Chemical Phosphorylation Reagent is most commonly used to phosphorylate the 5'-terminus of an oligonucleotide. Although this product is also successful in 3'-phosphorylation, 3'-Phosphate CPG allows direct preparation of oligonucleotides with a 3'-phosphate group. Chemical Phosphorylation Reagent II contains a DMT group on a side chain which is stable to base cleavage and can be left on the oligonucleotide for use in RP purification. The DMT group is later removed with aqueous acid and the side chain is eliminated after brief treatment with aqueous ammonium hydroxide to yield the 5'-phosphate.1 Solid CPR II is similar in performance to CPR II but it is easier to prepare aliquots since it is a powder. Many researchers treat synthesis supports with a hindered base (e.g., diethylamine, diisopropylethylamine, or DBU) post-synthesis to eliminate and remove the cyanoethyl phosphate groups. In this way, the acrylonitrile formed in situ is removed from the support and is not available to alkylate dT residues at the N3 position in the oligos. Since the sulfonylethyl group in 3'-Phosphate CPG is also susceptible to ß-elimination leading to oligo cleavage, this technique is not compatible with 3'-phosphate CPG. Using CPR II CPG, which is base labile but does not support ß-elimination, the cyanoethyl groups can be removed from the oligo prior to cleavage and base deprotection. ABI-style vials and columns are supplied unless otherwise requested.
Coupling: No changes needed from standard method recommended by synthesizer manufacturer.
Deprotection: As required by nucleobases. Complete deprotection to the 5’-phosphate requires a minimum of 17 hr @ RT or 4 hr @ 55 °C in ammonium hydroxide, 30 minutes at 65 °C in AMA (Ammonium hydroxide/40% methylamine 1:1 v/v), or 8 hr @ RT in K2CO3/MeOH. Note: the DMT group is removed during deprotection and is not available for reverse phase purification - e.g., GlenPak™ purification.