Enhancement of nucleic acid polymerization by aromatic compounds

Patent metadata

Field	Value
Publication number	US-11970731-B2
Application number	US-201816959985-A
Country	US
Kind code	B2
Filing date	Dec 27, 2018
Priority date	Jan 5, 2018
Publication date	Apr 30, 2024
Grant date	Apr 30, 2024

Abstract

The invention relates to compounds, methods and compositions for improving on nucleic acid polymerization, including DNA replication by in vitro primer extension to generate, for example, polymers for nanopore-based single molecule sequencing of a DNA template. A nucleic acid polymerase reaction composition is provided with polymerization enhancement moieties, which allows enhanced DNA polymerase activity with nucleotide analogs, resulting in improved length of primer extension products for sequencing applications.

First claim

What is claimed is:

1. A method of enhancing a nucleic acid polymerase reaction, the method comprising:
a. forming a nucleic acid polymerase reaction composition comprising:
i. a template nucleic acid,
ii. a nucleic acid polymerase,
iii. a mixture of nucleotides or nucleotide analogs, and
iv. at least one compound of formula (I); and

b. incubating the nucleic acid polymerase reaction composition under conditions allowing a nucleic acid polymerization reaction, wherein the at least one compound of formula (I) increases the processivity, rate, or fidelity of the nucleic acid polymerase reaction; wherein the compound of formula (I) is represented by:

wherein, independently at each occurrence:
m is 1, 2 or 3;
n is 0, 1 or 2;
p is 0, 1 or 2;
Ar1 is optionally substituted aryl;
Ar2 is selected from 5- and 6-membered monocyclic aromatic rings and 9- and 10-membered fused bicyclic rings comprising two 5- and/or 6-membered monocyclic rings fused together, where at least one of the two monocyclic rings is an aromatic ring, where
Ar2 is optionally substituted with one or more substituents selected from halide, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, E-CO 2 R 0 , E-CONH 2 , E-CHO, E-C(O)NH(OH), E-N(R 0 ) 2 , and E-OR 0 , where
E is selected from a direct bond and C 1 -C 6 alkylene; and
R 0 is selected from H, C 1 -C 6 alkyl and C 1 -C 6 haloalkyl,
M is selected from hydrogen, halogen and C 1 -C 4 alkyl; and
L is a linking group;
or a solvate, hydrate, tautomer, chelate or salt thereof.

2. The method of claim 1 , wherein Ar1 is monocyclic carbocyclic aryl.

3. The method of claim 1 , wherein Ar1 is monocyclic heterocyclic aryl.

4. The method of claim 1 , wherein Ar1 is bicyclic aryl.

5. The method of claim 1 , wherein Ar1 is tricyclic aryl.

6. The method of claim 1 , wherein Ar1 is unsubstituted aryl.

7. The method of claim 1 , wherein Ar1 is substituted aryl.

8. The method of claim 1 , wherein Ar2 is a 5-membered monocyclic aromatic ring selected from the group consisting of thiophene, 1,2-thiazole, 1,3-thiazole, furan, 1,2-oxazole, 1,3-oxazole, 1H-pyrrole, 1H-pyrazole, oxadiazole, thiadiazole, 1,2,4-triazole, 1,2,3-triazole and 1H-imidazole.

9. The method of claim 1 , wherein Ar2 is a 6-membered monocyclic aromatic ring selected from the group consisting of benzene, pyridine, pyridazine, pyrimidine and pyrazine.

10. The method of claim 1 , wherein Ar2 is a 9-membered fused bicyclic aromatic ring system selected from the group consisting of benzofuran, 1,3-benzoxazole, furo[3,2-b]pyridine, furo[3,2-c]pyridine, furo[2,3-c]pyridine, furo[2,3-b]pyridine, indole, 1H-benzimidazole, 1H-pyrrolo[3,2-b]pyridine, 1H-pyrrolo[3,2-c]pyridine, 1H-pyrrolo[2,3-c]pyridine, 1H-pyrrolo[2,3-b]pyridine, benzothiophene, 1,3-benzothiazole, thienol[3,2-b]pyridine, thieno[3,2-c]pyridine, thieno[2,3-c]pyridine, benzoxadiazole, benzothiadiazole, benzisoxazole, benzotriazole and thieno[2,3-b]pyridine.

11. The method of claim 1 , wherein Ar2 is a 10-membered fused bicyclic aromatic ring system selected from the group consisting of naphthylene, quinoline, quinazoline, quinoxaline, 1,5-naphthyridine, 1,6-naphthyridine, 1,7-naphthyridine, 1,8-naphthyridine, isoquinoline, phthalazine, 2,6-naphthyridine and 2,7-naphthyridine.

