selected from internucleotide phosphodiester bonds, substituted or unsubstituted C ₁ -C ₁₀ alkyl and substituted or unsubstituted C ₁ -C ₁₀ heteroalkyl containing at least one hetero atom, ,

R ₁₈ is hydroxy or P(OR ₁₅ )(N(R ₁₆ R ₁₇ ), or a support or nucleic acid to which a reporter represented by Formula 1 or Formula 2 is bound,

Y is CR ₃₅ and each R ₃₅ is hydrogen, deuterium, substituted or unsubstituted C ₁ -C ₁₀ alkyl, substituted or unsubstituted C ₁ -C ₁₀ heteroalkyl comprising at least one heteroatom, fluoro (-F), chloro (-Cl), bromo (-Br), iodo (-I),

n is an integer from 1 to 4.

The deoxyribonucleoside represented by Formula 3 may be specifically represented by Formula 4 below.

[Formula 4]

Also according to another aspect of the present invention there is provided an oligonucleotide comprising a reporter and a quencher as defined herein.

Also, according to another aspect of the present invention, there is provided a composition for detecting a nucleic acid comprising an oligonucleotide as defined herein.

In addition, according to another aspect of the present invention, there is provided a support for detecting a nucleic acid comprising a reporter as defined herein, a support, and a linker connecting the reporter and the support.

In addition, according to another aspect of the present invention, (a) preparing a reaction mixture comprising a target nucleic acid, a reagent necessary for amplifying the target nucleic acid, and an oligonucleotide as defined herein, (b) in the reaction mixture There is provided a nucleic acid detection method comprising amplifying a target nucleic acid by a polymerase chain reaction and (c) measuring the fluorescence intensity of the reaction mixture.

Since the reporter according to the present invention has excellent water solubility and fluorescence intensity, it can be usefully used to label various targets including biomolecules.

1 is a graph showing Real-Time PCR results repeated twice using a double-labeled probe according to DLP1 and DLP4 for a CT (Chlamydia trachomatis) target.
2 is a graph showing the results of Real-Time PCR repeated twice using a double-labeled probe according to DLP2 and DLP5 for a CT (Chlamydia trachomatis) target.
3 is a graph showing Real-Time PCR results repeated twice using double-labeled probes according to DLP3 and DLP6 for a CT (Chlamydia trachomatis) target.
4 to 9 show real-time PCR results (linearity) using double-labeled probes according to DLP1 to DLP6, respectively.

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

Also, unless the context specifically dictates otherwise, a term in the singular includes its plural form, and a plural term may also include its singular form.

Reporter for novel nucleic acid labeling

According to one aspect of the present invention, a reporter for labeling a nucleic acid represented by the following Chemical Formula 1 or Chemical Formula 2 is provided.

[Formula 1]

[Formula 2]

here,

Ar ₁ is a substituted or unsubstituted C ₆ -C ₂₀ aryl or C ₆ -C ₂₀ heteroaryl containing at least one hetero atom,

Z ₁ is NR ₅ R ₆ or OR ₇ ,

Z ₂ is NR ₈ or O,

X is O or S;

Y is CR ₉ R ₁₀ , NR ₁₁ , O or S;

R ₁ To R ₁₁ are each independently hydrogen, deuterium, substituted or unsubstituted C ₁ -C ₁₀ alkyl, substituted or unsubstituted C ₁ -C ₁₀ heteroalkyl including at least one hetero atom, substituted or unsubstituted substituted C ₂ -C ₁₀ alkenyl, substituted or unsubstituted C ₂ -C ₁₀ alkynyl, substituted or unsubstituted C ₃ -C ₂₀ cycloalkyl, substituted or unsubstituted C ₃ -C ₂₀ cycloalkenyl, substituted or unsubstituted C ₂ -C ₂₀ heterocycloalkyl, hydroxy, oxido (—O ^— ), substituted or unsubstituted C ₁ -C ₁₀ alkoxy, substituted or unsubstituted C ₃ -C ₂₀ cycloalkyloxy, substituted or unsubstituted C ₅ -C ₂₀ aryloxy, substituted or unsubstituted C ₂ -C ₂₀ heteroaryloxy, substituted or unsubstituted C ₅ -C ₂₀ aryl, substituted or unsubstituted C ₂ -C ₂₀ hetero aryl, substituted or unsubstituted C ₅ -C ₂₀ aralkyl, substituted or unsubstituted C ₁ -C ₁₀ alkylthio, substituted or unsubstituted C ₅ -C ₂₀ arylthio, substituted or unsubstituted C ₃ -C ₂₀ cycloalkylthio, substituted or unsubstituted C ₂ -C ₂₀ heteroarylthio, substituted or unsubstituted acylamino, acyloxy, substituted or unsubstituted phosphino, carboxylate (-CO ₂ ^- ), trifluoro Methylsulfonyl (-SO ₂ CF ₃ ), substituted or unsubstituted ammonium, nitro, sulfonic acid (-SO ₃ H), sulfonate, substituted sulfonyl, substituted sulfonic acid ester, substituted or unsubstituted sulfone Amide, substituted thioketone, trihalomethyl (-CF ₃ , -CCl ₃ , -CBr ₃ , -CI ₃ ), haloformyl (-COCl, -COBr, -COI), formyl (-CHO) , acyl, carboxyl, substituted ester, substituted or unsubstituted aminocarbonyl, nitro, nitroso (-N=O), fluoro (-F), chloro (-Cl), bromo (-Br ), iodo (-I), substituted or unsubstituted germanium, substituted or unsubstituted boron, substituted or unsubstituted aluminum, substituted or unsubstituted silyl, substituted or unsubstituted amide, carbamate, carboxylate, substituted or unsubstituted phosphine, substituted or unsubstituted phosphoric acid, phosphate, phosphonic acid, phosphonate, nite It may be a functional group selected from reyl, hydrazine, acetal, ketal and polyalkylene oxide, or two adjacent functional groups may combine with each other to form a ring.

In addition, when any functional group of R ₁ to R ₁₁ is a substituted functional group, an arbitrary substituent other than hydrogen may be bonded to at least one arbitrary carbon of the functional group. The substituent is deuterium, substituted or unsubstituted C ₁ -C ₁₀ alkyl, substituted or unsubstituted C ₁ -C ₁₀ heteroalkyl comprising at least one hetero atom, substituted or unsubstituted C ₂ -C ₁₀ alkenyl , substituted or unsubstituted C ₂ -C ₁₀ alkynyl, substituted or unsubstituted C ₃ -C ₂₀ cycloalkyl, substituted or unsubstituted C ₃ -C ₂₀ cycloalkenyl, substituted or unsubstituted C ₂ -C ₂₀ heterocycloalkyl, hydroxy, oxido (-O ^- ), substituted or unsubstituted C ₁ -C ₁₀ alkoxy, substituted or unsubstituted C ₃ -C ₂₀ cycloalkyloxy, substituted or unsubstituted C ₅ - C ₂₀ aryloxy, substituted or unsubstituted C ₂ -C ₂₀ heteroaryloxy, substituted or unsubstituted C ₅ -C ₂₀ aryl, substituted or unsubstituted C ₂ -C ₂₀ heteroaryl, substituted or unsubstituted C ₅ -C ₂₀ aralkyl, substituted or unsubstituted C ₁ -C ₁₀ alkylthio, substituted or unsubstituted C ₅ -C ₂₀ arylthio, substituted or unsubstituted C ₃ -C ₂₀ cycloalkylthio, substituted or unsubstituted substituted C ₂ -C ₂₀ heteroarylthio, substituted or unsubstituted acylamino, acyloxy, substituted or unsubstituted phosphino, carboxylate (-CO ₂ ^- ), trifluoromethylsulfonyl (-SO ₂ CF ₃ ), substituted or unsubstituted ammonium, nitro, sulfonic acid (-SO ₃ H), sulfonic acid salt, substituted sulfonyl, substituted sulfonic acid ester, substituted or unsubstituted sulfonamide, substituted thioketone, trihalomethyl (-CF ₃ , -CCl ₃ , -CBr ₃ , -CI ₃ ), haloformyl (-COCl, -COBr, -COI), formyl (-CHO), acyl, carboxyl, substituted Ester, substituted or unsubstituted aminocarbonyl, nitro, nitroso (-N=O), fluoro (-F), chloro (-Cl), bromo (-Br), iodo (-I) , substituted or unsubstituted germanium, substituted or unsubstituted boron, substituted or unsubstituted aluminum, substituted or unsubstituted silyl, substituted or unsubstituted amide, carbame phosphate, carboxylate, substituted or unsubstituted phosphine, substituted or unsubstituted phosphoric acid, phosphate, phosphonic acid, phosphonate, nitrile, hydrazine, acetal, ketal , polyalkylene oxide, and -L ₁ -R ₁₂ .

A reporter according to various embodiments of the present invention has at least one of R ₁ to R ₁₁ having a functional group represented by -L ₁ -R ₁₂ , and the reporter is a target biomolecule through a functional group represented by -L ₁ -R ₁₂ . It is possible to bind and label (eg, a nucleic acid).

In an embodiment, among the reporters represented by Formula 1 or Formula 2, Y may be CR ₉ R ₁₀ , and at least one of R ₉ and R ₁₀ may be -L ₁ -R ₁₂ .

L ₁ is a linker connecting the parent represented by Formula 1 or Formula 2 and R ₁₂ , and a single bond (ie, no element or functional group exists between the parent represented by Formula 1 or Formula 2 and R ₁₂ ) or saturated or unsaturated, substituted or unsubstituted, branched or unbranched alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl and heterocycloalkyl. For example, when L ₁ is not a single bond, L ₁ is (1) a substituted or unsubstituted C ₁ -C ₁₀ alkyl, a substituted or unsubstituted C ₁ -C ₁₀ hetero atom including at least one hetero atom alkyl, substituted or unsubstituted C ₂ -C ₁₀ alkenyl, substituted or unsubstituted C ₂ -C ₁₀ alkynyl, substituted or unsubstituted C ₃ -C ₂₀ cycloalkyl, substituted or unsubstituted C ₃ -C ₂₀ cycloalkenyl or substituted or unsubstituted C ₂ -C ₂₀ heterocycloalkyl, or (2) substituted or unsubstituted C ₁ -C ₁₀ alkyl, substituted or unsubstituted C ₁ containing at least one hetero atom -C ₁₀ heteroalkyl, substituted or unsubstituted C ₂ -C ₁₀ alkenyl, substituted or unsubstituted C ₂ -C ₁₀ alkynyl, substituted or unsubstituted C ₃ -C ₂₀ cycloalkyl, substituted or unsubstituted It may include at least one selected from C ₃ -C ₂₀ cycloalkenyl and substituted or unsubstituted C ₂ -C ₂₀ heterocycloalkyl.

