Derivatives of 7-deaza -2′-deoxyguanosine-5'-triphosphate, preparation and use thereof

• US 6,906,185 B1
• Filed: 03/20/1998
• Issued: 06/14/2005
• Est. Priority Date: 03/20/1997
• Status: Expired due to Term

• Current Assignee: GE Healthcare Bio-Sciences Corporation (General Electric Company)
• Original Assignee: Amersham Biosciences Corp. (General Electric Company)
• Inventors: McDougall, Mark, Fuller, Carl W., Kumar, Shiv
• Primary Examiner(s): MORAN, MARJORIE A
• Application Number:
  US09/045,732
• Time in Patent Office: 2,643 Days
• Field of Search: 536/23.1, 536/27.1, 536/25.3, 536/22.1, 536/27.12, 536/26.12, 536/26.1, 435/89
• US Class Current: 536/23.1
• CPC Class Codes: C07H 19/173 with 2-deoxyribosyl as the saccharide radical
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical

Abstract

Derivatives of 7-deaza-2′-deoxyguanosine are described. Also described is the use of such compounds in synthesis of nucleic acid polymers and in methods for determining a nucleotide base sequence.

Claims

1. Molecule comprising the following moiety:

wherein R₁ is C_1-10 alkyl group substituted by hydroxyl, C_1-4 alkoxy or halo; and

R₂ is hydrogen and R₇ is H or a mono-, di-, tri-phosphate or thiophosphate thereof.

2. The molecule of claim 1, wherein said molecule is a nucleic acid polymer.

3. The molecule of claim 2, wherein said nucleic acid is DNA.

4. A compound of the formula (II):

wherein R₁ is C_1-10 to alkyl group substituted by hydroxyl, C_1-4 alkoxy or halo;

R₂ is hydrogen; and

R₇ is H or a mono-, di-, or tri-phosphate or thiophosphate thereof.

5. A compound according to claim 4, wherein R₁ is a C_2-8 alkyl group substituted by hydroxyl, C_1-4 alkoxy or halo.

6. A compound according to any of the claims 4 or 5 wherein the compound of the formula (II) is present as a triphosphate.

7. A compound of claim 4 wherein R₇ is a di-, or tri-phosphate or thiophosphate thereof.

8. A compound of claim 4, wherein said compound is 7-Hydroxymethyl-7-deaza-2′

-deoxyguanosine or a mono-, di-, or tri-phosphate thereof.

9. A compound according to any one of claim 8, wherein said compound is a triphosphate.

10. A nucleotide sequence containing a compound of claim 8.

11. A deoxyribonucleic acid sequence containing a base of the formula:

wherein R₁ is a C_1-10 alkyl group substituted by hydroxyl, C_1-4 alkoxy or halo.

12. Method for determining the nucleotide base sequence of a DNA molecule comprising the steps of:

incubating a DNA molecule annealed with a primer molecule able to hybridize to said DNA molecule in a vessel containing a molecule comprising the following moiety of formula (II);

wherein R₁ is C_1-10 alkyl group substituted by hydroxyl, C_1-4 alkoxy or halo;

R₂ is hydrogen and R₇ is a tri-phosphate or thiophosphate thereof;

a DNA polymerase and at least one DNA synthesis terminating agent which terminates DNA synthesis at a specific nucleotide base in an incubating reaction; and

separating DNA products of the incubating reaction according to size whereby at least a part of the nucleotide base sequence of said DNA molecule can be determined.

13. The method of claim 12, wherein said molecule containing a moiety of formula (II) is a compound of formula (II).

14. The method of claim 12, wherein said moiety of formula (II) is 7-Hydroxymethyl-7-deaza-2′

-deoxyguanosine.

15. Method for elongation of an oligonucleotide sequence comprising the step of:

incubating an oligonucleotide sequence with a molecule comprising the following moiety of formula (II);

wherein R₁ is C_1-10 alkyl group substituted by hydroxyl, C_1-4 alkoxy or halo;

R₂ is hydrogen; and

R₇ is a tri-phosphate or thiophosphate thereof, and a DNA polymerase such that said molecule is added to the oligonucleotide sequence.

16. The method of claim 15, wherein said molecule containing a moiety of formula (II) is a compound of formula (II).

17. A compound of the formula (III):

wherein R₂ is hydrogen or hydroxyl and R₃ is C_2-10 alkynyl group substituted by hydroxyl, C_1-4 alkyl substituted amino, C_1-4 alkoxy or halo, and R₇ is a mono-, di-, or tri-phosphate or thiophosphate thereof.

18. A process for the preparation of a compound of the formula (II) wherein R₁ is C_1-10 alkyl group substituted by hydroxy, C_1-4 alkoxy or halo;

R₂ is hydrogen and R₇ is H or a mono-, di-, or tri-phosphate or thiophosphate thereof, which comprises;

(i) the deprotection of a compound of the formula (IV);

wherein R₁ is C_1-10 alkyl group substituted by hydroxy, amino, C_1-4 alkoxy or halo and R₄ is a protecting group, R₅ is hydrogen and R₆ is a protecting group which is the same or different to R₄, or

(ii) wherein R₁ is other than methyl the reduction of a compound of the formula (III)

wherein R₂ is hydrogen, R₃ is C_2-20 alkynyl group substituted by hydroxyl, amino, C_1-14 alkyl substituted amino, C_1-4 alkoxy or halo, and R₇ is H or a mono-di-, or tri-phosphate thereof;

(iii) and optionally thereafter preparing a mono-, di-, or triphosphate or thiophosphate.

19. A compound of formula (II):

wherein R₁ is an isopropyl group;

R₂ is hydrogen or hydroxyl; and

R₇ is a mono-, di- or tri-phosphate or thiophosphate thereof.

20. A method for determining the nucleotide base sequence of a DNA molecule comprising the steps of:

incubating a DNA molecule annealed with a primer molecule able to hybridize to said DNA molecule in a vessel containing a compound of formula (II);

wherein R₁ is an isopropyl group;

R₂ is hydrogen or hydroxyl; and

R₇ is a tri-phosphate or thiophosphate thereof;

a DNA polymerase, and at least one DNA synthesis terminating agent which terminates DNA synthesis at a specific nucleotide base in an incubating reaction; and

separating DNA products of the incubating reaction according to size whereby at least a part of the nucleotide base sequence of said DNA molecule can be determined.