(582o) Soft Immobilization of Redox Mediator for Electroenzymatic Synthesis

Enzyme-catalyzed reactions have emerged as an alternative strategy to conventional chemical synthesis for the production of pharmaceuticals, food, cosmetics, and even bulk chemicals. Particularly, oxidoreductases, one of the major classes of enzymes, are very promising in that they catalyze reactions involving oxygen insertion, hydrogen extraction, or electron transfer. In spite of their efficiency, however, the application of redox enzymes to chemical industries has been very limited; a major hindrance to their applications is that nicotinamide coenzymes, which most of them require as reduction or oxidation equivalents, are too expensive to be supplied in stoichiometric quantities. Therefore, efficient regeneration of the coenzymes is essential for successful applications of redox enzymes to industrial synthetic processes.

The reduced form of nicotinamide cofactor (nicotinamide adenine dinucleotide), NADH, is employed for more diverse enzymatic reactions than its counterpart (NAD⁺): reduction catalyzed by dehydrogenases; hydroxylations and epoxidations by oxygenases. To regenerate NADH in vitro, chemical, electrochemical, or enzymatic methods have been employed. Electrochemical regeneration, in which NAD⁺ is reduced by cathodic potential, is often favored owing to no requirement of impurities such as reducing agents, secondary enzyme, and co-substrate when compared with the other methods. Moreover, electrons are one of the cheapest redox equivalents available.

The NAD⁺/NADH redox pair has a formal potential of −0.32 V vs. the standard hydrogen electrode (pH 7), but direct electrochemical reduction of NAD⁺ requires a large overpotential. In addition, the direct reduction on unmodified electrodes mainly leads to the formation of  an inactive dimer and isomers. In order to overcome these limitations of the direct reduction, redox mediators capable of transferring electrons from electrodes to NAD⁺ were introduced. Among them, pentamethylcyclopentadienyl-2,2'-bipyridine-chloro-rhodium(III) [Cp*Rh(bpy)Cl]⁺, which is hydrolyzed in the form of [Cp*Rh(bpy)H₂O]²⁺ in aqueous medium, has been the most widely used, showing high regioselectivity (>99%) over a broad pH and temperature range.

For long-term operation of electroenzymatic processes, not only enzymes but also mediators should be immobilized. However, much effort has not been made to immobilize the organometallic mediator in an electrocatalytically-active form onto electrodes. It was found that substituents bearing nucleophilic moieties such as S- or N-containing groups on the bipyridine ligand interfered with the formation of the active Rh hydride complex, resulting in inactivation of the mediator. This means that immobilization methods based on covalent binding are not suitable for this redox mediator.

In this work, soft immobilization of the Rh complex mediator is carried out. The organometallic mediator is immobilized on solid electrode surfaces using non-covalent interactions. The electrochemical reactions were performed in a conventional three-electrode cell with a potentiostat (Gamry G750, USA): the working, counter, and reference electrodes were mediator-immobilized electrodes, a platinum wire, and Ag/AgCl (saturated KCl), respectively. NAD⁺-reducing activity and repeated usability of the immobilized mediator are examined using cyclic voltammetry and spectrophotometry. Cyclic voltammograms and amperometric responses are recorded with mediator-immobilized electrodes in the absence and in the presence of NAD⁺. The immobilized mediator is repeatedly used and its catalytic activity is measured after each use. Finally, the indirect electrochemical NADH regeneration is coupled with synthesis reactions catalyzed by dehydrogenases.

References

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