Drug therapeutics such as small molecules and peptides have high potential for the treatment of various diseases. However, most existing drugs pose serious threats to intact tissues due to their “always-on” pharmacological effect. To overcome this issue, prodrugs that only become cytotoxic in response to exogenous or endogenous stimuli, such as special disease markers, have been proposed.

An example for such a marker is hydrogen peroxide. As a relatively mild reactive oxygen species (ROS), H₂O₂ is recognized as a messenger molecule in various signaling processes. Additionally, in many enzymatic reactions, H₂O₂ is produced as a byproduct. However, excessive production of H₂O₂ is related to serious diseases including cardiovascular disorders, neurodegenerative diseases, inflammation, cancer, and diabetes. 

Thus, on the one hand, the determination of changes in the H₂O₂ level, e.g. via fluorescent H₂O₂-probes, is a valuable tool for the detection of pathological conditions. On the other hand, the increased H₂O₂ level in malfunctioning cells can be used for triggered drug release in such compartments without harming properly functioning cells. 

In the following, we present H₂O₂-sensitive self-immolative dioxoborolane linkers. Upon “detection” of hydrogen peroxide, the boronate protecting group is chemo-selectively converted to a phenol moiety followed by a 1,6-elimination reaction thus releasing the bound active species in a traceless manner. This reaction is thermodynamically driven by the release of CO₂ when a carbonate or carbamate ester linkage is employed.

Such dioxoborolane linkers, e.g. RL-4130, are used for example for the preparation of masked H₂O₂ probes releasing their active fluorophore upon trigger detection. Another application is the generation of liposomes, which disrupt upon H₂O₂ detection and then release the active cargo.

Payload bound to dioxoborolane linker and traceless release upon reaction with hydrogen peroxide and 1,6-elimination. 

The dioxoborolane motif is also provided as dendritic self-immolative linker (RL-4140) allowing to bind two payloads/reactive sites. In this case, the reaction of the dioxoborolane with H₂O₂ is followed by a 1,6-elimination and subsequent double 1,4-elimination leading to the release of the bound drugs. 

This bifunctional linker can be used, e.g. for the reversible cyclization of peptides, for instance via reaction of both handles with two Lys-side chain amines. Peptides are biological macromolecules whose versatile functions are largely determined by their conformational structures. As mentioned in the beginning, they emerged as important class of therapeutics, but their broad use is often hampered by their low cell-membrane penetration, lack of specificity, and poor in vivo stability. 

Cyclization of a peptide can lead to enhanced stability during circulation. Once inside the cell, the biologically active linear peptide can be released upon ring-opening via H₂O₂-triggered dual 1,4-elimination. 

Schematic illustration of the self-immolation of a dendritic dioxoborolane linker.

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Another linker suitable for reversible peptide cyclization is the azido-functionalized disulfide-based linker RL-4150.  As the overall red/ox potential in the human blood is oxidative, disulfide linkages remain intact during circulation. However, intracellularly, and notably inside tumor mass and cancer cells, the reductive potential is prevailing.

As an example, the azide of the linker can be clicked to one position of the peptide and the nitrophenylcarbonate is reacted with another functional group leading to cyclization. Reductive cleavage of the disulfide bond, e.g. by glutathione (GSH), followed by self-immolation, leads to ring-opening and release of the linear peptide. 

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