Process for the preparation of oligonucleotide compounds

• US 6,649,750 B1
• Filed: 01/05/2000
• Issued: 11/18/2003
• Est. Priority Date: 01/05/2000
• Status: Expired due to Term

• Current Assignee: Ionis Pharmaceuticals, Inc.
• Original Assignee: ISIS Pharmaceuticals Incorporated (Ionis Pharmaceuticals, Inc.)
• Inventors: Capaldi, Daniel C., Cole, Douglas L., Ravikumar, Vasulinga T.
• Primary Examiner(s): Wilson, James O.
• Assistant Examiner(s): Crane, Lawrence E
• Application Number:
  US09/477,878
• Time in Patent Office: 1,413 Days
• Field of Search: 536/23.31, 536/25.34
• US Class Current: 536/25.31
• CPC Class Codes: C07B 2200/11 Compounds covalently bound to a solid support
• 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

Abstract

Synthetic processes are provided wherein high purity oligomers are prepared using support bound phosphoramidite protocols starting with a nucleoside or larger synthon linked to a support media through a nucleosidic heterocyclic base moiety. Intermediates undergoing depurination at the support linkage site are removed during the wash cycle. Also provided are compositions useful in such processes.

Claims

1. A method of preparing an oligomeric compound having at least one moiety of formula:

wherein;

Q is an internucleoside linkage;

Bx is an optionally blocked heterocyclic base moiety;

Bxx is a purine or purine analog;

each R is, independently, hydrogen or an optionally protected substituent group;

L is a bifunctional linking moiety having the formula —

C(═

O)—

(CH₂)_nn—

C(═

O)—

where nn is from 4 to 25; and

SM is a support medium;

comprising the steps of;

(a) providing a compound of formula;

 

wherein;

T₁ is a 5′

-hydroxyl protecting group; and

T₂ is a hydroxyl blocking group, a nucleoside, a nucleotide, an oligonucleoside, an oligonucleotide or a conjugate group;

(b) removing said 5′

-hydroxyl protecting group to form a deprotected hydroxyl group by contacting same with a reagent effective to remove said protecting group;

(c) treating said deprotected hydroxyl group with a further compound having the formula;

 

wherein;

T₃ is a 5′

-hydroxyl protecting group, a nucleoside, a nucleotide, an oligonucleoside, an oligonucleotide or a conjugate group; and

T₄ is a reactive P^III species for forming an internucleoside linkage;

and an activating agent for a time and under conditions effective to form an extended oligomeric compound;

d) treating said extended oligomeric compound with a capping agent to form a capped compound;

e) treating said capped compound with an oxidizing agent; and

f) optionally repeating steps b through e one or more additional cycles to form said oligomeric compound.

2. The method of claim 1 further comprising treating said oligomeric compound with a reagent effective to form a deblocked oligomeric compound.

3. The method of claim 2 wherein said reagent is effective to cleave said ligomeric compound from the support medium.

4. The method of claim 2 further comprising treating said deblocked ligomeric compound with a reagent effective to cleave said oligomeric compound from the support medium.

5. The method of claim 2 wherein said deblocking reagent is a basic solution.

6. The method of claim 5 wherein said basic solution is concentrated ammonium hydroxide.

7. The method of claim 1, wherein said T₃ is said 5′

-hydroxyl protecting group, further comprising treating said oligomeric compound with a reagent effective to deprotect said 5′

-hydroxyl protecting group to give a free hydroxyl group.

8. The method of claim 1 wherein said activating agent is 1-H-tetrazole.

9. The method of claim 1 wherein said oxidizing agent is an oxaziridine.

10. The method of claim 9 wherein said oxidizing agent is 10-camphorsulphonyl oxazaridine, 2-phenylsulphonyl-3-phenyl oxazaridine, 2-(phenylsulphonyl)-3-(3-nitrophenyl)oxazaridine, or 8,8-dihalo-10-camphorsulphonyl oxazaridine.

