New Method Enables Faster Oxidation-Free Oligonucleotide Synthesis

Oligonucleotide synthesis is fundamental to genetic analysis, biological research, and nucleic acid therapeutics. However, current methods rely on moisture-sensitive building blocks and require a dedicated oxidation step with each chain elongation, making it operationally complex. To address this, researchers from Japan have developed a new approach using phosphorofluoridate building blocks, enabling efficient oxidation-free oligonucleotide synthesis. This advance simplifies the synthetic workflow, offering a faster and more reliable platform for nucleotide chemistry. 

The chemical synthesis of oligonucleotides (ONs) is central to modern molecular biology, diagnostics, and nucleic acid therapeutics. While there is an increasing demand for high-quality ONs, the conventional synthetic method is associated with long-standing challenges in terms of efficiency. The widely adopted P(III)-phosphoramidite-based ON synthesis requires an oxidation step after every nucleotide coupling cycle and uses moisture-sensitive building blocks, adding complexity to the workflow and slowing the process down.

Early studies for ON synthesis showed that pentavalent phosphorus [P(V)] chemistry could be used to form linkages between nucleotides, but practical limitations, including unstable intermediates, slow coupling, harsh deprotection, or poor performance during chain elongation, prevented these methods from replacing P(III)-based phosphoramidite chemistry.

In a recent study led by Associate Professor Noriko Saito-Tarashima from the Graduate School of Pharmaceutical Sciences, Tokushima University, Japan, along with Ms. Nana Mihara, a doctoral student from the same institute, investigated whether nucleoside 3′-phosphorofluoridates [P(V)–F] could be used as stable building blocks for ON synthesis without requiring a separate oxidation step. Their findings were made available online on June 19, 2026, and published in Volume 148, Issue 25 of the Journal of the American Chemical Society on July 01, 2026.

"The early development of ON chemistry served as the inspiration for this study. Around 70 years ago, a pioneering work demonstrated that ONs could be synthesized using P(V)-based chemistry. We wanted to know if our current approach could be applied to redesign this historically significant but underutilized chemistry," explains Dr. Saito-Tarashima while sharing their motivation for this study.

The P(V)–F blocks proved well suited for the objectives that the researchers focused on for this study, maintaining neutral chain growth during ON elongation, producing building blocks stable enough for isolation and storage, and achieving coupling efficiency comparable to standard P(III)-based phosphoramidite chemistry. The team successfully prepared P(V)–F corresponding to thymidine, 2′-deoxycytidine, 2′-deoxyadenosine, and 2′-deoxyguanosine, establishing a set of DNA monomers for the new platform.

A key feature of this method is the activation of the P(V)–F bond using a silicon-based additive, which facilitated the coupling or chain extension reaction between the nucleotides. The researchers assessed different bases and silicon-containing additives to find the optimal conditions that enabled efficient dinucleotide formation, and in one of the model reactions, the yield reached a quantitative level.

The study also compared the optimized P(V)–F coupling with conventional P(III)-phosphoramidite chemistry. The P(V)–F system coupled faster than the standard phosphoramidite approach, highlighting the potential of the new platform for efficient ON assembly.

Importantly, the underlying chemistry was not limited to solution-phase dinucleotide synthesis. The researchers adapted the approach to automated solid-phase ON synthesis using a standard DNA/RNA synthesizer. This step is critical for practical application, as automated synthesizers are the backbone of routine ON production. Although initial attempts showed inconsistent results, certain optimizations enabled successful ON synthesis.

Overall, the study provides a practical foundation for oxidation-free P(V)-based ON synthesis. Rather than replacing phosphoramidite chemistry immediately, the authors present the P(V)–F platform as a complementary approach that addresses key limitations of the conventional method.

"By offering a simpler and more robust way to synthesize ONs, our method could help improve the efficiency and reliability of producing these important molecules. In the future, this technology may support faster biological research, more advanced diagnostic technologies, and the development of next-generation nucleic acid-based therapies," concludes Dr. Saito-Tarashima.