Patent ID: 12215381

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

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 will have meanings commonly understood by those of ordinary skill in the art.

In addition, unless specifically indicated otherwise, terms in a singular form also include plural forms, and terms in a plural form should be understood to include singular forms as well.

Novel Reporter for Labeling Nucleic Acid

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

Wherein,Ar1may be substituted or unsubstituted C6-C20aryl or C6-C20heteroaryl containing at least one hetero atom,Z1may be NR5R6or OR7,Z2may be NR8or O,X may be O or S,Y may be CR9R10, NR11, O, or S, andR1to R11may each be independently a functional group selected from hydrogen, deuterium, substituted or unsubstituted C1-C10alkyl, substituted or unsubstituted C1-C10heteroalkyl containing at least one hetero atom, substituted or unsubstituted C2-C10alkenyl, substituted or unsubstituted C2-C10alkynyl, substituted or unsubstituted C3-C20cycloalkyl, substituted or unsubstituted C3-C20cycloalkenyl, substituted or unsubstituted C2-C20heterocycloalkyl, hydroxy, oxido (—O−), substituted or unsubstituted C1-C10alkoxy, substituted or unsubstituted C3-C20cycloalkyloxy, substituted or unsubstituted C5-C20aryloxy, substituted or unsubstituted C2-C20heteroaryloxy, substituted or unsubstituted C5-C20aryl, substituted or unsubstituted C2-C20heteroaryl, substituted or unsubstituted C5-C20aralkyl, substituted or unsubstituted C1-C10alkylthio, substituted or unsubstituted C5-C20arylthio, substituted or unsubstituted C3-C20cycloalkylthio, substituted or unsubstituted C2-C20heteroarylthio, substituted or unsubstituted acylamino, acyloxy, substituted or unsubstituted phosphino, carboxylate (—CO2−), trifluoromethylsulfonyl (—SO2CF3), substituted or unsubstituted ammonium, nitro, sulfonic acid (—SO3H), sulfonate, substituted sulfonyl, substituted sulfonic acid ester, substituted or unsubstituted sulfonamide, substituted thioketone, trihalomethyl (—CF3, —CCl3, —CBr3, or —CI3), haloformyl (—COCl, —COBr, or —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, nitrile, hydrazine, acetal, ketal, polyalkyleneoxide, and -L1-R12, or two adjacent functional groups are bonded to each other to form a ring.

In addition, when any functional group of R1to R11is a substituted functional group, any substituent other than hydrogen may be bonded to at least one carbon in the functional group. The substituent may be selected from deuterium, substituted or unsubstituted C1-C10alkyl, substituted or unsubstituted C1-C10heteroalkyl containing at least one hetero atom, substituted or unsubstituted C2-C10alkenyl, substituted or unsubstituted C2-C10alkynyl, substituted or unsubstituted C3-C20cycloalkyl, substituted or unsubstituted C3-C20cycloalkenyl, substituted or unsubstituted C2-C20heterocycloalkyl, hydroxy, oxido (—O−), substituted or unsubstituted C1-C10alkoxy, substituted or unsubstituted C3-C20cycloalkyloxy, substituted or unsubstituted C5-C20aryloxy, substituted or unsubstituted C2-C20heteroaryloxy, substituted or unsubstituted C5-C20aryl, substituted or unsubstituted C2-C20heteroaryl, substituted or unsubstituted C5-C20aralkyl, substituted or unsubstituted C1-C10alkylthio, substituted or unsubstituted C5-C20arylthio, substituted or unsubstituted C3-C20cycloalkylthio, substituted or unsubstituted C2-C20heteroarylthio, substituted or unsubstituted acylamino, acyloxy, substituted or unsubstituted phosphino, carboxylate (—CO2−), trifluoromethylsulfonyl (—SO2CF3), substituted or unsubstituted ammonium, nitro, sulfonic acid (—SO3H), sulfate, substituted sulfonyl, substituted sulfonic acid ester, substituted or unsubstituted sulfonamide, substituted thioketone, trihalomethyl (—CF3, —CCl3, —CBr3, or —CI3), haloformyl (—COCl, —COBr, or —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, nitrile, hydrazine, acetal, ketal, polyalkyleneoxide, and -L1-R12.

In reporters according to various embodiments of the present invention, at least one of R1to R11has a functional group represented by -L1-R12, and the reporters may bond to and label a target biomolecule (e.g., a nucleic acid) via a functional group represented by -L1-R12.

According to one embodiment, in the reporter represented by Formula 1 or 2, Y may be CR9R10, and at least one of R9and R10may be -L1-R12.

L1is a linker connecting a main body represented by Formula 1 or 2 and R12, and may be a single bond (i.e., there is no element or functional group between the main body represented by Formula 1 or 2 and R12), or at least one selected from a saturated or unsaturated, substituted or unsubstituted, or branched or unbranched alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, and heterocycloalkyl. For example, when L1is not a single bond, L1may include (1) substituted or unsubstituted C1-C10alkyl, substituted or unsubstituted C1-C10heteroalkyl containing at least one hetero atom, substituted or unsubstituted C2-C10alkenyl, substituted or unsubstituted C2-C10alkynyl, substituted or unsubstituted C3-C20cycloalkyl, substituted or unsubstituted C3-C20cycloalkenyl, or substituted or unsubstituted C2-C20heterocycloalkyl, or (2) at least one selected from substituted or unsubstituted C1-C10alkyl, substituted or unsubstituted C1-C10heteroalkyl containing at least one hetero atom, substituted or unsubstituted C2-C10alkenyl, substituted or unsubstituted C2-C10alkynyl, substituted or unsubstituted C3-C20cycloalkyl, substituted or unsubstituted C3-C20cycloalkenyl, and substituted or unsubstituted C2-C20heterocycloalkyl.

In addition, L1is a linker including at least one carbon, which is mentioned above, and any carbon of the linker may be a carbonyl carbon. The carbonyl may be at least one selected from aldehydes, ketones, carboxylic acids, esters, amides, enones, acyl halides, acid anhydrides, and imides.