11. The method of claim 1 wherein said reactive P^III species is a phosphoramidite.

12. The method of claim 1 wherein said heterocyclic base moiety is adeninyl, N⁶-benzoyladeninyl, 2-aminoadeninyl, cytosinyl, N⁴-benzoylcytosinyl, 5-methylcytosinyl, N⁴-benzoyl-5-methylcytosinyl, thyminyl, uracilyl, guaninyl or N²-isobutyrylguaninyl.

13. The method of claim 1 wherein said oligomeric compound comprises from about 10 to about 30 monomer subunits.

14. The method of claim 1 wherein said oligomeric compound comprises from about 15 to 25 monomer subunits.

15. The method of claim 1 wherein nn is from 6 to 20.

16. The method of claim 1 wherein nn is from 8 to 16.

17. The method of claim 1 wherein the bifunctional linking moiety attaches the support medium to the oligomeric compound at a heterocyclic base functional group.

18. The method of claim 17 wherein said functional group is a primary or secondary amino group.

19. The method of claim 17 wherein said functional group is a primary amino group.

20. The method of claim 1 wherein said internucleoside linkage is phosphodiester, phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphoramidate or alkylphosphonate (—

O—

P(jj)(O)—

O—

) where jj is C₁-C₁₂ alkyl.

21. The method of claim 1 wherein said oligomeric compound comprises from about 5 to about 50 monomer subunits.

22. The method of claim 1 wherein T₂ is a hydroxyl blocking group.

23. The method of claim 22 wherein said hydroxyl blocking group is base labile.

24. The method of claim 22 wherein said hydroxyl protecting group is of the formula —

C(═

O)R_d where R_d is C₁ to C₁₂ alkyl.

25. The method of claim 24 wherein R_d is CH₃.

26. The method of claim 1 wherein each said substituent group R is, independently, hydroxyl, C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, C₂-C₂₀ alkynyl, halogen, amino, thiol, keto, carboxyl, nitro, nitroso, nitrile, trifluoromethyl, trifluoromethoxy, O-alkyl, O-alkenyl, O-alkynyl, S-alkyl, S-alkenyl, S-alkynyl, NH-alkyl, NH-alkenyl, NH-alkynyl, N-dialkyl, O-aryl, S-aryl, NH-aryl, O-aralkyl, S-aralkyl, NH-aralkyl, N-phthalimido, imidazolyl, azido, hydrazino, hydroxylaamino, isocyanato, reporter molecule, conjugate, polyamine, polyamide, polyalkylene glycol, or polyether;

or each substituent group has one of formula I or II;

 

wherein;

Z₀ is O, S or NH;

J is a single bond, O or C(═

O);

E is C₁-C₁₀ alkyl, N(R₁)(R₂), N(R₁)(R₅), N═

C(R₁)(R₂), N═

C(R₁)(R₅) or has formula IV;

each R₆, R₇, R₉ and R₁₀ is, independently, hydrogen, C(O)R₁₁, substituted or unsubstituted C₁-C₁₀ alkyl, substituted or unsubstituted C₂-C₁₀ alkenyl, substituted or unsubstituted C₂-C₁₀ alkynyl, alkylsulfonyl, arylsulfonyl, or a conjugate group, wherein the substituent groups are selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl;

or optionally, R₉ and R₁₀, together form a phthalimido moiety with the nitrogen atom to which they are attached;

each R₁₁ is, independently, substituted or unsubstituted C₁-C₁₀ alkyl, trifluoromethyl, cyanoethyloxy, methoxy, ethoxy, t-butoxy, allyloxy, 9-fluorenylmethoxy, 2-(trimethylsilyl)-ethoxy, 2,2,2-trichloroethoxy, benzyloxy, butyryl, iso-butyryl, phenyl or aryl;

R₅ is T—

L₁,

T is a bond or a linking moiety;

