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

Abstract

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 k_off 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.