Cross-linking oligonucleotides

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): R 1 —B—(CH 2 ) q —(Y) r —(CH 2 ) m —A′  (Γ) wherein, R 1 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 Q 1 Q 2 and Q 3 or with a reactive precursor thereof suitable for nucleotide bond formation; Q 1 is hydroxy phosphate a diphosphate; Q 2 ═of or ═S; Q 3 is CH 2 —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.

What is claimed is:  
     
       1. An oligonucleotide having at least one nucleotide of the formula 
       
         
           R 1 —B—(CH 2 ) q —(Y) r —(CH 2 ) m —A′ 
         
       
       wherein 
       R 1  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 alkanephosphoate group;  
       B is a heterocyclic base selected from purine and pyrazolo [3,4-d]pyrimidine groups wherein the (CH 2 ) q  group is attached to the 7-position or 8 position of the purine and 3-position of the pyrazolo[3,4-d]pyrimidine groups and the R 1  group is attached to the 9-position of the purine and to the 1-position of the pyrazolo[3,4-d]pyrimidine groups;  
       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 (CH 2 ) m  and (CH 2 ) q  groups is directly linked to the —O—, —S—, —NR′—, NH—CO—, trifluoroacetamido and phtalimido  phthalimido groups and the other of said (CH 2 ) m  and (CH 2 ) q  groups is linked to the heterocyclic base with a carbon to carbon bond;  
       m is 1 to 8, inclusive;  
       q is 0 to 8, inclusive;  
       r is 0 or 1; and  
       A′ is a group selected from chloro, bromo, iodo, SO 2 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.  
     
     
       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 R 1  group is 1-(β-D-ribofuranosyl). 
     
     
       5. An oligonucleotide according to  claim 1  wherein the R 1  group is 1-(β-D-2-deoxyribofuranosyl). 
     
     
       6. An oligonucleotide according to  claim 1  wherein the R 1  group is 1-(β-D-2-O-methyl-ribofuranosyl). 
     
     
       7. An oligonucleotide according to  claim 1  wherein the group —(CH 2 ) q —(Y) p —(CH 2 ) m —A′ is 3-iodoacetamidopropyl, 3-(4-bromobutyramido)propyl, 4-iodoacetamidobutyl, or 4-(4-bromobutyramido)butyl. 
     
     
       8. A compound of the formula                    
       where R 1  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; 
       R 3  is (CH 2 ) q —(Y) r —(CH 2 ) m —A″ where A″ is a group selected from chloro, bromo, iodo, SO 2 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—, trifuluroacetamido and phtalimido  phthalimido groups where R′ is H or C 1-6  alkyl, and at least one of the (CH 2 ) m  and (CH 2 ) q  groups is directly linked to said —O—, —S—, —NR′—, NH—CO—, trifluoroacetamido and phtalimido  phthalimido groups and the other of said (CH 2 ) m  and CH 2 ) 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, SO 2 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 R 4  and R 6  is independently H, OR, SR, NHOR, NH 2 , or NH(CH 2 ) t NH 2 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 R 4  and R 6  is independently selected from a group consisting of H, OH and NH 2 . 
     
     
       10. A compound of the formula                    
       where R 1  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; 
       R 3  is (CH 2 ) q —(Y) r —(CH 2 ) 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 (CH 2 ) m  and (CH 2 ) q  groups is directly linked to said —O—, —S—, —NR′—, NH—CO—, trifluoroacetamido and phtalimido  phthalimido groups and the other of said (CH 2 ) m  and (CH 2 ) 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 R 4  and R6 is independently H, OR, SR, NHOR, NH 2 , or NH(CH 2 ) t NH 2  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 R 4  and R 6  is independently selected from a group consisting of H, OH and NH 2 . 
     
     
       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                    
       wherein R 1  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; 
       R 3  is (CH 2 ) q —(Y) r —(CH 2 ) 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 (CH 2 ) m  and (CH 2 ) q  groups is directly linked to said —O—, —S—, —NR′—, NH—CO—, trifluoroacetamido and phtalimido  phthalimido groups and the other of said (CH 2 ) m  and CH 2 ) 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 R 4  and R6 is independently H, OR, SR, NHOR, NH 2 , NH(CH 2 ) t NH 2  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 R 4  and R 6  is independently selected from a group consisting of H, OH and NH 2 . 
     
     
       15. An oligonucleotide in accordance with  claim 14  where the reporter group is biotin or 2,4-dinitrobenzene. 
     
     
       16. A compound having the formula                    
         wherein R   1    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;    
       
         R 
         3  
         is —W-X, wherein W is a chemical linker arm selected from the group consisting of C 
         1-12  
         alkylene, C 
         2-12  
         alkylene and C 
         2-12  
         alkynylene, and X is selected from the group consisting of OH, SH, NH 
         2  
         and chemically blocked derivatives thereof;  
       
         each of R   4    and R   6    is independently H, OR, SR, NHOR, NH   2   , or NH ( CH   2 ) t   NH   2    where R is H or C   1-6    alkyl and t is an integer from  0  to  12     
         with the proviso that when W is —CH   2   CH   2   —, then X is other than NH   2 . 
     
     
       17. A compound of  claim 16 , wherein W is C 1-12    alkylene and X is selected from the group consisting of OH, NH   2    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, NH   2    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 R 4    is NH   2    or OH and R   6    is H or NH   2   .   
     
     
       21. An oligonucleotide comprising at least one nucleotide unit of the formula                    
         wherein R   1    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;    
       
         R 
         3  
         is —W-X, 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 X is selected from the group consisting of OH, SH, NH 
         2  
         and chemically blocked derivatives thereof;  
       
         each of R   4    and R   6    is independently H, OR, SR, NHOR, NH   2   , or NH ( CH   2   )   t   NH   2    where R is H or C   1-6    alkyl and t is an integer from  0  to  12 .   
     
     
       22. An oligonucleotide of  claim 21 , wherein W is C 1-12    alkylene and X is selected from the group consisting of OH, NH   2    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, NH   2    and chemically blocked derivatives thereof.   
     
     
       24. An oligonucleotide of  claim 22 , wherein W is pentyl and X is NH- trityl.   
     
     
       25. A compound of  claim 10 , wherein the reporter group is selected from the group consisting of  3   H,    125   I,    35   S,    14   C and    32   P.   
     
     
       26. A compound of  claim 10 , wherein the reporter group is  3   H.   
     
     
       27. An oligonucleotide of  claim 13 , wherein the reporter group is selected from the group consisting of  3   H,    125   I,    35   S,    14   C and    32   P.   
     
     
       28. An oligonucleotide of  claim 13 , wherein the reporter group is  3   H.   
     
     
       29. A labeled oligonucleotide comprising at least one nucleotide unit of the formula                    
         wherein R   1    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;    
         R   3    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 R   4    and R   6    is independently H, OR, SR, NHOR, NH   2   , or NH ( CH   2   )   t   NH   2    where R is H or C   1-6   alkyl 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  3   H,    125   I,    35   S,    14   C and    32   P.   
     
     
       31. A labeled oligonucleotide of  claim 29 , wherein said reporter group is  3   H.   
     
     
       32. A compound having the formula                    
         wherein R   1    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;    
         R   3    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 R   4    and R   6    is independently H, OR, SR, NHOR, NH   2   , or NH ( CH   2 ) t   NH   2    where R is H or C   1-6   alkyl 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  3   H,    125   I,    35   S,    14   C and    32   P.   
     
     
       34. A compound of  claim 32 , wherein said reporter group is  3   H.