Synthesis with cofactor regeneration

Fig. 24 Electroenzymatic synthesis of L-lactate coupled with cofactor regeneration using a FAD-modified electrode...

Enzymes have been employed as catalysts in organic synthesis for many years, primarily for the construction of small chiral molecules. " Most synthetic chemists, however, perceive enzymes as being absolutely specific for their natural substrates and thus limited in their application to organic synthesis. The use of enzymes is also plagued by concerns of enzyme stability and requirements for the use of cofactors. Developments in biotechnology have, however, solved many of the problems associated with cofactor regeneration and enzyme stability. 

Sonoike, S., Itakura, T, Kitamura, M., and Aoki, S. (2012) One-pot chemoenzymatic synthesis of chiral 1,3-diols using an enantioselective aldol reaction with chiral Zn complex catalysts and enzymatic reduction using oxidoreductases with cofactor regeneration. Chem. Asian J., 7 (1), 64-74. 

An impressive one-pot six-step enzymatic synthesis of riboflavine from glucose on the laboratory scale has been reported with an overall yield of 35-50%. Six different enzymes are involved in the various synthesis steps, while two other enzymes take care for the in situ cofactor regenerations [12]. This example again shows that many more multi-enzyme cascade conversions will be developed in the near future, as a much greater variety of enzymes in sufficient amounts for organic synthetic purposes will become available through rapid developments in genomics and proteomics. 

The other four chapters of this volume are contributed by heads of department from the Institute of Enzyme Technology at Heinrich Heine University, Diisseldorf, of which Prof. Kula is the director. First of all, Privatdozent Dr. Werner Hummel concerns himself with New Alcohol Dehydrogenases for the Synthesis of Chiral Compounds . Up to just a few years ago, it was believed that due to the cofactor regeneration problem oxidoreductases could not be applied in practice. In his contribution, Dr. Hummel shows that an interesting pathway to chiral alcohols has been opened up by the provision of suffi-... 

Enzymatic synthesis of E-tm-leucine is another example of the use of isolated enzymes (Bommarius et al, 1995). An NADH-dependent leucine dehydrogenase was used as a catalyst for the reductive amination of the corresponding keto acid together with formate dehydrogenase (FDH) and formate as a cofactor regenerator (Fig. 19.5b Shaked and Whitesides, 1980 Wichmann et al, 1981). Furthermore, a unique membrane reactor system involving FDH and PEG-modihed-NAD for continuous NADH regeneration... 

Wild type cells often contain several enzymes which carry out the same reaction. Unfortunately, in many cases these enzymes produce compounds with opposite stereoselectivity. Therefore, whole cells in which such enzymes are present cannot be applied for the synthesis of enantiomerically pure products. To increase the stereoselectivity of a whole cell reaction, recombinant DNA techniques need to be applied. Very common is the overexpression of the enzyme, which catalyzes the particular reaction in a suitable heterologous host such as E. coli. The simultaneous overexpression of an enzyme which catalyzes the regeneration of the consumed cofactor is highly efficient. Ideally, growing cells should provide simultaneously the enzyme for the desired reaction as well as the cofactor regenerating enzyme. Such so-called designer cells seem to be very promising for technical applications. 

In summary, the concept of multienzyme reactions with integrated cofactor regeneration has been shown to be useful for sequential synthesis of rather complicated heterooligosaccharides. This conception opens up new perspectives for the synthesis of glycosides having up to three or four different glycosyl units in one-pot reactions. 

Figure 8 Synthesis of a-D-Galp-(l-3)-/3-D-Galp-(l-4)-0-D 22 with in situ cofactor regeneration.

Using an L-amino acid dehydrogenase in the presence of a formate dehydrogenase for cofactor regeneration, a prochiral keto acid is converted with high yield and en-antioselectivity. Furthermore, biocatalytic transaminations as well as Michael-addi-tions are important reactions for the large-scale synthesis of L-amino acids. 

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