P-31 NMR-studies on the interaction of morpholinyl anthracyclines and related compounds with d(CGTACG)2. Thermodynamic and kinetic parameters

31P NMR spectroscopy has been used to study the intercalation of the anthracyclines doxorubicin1, daunorubicin2, 4-demethoxydaunorubicin3, morpholinodoxorubicin4, methoxymorpholinodoxorubicin5 and 9-deoxydaunorubicin6 with the DNA fragment d(CGTACG)2. The individual phosphate resonances of the oligonucleotide were assigned in the free as well as in the intercalated species. The31P chemical shift variations allowed us to identify the intercalation sites, which resulted to be the same for all compoundsi.e. between the terminal CG base-pairs of the helix (two molecules of drugper duplex). The binding constants, the dissociation rate constants and ΔG # values have been determined in different conditions of ionic strength and temperature. The kinetic constant (k off) of the slow step of the anthracycline/duplex intercalation process has been directly measured by NOE exchange techniques. Binding constants depend on the ionic strength and on the self-association process so greatly, that their use to study by NMR anthracycline/DNA interactions is questionable. On the contrary, thek off are not affected by these phenomena and present an interesting trend for1–6, thus showing that the average lifetime of the drug in the intercalation site appears to be important for determining the cytotoxicity and the antimitotic activity.