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The ubiquitin–proteasome pathway degrades the majority of proteins in mammalian cells and plays an essential role in the generation of antigenic peptides presented by major histocompatibility class I molecules. Proteasome inhibitors are of great interest as research tools and drug candidates. Most work on proteasome inhibitors has focused on the inhibition of the chymotryptic-like (β5) sites; little attention has been paid to the inhibition of two other types of active sites, the trypsin-like (β2) and the caspase-like (β1). We report here the development of the first cell-permeable and highly selective inhibitors (4 and 5) of the proteasome’s caspase-like site. The selectivity of the compounds is directly and unambiguously established by Staudinger–Bertozzi labeling of proteasome subunits covalently modified with azide-functionalized inhibitor 5. This labeling reveals that the caspase-like site of the immunoproteasome (β1i …