L₁ is a conjugate group or a solid support material;

each R₁ and R₂ is, independently, H, a nitrogen protecting group, substituted or unsubstituted C₁-C₁₀ alkyl, substituted or unsubstituted C₂-C₁₀ alkenyl, substituted or unsubstituted C₂-C₁₀ alkynyl, wherein said substitution is OR₃, SR₃, NH₃⁺, N(R₃)(R₄), guanidino or acyl where said acyl is an acid amide or an ester;

or R₁ and R₂, together, are a nitrogen protecting group;

each R₃ and R₄ is, independently, H, C₁-C₁₀ alkyl, a nitrogen protecting group, or R₃ and R₄, together, are a nitrogen protecting group;

Z₄ is OX, SX, or N(X)₂;

each X is, independently, H, C₁-C₈ alkyl, C₁-C₈ haloalkyl, C(═

NH)N(H)R₅, C(═

O)N(H)R₅ or OC(═

O)N(H)R₅;

R₅ is H or C₁-C₈ alkyl;

Z₁, Z₂ and Z₃ form a ring system having from about 4 to about 7 carbon atoms or having from about 3 to about 6 carbon atoms and 1 or 2 hetero atoms wherein said hetero atoms are selected from oxygen, nitrogen and sulfur and wherein said ring system is aliphatic, unsaturated aliphatic, aromatic, or saturated or unsaturated heterocyclic;

Z₅ is alkyl or haloalkyl having 1 to about 10 carbon atoms, alkenyl having 2 to about 10 carbon atoms, alkynyl having 2 to about 10 carbon atoms, aryl having 6 to about 14 carbon atoms, N(R₁)(R₂), OR₁, halo, SR₁ or CN;

each q₁ is, independently, an integer from 1 to 10;

each q₂ is, independently, 0 or 1;

q₃ is 0 or an integer from 1 to 10;

q₄ is an integer from 1 to 10;

q₅ is from 0, 1 or 2; and

provided that when q₃ is 0, q₄ is greater than 1.

27. A method of preparing an oligomeric compound of formula:

wherein;

T₅ is H, a hydroxyl blocking group, a nucleoside, an oligonucleoside, a nucleotide an oligonucleotide or a conjugate group;

T₆ is H, a hydroxyl protecting group, a nucleoside, an oligonucleoside, a nucleotide an oligonucleotide or a conjugate group;

mm is from about 5 to about 50;

each Xa is, independently, O or S;

each Xb is, independently, OH, SH or NR_aR_b;

each R_a and R_b is, independently, H, a nitrogen protecting group, substituted or unsubstituted C₁-C₁₀ alkyl, substituted or unsubstituted C₂-C₁₀ alkenyl, substituted or unsubstituted C₂-C₁₀ alkynyl, wherein said substitution is by hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl or alkynyl;

Bxx is a purine or purine analog;

each Bx is, independently, an optionally blocked heterocyclic base moiety;

each R is, independently, hydrogen or an optionally protected substituent group;

L is a bifunctional linking moiety having the formula —

C(═

O)—

(CH₂)_nn—

C(═

O)—

where nn is from 4 to 25; and

SM is a support medium;

comprising the steps of;

(a) providing a compound of the formula;

 

wherein;

T₁ is a 5′

-hydroxyl protecting group;

(b) removing said 5′

-hydroxyl protecting group to form a deprotected hydroxyl group by contacting same with a reagent effective to remove said protecting groups;

(c) treating said deprotected hydroxyl group with a further compound having the formula;

 

wherein;

T₄ is a phosphoramidite;

and an activating agent for a time and under conditions effective to form an extended oligomeric compound;

d) treating said extended oligomeric compound with a capping agent to form a capped compound;

e) treating said capped compound with an oxidizing agent; and

f) optionally repeating steps b through e one or more times to form said oligomeric compound.

