Functionalized cyanine dyes (PEG)

• US 8,669,374 B2
• Filed: 08/25/2011
• Issued: 03/11/2014
• Est. Priority Date: 08/25/2010
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Inventors: Shen, Gene, Yue, Stephen
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US13/218,395
• Publication Number:
  US 20120058482A1
• Time in Patent Office: 929 Days
• Field of Search: 548/455, 435/6.1, 435/7.1, 435/7.2
• US Class Current: 548/455
• CPC Class Codes: C07D 209/14 Radicals substituted by nitrogen atoms, not forming part of a nitro radical
• CPC Class Codes: C07D 401/14 containing three or more hetero rings
• CPC Class Codes: C07D 405/14 containing three or more hetero rings
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07K 5/0815 with the first amino acid being basic
• CPC Class Codes: C07K 5/1019 with the first amino acid being basic
• CPC Class Codes: C09B 23/0025 the substituent being bound through an oxygen atom
• CPC Class Codes: C09B 23/0033 the substituent being bound through a sulfur atom
• CPC Class Codes: C09B 23/0066 the polymethine chain being part of a carbocyclic ring,(e.g. benzene, naphtalene, cyclohexene, cyclobutenene-quadratic acid)
• CPC Class Codes: C09B 23/06 three >CH- groups, e.g. carbocyanines
• CPC Class Codes: C09B 23/083 five >CH- groups
• CPC Class Codes: C09B 23/105 two >CH- groups
• CPC Class Codes: C09B 23/164 containing one nitrogen atom
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: G01N 2333/9126 DNA-directed DNA polymerase (2.7.7.7)
• CPC Class Codes: G01N 33/52 Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements immunological elements G01N33/54386; involving labelled immunochemicals G01N33/58; for haemoglobin or occult blood G01N33/72
• CPC Class Codes: G01N 33/582 with fluorescent label

Abstract

The invention provides a novel class of cyanine dyes that are functionalized with a linker moiety that facilitates their conjugation to other species and substituent groups which increase the water-solubility, and optimize the optical properties of the dyes. Also provided are conjugates of the dyes, methods of using the dyes and their conjugates and kits including the dyes and their conjugates.

Claims

1. A compound having the formula:

2. The compound according to claim 1 wherein at least two of R^f, R^g, R^h, and Rⁱ are alkylsulfonic acid.

3. The compound according to claim 1 wherein neither R^c nor R^d is unsubstituted alkyl.

4. The compound according to claim 1 wherein when two R^a moieties are not joined to form said ring substituted with said at least one SO₃H moiety, at least one R^a is SO₃H.

5. The compound according to claim 1 wherein when two R^e moieties are not joined to form said aryl ring substituted with said at least one SO₃H moiety, at least one R^e is SO₃H.

6. The compound according to claim 1 wherein two R^a moieties are joined, providing said compound with the substructure:

7. The compound according to claim 1 wherein two R^e moieties are joined, providing said compound with the substructure:

8. The compound according to claim 1 having the formula:

9. The compound according claim 1 having the formula:

10. The compound according to claim 1 having the formula:

11. The compound according to claim 1 having the formula:

12. The compound according to claim 1 having the formula:

13. The compound according to claim 1 wherein R^a, and R^e are independently selected from:

14. The compound according to claim 1 wherein R^m has a formula:

15. The compound according to claim 1 wherein R^m has the formula:

16. The compound according to claim 1 wherein R^m is a member selected from:

17. The compound according to claim 1 wherein 1, 2, 3 or 4 of R^f, R^g, R^h, Rⁱ, R^c and R^d is:

18. The compound according to claim 1 wherein both R^c and R^d are:

19. The compound according to claim 1 in which two, three, four, five or six of R^a, R^e, R^c, R^d, R^f, R^g, R^h, Ri, and R^m in any combination, include an independently selected R¹¹ moiety which is:

20. The compound according to claim 1 in which two, three, four, five or six of R^a, R^e, R^c, R^d, R^f, R^g, R^h, Ri, and R^m in any combination, include an independently selected dye moiety or dye linker moiety.

21. The compound according to claim 1 in which two, three, four-, five or six of R^a, R^e, R^j, R^c, R^d, R^f, R^g, R^h, Ri, and R^m′

in any combination, include an independently selected polyvalent moiety.

22. The compound according to claim 21 wherein said polyvalent moiety has bound thereto one or more member selected from a dye, a dye-linker moiety, and a nucleotide polyphosphate.

27. The compound according to claim 8 wherein R^b and R^j are independently selected from:

28. The compound according to claim 8 in which two, three, four, five or six of R^b and R^j in any combination, include an independently selected R¹¹ moiety which is:

29. The compound according to claim 8 in which two, three, four, five or six of R^b and R^j in any combination, include an independently selected dye moiety or dye linker moiety.

30. The compound according to claim 8 in which two, three, four, five or six of R^b and R^j in any combination, include an independently selected polyvalent moiety.

31. The compound according to claim 30 wherein said polyvalent moiety has bound thereto one or more member selected from a dye, a dye-linker moiety, and a nucleotide polyphosphate.

23. A method of monitoring an enzyme reaction, said method comprising:

(a) forming a reaction mixture by contacting said enzyme with a fluorescent dye that is a compound having the formula;

24. The method according to claim 23 wherein said enzyme is a DNA polymerase and said dye comprises a nucleic acid moiety which is said substrate for said enzyme.

25. The method according to claim 23 wherein said enzyme reaction is template directed DNA synthesis.

26. The method according to claim 23 wherein said reaction is a component of a single molecule DNA sequencing analysis.