Chemo-Enzymatic Synthesis of Purine 3′-O-Amino-Deoxyribonucleoside Triphosphates

3 ′ -O-amino-deoxyribonucleoside triphosphates (3 ′ -ONH 2 -dNTPs) are crucial reversible terminators for template-independent DNA synthesis. Owing to the structural complexity of purine bases, here we propose a chemo-enzymatic process for the synthesis of purine-type 3 ′ -ONH 2 -dNTPs. First, the aminoalkoxyl group was introduced into commercially available dA and dG via a chemical catalytic process, affording yields of 24.9% and 27.3%, respectively. Subsequently, to establish a green and scalable enzymatic cascade for phosphorylation of the substrates, we engineered two key phosphorylation enzymes (Dm-dNK and MrPPK2), improving the rate-limiting monophosphorylation of 3 ′ -ONH 2 -dG from 26% to 97.8% and achieving 59.9% conversion to the corresponding triphosphate. Lastly, the produced nucleotides exhibit performance equivalent to commercial counterparts in terminal deoxynucleotidyl transferase (TdT)-mediated DNA synthesis, supporting the construction of 120-nt strands with ∼99.7% stepwise accuracy. This work establishes an efficient and generalizable catalytic platform for producing modified dNTPs, advancing the practical application of enzymatic DNA synthesis technologies.