Engineering Enzyme‐Cleavable Oligonucleotides by Automated Solid‐Phase Incorporation of Cathepsin B Sensitive Dipeptide Linkers

Oligonucleotides containing cleavable linkers have emerged as versatile tools to achieve stimulus‐responsive and site‐specific cleavage of DNA. However, the limitations of previously reported cleavable linkers including photolabile and disulfide linkers have restricted their applications in vivo. Inspired by the cathepsin B‐sensitive dipeptide linkers in antibody–drug conjugates (ADCs) such as Adcetris, we have developed Val‐Ala‐02 and Val‐Ala‐Chalcone phosphoramidites for the automated synthesis of enzyme‐cleavable oligonucleotides. Cathepsin B digests Val‐Ala‐02 and Val‐Ala‐Chalcone linkers efficiently, enabling cleavage of oligonucleotides into two components or release of small‐molecule payloads. Based on the prior success of dipeptide linkers in ADCs, we believe that these dipeptide linker phosphoramidites will promote new clinical applications of therapeutic oligonucleotides.