Also, L ₁ is a linker comprising at least one carbon mentioned above, and any carbon in the linker may be a carbonyl carbon. The carbonyl may be at least one selected from an aldehyde, a ketone, a carboxylic acid, an ester, an amide, an enone, an acyl halide, an acid anhydride, and an imide.

In another case, L ₁ may have a structure in which adjacent linkers are interconnected by carbonyl carbons. In this case, L ₁ may include both a linker directly connected without a carbonyl carbon and a linker connected by a carbonyl carbon. For example, L ₁ may have a structure including an amide (aminocarbonyl) such as "-CH ₂ -CH ₂ -C(=O)-NH-CH ₂ -CH ₂ -CH=CH-CH ₂ -" have. In addition, L ₁ may have a structure including carbonylamino, such as "-CH ₂ -CH ₂ -NH-C(=O)-CH ₂ -CH ₂ -CH=CH-CH ₂ -".

R ₁₂ is a deoxyribonucleoside represented by the following formula (3).

[Formula 3]

here,

* represents the position at which the deoxyribonucleoside is bonded to L ₁ ,

B is a nucleobase. As a component of the deoxyribonucleoside represented by Formula 3, the nucleobase is a purine-based base such as adenine, guanine, hypoxanthine, xanthine, 7-methylguanine, and cytosine, thymine, uracil, 5,6-dihydrouracil, 5- It may be a pyrimidine-based base such as methylcytosine or 5-hydroxymethylcytosine.

Deoxyribonucleoside (thymidine) containing thymine as the nucleobase may be represented by the following formula (4).

[Formula 4]

In the deoxyribonucleoside represented by Formula 3 or Formula 4, R ₁₃ is selected from hydrogen, deuterium, P(OR ₁₅ )(N(R ₁₆ R ₁₇ ) and -L ₂ -R ₁₈ , and R ₁₄ is It may be an alcohol protecting group, hydroxy or P(OR ₁₅ )(N(R ₁₆ R ₁₇ ), or a support or nucleic acid to which a reporter represented by Formula 1 or Formula 2 is bound.

When R ₁₃ or R ₁₄ is P(OR ₁₅ )(N(R ₁₆ R ₁₇ ), R ₁₅ to R ₁₇ are each independently hydrogen, deuterium, substituted or unsubstituted C ₁ -C ₁₀ alkyl and at least one substituted or unsubstituted C ₁ -C ₁₀ heteroalkyl containing heteroatoms.

In addition, when any functional group of R ₁₅ to R ₁₇ is a substituted functional group, an arbitrary substituent other than hydrogen may be bonded to at least one arbitrary carbon of the functional group. The substituent is deuterium, substituted or unsubstituted C ₁ -C ₁₀ alkyl, substituted or unsubstituted C ₁ -C ₁₀ heteroalkyl comprising at least one hetero atom, substituted or unsubstituted C ₂ -C ₁₀ alkenyl , substituted or unsubstituted C ₂ -C ₁₀ alkynyl, substituted or unsubstituted C ₃ -C ₂₀ cycloalkyl, substituted or unsubstituted C ₃ -C ₂₀ cycloalkenyl, substituted or unsubstituted C ₂ -C ₂₀ heterocycloalkyl, hydroxy, oxido (-O ^- ), substituted or unsubstituted C ₁ -C ₁₀ alkoxy, substituted or unsubstituted C ₃ -C ₂₀ cycloalkyloxy, substituted or unsubstituted C ₅ - C ₂₀ aryloxy, substituted or unsubstituted C ₂ -C ₂₀ heteroaryloxy, substituted or unsubstituted C ₅ -C ₂₀ aryl, substituted or unsubstituted C ₂ -C ₂₀ heteroaryl, substituted or unsubstituted C ₅ -C ₂₀ aralkyl, substituted or unsubstituted C ₁ -C ₁₀ alkylthio, substituted or unsubstituted C ₅ -C ₂₀ arylthio, substituted or unsubstituted C ₃ -C ₂₀ cycloalkylthio, substituted or unsubstituted substituted C ₂ -C ₂₀ heteroarylthio, substituted or unsubstituted acylamino, acyloxy, substituted or unsubstituted phosphino, carboxylate (-CO ₂ ^- ), trifluoromethylsulfonyl (-SO ₂ CF ₃ ), substituted or unsubstituted ammonium, nitro, sulfonic acid (-SO ₃ H), sulfonic acid salt, substituted sulfonyl, substituted sulfonic acid ester, substituted or unsubstituted sulfonamide, substituted thioketone, trihalomethyl (-CF ₃ , -CCl ₃ , -CBr ₃ , -CI ₃ ), haloformyl (-COCl, -COBr, -COI), formyl (-CHO), acyl, carboxyl, substituted Ester, substituted or unsubstituted aminocarbonyl, nitro, nitroso (-N=O), fluoro (-F), chloro (-Cl), bromo (-Br), iodo (-I) , substituted or unsubstituted germanium, substituted or unsubstituted boron, substituted or unsubstituted aluminum, substituted or unsubstituted silyl, substituted or unsubstituted amide, carbame phosphate, carboxylate, substituted or unsubstituted phosphine, substituted or unsubstituted phosphoric acid, phosphate, phosphonic acid, phosphonate, nitrile, hydrazine, acetal, ketal , polyalkylene oxide, and -L ₁ -R ₁₂ .

When R ₁₃ is -L ₂ -R ₁₈ , L ₂ is a linker connecting O and R ₁₈ , a single bond (ie, no element or functional group exists between O and R ₁₈ ), or an internucleotide It may be an internucleotide phosphodiester bridge, saturated or unsaturated, substituted or unsubstituted, branched or unbranched alkyl or heteroalkyl.

For example, when L ₂ is not a single bond or an internucleotide phosphodiester bond, L ₂ is a substituted or unsubstituted C ₁ -C ₁₀ alkyl or a substituted or unsubstituted C containing at least one hetero atom. ₁ -C ₁₀ heteroalkyl.

In addition, L ₂ is a linker comprising at least one carbon mentioned above, and any carbon in the linker may be a carbonyl carbon. The carbonyl may be at least one selected from an aldehyde, a ketone, a carboxylic acid, an ester, an amide, an enone, an acyl halide, an acid anhydride, and an imide.

In another case, L ₂ may have a structure in which adjacent linkers are interconnected by carbonyl carbons. In this case, L ₂ may include both a linker directly connected without a carbonyl carbon and a linker connected by a carbonyl carbon. For example, L ₂ may have a structure including an amide (aminocarbonyl), such as "-CH ₂ -CH ₂ -C(=O)-NH-CH ₂ -CH ₂ -CH=CH-CH ₂ -" have. In addition, L ₂ may have a structure including carbonylamino, such as "-CH ₂ -CH ₂ -NH-C(=O)-CH ₂ -CH ₂ -CH=CH-CH ₂ -".

When L ₂ is an internucleotide phosphodiester bond, the deoxyribonucleosides represented by Chemical Formulas 3 and 4 may be represented by the following Chemical Formulas 3-1 and 4-1, respectively.

[Formula 3-1]

[Formula 4-1]

R ₁₈ may be hydroxy or P(OR ₁₅ )(N(R ₁₆ R ₁₇ ), or a support or nucleic acid to which a reporter represented by Formula 1 or Formula 2 is bound.

That is, when R ₁₃ is -L ₂ -R ₁₈ , the reporter represented by Formula 1 or Formula 2 may be coupled to a support or nucleic acid (RNA or DNA) via L ₂ .

The support is glass, cellulose, nylon, acrylamide gel, dextran, polystyrene, alginate, collagen, peptide, fibrin, hyaluronic acid, agarose, polyhydroxyethyl methacrylate, 　 polyvinyl alcohol, polyethylene glycol, 　 polyethylene Oxide, polyethylene glycol diacrylate, gelatin, matrigel (matrigel), polylactic acid, carboxymethyl cellulose, dextran, chitosan, may be made of at least one selected from latex and sepharose, may be in the form of beads or membranes have.

On the other hand, when R ₁₄ is an alcohol protecting group, the alcohol protecting group is acetal, acetyl, benzoyl, benzyl, β-methoxyethoxymethyl, dimethoxytrityl, methoxymethyl, methoxytrityl, p-methoxybenzyl, p-methoxyphenyl, methylthiomethyl, trityl, tetrahydropyranyl, tetrahydrofuran, trimethylsilyl, tert-butyldimethylsilyl, tri-iso-propylsiloxymethyl and tri It may be at least one selected from -iso-propylsilyl.

R ₁ to R ₁₁ may each independently exist as functional groups defined above, but in some embodiments, at least one of R ₁ to R ₁₁ is bonded to an adjacent substituent to a substituted or unsubstituted ring (eg, For example, a 4-membered ring, a 5-membered ring, a 6-membered ring or a ring consisting of more atoms, a fused ring in which a plurality of rings are fused, etc.) may be formed. The ring may also be an aliphatic or aromatic ring.

When at least one of R ₁ to R ₁₁ is bonded to an adjacent substituent to form a substituted or unsubstituted ring, at least one of R ₁ to R ₁₁ is adjacent to each other via C, N, O, S, Se or Si It may be bonded to each other with a substituent, or may be directly bonded to an adjacent substituent by a single bond.

In an embodiment, the reporter is represented by Formula 1, and when Z ₁ is NR ₅ R ₆ , R ₅ or R ₆ may be combined with R ₃ or R ₄ to form a substituted or unsubstituted ring. have.

The formation of a substituted or unsubstituted ring by R ₅ and R ₆ is independent of each other, and R ₅ and R ₃ (or R ₄ ) in one compound combine with each other to form a ring and R ₆ and R ₄ (or R ₃ ) A ring formed by bonding to each other may be present at the same time.

In another embodiment, the reporter is represented by Formula 1, and when Z ₁ is OR ₇ , R ₇ may be combined with R ₃ or R ₄ to form a substituted or unsubstituted ring.

