Oligonucleolotides having site specific chiral phosphorothioate internucleoside linkages

Abstract

Novel chiral compounds that mimic and/or modulate the activity of wild-type nucleic acids are disclosed. In general, the compounds are phosphorothioate oligonucleotides wherein the 5′, and the 3′-terminal internucleoside linkages are chirally Sp and internal internucleoside linkages are chirally Rp.

1. An oligomeric compound comprising a plurality of covalently-bound nucleosides; said compound having the formula:
   5′-T 1 —(Nu—Sp) n —(Nu—Lp) m —(NU—Sp) p —Nu—T 2 -3′ 
 
       wherein:
 T 1  and T 2  are each, independently, hydroxyl, a protected hydroxyl, a covalent attachment to a solid support, a nucleoside, an oligonucleoside, a nucleotide, an oligonucleotide, a conjugate group or a 5′ or 3′ substituent group;  
 each Sp is a chiral Sp phosphorothioate internucleoside linkage;  
 each Lp is, independently, a chiral Rp phosphorothioate internucleoside linkage, a racemic phosphorothioate internucleoside linkage or an internucleoside linkage other than a chiral phosphorothioate internucleoside linkage;  
 each n and m is, independently, from 1 to 100;  
 each p is from 0 to 100; where the sum of n, m and p is from 3 to about 200;  
 each Nu independently, has the formula: 
                 
 
  wherein:  
 Bx is a heterocyclic base moiety; and  
 R 1  is H, hydroxyl, a protected hydroxyl, a 2′-substituent group or a protected 2′-substituent group.  
 
     
     
       2. The oligomeric compound of  claim 1  wherein each R, is H or hydroxyl. 
     
     
       3. The oligomeric compound of  claim 1  wherein R 1  is C 1 -C 10  O-alkyl or C 1 -C 10  substituted O-alkyl. 
     
     
       4. The oligomeric compound of  claim 3  wherein R 1  is 2′-O-methoxyethyl or 2′-O-methyl. 
     
     
       5. The oligomeric compound of  claim 1  wherein each Nu is, independently, adenosine, guanosine, uridine, 5-methyluridine, cytidine, 5-methylcytidine or thymine. 
     
     
       6. The oligomeric compound of  claim 1  wherein the sum of n, m, and p is from 5 to about 50. 
     
     
       7. The oligomeric compound of  claim 1  wherein the sum of n, m, and p is from 8 to about 30. 
     
     
       8. The oligomeric compound of  claim 1  wherein the sum of n, m, and p is from 10 to about 25. 
     
     
       9. The oligomeric compound of  claim 1  wherein p is 1 or  2 . 
     
     
       10. The oligomeric compound of  claim 1  wherein n and p are each 1 and m is from 3 to about 20. 
     
     
       11. The oligomeric compound of  claim 1  wherein T 1  and T 2  are, independently, hydroxyl or a protected hydroxyl. 
     
     
       12. The oligomeric compound of  claim 1  wherein each Lp is an Rp phosphorothioate internucleoside linkage. 
     
     
       13. The oligomeric Compound of  claim 1  wherein at least one Lp is a racemic phosphorothioate internucleoside linkage. 
     
     
       14. The oligomeric Compound of  claim 1  wherein at least one Lp is an internucleoside linkage other than a chiral phosphorothioate internucleoside linkage. 
     
     
       15. The oligomeric Compound of  claim 1  wherein R 1  is a 2′-substituent group or a protected 2′-substituent group. 
     
     
       16. A pharmaceutical composition comprising a compound of  claim 1  and an acceptable pharmaceutical carrier. 
     
     
       17. A nucleoside having the formula: 
                 
 
       wherein:
 Bx is a heterocyclic base moiety;  
 R 4  is a hydroxyl protecting group;  
 R 1  is H, hydroxyl, a protected hydroxyl, a 2′-substituent group or a protected 2′-substituent group; and  
 R 2  is an Sp chiral auxiliary group.  
 
     
     
       18. The nucleoside of  claim 17  wherein said chiral auxiliary group has one of formulas I, II, III, IV, V or VI: 
                 
 
     
     
       19. The nucleoside of  claim 17  wherein Bx is adenosine, guanosine, uridine, 5-methyluridine, cytidine, 5-methylcytidine or thymine. 
     
