Mechanism of Production of Hydroxyl Radicals in the Copper-Catalyzed Oxidation of Dithiothreitol

We have undertaken detailed studies of the mechanisms involved in the production of OH radicals during the copper-catalyzed oxidation of dithiothreitol (DTT). Most of these studies were conducted in real time, detecting ^{\bullet}{\rm OH}$ based on its reaction with coumarin-3-carboxylic acid to produce the fluorescent derivative 7-hydroxycoumarin-3-carboxylic acid (7-OHCCA). Studies of the kinetics of oxidizing DTT show that production of ^{\bullet}{\rm OH}$ occurs in two stages: an initial lag period during which there is little production of 7-OHCCA, and a second reaction phase during which there is more rapid generation of ^{\bullet}{\rm OH}$. The duration of the initial reaction period depends on the concentrations of both DTT and ${\rm Cu}2^{+}$. During this initial stage, oxygen consumption is high, although an increasing concentration of Cu2+ decreases the oxygen consumption. The rate of production of ^{\bullet}{\rm OH}$ during the second phase of the reaction depends on the concentration of Cu2+, but little oxygen is consumed. A mechanism is proposed whereby a ${\rm Cu}^{2+}-{\rm DTT}$ complex is formed and catalyzes oxidation of free DTT via formation of an oxygen-containing intermediate. The second phase of the reaction begins after complete oxidation of free DTT and involves production of ${\rm O}_{2}^{\bullet -}$ and H2 O2 followed by generation of ^{\bullet}{\rm OH}$ via reduction of H2 O2 by cuprous ion.