The Liebermann-Burchard reaction: sulfonation, desaturation, and rearrangment of cholesterol in acid.

In the Liebermann-Burchard (LB) colorimetric assay, treatment of cholesterol with sulfuric acid, acetic anhydride, and acetic acid elicits a blue color. We studied the reactivity of cholesterol under LB conditions and provide definitive NMR characterization for approximately 20 products, whose structure and distribution suggest the following mechanistic picture. The major reaction pathways do not involve cholestadienes, i-steroids, or cholesterol dimers, as proposed previously. Instead, cholesterol and its acetate and sulfate derivatives undergo sulfonation at a variety of positions, often with skeletal rearrangements. Elimination of an SO(3)H group as H(2)SO(3) generates a new double bond. Repetition of this desaturation process leads to polyenes and ultimately to aromatic steroids. Linearly conjugated polyene cations can appear blue but form too slowly to account for the LB color response, whose chemical origin remains unidentified. Nevertheless, the classical polyene cation model is not excluded for Salkowski conditions (sulfuric acid), which immediately generate considerable amounts of cholesta-3,5-diene. Some rearrangements of cholesterol in H(2)SO(4) resemble the diagenesis pathways of sterols and may furnish useful lipid biomarkers for characterizing geological systems.