Rhodamine compounds and their use as fluorescent labels

• US 9,222,131 B2
• Filed: 05/02/2014
• Issued: 12/29/2015
• Est. Priority Date: 03/08/2013
• Status: Active Grant

• Current Assignee: Illumina Cambridge Ltd. (California) (Illumina Incorporated)
• Original Assignee: Illumina Cambridge Ltd. (California) (Illumina Incorporated)
• Inventors: Romanov, Nikolai Nikolaevich, Liu, Xiaohai
• Primary Examiner(s): SHAMEEM, GOLAM M
• Application Number:
  US14/268,690
• Publication Number:
  US 20140255920A1
• Time in Patent Office: 606 Days
• Field of Search: 549/356, 549/388, 549/394, 536/22.1, 435/6.1
• US Class Current: 1/1
• CPC Class Codes: C07D 311/82 Xanthenes
• CPC Class Codes: C07D 405/12 linked by a chain containing hetero atoms as chain links
• CPC Class Codes: C07D 405/14 containing three or more hetero rings
• CPC Class Codes: C07D 487/04 Ortho-condensed systems
• 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: C12Q 1/6869 Methods for sequencing

Abstract

The present invention relates to new rhodamine compounds and their use as fluorescent labels. The compounds may be used as fluorescent labels for nucleotides in nucleic acid sequencing applications.

Claims

1. A nucleotide or oligonucleotide labelled with a compound of formula (I) or a mesomer thereof:

2. The labelled nucleotide or oligonucleotide according to claim 1 wherein R₁₆ is alkyl and R₁₇ is alkyl.

3. The labelled nucleotide or oligonucleotide according to claim 1 wherein R₁, R₄, R₅, R₆, R₈ and R₉ are all H, R₂ and R₃ are H or CH₃.

4. The labelled nucleotide or oligonucleotide according to claim 1 wherein R₁₆ and R₁₇ are H.

5. The labelled nucleotide or oligonucleotide according to claim 1 wherein R₁ is linked to R₂ or R₈ via a chain of CH₂ groups to form a ring, and R₄ is linked to R₃ or R₉ via a chain of CH₂ groups to form a ring.

6. The labelled nucleotide or oligonucleotide according to claim 5 wherein R₁ and R₂ form a six membered ring, R₃ and R₄ form a six membered ring, R₅, R₆, R₈ and R₉ are H.

7. The labelled nucleotide or oligonucleotide according to claim 1 wherein one or more of R₁, R₄, R₁₆ and R₁₇ are alkyl groups substituted with an SO₃⁻

group.

8. The labelled nucleotide or oligonucleotide according to claim 1 wherein R₇ is OH.

9. The labelled nucleotide or oligonucleotide according to claim 1 wherein q is 3.

10. The labelled nucleotide or oligonucleotide according to claim 1 wherein R₁₃ is OH.

11. The labelled nucleotide or oligonucleotide according to claim 1 wherein R₁₃ is NR₁₄R₁₅ where R₁₄ is H or alkyl and R₁₅ is alkyl or a substituted alkyl.

12. The labelled nucleotide or oligonucleotide according to claim 11 wherein the compound is attached to a nucleotide or oligonucleotide via substituted alkyl group R₁₅.

13. The labelled nucleotide or oligonucleotide according to claim 1 wherein the label is attached to the C5 position of a pyrimidine base or the C7 position of a 7-deaza purine base through a linker moiety.

14. The labelled nucleotide or oligonucleotide according to claim 1, further comprising a 3′

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

15. A kit comprising two or more nucleotides wherein at least one nucleotide is a labelled nucleotide according to claim 1.

16. The kit according to claim 15 wherein two of the labelled nucleotides are excited using a single laser.

17. The kit according to claim 15 wherein a first of four nucleotides is a labelled nucleotide according to claim 1 and the second, third, and fourth nucleotides are each labelled with a different compound, wherein each compound has a distinct absorbance maximum and each of the compounds is distinguishable from the other three compounds.

18. The kit according to claim 15 wherein a first of four nucleotides is a labelled nucleotide according to claim 1 and two of the compounds have a distinct absorbance maximum above 600 nm.

19. A method of sequencing comprising incorporating a nucleotide according to claim 1 in a sequencing assay.

20. The method according to claim 19, wherein said sequencing assay is performed on an automated sequencing instrument wherein said automated sequencing instrument comprises two light sources operating at different wavelengths.