N-Arylhydroxamic acids as novel oxidoreductase substrates

N-Arylhydroxamic acids (AHAs) are promising novel N-OH mediators for oxidoreductase catalysis. They are electrochemically active compounds with a redox potential of 0.31-0.41 V vs. SCE. Representative oxidoreductases, e.g. fungal peroxidase from Coprinus cinereus (rCiP), catalyze the oxidation of AHAs with apparent bimolecular constants (k_ox) of 7.1·10³ to 1.5·10⁷ m^-1s^-1 at pH = 8.5 and 25°C. The limiting step in substrate oxidation was the reduction of compound II (Cpd II). The oxidation constants of N-hydroxyacetanilide (1a) and N-hydroxy-N-phenylbenzamide (2a), determined by a stopped-flow and steady-state method, were similar. The decrease in the reduction rate of Cpd II reduction rate decrease occurred at pK_a = 8.5 for 1a and 7.7 for 2a. The nitroxyl radical of 1a, an intermediate in the oxidation, shows decreasing stability at alkaline pH. The structure-activity relationships (SARs) of these AHAs were analyzed within the framework of Marcus cross-relationship, and by using ab initio quantum chemical calculations. A linear correlation of log(k_ox) vs. redox potential was only indicated for benzamides, as predicted by electron transfer theory. Acetamides showed the opposite tendency: the constant (k_ox) increased if the potential of substrate increased. The correlation between log(k_ox) and HOMO (Highest Occupied Molecular Orbital) energy revealed that for benzamides the reactivity decreased with decreasing HOMO energy, whereas for acetamides the reactivity increased if the HOMO energy decreased. The rather low reactivity of these AHAs and the unexpected dependence on the redox potential and the HOMO energy could be explained by the electronic structure of the AHAs and the substrates docking in the active center of the enzyme.