In another embodiment, the reporter is represented by Formula 2, and when Z ₂ is NR ₈ , R ₈ may be combined with R ₃ or R ₄ to form a substituted or unsubstituted ring.

When at least one of R ₁ to R ₁₁ is bonded to an adjacent substituent to form a substituted ring, an arbitrary substituent other than hydrogen may be bonded to any carbon of at least one of the rings. The substituent is deuterium, substituted or unsubstituted C ₁ -C ₁₀ alkyl, substituted or unsubstituted C ₁ -C ₁₀ heteroalkyl comprising at least one hetero atom, substituted or unsubstituted C ₂ -C ₁₀ alkenyl , substituted or unsubstituted C ₂ -C ₁₀ alkynyl, substituted or unsubstituted C ₃ -C ₂₀ cycloalkyl, substituted or unsubstituted C ₃ -C ₂₀ cycloalkenyl, substituted or unsubstituted C ₂ -C ₂₀ heterocycloalkyl, hydroxy, oxido (-O ^- ), substituted or unsubstituted C ₁ -C ₁₀ alkoxy, substituted or unsubstituted C ₃ -C ₂₀ cycloalkyloxy, substituted or unsubstituted C ₅ - C ₂₀ aryloxy, substituted or unsubstituted C ₂ -C ₂₀ heteroaryloxy, substituted or unsubstituted C ₅ -C ₂₀ aryl, substituted or unsubstituted C ₂ -C ₂₀ heteroaryl, substituted or unsubstituted C ₅ -C ₂₀ aralkyl, substituted or unsubstituted C ₁ -C ₁₀ alkylthio, substituted or unsubstituted C ₅ -C ₂₀ arylthio, substituted or unsubstituted C ₃ -C ₂₀ cycloalkylthio, substituted or unsubstituted substituted C ₂ -C ₂₀ heteroarylthio, substituted or unsubstituted acylamino, acyloxy, substituted or unsubstituted phosphino, carboxylate (-CO ₂ ^- ), trifluoromethylsulfonyl (-SO ₂ CF ₃ ), substituted or unsubstituted ammonium, nitro, sulfonic acid (-SO ₃ H), sulfonic acid salt, substituted sulfonyl, substituted sulfonic acid ester, substituted or unsubstituted sulfonamide, substituted thioketone, trihalomethyl (-CF ₃ , -CCl ₃ , -CBr ₃ , -CI ₃ ), haloformyl (-COCl, -COBr, -COI), formyl (-CHO), acyl, carboxyl, substituted Ester, substituted or unsubstituted aminocarbonyl, nitro, nitroso (-N=O), fluoro (-F), chloro (-Cl), bromo (-Br), iodo (-I) , substituted or unsubstituted germanium, substituted or unsubstituted boron, substituted or unsubstituted aluminum, substituted or unsubstituted silyl, substituted or unsubstituted amide, carbame phosphate, carboxylate, substituted or unsubstituted phosphine, substituted or unsubstituted phosphoric acid, phosphate, phosphonic acid, phosphonate, nitrile, hydrazine, acetal, ketal , polyalkylene oxide, and -L ₁ -R ₁₂ .

In addition, according to another aspect of the present invention, there is provided a reporter for labeling a nucleic acid represented by the following formula (5).

[Formula 5]

here,

R ₂₁ and R ₂₂ are each independently hydrogen, deuterium, substituted or unsubstituted C ₁ -C ₁₀ alkyl, substituted or unsubstituted C ₁ -C ₁₀ heteroalkyl containing at least one hetero atom, substituted or unsubstituted substituted C ₂ -C ₁₀ alkenyl, substituted or unsubstituted C ₂ -C ₁₀ alkynyl, substituted or unsubstituted C ₃ -C ₂₀ cycloalkyl, substituted or unsubstituted C ₃ -C ₂₀ cycloalkenyl, substituted or unsubstituted C ₂ -C ₂₀ heterocycloalkyl, substituted or unsubstituted C ₅ -C ₂₀ aryl and substituted or unsubstituted C ₂ -C ₂₀ heteroaryl,

R ₂₃ to R ₃₀ are each independently hydrogen, deuterium, substituted or unsubstituted C ₁ -C ₁₀ alkyl, substituted or unsubstituted C ₁ -C ₁₀ heteroalkyl containing at least one hetero atom, substituted or unsubstituted substituted C ₂ -C ₁₀ alkenyl, substituted or unsubstituted C ₂ -C ₁₀ alkynyl, substituted or unsubstituted C ₃ -C ₂₀ cycloalkyl, substituted or unsubstituted C ₃ -C ₂₀ cycloalkenyl, substituted or unsubstituted C ₂ -C ₂₀ heterocycloalkyl, hydroxy, oxido (—O ^— ), substituted or unsubstituted C ₁ -C ₁₀ alkoxy, substituted or unsubstituted C ₃ -C ₂₀ cycloalkyloxy, substituted or unsubstituted C ₅ -C ₂₀ aryloxy, substituted or unsubstituted C ₂ -C ₂₀ heteroaryloxy, substituted or unsubstituted C ₅ -C ₂₀ aryl, substituted or unsubstituted C ₂ -C ₂₀ hetero aryl, substituted or unsubstituted C ₅ -C ₂₀ aralkyl, substituted or unsubstituted C ₁ -C ₁₀ alkylthio, substituted or unsubstituted C ₅ -C ₂₀ arylthio, substituted or unsubstituted C ₃ -C ₂₀ cycloalkylthio, substituted or unsubstituted C ₂ -C ₂₀ heteroarylthio, substituted or unsubstituted acylamino, acyloxy, substituted or unsubstituted phosphino, carboxylate (-CO ₂ ^- ), trifluoro Methylsulfonyl (-SO ₂ CF ₃ ), substituted or unsubstituted ammonium, nitro, sulfonic acid (-SO ₃ H), sulfonate, substituted sulfonyl, substituted sulfonic acid ester, substituted or unsubstituted sulfone Amide, substituted thioketone, trihalomethyl (-CF ₃ , -CCl ₃ , -CBr ₃ , -CI ₃ ), haloformyl (-COCl, -COBr, -COI), formyl (-CHO) , acyl, carboxyl, substituted ester, substituted or unsubstituted aminocarbonyl, nitro, nitroso (-N=O), fluoro (-F), chloro (-Cl), bromo (-Br ), iodo (-I), substituted or unsubstituted germanium, substituted or unsubstituted boron, substituted or unsubstituted aluminum, substituted or unsubstituted silyl, substituted or unsubstituted substituted or unsubstituted amides, carbamates, carboxylates, substituted or unsubstituted phosphines, substituted or unsubstituted phosphoric acids, phosphates, phosphonic acids, phosphonates, It is a functional group selected from nitrile, hydrazine, acetal, ketal, polyalkylene oxide and -L ₁ -R ₁₂ , or two adjacent functional groups may combine with each other to form a ring.

In addition, R ₃₁ to R ₃₄ are each independently hydrogen, deuterium, substituted or unsubstituted C ₁ -C ₁₀ alkyl, substituted or unsubstituted C ₁ -C ₁₀ heteroalkyl containing at least one hetero atom, and -L It is selected from ₁ -R ₁₂ , and at least one of R ₃₁ to R ₃₄ may be -L ₁ -R ₁₂ . The definition for -L ₁ -R ₁₂ is the same as described above.

The reporter according to various embodiments of the present invention includes at least one of R ₃₁ to R ₃₄ having a functional group represented by -L ₁ -R ₁₂ , and the reporter is a target biomolecule through a functional group represented by -L ₁ -R ₁₂ . It is possible to bind and label (eg, a nucleic acid).

Y is CR ₃₅ and each R ₃₅ is hydrogen, deuterium, substituted or unsubstituted C ₁ -C ₁₀ alkyl, substituted or unsubstituted C ₁ -C ₁₀ heteroalkyl comprising at least one heteroatom, fluoro (-F), chloro (-Cl), bromo (-Br), iodo (-I), n is an integer of 1 to 4.

In addition, R ₃₅ may be each independently represented by the functional groups defined above, but in some embodiments, R ₃₅ is _a substituted or unsubstituted ring (eg, 4 an atomic ring, a 5-membered ring, a 6-membered ring or a ring composed of more atoms, a fused ring in which a plurality of rings are fused, etc.) may be formed. The ring may also be an aliphatic or aromatic ring.

When R ₃₅ is combined with R ₃₅ of neighboring Y to form a substituted ring, an arbitrary substituent other than hydrogen may be bonded to any carbon of at least one of the rings. The substituent is deuterium, substituted or unsubstituted C ₁ -C ₁₀ alkyl, substituted or unsubstituted C ₁ -C ₁₀ heteroalkyl comprising at least one hetero atom, substituted or unsubstituted C ₂ -C ₁₀ alkenyl , substituted or unsubstituted C ₂ -C ₁₀ alkynyl, substituted or unsubstituted C ₃ -C ₂₀ cycloalkyl, substituted or unsubstituted C ₃ -C ₂₀ cycloalkenyl, substituted or unsubstituted C ₂ -C ₂₀ heterocycloalkyl, hydroxy, oxido (-O ^- ), substituted or unsubstituted C ₁ -C ₁₀ alkoxy, substituted or unsubstituted C ₃ -C ₂₀ cycloalkyloxy, substituted or unsubstituted C ₅ - C ₂₀ aryloxy, substituted or unsubstituted C ₂ -C ₂₀ heteroaryloxy, substituted or unsubstituted C ₅ -C ₂₀ aryl, substituted or unsubstituted C ₂ -C ₂₀ heteroaryl, substituted or unsubstituted C ₅ -C ₂₀ aralkyl, substituted or unsubstituted C ₁ -C ₁₀ alkylthio, substituted or unsubstituted C ₅ -C ₂₀ arylthio, substituted or unsubstituted C ₃ -C ₂₀ cycloalkylthio, substituted or unsubstituted substituted C ₂ -C ₂₀ heteroarylthio, substituted or unsubstituted acylamino, acyloxy, substituted or unsubstituted phosphino, carboxylate (-CO ₂ ^- ), trifluoromethylsulfonyl (-SO ₂ CF ₃ ), substituted or unsubstituted ammonium, nitro, sulfonic acid (-SO ₃ H), sulfonic acid salt, substituted sulfonyl, substituted sulfonic acid ester, substituted or unsubstituted sulfonamide, substituted thioketone, trihalomethyl (-CF ₃ , -CCl ₃ , -CBr ₃ , -CI ₃ ), haloformyl (-COCl, -COBr, -COI), formyl (-CHO), acyl, carboxyl, substituted Ester, substituted or unsubstituted aminocarbonyl, nitro, nitroso (-N=O), fluoro (-F), chloro (-Cl), bromo (-Br), iodo (-I) , substituted or unsubstituted germanium, substituted or unsubstituted boron, substituted or unsubstituted aluminum, substituted or unsubstituted silyl, substituted or unsubstituted amide, carbame phosphate, carboxylate, substituted or unsubstituted phosphine, substituted or unsubstituted phosphoric acid, phosphate, phosphonic acid, phosphonate, nitrile, hydrazine, acetal, ketal , polyalkylene oxide, and -L ₁ -R ₁₂ .

