Synthesis of Allylic Phosphate Linked Dinucleotide Phosphoramidite: For the Application of Oligonucleotide Synthesis, Gene Assembly and Protein Expression^†

Comprehensive Summary Chain elongation via dinucleotide (dimer) block coupling was considered as an improved chemical technique capable of synthesizing high‐quality longer oligonucleotide for de novo DNA synthesis in synthetic biology. However, this dimer block‐wise approach was constrained by readily available dimer phosphoramidite with sufficient quality. Herein, through the usage of a one‐pot coupling‐oxidation‐deprotection cascade process for preparing the key precursors 3'‐hydroxyl dimers, then condensation with phosphorodiamidite, purification by flash column chromatography and precipation in methyl tert ‐butyl ether, a rationally designed dimer phosphoramidite bearing an internucleotide allyl phosphate and a β‐cyanoethyl phosphoramidite at the 3’‐hydroxyl was synthesized. All sixteen allylic dimer phosphoramidites 2a – p were smoothly prepared with overall yields exceeding 50% and HPLC purities ranging from 97.40% to 99.69%. With these allylic reagents, oligonucleotides were successfully synthesized using a modified solid‐phase phosphoramidite method and were completely deprotected under normal ammonialysis condition. Our results indicated that these dimer block‐wise synthesized oligonucleotides were of sufficient quality for gene assembly and protein expression, thus, the allylic phosphate linked dimer phosphoramidite can serve as a promising dimer reagent that will enable the applications of long oligonucleotides.