Diastereomer separation of phosphorothioate oligonucleotides via conformationally stabilizing ion pairs in reversed-phase liquid chromatography

Phosphorothioate (PS) modification is usually incorporated in oligonucleotide structures to improve nuclease resistance and biological activity. In another hand, PS modification introduces chiral centers and formation of diastereomers, leading to more challenging characterization. Several approaches have been reported to separate diastereomers, including ion-pairing reversed-phase liquid chromatography (IP-RPLC), hydrophilic interaction chromatography (HILIC), and anion exchange chromatography (AEX). The separation mechanisms are based on differences of hydrophobicity, charge and conformation between diastereomers. In this study, we showcase the power of alkyldiamines (ADs) as a new class of ion pairing reagents for oligonucleotide diastereomer separation using two FDA-approved small interfering RNA (siRNA) compounds, Lumasiran and Vutrisiran, and single-stranded sequences derived from the antisense strand of Lumasiran, as model compounds. The alkyldiamine ion-paring (AD-IP) systems showed significantly improved diastereomer separation compared with the widely used triethylamine acetate (TEAA) IP systems, varied selectivity for positional isomers and robust compatibility with denaturing conditions (e.g., high column temperature). This enhanced efficiency may be attributed to conformationally stabilizing effect of the ADs, confirmed by melting temperature (T_m) measurements. To best of our knowledge, this is the first time that ion pairing reagents with conformation-stabilizing effect are used for both single- and double-stranded oligonucleotide analysis. This work enriches the analytical toolkit for oligonucleotide characterization and may enable batch-to-batch diastereomer distribution monitoring in synthetic phosphorothioate oligonucleotides.