Heterocyclic compounds and their use for isolating nucleic acids

• US 6,403,786 B1
• Filed: 07/02/1999
• Issued: 06/11/2002
• Est. Priority Date: 09/12/1996
• Status: Expired due to Term

• Current Assignee: Roche Diagnostics GmbH (Roche Holding AG)
• Original Assignee: Roche Molecular Systems Inc (Roche Holding AG)
• Inventors: Von der Eltz, Herbert, Mühlegger, Klaus
• Primary Examiner(s): Gitomer, Ralph
• Assistant Examiner(s): Khare, Devesh
• Application Number:
  US09/254,644
• Time in Patent Office: 1,075 Days
• Field of Search: 536/26.8, 536/26.1, 536/23.1, 536/24.3, 514/49, 514/51
• US Class Current: 536/26.8
• CPC Class Codes: C07D 405/04 directly linked by a ring-member-to-ring-member bond
• CPC Class Codes: C07D 413/04 directly linked by a ring-member-to-ring-member bond
• CPC Class Codes: C07F 9/65586 at least one of the hetero rings does not contain nitrogen as ring hetero atom
• CPC Class Codes: C07H 19/04 Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
• CPC Class Codes: C07H 19/048 Pyridine radicals
• CPC Class Codes: C07H 19/06 Pyrimidine radicals
• CPC Class Codes: C07H 7/06 Heterocyclic radicals
• CPC Class Codes: C07J 51/00 Normal steroids with unmodified cyclopenta(a)hydrophenanthrene skeleton not provided for in groups C07J1/00 - C07J43/00

Abstract

The invention concerns compounds of the general formula (I) in which i:he residues R₁ to R₇ have the meanings given in the application as well as methods for their preparation. The compounds are in particular suitable as substrates for RNA or DNA polymerases and can thus be incorporated into RNA or DNA oligonucleotides and are especially suitable for labelling and detecting nucleic acids or for DNA sequencing.

Claims

1. A compound of the general formula

in which

R₁ and R₂ can be the same or different and represent hydrogen, oxygen, halogen, hydroxy, thio or substituted thio, amino or substituted amino, carboxy, lower alkyl, lower alkenyl, lower alkinyl, aryl, lower alkyloxy, aryloxy, aralkyl, aralkyloxy or a reporter group,

R₃ and R₄ each represent hydrogen, hydroxy, thio or substituted thio, amino or substituted amino, lower alkyloxy, lower alkenoxy, lower alkinoxy, a protecting group or a reporter group,

R₅ represents hydrogen, hydroxy, thio or substituted thio, amino or a substituted amino group, a reactive trivalent or pentavalent phosphorus group such as e.g. a phosphoramidite or H-phosphonate group, an ester or amide residue that can be cleaved in a suitable manner or a reporter group,

R₄ and R₅ together form a further bond between C-2′

and C-3′

or an acetal group,

R₆ represents hydrogen or a hydroxy, thio or substituted thio, amino or substituted amino group,

R₇ represents hydrogen, a monophosphate, diphosphate or triphosphate group or the alpha, beta or gamma thiophosphate analogue of this phosphoric acid ester or a protective group as well as possible tautomers and salts thereof,

X denotes methylene or methine substituted with halogen, hydroxy, thio or substituted thio, amino or substituted amino, carboxy, lower alkyl, lower alkenyl, lower alkinyl, aryl, lower alkyloxy, aryloxy, aralkyl, aralkyloxy or a reporter group, or oxygen and n=0 or 1,

Z denotes nitrogen or carbon provided that if Z denotes nitrogen, m is zero (0) and if X represents methylene, substituted methylene or substituted methine, Z cannot be carbon and if X denotes oxygen, Z cannot be nitrogen and if Z denotes carbon, R₂ cannot be hydrogen.

2. The compound as claimed in claim 1, in which R₁ can represent oxygen, R₂ can represent hydrogen or a reporter group, R₃ and R₄ can represent hydrogen, R₅ can represent hydroxy, hydrogen, a reactive trivalent or pentavalent phosphorus group, R₆ can represent hydrogen and R₇ can represent hydrogen, mono-, di-, or triphosphate groups.

3. An oligonucleotide comprising a compound of claim 2.

4. The compound as claimed in claim 1 wherein R₁ represents hydrogen, hydroxy, an amino group, a substituted amino group or a reporter group, R₂ represents an amino or substituted amino group or a reporter group, R₃ represents hydrogen, R₄ represents hydrogen, hydroxy, amino or substituted amino, lower alkyloxy, lower alkenoxy, lower alkinoxy, R₅ represents hydrogen, hydroxy, thio, an amino or substituted amino group, a phosphoramidite or a reporter group, R₄ and R₅ together represent an acetal group, R₆ represents hydrogen and R₇ represents a triphosphate group.

5. An oligonucleotide comprising a compound of claim 4.

6. The compound as claimed in claim 1 in which X denotes oxygen and at the same time Z represents carbon substituted with R₂ or Z denotes nitrogen and at the same time X represents methylene or methine substituted with amino or substituted amino, with carboxy or with a reporter group.

7. An oligonucleotide comprising a compound of claim 6.

8. The compound as claimed in claim 1 in which X=0 and Z represents methine substituted with amino or substituted amino, with carboxy or with a reporter group are a further preferred embodiment.

9. An oligonucleotide comprising a compound of claim 8.

10. The compound as claimed in claim 1, wherein the acetal group of the residues R₄ and R₅ is substituted with a reporter group.

11. An oligonucleotide comprising a compound of claim 10.

12. The compound as claimed in claim 1, wherein the reporter group denotes a hapten, a fluorophore, a metal-chelating group, a luminophore, a protein or an intercalator.

13. An oligonucleotide comprising a compound of claim 12.

14. The compound as claimed in claim 1, in which the reporter group is linked via a linker group.

15. An oligonucleotide comprising a compound of claim 14.

16. A method for synthesizing a nucleic acid comprising incorporating the compound of claim 1 into the nucleic acid by a DNA or RNA polymerase.

17. A method for synthesizing an oligonucleotide comprising incorporating the compound of claim 1 into the oligonucleotide wherein R₇ is a phosphoramidite or H-phosphonate.

18. An oligonucleotide comprising a compound of claim 1.

19. A nucleic acid comprising a compound of claim 1.