OPTIMIZED LARGE-SCALE SYNTHESIS OF PHOSPHOROTHIOATE OLIGONUCLEOTIDES ON PS-PEG SUPPORTS

Sulphurization is a crucial step during synthesis of phosphorothioate oligonucleotides. Insufficient reaction leads to inhomogeneous product s with phosphodiester defects and subsequently to destabilization of t he oligomers in biological media. To achieve a maximum extent of sulph ur incorporation, various sulphurizing agents have been investigated. Solely, the use of Beaucage reagent provided satisfactory results on P S-PEG supports. Based on our investigations in small scale synthesis ( 1 mu mol) with continuous-flow technique, upscaling to the 0.1-0.25 mm olar range has been achieved using a peptide synthesizer. The synthese s were performed in batch mode with standard phosphoramidite chemistry . Additionally, large scale synthesis of a phosphodiester oligonucleot ide has been carried out on PS-PEG with optimized protocols and compar ed to small scale synthesis on different supports. Products were analy sed by P-31 NMR, capillary gel electrophoresis and electrospray mass s pectrometry. An extent of sulphurization of 99% and coupling effiencie s of more than 99% were obtained and the products proved to have simil ar purity compared to small scale syntheses on CPG.