N-4 modified pyrimidine deoxynucleotides and oligonucleotide probes synthesized therewith

• US 5,591,584 A
• Filed: 04/26/1995
• Issued: 01/07/1997
• Est. Priority Date: 08/25/1994
• Status: Expired due to Term

• Current Assignee: Siemens Healthcare Diagnostics Incorporated (Siemens AG)
• Original Assignee: Chiron Corporation (Bayer AG)
• Inventors: Urdea, Michael S., Horn, Thomas, Chang, Chu-An
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): REES, DIANE
• Application Number:
  US08/429,196
• Time in Patent Office: 622 Days
• Field of Search: 435/5, 435/6, 435/91.1, 536/22.1
• US Class Current: 435/6.12
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• 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: Y02P 20/55 Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Abstract

Novel oligonucleotide probes are provided having the structure ##STR1## wherein: R² is lower alkyl; R³ is C₁ -C₁₂ alkylene containing 0 to 6 linkages selected from the group consisting of --O--, --S-- and --NR¹² -- wherein R¹² is hydrogen or lower alkyl; R⁵ is hydrogen or lower alkyl; R⁶ is selected from the group consisting of hydrogen, methyl, bromo and iodo; R⁷ is C₁ -C₁₂ alkylene containing 0 to 6 linkages selected from the group consisting of --O--, --S-- and --NR¹² --, and bound to R⁸ through an --O--, --S-- or --NR¹² -- moiety; and R⁸ is a protecting group that can be removed and replaced, without affecting the remainder of the compound, by reduction with a reducing agent. Methods for using the probes are provided as well.

Claims

1. A polynucleotide probe containing a plurality of modified deoxynucleotides each having the structural formula ##STR16## wherein:

R² is lower alkyl;

R³ is C₁ -C₁₂ alkylene containing 0 to 6 linkages selected from the group consisting of --O--, --S-- and --NR¹² -- wherein R¹² is hydrogen or lower alkyl;

R⁵ is hydrogen or lower alkyl;

R⁶ is selected from the group consisting of hydrogen, methyl, bromo and iodo;

R⁷ is C₁ -C₁₂ alkylene containing 0 to 6 linkages selected from the group consisting of --O--, --S-- and --NR¹² --, and bound to R⁸ through an --O--, --S-- or --NR¹² -- moiety; and

R⁸ is a protecting group that can be removed and replaced, without affecting the remainder of the compound, by reduction with a reducing agent.

2. A method of detecting a polynucleotide analyte in a sample, comprising the sequential steps of:

rendering any double-stranded polynucleotides in said sample single stranded;

contacting said sample with the polynucleotide probe of claim 1 under specifically hybridizing conditions for a time sufficient for nucleic acid complexes to form between the probe and the analyte;

isolating any such nucleic acid complexes; and

detecting the presence of the nucleic acid complexes to indicate the presence of the polynucleotide analyte.

14. The probe of claim 1, wherein R² is methyl.

15. The probe of claim 1, wherein R³ is --CH₂ CH₂ O)_m --wherein m is 1 or 2.

16. The probe of claim 15, wherein m is 2.

17. The probe of claim 1, wherein R⁵ is hydrogen.

18. The probe of claim 1, wherein R⁶ is methyl.

19. The probe of claim 1, wherein R⁶ is hydrogen.

20. The probe of claim 1, wherein R⁷ is --(CH₂)_p O-- in which p is an integer in the range of 1 to 6 inclusive.

21. The probe of claim 20, wherein p is 6.

22. The probe of claim 1, wherein R⁸ is --(CO)--CH₂ CH₂ --COCH₃.

23. The probe of claim 1, wherein:

R² is methyl;

R³ is --(CH₂ CH₂ O)_m -- wherein m is 1 or 2;

R⁵ is hydrogen;

R⁶ is methyl;

R⁷ is --(CH₂)_p --O--, wherein p is an integer in the range of 1 to 6 inclusive; and

R⁸ is --(CO)--CH₂ CH₂ --COCH₃.

3. A polynucleotide probe containing a plurality of modified deoxynucleotides each having the structural formula ##STR17## wherein:

R² is lower alkyl;

R³ is C₁ -C₁₂ alkylene containing 0 to 6 linkages selected from the group consisting of --O--, --S-- and --NR₁₂ -- wherein R¹² is hydrogen or lower alkyl;

R⁵ is hydrogen or lower alkyl;

R⁶ is selected from the group consisting of hydrogen, methyl, bromo and iodo;

R⁷ is C₁ -C₁₂ alkylene containing 0 to 6 linkages selected from the group consisting of --O--, --S-- and --NR¹² --, and bound to R⁸ through an --O--, --S-- or --NR¹² moiety; and

R⁸ is a protecting group that can be removed and replaced, without affecting the remainder of the compound, by reduction with a reducing agent.

4. The probe of claim 3, wherein R² is methyl.

5. The probe of claim 3, wherein R³ is --(CH₂ CH₂ O)_m -- wherein m is 1 or 2.

6. The probe of claim 5, wherein m is 2.

7. The probe of claim 3, wherein R⁵ is hydrogen.

8. The probe of claim 3, wherein R⁶ is methyl.

9. The probe of claim 3, wherein R⁶ is hydrogen.

10. The probe of claim 3, wherein R⁷ is --(CH₂)_p O-- in which p is an integer in the range of 1 to 6 inclusive.

11. The probe of claim 10, wherein p is 6.

12. The probe of claim 3, wherein R⁸ is --(CO)--CH₂ CH₂ --COCH₃ --.

13. The probe of claim 3, wherein:

R² is methyl;

R³ is --(CH₂ CH₂ O)_m -- wherein m is 1 or 2;

R⁵ is hydrogen;

R⁶ is methyl;

R⁷ is --(CH₂)_p --O--, wherein p is an integer in the range of 1 to 6 inclusive; and

R⁸ is --(CO)--CH₂ CH₂ --COCH₃.