Coumarin compounds and their uses as fluorescent labels

• US 10,533,211 B2
• Filed: 02/22/2019
• Issued: 01/14/2020
• Est. Priority Date: 12/22/2016
• Status: Active Grant

• Current Assignee: Illumina Cambridge Limited (Illumina Incorporated)
• Original Assignee: Illumina Cambridge Limited (Illumina Incorporated)
• Inventors: Romanov, Nikolai Nikolaevich
• Primary Examiner(s): Vajda, Kristin A
• Application Number:
  US16/282,783
• Publication Number:
  US 20190185918A1
• Time in Patent Office: 326 Days
• Field of Search: 435 611
• 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 a salt, or a mesomeric form thereof:

2. The compound of claim 1, wherein X is O.

3. The compound of claim 1, wherein X is S.

4. The compound of claim 1, wherein R¹ is substituted with —

C(O)OH.

5. The compound of claim 1, wherein the bond represented by a solid and dashed line is a double bond, and wherein R² is H, alkyl or a substituted alkyl.

6. The compound of claim 5, wherein R² is H, methyl, or —

CH₂—

SO₃H.

7. The compound of claim 1, wherein the bond represented by a solid and dashed line is a single bond, and wherein each of R² and R³ is H, alkyl or substituted alkyl.

8. The compound of claim 7, wherein R² is H, methyl, or —

CH₂—

SO₃H and R³ is H or methyl.

9. The compound of claim 1, wherein each of R⁴ and R⁵ is H or alkyl.

10. The compound of claim 1, wherein R⁶ is alkyl or substituted alkyl.

11. The compound of claim 10, wherein R⁶ is alkyl substituted with —

C(O)OR¹² or —

C(O)NR¹³R¹⁴, wherein R¹² is selected from the group consisting of optionally substituted alkyl, optionally substituted aryl, optionally substituted heteroaryl, and optionally substituted 3 to 7 membered cycloalkyl, and wherein each R¹³ and R¹⁴ is independently selected from H, optionally substituted alkyl, optionally substituted aryl, optionally substituted heteroaryl and optionally substituted 3 to 7 membered cycloalkyl.

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

13. The labeled nucleotide or oligonucleotide of claim 12, 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.

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

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

15. A kit comprising one or more nucleotides wherein at least one nucleotide is a labeled nucleotide claim 12.

16. The kit of claim 15, comprising two or more labeled nucleotides and wherein two of the labeled nucleotides are excited using a single laser.

17. A method of sequencing comprising incorporating a nucleotide according to claim 12 in a sequencing assay.

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

19. The method of claim 17, wherein the sequencing assay is performed on an automated sequencing instrument, and wherein the automated sequencing instrument comprises two light sources operating at different wavelengths.

20. The compound of claim 4, wherein the bond represented by a solid and dashed line is a double bond, and wherein R² is H, alkyl or a substituted alkyl.

21. The compound of claim 20, wherein each of R⁴ and R⁵ is H or alkyl, and R⁶ is alkyl.

22. The compound of claim 4, wherein the bond represented by a solid and dashed line is a single bond, wherein R² is H, alkyl or a substituted alkyl, and wherein R³ is H or alkyl.

23. The compound of claim 22, wherein each of R⁴ and R⁵ is H or alkyl, and R⁶ is alkyl.