Abstract(+)‐Yatakemycin is a new and potent member of a class of natural antitumor compounds that derive their biological activities from specific alkylation of adenine residues in the minor grooves of AT‐rich tracts. We have analyzed the covalent complex formed between (+)‐yatakemycin and the d(GACTAATTGAC)‐(GTCAATTAGTC) duplex, and have established that the ligand covalently binds to the A5 residue. For this purpose we used a hybrid approach based on 2D‐NMR spectroscopy and quantum mechanical (QM) calculations of 1H chemical shifts at the DFT/MPW1PW91 level. In this study we also show that the calculation of NMR parameters can be a useful tool for the structural characterization of ligand–receptor interactions.(© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2008)