Stereochemistry of oligo-based therapeutics

Oligonucleotide-based therapeutics represent a well-established class of treatments designed to modulate gene expression. Over the past three decades, intensive efforts have led to the development of a wide range of chemical modifications, among these, the substitution of one of the non-bridging oxygen atoms in the phosphodiester linkage with sulfur yields the phosphorothioate (PS) modification. The substitution of one of the non-bridging oxygen atoms with sulfur not only enhances nuclease resistance and improves the overall pharmacokinetic properties of the drug, but also introduces a stereogenic center, giving rise to two possible configurations, Rp and Sp, at every internucleotide linkage. Consequently, an oligonucleotide bearing n PS linkages, if not synthesized in a stereocontrolled fashion, exists as a mixture of 2n diastereoisomers, whose distribution can significantly influence the molecule’s biological activity. Despite the recognized importance and widespread use of the PS modification, the chirality at the phosphorus center remains a largely overlooked aspect in the context of therapeutic oligonucleotides. This is mainly due to the intrinsic complexity arising from the presence of multiple stereogenic centers in the backbone. For this reason, in this work, the stereochemistry of phosphorothioate oligonucleotides is investigated using an approach that combines spectroscopic (Section 1), synthetic (Section 2), and computational methods (Section 3).