In situ cofactor recycling

Groeger, H., Hummel, W., Rollmann, C., Chamouleau, E., Husken, H., Werner, H., Wunderlich, C., Abokitse, K., Drauz, K., and Buchholz, S. (2004) Preparative asymmetric reduction of ketones in a biphasic medium with an (S)-alcohol dehydrogenase under in situ-cofactor-recycling with a formate dehydrogenase. Tetrahedron, 60, 633-640. 

Kosjek, B., Nti-Gyabaah, J., Telari, K., Dunne, L., and Moore, J.C. (2008) Preparative asymmetric synthesis of 4,4-dimethoxytetrahydro-2H-pyran-3-ol with a ketone reductase and in situ cofactor recycling using glucose dehydrogenase. Org. Process Res. Dev., 12, 584-588. 

A Rhodococcus ruber strain reported by Faber and Kroutil et al. represents a further interesting wild-type organism since it shows a broad substrate spectrum as well as a (synthetically interesting) high isopropanol tolerance (up to 50% (v/v)), which contributes to a better solubility of hydrophobic ketones and serves at the same time as a cosubstrate for in situ cofactor recycling [83-85]. 

Besides wild-type strains, more recently the use of recombinant whole cells has gained increasing popularity for application in asymmetric ketone reduction. When overexpressing the ADFi only, in situ cofactor recycling based on a "substrate-coupled approach" represents a favorite approach as demonstrated in an early contribution by the Itoh group [86] utilizing a recombinant ADH from a Corynebacterium overexpressed in E. coli. This concept has been also applied by Daicel researchers in the presence of an E. coli catalyst with recombinant ADH from Candida parapsilosis. This biocatalyst catalyzes the reduction of p-ketoester 28 at a 36.6 g/1 substrate loading and fimiished the alcohol (R)-29 in 95.2% yield and with 99%ee (Scheme 23.12) [87]. 

As selected examples of more recently developed ketone reductions with recombinant whole cells following the concept of substrate-coupled in situ cofactor recycling, the work by the Schmid and Buehler group with a recombinant ADH from Thermus sp. [88] and by the Kroutil group with a DMSO-tolerant recombinant ADH from Paracoccus pantotrophus [89] shall be mentioned here. This recombinant whole-cell... 

The use of a recombinant whole-cell catalyst based on an ADH and a GDH also turned out to be a valuable approach for the synthesis of the a-hydroxy ester (R)-35, which serves as a key intermediate for clopidogrel (Scheme 23.17) [98]. To this end, Ema and Sakai et al. designed E. coli cells overexpressing an ADH from S. cerevisiae and a GDH for in situ cofactor recycling. This reduction runs at a high substrate loading of 198 g/1 and leads to (R)-35 with 86% conversion, in 82% yield and with... 

I 2 Neiv Trends in the In Situ Enzymatic Recycling of NAD(P) (H) Cofactors... 

It is possible to use isolated, partially purified enzymes (dehydrogenases) for the reduction of ketones to optically active secondary alcohols. However, a different set of complications arises. The new C H bond is formed by delivery of the hydrogen atom from an enzyme cofactor, nicotinamide adenine dinucleotide (phosphate) NAD(P) in its reduced form. The cofactor is too expensive to be used in a stoichiometric quantity and must be recycled in situ. Recycling methods are relatively simple, using a sacrificial alcohol, or a second enzyme (formate dehydrogenase is popular) but the real and apparent complexity of the ensuing process (Scheme 8)[331 provides too much of a disincentive to investigation by non-experts. 

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. 

Biocatalytic approaches to cofactor regeneration can be divided into coupled-enzyme methods and coupled-substrate methods.In the coupled-enzyme method, the oxidized cofactors (NAD+ and NADP+) are recycled in situ by performing an oxidation reaction using a second enzyme and an inexpensive auxiliary substrate. This second enzyme must employ the same cofactor, but neither enzyme should be able to accept the same substrate. 

In recent years, these facts have significantly prompted the research toward the development of new and more efficient in situ regeneration systems of the NAD(P)(H) cofactors [2, 3], which allow their use in catalytic instead of stoichiometric amounts, thus making the dehydrogenase-catalyzed processes acceptable from an economical point of view. Moreover, the recycling reactions can be also used to shift the equilibrium of thermodynamically unfavorable transformations toward product formation. 

As previously mentioned, an ADH can be used for the in situ recycling of NAD(P)H cofactors by exploiting the so-called substrate-coupled approach, that is, the coupHng of the reaction of interest with a secondary reaction running in the reverse direction... 

Solid-gas biocatalysis has not been restricted to the use of isolated enzymes. Whole cells are of particular interest where the in vivo recycling of cofactor can be achieved by addition of a cosubstrate. Dried Saccharomyces cerevisiae cells have been used to catalyze the continuous reduction of hexanal to hexanol with in situ regeneration of NADH via oxidation of ethanol in a solid-gas system [81]. The... 

---