A total synthesis of N-desmethyl thalassospiramide C, a unique strained macrocyclic proteobacterial depsipeptide, enabled a detailed crystallographic study of its covalent complex with cathepsin K, a member of a medicinally important family of cysteine proteases. The study provides support for the mechanism of action, and the insight gained can be used for structure-based drug design targeting these calpain proteases.