In addition, when any of the functional groups of R ₂₁ to R ₃₅ is a substituted functional group, an arbitrary substituent other than hydrogen may be bonded to at least one arbitrary carbon of the functional group. The substituent is deuterium, substituted or unsubstituted C ₁ -C ₁₀ alkyl, substituted or unsubstituted C ₁ -C ₁₀ heteroalkyl comprising at least one hetero atom, substituted or unsubstituted C ₂ -C ₁₀ alkenyl , substituted or unsubstituted C ₂ -C ₁₀ alkynyl, substituted or unsubstituted C ₃ -C ₂₀ cycloalkyl, substituted or unsubstituted C ₃ -C ₂₀ cycloalkenyl, substituted or unsubstituted C ₂ -C ₂₀ heterocycloalkyl, hydroxy, oxido (-O ^- ), substituted or unsubstituted C ₁ -C ₁₀ alkoxy, substituted or unsubstituted C ₃ -C ₂₀ cycloalkyloxy, substituted or unsubstituted C ₅ - C ₂₀ aryloxy, substituted or unsubstituted C ₂ -C ₂₀ heteroaryloxy, substituted or unsubstituted C ₅ -C ₂₀ aryl, substituted or unsubstituted C ₂ -C ₂₀ heteroaryl, substituted or unsubstituted C ₅ -C ₂₀ aralkyl, substituted or unsubstituted C ₁ -C ₁₀ alkylthio, substituted or unsubstituted C ₅ -C ₂₀ arylthio, substituted or unsubstituted C ₃ -C ₂₀ cycloalkylthio, substituted or unsubstituted substituted C ₂ -C ₂₀ heteroarylthio, substituted or unsubstituted acylamino, acyloxy, substituted or unsubstituted phosphino, carboxylate (-CO ₂ ^- ), trifluoromethylsulfonyl (-SO ₂ CF ₃ ), substituted or unsubstituted ammonium, nitro, sulfonic acid (-SO ₃ H), sulfonic acid salt, substituted sulfonyl, substituted sulfonic acid ester, substituted or unsubstituted sulfonamide, substituted thioketone, trihalomethyl (-CF ₃ , -CCl ₃ , -CBr ₃ , -CI ₃ ), haloformyl (-COCl, -COBr, -COI), formyl (-CHO), acyl, carboxyl, substituted Ester, substituted or unsubstituted aminocarbonyl, nitro, nitroso (-N=O), fluoro (-F), chloro (-Cl), bromo (-Br), iodo (-I) , substituted or unsubstituted germanium, substituted or unsubstituted boron, substituted or unsubstituted aluminum, substituted or unsubstituted silyl, substituted or unsubstituted amide, carbame phosphate, carboxylate, substituted or unsubstituted phosphine, substituted or unsubstituted phosphoric acid, phosphate, phosphonic acid, phosphonate, nitrile, hydrazine, acetal, ketal , polyalkylene oxide, and -L ₁ -R ₁₂ .

When the functional group as defined herein is alkenyl or alkynyl, the sp ² -hybrid carbon of alkenyl or sp-hybrid carbon of alkynyl is bonded directly or sp ² -hybrid carbon of alkenyl or sp-hybridized carbon of alkynyl sp ³ of the alkyl bonded to - may be indirectly bonded by a hybrid carbon.

A C _a -C _b functional group herein refers to a functional group having a to b carbon atoms. For example, C _a -C _b alkyl refers to saturated aliphatic groups having a to b carbon atoms, including straight chain alkyl and branched alkyl and the like. A straight chain or branched alkyl may have up to 40 in its main chain (eg, C ₁ -C ₁₀ straight chain, C ₃ -C ₁₀ grinding).

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

Also, as used herein, alkoxy refers to both an -O-(alkyl) group and an -O-(unsubstituted cycloalkyl) group, which is a straight chain or branched hydrocarbon having at least one ether group and 1 to 10 carbon atoms.

Specifically, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, n-hexoxy, 1,2-dimethylbutoxy, cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy, and the like.

In addition, as used herein, halogen means fluoro (-F), chloro (-Cl), bromo (-Br) or iodo (-I), and haloalkyl means alkyl substituted with the aforementioned halogen. For example, halomethyl means methyl (-CH ₂ X, -CHX ₂ or -CX ₃ ) in which at least one of the hydrogens of the methyl has been replaced by a halogen.

As used herein, aralkyl is a functional group in which aryl is substituted at the carbon of alkyl, and is a generic term for -(CH ₂ ) _n Ar. Examples of aralkyl include benzyl (—CH ₂ C ₆ H ₅ ) or phenethyl (—CH ₂ CH ₂ C ₆ H ₅ ) and the like.

As used herein, unless otherwise defined, aryl means an unsaturated aromatic ring comprising a single ring or multiple rings (preferably 1 to 4 rings) linked to each other by junctions or covalent bonds. Non-limiting examples of aryl include phenyl, biphenyl, o-terphenyl, m-terphenyl, p-terphenyl, 1-naphthyl, 2-naphthyl, 1-anthryl, 2- Anthryl, 9-anthryl, 1-phenanthrenyl, 2-phenanthrenyl, 3-phenanthrenyl, 4-phenanthrenyl, 9-phenanthrenyl, 1-pyrenyl, 2- pyrenyl and 4-pyrenyl;

Heteroaryl as used herein refers to a functional group in which one or more carbon atoms in the aryl as defined above are replaced by a non-carbon atom such as nitrogen, oxygen or sulfur. Non-limiting examples of heteroaryl, furyl (furyl), tetrahydrofuryl, pyrrolyl (phrrolyl), pyrrolidinyl (pyrrolidinyl), thienyl (thienyl), tetrahydrothienyl, oxazolyl (oxazolyl), isoxa Isoxazolyl, triazolyl, thiazolyl, isothiazolyl, pyrazolyl, pyrazolidinyl, oxadiazolyl, thiadiazolyl , imidazolyl, imidazolinyl, pyridyl, pyridaziyl, triazinyl, piperidinyl, morpholinyl, thio Morpholinyl, pyrazinyl, piperainyl, pyrimidinyl, naphthyridinyl, benzofuranyl, benzothienyl, indolyl, indolinyl , indolizinyl, indazolyl, quinolinyl, quinolinyl, isoquinolinyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxarinyl, pteridinyl ( pteridinyl), quinuclidinyl, carbazoyl, acridinyl, phenazinyl, phenothizinyl, phenoxazinyl, purinyl, benzimidazolyl, benzothiazolyl, etc. and their conjugated analogs there are

As used herein, a hydrocarbon ring (cycloalkyl) or a hydrocarbon ring including a hetero atom (heterocycloalkyl) 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- Morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl, 2 - Piperazinil, etc.

In addition, the hydrocarbon ring or the hydrocarbon ring including a hetero atom may have a form in which a hydrocarbon ring, a hydrocarbon ring including a hetero atom, aryl or heteroaryl is fused or covalently linked thereto.

Here, the polyalkylene oxide is a water-soluble polymer functional group, polyethylene glycol (PEG), polypropylene glycol (PPG), polyethylene glycol-polypropylene glycol (PEG-PPG) copolymer and N-substituted methacrylamide-containing polymers and copolymers.

The polyalkylene oxide may be additionally substituted as needed within the extent that 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 is hydroxy, alkyl ether (methyl ether, ethyl ether, propyl ether, etc.), carboxymethyl ether, carboxyethyl ether, benzyl ether, dibenzylmethylene ether, or It may be substituted with dimethylamine. In one embodiment, the polyalkylene oxide may be a polyalkylene oxide (mPEG) terminated with a methyl ether, wherein mPEG is represented by the formula -(CH ₂ CH ₂ O) _n CH ₃ , ethylene glycol The size of mPEG may vary depending on the size of n corresponding to the number of call repeat units.

In addition, the reporter represented by Formula 1, Formula 2, or Formula 5 may further include a counter ion. The counter ion is an organic or inorganic anion and may be appropriately selected in consideration of the solubility and stability of the reporter.

As an example of the counter ion of the reporter according to an embodiment of the present invention, phosphoric acid hexafluoride ion, halogen ion, phosphoric acid ion, perchlorate ion, periodate ion, antimony hexafluoride ion, tartrate hexafluoride ion Inorganic acid anions such as ions, fluoroboric acid ions and tetrafluoride ions, thiocyanate ions, benzenesulfonic acid ions, naphthalenesulfonic acid ions, p-toluenesulfonic acid ions, alkylsulfonic acid ions, benzenecarboxylate ions, alkylcarboxes and organic acid ions such as acid ions, trihaloalkylcarboxylic acid ions, alkylsulfonic acid ions, trihaloalkylsulfonic acid ions, and nicotinic acid ions. In addition, metal compound ions such as bisphenylditol, thiobisphenol chelate and bisdiol-α-dickentone, metal ions such as sodium and potassium, and the like and quaternary ammonium salts can also be selected as counter ions.

In addition, specific examples of the reporter represented by Formula 1, Formula 2, or Formula 5 are as follows. However, the following exemplary compounds are intended to help the understanding of the reporter as defined herein, and not to limit the scope of the reporter as defined herein.

