Molecules that have a reactive functional group within a macrocycle represent a class of covalent inhibitor. The relationship between reactivity and affinity for the target is cooperative and complicated. An understanding and characterization of this class of inhibitor are vital for the development of covalent inhibitors as drug candidates. Herein, we describe a systematic analysis of structure–activity relationships using a series of syringolin analogues, which are irreversible covalent inhibitors of proteasomes. We investigate the detailed mechanistic effects of the macrocycles on affinity and reaction rate.