Structural and Thermodynamic Analysis of Daunomycin Binding with Desoxyhexanucleotides with Different Base Sequences by NMR Spectroscopy

Abstract

Complexation of the anthracycline antibiotic daunomycin (DAU) with self-complementary desoxyhexanucleotides with different base sequences in the chain, 5′-d(CGTACG) and 5′-d(CGCGCG), is studied in aqueous solution. Homonuclear 2D-1H NMR spectroscopy (TOCSY and NOESY) and heteronuclear 1H–31P NMR spectroscopy (HMBC) are used for complete assignment of the nonexchangeable proton resonances and the phosphorus signals and for qualitative determination of preferable DAU binding sites, respectively. Daunomycin is preferably intercalated into the first d(CG)-site of each hexanucleotide, and the aminosaccharide ring of DAU lies in the minor groove of the hexamer duplex, partly overlapping the third base pair. Quantitative analysis of DAU complexation with desoxyhexanucleotides was done by analyzing the concentration and temperature dependences of the DAU proton chemical shifts; the equilibrium reaction constants and the thermodynamic parameters of the formation of the 1:1, 1:2, 2:1, and 2:2 DAU complexes with hexamers, as well as the limiting values of the DAU proton chemical shifts were determined in aqueous solution. The antibiotic preferably binds with the triplet sections of the nucleotide sequence containing two neighboring CG-pairs of nitrogen bases flanked by the AT-pair in 5′-d(CGTACG) compared to the triplets consisting of a sequence of three CG-pairs in the 5′-d(CGCGCG) hexamer. The specific binding of daunomycin depends on the character of the hydrophobic interactions of the aminosaccharide ring of the antibiotic in the minor groove of the DNA double helix. Calculations have been carried out to determine the most probable spatial structures of the 1:2 DAU–desoxyhexanucleotide complexes; the results agree well with X-ray diffraction data.