In another case, L1may have a structure in which neighboring linkers are interconnected via a carbonyl carbon. Wherein, L1may include both a linker directly connected without a carbonyl carbon and a linker connected by a carbonyl carbon. For example, L1may have a structure including an amide (aminocarbonyl), such as “—CH2—CH2—C(═O)—NH—CH2—CH2—CH—CH—CH2—.” In addition, L1may have a structure including a carbonylamino, such as “—CH2—CH2—NH—C(═O)—CH2—CH2—CH═CH—CH2—.”

R12is a deoxyribonucleoside represented by Formula 3.

Wherein,* indicates a location where the deoxyribonucleoside is bonded to L1,B is a nucleobase. As a component of the deoxyribonucleoside represented by Formula 3, the nucleobase may be a purine-based base such as adenine, guanine, hypoxanthine, xanthine, or 7-methylguanine; or a pyrimidine-based base such as cytosine, thymine, uracil, 5,6-dihydrouracil, 5-methylcytosine, or 5-hydroxymethylcytosine.

As the nucleobase, a deoxyribonucleoside including thymine (thymidine) may be represented by Formula 4 below.

In the deoxyribonucleoside represented by Formula 3 or 4, R13may be selected from hydrogen, deuterium, P(OR15)(N(R16R17), and -L2-R18, and R14may be an alcohol protecting group, hydroxy, or P(OR15)(N(R16R17), or a support or nucleic acid to which the reporter represented by Formula 1 or 2 is bound.

When R13or R14is P(OR15)(N(R16R17), R15to R17may each be independently selected from hydrogen, deuterium, substituted or unsubstituted C1-C10alkyl, and substituted or unsubstituted C1-C10heteroalkyl containing at least one hetero atom.

In addition, when any functional group in R15to R17is a substituted functional group, any substituent other than hydrogen may be bonded to at least one carbon of the functional group. The substituent may be selected from deuterium, substituted or unsubstituted C1-C10alkyl, substituted or unsubstituted C1-C10heteroalkyl containing at least one hetero atom, substituted or unsubstituted C2-C10alkenyl, substituted or unsubstituted C2-C10alkynyl, substituted or unsubstituted C3-C20cycloalkyl, substituted or unsubstituted C3-C20cycloalkenyl, substituted or unsubstituted C2-C20heterocycloalkyl, hydroxy, oxido (—O−), substituted or unsubstituted C1-C10alkoxy, substituted or unsubstituted C3-C20cycloalkyloxy, substituted or unsubstituted C5-C20aryloxy, substituted or unsubstituted C2-C20heteroaryloxy, substituted or unsubstituted C5-C20aryl, substituted or unsubstituted C2-C20heteroaryl, substituted or unsubstituted C5-C20aralkyl, substituted or unsubstituted C1-C10alkylthio, substituted or unsubstituted C5-C20arylthio, substituted or unsubstituted C3-C20cycloalkylthio, substituted or unsubstituted C2-C20heteroarylthio, substituted or unsubstituted acylamino, acyloxy, substituted or unsubstituted phosphino, carboxylate (—CO2−), trifluoromethylsulfonyl (—SO2CF3), substituted or unsubstituted ammonium, nitro, sulfonic acid (—SO3H), sulfate, substituted sulfonyl, substituted sulfonic acid ester, substituted or unsubstituted sulfonamide, substituted thioketone, trihalomethyl (—CF3, —CCl3, —CBr3, or —CI3), haloformyl (—COCI, —COBr, or —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, nitrile, hydrazine, acetal, ketal, polyalkyleneoxide, and -L1-R12.

When R13is -L2-R18, L2is a linker connecting O with R18, and may be a single bond (i.e., there is no element or functional group between O and R18), an internucleotide phosphodiester bond, or a saturated or unsaturated, substituted or unsubstituted, or branched or unbranched alkyl or heteroalkyl.

For example, when L2is neither a single bond nor an internucleotide phosphodiester bond, L2may be substituted or unsubstituted C1-C10alkyl, or substituted or unsubstituted C1-C10heteroalkyl containing at least one hetero atom.

In addition, L2is a linker including at least one carbon, which is mentioned above, and any carbon in the liker may be a carbonyl carbon. The carbonyl may be at least one selected from aldehydes, ketones, carboxylic acids, esters, amides, enones, acyl halides, acid anhydrides, and imides.

In another case, L2may have a structure in which neighboring linkers are interconnected via a carbonyl carbon. Wherein, L2may include both a linker directly connected without a carbonyl carbon and a linker connected by a carbonyl carbon. For example, L2may have a structure including an amide (aminocarbonyl), such as “—CH2—CH2—C(═O)—NH—CH2—CH2—CH—CH—CH2—.” In addition, L2may have a structure including a carbonylamino, such as “—CH2—CH2—CH═CH—CH2—.”

When L2is an internucleotide phosphodiester bond, the deoxyribonucleoside represented by Formula 3 or 4 may be represented by Formulas 3-1 or 4-1 below, respectively.

R18may be hydroxy or P(OR15)(N(R16R17), or a support or nucleic acid to which the reporter represented by Formula 1 or Formula 2 is bound.

That is, when R13is -L2-R18, the reporter represented by Formula 1 or 2 may be bound to a support or a nucleic acid (RNA or DNA) via L2.

The support may be prepared with at least one selected from glass, cellulose, nylon, acrylamide gel, dextran, polystyrene, alginate, collagen, peptides, fibrin, hyaluronic acid, agarose, polyhydroxyethyl methacrylate, polyvinyl alcohol, polyethylene glycol, polyethylene oxide, polyethylene glycol diacrylate, gelatin, Matrigel, polylactic acid, carboxymethyl cellulose, dextran, chitosan, latex, and Sepharose, and may be prepared in the form of beads or a membrane.

Meanwhile, when R14is an alcohol protecting group, the alcohol protecting group may be at least one selected from 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-iso-propylsilyl.

R1to R11may be independently present as the functional groups defined above, but in some embodiments, at least one of R1to R11may be bonded with an adjacent substituent, thereby forming substituted or unsubstituted ring (e.g., a 4-membered ring, a 5-membered ring, a 6-membered ring, a ring formed of more than 6 members, or a fusion ring formed by joining a plurality of rings). Alternatively, the ring may be an aliphatic or aromatic ring.

When at least one of R1to R11is bonded with an adjacent substituent to form substituted or unsubstituted ring, at least one of R1to R11may be bonded with an adjacent substituent via C, N, O, S, Se, or Si, or may be directly bonded with an adjacent substituent using a single bond.

