Coumarin compounds and their uses as fluorescent labels

• US 10,214,768 B2
• Filed: 12/21/2017
• Issued: 02/26/2019
• Est. Priority Date: 12/22/2016
• Status: Active Grant

• Current Assignee: Illumina Cambridge Ltd. (California) (Illumina Incorporated)
• Original Assignee: Illumina Cambridge Ltd. (California) (Illumina Incorporated)
• Inventors: Romanov, Nikolai
• Primary Examiner(s): Vajda, Kristin A
• Application Number:
  US15/851,014
• Publication Number:
  US 20180201981A1
• Time in Patent Office: 432 Days
• Field of Search: 546 80
• CPC Class Codes: C07D 491/04 Ortho-condensed systems
• CPC Class Codes: C07D 491/052 the oxygen-containing ring being six-membered
• CPC Class Codes: C07D 491/147 the condensed system containing one ring with oxygen as ring hetero atom and two rings with nitrogen as ring hetero atom
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/14 Pyrrolo-pyrimidine radicals
• CPC Class Codes: 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
• CPC Class Codes: C09B 57/02 Coumarine dyes
• CPC Class Codes: C09K 11/06 containing organic luminescent materials
• CPC Class Codes: C09K 2211/1018 Heterocyclic compounds
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes

Abstract

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

Claims

1. A compound of Formula (I), or salts, mesomeric forms thereof:

2. The compound of claim 1, wherein R¹ is

3. The compound of claim 2, wherein X is O.

4. The compound of claim 2, wherein X is S.

5. The compound of claim 1, wherein R¹ is

6. The compound of claim 1, wherein R¹ is

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

8. A nucleotide or oligonucleotide labeled with a compound according to claim 1.

9. The labeled nucleotide or oligonucleotide of claim 8, wherein the compound is covalently attached to the nucleotide or oligonucleotide via R⁶.

10. The labeled nucleotide or oligonucleotide of claim 8, wherein the compound is covalently attached to the nucleotide or oligonucleotide via R^10a, R^10b or R^10c, and wherein R^10a, R^10b or R^10c is a substituted alkyl.

11. The labeled nucleotide or oligonucleotide of claim 8, wherein the compound is attached to the C5 position of a pyrimidine base or the C7 position of a 7-deaza purine base of the nucleotide or oligonucleotide through a linker moiety.

12. The labeled nucleotide or oligonucleotide of claim 8, further comprising a 3′

-OH blocking group covalently attached to the ribose or deoxyribose sugar of the nucleotide or oligonucleotide.

13. A kit comprising one or more nucleotides wherein at least one nucleotide is a labeled nucleotide according to claim 8.

14. The kit of claim 13, comprising two or more labeled nucleotides.

15. The kit of claim 14, wherein two of the labeled nucleotides are excited using a single laser.

16. A method of sequencing comprising incorporating a nucleotide according to claim 8 in a sequencing assay.

17. The method of claim 16, further comprising detecting the nucleotide.

18. The compound of claim 1, wherein each R^10a, ^10b and R^10c is alkyl substituted with carboxyl, carboxylate, sulfo or sulfonate.

22. The compound of claim 2, wherein the bond represented by a solid and dashed line is a single bond, and wherein at least one of R² and R³ is alkyl.

23. The compound of claim 22, wherein at least one of R⁴ and R⁵ is alkyl.

24. The compound of claim 5, wherein R^10a is alkyl substituted with carboxyl, carboxylate, sulfo or sulfonate.

25. The compound of claim 24, wherein the bond represented by a solid and dashed line is a single bond, and wherein at least one of R² and R³ is alkyl.

26. The compound of claim 25, wherein at least one of R⁴ and R⁵ is alkyl.

19. A compound of Formula (I), or salts, mesomeric forms thereof:

20. The compound of claim 19, wherein R⁶ and R⁷ together with the atoms to which they are attached form an optionally substituted 6 membered heterocyclyl.

21. The compound of claim 19, having the structure