Synthesis and characterization of oligonucleotides containing 2'-deoxy-6-thioguanosin

6-Thioguanine is an anticancer drug that has been used for over 30 years in the treatment of human cancer, especially leukemias. Work conducted over the past few decades has shown that the incorporation of 6-thioguanine into nucleic acids, especially DNA, appears to be the mechanism by which 6-thioguanine exerts its anticancer actions. The development of synthetic methodology that allows the incorporation of 6-thioguanine, as 2'-deoxy-6-thioguanosine, into synthetic DNA has permitted the study of the structural consequences of 6-thioguanine incorporation as well as the study of the effects of 6-thioguanine incorporation on DNA-protein interactions vital for cell replication. It was shown that the incorporation of 2'-deoxy-6-thioguanosine into synthetic DNA requires a strategy that prevents the oxidative loss of sulfur, which is prevalent when normal DNA synthesis conditions are employed. Protection of the sulfur of 2'-deoxy-6-thioguanosine with the cyanoethyl group and deprotection in 1M NaOH/0.01 M NaSH permits the synthesis of 2'-deoxy-6-thioguanosine-containing DNA without 2'-deoxy-6-thioguanosine degradation. The results of this work indicate that the incorporation of 6-thioguanine for guanine in d(AAACs6GTTT) leads to several structural changes in the DNA octamer as detected by NMR spectroscopy. Spectroscopic evidence, both NMR and UV, indicated that the presence of 2'-deoxy-6-thioguanosine acts to strongly inhibit duplex formation in the octamer. It was also shown that 2'-deoxy-6-thioguanosine was very sensitive to oxidative degradation at elevated pH and temperature, even when incorporated into the DNA octamer. The addition of antioxidants such as dithiothreitol and ?-mercaptoethanol, were shown the effectively inhibit this degradation. Further NMR evidence indicated that 6-thioguanine incorporation leads to a change in the sugar conformation of deoxycytidine, the cross-strand partner of 2'-deoxy-6-thioguanosine, as well as a change in the overall structure of the entire octamer. The deoxyguanosine-containing octamer assumed a bent structure while the 2'-deoxy-6-thioguanosine-containing octamer did not contain evidence of bending. This work has also shown that 6-thioguanine incorporation can lead to disruptions in DNA-protein interactions required for DNA replication. These results provide strong evidence that the structural distortions induced by 6-thioguanine incorporation in DNA play a major role in the anticancer activity of 6-thioguanine.