# US 12,215,381 B2 · Reporter and uses thereof

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- Number: US 12,215,381 B2
- Document ID: US12215381B2
- Status: Granted
- Kind: B2
- Application number: 18/461,211
- Filed: 2023-09-05
- Published: 2024-04-25
- Granted: 2025-02-04
- Earliest claimed: 2021-03-10
- Assignees: Sfc Co., Ltd.
- Applicants: Sfc Co., Ltd.
- Inventors: Do Min Lee; Ho Young Si; Jae Hyung Jo; Goutam Masanta; Ju Man Song; Min Su Eum; Jong Tae Je
- Web page: https://patentgrep.com/patent/US12215381B2

## CPC

- C12Q 1/682: Signal amplification
- C07H 19/06: Pyrimidine radicals
- C07H 19/10: with the saccharide radical esterified by phosphoric or polyphosphoric acids
- C07H 21/00: Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
- C07H 21/04: with deoxyribosyl as saccharide radical
- C09B 23/00: Methine or polymethine dyes, e.g. cyanine dyes
- C09K 11/06: containing organic luminescent materials
- C12Q 1/6818: involving interaction of two or more labels, e.g. resonant energy transfer
- C12Q 1/6834: Enzymatic or biochemical coupling of nucleic acids to a solid phase
- C12Q 1/686: Polymerase chain reaction [PCR]
- G01N 21/6486: {Measuring fluorescence of biological material, e.g. DNA, RNA, cells (G01N21/6428 takes precedence)}
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C07H: SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS (derivatives of aldonic or saccharic acids C07C, C07D; aldonic acids, saccharic acids C07C59/105, C07C59/285; cyanohydrins C07C255/16; glycals C07D; compounds of unknown constitution C07G; polysaccharides, derivatives thereof C08B; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; sugar industry C13)
- C09B: ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES {, e.g. PIGMENTS}; MORDANTS; LAKES (fermentation or enzyme using processes to synthesise a desired chemical compound C12P)
- C09K: MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

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.

