Synthesis of phosphorothioamidites derived from 3'-thio-3'-deoxythymidine and 3'-thio-2',3'-dideoxycytidine and the automated synthesis of oligodeoxynucleotides containing a 3'-S-phosphorothiolate linkage

The synthesis of N4-benzoyl-5′-O-dimethoxytrityl-2′,3′-dideoxy-3′-thiocytidine and its phosphorothioamidite is described for the first time, together with a shortened procedure for the preparation of 5′-O-dimethoxytrityl-3′-deoxy-3′-thiothymidine and its corresponding phosphorothioamidite. The first fully automated coupling procedure for the incorporation of a phosphorothioamidite into a synthetic oligodeoxynucleotide has been developed, which conveniently uses routine activators and reagents. Coupling yields using this protocol were in the range of 85–90% and good yields of singularly modified oligonucleotides were obtained. Coupling yields were also equally good when performed on either a 0.2 or 1 µmol reaction column, thus facilitating large scale syntheses required for mechanistic studies.