NMR characterization of the diastereomeric composition of a model therapeutic oligonucleotide

There is an increasing demand from the pharmaceutical industry and regulatory agencies for analytical methods capable of accurately characterizing therapeutic oligonucleotides (ONs). In this work, we present the full NMR characterization of a model therapeutic ON (mtON) containing eight chemically modified nucleotide units and a phosphorothioate (PS) internucleotide linkage at each of its two extremities. Because of the presence of two P-chiral PS linkages and the lack of stereocontrol during conventional solid-phase synthesis, our 8-mer ON consists of a mixture of four diastereomers. NMR spectra acquired at 1 GHz and 600 MHz allowed the full assignment of the 1H, 13C, 31P and 19F signals of mtON, the confirmation of its primary structure, as well as the identification, and quantification of all the diastereomers.