Finally, we made the Cl-amidine analogue (Cl4) to see what effect this more reactive electrophile would have on the potency of the peptide. The IC50 values were similar to those of cP4_4, although without preincubation, the IC50 was slightly higher (Table ). Interestingly, when we determined Ki and kinact values using SPR, Cl4 had a weaker Ki but higher kinact (Figure , Table ). This is consistent with the larger chlorine atom sterically hindering binding, but increasing the rate of the covalent reaction step. This warhead was also confirmed to be more reactive by intact MS, which showed that Cl4 could covalently react twice with PADI4, once at the active site C645 and a second time at an unknown location (Figure ).