Factors Influencing Diastereomer Separations in Oligonucleotide Analysis

Significance of the Topic

This study addresses the challenge of resolving diastereomer mixtures generated by phosphorothioate (PS) modifications in synthetic oligonucleotides. Effective separation of these diastereomers is critical for accurate characterization, quality control, and downstream applications in therapeutic and research settings.

Goals and Study Overview

The primary objective was to evaluate how the hydrophobicity and concentration of ion-pairing (IP) agents affect the chromatographic resolution of diastereomer-containing oligonucleotides. Three single-stranded RNA sequences with zero, one, and two PS linkages were analyzed to compare separation performance under different IP conditions.

Methodology and Instrumentation

• Ion-pairing agents: weak triethylammonium acetate (TEAA) and strong hexylammonium acetate (HAA).
• Concentrations tested: 15 mM and 100 mM, all adjusted to pH 7.0.
• Chromatographic system: Waters ACQUITY H-Class Bio UPLC.
• Column: Waters ACQUITY UPLC OST BEH C18, 130 Å, 1.7 µm, 2.1 mm × 50 mm.
• Mobile phases: A = water with IP agent; B = 50:50 water/acetonitrile with IP agent; 15-minute linear gradient.

Main Results and Discussion

• At 15 mM TEAA (weak IP), oligonucleotides bearing one or two PS chiral centers exhibited partial diastereomer resolution (Rs ≈ 1.84), whereas those without PS modifications coeluted.
• Under the same conditions with 15 mM HAA (strong IP), all sequences coeluted, indicating suppression of hydrophobic contributions essential for diastereomer separation.
• Increasing TEAA to 100 mM further reduced resolution (Rs ≈ 0.72) but did not entirely eliminate partial diastereomer separation.
• Raising HAA concentration to 100 mM had minimal impact on separation, confirming dominance of ionic retention and lack of stereochemical discrimination.

Benefits and Practical Applications

These findings enable analysts to tailor IP-RPLC conditions for desired diastereomer resolution. By selecting an appropriately hydrophobic IP agent at optimized concentration, method developers can balance separation selectivity against run time and robustness. This strategy enhances quality control of PS-modified oligonucleotides and supports regulatory compliance.

Future Trends and Opportunities

Emerging directions include screening novel ion-pairing reagents with intermediate hydrophobicity, coupling IP-RPLC with high-resolution mass spectrometry for direct diastereomer identification, and developing advanced stationary phases designed for stereochemical selectivity. Automated method scouting and machine-learning–driven optimization may further improve throughput and reproducibility.

Conclusion

The type and concentration of ion-pairing agent critically influence diastereomeric separation in PS-modified oligonucleotide analysis. Weak IP agents at moderate concentration offer the best compromise for resolving stereoisomers, while strong agents or high concentrations prioritize ionic retention and suppress diastereomeric discrimination. These insights guide robust method development for analytical and preparative workflows.

References

• Stec W.; Zon G. Reversed-phase high-performance liquid chromatographic separation of diastereomeric phosphorothioate analogues of oligodeoxyribonucleotides and other backbone-modified congeners of DNA. J. Chromatogr. 1985, 326, 263.
• Gilar M. et al. Characterization of therapeutic oligonucleotides using liquid chromatography with on-line mass spectrometry detection. Oligonucleotides 2003, 13, 229.