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

Patent metadata

Field	Value
Publication number	US-10239909-B2
Application number	US-201615575743-A
Country	US
Kind code	B2
Filing date	May 23, 2016
Priority date	May 22, 2015
Publication date	Mar 26, 2019
Grant date	Mar 26, 2019

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.

First claim

The invention claimed is:

1. A compound of formula (I′) or mesomeric forms thereof:

wherein mCat+ or mAn− is an organic or inorganic positively/negatively charged counterion;
m is an integer 0-3;
x is an integer 0-2;
Ra 1 is H, SO 3 − , sulfonamide, halogen, hydroxy, alkoxy, amino or a further ring fused to an adjacent carbon atom where the ring is optionally substituted with one or more SO 3 − , sulfonamide, or halogen;
Rb 1 is SO 3 − , sulfonamide, halogen, hydroxy, alkoxy, amino, COOH or an amide or ester thereof;
n is 0-3;
each of Rc 1 and Rc 2 is independently alkyl or substituted alkyl;
each of Rd 1 and Rd 2 is independently H, alkyl, aryl, substituted alkyl, or substituted aryl; and
Re 1 is alkyl, substituted alkyl, aryl or substituted aryl; wherein either Rc 1 , Rb 1 or Re 1 comprises a COOH or COO − or an amide or ester thereof.

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

3. The compound according to claim 1 , wherein Re 1 is alkyl and Rb 1 is COOH or COO − or an amide or ester thereof.

4. The compound according to claim 1 , wherein Re 1 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 Re 1 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 Rb 1 is SO 3 − or sulfonamide.

8. The compound according to claim 1 , wherein each of Rc 1 and Rc 2 is methyl.

9. The compound according to claim 1 , wherein Rc 1 or Rc 2 is methyl, ethyl, propyl or —(CH 2 ) q SO 3 − where q is 1-6.

10. The compound according to claim 9 wherein either Rc 1 or Rc 2 is —(CH 2 ) 4 SO 3 − .

11. The compound according to claim 1 , wherein Ra 1 is H, SO 2 NH 2 or SO 3 − .

12. The compound according to claim 1 , wherein Ra 1 is a ring fused to an adjacent carbon atom, optionally substituted with one or more SO 3 − or sulfonamide.

13. The compound according to claim 1 , wherein each of Rd 1 and Rd 2 is independently H or methyl.

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

or a salt thereof, wherein mCat+ or mAn− is an organic or inorganic positively/negatively charged counterion;
m is an integer 0-3;
each n is independently 0-6;
Ra 1 is H, SO 3 − , sulfonamide, halogen, or a further ring fused to an adjacent carbon atom where the ring is optionally substituted with one or more SO 3 − , sulfonamide, or halogen;
Rb 1 is SO 3 − , sulfonamide, halogen, COOH or an amide or ester thereof;
n is 0-3; and
X is OH, or O − or —C(O)X is an ester or amide.

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 Re 1 —CH 2 —NH 2 :

wherein x is 0-2; Ra 1 is H, SO 3 − , sulfonamide, halogen, or a further ring fused to an adjacent carbon atom where the ring is optionally substituted with one or more SO 3 − , sulfonamide, or halogen;
Rb 1 is SO 3 − , sulfonamide, halogen or COOH;
n is 0-3;
each of Rc 1 and Rc 2 is independently alkyl or substituted alkyl;
each of Rd 1 and Rd 2 is independently H, alkyl, aryl, substituted alkyl, or substituted aryl; and
Re 1 is alkyl, substituted alkyl, aryl or substituted aryl; wherein either Rc 1 , Rb 1 or Re 1 comprises a COOH, or COO − or an amide or ester thereof.

26. The method according to claim 25 , wherein x is 1, and Re 1 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 Re 1 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:

and mesomeric forms thereof.

Assignees

• Illumina Cambridge Ltd

Inventors

• Romanov Nikolai
• Wu Xiaolin

Classifications

• C09B69/105

  containing a methine or polymethine dye · CPC title

• C07H19/10 Primary

  with the saccharide radical esterified by phosphoric or polyphosphoric acids · CPC title

• C07D401/06 Primary

  linked by a carbon chain containing only aliphatic carbon atoms · CPC title

• C07H19/14

  Pyrrolo-pyrimidine radicals · CPC title

• C09B23/06

  three >CH- groups, e.g. carbocyanines · CPC title