Nucleic acid labelling compounds

• US 8,497,064 B2
• Filed: 11/01/2011
• Issued: 07/30/2013
• Est. Priority Date: 03/12/2001
• Status: Expired due to Fees

• Current Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: McGall, Glenn H., Barone, Anthony D.
• Primary Examiner(s): Ward, Paul V.
• Application Number:
  US13/286,870
• Publication Number:
  US 20120053336A1
• Time in Patent Office: 637 Days
• Field of Search: 435/6, 536/23.1, 536/25.1, 544/320, 544/321, 549/430
• US Class Current: 435/6
• 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. The 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 represented by the structure

2. The nucleic acid labeling compound of claim 1, wherein:

L is selected from the group consisting of amido alkyl, functionalized alkyl, alkenyl alkyl, alkanoyl, and N-alkyl amido.

3. The nucleic acid labeling compound of claim 1, wherein:

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

CO(CH₂)₅NH—

, —

CO—

, —

NH—

, —

CO(O)—

, —

CO(NH)—

, —

(CH₂)_iO—

, —

(CH₂)_jNH—

, —

C(O)(CH₂)_hO—

, —

CO(CH₂)₅NHCO(CH₂)₅NH—

, —

NH(CH₂CH₂O)_kNH—

, and —

CO(CH₂)₅—

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

4. The nucleic acid labeling compound of claim 1, wherein:

Y₁ is selected from the group consisting of hydrogen, OR₉, wherein R₉ is hydrogen or alkyl, —

NR₁₆-L-(M)_m-Q, wherein R₁₆ is hydrogen, alkyl, or aryl, —

O-L-(M)_m-Q, and —

S-L-(M)_m-Q.

5. The nucleic acid labeling compound of claim 1, wherein:

Z₁ is selected from the group consisting of hydrogen, OR₁₀, wherein R₁₀ is hydrogen or alkyl, —

NR₁₆-L-(M)_m-Q, wherein R₁₆ is hydrogen or alkyl, —

O-L-(M)_m-Q, and —

S-L-(M)_m-Q.

6. The nucleic acid labeling compound of claim 1, wherein Q is:

a fluorescent group which absorbs light with a wavelength above 300 nm, and emits light with a wavelength above 310 nm, or

a fluorescent group which absorbs light with a wavelength above 400 nm, and emits light with a wavelength above 410 nm.

7. The nucleic acid labeling compound of claim 1, wherein:

A is H or H₄O₉P₃—

;

X₁ is O;

Y₁ is hydrogen, OR₉, wherein R₉ is hydrogen or alkyl, or —

NR₁₆-L-(M)_m-Q, wherein R₁₆ is hydrogen, alkyl, or aryl;

Z₁ is hydrogen, OR₁₀, wherein R₁₀ is hydrogen or alkyl, or —

NR₁₆-L-(M)_m-Q, wherein R₁₆ is hydrogen or alkyl;

L is selected from the group consisting of amido alkyl, functionalized alkyl, alkenyl alkyl, alkanoyl, and N-alkyl amido;

R₁₅ is hydrogen or alkyl;

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

CO(CH₂)₅NH—

, —

CO—

, —

NH—

, —

CO(O)—

, —

CO(NH)—

, —

(CH₂)_iO—

, —

(CH₂)_jNH—

, —

C(O)(CH₂)_hO—

, —

CO(CH₂)₅NHCO(CH₂)₅NH—

, —

NH(CH₂CH₂O)_kNH—

, and —

CO(CH₂)₅—

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

m is an integer from 1 to 15.

8. The nucleic acid labeling compound of claim 7, wherein:

L is N-alkyl amido; and

M is —

CH₂—

NR₃—

, wherein R₃ is hydrogen or alkyl.

9. The nucleic acid labeling compound of claim 7, wherein:

Y₁ is hydrogen or OH;

Z₁ is —

NH-L-(M)_m-Q;

L is —

(CH₂)_nNHC(O)—

, where n is an integer from 1 to 10;

M is —

NH(CH₂)_p—

, where p is an integer from 1 to 10;

R₁₅ is hydrogen or methyl; and

Q is biotin or a carboxyfluorescein.

10. The nucleic acid labeling compound of claim 9, wherein:

Y₁ is OH;

R₁₅ is hydrogen or methyl;

-L-(M)_m- is —

(CH₂)₄NH—

C(O)(CH₂)₅NH—

; and

Q is biotin or a carboxyfluorescein.

11. The nucleic acid labeling compound of claim 7, wherein:

Y₁ is —

NH-L-(M)_m-Q;

Z₁ is hydrogen or OH;

L is selected from the group consisting of amido alkyl, functionalized alkyl, alkenyl alkyl, alkanoyl, and N-alkyl amido;

M is —

NH(CH₂)_p—

, where p is an integer from 1 to 10;

R₁₅ is hydrogen or methyl; and

Q is biotin or a carboxyfluorescein.

12. The nucleic acid labeling compound of claim 7, wherein:

A is H or H₄O₉P₃—

;

X₁ is O;

Y₁ is hydrogen, OR₉, wherein R₉ is hydrogen or alkyl, or —

O-L-(M)_m-Q;

Z₁ is hydrogen, OR₁₀, wherein R₁₀ is hydrogen or alkyl, or —

O-L-(M)_m-Q;

L is alkylene; and

m is an integer from 1 to 10.