[Compound 1]

[Compound 2]

[Compound 3]

[Compound 4]

[Compound 5]

[Compound 6]

[Compound 7]

[Compound 8]

[Compound 9]

[Compound 10]

[Compound 11]

[Compound 12]

[Compound 13]

[Compound 14]

[Compound 15]

[Compound 16]

[Compound 17]

[Compound 18]

[Compound 19]

[Compound 20]

[Compound 21]

[Compound 22]

[Compound 23]

[Compound 24]

[Compound 25]

[Compound 26]

[Compound 27]

[Compound 28]

[Compound 29]

[Compound 30]

[Compound 31]

[Compound 32]

[Compound 33]

[Compound 34]

[Compound 35]

[Compound 36]

[Compound 37]

[Compound 38]

[Compound 39]

[Compound 40]

[Compound 41]

[Compound 42]

[Compound 43]

[Compound 44]

[Compound 45]

[Compound 46]

The biomolecule that is the target of the reporter represented by Formula 1, Formula 2 or Formula 5 disclosed herein is at least one selected from antibodies, lipids, proteins, peptides, carbohydrates, and nucleic acids (including DNA, RNA or nucleotides), preferably It may be a nucleic acid (including DNA, RNA or nucleotides).

Specific examples of lipids include fatty acids, phospholipids, lipopolysaccharides, and the like, and specific examples of carbohydrates include monosaccharides, disaccharides, and polysaccharides (eg, dextran).

In this case, the biomolecule is a functional group for reacting with any functional group of the reporter represented by Formula 1, Formula 2, or Formula 5 or a reactive group bound to the reporter represented by Formula 1, Formula 2, or Formula 5, amino, sulfhigh It may include at least one selected from sulphydryl, carbonyl, hydroxyl, carboxyl, phosphate and thiophosphate, or may have a derivative form thereof.

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

In addition, in addition to biomolecules, the reporter represented by Formula 1, Formula 2 or Formula 5 is a drug containing at least one selected from amino, sulfhydryl, carbonyl, hydroxyl, carboxyl, phosphate and thiophosphate; It can be used to label hormones (including receptor ligands), receptors, enzymes or enzyme substrates, cells, cell membranes, toxins, microorganisms or nano-biomaterials (polystyrene microspheres, etc.).

Oligonucleotides containing novel reporters, compositions for detecting nucleic acids, support for detecting nucleic acids

According to another aspect of the present invention, there is provided an oligonucleotide comprising at least one selected from a reporter represented by Formula 1, Formula 2, or Formula 5.

Oligonucleotide refers to a polymer of 1 to hundreds of nucleotides, and includes all of DNA, RNA, or PNA. In addition, analogs thereof, for example, those in which chemical modifications are made to the nucleotides, or those in which sugars are attached, include all those that can be easily modified by those skilled in the art, and all single-stranded or double-stranded ones means to include

The oligonucleotide preferably comprises a probe. The probe is more preferably a probe capable of complementary binding to a target nucleic acid, but is not limited thereto. Here, the probe may be selected from a nucleic acid, a peptide, a saccharide, an oligonucleotide, a protein, an antibody, or a combination thereof, but is not limited thereto.

In one embodiment, the oligonucleotide may comprise a quencher. For example, a reporter represented by Formula 1, Formula 2, or Formula 5 may be labeled at the 5' end of the oligonucleotide, and a quencher may be labeled at the 3' end of the oligonucleotide. A probe capable of complementary binding to a target nucleic acid may be positioned between the 5' end and the 3' end. In addition, the reporter represented by Formula 1, Formula 2, or Formula 5 may be labeled at an internal site other than the 5' end or 3' end of the oligonucleotide.

The maximum absorbance of the quencher usable herein may be 620 to 700 nm, preferably 660 to 680, and the absorbance range of the quencher may be 530 to 730 nm. In addition, the maximum absorbance and absorbance range of the quencher may be appropriately selected in consideration of the fluorescence properties of the reporter defined herein.

It is important that the probe is designed so that the reporter can be sufficiently quenched by the quencher while minimizing signal crosstalk. Accordingly, when designing a probe, it is confirmed that the reporter and the quencher labeled at the 5' end and the 3' end of the probe are compatible, respectively, depending on the type of the target biomolecule (eg, nucleic acid). something is needed

As the quencher, various known or commercially available quenchers (eg, BHQ0, BHQ1, BHQ2, BHQ3, BBQ650, DABCYL, TAMRA, MGBEclipse, Atto540Q, Atto575Q, Atto612Q, QSY7, QSY21, etc.) may be used. In addition, the quencher described in Korean Patent Application Laid-Open No. 10-2020-0067733 may be used as the quencher. A representative example of the quencher described in Korean Patent Application Laid-Open No. 10-2020-0067733 is as follows.

[Quorer 1]

[Quorer 2]

[Quencher 3]

[Quorer 4]

[Quorer 5]

[Quencher 6]

[Quorer 7]

[Quorer 8]

[Quorer 9]

In addition, the oligonucleotide according to the present invention may further include a minor groove binder (MGB) in order to improve binding to the nucleic acid.

The minor groove binder is a crescent-shaped probe capable of selectively non-covalently binding to a minor groove (eg, a shallow furrow in a DNA helix) contained in a nucleic acid such as DNA.

Such oligonucleotides can be used in various ways in chemical and biological fields. In particular, it may be usefully used in a real-time polymerase chain reaction or microassay, but is not limited thereto.

In addition, according to another aspect of the present invention, there is provided a composition for detecting a nucleic acid comprising the oligonucleotide.

The composition for detecting a nucleic acid according to an embodiment of the present invention includes an oligonucleotide including a reporter represented by Formula 1, Formula 2 or Formula 5, a minor groove binder, and a quencher at the same time, together with an enzyme, a solvent for reaction with a target biomolecule (buffers, etc.) and other reagents may be further included.

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

In addition, according to another aspect of the present invention, there is provided a support for detecting nucleic acids comprising a reporter represented by Formula 1, Formula 2 or Formula 5, a support, and a linker connecting the reporter and the support.

Accordingly, biomolecules in the sample can be immobilized on the support matrix through interaction with the reporter immobilized on the support.

The support matrix is glass, cellulose, nylon, acrylamide gel, dextran, polystyrene, alginate, collagen, peptide, fibrin, hyaluronic acid, agarose, polyhydroxyethyl methacrylate, polyvinyl alcohol, polyethylene glycol, It may be made of at least one selected from polyethylene oxide, polyethylene glycol diacrylate, gelatin, matrigel, polylactic acid, carboxymethyl cellulose, dextran, chitosan, latex and sepharose, and may be in the form of beads or membranes can

Glass, cellulose, nylon, acrylamide gel, dextran, polystyrene or resin may be used as the support, but is not necessarily limited thereto. Preferably, CPG (controlled pore glass) or polystyrene may be used as the support, and more preferably, CPG may be used.

Here, the linker is a part connecting the reporter and the support, and any material capable of connecting the reporter and the support may be used as the linker intended herein.

For example, the linker may be a substituted or unsubstituted C ₁ -C ₃₀ alkyl, a substituted or unsubstituted C ₂ -C ₃₀ heteroalkyl comprising at least one heteroatom, a substituted or unsubstituted C ₆ -C ₃₀ aryl and substituted or unsubstituted C ₃ -C ₃₀ heteroaryl, and the aforementioned functional groups may include at least one aminated carboxy group (or amide group).

Such a linker only connects the reporter and the support, but does not affect other reactions or fluorescence and quenching actions of the reporter or fluorophore.

Nucleic acid detection method

According to an embodiment of the present invention, a method for labeling a target nucleic acid by reacting a probe labeled with a reporter or a probe labeled with a reporter and a quencher can be implemented. In addition, a method for labeling a biomolecule using a target-specific interaction may be implemented by introducing an appropriate reactive group to the reporter according to the type of the target biomolecule. In addition, a method for identifying a biomolecule labeled with a reporter through electrophoresis may be implemented.

DNA microarray method

In the DNA microarray method, a target nucleic acid to be labeled is labeled with a dye, while a single-stranded probe nucleic acid having a nucleotide sequence complementary to the target nucleic acid is prepared, and the single-stranded target nucleic acid and the probe nucleic acid are denatured into a substrate. After hybridization above, the fluorescence of the target nucleic acid is measured.

As the probe nucleic acid to be immobilized on the substrate in this labeling method, in the case of examining gene expression, a library of cDNA such as cDNA, a library of genomes, or all genomes as a template, amplified by PCR, and prepared Can be used.

In addition, when investigating gene mutations, etc., various oligonucleotides corresponding to mutations can be synthesized based on a known sequence serving as a standard.

An appropriate method for immobilizing the probe nucleic acid on the substrate can be selected according to the type of the nucleic acid or the type of the substrate. For example, a method of electrostatic bonding to a substrate surface-treated with cations such as polylysine using the charge of DNA may be used.

A target nucleic acid denatured into a single chain is immobilized on a substrate and hybridized with an oligonucleotide. Here, the 5' end of the oligonucleotide is labeled with a fluorophore, and the 3' end of the oligonucleotide is labeled with at least one selected from reporters represented by Formula 1, Formula 2, or Formula 5. A probe capable of complementary binding to a target nucleic acid may be positioned between the 5' end and the 3' end.

Hybridization is preferably performed at room temperature to 70° C., and in the range of 2 to 48 hours. By hybridization, a target nucleic acid having a nucleotide sequence complementary to the probe nucleic acid is selectively combined with the probe nucleic acid. Thereafter, the substrate is cleaned and dried at room temperature.

At this time, the oligonucleotide is hybridized to the target nucleic acid by the probe, but the fluorophore at the 5' end remains quenched by the reporter at the 3' end.

Then, the oligonucleotide hybridized to the target nucleic acid is extended by a polymerase, the oligonucleotide is separated and degraded from the target nucleic acid by the exonuclease activity of the polymerase, and the fluorophore at the 5' end and the 3' end of the oligonucleotide of the reporters are dissociated from each other, thereby allowing the fluorophore to fluoresce.

At this time, it is possible to measure the amount of amplification of the target nucleic acid by measuring the generated fluorescence intensity.

PCR method

In the PCR method, a probe complementary to the nucleotide sequence of the target nucleic acid to be labeled is labeled with a reporter, and the target nucleic acid and the probe are reacted before or after amplification of the target nucleic acid to measure the fluorescence of the target nucleic acid.

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

When an enzyme synthesizes, a synthesis reaction is performed using nucleotides (dNTP, NTP) as raw materials.

