On the Electronic Structure of Ethidium

Abstract

The electronic structure of the common intercalating agent ethidium bromide (3,8-diamino-5-ethyl-6-phenylphenanthridinium bromide) is dominated by an interplay of electron donating and withdrawing effects mediated by its nitrogen atoms. X-ray crystallography, UV/Vis and IR absorption, fluorescence emission, and NMR spectroscopy are used to probe the electronic properties of the phenanthridinium "core" of ethidium as well as its exocyclic amines and 6-phenyl groups. Interestingly, despite its positive charge, most of ethidium's aromatic carbon and hydrogen atoms have high electron densities (compared to both 6-phenylphenanthridine and benzene). The data suggest that electron donation by ethidium's exocyclic amines dominates over the electron withdrawing effects of its endocyclic iminium in their combined influence on the electron densities of these atoms. Ethidium's nitrogen atoms are, conversely, electron deficient where the 5-position is the most electropositive, followed by the 3-amino, and lastly the 8-amino group. These results have been used to generate an empirically-based pi-electron density map of ethidium that may prove useful to understanding its nucleic acid binding specificity.