In one embodiment, the reporter may be represented by Formula 1, and when Z1is NR5R6, R5or R6may be bonded with R3or R4, thereby forming substituted or unsubstituted ring.

The formation of the substituted or unsubstituted rings by R5and R6is independent, and a ring formed by bonding R5and R3(or R4) and a ring formed by bonding R6and R4(or R3) may be simultaneously present in one compound.

In another embodiment, the reporter is represented by Formula 1, and when Z1is OR7, R7is bonded with R3or R4, thereby forming substituted or unsubstituted ring.

In still another embodiment, the reporter may be represented by Formula 2, when Z2is NR8, R8may be bonded with R3or R4, thereby forming substituted or unsubstituted ring.

When at least one of R1to R11is bonded with an adjacent substituent to form a substituted ring, any substituent other than hydrogen may be bonded to at least one carbon of the ring. The substituent may be selected from deuterium, substituted or unsubstituted C1-C10alkyl, substituted or unsubstituted C1-C10heteroalkyl containing at least one hetero atom, substituted or unsubstituted C2-C10alkenyl, substituted or unsubstituted C2-C10alkynyl, substituted or unsubstituted C3-C20cycloalkyl, substituted or unsubstituted C3-C20cycloalkenyl, substituted or unsubstituted C2-C20heterocycloalkyl, hydroxy, oxido (—O−), substituted or unsubstituted C1-C10alkoxy, substituted or unsubstituted C3-C20cycloalkyloxy, substituted or unsubstituted C5-C20aryloxy, substituted or unsubstituted C2-C20heteroaryloxy, substituted or unsubstituted C5-C20aryl, substituted or unsubstituted C2-C20heteroaryl, substituted or unsubstituted C5-C20aralkyl, substituted or unsubstituted C1-C10alkylthio, substituted or unsubstituted C5-C20arylthio, substituted or unsubstituted C3-C20cycloalkylthio, substituted or unsubstituted C2-C20heteroarylthio, substituted or unsubstituted acylamino, acyloxy, substituted or unsubstituted phosphino, carboxylate (−CO2−), trifluoromethylsulfonyl (−SO2CF3), substituted or unsubstituted ammonium, nitro, sulfonic acid (−SO3H), sulfate, substituted sulfonyl, substituted sulfonic acid ester, substituted or unsubstituted sulfonamide, substituted thioketone, trihalomethyl (—CF3, —CCl3, —CBr3, or —CI3), haloformyl (—COCI, —COBr, or —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, nitrile, hydrazine, acetal, ketal, polyalkyleneoxide, and -L1-R12.

In addition, according to another aspect of the present invention, a reporter for labeling a nucleic acid, represented by Formula 5 below, is provided.

Wherein,R21and R22may each be independently selected from hydrogen, deuterium, substituted or unsubstituted C1-C10alkyl, substituted or unsubstituted C1-C10heteroalkyl containing at least one hetero atom, substituted or unsubstituted C2-C10alkenyl, substituted or unsubstituted C2-C10alkynyl, substituted or unsubstituted C3-C20cycloalkyl, substituted or unsubstituted C3-C20cycloalkenyl, substituted or unsubstituted C2-C20heterocycloalkyl, substituted or unsubstituted C5-C20aryl, and substituted or unsubstituted C2-C20heteroaryl, andR23to R30may each be independently a functional group selected from hydrogen, deuterium, substituted or unsubstituted C1-C10alkyl, substituted or unsubstituted C1-C10heteroalkyl containing at least one hetero atom, substituted or unsubstituted C2-C10alkenyl, substituted or unsubstituted C2-C10alkynyl, substituted or unsubstituted C3-C20cycloalkyl, substituted or unsubstituted C3-C20cycloalkenyl, substituted or unsubstituted C2-C20heterocycloalkyl, hydroxy, oxido (—O−), substituted or unsubstituted C1-C10alkoxy, substituted or unsubstituted C3-C20cycloalkyloxy, substituted or unsubstituted C5-C20aryloxy, substituted or unsubstituted C2-C20heteroaryloxy, substituted or unsubstituted C5-C20aryl, substituted or unsubstituted C2-C20heteroaryl, substituted or unsubstituted C5-C20aralkyl, substituted or unsubstituted C1-C10alkylthio, substituted or unsubstituted C5-C20arylthio, substituted or unsubstituted C3-C20cycloalkylthio, substituted or unsubstituted C2-C20heteroarylthio, substituted or unsubstituted acylamino, acyloxy, substituted or unsubstituted phosphino, carboxylate (—CO2−), trifluoromethylsulfonyl (—SO2CF3), substituted or unsubstituted ammonium, nitro, sulfonic acid (—SO3H), sulfonate, substituted sulfonyl, substituted sulfonic acid ester, substituted or unsubstituted sulfonamide, substituted thioketone, trihalomethyl (—CF3, —CCl3, —CBr3, or —CI3), haloformyl (—COCI, —COBr, or —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, nitrile, hydrazine, acetal, ketal, polyalkyleneoxide, and -L1-R12, or two adjacent functional groups are bonded to each other to form a ring.

In addition, R31to R34may each be independently selected from hydrogen, deuterium, substituted or unsubstituted C1-C10alkyl, substituted or unsubstituted C1-C10heteroalkyl containing at least one hetero atom, and -L1-R12, and at least one of R31to R34may be -L1-R12. The definition of -L1-R12is the same as described above.

In reporters according to various embodiments of the present invention, at least one of R31to R34has a functional group represented by -L1-R12, and the reporters can bond to and label a target biomolecule (e.g., a nucleic acid) via a functional group represented by -L1-R12.

Y is CR35, each R35is selected from hydrogen, deuterium, substituted or unsubstituted C1-C10alkyl, substituted or unsubstituted C1-C10heteroalkyl containing at least one hetero atom, fluoro (—F), chloro (—Cl), bromo (—Br), and iodo (—I), and n is an integer of 1 to 4.

In addition, R35may be independently present as the functional groups defined above, but in some embodiments, R35may be bonded with R35of neighboring Y to form substituted or unsubstituted ring (e.g., a 4-membered ring, a 5-membered ring, a 6-membered ring, a ring formed of more than 6 members, or a fusion ring formed by joining a plurality of rings). In addition, the ring may be an aliphatic or aromatic ring.

