Antisense oligonucleotide alkylphosphonothioates and arylphospohonothioates
AssigneeHybridon, Inc.
InventorsA. Padmapriya, Sudhir Agrawal
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Abstract
The invention provides improved oligonucleotides having greater resistance to nucleolytic degradation by virtue of having alkylphosphonothioate or arylphosphonothioate internucleotide linkages.
Claims20
An antisense oligonucleotide having from about 8 to about 50 nucleotides, wherein three or more, but not all, nucleosides are connected by methylphosphonothioate linkages, wherein the sulfur of the methylphosphonothioate linkage is a non-bridging atom.
An antisense oligonucleotide having from about 8 to about 50 nucleotides, wherein two or more, but not all, nucleosides are connected by an alkylphosphonothioate or arylphosphonothioate linkage wherein the sulfur of the alkylphosphonothioate or arylphosphonothioate linkage is a non-bridging atom.
An antisense oligonucleotide having the nucleotide sequence 5'-ACACCCAATTCTGAAAATGG-3', wherein the two most 3' internucleotide linkages are methylphosphonothioate linkages, and wherein all other internucleotide linkages are phosphorothioate linkages.
An antisense oligonucleotide having the nucleotide sequence 5'-ACACCCAATTCTGAAAATGG-3', wherein the three most 3' internucleotide linkages are methylphosphonothioate linkages, and wherein all other internucleotide linkages are phosphorothioate linkages.
A method of incorporating into an antisense oligonucleotide an alkyl- or aryl-phosphonothioate internucleoside linkage, the method comprising the steps of (a) coupling together two nucleosides via an alkyl- or aryl-phosphonite linkage;
- and (b) oxidatively thiolating the alkyl- or aryl-phosphonite linkage with 3H-1,2-benzodithiol-3-one-1,1-dioxide to produce an alkyl or aryl-phosphonothioate linkage, wherein said coupling and thiolating steps are conducted on a solid support and the sulfur of the alkyl or aryl-phosphonothioate linkage is a non-bridging atom.
A method of making an antisense oligonucleotide having one or more alkyl- or aryl-phosphonothioate linkages at its 3' end, the method comprising the steps of:
- (a) coupling together two nucleosides via an alkyl- or aryl-phosphonite linkage;
- (b) oxidatively thiolating the alkyl- or aryl-phosphonite linkage with 3H-1,2-benzodithiol-3-one-1,1-dioxide to produce an alkyl- or aryl-phosphonothioate linkage;
- (c) repeating steps (a) and (b) for each additional alkyl- or aryl-phosphonothioate linkage to be added;
- and
- (d) sequentially adding as many nucleotides as desired in additional coupling steps, wherein said coupling and thiolating steps are conducted on a solid support and the sulfur of the alkyl or aryl-phosphonothioate linkage is a non-bridging atom.
A method of making an antisense oligonucleotide having one or more alkyl- or aryl-phosphonothioate linkages at its 5' end, the method comprising the steps of:
- (a) sequentially coupling together as many nucleotides as desired;
- (b) sequentially adding two nucleosides coupled together via an alkyl- or aryl-phosphonite linkage;
- (c) oxidatively thiolating the alkyl- or aryl-phosphonite linkage with 3-H-1,2-benzodithiol-3-one-1,1-dioxide to produce a alkyl- or aryl-phosphonothioate linkage;
- and
- (d) repeating steps (b) and (c) for each additional alkyl- or aryl-phosphonothioate linkage to be added,
- wherein said coupling and thiolating steps are conducted on a solid support and the sulfur of the alkyl or aryl-phosphonothioate linkage is a non-bridging atom.
A method of making an antisense oligonucleotide having one or more alkyl- or aryl-phosphonothioate linkages at its 5' and 3' ends, the method comprising the steps of:
- (a) coupling together two nucleosides via a alkyl- or aryl-phosphonite linkage;
- (b) oxidatively thiolating the alkyl- or aryl-phosphonite linkage with 3H-1,2-benzodithiol-3-one-1,1-dioxide to produce a alkyl- or aryl-phosphonothioate linkage;
- (c) repeating steps (a) and (b) for each additional alkyl- or aryl-phosphonothioate linkage to be added;
- and
- (d) sequentially adding as many nucleotides as desired in additional coupling steps;
- (e) sequentially adding two nucleosides coupled together via an alkyl- or aryl-phosphonite linkage;
- (f) oxidatively thiolating the alkyl- or aryl-phosphite linkage with 3H-1,2-benzodithiol-3-one-1,1-dioxide to produce an alkyl- or aryl-phosphonothioate linkage;
- and
- (g) repeating steps (e) and (f) for each additional alkyl- or aryl-phosphonothioate to be added,
- wherein said coupling and thiolating steps are conducted on a solid support and the sulfur of the alkyl or aryl-phosphonothioate linkage is a non-bridging atom.
Dependencies are inferred from the claim text.