Substituted benzoic acid amides as the modifiers of the ethanol bioelectrooxidation using NAD+-dependent alcohol dehydrogenase

Abstract

One of the methods for the alcohols determination is based on ethanol bioelectrooxidation with NAD+-dependent alcoholdehydrogenase. This enzyme has been used as a bioselective compound in ethanol electrochemical biosensors. The development issues of this type of biosensors are associated with the need to create special conditions for the NADH electrooxidation, the loss of catalytic activity and the instability of the active complex ADH-NAD+ on the electrode surface. Two substituted benzoic acid amides , 2,4-dichlorbenzamide and 2,4-dichlor-5-methylbenzamide, have been as modulators of alcohol dehydrogenase (ADH) activity. 2,4-dichlorbenzamide inhibited the rate of the forward ADH reaction by 30% and 2,4-dichlor-5-methylbenzamide - by 60%, respectively, in the concentration of 100 µM. This can stabilize the structure of the enzyme and temporarily prevent its inactivation in the paste electrode. The rate of the reverse ADH reaction was suppressed up to 80%. Inhibition of the reverse ADH reaction creates conditions for the accumulation of NADH and its subsequent regeneration. The studies have shown that the introduction of several substituents into the aromatic ring causes a more complex interaction of benzoic acid amides with ADH compared to unsubstituted amides.