Oligonucleotide Drug Synthesis and Analysis of Impurities

At present, most oligonucleic acid drugs are synthesized by solid-phase phosphoramide chemistry. The chemical synthesis is carried out in the direction of 3 '-5'.

A commonly used solid-phase carrier is a controlled microporous glass bead (CPG), which is covalently bound to the 3 '-OH of the initial nucleotide ribose by linker, while the 2' -OH of the ribose is protected with a protective agent such as tert-butyldimethylsilyl (TBDMS), and the 5 '-OH is protected with dimethoxytriphenylmethyl (DMT). In addition, due to the presence of primary amino groups in adenine, guanine, and cytosine, protection with acyl reagents (e.g. benzoyl) is also required.

Each cycle of solid-phase synthesis mainly involves four steps: deprotection, coupling, oxidation, and capping. Impurities produced in the synthesis (including the final deprotection) are of the following types:

Shorter sequences (N-X), such as N-1, N-2;

Longer sequences (N + X, e.g. N + G, N + A)

Phosphorothioate bond (PS) produces diastereomers and oxidizes to produce PO impurities;

Impurities caused by deamination of cytosine and 5-methylcytosine;

Other impurities, such as base depurine impurities, 2 '-5' linking impurities, sequence isomers, etc.;