The structural features of the covalent bonding of a novel CPI-lexitropsin conjugate 4 to a model duplex DNA has been examined by high field H-1-NMR analyses and restrained molecular dynamics calculations. Compound 4, that was designed for enhanced DNA binding compared with natural (+)CC-1065, exhibits an exceptional cytotoxic potency against KB human nasopharangeal-tumor cells in vitro of IC50 = 0.76 fg/L. Racemic 4 reacted readily with the duplex oligodeoxyribonucleotide d(CGCAATTGCG)(2) to form a single covalent adduct. The latter exhibits a new absorption band at 396 nm characteristic of the bound drug in addition to the duplex absorption at 258 nm. H-1-NMR analysis confirms by selective chemical shift changes and NOEs between protons in the drug and in the duplex that covalent bonding has taken place at A(4). The drug is aligned in a 5’- to 3’-direction at the AATT core in the minor groove resulting from the selective binding of one enantiomer of 4 and corresponding to the mode of binding of(-)-CC-1065. The stereochemistry at the site of attachment at the 4a position of the drug is (S), an inference that is corroborated by the restrained molecular dynamics simulation. The latter computations predict average total energies for the (R) and (S) drug-DNA adducts of +42 +/- 14.36 and -0.42 +/- 10.34 kcal/mol, respectively, signifying substantially greater stability for the latter diastereomer. The covalent adduct appears to be quite stable and showed no sign of reversibility such as has been observed with other CPI-based agents which may tentatively be attributed to the exceptionally snug fit of all parts of the drug within the minor groove.