When R35is bonded with R35of a neighboring Y to form a substituted ring, any substituent other than hydrogen may be bonded to at least one carbon of the ring. The substituent may be selected from deuterium, substituted or unsubstituted C1-C10alkyl, substituted or unsubstituted C1-C10heteroalkyl containing at least one hetero atom, substituted or unsubstituted C2-C10alkenyl, substituted or unsubstituted C2-C10alkynyl, substituted or unsubstituted C3-C20cycloalkyl, substituted or unsubstituted C3-C20cycloalkenyl, substituted or unsubstituted C2-C20heterocycloalkyl, hydroxy, oxido (—O−), substituted or unsubstituted C1-C10alkoxy, substituted or unsubstituted C3-C20cycloalkyloxy, substituted or unsubstituted C5-C20aryloxy, substituted or unsubstituted C2-C20heteroaryloxy, substituted or unsubstituted C5-C20aryl, substituted or unsubstituted C2-C20heteroaryl, substituted or unsubstituted C5-C20aralkyl, substituted or unsubstituted C1-C10alkylthio, substituted or unsubstituted C5-C20arylthio, substituted or unsubstituted C3-C20cycloalkylthio, substituted or unsubstituted C2-C20heteroarylthio, substituted or unsubstituted acylamino, acyloxy, substituted or unsubstituted phosphino, carboxylate (—CO2), trifluoromethylsulfonyl (—SO2CF3), substituted or unsubstituted ammonium, nitro, sulfonic acid (—SO3H), sulfate, substituted sulfonyl, substituted sulfonic acid ester, substituted or unsubstituted sulfonamide, substituted thioketone, trihalomethyl (—CF3, —CCl3, —CBr3, or —CI3), haloformyl (—COCI, —COBr, or —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, nitrile, hydrazine, acetal, ketal, polyalkyleneoxide, and -L1-R12.

In addition, when any functional group of R21to R35is a substituted functional group, any substituent other than hydrogen may be bonded to at least one carbon of the functional group. The substituent may be selected from deuterium, substituted or unsubstituted C1-C10alkyl, substituted or unsubstituted C1-C10heteroalkyl containing at least one hetero atom, substituted or unsubstituted C2-C10alkenyl, substituted or unsubstituted C2-C10alkynyl, substituted or unsubstituted C3-C20cycloalkyl, substituted or unsubstituted C3-C20cycloalkenyl, substituted or unsubstituted C2-C20heterocycloalkyl, hydroxy, oxido (—O−), substituted or unsubstituted C1-C10alkoxy, substituted or unsubstituted C3-C20cycloalkyloxy, substituted or unsubstituted C5-C20aryloxy, substituted or unsubstituted C2-C20heteroaryloxy, substituted or unsubstituted C5-C20aryl, substituted or unsubstituted C2-C20heteroaryl, substituted or unsubstituted C5-C20aralkyl, substituted or unsubstituted C1-C10alkylthio, substituted or unsubstituted C5-C20arylthio, substituted or unsubstituted C3-C20cycloalkylthio, substituted or unsubstituted C2-C20heteroarylthio, substituted or unsubstituted acylamino, acyloxy, substituted or unsubstituted phosphino, carboxylate (—CO2−), trifluoromethylsulfonyl (—SO2CF3), substituted or unsubstituted ammonium, nitro, sulfonic acid (—SO3H), sulfate, substituted sulfonyl, substituted sulfonic acid ester, substituted or unsubstituted sulfonamide, substituted thioketone, trihalomethyl (—CF3, —CCl3, —CBr3, or —CI3), haloformyl (—COCI, —COBr, or —COI), formyl (—CHO), acyl, carboxyl, substituted ester, substituted or unsubstituted aminocarbonyl, nitro, nitroso (—N═O), fluoro (—F), chloro (—CI), 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, nitrile, hydrazine, acetal, ketal, polyalkyleneoxide, and -L1-R12.

When the functional group defined in the present invention is alkenyl or alkynyl, the sp2-hybrid carbon of an alkenyl or the sp-hybrid carbon of an alkynyl is directly bonded, or indirectly bonded via the sp3-hybrid carbon of an alkyl bonded thereto.

In the present invention, the Ca-Cbfunctional group refers to a functional group having a to b carbon atoms. For example, Ca-Cbalkyl refers to a saturated aliphatic group, including a linear or branched alkyl having a to b carbon atoms. The linear or branched alkyl may have 40 or less carbon atoms in its main chain (e.g., C1-C40linear, or C3-C40branched).

Specifically, the alkyl may be 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, or n-octyl.

In addition, in the present invention, alkoxy is either of an —O-(alkyl) group and an —O-(unsubstituted cycloalkyl) group, and is linear or branched hydrocarbon having one or more ether groups and 1 to 10 carbon atoms.

Specific examples of the alkoxy include methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, n-hexoxy, 1,2-dimethylbutoxy, cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, and cyclohexyloxy, but the present invention is not limited thereto.

In addition, in the present invention, halogen means fluoro (—F), chloro (—Cl), bromo (—Br), or iodo (—I), and haloalkyl is alkyl substituted with the above-described halogen. For example, halomethyl means methyl in which at least one of the hydrogens is substituted with halogen (—CH2X, —CHX2or —CX3).

In the present invention, “aralkyl” is the generic term for —(CH2)nAr, which is a functional group in which a carbon of alkyl is substituted with aryl. Examples of the aralkyl include benzyl (—CH2C6H5) and phenethyl (—CH2CH2C6H5).

In the present invention, aryl is, unless defined otherwise, an unsaturated aromatic ring including a single ring, or multiple rings (preferably, 1 to 4 rings) conjugated or connected by covalent bonds. Non-limiting examples of the 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.

