Fluorescent dyes and their uses as biomarkers

• US 10,385,214 B2
• Filed: 09/28/2017
• Issued: 08/20/2019
• Est. Priority Date: 09/30/2016
• Status: Active Grant

• Current Assignee: Illumina Cambridge Limited (Illumina Incorporated)
• Original Assignee: Illumina Cambridge Limited (Illumina Incorporated)
• Inventors: Romanov, Nikolai
• Primary Examiner(s): Jackson, Shawquia
• Application Number:
  US15/719,216
• Publication Number:
  US 20180094140A1
• Time in Patent Office: 691 Days
• CPC Class Codes: C07D 417/04 directly linked by a ring-member-to-ring-member bond
• CPC Class Codes: C07D 491/04 Ortho-condensed systems
• CPC Class Codes: C09B 62/365 Specific dyes not provided for in groups C09B62/38 - C09B62/42
• CPC Class Codes: C09K 11/06 containing organic luminescent materials
• CPC Class Codes: C12Q 1/6869 Methods for sequencing

Abstract

The present application relates to new fluorescent dyes 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 mesomeric forms thereof:

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

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

OR⁷.

4. The compound of claim 1, wherein R⁷ is H or alkyl.

5. The compound of claim 1, wherein R is —

NR⁸R⁹.

6. The compound of claim 5, wherein each R⁸ and R⁹ is H.

7. The compound of claim 5, wherein R⁸ is H and R⁹ is alkyl or substituted alkyl.

8. The compound of claim 5, wherein both R⁸ and R⁹ are alkyl or substituted alkyl.

9. The compound of claim 7, wherein substituted alkyl is selected from alkyl substituted with carboxyl or sulfonyl hydroxide.

10. The compound of claim 1, wherein each R¹, R¹′

and R² is H.

11. The compound of claim 1, wherein at least one of R¹, R¹′

and R² is an alkyl.

12. The compound of claim 1, wherein each R³ and R⁴ is an alkyl.

13. The compound of claim 12, wherein each R³ and R⁴ is selected from methyl or ethyl.

14. The compound of claim 1, wherein R³ is H and R⁴ is an alkyl.

15. The compound of claim 1, wherein R¹ and R³ together with the atoms to which they are attached form an optionally substituted 3 to 7 membered heterocyclyl.

16. The compound of claim 15, wherein R¹ and R³ together with the atoms to which they are attached form an optionally substituted 6 membered heterocyclyl.

17. The compound of claim 15, wherein R⁴ is selected from H or alkyl.

18. The compound of claim 15, wherein at least one of R¹′

and R² is H.

19. The compound of claim 1, wherein R² and R⁴ together with the atoms to which they are attached form an optionally substituted 3 to 7 membered heterocyclyl.

20. The compound of claim 19, wherein R² and R⁴ together with the atoms to which they are attached form an optionally substituted 6 membered heterocyclyl.

21. The compound of claim 19, wherein R³ is selected from H or alkyl.

22. The compound of claim 19, wherein at least one of R¹′

and R¹ is H.

23. The compound of claim 1, wherein R¹ and R³ together with the atoms to which they are attached form an optionally substituted 3 to 7 membered heterocyclyl, and wherein R² and R⁴ together with the atoms to which they are attached form an optionally substituted 3 to 7 membered heterocyclyl.

24. The compound of claim 23, wherein R¹ and R³ together with the atoms to which they are attached form an optionally substituted 3 to 7 membered heterocyclyl, and wherein R² and R⁴ together with the atoms to which they are attached form an optionally substituted 6 membered heterocyclyl.

25. The compound of claim 15, wherein the heterocyclyl contains one heteroatom.

26. The compound of claim 24, wherein the heterocyclyl is substituted with one or more alkyl.

27. The compound of claim 1, wherein m is 0.

28. The compound of claim 1, wherein m is 1 and R⁵ is selected from halo, sulfo, aminosulfonyl, or sulfonyl halide.

29. The compound of claim 1, wherein n is 0.

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

31. The compound of claim 1, wherein the compound is covalently attached to a nucleotide or oligonucleotide via C(═

O)R, wherein R is —

OR⁷, and wherein R⁷ is H or a substituted alkyl.

32. The compound of claim 1, wherein the compound is covalently attached to a nucleotide or oligonucleotide via C(═

O)R, wherein R is —

NR⁸R⁹, and wherein at least one of R⁸ or R⁹ is a substituted alkyl.

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

34. The labeled nucleotide or oligonucleotide of claim 33, wherein the compound is covalently attached to the nucleotide or oligonucleotide via —

C(═

O)R, wherein R is —

OR⁷, and R⁷ is H or a substituted alkyl.

35. The labeled nucleotide or oligonucleotide of claim 33, wherein the compound is covalently attached to the nucleotide or oligonucleotide via —

C(═

O)R, wherein R is —

NR⁸R⁹, and wherein at least one of R⁸ or R⁹ is a substituted alkyl.

36. The labeled nucleotide or oligonucleotide of claim 33, 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 cleavable linker.

37. The labeled nucleotide or oligonucleotide of claim 33, further comprising a 3′

—

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

38. A kit comprising one or more nucleotides wherein at least one nucleotide is a labeled nucleotide according to claim 33.

39. The kit of claim 38, comprising two or more labeled nucleotides.

40. The kit of claim 39, wherein the two labeled nucleotides are excited using a single laser.

41. The kit of claim 38, further comprising a third and a fourth nucleotide, wherein each of the second, third and fourth nucleotide is labeled with a different compound, wherein each compound has a distinct absorbance maximum and each of the compound is distinguishable from the other compounds.

42. A method of sequencing comprising incorporating a labeled nucleotide according to claim 33 in a sequencing assay.

43. The method of claim 42, further comprising detecting the nucleotide.

44. The method of claim 42, 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.

45. A method of preparing a compound of Formula (Ia), comprising:

reacting a compound of Formula (IIa)

46. A method for preparation a compound of Formula (Ib), comprising:

converting a compound of Formula (Ia) to a compound of Formula (Ia′

) through carboxylic acid activation;