An Acid-Free Deprotection of 5′-Amino-Modified Oligonucleotides

The installation of reactive chemical handles onto synthetic oligonucleotides is essential for the generation of oligonucleotide conjugates, which are ubiquitous across healthcare, diagnostic, and research industries. This is typically accomplished by the incorporation of a monomethoxytrityl (MMTr)-protected primary amine, as it allows for purification via high-throughput reverse-phase cartridge purification and can be deprotected with standard laboratory reagents. While the MMTr group provides these advantages over alternative protecting groups, conventional deprotection methods rely on aqueous acids, which are inherently reversible in nature and are known to cause depurination of oligonucleotides. In this study, we demonstrate a novel equilibrium-driven deprotection strategy for MMTr-protected amino oligonucleotides, which simplifies the purification process while increasing synthetic efficiency and quality of the oligonucleotide. The method utilizes only water and heat to achieve the selective deprotection of MMTr groups, eliminating the need for acid treatment. This deprotection approach offers a safe, mild, operationally simple, and environmentally friendly alternative to conventional methods.