Modular and Automated Synthesis of Oligonucleotide-Small Molecule Conjugates for Cathepsin B Mediated Traceless Release of Payloads

Attachment of small molecules to oligonucleotides is a versatile tool in the development of therapeutic oligonucleotides. However, cleavable linkers in the oligonucleotide field are scarce, particularly with respect to the requirement of traceless release of payload in vivo. Herein, a cathepsin B-cleavable dipeptide phosphoramidite, Val-Ala(NB), is developed for the automated synthesis of oligonucleotide-small molecule conjugates. Val-Ala(NB) was protected by photolabile 2-nitrobenzyl to improve stability of the peptide linker during DNA synthesis. After photolysis, intracellular cathepsin B digests the dipeptide efficiently, releasing the payload-phosphate which is converted to the free payload by endogenous phosphatase enzymes. With the advantages of modular synthesis and stimuli-responsive drug release, we believe Val-Ala(NB) will be a commonly used cleavable linker in the development of oligonucleotide-drug conjugates.