Process for the preparation of oligonucleotides

• US 6,809,195 B1
• Filed: 08/16/2000
• Issued: 10/26/2004
• Est. Priority Date: 08/16/2000
• Status: Expired due to Term

• Current Assignee: Ionis Pharmaceuticals, Inc.
• Original Assignee: ISIS Pharmaceuticals Incorporated (Ionis Pharmaceuticals, Inc.)
• Inventors: Song, Quanlai, Sanghvi, Yogesh S.
• Primary Examiner(s): McGarry, Sean
• Assistant Examiner(s): EPPS -SMITH, JANET L
• Application Number:
  US09/640,279
• Time in Patent Office: 1,532 Days
• Field of Search: 536/25.31, 536/25.32, 536/25.33, 536/22.1, 536/23.1, 536/24.5, 536/25.3, 435/91.1, 435/6, 435/130, 585/833
• US Class Current: 536/25.3
• CPC Class Codes: C07H 19/04 Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
• 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

The present invention discloses methods for synthesizing oligomeric compounds. The methods include a modified phosphoramidite protocol wherein the oxidation and capping steps are combined into a single step. The methods result in increased efficiency and are especially amenable to the large scale synthesis of oligomeric compounds.

Claims

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

wherein;

X₂ is O or S;

X₁ is Pg—

O—

, Pg—

S—

, C₁-C₁₀ straight or branched chain alkyl, CH₃(CH₂)_nn—

O—

, R₂R₃N—

or a group remaining from coupling a chiral auxiliary;

nn is from 0 to 10;

Pg is CH₃, —

CH₂CH₂CN, —

C(CH₃)(CH₃)—

CCl₃, —

CH₂—

CCl₃, —

CH₂CH═

CH₂, CH₂CH₂SiCH₃, 2-yl-ethyl phenylsulfonate, δ

-cyanobutenyl, cyano p-xylyl, diphenylsilylethyl, 4-nitro-2-yl-ethylbenzene, 2-yl-ethyl-methyl sulfonate, methyl-N-trifluoroacetyl ethyl, acetoxy phenoxy ethyl, or a blocking group;

R₁ is, independently, hydrogen, a blocked hydroxyl group, or a sugar substituent group;

R₂ is, independently, hydrogen, a C₁-C₁₀ alkyl, a cycloalkyl, or aryl;

R₃ is, independently, hydrogen, a C₁-C₁₀ alkyl, a cycloalkyl, or aryl;

R₄ is, independently, N(L₁)L₂;

or optionally, R₂ and R₃, together with the nitrogen atom to which they are attached form a cyclic moiety;

each Bx is, independently, a heterocyclic base moiety; and

comprising the steps of;

(a) providing a 5′

-O-protected compound of the formula;

wherein;

T₁ is a hydroxyl protecting group; and

T₂ is a covalent attachment to a support media, a nucleoside bound to a support media, a nucleotide, an oligonucleoside or an oligonucleotide;

(b) treating said 5′

-O-protected compound with a deprotecting reagent for a time and under conditions effective to form a 5′

-deprotected compound;

(c) coupling said 5′

-O-deprotected compound with an activated phosphorus composition of the formula;

wherein;

T₃ is a hydroxyl protecting group, a nucleoside, nucleotide, an oligonucleoside or an oligonucleotide;

each L₁ and L₂ is, independently, C_1-6 straight or branched alkyl, or a C_5-7 cyclic aliphatic ring system;

or L₁ and L₂ are joined together to form a 4- to 13-membered heterocyclic ring system including the nitrogen atom to which L₁ and L₂ are attached; and

R₅ is X₁;

or R₄ and R₅ together with the phosphorus atom to which R₄ and R₅ are attached form a chiral auxiliary;

for a time and under conditions effective to form an extended compound having the formula;

(d) treating said extended compound with a mixture comprising an oxidizing reagent and a capping reagent in a single step and for a time and under conditions effective to form said oligomeric compound, and

(e) treating said oligomeric compound with a reagent for a time and under conditions effective to remove said blocking groups thereby forming a deblocked oligomeric compound.

2. The method of claim 1 wherein said reagent in step (e) is effective to cleave the oligomeric compound from the support media.

