Synthesis of Dinucleotide Non‐Symmetrical Triester Phosphate Phosphoramidites and Their Incorporation Within Oligonucleotides

In this protocol article, the synthesis of dinucleotide non-symmetrical triester phosphate phosphoramidites will be highlighted. Specifically, we use a selective transesterification starting with tris(2,2,2-trifluoroethyl) phosphate to afford a dinucleotide derivative phosphate ester. Substitution of the final trifluoroethyl group with various alcohols affords a dinucleotide triester phosphate with a hydrophobic group, which can then be deprotected and converted to a phosphoramidite for incorporation within oligonucleotides. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of a DMT- and TBS-protected unsymmetrical dinucleotide Basic Protocol 2: Synthesis of a DMT-protected unsymmetrical dinucleotide phosphotriester monoalcohols Basic Protocol 3: Synthesis of DMT-protected phenylethyl phosphotriester dinucleotide phosphoramidites Basic Protocol 4: Synthesis, purification, and characterization of RNAs containing triester phosphate modifications.