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) composed of eight chemically modified nucleotide units, with a phosphorothioate (PS) internucleotide linkage at each of its two termini. Due to the presence of two P-chiral PS linkages and the lack of stereocontrol during conventional solid-phase synthesis, the 8-mer ON exists as a mixture of four diastereomers. Using NMR spectra acquired at both 1 GHz and 600 MHz, we successfully assigned the ¹H, ¹³C, ³¹P and ¹⁹F signals of mtON, confirmed its primary structure, and identified and quantified the four diastereomers.