Polymethine compounds with long stokes shifts and their use as fluorescent labels

• US 10,239,909 B2
• Filed: 05/23/2016
• Issued: 03/26/2019
• Est. Priority Date: 05/22/2015
• Status: Active Grant

• Current Assignee: Illumina Cambridge Ltd. (California) (Illumina Incorporated)
• Original Assignee: Illumina Cambridge Ltd. (California) (Illumina Incorporated)
• Inventors: Romanov, Nikolai, Wu, Xiaolin
• Primary Examiner(s): Carcanague, Daniel R
• Application Number:
  US15/575,743
• Publication Number:
  US 20180111957A1
• Time in Patent Office: 1,037 Days
• Field of Search: 546201
• CPC Class Codes: C07D 401/06 linked by a carbon chain containing only aliphatic carbon atoms
• 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: C09B 23/06 three >CH- groups, e.g. carbocyanines
• CPC Class Codes: C09B 69/105 containing a methine or polymethine dye
• CPC Class Codes: C09K 11/06 containing organic luminescent materials
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes

Abstract

The present disclosure relates to new compounds and their use as fluorescent labels. The compounds may be used as fluorescent labels for nucleotides in nucleic acid sequencing applications. The labels are advantageous due to their long Stokes shifts.

Claims

1. A compound of formula (I′

) or mesomeric forms thereof;

2. The compound according to claim 1, wherein x is 1.

3. The compound according to claim 1, wherein Re₁ is alkyl and Rb₁ is COOH or COO⁻

or an amide or ester thereof.

4. The compound according to claim 1, wherein Re₁ is an alkyl or aryl group, each substituted with COOH or COO⁻

or an amide or ester thereof.

5. The compound according to claim 1, wherein Re₁ is an substituted aryl group comprising COOH or COO⁻

or an amide or ester thereof, and wherein there is an alkylene group between the aryl ring and the COOH or COO⁻

or an amide or ester thereof.

6. The compound according to claim 4, wherein n is 0.

7. The compound according to claim 4, wherein n is 1 and Rb₁ is SO₃⁻

or sulfonamide.

8. The compound according to claim 1, wherein each of Rc₁ and Rc₂ is methyl.

9. The compound according to claim 1, wherein Rc₁ or Rc₂ is methyl, ethyl, propyl or —

(CH₂)_qSO₃⁻

where q is 1-6.

10. The compound according to claim 9 wherein either Rc₁ or Rc₂ is —

(CH₂)₄SO₃⁻

.

11. The compound according to claim 1, wherein Ra₁ is H, SO₂NH₂ or SO₃⁻

.

12. The compound according to claim 1, wherein Ra₁ is a ring fused to an adjacent carbon atom, optionally substituted with one or more SO₃⁻

or sulfonamide.

13. The compound according to claim 1, wherein each of Rd₁ and Rd₂ is independently H or methyl.

14. The compound according to claim 1 which is represented by formula (II):

15. A nucleotide or oligonucleotide labelled with a compound according to claim 1.

16. The labelled nucleotide or oligonucleotide according to claim 15, wherein the compound is attached via an amide linkage formed from a COOH moiety.

17. The labelled nucleotide or oligonucleotide according to claim 15, 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.

18. The labelled nucleotide or oligonucleotide according to claim 15, further comprising a 3′

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

19. A kit comprising two or more nucleotides wherein at least one nucleotide is a labelled nucleotide according to claim 15.

20. The kit according to claim 19, wherein two nucleotides are labelled and can be measured by detection at the same wavelength.

21. The kit according to claim 19, comprising four nucleotides wherein a first nucleotide is the labelled nucleotide, a second nucleotide is labelled with a label that emits at the same wavelength as the first nucleotide, a third nucleotide is labelled with a mixture of labels and the fourth is unlabelled such that each of the four nucleotides is distinguishable from each other.

22. The kit according to claim 21, wherein the first nucleotide has a Stokes shift of greater than 100 nm and the second nucleotide has a Stokes shift of less than 50 nm.

23. The nucleotide according to claim 15, for use in sequencing, expression analysis, hybridisation analysis, genetic analysis, RNA analysis or protein binding assays.

24. The nucleotide according to claim 23, for use on an automated sequencing instrument wherein said automated sequencing instrument comprises two lasers operating at different wavelengths and a detection system having a single detection channel set to a fixed emission wavelength.

25. A method of synthesising a compound according to claim 1, comprising reacting a starting material of formula (X′

) or a salt thereof with Re₁—

CH₂—

NH₂;

26. The method according to claim 25, wherein x is 1, and Re₁ is an alkyl group substituted with COOH or COO⁻

or an amide or ester thereof.

27. The method according to claim 25, wherein x is 1, and Re₁ is an substituted aryl group comprising COOH or COO⁻

or an amide or ester thereof, and wherein there is an alkylene group between the aryl ring and the COOH or COO⁻

or an amide or ester thereof.

28. The compound according to claim 1, selected from the group consisting of: