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US 5,856,464 AGranted
Selective capping solution phase oligonucleotide synthesis
AssigneeLaJolla Pharmaceutical Company
InventorDouglas Alan Livingston
Life of this patent
Estimating the term…
Abstract
Improved methods for solution-phase synthesis of purified short-chain oligonucleotide coupling units, such as dimers, trimers, and tetramers, suitable for use in solution-phase or solid-phase oligonucleotide synthesis are provided.
Claims16
A method of preparing a coupled oligonucleotide, YZ, comprising the steps of:
- (a) providing a plurality of identical first nucleotidic segments, Y, in solution, each of said first segments having at least one reactive functional group, a.sup.(n), wherein n is a positive integer denoting the n-th Y reactive functional group;
- (b) providing a plurality of identical second nucleotidic segments, Z, in solution, each of said second segments having at least two non-identical reactive functional groups, b.sup.(m) and c.sup.(p), wherein m and p are positive integers denoting the m-th Z-b and p-th Z-c reactive functional groups, respectively, and wherein each b.sup.(m) reactive functional group is non-identical to each c.sup.(p) reactive functional group;
- (c) reacting said first and second segments in solution under conditions whereby said at least one Y reactive functional group, a.sup.(n), reacts with said at least two Z reactive functional groups, b.sup.(m) and c.sup.(p), to form a covalent internucleoside linkage joining Y and Z, thereby producing at least one first linkage isomer, YZ-b.sup.(m), which retains at least one b.sup.(m) residual reactive functional group, and at least one non-identical second linkage isomer, YZ-c.sup.(p), which retains at least one c.sup.(p) residual reactive functional group;
- (d) reacting a mixture of said YZ linkage isomers, or derivatives thereof, with a selective capping reagent, in solution, whereby said at least one b.sup.(m) residual reactive functional group of said at least one YZ-b.sup.(m) linkage isomer remains selectively unchanged, and said at least one c.sup.(p) residual reactive functional group of said at least one YZ-c.sup.(p) linkage isomer is selectively capped.
1 more element
A method of preparing a coupled oligonucleotide, YZ, comprising the steps of:
- (a) providing a plurality of identical first nucleotidic segments, Y, in solution, each of said first segments having a reactive functional group, a;
- (b) providing a plurality of identical second nucleotidic segments, Z, in solution, each of said second segments having two non-identical reactive functional groups, b and c;
- (c) reacting said first and second segments in solution under conditions whereby said Y reactive functional group, a, reacts with said Z reactive functional groups, b and c, to form a covalent internucleoside linkage joining Y and Z, thereby producing a first linkage isomer, YZ-b, which retains one b residual reactive functional group, and a non-identical second linkage isomer, YZ-c, which retains one c residual reactive functional group;
- (d) reacting a mixture of said YZ linkage isomers, or derivatives thereof, with a selective capping reagent, in solution, whereby said one b residual reactive functional group of said YZ-b linkage isomer remains selectively unchanged, and said one c residual reactive functional group of said YZ-c linkage isomer is selectively capped.
1 more element
A method of preparing a coupled oligonucleotide, YZ, comprising the steps of:
- (a) providing a plurality of identical first nucleotidic segments, Y, in solution, each of said first segments having a reactive functional group, a;
- (b) providing a plurality of identical second nucleotidic segments, Z, in solution, each of said second segments having two non-identical reactive functional groups, b and c;
- (c) reacting said first and second segments in solution under conditions whereby said Y reactive functional group, a, reacts with said Z reactive functional groups, b and c, to form a covalent internucleoside linkage joining Y and Z, thereby producing a first linkage isomer, YZ-b, which retains one b residual reactive functional group, and a non-identical second linkage isomer, YZ-c, which retains one c residual reactive functional group;
- (d) reacting a mixture of said first linkage isomer, YZ-b, and said second linkage isomer, YZ-c, with an oxidizing reagent in solution, thereby producing a first YZ linkage isomer derivative, YZ'-b, and a second YZ linkage isomer derivative, YZ'-c;
- (e) reacting a mixture of said YZ' linkage isomer derivatives with a selective capping reagent in solution, whereby said one b residual reactive functional group of said YZ'-b linkage isomer derivative remains selectively unchanged, and said one c residual reactive functional group of said YZ'-c linkage isomer derivative is selectively capped.
2 more elements
Dependencies are inferred from the claim text.