Cross-linking oligonucleotides

• US RE38,416 E1
• Filed: 10/19/2000
• Issued: 02/03/2004
• Est. Priority Date: 09/28/1988
• Status: Expired due to Term

• Current Assignee: Drug Royalty Trust
• Original Assignee: Epoch Biosciences Inc. (Ngen, Inc.)
• Inventors: Tabone, John C., Petrie, Charles R., Meyer, Rich B., Hurst, Gerald D.
• Primary Examiner(s): Wilson, James O.
• Assistant Examiner(s): Owens, Jr., Howard V.
• Application Number:
  US09/693,213
• Time in Patent Office: 1,202 Days
• Field of Search: 536/26.7, 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, 536/24.5
• US Class Current: 536/26.1
• CPC Class Codes: C07D 487/04 Ortho-condensed systems
• CPC Class Codes: C07H 19/04 Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
• CPC Class Codes: C07H 19/073 with 2-deoxyribosyl as the saccharide radical
• CPC Class Codes: C07H 19/23 Heterocyclic radicals containing two or more heterocyclic rings condensed among themselves or condensed with a common carbocyclic ring system, not provided for in groups C07H19/14 - C07H19/22
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C12Q 1/68 involving 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 1/6832 Enhancement of hybridisation reaction
• 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 a 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 (Γ):

R₁—B—(CH₂)_q—(Y)_r—(CH₂)_m—A′  (Γ)

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 Q₁Q₂ and Q₃ or with a reactive precursor thereof suitable for nucleotide bond formation;

Q₁ is hydroxy phosphate a diphosphate;

Q₂ ═of 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 to 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

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)_p—

(CH₂)_m—

A′

is 3-iodoacetamidopropyl, 3-(4-bromobutyramido)propyl, 4-iodoacetamidobutyl, or 4-(4-bromobutyramido)butyl.

8. A compound of the formula

where R₁ is H, or a 1-(β

-D-ribofuranosyl) or 1-(β

-D-2-D-ribofuranosyl) group which is optionally substituted on one or more of its hydroxyl function 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, SO₂R′

″

, 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—

, trifuluroacetamido and phtalimido phthalimido 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 phthalimido 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, SO₂R′

″

, 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)_tNH₂where R is H or C_1-6alkyl 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

where R₁ is H, or a 1-(β

-D-ribofuranosyl) or 1-(β

-D-2-deoxyribofuranosyl) group which is optionally substituted 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 phthalimido 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 phthalimido 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)_tNH₂ where R is H or C_1-6alkyl 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.

25. A compound of claim 10, wherein the reporter group is selected from the group consisting of ³H, ¹²⁵I, ³⁵S, ¹⁴C and ³²P.

26. A compound of claim 10, wherein the reporter group is ³H.

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

wherein R₁ is a 1-(β

-D-ribofuranosyl) or 1-(β

-D-2-deoxyribofuranosyl) group which is optionally substituted 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 phthalimido 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 phthalimido 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, NH(CH₂)_tNH₂ where R is H or C_1-6alkyl 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.

27. An oligonucleotide of claim 13, wherein the reporter group is selected from the group consisting of ³H, ¹²⁵I, ³⁵S, ¹⁴C and ³²P.

28. An oligonucleotide of claim 13, wherein the reporter group is ³H.

16. A compound having the formula

wherein R₁ is H, or a 1-(β

-D-ribofuranosyl) or 1-(β

-D-2-deoxyribofuranosyl) group which is optionally substituted 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 which precursor is suitable for internucleotide bond formation;

17. A compound of claim 16, wherein W is C_1-12 alkylene and X is selected from the group consisting of OH, NH₂ and chemically blocked derivatives thereof.

18. A compound of claim 16, wherein W is C_2-12 alkynylene and X is selected from the group consisting of OH, NH₂ and chemically blocked derivatives thereof.

19. A compound of claim 17, wherein W is pentyl and X is NH-trityl.

20. A compound of claim 16, wherein R₄ is NH₂ or OH and R₆ is H or NH₂.

21. An oligonucleotide comprising at least one nucleotide unit of the formula

wherein R₁ is a 1-(β

-D-ribofuranosyl) or 1-(β

-D-2-deoxyribofuranosyl) group which is optionally substituted 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 which precursor is suitable for internucleotide bond formation;

22. An oligonucleotide of claim 21, wherein W is C_1-12 alkylene and X is selected from the group consisting of OH, NH₂ and chemically blocked derivatives thereof.

23. An oligonucleotide of claim 21, wherein W is C_2-12 alkynylene and X is selected from the group consisting of OH, NH₂ and chemically blocked derivatives thereof.

24. An oligonucleotide of claim 22, wherein W is pentyl and X is NH-trityl.

29. A labeled oligonucleotide comprising at least one nucleotide unit of the formula

wherein R₁ is a 1-(β

-D-ribofuranosyl) or 1-(β

-D-2-deoxyribofuranosyl) group which is optionally substituted 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 which precursor is suitable for internucleotide bond formation;

R₃ is —

W-A, wherein W is a chemical linker selected from the group consisting of C_1-12 alkylene, C_2-12 alkenylene and C_2-12 alkynylene, and A is a reporter group; and

each of R₄ and R₆ is independently H, OR, SR, NHOR, NH₂, or NH(CH₂)_tNH₂ where R is H or C_1-6alkyl and t is an integer from 0 to 12.

30. A labeled oligonucleotide of claim 29, wherein said reporter group is selected from the group consisting of ³H, ¹²⁵I, ³⁵S, ¹⁴C and ³²P.

31. A labeled oligonucleotide of claim 29, wherein said reporter group is ³H.

32. A compound having the formula

wherein R₁ is H, or a 1-(β

-D-ribofuranosyl) or 1-(β

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

R₃ is —

W-A, wherein W is a chemical linker arm selected from the group consisting of C_1-12 alkylene, C_2-12 alkenylene and C_2-12 alkynylene, and A is a reporter group; and

each of R₄ and R₆ is independently H, OR, SR, NHOR, NH₂, or NH(CH₂)_tNH₂ where R is H or C_1-6alkyl and t is an integer from 0 to 12.

33. A compound of claim 32, wherein said reporter group is selected from the group consisting of ³H, ¹²⁵I, ³⁵S, ¹⁴C and ³²P.

34. A compound of claim 32, wherein said reporter group is ³H.