What special phosphoramidites are required for phosphorothioate synthesis?
The answer is none. The standard method of introducing phosphorothioates is to use standard phosphoramidites and sulfurization reagent in place of standard iodine oxidation. Sulfurization requires a contact time of at least one minute on the synthesizer. Sulfurization also needs to occur before capping, in the opposite order when compared to standard iodine oxidation. This is to prevent trace amounts of peroxides in capping reagents from performing undesired oxidation.
|Sulfurizing Reagent II, 40-4037
|0.05M Sulfurizing Reagent II in pyridine/acetonitrile, 40-4137
How long are phosphoramidites good for on the synthesizer?
It could be anywhere between one day and two or more weeks. The actual time frame will depend on a host of factors including the phosphoramidite, phosphoramidite concentration, solvent, instrument, humidity, temperature, water content and more. Special or rare phosphoramidites should generally be consumed within a 24-hour time frame, while standard DNA phosphoramidites should be good for at least a week. If a bottle of phosphoramidite that was coupling well initially starts showing reduced coupling efficiency, that’s a sign that it is time to replace that bottle.