Quencher and uses thereof

Patent metadata

Field	Value
Publication number	US-2022274960-A1
Application number	US-202217747863-A
Country	US
Kind code	A1
Filing date	May 18, 2022
Priority date	Nov 19, 2019
Publication date	Sep 1, 2022
Grant date	—

Abstract

The present invention relates to a quencher exhibiting a quenching effect on a fluorescent material exhibiting a luminescent property at an excited energy level, and various uses thereof.

First claim

What is claimed is:

1 . A quencher represented by Formula 1 below:

wherein
R 1 and R 2 are each conjugated with a and b, b and c, or c and d of Formula 2 below,

Ar 1 is selected from substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, and Formula 2,
n is an integer of 1 to 3, and
R 3 to R 19 are each independently selected from a functional group selected from 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 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 1 -C 10 alkoxy, substituted or unsubstituted aryloxy, substituted or unsubstituted C 1 -C 10 haloalkyl, a halogen, cyano, hydroxy, substituted or unsubstituted amino, substituted or unsubstituted amide, carbamate, sulfhydryl, nitro, carboxyl, carboxylate, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted aralkyl, quaternary ammonium, phosphoric acid, phosphate, substituted ketone, aldehyde, substituted ester, substituted sulfonyl, substituted or unsubstituted sulfonamide, acyl chloride, sulfonic acid, sulfonate, hydrazine, thiol, acetal, ketal, phosphonate (phosphite), hypophosphite, sulfohydroxy, sulfate, azido, guanidium, ketene, thiocarbonyl, aminothiocarbonyl, polyalkyleneoxide, a carboxyl derivative, dienophile, sulfonyl halide, epoxide, and phosphoramidite; and a reactive group capable of covalent bonding with a functional group selected from carboxyl, a carboxyl derivative, hydroxyl, haloalkyl, dienophile, aldehyde, ketone, sulfonyl halide, thiol, amine, sulfhydryl, alkene, epoxide, and phosphoramidite,
R 5 and R 6 are each independently present or connected to form a ring,
R 7 and R 8 are each independently present or connected to form a ring,
R 18 and R 19 are each independently present or connected to form a ring, and
L 1 and L 3 are linkers including a single bond or 1 to 40 non-hydrogen atoms, and L 2 and L 4 are linkers including 1 to 40 non-hydrogen atoms.

2 . The quencher of claim 1 , wherein R 1 and R 2 of Formula 1 are conjugated with a and b of Formula 2.

3 . The quencher of claim 1 , wherein R 1 and R 2 of Formula 1 are conjugated with b and c of Formula 2.

4 . The quencher of claim 1 , wherein R 1 and R 2 of Formula 1 are conjugated with c and d of Formula 2.

5 . The quencher of claim 1 , wherein at least one of R 3 to R 19 is a functional group selected from carboxyl, a carboxyl derivative, hydroxyl, haloalkyl, dienophile, aldehyde, ketone, sulfonyl halide, thiol, amine, sulfhydryl, alkene, epoxide, and phosphoramidite, or a reactive group capable of covalent bonding with the functional group.

6 . The quencher of claim 1 , wherein at least one of at least one of the substituents of R 3 to R 19 and Ar 1 is a functional group selected from carboxyl, a carboxyl derivative, hydroxyl, haloalkyl, dienophile, aldehyde, ketone, sulfonyl halide, thiol, amine, sulfhydryl, alkene, epoxide, and phosphoramidite, or a reactive group capable of covalent bonding with the functional group.

7 . The quencher of claim 1 , wherein the reactive group is selected from carboxyl, a carboxyl derivative, hydroxyl, haloalkyl, dienophile, aldehyde, ketone, sulfonyl halide, thiol, amine, sulfhydryl, alkene, epoxide, and phosphoramidite, and protected with a protective group.

8 . The quencher of claim 1 , wherein adjacent substituents of R 14 to R 17 are connected to each other to form a ring.

9 . The quencher of claim 1 , wherein at least one of the substituents of Ar 1 is a substituted amino group.

10 . The quencher of claim 9 , wherein the substituent binding to nitrogen of the amino group is connected with another substituent binding to the nitrogen to form a ring, or with another substituent, other than the amino group, to form a ring.

11 . The quencher of claim 1 , which is represented by Formula 3 below:

R 21 to R 23 are as defined above for R 3 to R 19 ,
at least one of R 3 to R 23 is a functional group selected from carboxyl, a carboxyl derivative, hydroxyl, haloalkyl, dienophile, aldehyde, ketone, sulfonyl halide, thiol, amine, sulfhydryl, alkene, epoxide, and phosphoramidite, or a reactive group capable of covalent bonding with the functional group, and
at least one of R 3 to R 23 is a substituted or unsubstituted amino group.

12 . The quencher of claim 11 , wherein at least one of R 20 to R 23 is a substituted amino group.

13 . The quencher of claim 11 , wherein R 21 is a substituted amino group.

14 . The quencher of claim 12 , wherein the substituent binding to nitrogen of the amino group forms a ring by connection with another substituent binding to the nitrogen, or by connection with another substituent, other than the amino group.

15 . An oligonucleotide comprising:
the quencher according to claim 1 ; a minor groove binder (MGB); and a fluorophore.

16 . The oligonucleotide of claim 15 , wherein the fluorophore is at least one selected from coumarin, cyanine, BODIPY, fluoroscein, rhodamine, pyrene, carbopyronine, oxazine, xanthane, thioxanthane, acridine, and a derivative thereof.

17 . A composition for detecting a nucleic acid comprising the oligonucleotide of claim 15 .

18 . A support for detecting a nucleic acid, comprising:
the quencher according to claim 1 ; a support; and a linker connecting the quencher with the support.

19 . The support of claim 18 , wherein the support is glass, cellulose, nylon, acrylamide gel, dextran, polystyrene or resin.

20 . The support of claim 18 , wherein the linker is selected from substituted or unsubstituted C 1 -C 30 alkyl, substituted or unsubstituted C 2 -C 30 heteroalkyl having at least one hetero atom, substituted or unsubstituted C 6 -C 30 aryl, and substituted or unsubstituted C 3 -C 30 heteroaryl.

21 . A method of detecting a nucleic acid, comprising:
(a) preparing a reaction mixture comprising a target nucleic acid, a reagent for amplifying the target nucleic acid, and the oligonucleotide of claim 15 ; (b) amplifying the target nucleic acid of the reaction mixture by a polymerase chain reaction; and (c) measuring the fluorescence intensity of the reaction mixture.

22 . The method of claim 21 , wherein, Step (b) comprises
(b-1) elongating an oligonucleotide hybridized to the target nucleic acid by a polymerase; (b-2) separating the quencher and the fluorophore of the oligonucleotide from the target nucleic acid by the exonuclease activity of the polymerase; and (b-3) emitting fluorescence from the fluorophore separated from the quencher.

23 . The method of claim 21 , further comprising:
(d) measuring the amplification amount of the target nucleic acid from the intensity of fluorescence measured in Step (c).

Assignees

• Sfc Co Ltd

Inventors

• Kim Tae Young
• Masanta Goutam
• Lee Do Min
• Song Ju Man
• Je Jong Tae

Classifications

• C09B23/083 Primary

  five >CH- groups · CPC title

• C07H21/00

  Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids · CPC title

• C09B23/06

  three >CH- groups, e.g. carbocyanines · CPC title

• C07D403/14

  containing three or more hetero rings · CPC title

• C07D403/06 Primary

  linked by a carbon chain containing only aliphatic carbon atoms · CPC title