At this time, if the nucleotide having a reporter is mixed with the normal nucleotides (dNTP, NTP) in an arbitrary ratio, a nucleic acid into which the dye is introduced in the ratio can be synthesized.

In addition, after introducing nucleotides having an amino group in an arbitrary ratio by PCR, a reporter can be bound to synthesize a nucleic acid into which the reporter is introduced.

When the enzyme is synthesizing, the synthesis reaction takes place using nucleotides as raw materials. In the case where the 3' OH of the nucleotide is changed to H at this time, the nucleic acid extension reaction does not occur anymore and the reaction is terminated at that point. do.

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

When a nucleic acid is synthesized by mixing a terminator with an ordinary nucleotide, the terminator is introduced with a certain probability and the reaction is terminated, so that nucleic acids of various lengths are synthesized.

When this is separated by size by gel electrophoresis, DNA is lined up in each order of length. Here, if each type of base of the terminator is labeled with a different reporter, a tendency depending on each base is observed at the end point (3' end) of the synthesis reaction. By reading the fluorescence information from the reporter labeled with the terminator, Base sequence information of the target nucleic acid can be obtained.

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

Moreover, PNA (peptide nucleic acid) can also be used as a probe. PNA is a nucleic acid in which the pentose and phosphate backbones, which are the basic backbone structures of nucleic acids, are replaced with polyamide backbones based on glycine. strongly coupled Through this, it can be used as a reagent for telomere research by applying it to telomere (telomere) PNA probes as well as conventional DNA analysis methods such as ISH (in-situ hybridization) method.

In the label, for example, double-stranded DNA is hybridized by contacting it with a reporter-labeled PNA having a nucleotide sequence complementary to all or part of the nucleotide sequence of the DNA, and heating the mixture to generate single-stranded DNA, the mixture can be carried out by slowly cooling to room temperature to prepare a PNA-DNA complex, and measuring its fluorescence.

In the above example, a method for measuring the fluorescence of a product by amplifying a target nucleic acid by PCR has been described. Needs to be.

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

For example, DNA labeled with donors and acceptors can be used. As a specific labeling method, the molecular beacon method, TaqMan-PCR method, cycling probe method, etc. which confirm the presence of a nucleic acid of a specific sequence are mentioned.

Other cover methods

In addition, the reporter of the present invention can be used for a method for labeling a target using specific binding.

That is, in the subject containing the target or the label of the subject modified by the modifying material, one of the binding material that specifically binds to the subject or the binding material that specifically binds to the modifying material is used as a reporter It can be labeled and the fluorescence from the labeled binding material can be measured.

Here, the antigen-antibody, hapten-anti-hapten antibody, biotin-avidin, Tag antigen, Tag antibody, lectin-glycoprotein or hormone-receptor may be used for the combination of the subject or the modifying material and the binding material.

Specifically, a specific antigen can be labeled through antigen-specific interaction of the antibody by reacting a binding material such as a reporter-labeled antibody with the antigen present in the substrate, solution, beads, or antibody.

Antigens include proteins, polysaccharides, nucleic acids, and peptides. In addition to antigens, haptens such as low molecular weight molecules such as FITC and dinitrophenyl groups can also be used. In this case, the combination of antigen (or hapten) and antibody includes GFP and anti-GFP antibody, FITC and anti-FITC antibody, and the like.

The labeled antigens can be used in various measurement methods such as immunostaining, ELISA, Western blotting or flow cytometry.

In addition, by using the reporter of the present invention, it is also possible to observe the intracellular signal transduction (signaling) phenomenon. Various enzymes and the like are involved in the internal signal transduction or the response of the cell accordingly. In a typical signal transduction phenomenon, it is known that a special protein kinase is activated, and thus protein phosphorylation is induced to initiate signal transduction.

Binding and hydrolysis of nucleotides (eg, ATP or ADP) play an important role in their activity, and by introducing a reporter into a nucleotide derivative, intracellular signal transduction can be observed with high sensitivity.

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

RNAi introduces double-stranded RNA (dsRNA) into cells to degrade mRNA of a target gene to suppress expression, and it is possible to observe RNAi phenomena by labeling the designed dsRNA with a reporter.

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

Hereinafter, specific embodiments of the present invention are presented. However, the examples described below are only for specifically illustrating or explaining the present invention, and the present invention is not limited thereto. In addition, among the reporters defined in the claims and detailed description of the present application, compounds whose synthesis method is not disclosed through the following preparations may be synthesized with reference to the following preparations.

Preparation Example 1.

(1) Synthesis of compound 1

Synthesis of intermediate 2

Intermediate 1 (synthesized with reference to Korean Patent Publication No. 10-2017-0009795) (30 g, 0.048 mol), N-hydroxysuccinimide (6.63 g, 0.058 mol), N,N'-dicyclohexylcarbodiimide (11.89 g, 0.058 mol), dichloromethane (600 mL) was added, stirred at room temperature for 2 hours, the resulting solid was filtered, and the filtrate was concentrated.

Synthesis of intermediate 4

Intermediate 2 (15 g, 0.021 mol), Intermediate 3 (synthesized with reference to International Patent Publication No. 2016-100401) (17.4 g, 0.025 mol), triethylamine (7.2 mL, 0.052 mol), tetrahydrofuran in a reactor (300 mL) and dichloromethane (300 mL) were added and stirred at room temperature for 1.5 hours. An aqueous sodium bicarbonate solution was added to the reactor, stirred vigorously, and the organic layer was separated. Sodium sulfate was added to the organic layer, stirred, filtered, and the filtrate was concentrated and purified by column.

Synthesis of compound 1

Intermediate 4 (8 g, 0.006 mol), 2-cyanoethyl N,N-diisopropylchlorophosphoamidite (4.06 g, 0.017 mol), triethylamine (5.0 ml, 0.037 mol), dichloromethane ( 160 mL) and stirred at room temperature for 1.5 hours. After adding water to the reactor and vigorously stirring, the organic layer was separated. Sodium sulfate was added to the organic layer, stirred, filtered, and the filtrate was concentrated and purified by column.

¹ H-NMR of the obtained compound 1 is as follows.

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.93-7.73 (m, 2H), 7.44-7.21 (m, 14H), 7.07-6.97 (m, 3H), 6.86-6.85 (m, 4H), 6.70-6.60 (m, 1H), 6.31 (br t, 1H), 5.75-5.71 (m, 1H), 5.53-5.46 (m, 1H), 4.53-4.15 (m, 4H), 3.77-3.11 (m, 18H), 2.69-2.62 (m,6H), 2.44-1.95 (m,9H), 1.47-1.05 (m, 34H), 0.94-0.80 (m, 1H), 0.70-0.50 (m, 1H),

(2) Synthesis of compound 2

Synthesis of intermediate 8

Intermediate 7 (synthesized with reference to International Patent Publication No. 2017-010852) (40.0 g, 0.139 mol), methyl iodide (39.5 g, 0.278 mol), and acetonitrile (400 mL) were added to the reactor and stirred under reflux for 16 hours did. After cooling, the resulting solid was filtered.

Synthesis of intermediate 10

Intermediate 8 (54.5 g, 0.127 mol), Intermediate 9 (synthesized with reference to US Patent No. 5760201) (50 g, 0.105 mol), triethyl amine (73.7 ml, 529.0 mol), acetonitrile (500) in a reactor mL), stirred under reflux for 2 hours, concentrated and purified by column.

Synthesis of intermediate 11

Intermediate 10 (40 g, 0.062 mol), 1N aqueous sodium hydroxide solution (630 mL), tetrahydrofuran (400 mL), and methanol (400 mL) were added to the reactor and stirred at room temperature for 16 hours. The reaction solution was acidified with hydrochloric acid, concentrated and extracted with dichloromethane. Sodium sulfate was added to the organic layer, stirred, filtered, and the filtrate was concentrated and purified by column.

Synthesis of intermediate 12

Intermediate 12 was synthesized in the same manner as in the synthesis of Intermediate 2 above.

Synthesis of intermediate 13

Intermediate 13 was synthesized in the same manner as in the synthesis of Intermediate 4 above.

Synthesis of compound 2

Compound 2 was synthesized in the same manner as in the synthesis of Compound 1.

¹ H-NMR of the obtained compound 2 is as follows.

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.14 (t, 2H), 7.93-7.88 (m, 1H), 7.44-7.03 (m, 19H), 7.06 (d, 1H), 6.85-6.83 (m, 4H) ), 6.74-6.59 (m, 3H), 6.31-6.17 (m, 3H), 5.39-5.30 (m, 1H), 4.54 (br t, 1H), 4.18 (m, 1H), 3.76-3.40 (m, 16H), 3.33-3.07 (m, 4H), 2.64-2.41 (m, 6H), 2.11 (t, 3H), 1.74-1.70 (m, 8H), 1.31-1.04 (m, 16H), 0.94-0.80 ( m, 1H), 0.60-0.40 (m, 1H)

(3) Synthesis of compound 3

Synthesis of intermediate 17

Intermediate 15 (synthesized with reference to International Patent Publication No. 2017-010852) (35 g, 108.0 mol), Intermediate 16 (synthesized with reference to European Patent Application Publication No. 1209205) (22.24 g, 119.0 mol), acetic acid ( 250 mL), stirred under reflux for 3 days, concentrated and purified by column.

Synthesis of intermediate 18

Intermediate 18 was synthesized in the same manner as in the synthesis of Intermediate 8.

Synthesis of intermediate 21

Intermediate 19 (synthesized with reference to Organic & Biomolecular Chemistry (2015), 13(30), 8169-8172) (164.6 g, 0.468 mol), Intermediate 20 (133.0 g, 0.515 mol), acetic anhydride (1.6 L) in a reactor was added and stirred at 110 °C for 2 hours. After cooling, the reaction solution was added to ethyl ether (6 L), stirred vigorously, and the resulting solid was filtered.

Synthesis of intermediate 22

Intermediate 18 (17.9 g, 0.039 mol), Intermediate 21 (24.9 g, 0.047 mol), and pyridine (180 mL) were placed in a reactor, stirred at 70° C. for 4 hours, concentrated and purified by column.

Synthesis of intermediate 23

Intermediate 23 was synthesized in the same manner as in the synthesis of Intermediate 2 above.

Synthesis of intermediate 24

Intermediate 24 was synthesized in the same manner as in the synthesis of Intermediate 4 above.

