Solution phase synthesis of oligonucleotides
AssigneeAvecia Limited
InventorsColin Bernard Reese, Quanlai Song
Life of this patent
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Abstract
A process for the synthesis in solution phase of a phosphorothioate triester is provided. The process comprises the solution phase coupling of an H-phosphonate with an alcohol in the presence of a coupling agent to form an H-phosphonate diester. The H-phosphonate diester is oxidised in situ with a sulfur transfer agent to produce the phosphorothioate triester. Preferably, the H-phosphonate and alcohol are protected nucleosides or oligonucleotides. Oligonucleotide H-phosphonates which can be used in the formation of phosphorothioate triesters are also provided.
Claims20
In an improved process for the synthesis of a deprotected oligonucleotide, said process comprising
- assembling a protected phosphorothioate triester oligonucleotide,
- deprotecting the phosphorothioate triester oligonucleotide to form a deprotected phosphodiester oligonucleotide, deprotected phosphorothioate oligonucleotide, or a deprotected oligonucleotide comprising both phosphodiester and phosphorothioate diester internucleotide linkages,
- the improvement being that said assembling comprises
- solution phase coupling, in the presence of a coupling agent,
- (i) a protected nucleoside or oligonucleotide H-phosphonate comprising a 3′- or 5′H-phosphonate function;
- with
- (ii) a protected nucleoside or oligonucleotide comprising a free 3′- or 5′-hydroxy function;
- to form an H-phosphonate diester and, in situ,
- reacting the H-phosphonate diester with a sulfur transfer agent to produce said phosphorothioate triester.
In an improved process for the synthesis of a deprotected oligonucleotide, said process comprising
- assembling a protected phosphorothioate triester oligonucleotide,
- deprotecting the phosphorothioate triester oligonucleotide to form a deprotected phosphodiester oligonucleotide, deprotected phosphorothioate oligonucleotide, or a deprotected oligonucleotide comprising both phosphodiester and phosphorothioate diester internucleotide linkages,
- the improvement being that said assembling comprises
- solution phase coupling
- (i) a protected nucleoside or oligonucleotide H-phosphonate comprising a 3′-H-phosphonate function;
- with
- (ii) a protected nucleoside or oligonucleotide comprising a free 5′-hydroxy function;
- to form an H-phosphonate diester and, in situ,
- reacting the H-phosphonate diester with a sulfur transfer agent to produce said phosphorothioate triester
- wherein said solution phase coupling is performed in the presence of a diaryl phosphorochloridate represented by formula (ArO)2POCl, wherein Ar represents phenyl, 2-chlorophenyl, 2,4,6-trichlorophenyl, or 2,4,6-tribromophenyl, and
- wherein said sulfur transfer agent is represented by the following formula
- wherein
- L represents a leaving group, and
- A represents an aryl group, a methyl group, a substituted alkyl group or an alkenyl group.
In an improved process for the synthesis of a purified, deprotected oligonucleotide, said process comprising
- assembling a protected phosphorothioate triester oligonucleotide,
- deprotecting the phosphorothioate triester oligonucleotide to form a deprotected phosphodiester oligonucleotide, deprotected phosphorothioate oligonucleotide, or a deprotected oligonucleotide comprising both phosphodiester and phosphorothioate diester internucleotide linkages,
- purifying said deprotected phosphodiester oligonucleotide, deprotected phosphorothioate oligonucleotide, or deprotected oligonucleotide comprising both phosphodiester and phosphorothioate diester internucleotide linkages,
- the improvement being that said assembling comprises
- solution phase coupling
- (i) a protected nucleoside or oligonucleotide H-phosphonate comprising a 3′-H-phosphonate function;
- with
- (ii) a protected nucleoside or oligonucleotide comprising a free 5′-hydroxy function;
- to form an H-phosphonate diester and, in situ,
- reacting the H-phosphonate diester with a sulfur transfer agent to produce said phosphorothioate triester
- wherein said solution phase coupling is performed in the presence of a diaryl phosphorochloridate represented by formula (ArO)2POCl, wherein Ar represents phenyl, 2-chlorophenyl, 2,4,6-trichlorophenyl, or 2,4,6-tribromophenyl, and
- wherein said sulfur transfer agent is represented by the following formula
- wherein
- L represents a leaving group, and
- A represents an aryl group, a methyl group, a substituted alkyl group or an alkenyl group.
Dependencies are inferred from the claim text.