Propargylethoxyamino nucleotides

• US 7,019,128 B2
• Filed: 07/09/2002
• Issued: 03/28/2006
• Est. Priority Date: 08/12/1996
• Status: Expired due to Fees

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applera Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Menchen, Steven M., Khan, Shaheer H., Rosenblum, Barnett B.
• Primary Examiner(s): LEWIS, PATRICK T
• Application Number:
  US10/192,776
• Publication Number:
  US 20030148470A1
• Time in Patent Office: 1,358 Days
• Field of Search: 536/26.26, 536/26.3, 536/26.7, 536/26.74, 536/26.8, 536/27.1, 536/27.13, 536/27.21, 536/27.6, 536/27.8, 536/27.81, 536/28.1, 536/28.3, 536/28.4, 536/28.5, 536/28.53, 536/28.54, 536/26.6, 536/25.3, 536/25.32, 536/26.71, 536/26.73, 536/27.11, 435/6, 435/89, 435/90, 435 911- 9153
• 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 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: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]

Abstract

Propargylethoxyamino nucleosides are disclosed having the structure
wherein R₁ and R₂ are —H, lower alkyl, or label; B is a 7-deazapurine, purine, or pyrimidine nucleoside base; W₁ is —H or —OH; W₂ is —OH or a moiety which renders the nucleoside incapable of forming a phosphodiester bond at the 3′-position; and W₃ is —PO₄, —P₂O₇, —P₃O₁₀, phosphate analog, or —OH. Additionaly, a primer extension method is provided employing the above propargylethoxyamino nucleosides.

Claims

1. A method for performing a primer extension reaction comprising:

annealing a polynucleotide primer to a portion of a template nucleic acid, and

extending the annealed primer in the presence of a compound having the structure;

 

wherein;

n is 1 or 2,

R1 and R2 taken separately are each selected from —

H, lower alkyl, protecting group and label,

B is a 7-deazapurine, purine, or pyrimidine nucleoside base,

wherein when B is purine or 7-deazapurine, the sugar moiety is attached at the N9-position of the purine or deazapurine, and when B is pyrimidine, the sugar moiety is attached at the N1-position of the pyrimidine, and

wherein if B is a purine, the adjacent triple-bonded carbon is attached to the 8-position of the purine, if B is 7-deazapurine, the adjacent triple-bonded carbon is attached to the 7-position of the 7-deazapurine, and if B is pyrimidine, the adjacent triple-bonded carbon is attached to the 5-position of the pyrimidine,

W1 is selected from —

H and —

OH,

W2 is —

OH or a moiety which renders the nucleoside incapable of forming a phosphodiester bond at the 3′

-position, and

W3 is selected from —

PO4, —

P2O7, —

P3O10, phosphate analog, and —

OH,

whereby an extended primer is formed comprising at least one of the compound.

2. The method of claim 1 wherein one of R1 and R2 is label and the other is —

H.

3. The method of claim 2 wherein W₃ is —

P₃O₁₀.

4. The method of claim 3 wherein W₁ is H.

5. The method of claim 4 wherein W₂ is —

OH or a moiety selected from —

H, azido, amino, fluoro and methoxy.

6. The method of claim 3 wherein W₂ is —

OH or a moiety selected from —

H, azido, amino, fluoro and methoxy.

7. The method of claim 2 wherein W₁ is H.

8. The method of claim 7 wherein W₂ is —

OH or a moiety selected from —

H, azido, amino, fluoro and methoxy.

9. The method of claim 2 wherein W₂ is —

OH or a moiety selected from —

H, azido, amino, fluoro and methoxy.

10. The method of claim 1 wherein W₃ is —

P₃O₁₀.

11. The method of claim 10 wherein W₁ is H.

12. The method of claim 11 wherein W₂ is —

OH or a moiety selected from —

H, azido, amino, fluoro and methoxy.

13. The method of claim 10 wherein W₂ is —

OH or a moiety selected from —

H, azido, amino, fluoro and methoxy.

14. The method of claim 1 wherein W₁ is H.

15. The method of claim 14 wherein W₂ is —

OH or a moiety selected from —

H, azido, amino, fluoro and methoxy.

16. The method of claim 1 wherein W₂ is —

OH or a moiety selected from —

H, azido, amino, fluoro and methoxy.

17. The method of claim 1 wherein R₁ is a label selected from a rhodamine dye and a fluorescein dye.

18. The method of claim 17 wherein W₁ is —

H and W₂ is —

H.

19. The method of claim 18 wherein the label is a rhodamine dye.

20. The method of claim 1 wherein the extending is mediated by a DNA polymerase.