Variations in the structure of TPCK (I), the reagent initially devised as a substrate-like alkylating agent for chymotrypsin, have led to inhibitors that either inactivate very rapidly or will otherwise increase the usefulness of this class of agents in structural work with serine proteases, for example, by inactivation of enzymes insensitive to TPCK. The essentiality of an α-substituent for rapid and exclusive alkylation of histidine in chymotrypsin is shown. Without it, alkylation of methionine-192 takes place, leading to chymotrypsin derivatives with residual hydrolytic activity. The substituent on the α-amino group may interact with chymotrypsin at the binding site for the N-terminal portion of a normal polypeptide substrate and, therefore, variations in this portion of the reagent are of added interest. For this purpose, the benzyloxycarbonyl derivatives ZPCK and ZPBK (II) are of particular value as synthetic intermediates since they can be deblocked, without loss of the halomethyl ketone grouping, to the amino compound (III), which is stable when not exposed to alkaline conditions.