3. The method of claim 2 wherein said reagent in step (e) is aqueous ammonium hydroxide.

4. The method of claim 1 further comprising treating said oligomeric compound with a further reagent for a time and under conditions effective to cleave the oligomeric compound from the support media.

5. The method of claim 1 further comprising treating said oligomeric compound with a deprotecting reagent for a time and under conditions effective to deprotect the T₃ hydroxyl protecting group.

6. The method of claim 1 wherein said mixture comprises from 0.02M to 0.2M oxidizing reagent.

7. The method of claim 6 wherein said mixture comprises from 0.1M to 0.2M oxidizing reagent.

8. The method of claim 1 wherein said oxidizing reagent transfers an oxygen atom.

9. The method of claim 8 wherein said oxidizing reagent is iodine, m-chloroperbenzoic acid, iodobenzene diacetate, tetra-n-butylammonium periodate, tert-butyl hydroperoxide, di-tert-butyl hydroperoxide, cumene hydroperoxide, hydrogen peroxide;

bis-trimethylsilyl peroxide;

dinitrogen tetroxide, oxone, molecular oxygen, (1S)-(+)(10-camphorsulfonyl)oxaziridine or a peracid.

10. The method of claim 9 wherein said oxidizing reagent is iodine, m-chloroperbenzoic acid, iodobenzene diacetate, tert-butyl hydroperoxide, di-tert-butyl hydroperoxide, hydrogen peroxide, oxone, molecular oxygen or a peracid.

11. The method of claim 1 wherein said oxidizing reagent transfers a sulfur atom.

12. The method of claim 11 wherein said oxidizing reagent is 3-amino-1,2,4-dithiazole-5-thione;

3-ethoxy-1,2,4-dithiazoline-5-one;

1,2,4-dithiazolidine-3,5-dione;

3-methyl-1,2,4-dithiazolin-5-one;

or dimethylthiuram disulfide.

13. The method of claim 12 wherein said oxidizing reagent is dimethylthiuram disulfide.

14. The method of claim 1 wherein said capping reagent comprises about one part by volume of either acetic anhydride in acetonitrile or tetrahydrofuran;

or chloroacetic anhydride in acetonitrile or tetrahydrofuran;

added to about one part by volume of either N-methylimidazole and pyridine in acetonitrile or tetrahydrofuran;

or t-butylphenoxyacetic anhydride in acetonitrile or tetrahydrofuran.

15. The method of claim 14 wherein said capping reagent comprises about one part by volume of 20% acetic anhydride in acetonitrile mixed with about one part by volume of a solution having 20% N-methylimidazole, 30% pyridine and 50% acetonitrile.

16. The method of claim 1 wherein said mixture comprises dimethylthiuram disulfide, acetic anhydride, acetonitrile, N-methyl imidazole and pyridine.

17. The method of claim 1 wherein said mixture comprises from about 0.05M to 0.2M dimethylthiuram disulfide, about 10% acetic anhydride, about 10% N-methyl imidazole and about 15% pyridine in a suitable solvent.

18. The method of claim 17 wherein said solvent is acetonitrile, toluene, ethyl acetate, tetrahydrofuran, dichloromethane, dichloroethane, dioxane, dimethylacetamide and dimethylformamide.

19. The method of claim 1 wherein said coupling of the 5′

-O-deprotected compound with the activated phosphorus composition is performed in the presence of an activating agent.

20. The method of claim 19 wherein said activating agent is 1-H-tetrazole or 4,5 dicyanoimidazole.

21. The method of claim 1 where said cyclic moiety is morpholino or phthalimido.

22. The method of claim 1 wherein each L₁ and L₂ is C_1-6alkyl.

23. The method of claim 22 wherein each L₁ and L₂ is isopropyl.

24. The method of claim 1 wherein L₁ and L₂ are joined together to form a heterocyclic ring system including the nitrogen atom to which said L₁ and L₂ are attached, wherein said ring system optionally includes at least one additional heteroatom selected from O, N and S.

