SYNTHESIS OF OLIGODEOXYNUCLEOTIDES CONTAINING 2-THIOPYRIMIDINE RESIDUES - A NEW PROTECTION SCHEME

A method is described for the incorporation of 2'-deoxy-2-thiouridine (d(S2)U) and 2'-deoxy-2-thiothymidine (d(S2)T) into oligodeoxynucleotides at predetermined positions. This requires N-3 orO(4)-acylation of d(S2)U and d(S2)T with toluoyl chloride. These base-protected thiopyrimidines are completely stable toward the aqueous iodine oxidation reagent used in the phosphoramidite DNA synthesis method. The toluoyl protecting group is removed during the standard post-synthetic ammonia treatment. This novel protection strategy allows d(S2)U and d(S2)T to be efficiently incorporated into oligodeoxynucleotides at predetermined sites without the usual problem of desulfurization and decomposition. Several 14-mers containing the Eco-Rl recognition site (dGGCGGAAXXCCGCC and dGGCGGAAXXCGCGG, where X represents dT, d(S2)U or d(S2)T) have been synthesized and characterized by base composition, thermal denaturation, CD spectroscopy and endonuclease substrate activity.