Oligonucleotide synthesis with alternative solvents
AssigneeIsis Pharmaceuticals, Inc.
InventorsMax N. Moore, Mark Andrade, Recaldo Carty, Anthony Scozzari, Achim Krotz
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Abstract
The invention provides for methods of manufacturing an oligonucleotide comprising a pentavalent phosphate triester. In particular, the method comprises providing a 5′ blocked-nucleoside, deblocking the 5′ blocked-nucleoside to form a 5′ OH-nucleoside, coupling the 5′ OH-nucleoside with a phosphoramidite to form and oligonucleotide comprising a trivalent phosphite triester; and oxidizing the oligonucleotide comprising a trivalent phosphite triester to the oligonucleotide comprising a pentavalent phosphate triester. In some embodiments, the wash between any of the steps above is with at least one solvent wash comprising a toluene.
Claims27
A method of manufacturing an oligonucleotide comprising a pentavalent phosphate triester, said method comprising:
- (a) providing a 5′ blocked-nucleoside;
- (b) deblocking the 5′ blocked-nucleoside to form a 5′ OH-nucleoside;
- (c) coupling the 5′ OH-nucleoside with a phosphoramidite of formula (IV)
- wherein Y is 0, S, CH2, CHF, CF2 or —CH═CH—;
- bg is a 5′-blocking group;
- R′2 is H, OZ, a 2′-substituent, or together with R′4 forms a bridge;
- R′4 is H, OZ, a 2′-substituent, or together with R′2 forma a bridge;
- Z is H or a removable protecting group;
- Bx is a nucleobase;
- pg is a phosphorus protecting group;
- each G is 0 or S;
- L is an amine leaving group;
- G1 is 0 or S;
- and
- q′ is 0 or 1;
- to form an oligonucleotide comprising a trivalent phosphite triester;
- and
- (d) oxidizing the oligonucleotide comprising a trivalent phosphite triester to form the oligonucleotide comprising a pentavalent phosphate triester;
- wherein at least a wash between any of the steps above is with a solvent wash comprising a toluene, pyridine, lutidine, hexane, cyclohexane, cyclohexene, a halogenated benzene, alkylated benzenes, a haloalkylbenzene, acetone, ethylacetate, methanol, ethanol, phenol, cyclic ethers, acyclic ethers, halogenated alkanes or mixtures thereof.
A method of manufacturing a compound of Formula I:
- wherein Y is O, S, CH2, CHF, CF2 or —CH═CH—;
- bg is a 5′-blocking group;
- n is a positive integer;
- each R′2 is, independently, H, OZ, a 2′-substituent, or together with R′4 forms a bridge;
- each R′4 is, independently, H, OZ, a 2′-substituent, or together with R′4 forms a bridge;
- each R′4 is, protecting group;
- each Bx is independently a nucleobase;
- each pg is independently a phosphorus protecting group;
- each G is 0 or S;
- each G, is 0 or S;
- each q′ is independently 0 or 1;
- and
- SS is a solid support;
- said process comprising:
- (a) contacting a compound of Formula II:
- with a deblocking agent to produce a compound of Formula III:
- (b) contacting the compound of Formula III with a first solvent wash;
- (c) contacting the compound of Formula III with a compound of Formula IV:
- wherein L is a leaving group, to form a compound of Formula V:
- (d) contacting the compound of Formula V with a second solvent wash;
- and
- (e) contacting the compound of Formula V with an oxidation reagent to form the compound of Formula I;
- wherein at least one of the first and second solvent wash comprises a toluene, pyridine, lutidine, hexane, cyclohexane, cyclohexene, a halogenated benzene, alkylated benzenes, a haloalkylbenzene, acetone, ethylacetate, methanol, ethanol, phenol, cyclic ethers, acyclic ethers, halogenated alkanes or mixtures thereof.
Dependencies are inferred from the claim text.