Synthesis of compound 3

Compound 3 was synthesized in the same manner as in the synthesis of Compound 1.

¹ H-NMR of the obtained compound 2 is as follows.

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.38 (t, 2H), 8.15 (t, 2H), 7.95-7.93 (m, 4H), 7.62-7.61 (m, 2H), 7.50-7.22 (m, 16H) ), 7.06 (d, 1H), 6.85-6.83 (m, 4H), 6.61 (t, 1H), 6.51-6.20 (m, 5H), 5.31-5.22 (m, 1H), 4.54 (br t, 1H) , 4.18 (m, 1H), 3.83 (s, 3H), 3.78-3.76 (m, 9H), 3.64-3.40 (m, 4H), 3.20-3.06 (m, 4H), 2.64-2.30 (m, 6H) , 2.07-1.98 (m, 11H), 1.26-1.05 (m, 16H), 0.94-0.80 (m, 1H), 0.60-0.40 (m, 1H),

Preparation Example 2. Synthesis of single-labeled probe (oligonucleotide)

Single-labeled oligonucleotides in which Compound 1, Compound 2 or Compound 3 are labeled at the 5' end as reporters were synthesized using MerMade ^™ 48X DNA Synthesizer and Universal UnyLinker Support (Chemgene, 500 Å) as CPG. The sequence of the single-labeled oligonucleotide is shown in Table 1 below.

division order SLP1 5'-compound 1-dt-TTT TTT TTT T-3' SLP2 5'-compound 2-dt-TTT TTT TTT T-3' SLP3 5'-compound 3-dt-TTT TTT TTT T-3'

After synthesis, single-labeled oligonucleotides were deprotected and purified by RP-HPLC. The absorption and emission spectra of the synthesized single-labeled oligonucleotide after purification were measured, and the measurement results (error rate less than 0.1%) of absorption/emission maximum wavelengths for Compounds 1 to 3 are shown in Table 2 below.

reporter Excitation _max (nm) Emission _max (nm) compound 1 602 628 compound 2 647 674 compound 3 689 716

Preparation Example 3. Synthesis of double-labeled probe (oligonucleotide)

5'-ATG CAA CAT TAA CCC GAG ATA CG-3' as forward primer for CT (Chlamydia trachomatis) using Universal UnyLinker Support (Chemgene, 500Å) and 5'-ACT CGG CTT GGG AAG AGC TT- as reverse primer 3' was synthesized and purified by HPLC on a 1 μmol scale, respectively.

The double-labeled probe is a CT (Chlamydia trachomatis) selective labeling probe, and the [Quor 7] induced CPG (synthesized with reference to Korean Patent Publication No. 10-2020-0067733) and a commercially available quencher, Black Hole Quencher® 2 and 3 (BHQ2 and BHQ3, LGC Biosearch Technologies) was used to synthesize 5'-TTG TCC ATA TCT TTG ATA CGA CGC CGC-quenchor-3' using CPG induced, followed by compound 1, compound 2 or compound 3 as a reporter. A double-labeled probe labeled with 1 umol scale was synthesized and purified by HPLC.

The form of the synthesized double-labeled probe is shown in Table 3, and the absorption wavelength regions of the quencher 7, BHQ2 and BHQ3 used as the quencher of the double-labeled probe are shown in Table 4 below.

division order DLP1 5'-Compound 1-dT-TTGTCCATATCTTTGATACGACGCGC-Quor 7-3' DLP2 5'-Compound 2-dT-TTGTCCATATCTTTGATACGACGCGC-Quor 7-3' DLP3 5'-Compound 3-dT-TTGTCCATATCTTTGATACGACGCGC-Quor 7-3' DLP4 5'-Compound 1-dT-TTGTCCATATCTTTGATACGACGCCGC-BHQ2-3' DLP5 5'-Compound 2-dT-TTGTCCATATCTTTGATACGACGCCGC-BHQ2-3' DLP6 5'-Compound 3-dT-TTGTCCATATCTTTGATACGACGCCGC-BHQ3-3'

division λ _Max (nm) ε (mol ^-1 cm ^-1 ) quencher 7 580 ~ 710 140,000 BHQ2 550 to 650 38,000 BHQ3 620 ~ 730 42,000

The structure of the CPG from which the quencher 7 was used in Preparation Example 3 was induced is as follows.

Experimental Example 1. Real-Time PCR experiment using double-labeled probe

Real-time PCR for CT (Chlamydia trachomatis) plasmid DNA with the composition shown in Table 5 using each double-labeled probe synthesized according to Preparation Example 3 was repeated twice twice (Biorad, CFX-96 used) ). The real-time PCR results are shown in FIGS. 1 to 2 .

division Content (μl) (Bioline)SensiFAST ^TM Probe No-ROX Mix (2X) 10 CT plasmid DNA (5x10^6, 5x10^4, 5x10^3 copies/μl) One CT F/R primer mix (10 pmol/μl) One CT Dual-labeled probe (7 pmol/μl) 3 Pure Water 5

1 to 3 showing the real-time PCR results, compared with DLP4 to DLP6 using BHQ2 or BHQ3, which are commercially available quenchers as quenchers of compounds 1 to 3, DLP 1 to using quencher 7 It can be seen that DLP3 exhibits a relatively low Ct value, and the final fluorescence amplification intensity is improved by about 5 to 10%.

Considering the Limit of Detection (LoD) of molecular diagnostics dealing with 1 to 2 copies of DNA or RNA contained in bacteria or viruses, when compounds 1 to 3 are used as reporters, quencher 7 is used as a quencher It can be expected that the ease of detection of the target will be improved as it is used.

In addition, referring to FIGS. 4 to 9 , compounds 1 to 3, which are reporters for nucleic acid labeling as defined herein, exhibit excellent linearity for all of the commercially available quencher BHQ2 or BHQ3 as well as quencher 7. can be checked

Therefore, the reporter for nucleic acid label defined herein is expected to be sufficiently usable not only for Chlamydia trachomatis nucleic acid label but also for various nucleic acid labels, and it can be seen that it can be easily applied without separate technical modification to previously commercialized quenchers. .

On the other hand, it is known that most of the previously commercially available reporters for labeling nucleic acids have a limitation in that substitution is possible only at the 5' end. On the other hand, as the reporter for nucleic acid label defined herein has a substituted form for dT, substitution is possible not only at the 5' end but also between the sequences, through which the distance between the reporter and the quencher is closely adjusted to increase the fluorescence amplification intensity. can contribute to improving

In the above, although some embodiments of the present invention have been described, those of ordinary skill in the art can add, change, delete or add components within the scope that does not depart from the spirit of the present invention described in the claims. The present invention may be variously modified and changed by such as, and it will be said that it is also included within the scope of the present invention.

Claims (15)

A reporter for labeling a nucleic acid represented by Formula 1 or Formula 2 below.
[Formula 1]

[Formula 2]

here,
Ar ₁ is a substituted or unsubstituted C ₆ -C ₂₀ aryl or C ₆ -C ₂₀ heteroaryl containing at least one hetero atom,
Z ₁ is NR ₅ R ₆ or OR ₇ ,
Z ₂ is NR ₈ or O,
X is O or S;
Y is CR ₉ R ₁₀ , NR ₁₁ , O or S;
R ₁ To R ₁₁ are each independently hydrogen, deuterium, substituted or unsubstituted C ₁ -C ₁₀ alkyl, substituted or unsubstituted C ₁ -C ₁₀ heteroalkyl including at least one hetero atom, substituted or unsubstituted substituted C ₂ -C ₁₀ alkenyl, substituted or unsubstituted C ₂ -C ₁₀ alkynyl, substituted or unsubstituted C ₃ -C ₂₀ cycloalkyl, substituted or unsubstituted C ₃ -C ₂₀ cycloalkenyl, substituted or unsubstituted C ₂ -C ₂₀ heterocycloalkyl, hydroxy, oxido (—O ^— ), substituted or unsubstituted C ₁ -C ₁₀ alkoxy, substituted or unsubstituted C ₃ -C ₂₀ cycloalkyloxy, substituted or unsubstituted C ₅ -C ₂₀ aryloxy, substituted or unsubstituted C ₂ -C ₂₀ heteroaryloxy, substituted or unsubstituted C ₅ -C ₂₀ aryl, substituted or unsubstituted C ₂ -C ₂₀ hetero aryl, substituted or unsubstituted C ₅ -C ₂₀ aralkyl, substituted or unsubstituted C ₁ -C ₁₀ alkylthio, substituted or unsubstituted C ₅ -C ₂₀ arylthio, substituted or unsubstituted C ₃ -C ₂₀ cycloalkylthio, substituted or unsubstituted C ₂ -C ₂₀ heteroarylthio, substituted or unsubstituted acylamino, acyloxy, substituted or unsubstituted phosphino, carboxylate (-CO ₂ ^- ), trifluoro Methylsulfonyl (-SO ₂ CF ₃ ), substituted or unsubstituted ammonium, nitro, sulfonic acid (-SO ₃ H), sulfonate, substituted sulfonyl, substituted sulfonic acid ester, substituted or unsubstituted sulfone Amide, substituted thioketone, trihalomethyl (-CF ₃ , -CCl ₃ , -CBr ₃ , -CI ₃ ), haloformyl (-COCl, -COBr, -COI), formyl (-CHO) , acyl, carboxyl, substituted ester, substituted or unsubstituted aminocarbonyl, nitro, nitroso (-N=O), fluoro (-F), chloro (-Cl), bromo (-Br ), iodo (-I), substituted or unsubstituted germanium, substituted or unsubstituted boron, substituted or unsubstituted aluminum, substituted or unsubstituted silyl, substituted or unsubstituted amide, carbamate, carboxylate, substituted or unsubstituted phosphine, substituted or unsubstituted phosphoric acid, phosphate, phosphonic acid, phosphonate, nite a functional group selected from reyl, hydrazine, acetal, ketal, polyalkylene oxide and -L ₁ -R ₁₂ , or two adjacent functional groups combine with each other to form a ring,
At least one of R ₁ to R ₁₁ is -L ₁ -R ₁₂ ,
L ₁ is a single bond, substituted or unsubstituted C ₁ -C ₁₀ alkyl, substituted or unsubstituted C ₁ -C ₁₀ heteroalkyl containing at least one hetero atom, substituted or unsubstituted C ₂ -C ₁₀ alkenyl, substituted or unsubstituted C ₂ -C ₁₀ alkynyl, substituted or unsubstituted C ₃ -C ₂₀ cycloalkyl, substituted or unsubstituted C ₃ -C ₂₀ cycloalkenyl and substituted or unsubstituted C ₂ -C ₂₀ comprising at least one selected from heterocycloalkyl,
R ₁₂ is a deoxyribonucleoside represented by the following formula (3).
[Formula 3]

