Investigation of the 'n-1' impurity in phosphorothioate oligodeoxynucleotides synthesized by the solid-phase beta-cyanoethyl phosphoramidite method using stepwise sulfurization.

Electrospray ionization mass spectrometry (ESI-MS) of reversed-phase HPLC-purified phosphorothioate oligodeoxynucleotides (S-ODNs), and the single-('n - 1') and double-nucleotide deletion ('n - 2') impurities subsequently isolated from them by preparative polyacrylamide gel electrophoresis (PAGE), has provided direct analytical data for the identification of both S-ODN products and their major oligomeric impurities. The 'n - 1' impurity seen by PAGE consists of a mixture of all possible single deletion sequences relative to the parent S-ODN (n-mer) and results from repetitive, though minor, imperfections in the synthesis cycle, such as incomplete detritylation, or incomplete coupling followed by incomplete capping or incomplete sulfurization. Therefore each possible 'n - 1', 'n - 2', and other short-mer sequence is present only in very low abundance. The conversion of the gel-isolated 'n - 1' impurity from phosphorothioate to phosphodiester followed by base composition-dependent anion-exchange chromatography allowed for independent confirmation of its heterogeneity and quantitation of its various components. ESI-MS of both S-ODN products and their gel-isolated impurities allowed for this first molecular identification of 'n - 1', 'n - 2' and other oligomeric impurities in S-ODNs obtained from state-of-the-art solid-phase synthesis and reversed-phase HPLC purification methods.