Synthesis, separation and characterization of the mono- and diazide analogs of ethidium bromide

Ethidium bromide is used to characterize nucleic acid secondary and tertiary structural properties and the biological consequences of drug interactions. The mono- and diazido analogs of ethidium have proven valuable as photoaffinity probes in chemical and biological studies on nucleic acids, since they render the ethidium-nucleic acid interaction covalent. Although both of these compounds have been synthesized previously, the published synthesis procedure for the monoazide is inadeqlate since a major portion of the product has been identified as the diazide analog. This lack of purity severely limits the usefulness for nucleic acid research. The procedure presented here for the synthesis, separation, purification and crystallization of these analogs should provide the quantities and quality of these important reagents needed to perform a variety of chemical and biological experiments.