Mechanochemical Synthesis of Short DNA Fragments

We demonstrate the first mechanochemical synthesis of DNA fragments by ball milling, enabling the synthesis of oligomers of controllable sequence and length using multi-step, one-pot reactions, without bulk solvent or the need to isolate intermediates. Mechanochemistry allowed for coupling of phosphoramidite monomers to the 5'-hydroxyl group of nucleosides, iodine/water oxidation of the resulting phosphite triester linkage, and removal of the 5'-dimethoxytrityl (DMTr) protecting group in situ in good yields (up to 60 % over three steps) to produce DNA dimers in a one-pot manner. H-Phosphonate chemistry under milling conditions enabled coupling and protection of the H-phosphonate linkage, as well as removal of the 5'-DMTr protecting group in situ, enabling a one-pot process with good yields (up to 65 % over three steps, or ca. 87 % per step). Sulfurization of the internucleotide linkage was possible using elemental sulfur (S8) or sulfur transfer reagents, yielding the target DNA phosphorothioate dimers in good yield (up to 80 % over two steps). This work opens the door to creation of solvent-free synthesis methodologies for DNA and RNA therapeutics.