Nucleic acid labeling compounds

• US 6,844,433 B2
• Filed: 06/02/2003
• Issued: 01/18/2005
• Est. Priority Date: 07/31/1998
• Status: Expired due to Term

• Current Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Barone, Anthony D., McGall, Glenn
• Primary Examiner(s): Barts, Samuel
• Assistant Examiner(s): Lewis, Patrick T.
• Application Number:
  US10/452,375
• Publication Number:
  US 20030232979A1
• Time in Patent Office: 596 Days
• Field of Search: 536/22.1, 536/24.2, 536/25.3, 536/25.32, 435/6, 544/224, 544/233, 544/235, 544/242
• US Class Current: 536/25.32
• CPC Class Codes: C07H 19/04 Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
• CPC Class Codes: C07H 19/052 Imidazole radicals
• 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 19/12 Triazine radicals
• 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

Abstract

Nucleic acid labeling compounds containing heterocyclic derivatives are disclosed. The heterocyclic derivative containing compounds are synthesized by condensing a heterocyclic derivative with a cyclic group (e.g. a ribofuranose derivative). The labeling compounds are suitable for enzymatic attachment to a nucleic acid, either terminally or internally, to provide a mechanism of nucleic acid detection.

Claims

1. A nucleic acid labeling compound of the following structure:

wherein A is H or a functional group that permits the attachment of the nucleic acid labeling compound to a nucleic acid;

X is O, S, NR₁ or CHR₂, wherein R₁ and R₂ are, independently, H, alkyl or aryl;

Y is H, N₃, F, OR₉, SR₉ or NHR₉, wherein R₉ is H, alkyl or aryl;

Z is H, N₃, F or OR₁₀, wherein R₁₀ is H, alkyl or aryl;

L is amino alkyl;

Q is a detectable moiety; and

M is a connecting group, wherein m is an integer ranging from 0 to about 3.

2. A nucleic acid labeling compound according to claim 1, wherein A is H or H₄O₉P₃—

;

X is O;

Y is H or OR₉, wherein R₉ is H, alkyl or aryl;

Z is H, N₃, F or OR₁₀, wherein R₁₀ is H, alkyl or aryl;

L is —

NH(CH₂)_nNH—

, wherein n is an integer ranging from about 2 to about 10;

Q is biotin or carboxyfluorescein; and

M is —

CO(CH₂)₅NH—

or —

CO(CH₂)₅NHCO(CH₂)₅NH—

, wherein m is 1 or 0.

3. A nucleic acid labeling compound according to claim 2, wherein Y is H or OH;

Z is H or OH;

L is —

NH(CH₂)₄NH—

;

Q is biotin; and

m is 0.

4. A nucleic acid labeling compound according to claim 2, wherein Y is H or OH;

Z is H or OH;

L is —

NH(CH₂)₄NH—

;

Q is 5-carboxyfluorescein; and

m is 0.

5. A nucleic acid labeling compound according to claim 2, wherein A is H;

Y is H;

Z is H;

L is —

NH(CH₂)₄NH—

;

Q is biotin; and

m is 0.

6. A nucleic acid labeling compound according to claim 2, wherein A is H₄O₉P₃—

;

Y is H;

Z is H;

L is —

NH(CH₂)₄NH—

;

Q is biotin; and

m is 0.

7. A nucleic acid labeling compound according to claim 2, wherein A is H;

Y is H;

Z is H;

L is —

NH(CH₂)₄NH—

;

Q is biotin;

M is —

CO(CH₂)₅NH—

; and

m is 1.

8. A nucleic acid labeling compound according to claim 2, wherein A is H₄O₉P₃—

;

Y is H;

Z is H;

L is —

NH(CH₂)₄NH—

;

Q is biotin;

M is —

CO(CH₂)₅NH—

; and

m is 1.

9. A nucleic acid labeling compound according to claim 1, wherein A is H;

Y is H;

Z is H;

L is —

NH(CH₂)₄NH—

;

Q is biotin;

M is —

CO(CH₂)₅NH—

; and

m is 2.

10. A nucleic acid labeling compound according to claim 1, wherein A is H₄O₉P₃—

;

Y is H;

Z is H;

L is —

NH(CH₂)₄NH—

;

Q is biotin;

M is —

CO(CH₂)₅NH—

; and

m is 2.

11. A nucleic acid labeling compound according to claim 2, wherein A is H;

Y is H;

Z is H;

L is —

NH(CH₂)₄NH—

;

Q is 5-carboxyfluorescein; and

m is 0.

12. A nucleic acid labeling compound according to claim 2, wherein A is H₄O₉P₃—

;

Y is H;

Z is H;

L is —

NH(CH₂)₄NH—

;

Q is 5-carboxyfluorescein; and

m is 0.