25. The method of claim 24 wherein said heterocyclic ring system is morpholino.

26. The method of claim 1 wherein each of said sugar substituent groups is, independently, C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, C₂-C₂₀ alkynyl, C₅-C₂₀ aryl, O-alkyl, O-alkenyl, O-alkynyl, O-aryl, O-aralkyl, O-alkylamino, O-alkylaminoalkyl (O-alkyl-N(H)alkyl), O-alkylaminodialkyl (O-alkyl-N-(alkyl)₂), O-alkylalkoxy (O-alkyl-O-alkyl), O-alkyl-(N-imidazole), thiol, S-alkyl, S-alkenyl, S-alkynyl, NH-alkyl, NH-alkenyl, NH-alkynyl, N-dialkyl, S-aryl, NH-aryl, S-aralkyl, NH-aralkyl, N-phthalimido, halogen keto, carboxyl, nitro, nitroso, nitrile, trifluoromethyl, trifluoromethoxy, N-imidazole, azido, hydrazino, hydroxylamino, isocyanato, sulfoxide, sulfone, sulfide, disulfide, silyl, heterocycle, carbocycle, polyamine, polyamide, polyalkylene glycol, or polyether;

or, alternatively, one or more substituent groups 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 one of formula III or IV;

each R₆, R₇, R₈, 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, a chemical functional group 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, R₇ and R₈, together form a phthalimido moiety with the nitrogen atom to which they are attached;

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

R_5′

is T-L,

T is a bond or a linking moiety;

L is a chemical functional group, a conjugate group or a support media;

each R_1′

and R_2′

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(R_3′

)(R_4′

), guanidino or acyl where said acyl is an acid amide or an ester;

or R_1′

and R_2′

, together, are a nitrogen protecting group or are joined in a ring structure that optionally includes an additional heteroatom selected from N and O;

or R_1′

, T and L, together, are a chemical functional group;

each R_3′

and R_4′

is, independently, H, C₁-C₁₀ alkyl, a nitrogen protecting group, or R_3′

and R_4′

, together, are a nitrogen protecting group;

or R_3′

and R_4′

are joined in a ring structure that optionally includes an additional heteroatom selected from N and O;

Z₄ is OX, SX, or N(X)₂;

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

NH)N(H)R_5′

, C(═

O)N(H)R_5′

or OC(═

O)N(H)R_5′

;

R_5′

is H or C₁-C₈ alkyl;

Z₁, Z₂ and Z₃ comprise 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(R_1′

)(R_2′

) OR_1′

, halo, SR_1′

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. The method of claim 1 wherein said X₁ is Pg—

O—

, Pg—

S—

, CH₃—

, CH₃—

O—

, morpholino or R₂R₃N—

where each R₂ and R₃ is, independently, hydrogen or C₁-C₁₀ alkyl.

28. The method of claim 1 wherein said Pg is CH₂CH₂CN, diphenylsilylethyl, δ

-cyanobutenyl, cyano p-xylyl, methyl-N-trifluoroacetyl ethyl or acetoxy phenoxy ethyl.

29. The method of claim 1 wherein said heterocyclic base moiety is adenine, N⁶-benzoyladenine, cytosine, N⁴-benzoylcytosine, 5-methylcytosine, N⁴-benzoyl-5-methylcytosine, thymine, uracil, guanine, N²-isobutyrylguanine or 2-aminoadenine.

30. The method of claim 1 wherein said support media bound nucleoside, nucleotide, oligonucleoside or oligonucleotide is blocked at reactive sites.

31. The method of claim 1 wherein said blocking groups are acid stable.

32. The method of claim 1 wherein said blocking groups are base labile.

33. The method of claim 1 wherein said deprotecting reagent is acidic, neutral or basic.

34. The method of claim 33 wherein said deprotecting reagent is dichloroacetic acid, trichloracetic acid, zinc bromide, AlC₃, TiC₄, (Et)AlCl, (i-Bu)₂AlCl, ceric ammonium nitrate, 1,1,1,3,3,3-hexafluoro-2-propanol or diethyloxomalonate.

35. The method of claim 34 wherein said deprotecting reagent is 2-5% dichloroacetic acid in dichloromethane or dichloroethane.

36. The method of claim 1 wherein said deprotecting reagent is a fluoride moiety.

37. The method of claim 36 wherein said fluoride moiety is BF₃-etherate.

38. The method of claim 1 wherein said oligomeric compound comprises from 5 to about 50 nucleosides.

39. The method of claim 1 wherein said oligomeric compound comprises from 8 to about 30 nucleosides.

40. The method of claim 1 wherein said oligomeric compound comprises from 15 to about 25 nucleosides.