Detecting the Major\nDegradation Products of Phosphorothioate\nOligonucleotides by Chemical Derivatization and Weak Anion Exchange\nChromatography

Novel polar cysteine analogues have been synthesized\nfor the derivatization\nof oligonucleotide depurination impurities that may be formed under\nacidic conditions. Depurination impurities belong to a group that\nincludes deamination and phosphate diester impurities, which are similar\nin chemical structure to each other and the parent oligonucleotide,\nand thus coelute by most chromatographic separation methods. The polar\ncysteine analogues react with depurination impurities and enable their\ncomplete separation from the parent oligonucleotide by weak anion\nexchange (WAX) chromatography. Optimized conditions for the derivatization\nreaction and the WAX analysis are presented. The ability of the WAX\nmethod to chromatographically separate deamination and phosphate diester\nimpurities is also demonstrated, and therefore, the combination of\nchemical derivatization and WAX chromatography permits detection and\nquantification of the three major degradation products of phosphorothioate\n(PS) oligonucleotides.