Investigation of the ‘n–1’ impurity in phosphorothioate oligodeoxynucleotides synthesized by the solid-phase β-cyanoethyl phosphoramidite method using stepwise sulfurization

Etectrospray ionization mass spectrometry (ESMMS) of reversed-phase HPLC-purrfted phosphorothioate oligodeoxynucleotides (S-ODNs), and the single- (‘n – 1’ ) and double-nucleotlde 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 ollgomeric impurities. The ‘n -’ 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 detritylatlon, 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 an Ionexchange chromatography allowed for independent confirmation of its heterogeneity and quantitatlon 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-ofthe- art solid-phase synthesis and reversed-phase HPLC purification methods.