An alternative and convenient strategy for generation of substantial quantities of singly 5'-32P-end-labeled double-stranded DNA for binding studies: development of a protocol for examination of functional features of (+)-CC-1065 and the duocarmycins that contribute to their sequence-selective DNA alkylation properties.

Development of an alternative strategy for securing substantial quantities of singly 5'-32P-end-labeled double-stranded DNA suitable for binding studies is described based on M13 cloning techniques and offers advantages of production of replenishable quantities of singly 5'-32P-end-labeled double-stranded DNA of homogenous length without need for DNA isolation (restriction fragment), dephosphorylation, and lengthy preparative gel electrophoresis procedures. The 32P label is introduced onto the free 5'-hydroxyl group of a chemically synthesized universal primer [5'-32P-d(GTAAAACGACGGCCAGT)-3'] which is used to initiate DNA synthesis on M13-derived single-stranded DNA templates. Following DNA synthesis, a restriction enzyme cleavage reaction produces a uniform length duplex suitable for agent binding studies. The strategy further permits the use of the Sanger dideoxynucleotide sequencing technique for direct and unambiguous identification of cleavage sites introduced by an agent on the end-labeled DNA. The use of the procedure in the examination of the DNA alkylation properties of (+)-CC-1065 (1) and a series of synthetic analogs is reviewed. From these studies a refined definition of the alkylation selectivity of (+)-CC-1065 is detailed. Employing agents possessing the parent 1,2,7,7a-tetrahydrocycloprop[1,2-c]indol-4-one (CI) alkylation subunit constituting the minimum pharmacophore of the CC-1065 alkylation subunit (CPI), comparative DNA alkylation studies illustrate that the activated cyclopropane is not obligatory for observation of the CI/CPI characteristic alkylation, highlight the relative nonselectivity of the alkylation event in the absence of noncovalent binding selectivity, illustrate a prominent role for agent binding selectivity for agents that possess such capabilities, and demonstrate that a sequence dependent autocatalytic phosphate activation of the alkylation event may not be uniquely responsible for the nonselective or selective alkylations. The ease with which the procedure may be extended to the rapid and convenient examination of additional agents is illustrated with the demonstration of the strikingly similar DNA alkylation properties of the duocarmycins (3-8) and (+)-CC-1065 (1) which suggest that the agents may be acting by a common mechanism.