Identification of acetaminophen polymerization products catalyzed by horseradish peroxidase

Horseradish peroxidase catalyzed the H2O2-dependent oxidation and polymerization of acetaminophen. Six acetaminophen polymers were isolated from horseradish peroxidase reaction mixtures by semipreparative high pressure liquid chromatography. Chemical structures were determined by a combination of electron impact and chemical ionization mass spectrometry and 500-MHz proton magnetic resonance spectroscopy. Two dimers, three trimers, and one tetramer were identified. The polymers formed primarily through a covalent bond between carbons ortho to the hydroxyl group, and to a lesser extent, between the carbon ortho to the hydroxyl group and the amino group of another acetaminophen molecule. Greater than 99% of the polymerization reaction products were quenched by the addition of 2.0 mM ascorbate. High acetaminophen concentration favored dimer formation, whereas low acetaminophen concentration favored formation of trimers and tetramers. Since approximately 1 mol of H2O2 was consumed per mol of covalent ligand formed between acetaminophen molecules, these products probably result from free radical termination reactions.