The type of linkage between a payload and a biomolecule can either be permanent or cleavable under certain well-defined circumstances. As payloads are typically highly cytotoxic, it would be fatal if they would be released from their carrier during circulation in plasma. Hence, the linker part should be stable to conditions like pH, redox potential or presence of proteases in plasma. However, at the desired location, e. g. after internalization, it is favorable that the linker is fragmentizing in order to release the drug molecule, ideally in a traceless manner.

Besides dipeptidic linkers (Val-Cit, Val-Ala), glucuronic acid capped linkers or chinoidic variations of the trimethyl lock, one approach to introduce a cleavable linker is the incorporation of a disulfide bond. Disulfides are stable at physiological pH during circulation due to the low reducing potential of blood. In contrast, the cytosol provides a significantly more reducing environment compared to the extracellular milieu, and the intracellular concentration of reducing agents such as glutathione (GSH) is typically 1000-fold greater, with the reductive potential in cancer cells being even higher.

Thus, disulfide-containing constructs represent versatile and effective vehicles for the delivery of bioactive molecules to their cellular targets and subsequent reductive release of the cargo.

For ease of synthesis, Iris Biotech offers pyridyl disulfides as building blocks for the preparation of disulfide-based self-immolative linkers. Pyridyl disulfides undergo a disulfide exchange reaction with sulfhydryl groups to form disulfide bonds over a broad pH range also suitable for physiological pH. During the reaction, a disulfide exchange occurs between the biomolecule’s thiol group and the reagent’s 2-pyridyldithiol group. As a result, pyridine-2-thione is released, which can be followed spectrophotometrically (λ_max = 343 nm) to monitor the progress of the reaction. The p-nitrophenylcarbonate activating group reacts preferably with amines or other nucleophiles and allows further derivatization, e.g. with the desired drug molecule.

Chemical conjugation of a pyridyldithiol to a sulfhydryl substituted biomolecule.

The variation in the linker’s chemical composition (disulfide ethoxycarbonyl-based (SSE) in the case of RL-3500 vs. disulfide benzyloxycarbonyl-based (SSB) for RL-3550) results in chemically tunable kinetics of the self-immolative cleavage due to different response rates towards GSH, showing higher rates for SSB-based self-immolative linkers compared to SSE-based ones. Thus, the choice of the linker allows the fine-tuning of the cleavage speed and payload release.

Disulfide-based self-immomlative linkers either based on an intramolecular cyclization cascade mechanism or an 1,6-elimination.

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