Drugs which contain a thiol functionality may be enzymatically or nonenzymatically oxidized to reactive metabolites, some of which cause adverse reactions. The synthesis of disulfide prodrugs to obviate unwanted drug side effects requires the development of novel catalysis. Herein, we describe the synthesis, structure-activity relationship, and mechanism investigations of the oxidation of model thiophenols with isoalloxazine disulfide formation catalysts. m-Nitrothiophenol reacts with the electron-deficient 8-cyano-N-3-(mercaptoalkyl)-10-phenylisoalloxazines and non-electron-deficient N-3-(mercaptoalkyl)-10-methylisoalloxazines to produce m-nitrothiophenol disulfide. m-Nitrothiophenol reacts with electron-deficient 8-cyano-10-phenyl-3-isoalloxazinepentanoic acid or 10-methyl-3-isoalloxazinepentanoic acid to form m-nitrothiophenol disulfide at a reduced rate, or not at all, respectively. Of the substituted isoalloxazines studied, electron-deficient isoalloxazines containing an N-3-mercaptoalkyl side chain were most efficient at catalyzing m-nitrothiophenol disulfide formation. Non-electron-deficient isoalloxazines without an N-3-alkyl mercaptan side chain did not catalyze m-nitrothiophenol oxidation. Electron-deficient isoalloxazines without N-3-alkyl mercaptan side chains catalyzed m-nitrothiophenol oxidation at 1/20 the rate for isoalloxazine 5. From kinetic and product studies, the differences in catalytic activity of 1-10 were judged to be due to changes in the chemical properties of the isoalloxazines and the ability to stabilize intramolecular thiol attack on the C(4a)-N(5) bond of the isoalloxazine. Electron-deficient isoalloxazines may be useful catalysts for the syntheses of disulfide prodrugs.