Sequence Modifiers are designed for use in automated synthesis. The carboxy-dT is hydrolyzed during deprotection and can be coupled directly to a molecule containing a primary amino group by a standard peptide coupling or via the intermediate N-hydroxysuccinimide (NHS) ester. Amino-Modifier dA, Amino-Modifier dC, N2-Amino-Modifier dG and both Amino-Modifier dT products can be added in place of a dA, dC, dG and dT residue, respectively, during oligonucleotide synthesis. Corresponding Amino-Modifier supports can replace their respective deoxynucleoside supports. After deprotection, the primary amine on the C6 analogues is separated from the oligonucleotide by a spacer arm with a total of 7 -10 atoms and can be labeled or attached to an enzyme. The C2 analogue is more suitable for the attachment of molecules designed to react with the oligonucleotide.
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.