Fluorescent dye

• US 6,706,879 B2
• Filed: 02/28/2002
• Issued: 03/16/2004
• Est. Priority Date: 02/28/2001
• Status: Expired due to Term

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Stratagene California (Agilent Technologies, Inc.)
• Inventors: Anderson, Jack, Braman, Jeffrey Carl
• Primary Examiner(s): POWERS, FIONA
• Application Number:
  US10/087,072
• Publication Number:
  US 20030088109A1
• Time in Patent Office: 747 Days
• Field of Search: 546/88, 548/120, 548/151, 548/218, 548/302.1, 548/433, 864/8, 435/6, 436/94, 436/172, 436/800
• US Class Current: 546/88
• CPC Class Codes: C07H 19/06 Pyrimidine radicals
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/16 Purine radicals
• CPC Class Codes: C07H 19/20 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C09B 23/0075 the polymethine chain being part of an heterocyclic ring
• CPC Class Codes: C09B 23/06 three >CH- groups, e.g. carbocyanines
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: Y10S 436/80 Fluorescent dyes, e.g. rhodamine
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

The invention relates to fluorescent dyes. More particularly, the invention relates to fluorescent cyanine dyes, and especially to water soluble fluorescent cyanine dyes that contain additional sites for attachment to biomolecules. The invention provides a group of novel, water soluble fluorescent cyanine dyes that have distinct fluorescence characteristics that permit their use in any assay or method suited to water soluble fluorescent dyes, and especially to assays requiring a plurality of distinguishable fluorescent markers. The invention further relates to nucleotides, nucleosides, polynucleotides and polypeptides labeled with novel fluorescent cyanine dyes according to the invention, and methods of using them.

Claims

1. A fluorescent cyanine dye having the formula:

2. The fluorescent cyanine dye of claim 1 wherein one or both of Y₁ and Y₂ are N.

3. The fluorescent cyanine dye of claim 2 wherein one or both of X₁ and X₂ are S.

4. The fluorescent cyanine dye of claim 2 wherein one or both of X₁ and X₂ are O.

5. The fluorescent cyanine dye of claim 2 wherein one or both of X₁ and X₂ are CH₂.

6. The fluorescent cyanine dye of claim 2 wherein one or both of X₁ and X₂ are (CH═

CH).

7. The fluorescent cyanine dye of claim 2 wherein one or both of Y₁ and Y₂ are S.

8. The fluorescent cyanine dye of claim 1 wherein Z₁ and Y₂ are S.

9. The fluorescent cyanine dye of claim 1 wherein Y₁ and Z₂ are S.

10. The fluorescent cyanine dye of claim 1 wherein Q is CH₂.

11. The fluorescent cyanine dye of claim 1 wherein Q is C(CH₃)₂.

12. A composition comprising a fluorescent cyanine dye of claim 1.

34. A fluorescent cyanine dye of any one of claims 1, 13, 16, 19, 22, 25 or 28 that comprises a succinimide ester linked to a heterocyclic nitrogen.

35. A nucleoside or nucleotide labeled with a flourescent cyanine dye of any one of claims 1, 13, 16, 19, 22, 25 or 28.

36. A polynucleotide labeled with a flourescent cyanine dye of any one of claims 1, 13, 16, 19, 22, 25 or 28.

37. A polypeptide labeled with a flourescent cyanine dye of any one of claims 1, 13, 16, 19, 22, 25 or 28.

38. A method of labeling a nucleotide or nucleoside, said method comprising contacting a fluorescent cyanine dye of claim 1 with said nucleotide or nucleoside.

39. A method of labeling a nucleic acid, said method comprising contacting a fluorescent cyanine dye of claim 1 with said nucleic acid.

40. The method of claim 39 wherein said nucleic acid comprises an allyl-amine-modified nucleotide, and said dye comprises an NHS group.

41. A method of labeling a polypeptide, said method comprising contacting a fluorescent cyanine dye of claim 1 with said polypeptide.

42. A method of labeling a nucleic acid, said method comprising contacting said nucleic acid with a cis-platinum complex comprising a fluorescent cyanine dye of claim 1.

43. A method of determining a nucleic acid sequence, said method comprising performing a nucleic acid sequencing reaction in the presence of a labeled nucleotide of claim 35.

44. The method of claim 43, wherein said contacting is performed in the presence of a second nucleotide comprising a fluorescent dye that is spectrally distinct from the dye on said first nucleotide.

45. A method of determining a nucleic acid sequence, said method comprising determining a nucleic acid sequence on a nucleic acid comprising a fluorescent cyanine dye of claim 1.

46. A method of detecting a polynucleotide, said method comprising detecting a polynucleotide comprising a labeled nucleotide of claim 35.

47. A method of detecting a polynucleotide, said method comprising detecting a polynucleotide comprising a fluorescent cyanine dye of claim 1.

48. The method of claim 47, wherein said detecting is performed on a nucleic acid microarray.

49. A method of detecting a polypeptide, said method comprising detecting a polypeptide comprising a fluorescent cyanine dye of claim 1.

13. A fluorescent cyanine dye having the formula:

wherein;

n≧

1;

Q is selected from the group consisting of O, S, CH₂, (CH═

CH) and C(CH₃)₂;

R₁-R₄ are the same or different and are selected from the group consisting of H, methyl, ethyl and (CH₂)_qV, wherein q is an integer from 1 to 25 and V is a reactive group or H, and at least one of R1-R4 has a reactive group;

each of W_1-8 is the same or different and may be H or a hydrophilic moiety; and

at least one occurrence of W is a hydrophilic moiety.