In the present invention, heteroaryl is a functional group in which one or more carbon atoms in the aryl defined above are substituted with a non-carbon atom such as nitrogen, oxygen or sulfur. Non-limiting examples of the heteroaryl include furyl, tetrahydrofuryl, pyrrolyl, pyrrolidinyl, thienyl, tetrahydrothienyl, oxazolyl, isoxazolyl, triazolyl, thiazolyl, isothiazolyl, pyrazolyl, pyrazolidinyl, oxadiazolyl, thiadiazolyl, imidazolyl, imidazolinyl, pyridyl, pyridaziyl, triazinyl, piperidinyl, morpholinyl, thiomorpholinyl, pyrazinyl, piperainyl, pyrimidinyl, naphthyridinyl, benzofuranyl, benzothienyl, indolyl, indolinyl, indolizinyl, indazolyl, quinolizinyl, quinolinyl, isoquinolinyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, pteridinyl, quinuclidinyl, carbazoyl, acridinyl, phenazinyl, phenothizinyl, phenoxazinyl, purinyl, benzimidazolyl, benzothiazolyl, and analogs conjugated therewith.

In the present invention, unless defined otherwise, a hydrocarbon ring (cycloalkyl) or a hydrocarbon ring having a hetero atom (heterocycloalkyl) may be understood as a cyclic structure of an alkyl or heteroalkyl, respectively.

Non-limiting examples of the cycloalkyls include cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, and cycloheptyl. Non-limiting examples of the heterocycloalkyls include 1-(1,2,5,6-tetrahydropyrinyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothiene-2-yl, tetrahydrothiene-3-yl, 1-piperazinyl, and 2-piperazinyl.

In addition, the cycloalkyl or heterocycloalkyl may have a form in which cycloalkyl, heterocycloalkyl, aryl or heteroaryl is conjugated or connected by a covalent bond.

Wherein, the polyalkyleneoxide is a water-soluble polymer functional group, and examples of such polyalkyleneoxides include polyethylene glycol (PEG), polypropylene glycol (PPG), a polyethylene glycol-polypropylene glycol (PEG-PPG) copolymer, and N-substituted methacrylamide-containing polymers and copolymers.

The polyalkyleneoxide may be additionally substituted, as needed, as long as the characteristics of the polymer are maintained. For example, the substitution may be a chemical bond for increasing or decreasing the chemical or biological stability of the polymer. As a specific example, any carbon or terminal carbon in the polyalkyleneoxide may be substituted with hydroxy, alkyl ether (methyl ether, ethyl ether, propyl ether or the like), carboxymethyl ether, carboxyethyl ether, benzyl ether, dibenzylmethylene ether, or dimethylamine. In one embodiment, the polyalkyleneoxide may be polyethyleneoxide terminated with methyl ether (mPEG), wherein mPEG is represented by the formula —(CH2CH2O)nCH3, whose size may change depending on the size of n corresponding to the number of ethylene glycol repeat units.

In addition, the reporters represented by Formulas 1, 2, and 5 may have a structure further including a counter ion. The counter ion, which is an organic or inorganic anion, may be suitably selected in consideration of the solubility and stability of the reporter.

Examples of the counter ions of the reporter according to one embodiment of the present invention include inorganic anions such as a phosphoric acid hexafluoride ion, a halogen ion, a phosphoric acid ion, a perchloric acid ion, a periodic acid ion, an antimony hexafluoride ion, a tartaric acid hexafluoride ion, a fluoroboric acid ion, and a tetrafluoride ion; and organic anions such as a thiocyanate ion, a benzenesulfonic acid ion, a naphthalenesulfonic acid ion, a p-toluenesulfonic acid ion, an alkylsulfonic acid ion, a benzenecarboxylic acid ion, an alkylcarboxylic acid ion, a trihaloalkylcarboxylic acid ion, an alkyl sulfonic acid ion, a trihaloalkylsulfonic acid ion, and a nicotinic acid ion. In addition, metal compound ions such as bisphenylditol, thiobisphenol chelate, and bisdiol-α-diketone, metal ions such as sodium and potassium, and quaternary ammonium salts may also be selected as the counter ions.

Specific examples of the reporters represented by Formulas 1, 2, and 5 are as follows. However, the following exemplary compounds are provided to help understanding the reporters defined herein and are not intended to limit the scope of the reporters defined herein.

A biomolecule targeted by the reporter represented by Formula 1, 2, or 5 disclosed herein may be at least one selected from an antibody, a lipid, a protein, a peptide, a carbohydrate, a nucleic acid (including DNA, RNA or a nucleotide), and preferably, a nucleic acid (including DNA, RNA or a nucleotide).

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

Wherein, a biomolecule may include at least one selected from amino, sulfhydryl, carbonyl, hydroxyl, carboxyl, phosphate and thiophosphate, or a derivative thereof as any functional group of a reporter represented by Formula 1, 2, or 5 or a functional group for reacting with a reactive group binding to the reporter represented by Formula 1, 2, or 5.

In addition, the biomolecule may be an oxy or deoxy polynucleotide which includes at least one selected from amino, sulfhydryl, carbonyl, hydroxyl, carboxyl, phosphate and thiophosphate, or a derivative thereof.

Moreover, in addition to biomolecules, the reporter represented by Formula 1, 2, or 5 may be used to label a drug, a hormone (including a receptor ligand), a receptor, an enzyme or an enzyme substrate, cells, a cell membrane, a toxin, a microorganism or a nano-biomaterial (a polystyrene microsphere, etc.) including at least one selected from amino, sulfhydryl, carbonyl, hydroxyl, carboxyl, phosphate and thiophosphate.

Oligonucleotide, Composition for Detecting Nucleic Acid, and Support for Detecting Nucleic Acid, Including Novel Reporter

According to another aspect of the present invention, an oligonucleotide including at least one selected from the reporters represented by Formulas 1, 2, and 5. The oligonucleotide refers to a polymer of one to several hundred nucleotides, and includes all of DNA, RNA, and PNA. In addition, examples of such oligonucleotides include those that can be easily modified by one of ordinary skill in the art, such as analogs thereof, for example, those in which chemical modifications have been applied to the nucleotides, or those in which sugars are linked, and encompasses single-stranded or double-stranded ones.

The oligonucleotide preferably includes a probe. Such a probe is more preferably a probe that is capable of complementarily binding to a target nucleic acid, but the present invention is not limited thereto. Wherein, the probe may be selected from a nucleic acid, a peptide, a saccharide, an oligonucleotide, a protein, an antibody, or a combination thereof, but the present invention is not limited thereto.

