Substituted coumarin dyes and uses as fluorescent labels

Patent metadata

Field	Value
Publication number	US-11981964-B2
Application number	US-202117385232-A
Country	US
Kind code	B2
Filing date	Jul 26, 2021
Priority date	Jul 28, 2020
Publication date	May 14, 2024
Grant date	May 14, 2024

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 (II) or (II′), or a salt or mesomeric form thereof:

wherein X is S, O, or NR a ;
Y is O or NH;
each R 1 , R 2 , R 3 and R 5 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 hydroxy alkyl, (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, optionally substituted C 6 -C 10 aryl, optionally substituted 5 to 10 membered heteroaryl, or optionally substituted 3 to 10 membered heterocyclyl;
R 4 is —NR b R c , halo, —OR d or —OS(O) 2 R d ;
each of R 6 and R 7 is independently H, carboxyl, —C(O)NR b R c , —C(O)OR d , C 1 -C 6 alkyl, substituted C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, halo, optionally substituted amino, N-sulfonamido, sulfonyl, S-sulfonamido, hydroxy, cyano, nitro, optionally substituted phenyl, optionally substituted C 3 -C 8 carbocyclyl, optionally substituted 5 to 10 membered heteroaryl, or optionally substituted 3 to 10 membered heterocyclyl; or R 6 and R 7 in Formula (II) together with the atoms to which they are attached form an optionally substituted C 5 -C 8 carbocyclyl or an optionally substituted 5 to 8 membered heterocyclyl;
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; or R b and R c together with the nitrogen atom to which they are attached form an optionally substituted 4 to 10 membered heterocyclyl; and
each R d is independently optionally substituted C 1 -C 6 alkyl or optionally substituted phenyl; provided that
at least one of R 6 and R 7 , or the optionally substituted C 5 -C 8 carbocyclyl or optionally substituted 5 to 8 membered heterocyclyl formed from R 6 and R 7 and the atoms to which they are attach, comprises a carboxyl; or
R 4 is —NR b R c and at least one of R b and R c comprises a carboxyl or —C(O)OR d .

2. The compound of claim 1 , wherein R 4 is halo or —OS(O) 2 R d , and wherein R d is C 1 -C 6 alkyl or C 1 -C 6 haloalkyl.

3. The compound of claim 1 , wherein R 4 is —NR b R c and the compound is also represented by Formula (IIa) or (IIa′):

or a salt or a mesomeric form thereof.

4. The compound of claim 3 , wherein each of R b and R c is independently H, C 1 -C 6 alkyl, or substituted C 1 -C 6 alkyl, or R b is H and R c is C 1 -C 6 alkyl or substituted C 1 -C 6 alkyl.

5. The compound of claim 4 , wherein the substituted C 1 -C 6 alkyl is independently substituted with carboxyl, —C(O)OR d , sulfo (—SO 3 H), sulfonate (—SO 3 − ), —O—SO 3 − or —SO 2 NR e R f , and wherein each of R e and R f is independently H or C 1 -C 6 alkyl.

6. The compound of claim 4 , wherein each of R b and R c is ethyl.

7. The compound of claim 4 , wherein each of R b and R c is independently C 1 -C 6 alkyl substituted with carboxyl, —C(O)OR d , —SO 3 H, —SO 3 − , or —SO 2 NH 2 .

8. The compound of claim 4 , wherein R b is H and R c is C 1 -C 6 alkyl or C 1 -C 6 alkyl substituted with carboxyl, —C(O)OR d , —SO 3 H, —SO 3 − , —O—SO 3 − or —SO 2 NH 2 .

9. The compound of claim 3 , wherein R b and R c together with the nitrogen atom to which they are attached form an optionally substituted 4, 5 or 6 membered heterocyclyl.

10. The compound of claim 1 , wherein Y is O.

11. The compound of claim 1 , wherein X is S or O.

12. The compound of claim 1 , wherein R 1 is H or C 1 -C 6 alkyl.

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

14. The compound of claim 1 , wherein R 3 is H or C 1 -C 6 alkyl.

15. The compound of claim 1 , wherein R 5 is H.

16. The compound of claim 1 , wherein each of R 6 and R 7 is independently H, C 1 -C 6 alkyl, substituted C 1 -C 6 alkyl, optionally substituted phenyl, carboxyl or —C(O)OR d .

17. The compound of claim 16 , wherein one of R 6 or R 7 is H or C 1 -C 6 alkyl, and the other one of R 6 or R 7 is carboxyl, —C(O)OR d , C 1 -C 6 alkyl, substituted C 1 -C 6 alkyl, or phenyl optionally substituted with carboxyl, —C(O)OR d or sulfo.

18. The compound of claim 16 , wherein one of R 6 or R 7 is H or methyl, and the other one of R 6 or R 7 is carboxyl, —C(O)OR d , methyl, phenyl, phenyl substituted with carboxyl or —C(O)OR d , or C 1 -C 6 alkyl substituted with carboxyl or C(O)OR d .

19. The compound of claim 1 , wherein R 6 and R 7 together with the atoms to which they are attached form a C 5 -C 6 carbocyclyl.

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

and C 1 -C 6 alkyl carboxylic esters, salts and mesomeric forms thereof.

21. A nucleotide or oligonucleotide labeled with a compound according to claim 3 .

22. A kit comprising a first type of labeled nucleotide according to claim 21 .

23. Use of a labeled nucleotide or oligonucleotide of claim 21 , in sequencing, expression analysis, hybridization analysis, genetic analysis, RNA analysis, or a protein binding assay.

24. 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 labeled nucleotides, wherein at least one of said labeled nucleotide is a nucleotide of claim 21 , 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; and
(c) performing one or more fluorescent measurements to determine the identity of the incorporated nucleotide.

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

• C12Q1/6876 Primary

  Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes · CPC title

• C07D405/14

  containing three or more hetero rings · CPC title

• C07D407/04 Primary

  directly linked by a ring-member-to-ring-member bond · CPC title

• C07D409/04

  directly linked by a ring-member-to-ring-member bond · CPC title