Substituted coumarin dyes and uses as fluorescent labels

Patent metadata

Field	Value
Publication number	US-12351871-B2
Application number	US-202418630722-A
Country	US
Kind code	B2
Filing date	Apr 9, 2024
Priority date	Jul 28, 2020
Publication date	Jul 8, 2025
Grant date	Jul 8, 2025

Abstract

The present application relates to substituted coumarin derivatives and their uses as fluorescent labels. These compounds may be used as fluorescent labels for nucleotides in nucleic acid sequencing applications.

First claim

What is claimed is:

1. A compound of Formula (I), or a salt or mesomeric form thereof:

wherein R 1 is

 each optionally substituted with one or more substituents independently selected from the group consisting of carboxyl, —C(O)NR b R c , —C(O)OR d , C 1 -C 6 alkyl, substituted C 1 -C 6 alkyl, halo, optionally substituted amino, hydroxy, sulfo, sulfonate, sulfate, N-sulfonamido, and S-sulfonamido;
R 2 is H, optionally substituted C 1 -C 6 alkyl, or phenyl optionally substituted with one or more substituents selected from the group consisting of carboxyl,
—C(O)NR b R c , —C(O)OR d , optionally substituted C 1 -C 6 alkyl, halo, cyano, nitro, sulfonyl, sulfino, sulfo, sulfonate, optionally substituted amino, and hydroxy;
each R 3 , R 4 and R 7 is independently H, optionally substituted C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 1 -C 6 haloalkyl, C 1 -C 6 haloalkoxy, C 1 -C 6 hydroxyalkyl, (C 1 -C 6 alkoxy)(C 1 -C 6 alkyl), optionally substituted amino, halo, cyano, hydroxy, nitro, sulfonyl, sulfino, sulfo, sulfonate, S-sulfonamido, N-sulfonamido, optionally substituted C 3 -C 10 carbocyclyl, or optionally substituted 3 to 10 membered heterocyclyl;
R 5 is H and R 6 is H, C 1 -C 6 alkyl, or substituted C 1 -C 6 alkyl, or both R 5 and R 6 are C 1 -C 6 alkyl, or R 5 and R 6 together with the nitrogen atom to which they are attached form an optionally substituted 4 to 10 membered heterocyclyl;
X is O, S, or NR a ;
R a is H or C 1 -C 6 alkyl;
each R b and R c is independently H, C 1 -C 6 alkyl, or substituted C 1 -C 6 alkyl; and
R d is optionally substituted C 1 -C 6 alkyl or optionally substituted phenyl; provided that
at least one of R 6 , or the optionally substituted 4 to 10 membered heterocyclyl formed from R 5 and R 6 and the nitrogen atom to which they are attached, comprises a carboxyl or —C(O)OR d ; or
R 1 comprises a carboxyl or —C(O)OR d .

2. The compound of claim 1 , wherein R 1 is

each substituted with carboxyl.

3. The compound of claim 2 , wherein R 1 is

4. The compound of claim 1 , wherein each of R 5 and R 6 is C 1 -C 6 alkyl.

5. The compound of claim 1 , wherein R 5 is H and R 6 is substituted C 1 -C 6 alkyl.

6. The compound of claim 5 , wherein the C 1 -C 6 alkyl is substituted with carboxyl, —C(O)OR d , sulfo (—SO 3 H), sulfonate (—SO 3 − ), sulfate (—O—SO 3 − ) or an optionally substituted amino.

7. The compound of claim 1 , wherein R 1 is

each unsubstituted or substituted with sulfo (—SO 3 H).

8. The compound of claim 7 , wherein R 5 is H and R 6 is C 1 -C 6 alkyl substituted with carboxyl or —C(O)OR d .

9. The compound of claim 7 , wherein R 5 and R 6 and the nitrogen atom to which they are attached form a 5 or 6 membered heterocyclyl substituted with carboxyl or —C(O)OR d .

10. The compound of claim 1 , wherein R 2 is H.

11. The compound of claim 1 , wherein R 2 is C 1 -C 6 alkyl.

12. The compound of claim 1 , wherein each of R 3 , R 4 and R 7 is H.

13. The compound of claim 1 , selected from the group consisting of:

salts and mesomeric forms thereof.

14. A nucleotide or oligonucleotide labeled with a compound according to claim 1 , wherein the compound is attached the nucleotide or oligonucleotide via a carboxyl group of the compound.

15. The labeled nucleotide or oligonucleotide of claim 14 , wherein the compound is attached to the C5 position of a pyrimidine base or the C7 position of a 7-deaza purine base through a linker moiety.

16. The labeled nucleotide or oligonucleotide of claim 14 , further comprising a 3′ OH blocking group covalently attached to the ribose or deoxyribose sugar of the nucleotide.

17. The nucleotide or oligonucleotide of claim 14 , wherein the nucleotide or oligonucleotide is an oligonucleotide hybridized to at least a portion of a target polynucleotide, and wherein the target polynucleotide is immobilized on a solid support.

18. A kit comprising a first type of labeled nucleotide according to claim 14 .

19. The kit of claim 18 , wherein the kit comprises four types of nucleotides, wherein each of the first, the second, the third, and the fourth type of nucleotides is labeled with a different compound, wherein each label has a distinct absorbance maximum that is distinguishable from the other labels.

20. The kit of claim 18 , wherein the kit comprises four types of nucleotides, the second type of nucleotide is labeled with a second label, the third type of nucleotide is labeled with a third label, and the fourth type of nucleotide is unlabeled (dark).

21. The kit of claim 18 , wherein the kit comprises four types of nucleotides, the second type of nucleotide is labeled with a second label, the third type of nucleotide is labeled with a mixture of two labels, and the fourth type of nucleotide is unlabeled (dark).

22. A method of determining the sequence of a single-stranded target polynucleotide, comprising:
(a) contacting a primer polynucleotide/target polynucleotide complex with one or more different types of nucleotides, wherein at least one type of nucleotides is the label nucleotide of claim 15 , wherein each nucleotide comprises a 3′ hydroxy blocking group covalently attached to the deoxyribose sugar of the nucleotide, and wherein the primer polynucleotide is complementary to at least a portion of the target polynucleotide;
(b) incorporating a labeled nucleotide into the primer polynucleotide;
(c) performing one or more fluorescent measurements to determine the identity of the incorporated nucleotide; and
(d) removing the label and the 3′ blocking group from the nucleotide incorporated into the primer polynucleotide.

23. The method of claim 22 , further comprising (e) washing the removed label and the 3′ blocking group away from the primer polynucleotide, and wherein steps (a) to (e) are repeated until a sequence of at least a portion of the template polynucleotide strand is determined.

24. The method of claim 22 , wherein the label and the 3′ blocking group from the nucleotide incorporated into the primer polynucleotide are removed in a single chemical reaction.

25. The method of claim 22 , wherein the method is performed on an automated sequencing instrument, and wherein the automated sequencing instrument comprises two light sources operating at different wavelengths.

Assignees

• Illumina Cambridge Ltd

Inventors

• Romanov Nikolai Nikolaevich
• Callingham Michael
• Anastasi Carole
• Mccauley Patrick
• Hynes Niall
• Crake Natasha
• Wu Xiaolin
• Liu Xiaohai

Classifications

• C07D407/02

  containing two hetero rings · CPC title

• G01N2021/6439

  with indicators, stains, dyes, tags, labels, marks · CPC title

• G01N21/6428

  Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" (in vivo A61B5/00; immunoassay G01N33/53) · CPC title

• C12Q1/6869

  Methods for sequencing · CPC title

• C09B57/02

  Coumarine dyes · CPC title