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+/NADPH) and nonphosphorylated (NAD+/NADH) forms ( Through the process of natural selection, Nature has evolved well-adapted macromolecular structures that interact with biological small molecules. Oxidoreductases, for example, rely on the nicotinamide coenzymes to supply them with the redox equivalents required to sustain their catalytic cycles. Two forms of natural coenzymes exist: the phosphorylated (NADP/NADPH) and nonphosphorylated (NAD/NADH) forms ( Figure 1 A). Nicotinamide coenzymes essentially contain two structural motifs, the nicotinamide moiety conferring their electrochemical function (i.e., serving as an electron source or sink in the form of a hydride) and the adenosine dinucleotide moiety conferring the separation between anabolic and catabolic pathways. NADP is involved in anabolic redox processes, whereas NAD is mostly found in processes dealing with energy metabolism. While this separation between different metabolic pathways is essential for cellular survival, it is irrelevant in chemical applications of redox enzymes. Therefore, there is a renewed interest in the design of simple synthetic analogues of the natural nicotinamide coenzymes. The laboratory-based design and synthesis of small molecule biomimetics that can functionally substitute (or even outperform) those available in Nature is a major challenge. The development of biomimetics that can be synthesized easily and exploited widely would be a game changer in establishing new manufacturing technologies that would be too expensive using natural biological molecules. The natural coenzymes NAD(P)H are prohibitively expensive and chemically too unstable for stoichiometric use in fine and specialty chemicals manufacture. This has prevented their general uptake as a source of reducing equivalents in biocatalytic oxidoreductase-catalyzed reactions and has led to the development of in situ regeneration systems to replenish NAD(P)H (e.g., using enzymatic, photochemical, and electrochemical approaches (1-5) ) or the use of hydrogen-borrowing biocatalytic cascades. (6-9) In turn, the limited stability and expense of natural nicotinamide coenzymes have driven a search for more stable synthetic nicotinamide coenzyme analogues that can interface generally with biological oxidoreductase catalysts. (10)

+, this hydride is then transferred in the oxidative half-reaction from the flavin N5 position to the activated alkene substrate ( Nicotinamide-dependent biocatalysts have wide-ranging potential in biocatalytic transformations. Ene reductases (ERs) from the Old Yellow Enzyme family (EC 1.3.1.31) are particularly attractive as they are a group of broad specificity biocatalysts that catalyze the asymmetric reduction of activated C═C bonds, forming up to two new stereogenic centers at the expense of the natural nicotinamide coenzyme NAD(P)H as an electron source. (11-13) In particular, α,β-unsaturated carbonyl compounds (e.g., enals and enones) and nitroalkenes are excellent substrates. (14-17) In general, α,β-unsaturated diesters as well as α,β-unsaturated diacids are also reduced by ERs. (18) In contrast, the efficient reduction of α,β-unsaturated monoacids or monoesters requires an additional electron-withdrawing group in the α- or β-position in order to activate the alkene moiety. (19-22) The ability to form new stereogenic centers and the wide acceptance of different substrate types are driving the exploitation of ERs toward novel applications in redox biocatalysis and implementation in key industrial processes. (22-25) ERs have been studied extensively over the past decade, and there is detailed information known, such as their structure, reaction mechanism, substrate scope, kinetic properties, and biocatalytic approaches. (26, 27, 13) The catalytic cycle of ER-catalyzed reactions can be divided into two separated half-reactions: in the reductive half-reaction, a hydride is transferred from NAD(P)H to the enzyme-bound flavin (flavin mononucleotide; FMN). After release of oxidized NAD(P), this hydride is then transferred in the oxidative half-reaction from the flavin N5 position to the activated alkene substrate ( Figure 1 B). (28-30)

Figure 1 Figure 1. (A) Structure of NAD(P)H and synthetic nicotinamide biomimetic mNADHs (1–5) and (B) the catalytic cycle of ER-catalyzed reactions.