2-aminopyrimidin-4-one nucleic acid labeling compounds

• US 7,468,243 B2
• Filed: 03/12/2002
• Issued: 12/23/2008
• Est. Priority Date: 03/12/2001
• 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): Wilson; James O.
• Assistant Examiner(s): Ward; Paul V.
• Application Number:
  US10/097,113
• Publication Number:
  US 20020182625A1
• Time in Patent Office: 2,478 Days
• Field of Search: 536/23.1, 536/29.1, 536/25.1, 544/320, 544/321, 435/6, 549/430
• US Class Current: 435/6.16
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: C07D 239/47 One nitrogen atom and one oxygen or sulfur atom, e.g. cytosine
• CPC Class Codes: C07H 19/06 Pyrimidine radicals
• CPC Class Codes: C07H 7/00 Compounds containing non-saccharide radicals linked to saccharide radicals by a carbon-to-carbon bond
• CPC Class Codes: C07H 7/06 Heterocyclic radicals
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence

Abstract

Nucleic acid labeling compounds are disclosed having the formula:

These compounds are useful for attaching a detectable label to a nucleic acid.

Claims

1. A nucleic acid labeling compound having the formula:

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

;

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

z is hydrogen, N₃, F or OR₁₀, wherein R₁₀ is hydrogen, alkyl or aryl; and

L is amido alkyl and M is -(M₁)_a-(M₂)_b- wherein a and b are independently integers from 0 to 5, and wherein M₁ and M₂ are connecting groups.

3. The nucleic acid labeling compound of claim 2, wherein L is —

(CH₂)_nC(O)NR₃—

, wherein R₃ is hydrogen, alkyl or aryl and n is an integer ranging from 1 to 10;

M₁ is —

(CH₂)_iO— and

M₂ is —

(CH₂)_iNH—

, wherein i and j are independently integers from 1 to 5.

4. The nucleic acid labeling compound of claim 3, wherein Y is H or OH;

Z is H or OH;

-L is —

CH₂C(O)NH—

;

M₁ is —

(CH₂CH₂O)₃— and

M₂ is —

CH₂CH₂NH—

; and

Q is biotin or a carboxyfluorescein.

5. The nucleic acid labeling compound of claim 4, wherein Y is OH;

Z is OH;

-L-(M)_m - is —

CH₂C(O)NH—

(CH₂CH₂O)₃—

CH₂CH₂NH—

; and

Q is biotin.

6. The nucleic acid labeling compound of claim 4 wherein Y is OH;

Z is OH;

-L-(M)_m - is —

CH₂C(O)NH—

(CH₂CH₂O)₃—

CH₂CH₂NH—

; and

Q is a carboxyfluorescein.

7. The nucleic acid labeling compound of claim 1, wherein A is H₄O₉P₃—

;

Y is H or OR₉, wherein R₉is hydrogen, alkyl or aryl;

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

L is amido alkyl and M is -(M₁)_a-(M₂)_b- (M₃)_c-(M₄)_d - wherein a, b, c, and d are independently integers from 0 to 5 and the sum of a, b, c, and d is not greater than 15, and wherein M₁, M₂, M₃, and M₄ are connecting groups.

8. The nucleic acid labeling compound of claim 7, wherein L is —

(CH₂)_nC(O)NR₃—

, wherein R₃ is hydrogen, alkyl or aryl and n is an integer ranging from 1 to 10;

M₁, M₂, M₃, and M₄ are each independently —

C(O)(CH₂)_kO—

, —

(CH₂)_i O—

or —

(CH₂)_jNH—

, wherein i, j and k are independently integers from 1 to 5.

9. The nucleic acid labeling compound of claim 8, wherein L is —

CH₂C(O)NR₃—

, wherein R₃ is hydrogen or, alkyl;

M₁ is —

(CH₂)₂NH—

, M₂ is —

C(O)(CH₂)₂O—

, M₃ is —

(CH₂CH₂O)₃—

, and M₄ is —

(CH₂)₂NH—

.

10. The nucleic acid labeling compound of claim 9 wherein Y is OH;

Z is OH;

L-(M)_m - is —

CH₂C(O)NH—

CH₂CH₂NH—

C(O)CH₂CH₂O—

(CH₂CH₂O)₃—

CH₂CH₂NH—

; and

Q is biotin.

11. The nucleic acid labeling compound of claim 9, wherein Y is OH;

Z is OH;

-L-(M)_m - is —

CH₂C(O)NH—

CH₂CH₂NH—

;

m is 1; and

Q is a carboxyfluorescein.

