Process for the preparation of phosphorothioate oligonucleotides
AssigneeAvecia Limited
InventorsBen James Mellor, Donald Alfred Wellings, Mark Edward Douglas
Life of this patent
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Abstract
A process for the synthesis of phosphorothioate oligonucleotides is provided which comprises assembling an oligonucleotide bound to a solid support in the presence of acetonitrile; prior to cleaving the oligonucleotide from the solid support removing the acetonitrile; and cleaving the oligonucleotide from the solid support. The process is particularly suited to the large scale synthesis of nucleotides. The acetonitrile may be removed from the solid support by one or both of drying and by washing with solvents. Preferred washing solvents comprise trialkylamines.
Claims18
A process for the large-scale synthesis of phosphorothioate oligonucleotides which comprises:
- a) assembling an oligonucleotide bound to a solid support in the presence of acetonitrile;
- and
- b) cleaving the oligonucleotide from the solid support;
- characterised in that the concentration of acetonitrile is reduced to less than 10% by weight of the oligonucleotide plus solid support prior to the cleavage of the oligonucleotide from the solid support.
A process for the synthesis of phosphorothioate oligonucleotides which comprises:
- a) assembling an oligonucleotide bound to a solid support in the presence of acetonitrile;
- b) prior to cleaving the oligonucleotide from the solid support, washing the oligonucleotide bound to a solid support with a washing regime employing one or more solvent washes;
- and
- c) cleaving the oligonucleotide from the solid support;
- characterised in that the final wash of the washing regime employs a solvent other than acetonitrile or dioxane.
A process for the synthesis of phosphorothioate oligonucleotides which comprises:
- a) assembling an oligonucleotide bound to a solid support in the presence of acetonitrile;
- b) prior to cleaving the oligonucleotide from the solid support, washing the oligonucleotide bound to a solid support with a washing regime employing one or more solvent washes;
- and
- c) cleaving the oligonucleotide from the solid support;
- characterised in that the final wash of the washing regime employs as solvent wash a solution comprising a trialkylamine.
A process for the synthesis of phosphorothioate oligonucleotides comprising assembling an oligonucleotide bound to a solid support in the presence of acetonitrile;
- air drying the supported oligonucleotide;
- contacting the dried supported oligonucleotide with a trialkylamine, preferably triethylamine, for sufficient time to deprotect the oligonucleotide, and subsequently cleaving the oligonucleotide from the solid support.
A process according to any preceding claim, wherein the acetonitrile concentration is reduced to less than 5%, and especially less than about 1%, by weight of the oligonucleotide plus solid support.
A process according to any preceding claim which is operated at or above a batch size of 10 mmol of oligonucleotide.
A process for the preparation of an oligonucleotide which comprises coupling a nucleoside or oligonucleotide phosphoramidite with a nucleoside or oligonucleotide comprising a free hydroxy group supported on a solid support to form an oligonucleotide phosphite triester, characterised in that the solid support is a microporous support.
A process according to any preceding claim, wherein the solid support is an amine-functionalised support derived from supports prepared by copolymerisation of acryloyl-sarcosine methyl ester, N,N-dimethylacrylamide and bis-acryloylethylenediamine.
A process according to any preceding claim, wherein the oligonucleotide is bound to the solid support via a cleavable linker selected from the group consisting of urethane, oxalyl, succinyl, and amino-derived linkers.
A process according to any preceding claim wherein the oligonucleotide is cleaved from the solid support by contact with a cleaving reagent.
A process according to any preceding claim which is employed to prepare oligonucleotides having from 1 to 100 internucleoside linkages.
A process according to any preceding claim which is employed to prepare compounds wherein at least 50% of the internucleoside linkages are phosphorothioated.
Dependencies are inferred from the claim text.