     
       20. The nucleoside of  claim 17  wherein each R 1  is H or hydroxyl. 
     
     
       21. The nucleoside of  claim 17  wherein R 1  is C 1 -C 10  alkyl or C 1 -C 10  substituted alkyl. 
     
     
       22. The nucleoside of  claim 21  wherein R 1  is 2′-O-methoxyethyl or 2′-O-methyl. 
     
     
       23. The nucleoside of  claim 17  wherein at least one R 1  is 2′-O-methoxyethyl or 2′-O-methyl. 
     
     
       24. The nucleoside of  claim 17  wherein R 1  is a 2′-substituent group or a protected 2′-substituent group. 
     
     
       25. A method of preparing an oligomeric compound of formula:
   5′-T 1 —(Nu—Sp) n —(NU—Lp) m —(NU—Sp) p —Nu—T 2 -3′ 
 
       wherein:
 each T 1  and T 2  is, independently, hydroxyl, a protected hydroxyl, a covalent attachment to a solid support, a nucleoside, an oligonucleotide, a nucleotide or an oligonucleotide, a conjugate group or a 5′ or 3′ substituent group;  
 each Sp is an Sp phosphorothioate internucleoside linkage;  
 each Lp is, independently, an Rp phosphorothioate internucleoside linkage, a racemic phosphorothioate internucleoside linkage or an internucleoside linkage other than a chiral Rp phosphorothioate internucleoside linkage;  
 each n and m is, independently, from 1 to 100;  
 each p is from 0 to 100 where the sum of n, m and p is from 3 to about 200:  
 each Nu, independently, has the formula: 
                 
 
  wherein  
 Bx is a heterocyclic base moiety; and  
 R 1  is H, hydroxy, a protected hydroxyl, a 2′-substituent group or a protected 2′-substituent group;  
 comprising the steps of: 
 (a) providing a compound of formula: 
                 
 
 wherein:  
 
 R 4  is a labile hydroxyl protecting group;  
 R 3  is a covalent attachment to a solid support; 
 (b) deblocking said labile hydroxyl protecting group to form a deblocked hydroxyl group;  
 (c) optionally treating said deblocked hydroxyl group with a further compound having the formula: 
                 
 
 wherein:  
 
 R 2  is an Sp chiral auxiliary group;  
 and a condensing reagent to form an extended compound: 
 (d) optionally repeating steps (b) and (c);  
 (e) treating said deblocked hydroxyl group with a compound having the formula: 
                 
 
 wherein:  
 
 R 5  is an Rp chiral auxiliary group or an activated phosphorus group;  
 and a condensing reagent to form a further extended compound; 
 (f) optionally repeating steps (e) and (f) to add further nucleosides;  
 (g) deblocking said labile hydroxyl protecting group to form a deblocked hydroxyl group;  
 (h) treating said deblocked hydroxyl group with a further compound having the formula: 
                 
 
 wherein:  
 
 R 1  is an Sp chiral auxiliary group;  
 and a condensing reagent to form a protected oligomeric compound; and 
 (i) optionally repeating steps (h) and (i) to add at additional nucleosides thereby forming a further protected oligomeric compound.  
 
 
     
     
       26. The method of  claim 25  further comprising the step of deblocking the product of step (i). 
     
     
       27. The method of  claim 25  wherein said Sp chiral auxiliary group has one of formulas I, II or III: 
                 
 
     
     
       28. The method of  claim 25  wherein said Rp chiral auxiliary group has one of formulas IV, V or VI: 
                 
 
     
     
       29. The method of  claim 25  further comprising one or more capping steps after steps c, d, e, f, h, or i; said capping steps comprising treatment with a capping agent. 
     
     
       30. The method of  claim 25  further comprising one or more oxidation steps; said oxidation steps comprising treatment with an oxidizing agent. 
     
     
       31. The method of  claim 25  wherein said labile hydroxyl protecting group is dimethoxytrityl, monomethoxy trityl, trityl or 9-phenyl-xanthene. 
     
     
       32. The method of  claim 25  wherein said heterocyclic base moiety is a purine or a pyrimidine. 
     
     
       33. The method of  claim 32  wherein said purine or pyrimidine is, independently, adenosine, guanosine, uridine, 5-methyluridine, cytidine, 5-methylcytidine or thymine. 
     