12. The nucleic acid labeling compound of claim 8, wherein L is —

(CH₂)₂C(O)NR₃—

, wherein R₃ is hydrogen or, alkyl;

M₁ is —

(CH₂)₂NH—

, M₂ is —

C(O)(CH₂)₂O—

, M₃ is —

(CH₂CH₂O)₃— and

M₄ is (CH₂)₂NH—

.

13. The nucleic acid labeling compound of claim 12, wherein Y is OH;

Z is OH;

L-(M)_m - is —

CH₂C(O)NH—

CH₂CH₂NH—

C(O)CH₂CH₂O—

(CH₂CH₂O)₃—

CH₂CH₂NH—

; and

Q is biotin.

14. The nucleic acid labeling compound of claim 12, wherein Y is OH;

Z is OH;

-L-(M)_m - is —

CH₂CH₂C(O)NH—

CH₂CH₂NH—

;

m is 1; and

Q is a carboxyfluorescein.

15. The nucleic acid labeling compound of claim 1, wherein A is H₄O₉P₃—

;

Y is H or OR₉, wherein R₉is hydrogen, alkyl or aryl;

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

L is functionalized alkyl and M is -(M₁)_a- (M₂)_b-(M₃)_c-(M₄)_d - wherein a, b, c, and d are independently integers from 0 to 5 and the sum of a, b, c, and d is not greater than 15, and wherein M₁, M₂, M₃, and M₄ are connecting groups.

16. The nucleic acid labeling compound of claim 15, wherein L is —

(CH₂)_nO—

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

M₁, M₂, M₃, and M₄ are each independently —

C(O)(CH₂)_kO—

, —

(CH₂)_iO—

or —

(CH₂)_jNH—

, wherein i, j and k are independently integers from 1 to 5.

17. The nucleic acid labeling compound of claim 16, wherein L is —

CH₂O—

;

M₁ is —

(CH₂)₂NH—

, M₂ is —

C(O)(CH₂)₂O—

, M₃ is —

(CH₂CH₂O)₃— and

M₄ is —

(CH₂)₂NH—

.

18. The nucleic acid labeling compound of claim 17, wherein Y is OH;

Z is OH;

L-(M)_m - is —

CH₂O—

CH₂CH₂NH—

C(O)(CH₂)₂O—

(CH₂CH₂O)₃—

CH₂CH₂NH—

; and

Q is biotin.

19. The nucleic acid labeling compound of claim 17, wherein Y is OH;

Z is OH;

L-(M)_m - is —

CH₂O—

CH₂CH₂NH—

;

m is 1; and

Q is a carboxyfluorescein.

20. The nucleic acid labeling compound of claim 15, wherein L is —

(CH₂)_nNR₃—

, wherein R₃ is hydrogen, alkyl or aryl and n is an integer ranging from 1 to 10;

M₁, M₂, M₃, and M₄ are each independently —

C(O)(CH₂)_kO—

, —

(CH₂)_iO—

or —

(CH₂)_jNH—

, wherein i, j and k are independently integers from 1 to 5.

21. The nucleic acid labeling compound of claim 20, wherein L is —

(CH₂)₆NH—

;

M₁ is —

(CH₂)₂NH—

, M₂ is —

C(O)(CH₂)₂O—

, and M₃ is —

(CH₂CH₂O)₃—

.

22. The nucleic acid labeling compound of claim 21, wherein Y is OH;

Z is OH;

-L-(M)_m - is —

(CH₂)₆NH—

C(O)(CH₂)₂O—

(CH₂CH₂O)₃—

CH₂CH₂NH—

; and

Q is biotin.

23. The nucleic acid labeling compound of claim 21, wherein Y is OH;

Z is OH;

L is —

(CH₂)₆NH—

;

m is 0; and

Q is a carboxyfluorescein.

24. The nucleic acid labeling compound of claim 1, wherein A is H₄O₉P₃—

;

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

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

L is alkenyl alkyl and M is -(M₁)_a- (M₂)_b-(M₃)_c-(M₄)_d - wherein a, b, c, and d are independently integers from 0 to 5 and the sum of a, b, c, and d is not greater than 15, and wherein M₁, M₂, M₃, and M₄ are connecting groups.

25. The nucleic acid labeling compound of claim 7, wherein L is—

structure —

CH═

CH—

(CH₂)_nC(O)N(R₆)—

, wherein R₆ is hydrogen, alkyl or aryl and n is an integer ranging from 0 to 10;

M₁, M₂, M₃, and M₄ are each independently —

C(O)(CH₂)_kO—

, —

(CH₂)_iO—

or —

(CH₂)_jNH—

, wherein i, j and k are independently integers from 1 to 5.

