Sequence-selective guanine reactivity by duocarmycin A.

High selectivity for covalent reaction at adenine N-3 within duplex DNA is a distinguishing feature of the CC-1065 and duocarmycin classes of natural products. Studies of the base and sequence selectivity exhibited by duocarmycins and CC-1065-based alkylating agents have focused on characterization of the predominant covalent adenine adducts that are formed. While information about minor DNA reaction products could provide valuable insights to our understanding the DNA recognition and reactivity properties of these agents, little characterization of such adducts by these agents has appeared in the literature. To broaden our structure-reactivity understanding of these DNA alkylating compounds, comparative investigations of the covalent sequence selectivity exhibited by compounds containing altered cyclopropapyrroloindole (CPI) alkylating subunits such as duocarmycin A were undertaken using the DNA polymerase inhibition assay. We were surprised to identify with this assay a DNA sequence with an unusual propensity for covalent reaction with duocarmycin A at a guanine nucleotide. Using the heat strand breakage assay with a duplex oligonucleotide containing this interesting sequence, we confirmed the site of alkylation to be the indicated guanine in the sequence 5'-CGCGTTG*GGAG-3'. The trimethoxyindole-CPI analog of duocarmycin A does not alkylate this guanine, suggesting that there are interesting features to the duplex recognition/reactivity exhibited by duocarmycin A. Herein we describe our identification of the first DNA sequence which covalently reacts with duocarmycin A at a guanine nucleotide in the absence of additional minor groove binding agents.