here,
* represents the position at which the deoxyribonucleoside is bonded to L ₁ ,
B is a nucleobase,
R ₁₃ is selected from hydrogen, deuterium, P(OR ₁₅ )(N(R ₁₆ R ₁₇ ) and -L ₂ -R ₁₈ ,
R ₁₄ is an alcohol protecting group, hydroxy or P(OR ₁₅ )(N(R ₁₆ R ₁₇ ), or a support or nucleic acid to which the reporter is bound,
R ₁₅ to R ₁₇ are each independently selected from hydrogen, deuterium, substituted or unsubstituted C ₁ -C ₁₀ alkyl and substituted or unsubstituted C ₁ -C ₁₀ heteroalkyl containing at least one hetero atom,
L ₂ is a single bond or is selected from internucleotide phosphodiester bonds, substituted or unsubstituted C ₁ -C ₁₀ alkyl and substituted or unsubstituted C ₁ -C ₁₀ heteroalkyl containing at least one hetero atom, ,
R ₁₈ is hydroxy or P(OR ₁₅ )(N(R ₁₆ R ₁₇ ), or a support or nucleic acid to which a reporter represented by Formula 1 or Formula 2 is bound.
According to claim 1,
L ₁ is a linker comprising at least one carbon,
any carbon in the linker is a carbonyl carbon;
A reporter for labeling nucleic acids.
According to claim 1,
Y is CR ₉ R ₁₀ ,
at least one of R ₉ and R ₁₀ is -L ₁ -R ₁₂ ;
A reporter for labeling nucleic acids.
According to claim 1,
R ₁₂ is deoxyribonucleoside represented by the following formula (4),
A reporter for labeling nucleic acids.
[Formula 4]

A reporter for labeling a nucleic acid represented by the following formula (5).
[Formula 5]

here,
R ₂₁ and R ₂₂ are each independently hydrogen, deuterium, substituted or unsubstituted C ₁ -C ₁₀ alkyl, substituted or unsubstituted C ₁ -C ₁₀ heteroalkyl containing at least one hetero atom, substituted or unsubstituted substituted C ₂ -C ₁₀ alkenyl, substituted or unsubstituted C ₂ -C ₁₀ alkynyl, substituted or unsubstituted C ₃ -C ₂₀ cycloalkyl, substituted or unsubstituted C ₃ -C ₂₀ cycloalkenyl, substituted or unsubstituted C ₂ -C ₂₀ heterocycloalkyl, substituted or unsubstituted C ₅ -C ₂₀ aryl and substituted or unsubstituted C ₂ -C ₂₀ heteroaryl,
R ₂₃ to R ₃₀ are each independently hydrogen, deuterium, substituted or unsubstituted C ₁ -C ₁₀ alkyl, substituted or unsubstituted C ₁ -C ₁₀ heteroalkyl containing at least one hetero atom, substituted or unsubstituted substituted C ₂ -C ₁₀ alkenyl, substituted or unsubstituted C ₂ -C ₁₀ alkynyl, substituted or unsubstituted C ₃ -C ₂₀ cycloalkyl, substituted or unsubstituted C ₃ -C ₂₀ cycloalkenyl, substituted or unsubstituted C ₂ -C ₂₀ heterocycloalkyl, hydroxy, oxido (—O ^— ), substituted or unsubstituted C ₁ -C ₁₀ alkoxy, substituted or unsubstituted C ₃ -C ₂₀ cycloalkyloxy, substituted or unsubstituted C ₅ -C ₂₀ aryloxy, substituted or unsubstituted C ₂ -C ₂₀ heteroaryloxy, substituted or unsubstituted C ₅ -C ₂₀ aryl, substituted or unsubstituted C ₂ -C ₂₀ hetero aryl, substituted or unsubstituted C ₅ -C ₂₀ aralkyl, substituted or unsubstituted C ₁ -C ₁₀ alkylthio, substituted or unsubstituted C ₅ -C ₂₀ arylthio, substituted or unsubstituted C ₃ -C ₂₀ cycloalkylthio, substituted or unsubstituted C ₂ -C ₂₀ heteroarylthio, substituted or unsubstituted acylamino, acyloxy, substituted or unsubstituted phosphino, carboxylate (-CO ₂ ^- ), trifluoro Methylsulfonyl (-SO ₂ CF ₃ ), substituted or unsubstituted ammonium, nitro, sulfonic acid (-SO ₃ H), sulfonate, substituted sulfonyl, substituted sulfonic acid ester, substituted or unsubstituted sulfone Amide, substituted thioketone, trihalomethyl (-CF ₃ , -CCl ₃ , -CBr ₃ , -CI ₃ ), haloformyl (-COCl, -COBr, -COI), formyl (-CHO) , acyl, carboxyl, substituted ester, substituted or unsubstituted aminocarbonyl, nitro, nitroso (-N=O), fluoro (-F), chloro (-Cl), bromo (-Br ), iodo (-I), substituted or unsubstituted germanium, substituted or unsubstituted boron, substituted or unsubstituted aluminum, substituted or unsubstituted silyl, substituted or unsubstituted substituted or unsubstituted amides, carbamates, carboxylates, substituted or unsubstituted phosphines, substituted or unsubstituted phosphoric acids, phosphates, phosphonic acids, phosphonates, It is a functional group selected from nitrile, hydrazine, acetal, ketal, polyalkylene oxide and -L ₁ -R ₁₂ , or two adjacent functional groups combine with each other to form a ring,
R ₃₁ to R ₃₄ are each independently hydrogen, deuterium, substituted or unsubstituted C ₁ -C ₁₀ alkyl, substituted or unsubstituted C ₁ -C ₁₀ heteroalkyl containing at least one hetero atom, and -L ₁ - selected from R ₁₂ , wherein at least one of R ₃₁ to R ₃₄ is -L ₁ -R ₁₂ ,
L ₁ is a single bond, substituted or unsubstituted C ₁ -C ₁₀ alkyl, substituted or unsubstituted C ₁ -C ₁₀ heteroalkyl containing at least one hetero atom, substituted or unsubstituted C ₂ -C ₁₀ alkenyl, substituted or unsubstituted C ₂ -C ₁₀ alkynyl, substituted or unsubstituted C ₃ -C ₂₀ cycloalkyl, substituted or unsubstituted C ₃ -C ₂₀ cycloalkenyl and substituted or unsubstituted C ₂ -C ₂₀ comprising at least one selected from heterocycloalkyl,
R ₁₂ is a deoxyribonucleoside represented by the following formula (3),
[Formula 3]

here,
* represents the position at which the deoxyribonucleoside is bonded to L ₁ ,
B is a nucleobase,
R ₁₃ is selected from hydrogen, deuterium, P(OR ₁₅ )(N(R ₁₆ R ₁₇ ) and -L ₂ -R ₁₈ ,
R ₁₄ is an alcohol protecting group, hydroxy or P(OR ₁₅ )(N(R ₁₆ R ₁₇ ), or a support or nucleic acid to which the reporter is bound,
R ₁₅ to R ₁₇ are each independently selected from hydrogen, deuterium, substituted or unsubstituted C ₁ -C ₁₀ alkyl and substituted or unsubstituted C ₁ -C ₁₀ heteroalkyl containing at least one hetero atom,
L ₂ is a single bond or is selected from internucleotide phosphodiester bonds, substituted or unsubstituted C ₁ -C ₁₀ alkyl and substituted or unsubstituted C ₁ -C ₁₀ heteroalkyl containing at least one hetero atom, ,
R ₁₈ is hydroxy or P(OR ₁₅ )(N(R ₁₆ R ₁₇ ), or a support or nucleic acid to which a reporter represented by Formula 1 or Formula 2 is bound,
Y is CR ₃₅ and each R ₃₅ is hydrogen, deuterium, substituted or unsubstituted C ₁ -C ₁₀ alkyl, substituted or unsubstituted C ₁ -C ₁₀ heteroalkyl comprising at least one heteroatom, fluoro (-F), chloro (-Cl), bromo (-Br), iodo (-I),
n is an integer from 1 to 4.
6. The method of claim 5,
L ₁ is a linker comprising at least one carbon,
any carbon in the linker is a carbonyl carbon;
A reporter for labeling nucleic acids.
6. The method of claim 5,
R ₁₂ is deoxyribonucleoside represented by the following formula (4),
A reporter for labeling nucleic acids.
[Formula 4]

a reporter according to any one of claims 1 to 7; and
quencher;
Oligonucleotides comprising a.
9. The method of claim 8,
An oligonucleotide further comprising a minor groove binder (MGB) interposed between the reporter and the quencher.
A composition for detecting a nucleic acid comprising the oligonucleotide according to claim 8.
a reporter according to any one of claims 1 to 7;
support; and
a linker connecting the reporter and the support;
containing,
A support for detecting nucleic acids.
12. The method of claim 11,
The support is glass, cellulose, nylon, acrylamide gel, dextran, polystyrene or resin,
A support for detecting nucleic acids.
(a) preparing a reaction mixture comprising a target nucleic acid, a reagent necessary for amplifying the target nucleic acid, and the oligonucleotide according to claim 8;
(b) amplifying a target nucleic acid in the reaction mixture by a polymerase chain reaction; and
(c) measuring the fluorescence intensity of the reaction mixture;
containing,
Nucleic acid detection method.
14. The method of claim 13,
The step (b) is,
(b-1) extending the oligonucleotide hybridized to the target nucleic acid by a polymerase;
(b-2) separating the reporter and the quencher of the oligonucleotide from the target nucleic acid by the exonuclease activity of the polymerase; and
(b-3) emitting the fluorescence of the reporter separated from the quencher;
containing,
Nucleic acid detection method.
14. The method of claim 13,
Further comprising the step (d) of measuring the amount of amplification of the target nucleic acid from the fluorescence intensity measured in step (c),
Nucleic acid detection method.

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