26. The nucleic acid labeling compound of claim 2, wherein L is —

CH═

CH—

C(O)N(H);

M₁ is —

(CH₂)₂NH—

, M₂ is —

C(O)(CH₂)₂O—

, M₃ is (CH₂CH₂O)₃— and

M₄ is —

(CH₂)₂NH—

.

27. The nucleic acid labeling compound of claim 12, wherein -L-(M)_m - is —

CH═

CH—

C(O)NH—

CH₂CH₂NH—

C(O)CH₂CH₂O—

(CH₂CH₂O)₃—

CH₂CH₂NH—

;

Y is OH;

Z is OH; and

Q is biotin.

28. The nucleic acid labeling compound of claim 12, wherein Y is OH;

Z is OH;

L-(M)hd m - is —

CH═

CH—

C(O)NH—

CH₂CH₂NH—

;

m is 1; and

Q is a carboxyfluorescein.

29. The nucleic acid labeling compound of claim 1 having the formula:

30. A nucleic acid labeling compound of claim 1 having the formula:

31. The nucleic acid labeling compound according to claim 24, wherein Y is H or OH;

Z is H or OH;

L is structure —

CH═

CH—

(CH₂)_nC(O)N(R₆)—

;

M₁ is —

(CH₂)_jNH—

;

M₂ is —

C(O)(CH₂)₅NH—

, c and d are each 0; and

Q is biotin or a carboxyfluorescein.

32. The nucleic acid labeling compound of claim 31, wherein L is —

CH═

CH—

C(O)N(H); and

M₁ is —

(CH₂)₂NH—

.

33. A nucleic acid labeling compound according to claim 31, wherein Y is OH;

Z is OH;

L is —

CH═

CH—

C(O)N(H)—

; and

Q is biotin.

34. A nucleic acid labeling compound according to claim 1, wherein:

A is selected from the group consisting of H;

monophosphate;

diphosphate;

triphosphate;

(R₂N)(R′

O)P, wherein R is linear, branched or cyclic alkyl, and R′

is a protecting group; and

HP(O)O—

HNR, wherein R is linear, branched or cyclic alkyl;

L is selected from the group consisting of —

C(O)—

NR—

, wherein R is H, alkyl, or aryl;

—

R′

C(O)NR—

, wherein R is H, alkyl, or aryl, and R′

is alkyl or aryl;

—

(CH₂)_nC(O)NH—

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

—

C(O)NRR′

—

, wherein R is H, alkyl, or aryl, and R′

is alkylene or arylene;

—

C(O)NH(CH₂)_nR—

, wherein R is —

O—

, —

NR′

—

, or —

C(O)—

, and R′

is H, alkyl, or aryl, and n is an integer ranging from 2 to 10;

—

(CH₂)_nR—

, wherein n is an integer ranging from 1 to 10, and R is —

O—

, —

S—

, —

NH—

, or —

C(O)—

;

—

CH═

CH—

R—

, wherein R is alkylene or arylene;

—

CH═

CH(CH₂)_n—

R—

, wherein n is an integer ranging from 1 to 10, R is —

O—

, —

NR′

—

, —

C(O)—

, or —

C(O)NR′

—

, and R′

is H, alkyl, or aryl;

—

(CH₂)_nC(O)—

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

—

O(CH₂)_nR—

, wherein n is an integer ranging from 2 to 10, and R is —

O—

, —

S—

, —

NH—

, or —

C(O)—

;

—

S(CH₂)_nR—

, wherein n is an integer ranging from 2 to 10, and R is —

O—

, —

S—

, —

NH—

, or —

C(O)—

; and

—

NH(CH₂)_nNH—

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

Q is selected from the group consisting of radiolabels, magnetic particles, colloidal gold, fluorescent labels, chemiluminescent labels, bioluminescent labels, spin labels, dyes, and colored glass or plastic beads;

M is selected from the group consisting of amino alkyl groups, —

C(O)(CH₂)₅NH—

, —

C(O)—

, —

NH—

, —

C(O)O—

, —

C(O)NH—

, —

(CH₂)_nO—

, —

(CH₂)_nNH—

, —

C(O)(CH₂)_nO—

, —

C(O)(CH₂)₅NHC(O)(CH₂)₅NH—

, —

NH(CH₂CH₂O)_nNH—

, and —

C(O)(CH₂)₅—

, wherein n is an integer ranging from 1 to 5; and

m is an integer ranging from 0 to 5.

35. A method of synthesizing a labeled nucleic acid comprising attaching a nucleic acid labeling compound of claims 2, 7, 12, 15, 20, or to a nucleic acid.