In one embodiment, the oligonucleotide may include a quencher. For example, the 5′ end of the oligonucleotide may be labeled with the reporter represented by Formula 1, 2, or 5, and the 3′ end thereof may be labeled with the quencher. A probe complementarily binding to a target nucleic acid may be located between the 5′ end and the 3′ end. In addition, the reporter represented by Formula 1, 2, or 5 may also be labeled at an internal site, other than the 5′ or 3′ end of the oligonucleotide.

The maximum absorbance of the quencher usable in the present invention may be 620 to 700 nm, and preferably, 660 to 680 nm, 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 considering the fluorescence properties of the reporter defined herein.

It is important that the probe is designed such that the reporter can be sufficiently quenched by the quencher while minimizing signal crosstalk. Accordingly, when designing a probe, depending on the type of target biomolecule (e.g., a nucleic acid), it is necessary to confirm that the reporter and the quencher, which are labeled at the 5′ end and the 3′ end of the probe, are compatible with each other.

As the quencher, various known or commercially available quenchers (e.g., BHQ0, BHQ1, BHQ2, BHQ3, BBQ650, DABCYL, TAMRA, MGBEclipse, Atto540Q, Atto575Q, Atto612Q, QSY7, and QSY21) may be used. In addition, as the quencher, the quenchers disclosed in Korean Unexamined Patent Application Publication No. 10-2020-0067733 may be used. Representative examples of the quenchers disclosed in Korean Unexamined Patent Application Publication No. 10-2020-0067733 are as follows.

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

The MGB is a crescent-shaped probe that can selectively bind non-covalently to a minor groove (e.g., shallow furrow in the DNA helix) included in a nucleic acid such as DNA.

Such an oligonucleotide may be used in various ways in the fields of chemistry and biology. Particularly, it may be useful for real-time PCR or a microarray, but the present invention is not limited thereto.

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

The composition for detecting a nucleic acid according to one embodiment of the present invention may further include an enzyme, a solvent (buffer, etc.) and other reagents, which are used for a reaction with a target biomolecule, in addition to an oligonucleotide including the reporter represented by Formula 1, 2, or 5, an MGB and a quencher at the same time.

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

In addition, according to still another aspect of the present invention, a support for detecting a nucleic acid, which includes the reporter represented by Formula 1, 2, or 5, a support, and a linker that connects the reporter and the support, is provided.

Accordingly, a biomolecule in a sample may be fixed on a support matrix through interaction with the reporter fixed on the support.

The support matrix may be manufactured with at least one selected from glass, cellulose, nylon, acrylamide gel, dextran, polystyrene, resin, alginate, collagen, peptides, fibrin, hyaluronic acid, agarose, polyhydroxyethylmethacrylate, polyvinyl alcohol, polyethylene glycol, polyethyleneoxide, polyethylene glycol diacrylate, gelatin, Matrigel, polylactic acid, carboxymethylcellulose, chitosan, latex, and Sepharose, and have a form of beads or a membrane.

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

Wherein, 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 a linker intended by the present invention.

For example, the linker may be selected from substituted or unsubstituted C1-C30alkyl, substituted or unsubstituted C2-C30heteroalkyl containing at least one hetero atom, substituted or unsubstituted C6-C30aryl, and substituted or unsubstituted C3-C30heteroaryl, and the above-mentioned functional groups may include at least one aminated carboxyl group (or amine group).

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

Method of Detecting Nucleic Acid

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

DNA Microarray

A DNA microarray is for measuring the fluorescence of a target nucleic acid by preparing a single-stranded probe nucleic acid which labels a target nucleic acid through a reaction with a dye and has a complementary base sequence to the target nucleic acid, and hybridizing the probe nucleic acid with the target nucleic acid denatured into a single strand on a substrate.

In the labeling method, when gene expression is investigated, as the probe nucleic acid immobilized on the substrate, cDNA, which is prepared by amplifying a cDNA library, genome library, or any of all genomes as a template through PCR, may be used.

In addition, for investigation of gene mutations, various oligonucleotides corresponding to mutations may be synthesized based on a known sequence serving as a reference and used.

A proper method for immobilizing the probe nucleic acid on the substrate may be selected according to the type of nucleic acid or substrate. For example, a method for electrostatic binding to a substrate surface-treated with a cation such as polylysine using the charge of DNA may also be used.

The target nucleic acid denatured into a single strand is immobilized on the substrate, and hybridized with the oligonucleotide. Wherein, the 5′ end of the oligonucleotide is labeled with at least one selected from the reporters represented by Formulas 1, 2, or 5, and the 3′ end thereof is labeled with a quencher. Between the 5′ end and the 3′ end, a probe that is able to complimentarily bind to the target nucleic acid may be located.

Hybridization is preferably performed at room temperature to 70° C. for approximately 2 to 48 hours. Through hybridization, a target nucleic acid having a complementary base sequence with the probe nucleic acid is selectively bound to probe nucleic acid. Afterward, the substrate is washed and dried at room temperature.

Wherein, the oligonucleotide is hybridized to the target nucleic acid by the probe, but the fluorophore at the 5′ end is present in a quenched state by the quencher at the 3′ end.

Subsequently, the oligonucleotide hybridized to the target nucleic acid is elongated by a polymerase, separated from the target nucleic acid due to the exonuclease activity of the polymerase, and degraded. The fluorophore at the 5′ end of the oligonucleotide and the quencher at the 3′ end thereof are separated from each other, and thus the fluorophore may exhibit fluorescence.

Wherein, the intensity of the generated fluorescence is measured to measure the amplification amount of the target nucleic acid.

PCR Method

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

Specifically, the elongation reaction of the target nucleic acid is carried out by an enzyme (DNA polymerase or RNA polymerase), and Wherein, a double-stranded nucleic acid sequence formed of the target nucleic acid and a primer consisting of an oligonucleotide is recognized by the enzyme to carry out the elongation reaction from the recognition site, and only a target gene area is amplified.

When synthesis is performed by the enzyme, the synthesis reaction is carried out using nucleotides (dNTP and NTP) as raw materials.

Wherein, by mixing common nucleotides (dNTP and NTP) with reporter-bearing nucleotides in an arbitrary ratio, a nucleic acid into which the equivalent amount of dye is introduced may be synthesized.

In addition, a nucleic acid into which a reporter is introduced may be synthesized by bonding the reporter after introducing nucleotides having an amino group in an arbitrary ratio by PCR.