28. The method of claim 27 wherein each substituent group R is, independently, hydroxyl, C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, C₂-C₂₀ alkynyl, halogen, amino, thiol, keto, carboxyl, nitro, nitroso, nitrile, trifluoromethyl, trifluoromethoxy, O-alkyl, O-alkenyl, O-alkynyl, S-alkyl, S-alkenyl, S-alkynyl, NH-alkyl, NH-alkenyl, NH-alkynyl, N-dialkyl, O-aryl, S-aryl, NH-aryl, O-aralkyl, S-aralkyl, NH-aralkyl, N-phthalimido, imidazolyl, azido, hydrazino, hydroxylamino, isocyanato, reporter molecule, conjugate, polyamine, polyamide, polyalkylene glycol, or polyether;

or each substituent group has one of formula I or II;

 

wherein;

Z₀ is O, S or NH;

J is a single bond, O or C(═

O);

E is C₁-C₁₀ alkyl, N(R₁)(R₂), N(R₁)(R₅), N═

C(R₁)(R₂), N═

C(R₁)(R₅) or has formula IV;

each R₆, R₇, R₉ and R₁₀ is, independently, hydrogen, C(O)R₁₁, substituted or unsubstituted C₁-C₁₀ alkyl, substituted or unsubstituted C₂-C₁₀ alkenyl, substituted or unsubstituted C₂-C₁₀ alkynyl, alkylsulfonyl, arylsulfonyl, or a conjugate group, wherein the substituent groups are selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl;

or optionally, R₉ and R₁₀, together form a phthalimido moiety with the nitrogen atom to which they are attached;

each R₁₁, is, independently, substituted or unsubstituted C₁-C₁₀ alkyl, trifluoromethyl, cyanoethyloxy, methoxy, ethoxy, t-butoxy, allyloxy, 9-fluorenylmethoxy, 2-(trimethylsilyl)-ethoxy, 2,2,2-trichloroethoxy, benzyloxy, butyryl, iso-butyryl, phenyl or aryl;

R₅is T—

L₁,

T is a bond or a linking moiety;

L₁ is a conjugate group or a solid support material;

each R₁ and R₂ is, independently, H, a nitrogen protecting group, substituted or unsubstituted C₁-C₁₀ alkyl, substituted or unsubstituted C₂-C₁₀ alkenyl, substituted or unsubstituted C₂-C₁₀ alkynyl, wherein said substitution is OR₃, SR₃, NH₃⁺, N(R₃)(R₄), guanidino or acyl where said acyl is an acid amide or an ester;

or R₁ and R₂, together, are a nitrogen protecting group;

each R₃ and R₄ is, independently, H, C₁-C₁₀ alkyl, a nitrogen protecting group, or R₃ and R₄, together, are a nitrogen protecting group;

Z₄ is OX, SX, or N(X)₂;

each X is, independently, H, C₁-C₈ alkyl, C₁-C₈ haloalkyl, C(═

NH)N(H)R₅, C(═

O)N(H)R₅ or OC(═

O)N(H)R₅;

R₅ is H or C₁-C₈ alkyl;

Z₁, Z₂ and Z₃ form a ring system having from about 4 to about 7, carbon atoms or having from about 3 to about 6 carbon atoms and 1 or 2 hetero atoms wherein said hetero atoms are selected from oxygen, nitrogen and sulfur and wherein said ring system is aliphatic, unsaturated aliphatic, aromatic, or saturated or unsaturated heterocyclic;

Z₅ is alkyl or haloalkyl having 1 to about 10 carbon atoms, alkenyl having 2 to about 10 carbon atoms, alkynyl having 2 to about 10 carbon atoms, aryl having 6 to about 14 carbon atoms, N(R₁)(R₂) OR₁, halo, SR₁ or CN;

each q₁ is, independently, an integer from 1 to 10;

each q₂ is, independently, 0 or 1;

q₃ is 0 or an integer from 1 to 10;

q₄ is an integer from 1 to 10;

q₅ is from 0, 1 or 2; and

provided that when q₃ is 0, q₄ is greater than 1.

