3'-Deoxyribonucleotide derivatives

• US 6,265,569 B1
• Filed: 03/05/1999
• Issued: 07/24/2001
• Est. Priority Date: 07/07/1997
• Status: Expired due to Fees

• Current Assignee: WAKO Pure Chemical Industries Limited (Fujifilm Holdings Corporation), Nippon Genetech Co., Ltd., The Institute of Physical and Chemical Research
• Original Assignee: The Institute of Physical and Chemical Research
• Inventors: Ozawa, Kaori, Hayashizaki, Yoshihide, Tanaka, Takumi, Fujio, Kazunari
• Primary Examiner(s): Gitomer, Ralph
• Assistant Examiner(s): Khare, Devesh
• Application Number:
  US09/263,520
• Time in Patent Office: 872 Days
• Field of Search: 536/26.26, 536/26.3, 536/26.6, 536/26.7, 536/26.8, 536/28.1, 536/28.4, 536/23.1, 536/24.3
• US Class Current: 536/26.26
• CPC Class Codes: C07H 19/04 Heterocyclic radicals containing only nitrogen atoms as ring hetero atom

Abstract

Disclosed are 3′-deoxyribonucleotide derivatives represented by the following general formula [I]:

Q—V—(CH₂)_n—NH—R  [I]

wherein Q represents a 3′-deoxyribonucleotide residue, n represents an integer not less than 4, V represents —C≡C— or —CH═CH—, and R represents a fluorescent group.

The above 3′-deoxyribonucleotide derivatives are derivatives with improved rates for incorporation using RNA polymerases, which are useful as terminators in the DNA sequence determination methods utilizing RNA polymerases.

Claims

1. A 3′

-deoxyribonucleotide derivative represented by the following general formula [I];

2. The 3′

-deoxyribonucleotide derivative of claim 1, wherein n represents an integer of 4-10.

3. The 3′

-deoxyribonucleotide derivative of claim 1, wherein n represents an integer of 4-8.

4. The 3′

-deoxyribonucleotide derivative of claim 1, wherein n represents 4 or 6.

5. The 3′

-deoxyribonucleotide derivative of the claim 1, wherein R is represented by the following general formula [VII];

wherein W represents a carboxyl group, X represents —

O—

, —

S—

, —

NR′

—

where R′

represents hydrogen atom, a lower alkyl group, an aralkyl group or an aryl group, or —

CH₂—

, one of the ring A and the ring B represents

and the other one represents

where Z represents O or ═

N⁺R₁R₂, and Y represents OH or —

NR₁R₂ where R₁ and R₂ each independently represent hydrogen atom or a lower alkyl group, or both of R₁ and R₂ represent a trimethylene group (provided that R₁ and R₂ represent two trimethylene groups, of which other end is bonded to either one of the carbon atoms next to the nitrogen atom to which the trimethylene group is bonded, the broken line —

—

—

—

—

—

in the ring C;

represents a bond present at a position decided by the structures of the ring A and the ring B, and the rings A, B and C and the benzene ring having W may optionally have one or more additional substituents.

6. The 3′

-deoxyribonucleotide derivative of claim 5, wherein X is —

O—

.

7. The 3′

-deoxyribonucleotide derivative of claim 6, wherein Z represents ═

N⁺R₁R₂, and Y represents —

NR₁R₂.

8. The 3′

-deoxyribonucleotide derivative of claim 7, wherein R₁ and R₂ each independently represent hydrogen atom or a lower alkyl group having 1 to 6 carbon atoms, or both of R₁ and R₂ represent a trimethylene group.

9. The 3′

-deoxyribonucleotide derivative of claim 8, wherein at least one of the rings A, B and C and the benzene ring having W has one or more lower alkyl group having 1 to 6 carbon atoms as additional substituents.

10. The 3′

-deoxyribonucleotide derivative of claim 7, wherein at least one of the rings A, B and C and the benzene ring having W has one or more lower alkyl group having 1 to 6 carbon atoms as additional substituents.

11. The 3′

-deoxyribonucleotide derivative of claim 6, wherein at least one of the rings A, B and C and the benzene ring having W has one or more lower alkyl group having 1 to 6 carbon atoms as additional substituents.

12. The 3′

-deoxyribonucleotide derivative of claim 5, wherein Z represents ═

N⁺R₁R₂, and Y represents —

NR₁R₂.

13. The 3′

-deoxyribonucleotide derivative of claim 12, wherein R₁ and R₂ each independently represent hydrogen atom or a lower alkyl group having 1 to 6 carbon atoms, or both of R₁ and R₂ represent a trimethylene group.

14. The 3′

-deoxyribonucleotide derivative of claim 13, wherein at least one of the rings A, B and C and the benzene ring having W has one or more lower alkyl group having 1 to 6 carbon atoms as additional substituents.

15. The 3′

-deoxyribonucleotide derivative of claim 12, wherein at least one of the rings A, B and C and the benzene ring having W has one or more lower alkyl group having 1 to 6 carbon atoms as additional substituents.

16. The 3′

-deoxyribonucleotide derivative of claim 5, wherein at least one of the rings A, B and C and the benzene ring having W has one or more lower alkyl group having 1 to 6 carbon atoms as additional substituents.

17. The 3′

-deoxyribonucleotide derivative of claim 5, wherein V is —

CH═

CH—

.

18. The 3′

-deoxyribonucleotide derivative of claim 5, wherein V is —

C≡

C—

.

19. The 3′

-deoxyribonucleotide derivative of claim 1, wherein V is —

CH═

CH—

.

20. The 3′

-deoxyribonucleotide derivative of claim 1, wherein V is —

C≡

C—

.

21. A terminator composed of the 3′

-deoxyribonucleotide derivative of claim 1, which is used for methods for determining nucleotide sequences utilizing RNA polymerases.