The examination of shortened, simplified, and extended analogs of duocarmycin SA is described and constitutes a detailed study of the role of linked DNA binding subunit. In addition to enhancing the DNA binding affinity and selectivity through minor groove noncovalent contacts, the studies in conjunction with those of the accompanying article illustrate that an extended rigid N2 amide substituent is required for catalysis of the DNA alkylation reaction. This activation for DNA alkylation is independent of pH, and we propose it results from a binding-induced conformational change in the agents which increases their inherent reactivity. The ground state destabilization of the substrate results from a twist in the linking amide that disrupts the vinylogous amide stabilization of the alkylation subunit and activates the agent for nucleophilic addition. This leads to preferential activation of the agents for DNA alkylation within the narrower, deeper AT-rich minor groove sites where the inherent twist in the linking amide and helical rise of the bound conformation is greatest. Thus, shape-selective recognition (preferential AT-rich noncovalent binding) and shape-dependent catalysis (induced twist in linking N2 amide) combine to restrict SN2 alkylation to accessible adenine N3 nucleophilic sites within the preferred binding sites. Additional ramifications of this DNA binding-induced conformational change on the reversibility of the DNA alkylation reaction are discussed. The results of the study illustrate the importance of the C5‘ methoxy group and the C6 methyl ester of duocarmycin SA, and a previously unrecognized role for these substituents is proposed.

The synthesis and examination of two unique classes of duocarmycin SA analogs are described which we refer to as reversed and sandwiched analogs. Their examination established both the origin of the DNA alkylation selectivity and that both enantiomers of this class of natural products are subject to the same polynucleotide recognition features. The most beautiful demonstration of this is the complete switch in the enantiomeric alkylation selectivity of the reversed analogs which is only consistent with the noncovalent binding model and incompatible with alkylation site models of the origin of the DNA alkylation selectivity. In addition, dramatic alterations in the rates of DNA alkylation were observed among the agents and correlate with the presence or absence of an extended, rigid N2 amide substituent. This has led to the proposal of a previously unrecognized source of catalysis for the DNA alkylation reaction which was introduced in the preceding paper of this issue (J. Am. Chem. Soc. 1997, 119, 4977−4986).

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