Pyrimidine derivatives for labeled binding partners

• US 6,951,931 B2
• Filed: 11/14/2002
• Issued: 10/04/2005
• Est. Priority Date: 11/07/1997
• Status: Expired due to Term

• Current Assignee: Ionis Pharmaceuticals, Inc.
• Original Assignee: ISIS Pharmaceuticals Incorporated
• Inventors: Matteucci, Mark D., Lin, Kuei-Ying
• Primary Examiner(s): Wilson, James O.
• Assistant Examiner(s): Lewis, Patrick T.
• Application Number:
  US10/294,181
• Publication Number:
  US 20030176677A1
• Time in Patent Office: 1,055 Days
• Field of Search: 536/22.1, 536/23.1, 536/24.3, 536/25.3, 536/25.31, 536/25.32, 536/25.34, 536/25.4, 536/25.33, 435/6, 435/87, 435/90, 544/242, 544/245, 544/249, 514/256, 514/267
• US Class Current: 536/24.3
• CPC Class Codes: C07H 19/06 Pyrimidine radicals
• 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: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 2525/101 incorporating non-naturally occurring nucleotides, e.g. inosine
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: Y02P 20/55 Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Abstract

Compounds having structure (1)
wherein R¹ is —H a protecting group, a linker or a binding partner; and R² and R³⁴ are as defined in the specification. The invention also provides intermediates and methods make the structure (1) compounds, as well as methods to use the compounds as labels in diagnostic assays and to enhance binding to complementary bases.

Claims

1. A pair of primers for amplification or detection of a nucleic acid, at least one of said primers comprising a compound of the formula B_x—

R¹, or a tautomer, solvate or salt thereof, wherein R¹ is a binding partner and B_x is of the subformula;

wherein

R² is A(Z)_X1, wherein A is a spacer and Z independently is a label bonding group optionally bonded to a detectable label, but R² is not an amine, protected amine, nitro or cyano;

each

R²⁷ is independently —

CH═

, —

N═

, —

C(C_1-8 alkyl)= or —

C(halogen)=, but no adjacent R²⁷ are both —

N═

, or two adjacent R²⁷ are taken together to form a ring having the structure;

where R^a is independently —

CH, —

N═

, —

C(C_1-8alkyl)= or —

C(halogen)=, but no adjacent R^a are both —

N═

; and

R³⁴ is —

O—

, —

S—

, or —

N(CH₃)—

; and

X1 is 1, 2 or 3.

2. The pair of primers of claim 1, wherein R¹ is an oligomer.

3. The pair of primers of claim 1, wherein R¹ is a polymer.

4. The pair of primers of claim 1, wherein R¹ is an oligonucleotide.

5. The pair of primers of claim 1, wherein B_x is a cytosine surrogate for supplemental Watson-Crick base pairing.

6. The pair of primers of claim 1, wherein Z is capable of hydrogen bonding to a guanine in a complementary oligonucleotide strand.

7. The pair of primers of claim 1, wherein Z is bonded to a detectable label.

8. A pair of primers for amplification or detection of nucleic acid, each of said pair of primers comprising a compound of the formula B_x—

R¹, or a tautomer, solvate or salt thereof, wherein R¹ is a binding partner and B_x is of the subformula;

wherein

R² is A(Z)_X1, wherein A is a spacer and Z independently is a label bonding group optionally bonded to a detectable label, but R² is not an amine, protected amine, nitro or cyano;

each

R²⁷ is independently —

CH═

, —

N═

, —

C(C_1-8 alkyl)= or —

C(halogen)=, but no adjacent R²⁷ are both —

N═

, or two adjacent R²⁷ are taken together to form a ring having the structure;

where R^a is independently —

CH═

, —

N═

, —

C(C_1-8alkyl)= or —

C(halogen)=, but no adjacent R^a are both —

N═

; and

R³⁴ is —

O—

, —

S—

, or —

N(CH₃)—

; and

X1 is 1, 2 or 3.

9. The pair of primers of claim 8, wherein R¹ is an oligomer.

10. The pair of primers of claim 8, wherein R¹ is a polymer.

11. The pair of primers of claim 8, wherein R¹ is an oligonucleotide.

12. The pair of primers of claim 8, wherein B_x is a cytosine surrogate for supplemental Watson-Crick base pairing.

13. The pair of primers of claim 8, wherein Z is capable of hydrogen bonding to a guanine in a complementary oligonucleotide strand.

14. The pair of primers of claim 8, wherein Z is bonded to a detectable label.

15. A method for amplification of nucleic acid, the method comprising:

selecting at least one primer for use in said method, the primer comprising a compound of the formula B_x—

R¹, or a tautomer, solvate or salt thereof, wherein R¹ is a binding partner and B_x is of the subformula;

wherein

R² is A(Z)_X1, wherein A is a spacer and Z independently is a label bonding group optionally bonded to a detectable label, but R² is not an amine, protected amine, nitro or cyano;

each

R²⁷ is independently —

CH═

, —

N═

, —

C(C_1-8 alkyl)= or —

C(halogen)=, but no adjacent R²⁷ are both —

N═

, or two adjacent R²⁷ are taken together to form a ring having the structure;

where R^a is independently —

CH═

, —

N═

, —

C(C_1-8alkyl)= or —

C(halogen)=, but no adjacent R^a are both —

N═

; and

R³⁴ is —

O—

, —

S—

, or —

N(CH₃)—

; and

X1 is 1, 2 or 3; and

performing at least one step for amplifying or detecting a nucleic acid sequence.

16. The method of claim 15, wherein R¹ is an oligomer.

17. The method of claim 15, wherein R¹ is a polymer.

18. The method of claim 15, wherein R¹ is an oligonucleotide.

19. The method of claim 15, wherein B_x is a cytosine surrogate for supplemental Watson-Crick base pairing.

20. The method of claim 15, wherein Z is capable of hydrogen binding to a guanine in a complementary oligonucleotide strand.

21. The method of claim 15, wherein Z is bonded to a detectable label.

22. The method of claim 15, wherein said primer is a member of a set of primers, and both members of the set of primers are independently of the formula B_x—

R¹, or a tautomer, solvate or salt thereof, wherein R¹ is a binding partner and B_x is of the subformula;

wherein the variables are as defined in claim 15.