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.
Cathepsin B‐sensitive dipeptide phosphoramidites were developed for automated synthesis of enzyme‐cleavable oligonucleotides. The enzymatic cleavage of Val‐Ala‐02 by cathepsin B is efficient in the context of single‐stranded DNA, but is sterically hindered by double‐stranded DNA and short hairpin loops. The design of Val‐Ala‐Chalcone allows the construction of oligonucleotide–drug conjugates which release their small‐molecule payload after enzymatic cleavage.
Keywords: Cathepsin B; DNA Breakage; Dipeptide Linker; Oligonucleotide–Drug Conjugates; Solid-Phase Synthesis.
© 2021 The Authors. Published by Wiley-VCH GmbH.