Oxidation process
View Patent ↗A process for the preparation of an oligonucleotide having at least one phosphate internucleotide linkage I provided. The process comprises the steps of: a) forming a nascent oligonucleotide comprising a phosphorus (III) internucleotide linkage; and b) oxidation of the nascent oligonucleotide with aqueous iodine solution thereby to form a phosphorus (V) internucleotide linkage; wherein the oxidation is carried out in the presence of a base, the conjugate acid of said base having a pKa higher than the pKa of the conjugate acid of pyridine. Preferably the base is N-methylimidazole.
1. A process for the preparation of an oligonucleotide having at least one phosphate internucleotide linkage comprising the steps of:
a) forming a nascent oligonucleotide comprising a phosphorus (III) internucleotide linkage; and
b) oxidation of the nascent oligonucleotide with aqueous iodine solution thereby to form a phosphorus (V) internucleotide linkage; wherein the oligonucleotide is prepared using solid phase chemistry, and the oligonucleotide is washed with a washing solution comprising the base immediately before oxidation;
wherein the oxidation is carried out in the presence of a base, the conjugate acid of said base having a pKa higher than the pKa of the conjugate acid of pyridine.
2. A process according to claim 1 , wherein the phosphorus (III) internucleotide linkage is formed by coupling a nucleoside phosphoramidite to a free hydroxyl or thiol group attached to a nascent oligonucleotide.
3. A process according to claim 2 , wherein the nucleoside phosphoramidite is a nucleoside 3′-(betacyanoethyl) phosphoramidite.
4. A process according to claim 1 , wherein the pKa of the conjugate acid of the base employed is 5.4 or greater and less than 12.
5. A process according to claim 4 , wherein the pKa of the conjugate acid of the base employed is from 6.5 to 7.5.
6. A process according to claim 1 , wherein the washing solution comprises up to 50% v/v base.
7. A process according to claim 1 , wherein the washing solution comprises 20% v/v N-methylimidazole and 30% v/v pyridine in acetonitrile.
8. A process according to claim 1 , wherein the base is N-methylimidazole.
9. A process according to claim 1 , wherein the oligonucleotide which is oxidised comprises an acid labile protecting group.
10. A process according to claim 9 , wherein the acid labile protecting group is located at the 5′-position of an oligoribonucleotide or an oligodeoxyribonucleotide.
11. A process according to claim 9 or 10 , wherein the acid labile protecting group is a dimethoxytrityl group.
12. A process according to claim 1 , wherein the oligonucleotide prepared comprises 20% or more phosphate internucleotide linkages.
13. A process according to claim 12 , wherein the oligonucleotide comprises 50% or more phosphate internucleotide linkages.
14. A process according to claim 12 , wherein the oligonucleotide comprises 100% phosphate internucleotide linkages.
15. A process according to claim 6 , wherein the washing solution comprises from 10-30% v/v base.