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 micro mol reaction column, thus facilitating large scale syntheses required for mechanistic studies.

NMR and UV studies of 3'-S-phosphorothiolate modified DNA in a DNA : RNA hybrid dodecamer duplex; implications for antisense drug design.

High-resolution NMR spectroscopy has been used to establish the conformational consequences of the introduction of a single 3[prime or minute]-S-phosphorothiolate link in the DNA strand of a DNA : RNA hybrid. These systems are of interest as potential antisense therapeutic agents. Previous studies on similarly modified dinucleotides have shown that the conformation of the sugar to which the sulfur is attached shifts to the north (C(3[prime or minute])-endo/C(2[prime or minute])-exo). Comparisons made between NOESY cross-peak intensities, and coupling constants from PE-COSY spectra, for both non-modified and modified duplexes confirm that this conformational shift is also present in the double helical oligonucleotide system. In addition it is noted that in both the dinucleotides and the modified duplex, the conformation of the sugar ring 3[prime or minute] to the site of modification is also shifted to the north. That this pattern is observed in the small monomeric system as well as the larger double helix is suggestive of some pre-ordering of the sequences. The conclusion is supported by consideration of the (1)H chemical shifts of the heterocyclic bases near the site of the modification. The enhanced stability that these conformational changes should bring was confirmed by UV thermal melting studies. Subsequently a series of singly and doubly 3[prime or minute]-S-phosphorothiolate-modified duplexes were investigated by UV. The results are indicative of an additive effect of the modification with thermodynamic benefit being derived from alternate spacing of two modified linkers.

Probing the effect of a 3'-S-phosphorothiolate link on the conformation of a DNA:RNA hybrid; implications for antisense drug design.

The effects of a single 3'-S-phosphorothiolate link in the DNA strand of a DNA:RNA dodecamer duplex is described; the sulfur induces a conformational shift in the (attached) sugar pucker, as shown by 1H NMR studies, and increases the thermal stability of the duplex compared to the non-modified system.