Our repertoire of NHS ester derivatives has been expanded to include the NHS-Carboxy-dT-CE Phosphoramidite. By making a dT analog of the Carboxy-Modifier C10, it is possible to label one or multiple sites within an oligonucleotide. This opens up the possibility to label any number of different dyes or molecules within an oligonucleotide when the phosphoramidite is unavailable. Doing so is straightforward and may be done manually off the synthesizer or even in a fully-automated manner on the DNA synthesizer.
We have never found conditions which allow the TFA group to be removed from an amino-modifier while the oligonucleotide remains attached to the support. We are able to solve this problem by using a 9-fluorenylmethoxycarbonyl (Fmoc) protecting group. The Fmoc group is removed using a two step procedure, the first to remove the cyanoethyl protection groups and flush the formed acrylonitrile from the synthesis column using 1% diisopropylamine in acetonitrile, and the second to remove the Fmoc group using 10% piperidine in DMF. The amino group so formed on the column can be reacted with a variety of activated esters. We offer Fmoc-Amino-Modifier C6 dT Phosphoramidite as a nucleosidic option and Amino-Modifier Serinol Phosphoramidite as a non-nucleosidic alternative. We also offer S-Bz-Thiol-Modifier C6-dT to join the ranks of thiol-modifiers for oligonucleotide synthesis. Thiol-Modifier C6-dT can be added as usual at the desired locations within a sequence.
Coupling: No changes needed from standard method recommended by synthesizer manufacturer.
Deprotection: To label amine on column, treat support successively with 1 ml each for 10 minutes, 10% diethylamine/ACN (to remove cyanoethyl protection) and 20% piperidine/DMF to remove Fmoc protection. Rinse support with DMF and ACN to remove piperidine. Dissolve NHS ester (5-10 fold excess) in 0.5 ml appropriate solvent containing 1% diisopropylethylamine and incubate for 2-4 hours @ 35 oC with CPG to label amine. Rinse support to remove excess label and cleave/deprotect as normal or with conditions compatible with coupled label.