Separation and characterization of diastereoisomers of antisense oligodeoxyribonucleoside phosphorothioates

Diastereoisomers of oligodeoxyribonucleoside phosphorothioates (OPT) up to a tetramer were effectively separated with reversed-phase high-performance liquid chromatography (reversed-phase HPLC) under optimized conditions. The diastereoisomers of OPT resulted in different retention times on the reversed-phase HPLC. From the results, we found that there were certain rules in the elution order of the diastereoisomers. The configurational sequence of the diastereoisomers was determined by digestion with nuclease P1, snake venom phosphodiesterase, and alkaline phosphatase. The diastereoisomers were studied by CD spectroscopy with respect to their conformation in aqueous media. We found that a large variation in the conformation of diastereoisomers exists. Results suggest that much attention should be paid to the diastereoisomerism in antisense molecules having chiral internucleotide linkages such as OPT.