Automated chemical synthesis of oligonucleotides is of fundamental importance for the production of primers for the polymerase chain reaction (PCR), for oligonucleotide-based drugs, and for numerous other medical and biotechnological applications. The highly optimised automised chemical oligonucleotide synthesis relies upon phosphoramidites as the phosphate precursors and one of the drawbacks of this technology is the poor bench stability of phosphoramidites. Here, we report on the development of an on-demand flow synthesis of phosphoramidites from their corresponding alcohols, which is accomplished with short reaction times, near-quantitative yields and without the need of purification before being submitted directly to automated oligonucleotide synthesis. Sterically hindered as well as redox unstable phosphoramidites are synthesised using this methodology and the subsequent couplings are near-quantitative for all substrates. The vision for this technology is direct integration into DNA synthesisers thereby omitting manual synthesis and storage of phosphoramidites.

寡核苷酸的自动化化学合成对于生产用于聚合酶链反应（PCR），基于寡核苷酸的药物以及许多其他医学和生物技术应用的引物至关重要。高度优化的自动化化学寡核苷酸合成依赖于亚磷酰胺作为磷酸盐前体，该技术的缺点之一是亚磷酰胺的较差的工作台稳定性。在这里，我们报道了从其相应的醇按需流式合成亚磷酰胺的进展，该过程以较短的反应时间，接近定量的产率完成，无需进行纯化即可直接直接进行自动化寡核苷酸合成。使用这种方法可以合成位阻以及氧化还原不稳定的亚磷酰胺，随后的偶联对于所有底物都接近定量。该技术的愿景是直接整合到DNA合成器中，从而省去了人工合成和储存亚磷酰胺的过程。