Propargylethoxyamino nucleotide primer extensions

• US 6,504,024 B2
• Filed: 06/14/2001
• Issued: 01/07/2003
• Est. Priority Date: 08/12/1996
• Status: Expired due to Term

• Current Assignee: PE Corp. (Thermo Fisher Scientific Incorporated)
• Original Assignee: PE Corp. (Thermo Fisher Scientific Incorporated)
• Inventors: Menchen, Steven M., Khan, Shaheer H., Rosenblum, Barnett B.
• Primary Examiner(s): Wilson, James O.
• Application Number:
  US09/881,898
• Publication Number:
  US 20020045180A1
• Time in Patent Office: 572 Days
• Field of Search: 536/26.26, 536/26.3, 536/26.6, 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, 536/28.53
• US Class Current: 536/26.26
• 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. Additionally, a primer extension method is provided employing the above propargylethoxyamino nucleosides.

Claims

1. A propargylethoxyamino nucleotide having the structure:

wherein;

n is 1 or 2;

R₁ and R₂ are independently selected from H, C₁-C₈ lower alkyl, and a protecting group, or R₁ and R₂ taken together is a protecting group;

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

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

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

W₁ is H or OH;

W₂ is selected from H, OH, and a moiety which renders the nucleoside incapable of forming a phosphodiester bond at the 3′

-position; and

W₃ is selected from OH, phosphate, diphosphate, triphosphate, an activated phosphate, and a phosphate analog.

2. The propargylethoxyamino nucleotide of claim 1 wherein R₁ is H, and R₂ is trifluoroacetyl.

3. The propargylethoxyamino nucleotide of claim 1 wherein R₁ and R₂ taken together is phthalimido.

4. The propargylethoxyamino nucleotide of claim 1 wherein W₁ and W₂ are each H.

5. The propargylethoxyamino nucleotide of claim 1 wherein W₂ is selected from azido, amino, fluoro, and methoxy.

6. The propargylethoxyamino nucleotide of claim 1 wherein B is selected from adenine, guanine, cytosine, uracil, thymine, deazaadenine, and deazaguanosine.

7. A labelled propargylethoxyamino nucleotide having the structure:

wherein;

n is 1 or 2;

L is a label;

R₁ is H or C₁-C₈ lower alkyl;

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

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

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

W₁ is H or OH;

W₂ is selected from H, OH, and a moiety which renders the nucleoside incapable of forming a phosphodiester bond at the 3′

-position; and

W₃ is selected from OH, phosphate, diphosphate, triphosphate, and phosphate analog.

8. The labelled propargylethoxyamino nucleotide of claim 7 wherein L is a fluorescein-type dye selected from 6-carboxyfluorescein, 5-carboxyfluorescein, 6-carboxy-4,7,2′

,7′

-tetrachlorofluorescein, 6-carboxy-4,7,2′

,4′

,5′

,7′

-hexachlorofluorescein, 5-(and

     6)carboxy-4′

,5′

-dichloro-2′

7′

-dimethoxyfluorescein, and 5-carboxy-2′

,4′

,5′

,7′

-tetrachlorofluorescein.

9. The labelled propargylethoxyamino nucleotide of claim 7 wherein L is a rhodamine-type dye selected from tetramethylrhodamine, 4,7-dichlorotetramethyl rhodamine, rhodamine ROX, rhodamine R6G, and rhodamine R110.

10. The labelled propargylethoxyamino nucleotide of claim 7 wherein W₁ and W₂ are each H.

11. The labelled propargylethoxyamino nucleotide of claim 7 wherein W₂ is selected from azido, amino, fluoro, and methoxy.

12. The labelled propargylethoxyamino nucleotide of claim 7 wherein B is selected from adenine, guanine, cytosine, uracil, thymine, deazaadenine, and deazaguanosine.