Coupling: No changes needed from standard method recommended by synthesizer manufacturer. To avoid any exchange of the iPr-Pac group on the dG with acetyl, use the UltraMild Cap Mix A (40-4210-xx/ 40-4212-xx).
Deprotection: UltraMILD deprotection: 0.05M Potassium Carbonate in Methanol, 4 hours at Room Temperature to leave the methyl phosphotriester intact.
Storage: Refrigerated storage, maximum of 2-8°C, dry
QUESTION: Can Me phosphoramidites (10-1300, etc.) be used to produce methyl triester oligonucleotides?
RESPONSE:Possibly, but we are unaware of a method. Some people remember that the methyl phosphoramidites were used to prepare oligos originally and that thiophenol was used to remove the methyl group prior to ammonium hydroxide deprotection. This might suggest that omitting the thiophenol step would lead to the methyl triesters. Wrong. Thiophenol was used to remove the methyl group specifically prior to the ammonium hydroxide step to avoid chain scission. If the methyl triesters are treated with ammonium hydroxide, they are predominantly hydrolyzed to the phosphodiesters but a small percentage of the linkages are also hydrolyzed to eliminate either the 3'or 5' alcohol (chain scission).
While working with dT-Me Phosphoramidite and deprotecting under UltraMild conditions, it was clear that the methyl triester group was essentially unreacted over 24 hours in potassium carbonate in methanol. We have now introduced UltraMild Me Phosphoramidite monomers and, finally, methyl triester oligonucleotides can be simply produced.
The table below shows pack size data and, for solutions, dilutions and approximate couplings based on normal priming procedures. Please link for more detailed usage information with the various synthesizers.