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How do you synthesize oligos with a phosphorothioate backbone?
How do you control the placement of the phosphorothioate and phosphodiester linkages when a chimeric backbone is requested?
The addition of a new base to an oligo takes place in four steps:
1) The trityl group is removed from the 5' oxygen
2) The two bases are coupled together
3) The phosphate is oxidized, stablizing the bond between the two bases
4) Any oligos which did not participate in the coupling reaction are capped (to block addition of subsequent bases).
The difference between the synthesis of a normal oligo with a full phosphodiester backbone and a oligo with a partial or full phophorothioate backbone is the choice of oxidizing agent used in step 3. A phophodiester bond is produced by using iodine and water to add a fourth oxygen to the phosphate. A phosphorothioate bond is produced by using Beaucage reagent to add a sulfer to the phosphate. Once either the sulfer or the oxygen has been attached to the phosphate, the bond is stablized and will not be affected by the subsequent cycles of chemistry. By switching back and forth between the two oxidizing agents, a chimeric backbone can be constructed.