Cross-linking oligonucleotides

• US 5,824,796 A
• Filed: 11/04/1994
• Issued: 10/20/1998
• Est. Priority Date: 09/28/1988
• Status: Expired due to Term

• Current Assignee: Epoch Pharmaceuticals, Inc.
• Original Assignee: Epoch Pharmaceuticals, Inc.
• Inventors: Meyer, Rich B., Tabone, John C., Petrie, Charles R., Hurst, Gerald D.
• Primary Examiner(s): Kunz, Gary L.
• Application Number:
  US08/334,490
• Time in Patent Office: 1,446 Days
• Field of Search: 536/26.1, 536/26.12, 536/26.13, 536/26.14, 536/26.8, 536/27.6, 536/27.81, 536/28.5, 536/28.54, 536/26.7, 536/24.5
• US Class Current: 536/26.7
• CPC Class Codes: C07D 487/04 Ortho-condensed systems
• CPC Class Codes: C07H 19/06 Pyrimidine radicals
• 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: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/708 for papilloma

Abstract

This invention is directed to novel substituted nucleotide bases with a crosslinking arm which accomplish crosslinking between specific sites on adjoining strands of oligonucleotides or oligodeoxynucleotides. The invention is also directed to oligonucleotides comprising at least one of these crosslinking agents and to the use of the resulting novel oligonucleotides for diagnostic and therapeutic purposes. The crosslinking agents of the invention are of the following formula (I'"'"'):

R.sub.1 --B--(CH.sub.2).sub.q --(Y).sub.r --(CH.sub.2).sub.m --A'"'"'(I'"'"')

wherein,

R₁ is hydrogen, or a sugar moiety or analog thereof optionally substituted at its 3'"'"' or its 5'"'"' position with a phosphorus derivative attached to the sugar moiety by an oxygen and including groups Q₁ Q₂ and Q₃ or with a reactive precursor thereof suitable for nucleotide bond formation;

Q₁ is hydroxy, phosphate or diphosphate;

Q₂ is ═O or ═S;

Q₃ is CH₂ --R'"'"', S--R'"'"', O--R'"'"', or N--R'"'"'R";

each of R'"'"' and R" is independently hydrogen or C_1-6 alkyl;

B is a nucleic acid base or analog thereof that is a component of an oligonucleotide;

Y is a functional linking group;

each of m and q is independently 0 to 8, inclusive;

r is 0 or 1; and

A'"'"' is a leaving group.

This invention is also directed to novel 3,4-disubstituted and 3,4,-trisubstituted pyrazolo 3,4-d!-pyrimidines and to the use of these nucleic acid bases in the preparation of oligonucleotides. The invention includes nucleosides and mono- and oligonucleotides comprising at least one of these pyrazolopyrimidines, and to the use of the resulting novel oligonucleotides for diagnostic purposes.

Claims

1. An oligonucleotide having at least one nucleotide of the formula

View Dependent Claims (2, 3, 4, 5, 6, 7)

2. An oligonucleotide according to claim 1 wherein B is selected from adenine-8-yl, guanine-8-yl, 4-aminopyrazolo 3,4-d!pyrimidin-3-yl, and 4-amino-6-oxopyrazolo 3,4-d!pyrimidin-3-yl groups.

3. An oligonucleotide according to claim 1 wherein m is 1, 2 or 3;

q is 2, 3, or 4; and

r is 1.

4. An oligonucleotide according to claim 1 wherein the R₁ group is 1-(β

-D-ribofuranosyl).

5. An oligonucleotide according to claim 1 wherein the R₁ group is 1-(β

-D-2-deoxyribofuranosyl).

6. An oligonucleotide according to claim 1 wherein the R₁ group is 1-(β

-D-2-O-methyl-ribofuranosyl).

7. An oligonucleotide according to claim 1 wherein the group --(CH₂)_q --(Y)_r --(CH₂)_m --A'"'"' is 3-iodoacetamidopropyl, 3-(4-bromobutyramido)propyl, 4-iodoacetamidobutyl, or 4-(4-bromobutyramido)butyl.

