The preparation and evaluation of both enantiomers of 5 are described and it constitutes an analogue of CBI-TMI (4), the duocarmycins, and CC-1065 in which the amide linking the alkylation and DNA binding subunits has been replaced by a methylene. The agent proved remarkably stable to acid-catalyzed solvolysis consistent with alkylation subunit stabilization derived from a fully engaged vinylogous amide. It was found to exhibit an acid-catalyzed solvolysis half-life (t(1/2)) of 80 h, 824 h, and ca. 30 500 h (3.3 days, 34 days, and ca. 3.5 years) at pH 1, 2, and 3, respectively, and to be completely stable at pH 7. The removal of the linking amide resulted in a 10(5)-fold loss in cytotoxic potency and the complete loss of DNA alkylation capabilities providing an agent that is >10(6)x less effective than 4 and >10(2)x less effective than even N-BOC-CBI or N-Ac-CBI. These observations highlight the critical importance of the linking amide and implicate a fundamental role in DNA alkylation catalysis. Thus, rather than enhancing DNA alkylation by facilitating C4 carbonyl protonation (acid catalysis), the removal of the linking amide abolished the capabilities for DNA alkylation. This is consistent with the intimate participation of the linking amide in catalysis derived from a DNA binding-induced conformation change that serves to disrupt the alkylation subunit cross-conjugated vinylogous amide stabilization activating the agents for nucleophilic attack.