Cofactor recycling systems

Each reaction was performed with a CYP biocatalyst concentration of 1 pM (1000 nmol L 1), in the presence of a corresponding CYP reaction mix containing reduced nicotinamide cofactor and a cofactor recycling system at 30 °C, with agitation to promote oxygen transfer to the reaction solution. 

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. 

It is only recently that isolated enzymes have been used in the presence of appropriate cofactor recycling systems.14 Not long ago, application of the whole-cell system was the only way to get high yields and high ee in enzyme-catalyzed organic synthesis. 

Maurer, S. C., Schulze, H., Schmid, R. D., and Urlacher, V. 2003. Immobilisation of P450BM-3 and an NADP(+) cofactor recycling system Towards a technical application of heme-containing monooxygenases in fine chemical synthesis. Adv. Synth. Catalys.,345, 802-810. 

For these reasons, recent efforts have been aimed at cheap, effident cofactor recycle systems for use with cell-free alcohol dehydrogenase preparations. Regeneration of the nicotinamide adenine dinudeotide cofactor required for alcohol synthesis can be catalyzed by a second enzyme or reduced by the same enzjme, provided the overall equilibrium is favorable. 

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). 

Leucine dH is the enzyme used as the biocatalyst in the process commercialized by Degussa AG (Hanau, Germany) to produce v-tert-leucine (L-Tle) [24]. This unnatural amino acid has found widespread use in peptidomimetic drugs in development, and the demand for this unique amino acid continues to increase [44]. This process, which has been the subject of much study, requires a cofactor recycling system (Scheme 3. R = (CHj)jC) [45.46]. Similar to phenylalanine dH, leucine dH has been used to prepare numerous unnatural amino acids because of its broad substrate specificity [33.47.48]. 

An alternative method for driving the reaction equilibrium of transaminations was developed by Hohne et al. (Figure 14.44) [65]. Alanine served as the amine donor, and pyruvate decarboxylase was used to remove the pyruvate coproduct by decarboxyl ation to acetaldehyde. An advantage of pyruvate decarboxylase over lactate dehydro genase is that it requires no cofactor recycling system, and the high volatility of the coproducts allows for the desired shift of equilibrium. 

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. 

In every catalytic process, the operational stability of the catalyst under process conditions is a key parameter. It is described by the dimensionless total turnover number (TTN), which is determined by the moles of product formed by the amount of catalyst spent and - in other words - it stands for the amount of product which is produced by a given amount of catalyst during its whole lifetime. If the TONs of repetitive batches of a reaction are measured until the catalyst is dead, the sum of all TONs would equal to the TTN. TTNs are also commonly used to describe the efficiency of cofactor recycling systems. 

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. 

In summary, many different cofactor recycling systems have been estabhshed. In vitro as well as in vivo systems display remarkable performance and they range from simple approach based on two single enzymatic to fusion-protein strategies. 

Scheme 3.19 Metalloenzyme ATHase S112A combined in a chemo-enzymatic cofactor recycling system for the oxygenation reaction catalyzed by the hydroxybiphenyl monooxygenase from P. azaleica.

Alternatively, 41 can be formed from glycerol by successive phosphorylation and oxidation effected by a combination of glycerol kinase and glycerol phosphate dehydrogenase, tvith an integrated double ATP/NAD+ cofactor recycling system [148]. 

Hogan, M.C. Woodley, J.M. (2000). Modelling of two enzyme reactions in a linked cofactor recycle system for chiral lactones synthesis. Chemical Engineering Science, Vol. 55, No.ll, (June 2000), pp 2001-2008... 

Fischer and Pietruszka have reported the efficient synthesis of both enantiomers of ethyl 5-hydroxyhept-6-enoate (3, Scheme 4.3) using isolated alcohol dehydrogenases (ADHs) and 2-propanol as cofactor recycling system in a coupled-substrate approach [33]. 6-Hydroxy esters have been used as key intermediates for a variety... 

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]. 

Methyl isobutyl ketone was notably reduced in high yield and with excellent stereoselectivity to (f )-4-methyl-2-butylamine (85% isolated yield and 99.8% ee) by the engineered enzyme, combined with a GDH cofactor recycling system (Figure 13.37) [35]. 

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

When the GDH cofactor recycle system was used with toluene cosolvent, 145 g/L of substrate was tolerable with a space-time yield of 51.6 g/L/day. 

CRED enzymes with yields more than 90% and ee s up to 99% using GDH as the cofactor recycle system as shown in Scheme 6.23 [36]. 

Merck scientists reported a practical enantioselective synthesis of cis-2,5-disubstituted pyrrolidines where the key chiral step 3 is a dynamic kinetic bioreduction as shown in Scheme 6.29. The bioreduction is performed at pH 7 and 30 °C using GDH as the cofactor recycle system yielding 99% ee and more than 90% assayed yield [43]. 

Nakamura K, Yamanaka R. Light mediated cofactor recycling system in biocatalytic asymmetric reduction of ketone. /. Chem. Soc. Chem. Commun. 2002 16 1782-1783. 

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