4.17.1.5.4. 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 nucleoside 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-boud oligonucleotides with solutions of bases in an organic solvent, for instance, with 50% triethylamine in acetonitrile or 10% diethylamine in acetonitrile.
The solid support-bound oligonucleotides are deprotected using one of the two general approaches.

•  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 by treatment with aqueous ammonium hydroxide. This removes all remaining protection groups from 2'-deoxyoligonucleotides, resulting in a reaction mixture containing the desired product. The fully deprotected product is purified by desalting using ethanol precipitation, or size exclusion chromatography, or reverse-phase HPLC.
• 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. Finally, the product is desalted
• For some applications, additional reporter groups may be attached to an oligonucleotide using a variety of post-synthetic procedures.

4.17.1.5.5. Characterization

As with any other organic compound, it is prudent to characterize synthetic oligonucleotides upon their preparation. In more complex cases oligonucleotides are characterized after their deprotection and after purification. In day-by-day practice, it is sufficient to obtain the molecular mass of an oligonucleotide by recording its mass spectrum. Two methods are currently widely used for characterization of oligonucleotides: electrospray mass spectrometry (ES MS) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF).