Analytical techniques for characterizing diastereomers of phosphorothioated oligonucleotides

•Overviewed configuration, properties, synthesis of PS oligonucleotide diastereomers.•Reviewed analytical methods for PS oligonucleotide diastereomer characterization.•Included LC (IP-RP, AEX, MMC, hybrid), CE, spectroscopy, and other methods.•Discussed factors influencing the IP-RP diastereomer selectivity and retention. Oligonucleotides have emerged as powerful therapeutics for treating diverse diseases. To fully unlock the therapeutic potential of oligonucleotides, there is still a great need to further improve their drug-like properties. Numerous chemical modifications have been explored to achieve this goal, with phosphorothioation being one of the most widely used strategies. However, phosphorothioate modification produces diastereomers that are reported to have different properties and performances, demanding detailed characterization of these diastereomers. Here we provide an overview of phosphorothioated oligonucleotide diastereomers, covering their origin and configurations, physicochemical and pharmacological properties, and stereo-selective chemical synthesis, followed by a summary of currently available analytical techniques for characterizing these diastereomers, with a focus on liquid chromatography-based approaches, including ion-pair reversed-phase liquid chromatography, anion exchange chromatography, mixed-mode chromatography, and hybrid approaches. Non-chromatographic techniques, such as capillary electrophoresis, spectroscopy and other methods, are also being reviewed.