Reduction cofactor recycling

The asymmetric reduction of prochiral functional groups is an extremely useful transformation in organic synthesis. There is an important difference between isolated enzyme-catalyzed reduction reactions and whole cell-catalyzed transformations in terms of the recycling of the essential nicotinamide adenine dinucleotide (phosphate) [NAD(P)H] cofactor. For isolated enzyme-catalyzed reductions, a cofactor recycling system must be introduced to allow the addition of only a catalytic amount (5% mol) of NAD(P)H. For whole cell-catalyzed reductions, cofactor recycling is automatically achieved by the cell, and the addition of a cofactor to the reaction system is normally not required. 

In respect of designing an economic production process, the stoichiometric cofactor required in carbonyl reductions or the respective oxidation reactions needs to be minimized that is, enabled by recycling of the cofactor. The measure for the efficiency of the recycling process is the total turnover number (TTN), which describes the moles of product synthesized in relation to the moles of cofactor needed. The different approaches in cofactor recycling were recently reviewed by Goldberg et at. [12]. 

Groger, H., Hummel, W., Rollmann, C. et al. (2004) Preparative asymmetric reduction of ketones in a biphasic medium with an (5)-alcohol dehydrogenase under in f/w-cofactor-recycling with a formate dehydrogenase. Tetrahedron, 60 (3), 633-640. 

Biosynthetic production of thymidine is overall a complex process combining the controlled introduction of a novel biotransformation step into a biological system with selective enhancement or knock-out of a series of existing metabolic steps. Metabolic engineering to enhance cofactor recycling at both ribonucleotide reduction and dUMP methylation steps has important parallels in other systems, as whole-cell biotransformations are frequently employed as a means to supply, in situ, high-cost and usually labile cofactors. 

The use of water-miscible organic solvent-water mixtures is a particularly attractive method for use with cofactor-dependent enzymes due to its simphcity. The high water content can allow dissolution of both enzyme and cofactor, whilst the water-miscible solvent can provide a dual role in both substrate dissolution and as a cosubstrate for cofactor recycling (substrate-coupled cofactor recycling).The asymmetric reduction of a ketone intermediate of montelukast using an engineered ADH in the presence of 50 % v/v isopropanol offers a powerful demonstration of this methodology (Scheme 1.55). 

Naturally occurring redox enzymes have been successfully exploited for asymmetric synthesis for some years.1 Although impressive chemo-, regio-, and enantioselectivities have been achieved in some cases, these biocatalysts have prescribed selectivity and often require expensive cofactors that must be recycled for preparative work. Catalytic antibodies offer an attractive alternative, since they are not limited a priori by Nature s choices. Thus the need for cofactor recycling can be circumvented through the use of inexpensive oxidants and reductants, and, as we have seen above, selectivity can be tailored through appropriate hapten design. 

Fig. 39 Conversion of 5-(l,3-dioxolan-2-yl)-2-oxo-pentanoid acid to allysine ethylene acetal by reductive amination using phenylalanine dehydrogenase (PDH) and formate dehydrogenase (FDH) for cofactor recycling...

Much higher productivities can be obtained using isolated enzymes or cell extracts [118]. This approach is therefore highly preferred. Because of the importance of whole cell technology for biocatalytic reduction a few examples will be given. However the main part of this chapter will be devoted to industrial examples of bioreduction involving isolated enzymes and cofactor recycling. 

Formate dehydrogenase in conjunction with polyethyleneglycol-immobilized nicotinamide adenine dinudeotide has been used to good effect as a cofactor recycle system (39). The alcohol dehydrogenase from Thermoanaerobium hrockii catalyzed the reduction of ketones independently when driven by the cooxidation of isopropanol (40,41). 

A variation on the transamination approach that also starts with an a-keto acid substrate is to perform a reductive amination catalyzed by amino acid dehydrogenases (dHs) (Scheme 9.31) in combination with the formate dH cofactor recycling system, although other reducing systems can be used. " The generation of carbon dioxide from formate drives the coupled reactions to completion. 

An important technical issue is the large-scale applicability of co-factor-dependent enzymatic systems. It is generally accepted that, e.g., NADH-requiring oxidoreductases can easily be used in whole-cell biocatalysis such as baker s yeast-mediated reductions, where the cofactor recycling step is simultaneously performed within the intact cell, driven by the reduction equivalents introduced via the external carbon and energy source (glucose). 

Simultaneous biocatalytic oxidation and reduction was also reported for the resolution of secondary alcohols through combination of molecular oxygen and stereoselective reducing agent (alcohol dehydrogenase) with a cofactor-recycling... 

The synthesis of optically pure L-phenylglycine via the deracemization of mandelic acid was reported via three steps (racemization, enantioselective oxidation and stereoselective reductive amination). Racemization by mandelate racemase combined with simultaneous oxidation and reduction reactions with cofactor recycling gave the amino acid in 97% ee and 94% yield (Scheme 4.43) [96]. 

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. 

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]. 

J ,5J )-Dihydrocarvone, a minor component of essential oils produced by plants, has been recently used in the synthesis of antimalarial drugs [91]. (+)-Dihy-drocarvone was prepared via reduction of (5i )-carvone with pentaerythritol tetrani-trate reductase (PETNR) from Enterohacter cloacae st. PB2 at the expense of NADPH in the presence of glucose/GDH cofactor recycling system in 88% yield with 95% de (Figure 13.14) [43]. It is important to note that (5S)-carvone was reduced to the diastereomeric (2 R, 5 S)-dihydrocarvone in 88% de. 

A rare case of enzyme catalyzing imine reduction reaction (see also Section 13.4.4) is the stereoselective reduction of dihydrofolic acid to (6S)-tetrahydrofolic acid by dihydrofolate reductase (DHFR) at the expense of NADPH. This biocatalytic step was employed in the synthesis of (S)-leucovorin [(6S)-5-formyl-5,6,7,8-tetrahydro-folate], a drug used in cancer chemotherapy. DHFR produced by E. coli was combined with a GDH/glucose cofactor recycling system and yielded (6S)-tetrahy-drofolic acid, which upon formylation furnished L-leucovorin with >99.5% de (Figure 13.31) [37-39]. 

NAD(P)H cofactor recycle systems for CRED mediated ketone reductions. 

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