The synthesis of labelled oligonucleotides has become a standard procedure in many laboratories and many labelling reagents, e.g., biotin and fluorescein, are now available as β-cyanoethyl (CE) phosphoramidites. Labels which are currently available as CE phosphoramidites have one common property - they must be stable to the strongly alkaline conditions required for removal of the base protecting groups. This property is lacking in several interesting dyes and labels. We sought an alternative protecting scheme for the normal CE phosphoramidites which would allow UltraMILD deprotection and would not react with a wider variety of tags and labels. A set of monomers using phenoxyacetyl (Pac) protected dA and 4-isopropyl-phenoxyacetyl (iPr-Pac) protected dG, along with acetyl protected dC, met the desired criteria for UltraMILD deprotection.
We recommend the use of phenoxyacetic anhydride (Pac2O) in Cap A. This modification removes the possibility of exchange of the iPr-Pac protecting group on the dG with acetate from the acetic anhydride capping mix. Cleavage and deprotection can be carried out in 2 hours at room temperature with ammonium hydroxide or 4 hours with 0.05M potassium carbonate in methanol.