Rhodamine compounds and their use as fluorescent labels

Patent metadata

Field	Value
Publication number	US-9222131-B2
Application number	US-201414268690-A
Country	US
Kind code	B2
Filing date	May 2, 2014
Priority date	Mar 8, 2013
Publication date	Dec 29, 2015
Grant date	Dec 29, 2015

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.

First claim

The invention claimed is:

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

Wherein M +/− is a common counter ion,
k is an integer of from 0 to 6,
q is an integer of from 1 to 6,
R 1 is H or an alkyl, aryl or substituted alkyl or substituted aryl group,
R 2 is H, alkyl or substituted alkyl group, halogen, carboxy, carboxamide, hydroxy- or alkoxy group, or R 2 together with R 1 or R 5 is a carbon or heterosubstituted chain forming a ring,
R 3 is H, alkyl or substituted alkyl group, halogen, carboxy, carboxamide, hydroxy- or alkoxy group or R 3 together with R 4 or R 6 is a carbon or heterosubstituted chain forming a ring,
R 4 is H or an alkyl, aryl or substituted alkyl or substituted aryl group,
R 5 and R 6 are H, alkyl or substituted alkyl group, halogen, hydroxy- or alkoxy group,
R 8 is H, halogen, hydroxy- or alkoxy group, alkyl or substituted alkyl group or together with R 1 is a carbon or heterosubstituted carbon chain forming a ring,
R 9 is H, halogen, hydroxy- or alkoxy group, alkyl or substituted alkyl group or together with R 4 is a carbon or heterosubstituted carbon chain forming a ring,
R 7 is OR 11 or NR 11 R 12 where R 11 and R 12 are independently H, alkyl or a substituted alkyl,
R 13 is OR 14 or NR 14 R 15 where R 14 and R 15 are independently H, alkyl or a substituted alkyl; aryl or a substituted aryl, and
R 16 and R 17 are independently H or an alkyl, aryl or substituted alkyl or substituted aryl group.

2. The labelled nucleotide or oligonucleotide according to claim 1 wherein R 16 is alkyl and R 17 is alkyl.

3. The labelled nucleotide or oligonucleotide according to claim 1 wherein R 1 , R 4 , R 5 , R 6 , R 8 and R 9 are all H, R 2 and R 3 are H or CH 3 .

4. The labelled nucleotide or oligonucleotide according to claim 1 wherein R 16 and R 17 are H.

5. The labelled nucleotide or oligonucleotide according to claim 1 wherein R 1 is linked to R 2 or R 8 via a chain of CH 2 groups to form a ring, and R 4 is linked to R 3 or R 9 via a chain of CH 2 groups to form a ring.

6. The labelled nucleotide or oligonucleotide according to claim 5 wherein R 1 and R 2 form a six membered ring, R 3 and R 4 form a six membered ring, R 5 , R 6 , R 8 and R 9 are H.

7. The labelled nucleotide or oligonucleotide according to claim 1 wherein one or more of R 1 , R 4 , R 16 and R 17 are alkyl groups substituted with an SO 3 − group.

8. The labelled nucleotide or oligonucleotide according to claim 1 wherein R 7 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 R 13 is OH.

11. The labelled nucleotide or oligonucleotide according to claim 1 wherein R 13 is NR 14 R 15 where R 14 is H or alkyl and R 15 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 R 15 .

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.

Assignees

• Illumina Cambridge Ltd

Inventors

• Romanov Nikolai Nikolaevich
• Liu Xiaohai

Classifications

• C07D405/12

  linked by a chain containing hetero atoms as chain links · CPC title

• C07D491/147

  the condensed system containing one ring with oxygen as ring hetero atom and two rings with nitrogen as ring hetero atom · CPC title

• C07D487/04

  Ortho-condensed systems · CPC title

• C12Q1/6869 Primary

  Methods for sequencing · CPC title

• C07D405/14 Primary

  containing three or more hetero rings · CPC title