The use of 31P NMR and principal components analysis to determine sameness or differences between diastereomeric composition of phosphorothioate oligonucleotides

Abstract

Synthetic oligonucleotides often contain chemically modified backbones to prevent degradation and improve bioactivity. Phosphorothioate (PS) linkage in place of phosphodiester is the most used one. Substituting an oxygen for a sulfur atom introduces a stereogenic element on each PS, which leads to an exponentially large number of diastereomers, if the synthetic process is not stereoselective. Variable conditions during synthesis may lead to unpredictable variation in the manifold of diastereomers, with heavy fallouts in bioactivity. We developed an analytical protocol based on ³¹P-NMR followed by principal components analysis (PCA) to fingerprint each manifold. We assess the precision of this method by investigating repeatability and reproducibility. By working on two instruments at different magnetic fields, we also assess the problem of robustness and ultimately the possibility of exchanging data between laboratories.