Figure 8.

Jump-dilution inhibition experiment for nitrile 1b and alkyne 2b with cathepsin B to prove the irreversible inhibition by 2b. The inhibitors were preincubated in a volume of 30 or 180 μL in the presence of enzyme as schematically shown in Figure S46. The highly concentrated solution was diluted to 180 μL immediately before measurement. Subsequently, the reaction was started by substrate addition so that equal enzyme and inhibitor concentrations were achieved at the start of each measurement. The measurement was performed as a duplicate determination in assay buffer (pH 6.0) containing 200 μM Z-RR-AMC, 25 ng/mL cathepsin B, and 1.5% DMSO. The concentrations indicated refer to the final concentrations during the measurement after dilution.