A branching monomer is required to construct comb-like oligonucleotide probes. The developers of the comb system from Chiron Corporation evaluated1 several protecting groups for the branch point and chose levulinyl (LEV), which is specifically removed using a reagent containing hydrazine hydrate, acetic acid and pyridine.
Coupling: dC Brancher reacts in a manner identical to normal phosphoramidites.
Deprotection: Levulinyl Deprotection: The levulinyl protecting group can be selectively removed without cleavage of the oligonucleotide from the CPG by treatment with 0.5 M Hydrazine hydrate in 1:1 pyridine/acetic acid. [Note hydrazine hydrate is a violent poison that is both volatile and readily absorbed through skin] Fit the column with syringes and push the solution back and forth across the column. Let sit for 15 minutes at room temperature. Rinse the column with 1.5 mL of 1:1 pyridine/acetic acid (3x) and then 1.5 mL of ACN (3x). Dry CPG under a stream of argon and proceed with the synthesis of the branching sequence. If a non-branching control is desired, simply deprotect in ammonium hydroxide as required by the nucleobases.