Formaldehyde-induced alkylation of a 2 '-aminoglucose rebeccamycin derivative to both A center dot T and G center dot C base pairs in DNA

Rebeccamycin derivatives represent a promising class of antitumor agents. I n this series, two glycosylated indolocarbazoles, NB-506 and NSC-655649, ar e currently undergoing clinical trials. Their anticancer activities are ass ociated with their capacities to interact with DNA and to inhibit DNA topoi somerases. Previous studies revealed that the planar indolocarbazole chromo phore can intercalate into DNA, locating the appended carbohydrate residue in one of the two helical grooves, probably the minor groove as is the case with the anthracyclines and other DNA-binding antibiotics. The sugar resid ue contributes significantly to the DNP; binding free energy of NB-506. How ever, the exact positioning of the glycosyl residue of rebeccamycin derivat ives in the drug-DNA complex remains poorly understood. To better understan d how glycosylated indolocarbazoles interact with DNA, we investigated the interaction of a rebeccamycin derivative (85) bearing a 2'-amino group on t he sugar residue. We show that the presence of the 2'-amino function permit s the formation of covalent drug-DNA complexes in the presence of formaldeh yde. Complementary biochemical and spectroscopic measurements attest that 8 5 reacts covalently with the 2-amino group of guanines exposed in the minor groove of the double helix, as is the case with daunomycin. In contrast to daunomycin, 85 also forms cross-links with an oligonucleotide containing o nly A.T base pairs. The covalent binding to A.T base pairs was detected usi ng a gel mobility shift assay and was independently confirmed by thermal de naturation studies and by fluorescence measurements using a series of synth etic polynucleotides. The HCHO-mediated alkylation reaction of the drug wit h A.T base pairs apparently involves the 6-amino group of adenines exposed in the major groove whereas the covalent attachment to G.C base pairs impli cates the 2-amino group of guanines situated in the opposite minor groove. Therefore, the results suggest that either the drug is able to switch groov es in response to sequence or it can simultaneously bind to both the minor and major grooves of the double helix. This study will help to guide the ra tional design of new DNA-binding antitumor indolocarbazole drugs and also p rovides a general experimental approach for probing minor versus major groo ve interactions between small molecules and DNA.