     
       34. The method of  claim 25  wherein the sum of n, m, and p is from 5 to about 50. 
     
     
       35. The method of  claim 25  wherein the sum of n, m, and p is from 8 to about 30. 
     
     
       36. The method of  claim 25  wherein the sum of n, m, and p is from 10 to about 25. 
     
     
       37. The method of  claim 25  wherein T 1  and T 2  are, independently hydroxyl or a protected hydroxyl. 
     
     
       38. The method of  claim 25  wherein each Lp is an Rp phosphorothioate internucleoside linkage. 
     
     
       39. The method of  claim 25  wherein at least one Lp is a racemic phosphorothioate internucleoside linkage. 
     
     
       40. The method of  claim 25  wherein n and p are each 1 and m is from 3 to about 20. 
     
     
       41. The method of  claim 25  wherein n and p are each 2 and m is from 3 to about 20. 
     
     
       42. The method of  claim 25  wherein p is 0. 
     
     
       43. The method of  claim 25  wherein at least one R 1  is a 2′-substituent group or a protected 2′-substituent group. 
     
     
       44. The method of  claim 25  wherein said activated phosphorus group is a phosphoramidite, an H-phosphonate or a phosphate triester. 
     
     
       45. The method of  claim 25  wherein said covalent attachment to a solid support is a sarcosinyl-succinonyl linker. 
     
     
       46. A method of modulating the production or activity of a protein in an organism, comprising contacting said organism with a compound of  claim 1 , wherein said protein is protein kinase C, ICAM-1, VCAM-1, PECAM-1, ELAM-1, H-ras, K-ras, AP-1, a Jun N-terminal kinase, or a matrix metalloproteinase. 
     
     
       47. A method of treating an organism having a disease characterized by the undesired production of a protein, comprising contacting said organism with a compound of  claim 1 , wherein said disease is psoriasis, an inflammatory disorder of the skin, an infectious disease of the skin, or skin cancer. 
     
     
       48. The oligomeric compound of  claim 47  wherein R 1  is 2′-O-methoxyethyl or 2′-O-methyl. 
     
     
       49. A compound of formula: 
                 
 
       wherein:
 R 62  is H or a hydroxyl protecting group;  
 R 1  is H, hydroxyl, a protected hydroxyl, a 2′-substituent group or a protected 2′-substituent group;  
 B is a heterocyclic base moiety; and  
 R 61  is a chiral auxiliary selected from formulas I-VI: 
                 
 
 
     
     
       50. The oligomeric compound of  claim 49  wherein each R 1  is H or hydroxyl. 
     
     
       51. The oligomeric compound of  claim 49  wherein R 1  is C 1 -C 10  O-alkyl or C 1 -C 10  substituted O-alkyl. 
     
     
       52. The oligomeric compound of  claim 51  wherein R 1  is 2′-O-methoxyethyl or 2′-O-methyl. 
     
     
       53. The oligomeric compound of  claim 49  wherein each Nu is, independently, adenosine, guanosine, uridine, 5-methyluridine, cytidine, 5-methylcytidine or thymine. 
     
     
       54. A compound having the formula: 
                   
       wherein:
 B is a heterocyclic base moiety;  
 q is 0 to about 50;  
 R 62  is H or a hydroxyl protecting group;  
 R 1  is H, hydroxyl, a protected hydroxyl, a 2′-substituent group or a protected) 2′-substituent group;  
 R 64  is H, a hydroxyl protecting group, or a linker to a solid support;  
 R 63  is a radical selected from the group consisting of 
                 
 
 
       
        
       
     
     
       55. The oligomeric  compound of  claim 54  wherein each R 1  is H or hydroxyl. 
     
     
       56. The oligomeric  compound of  claim 54  wherein R 1  is C 1 -C 10  O-alkyl or C 1 -C 10  substituted O-alkyl. 
     
     
       57. The oligomeric  compound of  claim 54  wherein R 1  is 2′-O-methoxyethyl or 2′-O-methyl. 
     
     
       58. The oligomeric  compound of  claim 54  wherein each Nu is, independently, adenosine, guanosine, uridine. 5-methyluridine, cytidine 5-methylcytidine or thymine. 
     
     
       59. The oligomeric  compound of  claim 54  wherein q is 5 to about 50. 
     
     
       60. The oligomeric  compound of  claim 54  wherein q is from 8 to about 30. 
     
     
       61. The oligomeric  compound of  claim 54  wherein q is from 10 to about 25. 
     
     
       62. The oligomeric  compound of  claim 54  wherein q is 1. 
     
     
       63. The oligomeric compound of  claim 54  wherein q is 0. 
     
     
       64. A compound having the formula:
                   
       
         wherein:  
         
           B is a heterocyclic base moiety;  
         
         
           q is  1  to about  50 ;  
         
         
           R 
           62  
           is H or a hydroxyl protecting group;  
         
           R   1    is H, hydroxyl, a protected hydroxyl, a  2 ′ - substituent group or a protected  2 ′ - substituent group;    
         
           R 
           64  
           is H, a hydroxyl protecting group, or a linker to a solid support;  
         
         
           R 
           63  
           is a radical selected from the group consisting of