When synthesis is performed by the enzyme, the synthesis reaction is carried out using nucleotides as raw materials, and Wherein, when a material in which the 3′ OH of the nucleotide is substituted with His used, a nucleic acid is no longer elongated, and at this point of time, the reaction ends.

This nucleotide, that is, dideoxynucleotide triphosphate (ddNTP) is called a terminator.

When a terminator is mixed with common nucleotides to synthesize a nucleic acid, the terminator is introduced with a certain probability to end the reaction, so nucleic acids of various lengths are synthesized.

When the above are separated by size through gel electrophoresis, DNA is lined up in order of length. Wherein, when labeled with a different reporter for each type of terminator base, at the end point (3′ end) of the synthesis reaction, a dependency on each base is observed, and by reading fluorescence information starting with the reporter attached to the terminator, base sequence information of the target nucleic acid may be obtained.

In addition, instead of the terminator, primers previously labeled with the reporter may be used for hybridization with a target nucleic acid.

In addition, as a probe, a peptide nucleic acid (PNA) may also be used. PNA is obtained by replacing the pentose phosphate backbone, which is the basic skeleton of a nucleic acid, with a polyamide backbone composed of glycine as a unit, and PNA has a 3D structure highly similar to nucleic acids, and is very specific for a nucleic acid having a complementary base sequence and strongly binds thereto. Accordingly, PNA may also be used as a reagent for telomere research by applying a telomere PNA probe, in addition to a conventional DNA analysis method such as in-situ hybridization (ISH).

For labeling, for example, double-stranded DNA is brought into contact with PNA having a base sequence complementary to all or a part of the base sequence of the DNA and labeled with a reporter for hybridization, the mixture is heated to generate single-stranded DNA, and slowly cooled to room temperature to prepare a PNA-DNA complex, and then fluorescence is measured.

In the above example, a method of amplifying a target nucleic acid through PCR and measuring the fluorescence of a product has been described, but in this method, it is necessary to identify the size of the product through electrophoresis and then investigate the amount of amplification product by measuring fluorescence intensity.

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

For example, DNA labeled with a donor and an acceptor may be used. A specific labeling method may be a molecular beacon method, a TaqMan-PCR method, or a cycling probe method, which is used to confirm the presence of a nucleic acid having a specific sequence.

Other Labeling Methods

In addition, the reporter of the present invention may also be used in a method of labeling a target using specific binding.

That is, in the labeling of a sample including a target or a sample modified by a modifying material, one of a binding material specifically binding to the sample and a binding material specifically binding to the modifying material may be labeled with a reporter, and fluorescence may be measured from the labeled binding materials.

Wherein, for the combination of the sample or modifying material with the binding material, antigen-antibody, hapten-anti-hapten antibody, biotin-avidin, a Tag antigen, a Tag antibody, lectin-glycoprotein, or hormone-receptor may be used.

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

An antigen may be a protein, a polysaccharide, a nucleic acid, or a peptide, and other than the antigen, a hapten such as a low-molecular-weight molecule, for example, FITC or a dinitrophenyl group may also be used. Wherein, as an antigen (or hapten)-antibody combination, there are GFP and anti-GFP antibodies, FITC and anti-FITC antibodies and the like.

Labeled antigens may be used in various measurement methods including immunostaining, ELISA, Western blotting or flow cytometry.

In addition, an intracellular signaling phenomenon may be observed using the reporter of the present invention. Various enzymes are involved in internal signaling or cell responses according to the signaling. In a representative signaling phenomenon, it is known that a special protein kinase is activated, thereby inducing protein phosphorylation to initiate signaling.

Binding and hydrolysis of a nucleotide (e.g., ATP or ADP) play a critical role in its activity, and an intracellular signaling phenomenon may be observed with high sensitivity by introducing a reporter into a nucleotide derivative.

In addition, the reporter of the present invention may also be used in observation of a gene expression phenomenon using RNA interference (RNAi).

RNAi is inhibition of expression by degradation of mRNA of a target gene by introducing double-stranded RNA (dsRNA) into cells, and thus it is possible to observe the RNAi phenomenon by labeling designed dsRNA with a reporter.

In addition, since the reporter of the present invention has a reactive group capable of labeling a target nucleic acid or target protein in tissue or cells, it may be used as a dye for confirming the transcription level of a target nucleic acid or the expression level of a target protein.

Hereinafter, specific examples of the present invention are presented. However, the following examples are only for exemplifying or explaining the present invention in detail, and the present invention is not limited thereto. In addition, among the reporters defined in the claims and detailed description of the present invention, compounds whose synthesis methods are not disclosed through the following preparation examples may be synthesized with reference to the following preparation examples.

Preparation Example 1

(1) Synthesis of Compound 1

Synthesis of Intermediate 2

Intermediate 1 (synthesized with reference to Korean Unexamined Patent Application 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), and dichloromethane (600 mL) were put into a reactor and stirred at room temperature for 2 hours, and 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 laid-open Patent Application Publication No. 2016-100401) (17.4 g, 0.025 mol), triethylamine (7.2 mL, 0.052 mol), tetrahydrofuran (300 mL), and dichloromethane (300 mL) were put into a reactor and stirred at room temperature for 1.5 hours. An aqueous sodium bicarbonate solution was put into the reactor and stirred vigorously and then the organic layer was separated. After adding sodium sulfate to the organic layer and stirring, the solid was filtered, and the filtrate was concentrated and purified by column chromatography.

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), and dichloromethane (160 mL) were put into a reactor and stirred at room temperature for 1.5 hours. After adding water to the reactor and stirring vigorously, the organic layer was separated. After adding sodium sulfate to the organic layer and stirring, the solid was filtered and the filtrate was concentrated and purified by column chromatography.

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

1H NMR (400 MHZ, CDCl3) 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 laid-open Patent Application Publication No. 2017-010852X (40.0 g, 0.139 mol), methyl iodide (39.5 g, 0.278 mol), and acetonitrile (400 mL) were put into a reactor, and stirred for 16 hours under reflux. 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 Publication No. 5760201) (50 g, 0.105 mol), triethyl amine (73.7 ml, 529.0 mol), and acetonitrile (500 mL) were put into a reactor, stirred for 2 hours under reflux and then concentrated and purified by column chromatography.

