Solution phase synthesis of oligonucleotides
AssigneeAvecia Limited
InventorsColin Bernard Reese, Quanlai Song
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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 oxidized 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.
Claims24
A process for the preparation of a phosphorothioate triester which comprises the solution phase coupling of an H-phosphonate with an alcohol in the presence of a coupling agent thereby to form an H-phosphonate diester and, in situ, reacting the H-phosphonate diester with a sulfur transfer agent to produce a phosphorothioate triester.
An H-phosphonate having the general chemical formula:
- wherein
- each B independently is a base selected from A, G, T, C or U;
- each Q independently is H or OR′ wherein R′ is alkyl, substituted alkyl, alkenyl or a protecting group;
- each R independently is an aryl, methyl, substituted alkyl or alkenyl group;
- W is H, a protecting group or an H-phosphonate group of formula
- in which
- M+ is a monovalent cation;
- each X independently represent O or S;
- each Y represents S;
- Z is H, a protecting group or an H-phosphonate group of formula
- in which
- M+ is a monovalent cation;
- and
- n is an integer;
- provided that when W is H or a protecting group, that Z is a positive H-phosphonate group, and that when Z is H or a protecting group, that W is an H-phosphonate group.
An H-phosphonate according to either of claim 9 or 10, wherein n is 1, 2 or 3.
A process for the preparation of a deprotected phosphodiester oligonucleotide, deprotected phosphorothioate oligonucleotide or a deprotected oligonucleotide comprising both phosphodiester and phosphorothioate diester internucleotide linkages which comprises:
- (a) solution phase coupling of a protected nucleoside or oligonucleotide H-phosphonate comprising a 3′ or 5′-H-phosphonate function with a protected nucleoside or oligonucleotide comprising a free 3′ or 5′-hydroxy function in the presence of a coupling agent thereby to form an H-phosphonate diester and, in situ, reacting the H-phosphonate diester with a sulfur transfer agent to produce a phosphorothioate triester;
- and
- (b) deprotecting the phosphorothioate triester produced in (a) thereby to form a deprotected phosphodiester oligonucleotide, deprotected phosphorothioate oligonucleotide or a deprotected oligonucleotide comprising both phosphodiester and phosphorothioate diester internucleotide linkages.
Dependencies are inferred from the claim text.