29. The method of claim 27 further comprising treating said oligomeric compound with a reagent effective to form a deblocked oligomeric compound.

30. The method of claim 29 wherein said reagent is effective to cleave said oligomeric compound from the support medium.

31. The method of claim 29 further comprising treating said deblocked oligomeric compound with a reagent effective to cleave said oligomeric compound from the support medium.

32. The method of claim 27 wherein said deblocking reagent is a basic solution.

33. The method of claim 32 wherein said basic solution is concentrated ammonium hydroxide.

34. The method of claim 27, wherein said T₅ is said 5′

-hydroxyl protecting group, further comprising treating said oligomeric compound with a reagent effective to deprotect said 5′

-hydroxyl protecting group.

35. The method of claim 27 wherein said activating agent is 1-H-tetrazole.

36. The method of claim 27 wherein said oxidizing agent is an oxaziridine.

37. The method of claim 36 wherein said oxidizing agent is 10-camphorsulphonyl oxazaridine, 2-phenylsulphonyl-3-phenyl oxazaridine, 2-(phenylsulphonyl)-3-(3-nitrophenyl)oxazaridine, or 8,8-dihalo-10-canphorsulphonyl oxazaridine.

38. The method of claim 27 wherein said heterocyclic base moiety is adeninyl, N⁶-benzoyladeninyl, 2-aminoadeninyl, cytosinyl, N⁴-benzoylcytosinyl, 5-methylcytosinyl, N⁴-benzoyl-5-methylcytosinyl, thyminyl, uracilyl, guaninyl or N²-isobutyrylguaninyl.

39. The method of claim 27 wherein nn is from 6 to 20.

40. The method of claim 27 wherein nn is from 8 to 16.

41. The method of claim 27 wherein the covalent attachment of the bifunctional linking moiety to said Bxx is through an amide bond formed between an exocyclic amino group on Bxx and an acyl group on said bifunctional linking moiety.

42. The method of claim 41 wherein Bxx is a purine.

43. The method of claim 41 wherein Bxx is a purine analog.

44. The method of claim 43 wherein said purine analog is xanthine, hypoxanthine, 2-aminoadenine, 7-methylguanine, 7-methyladenine, a 6-methyl derivative of adenine or guanine, a 2-propyl derivative of adenine or guanine, an 8-halo, 8-amino, 8-thiol, 8-thioalkyl, or an 8-hydroxyl substituted adenine or guanine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, or 3-deazaadenine.

45. The method of claim 44 wherein said purine analog has a primary or secondary amino functionality integral with or exocyclic to the purine ring system.

46. The method of claim 27 wherein T₆ is said hydroxyl protecting group.

47. The method of claim 46 wherein said hydroxyl protecting group is base labile.

48. The method of claim 46 wherein said hydroxyl protecting group is of the formula —

C(═

O)R_d where R_d is C₁ to C₁₂ alkyl.

49. The method of claim 48 wherein R_d is CH₃.

50. The method of claim 27 wherein mm is from about 10 to about 30.

51. The method of claim 27 wherein mm is from about 15 to 25.

52. The method of claim 27 wherein said support medium is an insoluble solid support.

53. The method of claim 27 wherein said support medium is a soluble polymeric support.

54. The method of claim 53 wherein said polymeric support is monomethoxy polyethylene glycol.

55. The method of claim 54 wherein said polymeric support is poly(N-acryloyl-morpholine).

56. The method of claim 27 wherein said purine analog is xanthine, hypoxanthine, 2-aminoadenine, 7-methylguanine, 7-methyladenine, a 6-methyl derivative of adenine or guanine, a 2-propyl derivative of adenine or guanine, an 8-halo, 8-amino, 8-thiol, 8-thioalkyl, or an 8-hydroxyl substituted adenine or guanine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, or 3-deazaadenine.

57. The method of claim 56 wherein said purine analog has a primary or secondary amino functionality integral with or exocyclic to the purine ring system.