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Macrocycles are abundantly used by nature to enable cell-permeable bioactive molecules. Synthetic non-peptidic macrocycles are also increasingly considered as modalities for difficult-to-bind proteins but guidelines for macrocyclization are only beginning to emerge. Macrocycles are thought to constrain the available conformations but also to allow for residual flexibility, the latter being poorly understood. Here we show that even medium-sized macrocycles display an unexpected high conformational plasticity, even when bound to their protein target. Minor modification of the linker region of macrocycles can shift the conformational ensemble to distinct conformational subclasses, each constituting distinct three-dimensional scaffolds for further optimization. This led to several new ligands with improved affinity and beneficial physicochemical parameters for the FK506-binding protein 51, a promising target for depression, obesity and chronic pain. Importantly, none of the beneficial modifications could have been identified by classical medicinal chemistry as they only work in the macrocyclic context. Our results show that macrocyclization can do more than keeping loose ends together but rather provide a platform for multiple series of macrocycles with distinct binding modes., (© 2025 The Author(s). Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

The intracellular delivery of cargos via cell penetrating peptides (CPPs) holds significant promise as a drug delivery vehicle, but a major issue is their lack of cell type specificity, which can lead to detrimental off-target effects. We use an ADEPT-like concept to introduce conditional and selective activation of cellular uptake by using the lysine-rich, cationic, and amphiphilic L17E peptide as a model CPP. By masking the lysine residues of the L17E peptide with enzyme-cleavable acetyl protecting groups, the delivery of the covalently conjugated fluorophore TAMRA to HeLa cells was diminished. Recovery of cellular uptake could be achieved by deacetylation of the masked acetylated L17E peptide using the NAD-dependent sirtuin 2 (SirT2) deacetylase in vitro . Finally, trastuzumab-SirT2 and anti-B7H3-SirT2 antibody-enzyme conjugates were generated for the conditional and selective delivery of a cryptophycin cytotoxin by the L17E peptide. While the masked peptide still demonstrated some cytotoxicity, selective cell killing mediated by the antibody-enzyme conjugates was observed.