Binding Affinity and Site Selectivity of Daunomycin Analogues

We have tested a series of daunomycin analogues for binding affinity to a group of oligonucleotides that contain binding sites specific for daunomycin and that were previously screened for relative binding affinity for daunomycin. The series of drugs differed from daunomycin in the sugar moiety, including substitution of a hydroxyl group for the charged amino group and replacement of the 2′-OH by an iodo substituent. Data were analyzed by Scatchard plots and association constants were estimated from the y-intercept at saturating levels of oligonucleotide. Because of the solubility problems associated with these compounds, Scatchard plots could not be extended to high levels of binding. A second method of analysis of the fluorescence data confirmed the semiquantitative association constants obtained from the intercepts of the Scatchard plots. The association constants were in the range of 10⁵-10⁷ M⁻¹. When compared with daunomycin, the compounds with hydroxyl substituted for the amino group in the sugar ring generally bound less well to the oligonucleotides, by factors of up to several hundred. Much of the binding lost upon removal of the charged amino group was restored, however, with compounds containing an iodo substituent on the sugar ring. Changing the iodo-substituted sugar from the natural L-form to the D-form diminished binding by 6-50-fold, depending on sequence. This result implies a stereospecific interaction of the natural sugar with the DNA chain. A positively cooperative curve was observed in the Scatchard plot for the D-form sugar. Binding constants for a given analogue to a set of sequences varied by roughly 5-fold in all cases, but there was no sequence that was consistently preferred from one analogue to another.