12. The method of claim 1 , wherein the substitution on Ar2 includes carboxylic acid.

13. The method of claim 1 , wherein substitution on Ar2 includes trifluormethyl.

14. The method of claim 1 , wherein substitution on Ar2 includes hydroxyl.

15. The method of claim 1 , wherein substitution on Ar2 includes at least two of hydroxyl, carboxylic acid and trifluoromethyl.

16. The method of claim 15 , wherein the at least one compound of formula (I) is described by a formula selected from:

17. The method of claim 1 , wherein the compound of formula (I) is in a form of a chelate.

18. The method of claim 17 , wherein the chelate is a copper chelate.

19. The method of claim 1 , wherein the compound of formula (I) has a log P of at least 4.9.

20. The method of claim 1 , wherein the compound of formula (I) is selected from:
4,4′-(pyridine-2,6-diylbis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid);
4,4′-(pyridine-3,5-diylbis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid);
4,4′-(1,3-phenylenebis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid);
4,4′-((9H-carbazole-3,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid);
4,4′-((9H-carbazole-3,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))dianiline;
4,4′-((9H-carbazole-3,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))dibenzoic acid;
3,6-bis(1-(4-methoxyphenyl)-1H-1,2,3-triazol-4-yl)-9H-carbazole;
4,4′-((4-methoxypyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid);
4,4′-((4-carboxypyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid);
4,4′-((4-nitropyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid);
5,5′-((4-cyanopyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid);
4,4′-((4-methylpyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid);
4,4′-((4-(ethoxycarbonyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid);
5,5′-((4-(ethoxycarbonyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid);
4,4′-((4-(methoxycarbonyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid);
4,4′-((4-(ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid);
4,4′-((4-(methylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid);
4,4′-((4-carbamoylpyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid);
4,4′-(pyrazine-2,6-diylbis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid);
4,4′-(1,4-phenylenebis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid);
4,4′-(1,3-phenylenebis(1H-1,2,3-triazole-4,1-diyl))dianiline;
4,4′-(1,3-phenylenebis(1H-1,2,3-triazole-4,1-diyl))dibenzoic acid;
4-(4-(3-(1-(4-methoxyphenyl)-1H-1,2,3-triazol-4-yl)phenyl)-1H-1,2,3-triazol-1-yl)benzoic acid;
4,4′-(pyridine-2,6-diylbis(1H-1,2,3-triazole-4,1-diyl))dianiline;
4,4′-(pyridine-2,6-diylbis(1H-1,2,3-triazole-4,1-diyl))dibenzoic acid;
2,6-bis(1-(4-methoxyphenyl)-1H-1,2,3-triazol-4-yl)pyridine;
4-(4-(3-(1-(4-carboxyphenyl)-1H-1,2,3-triazol-4-yl)phenyl)-1H-1,2,3-triazol-1-yl)-2-hydroxybenzoic acid;
4-(4-(3-(1-(4-methoxyphenyl)-1H-1,2,3-triazol-4-yl)phenyl)-1H-1,2,3-triazol-1-yl)benzoic acid;
4,4′-((3,5-dimethylpyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid);
4,4′-((413-pyridine-2,6-diyl)bis(5-iodo-1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid);
4,4′-((4-acetamidopyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid);
4,4′-((9-acetyl-9H-carbazole-3,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid);
4,4′-(pyridine-2,6-diylbis(1H-1,2,3-triazole-4,1-diyl))bis(N,2-dihydroxybenzamide);
4,4′-(pyridine-2,6-diylbis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzamide);
4,4′-((4-carboxypyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid);
4,4′-((1,10-phenanthroline-2,9-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid);
4,4′-((4-(trifluoromethyl)pyridine-2,6-diyl)bis(

Assignees

• Roche Sequencing Solutions Inc

Inventors

• Kokoris Mark Stamatios
• Tabone John
• Nabavi Melud
• Jacobs Aaron
• O'Connell Dylan
• Goodman Drew
• Merrill Lacey
• Chandrasekar Jagadeeswaran
• Berg Kendall
• Vellucci Samantha
• Vellucci Jessica

Classifications

• C12Q1/6848 Primary

  characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction · CPC title

• C07D249/06 Primary

  with aryl radicals directly attached to ring atoms · CPC title

• C07D401/14

  containing three or more hetero rings · CPC title

• C07D403/14

  containing three or more hetero rings · CPC title

• C07D471/04

  Ortho-condensed systems · CPC title