The Oxidative Addition Reaction between Compounds of Resorufin (7-Hydroxy-3H-phenoxazin-3-one) and 2-Mercaptoethanol

2-Mercaptoethanol reacts with the methanesulphonate, the dimethylcarbamate, and the ethyl ether of resorufin (7-hydroxy-3H-phenoxazin-3-one) at 25°C and pH 7.4 to give colored products with maximal absorbances at 433, 442, and 481 nm, respectively. NMR spectra of the products show that the hydrogen atom on C-2 has been specifically replaced by the -SCH2CH2OH group. It is concluded that the thiol adds to the quinonimine ring and that the first-formed product then undergoes spontaneous oxidation to reform the resorufin structure. The implications of these studies toward biochemical work with resorufin derivatives is discussed.