## Claims

### Claim 1

A reporter for labeling a nucleic acid represented by Formula 5 below:
wherein,
R21 and R22 are each independently selected from hydrogen, deuterium, substituted or unsubstituted C1-C10 alkyl, substituted or unsubstituted C1-C10 heteroalkyl containing at least one hetero atom, substituted or unsubstituted C2-C10 alkenyl, substituted or unsubstituted C2-C10 alkynyl, substituted or unsubstituted C3-C20 cycloalkyl, substituted or unsubstituted C3-C20 cycloalkenyl, substituted or unsubstituted C2-C20 heterocycloalkyl, substituted or unsubstituted C5-C20 aryl, and substituted or unsubstituted C2-C20 heteroaryl,
R23 to R30 are each independently a functional group selected from hydrogen, deuterium, substituted or unsubstituted C1-C10 alkyl, substituted or unsubstituted C1-C10 heteroalkyl containing at least one hetero atom, substituted or unsubstituted C2-C10 alkenyl, substituted or unsubstituted C2-C10 alkynyl, substituted or unsubstituted C3-C20 cycloalkyl, substituted or unsubstituted C3-C20 cycloalkenyl, substituted or unsubstituted C2-C20 heterocycloalkyl, hydroxy, oxido (—O−), substituted or unsubstituted C1-C10 alkoxy, substituted or unsubstituted C3-C20 cycloalkyloxy, substituted or unsubstituted C5-C20 aryloxy, substituted or unsubstituted C2-C20 heteroaryloxy, substituted or unsubstituted C5-C20 aryl, substituted or unsubstituted C2-C20 heteroaryl, substituted or unsubstituted C5-C20 aralkyl, substituted or unsubstituted C1-C10 alkylthio, substituted or unsubstituted C5-C20 arylthio, substituted or unsubstituted C3-C20 cycloalkylthio, substituted or unsubstituted C2-C20 heteroarylthio, substituted or unsubstituted acylamino, acyloxy, substituted or unsubstituted phosphino, trifluoromethylsulfonyl (—SO2CF3), substituted or unsubstituted ammonium, nitro, 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, 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, substituted or unsubstituted phosphine, nitrile, hydrazine, acetal, ketal, polyalkyleneoxide, and -L1-R12, or two adjacent functional groups are bonded to each other to form a ring,
R31 to R34 are each independently selected from hydrogen, deuterium, substituted or unsubstituted C1-C10 alkyl, substituted or unsubstituted C1-C10 heteroalkyl containing at least one hetero atom, and -L1-R12, and at least one of R31 to R34 is -L1-R12,
L1 includes a single bond or at least one selected from substituted or unsubstituted C1-C10 alkyl, substituted or unsubstituted C1-C10 heteroalkyl containing at least one hetero atom, substituted or unsubstituted C2-C10 alkenyl, substituted or unsubstituted C2-C10 alkynyl, substituted or unsubstituted C3-C20 cycloalkyl, substituted or unsubstituted C3-C20 cycloalkenyl, and substituted or unsubstituted C2-C20 heterocycloalkyl, and
R12 is a deoxyribonucleoside represented by Formula 3 below,
Wherein,
* indicates a location where the deoxyribonucleoside is bonded to L1,
B is a nucleobase,
R13 is selected from hydrogen, deuterium, P(OR15)(N(R16R17), and -L2-R18,
R14 is an alcohol protecting group, hydrogen or P(OR15)(N(R16R17), or a support or nucleic acid to which the reporter is bound, at least one of R13 and R14 is P(OR15)(N(R16R17),
R15 to R17 are each independently selected from hydrogen, deuterium, substituted or unsubstituted C1-C10 alkyl, and substituted or unsubstituted C1-C10 heteroalkyl containing at least one hetero atom,
L2 is a single bond, or selected from an internucleotide phosphodiester bond, substituted or unsubstituted C1-C10 alkyl and substituted or unsubstituted C1-C10 heteroalkyl containing at least one hetero atom,
R18 is hydroxy or P(OR15)(N(R16R17), or a support or nucleic acid to which the reporter is bound,
Y is CR35, each R35 is selected from hydrogen, deuterium, substituted or unsubstituted C1-C10 alkyl, substituted or unsubstituted C1-C10 heteroalkyl containing at least one hetero atom, fluoro (—F), chloro (—Cl), bromo (—Br), and iodo (—I), and
n is an integer of 1 to 4,
when any functional group of R21 to R35 is a substituted functional group, any substituent other than hydrogen bonded to at least one carbon of the functional group,
wherein the substituent is selected from deuterium, C1-C10 alkyl, C1-C10 heteroalkyl containing at least one hetero atom, C2-C10 alkenyl, C2-C10 alkynyl, C3-C20 cycloalkyl, C3-C20 cycloalkenyl, C2-C20 heterocycloalkyl, hydroxy, oxido (—O−), C1-C10 alkoxy, C3-C20 cycloalkyloxy, C5-C20 aryloxy, C2-C20 heteroaryloxy, C5-C20 aryl, C2-C20 heteroaryl, C5-C20 aralkyl, C1-C10 alkylthio, C5-C20 arylthio, C3-C20 cycloalkylthio, C2-C20 heteroarylthio, acylamino, acyloxy, phosphino, trifluoromethylsulfonyl (—SO2CF3), ammonium, nitro, substituted sulfonyl, substituted sulfonic acid ester, sulfonamide, substituted thioketone, trihalomethyl (—CF3, —CCl3, —CBr3, or —CI3), haloformyl (—COCl, —COBr, or —COI), formyl (—CHO), acyl, substituted ester, aminocarbonyl, nitro, nitroso (—N═O), fluoro (—F), chloro (—Cl), bromo (—Br), iodo (—I), germanium, boron, aluminum, silyl, amide, carbamate, phosphine, nitrile, hydrazine, acetal, ketal, polyalkyleneoxide, and -L1-R12.

### Claim 2 (depends on 1)

The reporter of claim 1, wherein L1 is a linker including at least one carbon, and any carbon of the linker is a carbonyl carbon.

### Claim 3 (depends on 1)

The reporter of claim 1, wherein R12 is a deoxyribonucleoside represented by Formula 4 below:

### Claim 4 (depends on 1)

An oligonucleotide, comprising:
the reporter according to claim 1; and
a quencher.

### Claim 5 (depends on 4)

The oligonucleotide of claim 4, further comprising:
a minor groove binder (MGB) interposed between the reporter and the quencher.

### Claim 6 (depends on 4)

A composition for detecting a nucleic acid, comprising the oligonucleotide of claim 4.

### Claim 7 (depends on 1)

A support for detecting a nucleic acid, comprising:
the reporter of claim 1;
a support; and
a linker connecting the reporter and the support, through any position of the compound of Formula 3.

### Claim 8 (depends on 7)

The support of claim 7, wherein the support is glass, cellulose, nylon, acrylamide gel, dextran, polystyrene, or resin.

### Claim 9 (depends on 4)

A method of detecting a nucleic acid, comprising:
(a) preparing a reaction mixture including a target nucleic acid, a reagent required for amplifying the target nucleic acid, and the oligonucleotide of claim 4;
(b) amplifying the target nucleic acid in the reaction mixture through polymerase chain reaction; and
(c) measuring a fluorescence intensity of the reaction mixture.

### Claim 10 (depends on 9)

The method of claim 9, wherein (b) includes
(b-1) elongating 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) allowing the reporter separated from the quencher to emit fluorescence.

### Claim 11 (depends on 9)

The method of claim 9, further comprising:
(d) measuring an amplification amount of the target nucleic acid from the fluorescence intensity measured in (c).

### Claim 12 (depends on 1)

An oligonucleotide, comprising:
the reporter according to claim 1.