Synthetic oligonucleotides are extremely promising candidates for biopharmaceuticals in a wide range of diseases. This is why nucleic acid therapeutics such as silencing RNA (siRNA), messenger RNA (mRNA) and antisense oligonucleotides (ASOs) are gaining more and more attention in current research. Phosphoramidites are the essential part of the chemical synthesis of oligonu-cleotides, short fragments of nucleotides and analogues. To prevent any side reactions on residual reactive sites such as hydroxyl and amino groups during oligonucleotide synthesis, these groups need to be protected.
Therefore, the highly reactive native nucleotides are modified with four different protecting groups. Benzoyl (Bz) or isobutyryl (iBu) groups protect the amino group of the base. This is only required for the nucleobases adenine, guanine and cyto-sine as they all have primary amino groups which other-wise would attach to another nucleotide. For thymine, this protecting group is not required since it only has a less reactive secondary amine group. The 5’-hydroxyl group of the sugar moiety is protected by a dimethoxytrityl (DMT) group. Inhibiting side reactions at the 3’-hydroxyl group of the sugar moiety is performed by two groups: a diiso-propylamino and a 2-cyanoethyl (CE) group are attached to the phosphate atom. Figure 1 shows the protecting groups using the example of an adenosine based phos-phoramidite.
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