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

Cheng Jin, Siqi Li, Katherine A. Vallis, Afaf H. El-Sagheer and Tom Brown. RSC Chemical Biology

a cathepsin B-cleavable Val-Ala(NB) phosphoramidite monomer has been developed for the efficient automated synthesis of oligonucleotide-small molecule conjugates. Cathepsin B releases small molecule-phosphate derivatives from oligonucleotides which are converted to the free small molecules by cellular phosphatase enzymes. To demonstrate the utility of our methodology, we have shown that the microtubule destabilizing reagent combretastatin A- 4 (CA4) is released from an oligonucleotide after intracellular cathepsin B and phosphatase mediated cleavage of the dipeptide in lysosomes where cathepsin B largely localizes. To broaden the scope of the methodology to molecules that do not contain hydroxyl groups, we have carried out the solid-phase conjugation of small molecules containing amino groups to oligonucleotides via the Val- Ala(NB) linker by carbamate formation.


The reversible attachment of small molecules to oligonucleotides provides versatile tools for the development of improved oligonucleotide therapeutics. However, cleavable linkers in the oligonucleotide field are scarce, particularly with respect to the requirement for traceless release of the payload in vivo. Herein, we describe a cathepsin B-cleavable dipeptide phosphoramidite, Val-Ala(NB) for the automated synthesis of oligonucleotide-small molecule conjugates. Val-Ala(NB) was protected by a photolabile 2-nitrobenzyl group to improve the stability of the peptide linker during DNA synthesis. 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 potentially valuable cleavable linker for use in oligonucleotide-drug conjugates.