Dynamic covalent chemistry (DCvC) has emerged as a versatile synthetic tool for devising stable, stimuli-responsive linkers or conjugates. The interplay of binding affinity, association and dissociation constants exhibits a strong influence on the selectivity of the reaction, the conversion rate, as well as the stability in aqueous solutions. Nevertheless, dynamic covalent interactions often exhibit fast binding and fast dissociation events or vice versa, affecting their conversion rates or stabilities. To overcome the limitation in linker design, we reported herein dual responsive dynamic covalent peptide tags combining a pH responsive boronate ester with fast association and dissociation rates, and a redox-active disulfide with slow formation and dissociation rate. Precoordination by boronic acid–catechol interaction improves self-sorting and selectivity in disulfide formation into heterodimers. The resulting bis-peptide conjugate exhibited improved complex stability in aqueous solution and acidic tumor-like extracellular microenvironment. Furthermore, the conjugate responds to pH changes within the physiological range as well as to redox conditions found inside cancer cells. Such tags hold great promise, through cooperative effects, for controlling the stability of bioconjugates under dilution in aqueous media, as well as designing intelligent pharmaceutics that react to distinct biological stimuli in cells.

The cysteine protease cathepsin S (CatS) is overexpressed in many tumors. It is known to be involved in tumor progression as well as antigen processing in antigen‐presenting cells (APC). Recent evidence suggests that silencing CatS improves the anti‐tumor immune response in several cancers. Therefore, CatS is an interesting target to modulate the immune response in these diseases. Here, we present a series of covalent‐reversible CatS inhibitors based on the α‐fluorovinylsulfone and ‐sulfonate warheads. We optimized two lead structures by molecular docking approaches, resulting in 22 final compounds which were evaluated in fluorometric enzyme assays for CatS inhibition and for selectivity towards the off‐targets CatB and CatL. The most potent inhibitor in the series has subnanomolar affinity (Ki=0.08 nM) and more than 100,000‐fold selectivity towards cathepsins B and L. These new reversible and non‐cytotoxic inhibitors could serve as interesting leads to develop new immunomodulators in cancer therapy.

Dynamic covalent chemistry (DCvC) has emerged as a versatile synthetic tool for devising stable, stimuli-responsive bioconjugates. The interplay of binding affinity, association and dissociation constants exhibits a strong influence on the selectivity of the reaction, the conversion rate, as well as the stability in aqueous solutions. Nevertheless, dynamic covalent interactions often exhibit fast binding in combination with fast dissociation events and vice versa. To overcome the intrinsic limitation, we have designed dynamic covalent peptide tags combining two different pairs of dynamic covalent interactions with different reaction kinetics:(1) the fast association of boronic acid and catechol that forms pH-sensitive and rapidly dissociating boronate esters, and (2) the slower formation of a redox-active disulfide bond with slow dissociation rate. Pre-coordination of the thiols of the cysteine residues by the fast boronic acid-catechol interaction primarily yields the heterodimers proving selectivity and self-sorting capability of the reaction, with improved complex stability in aqueous solution and even in the acidic tumor-like extracellular microenvironment. The resulting bis-peptide conjugate responds to pH changes within the physiological range as well as to a redox environment that is similar to certain conditions found inside cancer cells. We believe that such tags hold great promise, through cooperative effects, for controlling the stability of bioconjugates under dilution in aqueous media, as well as designing intelligent pharmaceutics that react to distinct biological stimuli in cellular environments.

Dynamic covalent chemistry has emerged as a versatile synthetic tool for devising stable, stimuli-responsive bioconjugates. Nevertheless, dynamic covalent interactions often exhibit fast binding and dissociation events or vice versa, affecting their conversion rates or stabilities. To overcome this, we designed dual responsive peptide tags combining: (1) a pH responsive boronate ester with fast association and dissociation rates, and (2) a redox-active disulfide with slow formation and dissociation rate. Pre-coordination by boronic acid–catechol interaction improves self-sorting and selectivity in disulfide formation into heterodimers. The resulting bis-peptide conjugate exhibited improved complex stability in aqueous solution and acidic tumor-like extracellular microenvironment. The conjugate responds to pH changes and to a redox environment that is similar to certain conditions inside cancer cells. Such tags hold great promise for controlling the stability of bioconjugates under dilution in aqueous media, as well as designing intelligent pharmaceutics that react to distinct biological stimuli in cells.