Oligonucleotide Synthesis - Post-synthetic Processing

Post-synthetic Processing

After the completion of the chain assembly, the solid support-bound oligonucleotide is fully protected:

• The 5'-terminal 5'-hydroxy group is protected with DMT group;
• The internucleosidic phosphate or phosphorothioate moieties are protected with 2-cyanoethyl groups;
• The exocyclic amino groups in all nucleic bases except for T and U are protected with acyl protecting groups.

To furnish a functional oligonucleotide, all the protecting groups have to be removed. The N-acyl base protection and the 2-cyanoethyl phosphate protection may be, and is often removed simultaneously by treatment with inorganic bases or amines. However, the applicability of this method is limited by the fact that the cleavage of 2-cyanoethyl phosphate protection gives rise to acrylonitrile as a side product. Under the strong basic conditions required for the removal of N-acyl protection, acrylonitrile is capable of alkylation of nucleic bases, primarily, at the N3-position of thymine and uracil residues to give the respective N3-(2-cyanoethyl) adducts via Michael reaction. The formation of these side products may be avoided by treating the solid support-bound oligonucleotides with solutions of bases in an organic solvent, for instance, with 50% triethylamine in acetonitrile or 10% diethylamine in acetonitrile. This treatment is strongly recommended for medium- and large scale preparations and is optional for syntheses on small scale where the concentration of acrylonitrile generated in the deprotection mixture is low.

Regardless of whether the phosphate protecting groups were removed first, the solid support-bound oligonucleotides are deprotected using one of the two general approaches.

• (1) Most often, 5'-DMT group is removed at the end of the oligonucleotide chain assembly. The oligonucleotides are then released from the solid phase and deprotected (base and phosphate) by treatment with aqueous ammonium hydroxide, aqueous methylamine, their mixtures, gaseous ammonia or methylamine or, less commonly, solutions of other primary amines or alkalies at ambient or elevated temperature. This removes all remaining protection groups from 2'-deoxyoligonucleotides, resulting in a reaction mixture containing the desired product. If the oligonucleotide contains any 2'-O-protected ribonucleotide residues, the deprotection protocol includes the second step where the 2'-O-protecting silyl groups are removed by treatment with fluoride ion by various methods. The fully deprotected product is used as is, or the desired oligonucleotide can be purified by a number of methods. Most commonly, the crude product is desalted using ethanol precipitation, size exclusion chromatography, or reverse-phase HPLC. To eliminate unwanted truncation products, the oligonucleotides can be purified via polyacrylamide gel electrophoresis or anion-exchange HPLC followed by desalting.
• (2) The second approach is only used when the intended method of purification is reverse-phase HPLC. In this case, the 5'-terminal DMT group that serves as a hydrophobic handle for purification is kept on at the end of the synthesis. The oligonucleotide is deprotected under basic conditions as described above and, upon evaporation, is purified by reverse-phase HPLC. The collected material is then detritylated under aqueous acidic conditions. On small scale (less than 0.01–0.02 mmol), the treatment with 80% aqueous acetic acid for 15–30 min at room temperature is often used followed by evaporation of the reaction mixture to dryness in vacuo. Finally, the product is desalted as described above.
• For some applications, additional reporter groups may be attached to an oligonucleotide using a variety of post-synthetic procedures.