14. A composition comprising the dye of claim 13.

15. A fluorescent cyanine dye having the formula:

wherein R₁-R₄ are the same or different and are selected from the group consisting of H, methyl, ethyl and (CH₂)_qV, wherein q is an integer from 1 to 25 and V is a reactive group or H, and at least one of R₁-R₄ has a reactive group.

16. A fluorescent cyanine dye having the formula:

wherein;

n≧

1;

Q is selected from the group consisting of O, S, CH₂, (CH═

CH) and C(CH₃)₂;

R1-R4 are the same or different and are selected from the group consisting of H, methyl, ethyl and (CH₂)_qV, wherein q is an integer from 1 to 25 and V is a reactive group or H, and at least one of R1-R4 has a reactive group;

each of W_1-8 is the same or different and may be H or a hydrophilic moiety; and

at least one occurrence of W is a hydrophilic moiety.

17. A composition comprising the dye of claim 16.

18. A fluorescent cyanine dye having the formula:

wherein R₁-R₄ are the same or different and are selected from the group consisting of H, methyl, ethyl and (CH₂)_qV, wherein q is an integer from 1 to 25 and V is a reactive group or H, and at least one of R₁-R₄ has a reactive group.

19. A fluorescent cyanine dye having the formula:

wherein;

n≧

1;

Q is selected from the group consisting of O, S, CH₂, (CH═

CH) and C(CH₃)₂;

R1-R4 are the same or different and are selected from the group consisting of H, methyl, ethyl and (CH₂)_qV, wherein q is an integer from 1 to 25 and V is a reactive group or H, and at least one of R1-R4 has a reactive group;

each of W_1-8 is the same or different and may be H or a hydrophilic moiety; and

at least one occurrence of W is a hydrophilic moiety.

20. A composition comprising the dye of claim 19.

21. A fluorescent cyanine dye having the formula:

wherein R₁-R₄ are the same or different and are selected from the group consisting of H, methyl, ethyl and (CH₂)_qV, wherein q is an integer from 1 to 25 and V is a reactive group or H, and at least one of R₁-R₄ has a reactive group.

22. A fluorescent cyanine dye having the formula:

wherein;

n≧

1;

Q is selected from the group consisting of O, S, CH₂, (CH═

CH) and C(CH₃)₂;

R₁-R₄ are the same or different and are selected from the group consisting of H, methyl, ethyl and (CH₂)_qV, wherein q is an integer from 1 to 25 and V is a reactive group or H, and at least one of R₁-R₄ has a reactive group;

each of W_1-8 is the same or different and may be H or a hydrophilic moiety; and

at least one occurrence of W is a hydrophilic moiety.

23. A composition comprising the dye of claim 22.

24. A fluorescent cyanine dye having the formula:

wherein R₁-R₄ are the same or different and are selected from the group consisting of H, methyl, ethyl and (CH₂)_qV, wherein q is an integer from 1 to 25 and V is a reactive group or H, and at least one of R₁-R₄ has a reactive group.

25. A fluorescent cyanine dye having the formula:

wherein;

n≧

1;

Q is selected from the group consisting of O, S, CH₂, (CH═

CH) and C(CH₃)₂;

R₁-R₄ are the same or different and are selected from the group consisting of H, methyl, ethyl and (CH₂)_qV, wherein q is an integer from 1 to 25 and V is a reactive group or H, and at least one of R₁-R₄ has a reactive group;

each of W_1-8 is the same or different and may be H or a hydrophilic moiety; and

at least one occurrence of W is a hydrophilic moiety.

26. A composition comprising the dye of claim 25.

27. A fluorescent cyanine dye having the formula:

wherein R₁-R₄ are the same or different and are selected from the group consisting of H, methyl, ethyl, C(CH₃)₂ and (CH₂)_qV, wherein q is an integer from 1 to 25 and V is a reactive group or H, and at least one of R₁-R₄ has a reactive group.

28. A fluorescent cyanine dye having the formula:

wherein;

n≧

1;

Q is selected from the group consisting of O, S, CH₂, (CH═

CH) and C(CH₃)₂;

R₁-R₄ are the same or different and are selected from the group consisting of H, methyl, ethyl and (CH₂)_qV, wherein q is an integer from 1 to 25 and V is a reactive group or H, and at least one of R₁-R₄ has a reactive group;

each of W_1-8 is the same or different and may be H or a hydrophilic moiety; and

at least one occurrence of W is a hydrophilic moiety.

29. A composition comprising the dye of claim 28.

30. A fluorescent cyanine dye having the formula:

wherein R₁-R₄ are the same or different and are selected from the group consisting of H, methyl, ethyl and (CH₂)_qV, where in q is an integer from 1 to 25 and V is a reactive group or H, and at least one of R₁-R₄ has a reactive group.

32. A composition comprising the dye of claim 30.

31. A fluorescent cyanine dye having the formula:

wherein;

n≧

1;

Q is selected from the group consisting of O, S, CH₂, (CH═

CH) and C(CH₃)₂;

R₁-R₄ are the same or different and are selected from the group consisting of H, methyl, ethyl and (CH₂)_qV, wherein q is an integer from 1 to 25 and V is a reactive group or H, and at least one of R₁-R₄ has a reactive group;

each of W_1-8 is the same or different and may be H or a hydrophilic moiety; and

at least one occurrence of W is a hydrophilic moiety.

33. A fluorescent cyanine dye having the formula:

wherein R₁-R₄ are the same or different and are selected from the group consisting of H, methyl, ethyl and (CH₂)_qV, wherein q is an integer from 1 to 25 and V is a reactive group or H, and at least one of R₁-R₄ has a reactive group.