A study of the solvolysis pH-rate profiles for two key reactive CC-1065/duocarmycin alkylation subunit analogues is detailed. Unlike the authentic alkylation subunits and N-BOC-CBI (4) which are too stable to establish complete solvolysis pH-rate profiles, the analogues N-BOC-CBQ (5) and N-BOC-CNA (6) are reactive throughout the pH range of 2-12. Moreover, they possess progressively diminished vinylogous amide conjugation resulting in a corresponding progressively increasing reactivity adopting and reflecting conformations analogous to that proposed for DNA-bound CC-1065. For both, the acid-catalyzed reaction was observed only at the lower pH of 2-5, and the uncatalyzed solvolysis reaction rate dominated at pH >/=6, indicating that the CC-1065 and duocarmycin DNA alkylation reaction observed at pH 7.4 need not be an acid-catalyzed reaction. The studies provide further strong evidence that catalysis for the DNA alkylation reaction (pH 7.4) is derived from a DNA binding-induced conformational change in the agents that disrupts the stabilizing alkylation subunit vinylogous amide conjugation activating the agents for nucleophilic attack independent of pH.