Biocatalytic reduction system

This example and the next one (Sect. 15.1.4.5) using G. candidum show that the biocatalytic reduction system is very beneficial for the reduction of aliphatic ketones over a non-enzymatic system where no report on highly enantioselective (> 99% ee) reduction of unfunctionalized dialkyl ketones can be found, to the best of our knowledge. 

Asako, H., Shimizu, M Makino, Y and Itoh, N. (2010) Biocatalytic reduction system for the production of chiral methyl (R)/(S)-4-bromo-3-hydroxybutyrate. Tetrahedron Lett.. 51, 2664—2666. 

Formate is one of the most representative hydrogen sources for the biocatalytic reduction because CO2 formed by the oxidation of formate is released easily from the reaction system [4]. For example, for the reduction of aromatic ketones by the... 

Development of new reduction systems that reduce sterically hindered compounds The reported examples of reduction of carbonyl compounds are usually for the substrates that can be easily reduced such as methyl ketones. Since the demand for reduction of various types of compounds is increasing, investigation of new biocatalytic reductions is required. Photosynthetic organisms are not investigated yet, and they may have new type of enzymes, which can reduce sterically hindered compounds. 

The biocatalytic reduction of carboxylic acids to their respective aldehydes or alcohols is a relatively new biocatalytic process with the potential to replace conventional chemical processes that use toxic metal catalysts and noxious reagents. An enzyme known as carboxylic acid reductase (Car) from Nocardia sp. NRRL 5646 was cloned into Escherichia coli BL21(DE3). This E. coli based biocatalyst grows faster, expresses Car, and produces fewer side products than Nocardia. Although the enzyme itself can be used in small-scale reactions, whole E. coli cells containing Car and the natural cofactors ATP and NADPH, are easily used to reduce a wide range of carboxylic acids, conceivably at any scale. The biocatalytic reduction of vanillic acid to the commercially valuable product vanillin is used to illustrate the ease and efficiency of the recombinant Car E. coli reduction system." A comprehensive overview is given in Reference 6, and experimental details below are taken primarily from Reference 7. 

The versatility of these reduction systems are demonstrated by the next few examples, chosen to show that a wide range of functional groups are tolerated by these biocatalysts and how the biotransformations can be applied to synthesize intermediates in API production. Zhu et from Biocatalytics have devel-... 

In a further example, phenyl boronic add-functionalized CdSe/ZnS QDs were used to bind NADor NADH via the boronic acid ligand to form boronate esters. The quenching of the QDs luminescence by the NAD + cofactor, via an ET quenching route, was substantially more efficient than was the quenching of QDs by NADH. This difference in quenching between NAD and NADH enabled QDs to be used for the luminescence analysis of NAD -dependent enzymes and their substrates, such as the AlcDH/ethanol system [141] (Figure 6.31a). The biocatalytic reduction of the... 

In 2006, Kosjek et al. reported a similar methodology for the biocatalytic reduction of a,jS-unsaturated ketones, providing the corresponding chiral allyhc alcohols in both high enantio- and diastereoselectivities, as depicted in Scheme 3.10. The method employed the enzyme KRED 108 including an NADPH cofactor recycling system using KRED 104/2-propanol. 

Fig. 20 Bioelectrocatalytic reduction systems using (a) AlcDH and FNR for the biocatalyzed transformation and the regeneration of NADPH, respectively, and (b) using AlcDH for both processes, where the biocatalytic cycle Substrate 1/Product 1 performs the mediating function for the NADPH regeneration and the second biocatalytic cycle results in the formation of the aim product 2.

Figure 8.15 Biocatalytic reductions in a recirculating diphasic flow system MS. Source By courtesy ofThe Royal Society of Chemist [57].

Solvent Systems for Biocatalytic Reductions 243 Table 9.3 Influence of (co)solvents in the bioreduction of 2 -chloroacetophenone. 

The use of biphasic aqueous-organic systems in biocatalytic reductions is of great interest because the enzyme and its cofactor are dissolved in the aqueous phase, where the reaction takes place, while the hydrophobic substrate and product are mostly located in organic solvent layer and partitioned into the aqueous phase. This distribution reduces the concentrations of toxic substrate and product around the enzyme in aqueous layer and relieves the enzyme from substrate and product inhibition. Other distinctive features of this biphasic system are simple separation, easy regeneration of the enzyme, and easy recovery of the products. However, in this system, the reaction rates are relatively low because of a low rate of mass-transfer across the interface. Although this hindrance can be eliminated by intensive agitation, the increased interface often results in faster denaturation and inactivation of the enzyme. 

There is a range of process strategies for the scale-up of a given biocatalytic reduction. There is a clear need for guidance and comparison of systems to help formulate a decision-making framework on a more quantitative basis. 

An alternative was provided by Codexis that developed a route to a chiral precursor to ezetimibe based on the asymmetric biocatalytic reduction of 5-[(4S)-2-oxo-4-phenyl(l,3-oxazolidin-3-yl)]-l-(4-fluorophenyl)pentane-l,5-dione to (4S)-3-[(5S)-5-(4-fluorophenyl)-5-hydroxy-pentanoyl]-4-phenyl-l,3-oxazolidin-2-one (Figure 13.5a). Ketoreductases from LactohaciUus sp. identified as potential catalysts were improved via protein engineering and the best mutant was implemented in a process running at 100 g/1 with a coupled-enzyme cofactor recycling system (GDH and glucose), allowing formation of the alcohol with >99.9% ee [20]. 

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