Process for the manufacture of oligonucleotides
Inventors
Wyrzykiewicz, Tadeusz Krzysztof • Cramer, Hagen • Zhu, Huihe • Finn, Kevin James
Assignees
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Publication Number
US-8513404-B2
Publication Date
2013-08-20
Expiration Date
Abstract
A process for manufacturing an oligonucleotide which comprises removing β-eliminating phosphorus-protecting groups, in particular β-cyanoethyl protective groups from a protected oligonucleotide, wherein said removing comprises contacting the protected oligonucleotide with an amine solution in a solvent which preferably does not consist of pyridine, wherein the conjugate acid of the amine has preferably a pKa of greater than 11.5, and wherein the concentration of the amine in the solution is less than 0.5 mole/liters.
Core Innovation
The invention relates to a process for manufacturing an oligonucleotide by removing β-eliminating phosphorus-protecting groups from a protected oligonucleotide attached to a solid support. The removal comprises contacting the protected oligonucleotide in a device having inlet and outlet openings with an amine solution in a solvent which does not consist of pyridine, and the conjugate acid of the amine has a pKa greater than 11.5.
The process uses an amine concentration of less than 0.5 mole/liters and emphasizes avoiding formation of cyanoethyl adducts to nucleobases during deprotection of β-eliminating phosphorus-protecting groups, particularly β-cyanoethyl protective groups. The protected oligonucleotide can be contacted by passing the amine through a column or device via inlet and outlet openings without cleaving it from the support.
Amine options include DBU, with DBN, TMG, and TBD described as alternatives. The process is applicable to manufacturing oligonucleotides of different chemistries, including DNA, RNA, LNA, BNA, ENA, and UNA, and to protected phosphate and phosphorothioate linkages including phosphate, phosphorthioate, phosphordithioate, phosphoramidate, and thiophosphoramidate.
Representative examples and comparative examples use HPLC, LC-MS, IP-HPLC, and RP-HPLC, showing substantially complete deprotection while reducing or avoiding cyanoethyl adduct formation. Specific examples compare outcomes to cases where the amine contacting step is omitted.
Claims Coverage
The independent claims are clm-00001 and clm-00002. Across these claims, the inventive content is focused on contacting a protected oligonucleotide bearing β-eliminating phosphorus-protecting groups with an amine solution under solvent, pKa, and concentration constraints, with one claim further specifying a solid-support format in a device having inlet and outlet openings.
Device contacting of β-eliminating deprotection using non-pyridine amine solution
Removing β-eliminating phosphorus-protecting groups from a protected oligonucleotide attached to a solid support by contacting the protected oligonucleotide in a device having inlet and outlet openings with an amine solution in a solvent which does not consist of pyridine.
High-pKa conjugate acid amine for selective deprotection
Using an amine whose conjugate acid has a pKa greater than 11.5 for contacting the protected oligonucleotide during removal of β-eliminating phosphorus-protecting groups.
Low amine concentration for controlled β-eliminating deprotection
Maintaining the concentration of the amine in the solution at less than 0.5 mole/liters during removal of β-eliminating phosphorus-protecting groups.
β-cyanoethyl selection within β-eliminating phosphorus protection
Characterizing the β-eliminating phosphorus-protecting groups as β-cyanoethyl protective groups for the removal step.
Concentration-constrained non-pyridine amine contacting without device limitation
Removing β-eliminating phosphorus-protecting groups from a protected oligonucleotide by contacting the protected oligonucleotide with an amine solution in a solvent which does not consist of pyridine, where the conjugate acid of the amine has a pKa greater than 11.5 and the concentration of the amine in the solution is from 0.03 to 0.25 mole/liters.
Overall, the claims cover manufacturing an oligonucleotide by removing β-eliminating phosphorus-protecting groups using a non-pyridine solvent with an amine having a conjugate acid pKa greater than 11.5 at a constrained amine concentration, with clm-00001 further specifying contacting in a device with inlet and outlet openings and a solid-support attachment, and dependents narrowing to β-cyanoethyl protective groups and additional condition ranges.
Stated Advantages
Substantially complete deprotection while reducing or avoiding cyanoethyl adduct formation.
Documented Applications
Manufacturing oligonucleotides using deprotection of β-eliminating phosphorus-protecting groups, including β-cyanoethyl protective groups, in solid-phase formats using devices or columns with inlet and outlet openings.
Deprotection for oligonucleotide chemistries including DNA, RNA, LNA, BNA, ENA, and UNA, and for protected phosphate and phosphorothioate linkages including phosphate, phosphorthioate, phosphordithioate, phosphoramidate, and thiophosphoramidate.
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