A Simplified One-Pot Platform for the Synthesis of Deoxyribonucleoside 5'-Triphosphates from Unprotected Nucleosides

Deoxyribonucleoside 5'-triphosphates (dNTPs) are essential biomolecules with broad applications in molecular biology, biotechnology, and pharmaceutical research. Their chemical synthesis, however, is often complicated by the formation of complex mixtures of phosphorylated byproducts, resulting in reduced yields and demanding purification procedures. This limitation has traditionally been addressed using protecting groups to improve regioselectivity and prevent undesired phosphorylation at hydroxyl groups other than the 5' position. However, protection and deprotection strategies add synthetic steps, cost, and operational complexity to the overall process. This protocol describes a simplified one-pot platform for the chemical synthesis of deoxyribonucleoside 5'-triphosphates directly from unprotected deoxyribonucleosides. The procedure is based on a modified Ludwig approach employing phosphorus oxychloride and bis(tributylammonium) pyrophosphate as the key phosphorylating reagents, without requiring prior hydroxyl protection or isolation of monophosphate or diphosphate intermediates. The synthesis of deoxyadenosine 5'-triphosphate (dATP) from unprotected 2'-deoxyadenosine is presented as a representative example of the complete workflow. The procedure involves sequential reagent addition in a single reaction vessel, followed by liquid-liquid extraction and reversed-phase chromatographic analysis. A key feature of this method is its operational simplicity and compatibility with unprotected deoxyribonucleoside substrates. Using dATP synthesis as a proof of concept, the method achieved a chromatographic yield of 67% under controlled anhydrous conditions. The same reaction platform may be adapted to other canonical deoxyribonucleosides following substrate-specific optimization of reagent stoichiometry, reaction conditions, and chromatographic analysis parameters. The protocol requires minimal specialized equipment and basic anhydrous handling techniques, thereby reducing the technical barriers associated with laboratory-scale dNTP synthesis.