Cofactor regeneration systems reactions

Biocatalytic ledox reactions offer great synthetic utility to organic chemists. The majority of oxidase-catalyzed preparative bioconversions are still performed using a whole-ceU technique, despite the fact that the presence of more than one oxidoreductase in cells often leads to product degradation and lower selectivity. Fortunately, several efficient cofactor regeneration systems have been developed (160), making some cell-free enzymatic bioconversions economically feasible (161,162). 

Figure 19.5. Cofactor regeneration systems for NAD(P)H-dependent enzyme reactions. The enzyme-coupled one involving GDH (a), that involving FDH (b), and the substrate-coupled one (c). AR1, aldehyde reductase from S. salmonicolor Leu DH, leucine dehydrogenase ADH, sec-alcohol dehydrogenase.

CRS = cofactor regeneration system Figure 20.5 Overview of monooxygenase reactions in synthetic chemistry. 

This class of reagents holds the most promise for rapid development in the near future as most reactions are asymmetric. The problems being overcome are the tight substrate specificity of many enzymes and the need for cofactor regeneration. Systems are now being developed for asymmetric synthesis rather than resolution approaches. Some of these reactions are discussed in Chapter 13. 

The use of isolated enzymes to form or cleave P-O bonds is an important application of biocatalysts. Restriction endonucleases, (deoxy)ribonucleases, DNA/ RNA-ligases, DNA-RNA-polymerases, reverse transcriptases etc. are central to modern molecular biology(1). Enzyme catalyzed phosphoryl transfer reactions have also found important applications in synthetic organic chemistry. In particular, the development of convenient cofactor regeneration systems has made possible the practical scale synthesis of carbohydrates, nucleoside phosphates, nucleoside phosphate sugars and other natural products and their analogs. This chapter gives an overview of this field of research. 

Several cofactor regeneration systems were based on FDH. The substrate formate is an inexpensive, stable, and innocuous compoimd, while CO2, which is produced by FDH, can be easily removed from the reaction by evaporation. A general drawback of FDH is, however, its low specific activity [262]. More stable FDH variants have been engineered and successfully applied... 

SAM and related cofactors, isolated or produced by total synthesis, are highly priced and their stoichiometric consumption requires one or even more equivalents. An economically feasible cofactor-dependent biocatalysis thus rehes on the substoichiometric or even catalytic use of the cofactor associated with a cofactor regeneration system, ideally in a cascaded reaction sequence. Such enzymatic conversions are assisted by an additional recycling reaction which restores the... 

Instead, the orthogonal multienzymatic reactions are always cascade processes by definition. However, this type of multienzymatic processes has been largely investigated in the past especially for the development of enzymecofactor regeneration systems. These studies not only allowed the wide exploitation of cofactor-dependent enzymes, such as NAD(P)H-dependent dehydrogenases, by making their reactions economically feasible but were also useful in identifying relevant process options for the development of effective multienzymatic reaction systems (3). 

In fact, successful deracemization was not achieved when using a cell-free extract of the Alcaligenes cells, which showed NADH-dependent (R)-selective ADHs active in the oxidation reaction, coupled to a NADH-dependent (S)-selective ADH for the reduction reaction. Instead, it was easily achieved when using microbial whole cells in the biooxidation reaction or when combining the same cell-free extract with a NADPH-dependent ADH. In both cases, possible short circuits between the two cofactor regeneration systems were therefore avoided, thanks to the compartmen-talization of one of the involved biocatalysts in the cells or to the use of enzymes with different cofactor specificity. 

However, when biocatalysts showing a sufficient cofactor specificity are not naturally available, possible undesired interferences between the cofactor regeneration systems can be circumvented by different approaches still maintaining the one-pot fashion of the multienzymatic process. For example, in the biocatalyzed synthesis of 12-ketoursodeoxycholic acid, the performances of the investigated cascade system, in which five enzymes were involved in concurrent oxidation and reduction reactions at different sites of the starting substrate cholic acid, were significantly improved by simple compartmentalization of the oxidative and reductive enzymes in two membrane reactors (Scheme 11.4b) [11]. 

Chapters 8-12 will present the improvements brought about by the optimization of reaction conditions (e.g., the choice of the cofactor regeneration system, the nature of the solvent, and the employment of in situ product removal technologies)... 

Enzymatic cofactor regeneration can be subdivided into two categories the enzyme-coupled approach, where two different enzymes are used (one for the production reaction, and one for the regeneration reaction) and the substrate-coupled approach, where one and the same enzyme is used for both production and regeneration (E = E2). The most convenient and commonly used enzymatic regeneration systems are summarized in Table 43.1. 

---