Recent advances in polymerase engineering have made it possible to copy information back and forth between DNA and artificial genetic polymers composed of TNA (α-L-threofuranosyl-(3',2') nucleic acid). This property, coupled with enhanced nuclease stability relative to natural DNA and RNA, warrants further investigation into the structural and functional properties of TNA as an artificial genetic polymer for synthetic biology. Here, we report a highly optimized chemical synthesis protocol for constructing multigram quantities of TNA nucleosides that can be readily converted to nucleoside 2'-phosphoramidites or 3'-triphosphates for solid-phase and polymerase-mediated synthesis, respectively. The synthetic protocol involves 10 chemical transformations with three crystallization steps and a single chromatographic purification, which results in an overall yield of 16-23% depending on the identity of the nucleoside (A, C, G, T).
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