For many years, Glen Research has supplied methyl phosphoramidites in addition to ß-cyanoethyl (CE) phosphoramidites for the few situations where the more labile cyanoethyl group is not an advantage. Some of our customers, probably remembering that the methyl group was removed specifically with thiophenol, have tried to use these monomers to prepare the interesting, uncharged, and nuclease-resistant methyl phosphotriester linkage. Unfortunately, this linkage is labile to ammonium hydroxide and the regular phosphodiester linkage is formed (along with a small amount of chain scission). We offer UltraMild methyl phosphoramidites for this application. Oligos produced from these monomers can be deprotected with potassium carbonate in methanol to produce methyl phosphotriester linkages. Since these linkages are diastereomeric and uncharged, the oligos may be hard to handle. Consequently, it is likely that chimeras will be produced using these monomers along with the regular UltraMild CE phosphoramidites. If many dG residues are included in the oligonucleotide, we recommend the use of phenoxyacetic anhydride (Pac2O) in Cap A. This modification removes the possibility of exchange of the isopropyl-phenoxyacetate (iPr-Pac) protecting group on the dG with acetate from the acetic anhydride capping mix.
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 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.