Caspases are cysteine proteases that are essential for cytokine maturation and 
apoptosis. To facilitate the dissection of caspase function in vitro and in 
vivo, we have synthesized irreversible caspase inhibitors with biotin attached 
via linker arms of various lengths (12a-d) and a 2,4-dinitrophenyl labeled 
inhibitor (13). Affinity labeling of apoptotic extracts followed by blotting 
reveals that these affinity probes detect active caspases. Using the strong 
affinity of avidin for biotin, we have isolated affinity-labeled caspase 6 from 
apoptotic cytosolic extracts of cells overexpressing procaspase 6 by treatment 
with 12c, which contains biotin attached to the N(epsilon)-lysine of the 
inhibitor by a 22.5 A linker arm, followed by affinity purification on monomeric 
avidin-sepharose beads. Compound 13 has proven sufficiently cell permeable to 
rescue cells from apoptotic execution. These novel caspase inhibitors should 
provide powerful probes for the study of the active caspase proteome during 
apoptosis both in vitro and in vivo.