13. The nucleic acid labeling compound of claim 12, wherein:

Y₁ is hydrogen or OH;

Z₁ is —

O-L-(M)_m-Q;

L is —

(CH₂)_n—

, where n is an integer from 1 to 12;

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

CO(CH₂)₅NH—

, —

CO—

, —

NH—

, —

CO(O)—

, —

CO(NH)—

, —

(CH₂)_iO—

, —

(CH₂)_jNH—

, —

C(O)(CH₂)_hO—

, —

CO(CH₂)₅NHCO(CH₂)₅NH—

, —

NH(CH₂CH₂O)_kNH—

, and —

CO(CH₂)₅—

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

R₁₅ is hydrogen or methyl; and

Q is biotin or a carboxyfluorescein.

14. The nucleic acid labeling compound of claim 13, wherein:

M is —

NH—

.

15. The nucleic acid labeling compound of claim 14, wherein:

Y₁ is OH;

R₁₅ is hydrogen or methyl;

-L-(M)_m- is —

(CH₂)₆—

NH—

; and

Q is biotin or a carboxyfluorescein.

16. The nucleic acid labeling compound of claim 7, wherein:

Y₁ is —

O-L-(M)_m-Q;

Z₁ is hydrogen or OH;

L is —

(CH₂)_n—

, where n is an integer from 1 to 12;

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

CO(CH₂)₅NH—

, —

CO—

, —

NH—

, —

CO(O)—

, —

CO(NH)—

, —

(CH₂)_iO—

, —

(CH₂)_jNH—

, —

C(O)(CH₂)_hO—

, —

CO(CH₂)₅NHCO(CH₂)₅NH—

, —

NH(CH₂CH₂O)_kNH—

, and —

CO(CH₂)₅—

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

R₁₅ is hydrogen or methyl; and

Q is biotin or a carboxyfluorescein.

17. The nucleic acid labeling compound of claim 16, wherein:

Z₁ is OH;

R₁₅ is hydrogen or methyl;

-L-(M)_m- is —

(CH₂)₆—

NH—

; and

Q is biotin or a carboxyfluorescein.

18. The nucleic acid labeling compound of claim 7, wherein:

A is H or H₄O₉P₃—

;

X₁ is O;

Y₁ is hydrogen, OR₉, wherein R₉ is hydrogen or alkyl, or —

S-L-(M)_m-Q;

Z₁ is hydrogen, OR₁₀, wherein R₁₀ is hydrogen or alkyl, or —

S-L-(M)_m-Q;

L is alkylene; and

m is an integer from 1 to 10.

19. The nucleic acid labeling compound of claim 18, wherein:

Y₁ is hydrogen or OH;

Z₁ is —

S-L-(M)_m-Q;

L is —

S—

(CH₂)_n—

, where n is an integer from 1 to 10;

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

CO(CH₂)₅NH—

, —

CO—

, —

NH—

, —

CO(O)—

, —

CO(NH)—

, —

(CH₂)_iO—

, —

(CH₂)_jNH—

, —

C(O)(CH₂)_hO—

, —

CO(CH₂)₅NHCO(CH₂)₅NH—

, —

NH(CH₂CH₂O)_kNH—

, and —

CO(CH₂)₅—

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

R₁₅ is hydrogen or methyl; and

Q is biotin or a carboxyfluorescein.

20. The nucleic acid labeling compound of claim 19, wherein:

Y₁ is OH;

R₁₅ is hydrogen or methyl;

-L-(M)_m- is —

S—

(CH₂)₂—

NH—

; and

Q is biotin or a carboxyfluorescein.

21. The nucleic acid labeling compound of claim 7, wherein:

Y₁ is —

S-L-(M)_m-Q;

Z₁ is hydrogen or OH;

L is —

S—

(CH₂)_n—

, where n is an integer from 1 to 10;

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

CO(CH₂)₅NH—

, —

CO—

, —

NH—

, —

CO(O)—

, —

CO(NH)—

, —

(CH₂)_iO—

, —

(CH₂)_jNH—

, —

C(O)(CH₂)_hO—

, —

CO(CH₂)₅NHCO(CH₂)₅NH—

, —

NH(CH₂CH₂O)_kNH—

, and —

CO(CH₂)₅—

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

R₁₅ is hydrogen or methyl; and

Q is biotin or a carboxyfluorescein.

22. The nucleic acid labeling compound of claim 21, wherein:

Z₁ is OH;

R₁₅ is hydrogen or methyl;

-L-(M)_m- is —

S—

(CH₂)₂—

NH—

; and

Q is biotin or a carboxyfluorescein.

23. A nucleic acid derivative produced by the process of:

attaching at least one nucleic acid to a support; and

coupling the at least one nucleic acid to the nucleic acid labeling compound of claim 1.

24. The nucleic acid derivative produced by the process of claim 23, wherein either a terminal transferase or a ligase performs the coupling step.

25. A hybridization product, comprising the nucleic acid derivative of claim 23 bound to a complementary probe.

26. The hybridization product of claim 25, wherein the complementary probe is attached to a glass chip.