13. The nucleic acid labeling compound according to claim 6, 8, 10 or 12 having the formula:

wherein A, X, Y, Z, L, M, N, m and Q are as defined herein above.

14. The nucleic acid labeling compound according to claim 6, 8, 10 or 12 having the formula:

wherein A, X, Y, Z, L, M, N, m and Q are as defined herein above.

15. The nucleic acid labeling compound according to claim 6, 8, 10 or 12 having the formula:

wherein A, X, Y, Z, L, M, N, m and Q are as defined herein above.

27. A method of synthesizing a labeled nucleic acid comprising attaching a nucleic acid labeling compound according to claim 2 to a nucleic acid.

28. A method of synthesizing a labeled nucleic acid comprising attaching a nucleic acid labeling compound according to claim 13 to a nucleic acid.

29. A method of synthesizing a labeled nucleic acid comprising attaching a nucleic acid labeling compound according to claim 14 to a nucleic acid.

30. A method of synthesizing a labeled nucleic acid comprising attaching a nucleic acid labeling compound according to claim 15 to a nucleic acid.

16. A nucleic acid labeling compound of the following structure:

wherein A is H or a functional group that permits the attachment of the nucleic acid labeling compound to a nucleic acid;

X is O, S, NR₁ or CHR₂, wherein R₁ and R₂ are, independently, H, alkyl or aryl;

Y is H, N₃, F, OR₉, SR₉ or NHR₉, wherein R₉ is H, alkyl or aryl;

Z is H, N₃, F or OR₁₀, wherein R₁₀ is H, alkyl or aryl;

L is alkynyl alkyl;

Q is a detectable moiety; and

M is a connecting group, wherein m is an integer ranging from 0 to about 3.

17. A nucleic acid labeling compound according to claim 16, wherein A is H or H₄O₉P₃—

;

X is O;

Y is H or OR₉, wherein R₉ is H, alkyl or aryl;

Z is H, N₃, F or OR₁₀, wherein R₁₀ is H, alkyl or aryl;

L is —

C≡

CH₂NH—

;

wherein n is an integer ranging from about 1 to about 10;

Q is biotin or carboxyfluorescein;

M is —

CO(CH₂)₅NH—

; and

m is 1 or 0.

18. A nucleic acid labeling compound according to claim 17, wherein Y is H or OH;

Z is H or OH;

L is —

C≡

CH₂NH—

;

Q is biotin; and

m is 1.

19. A nucleic acid labeling compound according to claim 17, wherein Y is H or OH;

Z is H or OH;

L is —

C≡

CH₂NH—

;

Q is 5-carboxyfluorescein; and

m is 1.

20. A nucleic acid labeling compound according to claim 17, wherein A is H;

Y is OH;

Z is OH;

L is —

C≡

CH₂NH—

;

Q is biotin;

M is —

CO(CH₂)₅NH—

; and

m is 1.

21. A nucleic acid labeling compound according to claim 17, wherein A is H₄O₉P₃—

;

Y is OH;

Z is OH;

L is —

C≡

CH₂NH—

;

Q is biotin;

M is —

CO(CH₂)₅NH—

; and

m is 1.

22. A nucleic acid labeling compound according to claim 17, wherein A is H;

Y is OH;

Z is OH;

L is —

C≡

CH₂NH—

;

Q is 5-carboxyfluorescein;

M is —

CO(CH₂)₅NH—

; and

m is 1.

23. A nucleic acid labeling compound according to claim 17, wherein A is H₄O₉P₃—

;

Y is OH;

Z is OH;

L is —

C≡

CH₂NH—

;

Q is 5-carboxyfluorescein;

M is —

CO(CH₂)₅NH—

; and

m is 1.

24. The nucleic acid labeling compound according to claim 21 or 23 having the formula:

wherein A, X, Y, Z, L, M, N, m and Q are as defined herein above.

25. The nucleic acid labeling compound according to claim 21 or 23 having the formula:

wherein A, X, Y, Z, L, M, N, m and Q are as defined herein above.

26. The nucleic acid labeling compound according to claim 21 or 23 having the formula:

wherein A, X, Y, Z, L, M, N, m and Q are as defined herein above.

31. A method of synthesizing a labeled nucleic acid comprising attaching a nucleic acid labeling compound according to claim 17 to a nucleic acid.

32. A method of synthesizing a labeled nucleic acid comprising attaching a nucleic acid labeling compound according to claim 24 to a nucleic acid.

33. A method of synthesizing a labeled nucleic acid comprising attaching a nucleic acid labeling compound according to claim 25 to a nucleic acid.

34. A method of synthesizing a labeled nucleic acid comprising attaching a nucleic acid labeling compound according to claim 26 to a nucleic acid.