Interdisciplinary Nanoscience Center, iNANO, Aarhus University, Aarhus C, Denmark.
Department of Chemistry, Aarhus University, Aarhus C, Denmark.
Nat Commun. 2021 May 12;12(1):2760. doi: 10.1038/s41467-021-22945-z.
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聚合酶链反应 (PCR) 引物的生产、寡核苷酸药物的生产以及许多其他医学和生物技术应用都具有重要意义。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前体，而该技术的一个缺点是亚磷酰胺的台面稳定性差。在这里，我们报告了一种按需从相应醇合成亚磷酰胺的流动合成方法，该方法具有反应时间短、接近定量的产率且无需在直接提交给自动化寡核苷酸合成之前进行纯化。使用这种方法可以合成空间位阻大的以及氧化还原不稳定的亚磷酰胺，并且所有底物的偶联反应都接近定量。这项技术的愿景是直接集成到 DNA 合成仪中，从而省去了亚磷酰胺的手动合成和储存。
