Efficient Automated Solid‐Phase Synthesis of DNA and RNA 5′‐Triphosphates

Abstract

A fast, high-yielding and reliable method for the synthesis of DNA- and RNA 5'-triphosphates is reported. After synthesizing DNA or RNA oligonucleotides by automated oligonucleotide synthesis, 5-chloro-saligenyl-N,N-diisopropylphosphoramidite was coupled to the 5'-end. Oxidation of the formed 5'-phosphite using the same oxidizing reagent used in standard oligonucleotide synthesis led to 5'-cycloSal-oligonucleotides. Reaction of the support-bonded 5'-cycloSal-oligonucleotide with pyrophosphate yielded the corresponding 5'-triphosphates. The 5'-triphosphorylated DNA and RNA oligonucleotides were obtained after cleavage from the support in high purity and excellent yields. The whole reaction sequence was adapted to be used on a standard oligonucleotide synthesizer.

Prodrugs of Nucleoside Triphosphates as a Sound and Challenging Approach: A Pioneering Work That Opens a New Era in the Direct Intracellular Delivery of Nucleoside Triphosphates

“…To circumvent this handicap, prodrugs of the phosphorylated metabolites (nucleotides) have been reported by af ew research groups. The idea is to mask the chargeso ft he phosphate group with lipophilic moieties so as to facilitate cell penetration, and, once inside the cells, release the nucleoside monophosphates using the cell machinery to finally yield the active metabolites (the nucleoside triphosphates)b ys uccessive di-and tri-phosphorylation events (Figure 1).To bypass the first phosphorylation step, the group of McGuigan developedasuccessful strategy namedt he ProTide approach, kinase bypass or phosphoramidate strategy, [7,8] in which lipophilic prodrugs of nucleoside monophosphates were prepared by the introduction of ap hosphoramidate group at the 5'-position of the nucleoside. [9] In these phosphoramidate prodrugs, the double anionic charges of the nucleoside monophosphates are masked by an a-amino acid ester and aryloxy groups (Figure2).…”

“…To bypass the first phosphorylation step, the group of McGuigan developedasuccessful strategy namedt he ProTide approach, kinase bypass or phosphoramidate strategy, [7,8] in which lipophilic prodrugs of nucleoside monophosphates were prepared by the introduction of ap hosphoramidate group at the 5'-position of the nucleoside. [9] In these phosphoramidate prodrugs, the double anionic charges of the nucleoside monophosphates are masked by an a-amino acid ester and aryloxy groups (Figure2).…”

Stealth fluorescence labeling for live microscopy imaging of mRNA delivery

“…1), which are prepared from commercially available 5-chlorosalicylic acid and pyrophosphoric acid. This method was developed by Meier et al 48 to achieve efficient 5′-triphosphorylation of short solid support-bound DNA and RNA oligonucleotides in moderate to good yields. Interestingly, it was originally used for solution-based synthesis of unmodified ribonucleosides and deoxyribonucleosides, 49 but has progressively been combined with solid supports to facilitate purification of the products.…”

Solid‐Phase Synthesis of DNA and RNA 5′‐O‐Triphosphates Using cycloSal Chemistry

“…By using AMA for just 2 hr at room temperature, standard base protected oligonucleotides are fully deprotected and the 5′‐ O ‐triphosphate group stays intact. Subsequent desilylation with triethylammonium trihydrofluoride (TEA‐3HF) is possible, but still a few percent the 5′‐ O ‐di‐ and 5′‐ O ‐monophosphorylated species are formed (Sarac and Meier, ). TBAF remains the optimal desilylation reagent because of its neutral to slightly basic pH.…”

“…The filtration should be done as quickly as possible, otherwise Arbuzov rearrangement will occur, lowering the yield (Sarac and Meier, 2015).…”