SPIN-LABELED OXAZOLOPYRIDOCARBAZOLE AS A PROBE FOR STUDYING NONINTERCALATING DNA GROOVE BINDING LIGANDS

A spin-label (P-OPC) composed of the nitroxide-containing ring proxyl linked at the C1 position of the intercalating fluorescent chromophore oxazolopyridocarbazole (OPC) has been synthesized. The spin-labeled OPC was found to interact with DNA and polynucleotides according to an external minor groove binding mode with association constant values Kapp ranging from 105 to 106 M−1. External binding was obvious from the inability of P-OPC to increase the length of sonicated DNA upon binding, the low unwinding angle (9.6°) of circular PM2 DNA, and the low energy transfer from DNA bases to bound chromophore. Binding of P-OPC to DNA or polynucleotide results in a strong immobilization of the proxyl moiety, resulting in the appearance of an asymmetric and broad ESR spectrum with a maximal hyperfine splitting of 56.5 G. In the equilibrium conditions, the occurrence of superimposed ESR spectra related to the P-OPC fraction undergoing rapid motion and to the P-OPC fraction immobilized allows the estimation of the concentrations of free and DNA-bound spin-label. The external mode of binding to DNA as well as the characteristics of the ESR spectra make P-OPC suitable for the determination of DNA binding parameters of nonintercalating ligands using competition experiments. The measurement of the binding constants of distamycin A to polyfd(A-T)] and poly[d(G-C)] is taken as an example. © 1990, American Chemical Society. All rights reserved.