Synthesis of Intermediate 11

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

Synthesis of Intermediate 12

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

Synthesis of Intermediate 13

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

Synthesis of Compound 2

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

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

1H NMR (400 MHZ, CDCl3) 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 Application 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), and citric acid (250 mL) were put into a reactor, stirred for 3 days under reflux, and then concentrated and purified by column chromatography.

Synthesis of Intermediate 18

Intermediate 18 was synthesized in the same manner as in 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), and acetic anhydride (1.6 L) were put into a reactor and stirred at 110° C. for 2 hours. After cooling, the reaction solution was added to ethyl ether (6 L) and 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 put into a reactor, stirred at 70° C. for 4 hours and then concentrated and purified by column chromatography.

Synthesis of Intermediate 23

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

Synthesis of Intermediate 24

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

Synthesis of Compound 3

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

1H-NMR of the obtained Compound 3 is as follows.

1H NMR (400 MHZ, CDCl3) 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)

A single-labeled oligonucleotide in which Compound 1, 2 or 3 as a reporter was labeled at the 5′ end was synthesized using the MerMade™ 48×DNA Synthesizer and Universal UnyLinker Support (Chemgene, 500 Å) as CPG. The sequence of the single-labeled oligonucleotide is shown in Table 1 below.

TABLE 1ClassificationSequenceSLP15′-Compound 1-dt-TTT TTT TTT T-3′SLP25′-Compound 2-dt-TTT TTT TTT T-3′SLP35′-Compound 3-dt-TTT TTT TTT T-3′

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

TABLE 2ReporterExcitationmax(nm)Emissionmax(nm)Compound 1602628Compound 2647674Compound 3689716

Preparation Example 3. Synthesis of Dual-Labeled Probe (Oligonucleotide)

Using the Universal UnyLinker Support (Chemgene, 500 Å), each of 5′-ATG CAA CAT TAA CCC GAG ATA CG-3′ as a forward primer and 5′-ACT CGG CTT GGG AAG AGC TT-3′ as a reverse primer forChlamydia trachomatis(CT) was synthesized at 1 μmol scale and purified by HPLC.

The dual-labeled probe is a CT-selective labeled probe, and 5′-TTG TCC ATA TCT TTG ATA CGA CGC CGC-quencher-3′ was synthesized using [Quencher 7]-attached CPG (synthesized with reference to Korean Unexamined Patent Application Publication No. 10-2020-0067733) and CPG to which Black Hole Quenchers 2 and 3 (BHQ2 and BHQ3, LGC Biosearch Technologies), which are commercially available quenchers, are attached, and then dual-labeled probes labeled with Compound 1, 2, or 3 were synthesized as reporters at 1 μmol scale each and then subjected to HPLC purification.

The forms of the synthesized dual-labeled probes are shown in Table 3 below, and the absorption wavelength ranges of Quencher 7, BHQ2, and BHQ3, used as the quenchers of the dual-labeled probes, are shown in Table 4 below.

TABLE 3Classi-ficationSequenceDLP15′-Compound 1-dT-TTGTCCATATCTTTGATACGACGCCGC-Quencher 7-3′DLP25′-Compound 2-dT-TTGTCCATATCTTTGATACGACGCCGC-Quencher 7-3′DLP35′-Compound 3-dT-TTGTCCATATCTTTGATACGACGCCGC-Quencher 7-3′DLP45′-Compound 1-dT-TTGTCCATATCTTTGATACGACGCCGC-BHQ2-3′DLP55′-Compound 2-dT-TTGTCCATATCTTTGATACGACGCCGC-BHQ2-3′DLP65′-Compound 3-dT-TTGTCCATATCTTTGATACGACGCCGC-BHQ3-3′

TABLE 4ClassificationλMax(nm)ε (mol−1· cm−1)Quencher 7580~710140,000BHQ2550~65038,000BHQ3620~73042,000

The structure of the Quencher 7-attached CPG used in Preparation Example 3 is as follows.

Experimental Example 1. Real-Time PCR Experiment Using Dual-Labeled Probe

Real-Time PCR was repeatedly performed twice on CT plasmid DNA using each dual-labeled probe synthesized according to Preparation Example 3 with the composition shown in Table 5 (using CFX-96, Biorad). The real-time PCR results are shown inFIGS.1and2.

TABLE 5ClassificationContent (μl)(Bioline)SensiFAST ™ Probe No-ROX Mix (2X)10CT plasmid DNA (5 × 10{circumflex over ( )}6, 5 × 10{circumflex over ( )}4, 5 × 10{circumflex over ( )}31copies/μl)CT F/R primer mix (10 pmole/μl)1CT Dual-labeled probe (7 pmole/μl)3Pure Water5

Referring toFIGS.1to3showing the real-time PCR results, compared with DLP4 to DLP6 using commercially-available BHQ2 or BHQ3 as one of the quenchers of Compounds 1 to 3, it can be seen that DLP 1 to DLP3 using Quencher 7 show relatively low Ct values, and the final fluorescence amplification intensity is improved approximately 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, it can be expected that the ease of detecting the target will be improved by using Quencher 7 as a quencher.

In addition, referring toFIGS.4to9, it can be confirmed that Compounds 1 to 3 as reporters for labeling a nucleic acid defined herein exhibit excellent linearity not only for commercially available quencher BHQ2 or BHQ3 but also for Quencher 7.

Accordingly, the reporter for labeling a nucleic acid defined herein is expected to be sufficiently used to label not only CT nucleic acids but also various nucleic acids, and it can be seen that the reporter for labeling a nucleic acid defined herein can also be easily applied to existing commercially available quenchers without separate technical modifications.

Meanwhile, it is known that most existing, commercially-available reporters for labeling a nucleic acid have limitations in that substitutions can only be made at the 5′ end. On the other hand, as the reporter for labeling a nucleic acid defined herein has a substituted form at dT, substitution can be made not only at the 5′ end but also internal position of the sequence and thus can be contributed to improve the intensity of fluorescence amplification by closely adjusting the distance between a reporter and a quencher.

Although embodiments of the present invention have been described above, it will be understood by those of ordinary skill in the art that the present invention may be modified and altered in various ways by adding, altering, or deleting a component without departing from the spirit of the present invention defined in the appended claims, and such modifications and alterations are also be included in the scope of the present invention.