Structure and dynamics of intercalation complexes of anthracyclines with d(CGATCG)2 and d(CGTACG)2. 2D-1H and 31P NMR investigations

The interaction of (2S )-2-methoxymorpholinodoxorubicin 4 and morpholinodoxorubicin 5 with the hexanucleotides d(CGATCG)2 and d(CGTACG)2 has been studied by a combined use of 2D-1H and 31P NMR techniques and molecular dynamics (MD) calculations, in comparison with doxorubicin 1, daunorubicin 2 and idarubicin 3. Both 1H and 31P chemical shifts of imino protons and phosphates respectively have been shown to be a sensitive probe for the intercalation sites (two anthracycline molecules at the CpG sites). A relevant number of NOE interactions allowed the structure of the complexes in solution to be derived through restrained MD calculations, which were compared with those obtained by X-ray analysis. In all the complexes the aglycone was shown to be located in the middle of the double helix, orthogonally oriented with respect to the base pairs, with ring D extending out of the helix on the major groove and ring A, with 9H8 conformation, between guanines G2 and G12. The daunosamine and morpholino moieties lie in the center of the minor groove, with slightly different positions than in the X-ray structures. In all the complexes the daunosamine ring is located at the A3 (T3) level, while the morpholino ring displays NOE interactions with the fourth unit T4 (A4). The deformations of the double helix are revealed by an increased distance between protons of the C5 and G6 units and by conformational changes at the level of the α, ζ, ε and δ angles of the phosphoribose backbone. The variation of the 31P chemical shifts is interpreted in terms of conformational equilibria leading to different populations of conformers. This is clearly shown from the values of the α and ζ torsion angles, monitored during the MD, which indicate a relevant population of trans forms for ζ and also for α angles of C5pG6 and G2pT3 (G2pA3) units, while the other phosphates exist entirely in the α, ζ, gauche,gauche conformation. The dissociation rate constants were measured by 2D 31P NOESY-exchange experiments for 1 and 4. The decrease of koff for 4, corresponding to a ten-fold increase of the residence time of the drug in the intercalation sites, is in line with the higher activity found for methoxymorpholinodoxorubicin 4.