8. A compound of the formula ##STR10## where R₁ is H, or a 1-(β

-D-ribofuranosyl) or 1-(β

-D-2-deoxyribofuranosyl) group which is optionally substitituted on one or more of its hydroxyl functions with a Z group wherein Z independently is methyl or a phosphate, thiophosphate alkylphosphate or alkanephosphonate group, or a reactive precursor of said phosphate, thiophosphate, alkylphosphate or alkanephosphonate group which precursor is suitable for internucleotide bond formation;

R₃ is (CH₂)_q --(Y)_r --(CH₂)_m --A" where A" is a group selected from chloro, bromo, iodo, SO₂ R'"'"'", S⁺ R'"'"'"R"" and a radical which activates the carbon to which it is attached for nucleophilic substitution, where each of R'"'"'" and R"" is independently C_1-6 alkyl or aryl or R'"'"'" and R"" together form a C_1-6 alkylene bridge, or A" is an intercalator group, a metal ion chelator or a reporter group;

Y is a functional linking group selected from a group consisting of --O--, --S--, --NR'"'"'--, --NH--CO--, trifluoroacetamido and phtalimido groups where R'"'"' is H or C_1-6 alkyl, and at least one of the (CH₂)_m and (CH₂)_q groups is directly linked to said --O--, --S--, --NR'"'"'--, NH--CO--, trifluoroacetamido and phtalimido groups and the other of said (CH₂)_m and (CH₂)_q groups is linked to the heterocyclic base with a carbon to carbon bond;

each of m and q is independently 0 to 8, inclusive;

r is 0 or 1 provided that when A" is a group selected from chloro, bromo, iodo, SO₂ R'"'"'", S⁺ R'"'"'"R"" and a radical which activates the carbon to which it is attached for nucleophilic substitution, then m is not 0;

each of R₄ and R₆ is independently H, OR, SR, NHOR, NH₂, or NH(CH₂)_t NH₂ where R is H or C_1-6 alkyl and t is an integer from 0 to 12.

9. A compound in accordance with claim 8 where each of R₄ and R₆ is independently selected from a group consisting of H, OH and NH₂.

10. A compound of the formula ##STR11## where R₁ is H, or a 1-(β

-D-ribofuranosyl) or 1-(β

-D-deoxyribofuranosyl) group which is optionally substitituted on one or more of its hydroxyl functions with a Z group wherein Z independently is methyl or a phosphate, thiophosphate, alkylphosphate or alkanephosphonate group, or a reactive precursor of said phosphate, thiophosphate, alkylphosphate or alkanephosphonate group which precursor is suitable for internucleotide bond formation;

R₃ is (CH₂)_q --(Y)_r --(CH₂)_m --A" and A" is a reporter group;

Y is a functional linking group selected from a group consisting of --O--, --S--, --NR'"'"'--, --NH--CO--, trifluoroacetamido and phtalimido groups where R'"'"' is H or C_1-6 alkyl, and at least one of the (CH₂)_m and (CH₂)_q groups is directly linked to said --O--, --S--, --NR'"'"'--, NH--CO--, trifluoroacetamido and phtalimido groups and the other of said (CH₂)_m and (CH₂)_q groups is linked to the heterocyclic base with a carbon to carbon bond;

each of m and q is independently 0 to 8, inclusive;

r is 0 or 1, and

each of R₄ and R6 is independently H, OR, SR, NHOR, NH₂, or NH(CH₂)_t NH₂ where R is H or C_1-6 alkyl and t is an integer from 0 to 12.

11. A compound in accordance with claim 10 where each of R₄ and R₆ is independently selected from a group consisting of H, OH and NH₂.

12. A compound in accordance with claim 11 where the reporter group is biotin or 2,4-dinitrobenzene.

13. An oligonucleotide having at least one nucleotide of the formula ##STR12## wherein R₁ is a 1-(β

-D-ribofuranosyl) or 1-(β

-D-2-deoxyribofuranosyl) group which is optionally substitituted on one or more of its hydroxyl functions with a Z group wherein Z independently is methyl or a phosphate, thiophosphate, alkylphosphate or alkanephosphonate group;

R₃ is (CH₂)_q --(Y)_r --(CH₂)_m --A and A is a reporter group;

Y is a functional linking group selected from a group consisting of --O--, --S--, --NR'"'"'--, --NH--CO--, trifluoroacetamido and phtalimido groups where R'"'"' is H or C_1-6 alkyl, and at least one of the (CH₂)_m and (CH₂)_q groups is directly linked to said --O--, --S--, --NR'"'"'--, NH--CO--,trifluoroacetamido and phtalimido groups and the other of said (CH₂)_m and (CH₂)_q groups is linked to the heterocyclic base with a carbon to carbon bond;

each of m and q is independently 0 to 8, inclusive;

r is 0 or 1, and

each of R₄ and R6 is independently H, OR, SR, NHOR, NH₂, or NH(CH₂)_t NH₂ where R is H or C_1-6 alkyl and t is an integer from 0 to 12.

14. An oligonucleotide in accordance with claim 13 where each of R₄ and R₆ is independently selected from a group consisting of H, OH and NH₂.

15. An oligonucleotide in accordance with claim 14 where the reporter group is biotin or 2,4-dinitrobenzene.