Substrate-coupled approach

Figure 5.9 NAD+/NADH cofactor regeneration a the coupled enzyme approach b the coupled substrate approach c, d examples [42,43] of the two approaches.

For the production of sulcatol (3, 6-methyl-5-hepten-2-ol). 71 brockii alcohol dehydrogenase has been used as a soluble enzyme, immobilized onto solid supports (PAN or Eupergit)26 113 237 and, recently, in a continuously working membrane reactor250. Generally the coupled substrate approach is used. 

YADH and HLADH are less useful for the asymmetric reductirai of open-chain ketones, but this gap is efficiently covered by a range of alcohol dehydrogenases from mesophiUc bacteria, such as Rhodococcus (ADH-A) and Lactobacillus (LBADH, LKADH), and thermophilic Thermoanaerobacter [802] and Thermo-anaerobium (TBADH) strains (Scheme 2.119) [296, 803-806]. Some of these enzymes are remarkably thermostable (up to 85°C) and can tolerate the presence of organic solvents such as /sopropanol, which serves as hydrogen-donor for NADP-recycling in a coupled-substrate approach [807-809]. 

Another potential bottleneck to be overcome is the inherent equilibrium problem associated with the coupled substrate approach to biocatalytic carbonyl reduction and in situ product removal allowed the isolation of the pure (S)-2-bromo-2-cyclohexen-l-ol in 88% yield and with 99.8% enantiomeric excess [135]. 

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... 

ADH from Lactobacillus brevis (IBADH) was used for the synthesis of a statin side chain, used as an alternative key intermediate in the synthesis of atorvastatin (Figure 13.2). In this variant, the NADPH-dependent enzyme was highly regio-and stereoselective, reducing fert-butyl 6-chloro-3,5-dioxohexanoate to tert-butyl (S)-6-chloro-5-hydroxy-3-oxohexanoate in 72% yield and with >99.5% ee. The cofactor was recycled in a coupled-substrate approach [1], where isopropanol was concomitantly oxidized to acetone at the expense of NADP, thereby driving the reaction toward product formation. Crude cell extract of recombinant LBADH expressed in E. coli was used in a fed-batch system on 8 1 scale, and a TTN of 2x10 could be calculated for IBADH [13-15]. 

ADHs were screened for this purpose and ADH-A from Rhodoccocus ruber could furnish the (S)-enantiomer with perfect enantiopurity (>99% ee) and 93% isolated yield. Reducing equivalents were provided by isopropanol in a coupled-substrate approach and allowed the use of catalytic amounts of NADH. The product from the enzymatic reduction required stereoinversion of the stereogenic center en route to the more active (R)-form of ramatroban. This was performed by converting the alcohol into the amine via the azide form, in a combined Mitsunobu-Staudinger one-pot reaction at low temperature [27]. 

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. 

The coupled substrate method is perhaps the simplest approach to asymmetric ketone reduction, using a single recombinant ADH to perform the oxidation of a cheap auxiliary... 

A related approach, developed almost simultaneously by Neuss and co-workers at Eli Lilly (25) and by Kutney et al. in Vancouver (26), utilized as coupling substrate the chloroindoline alkene 17, which, through equilibrium with the corresponding chloroindolenine, was readily prepared by chlorination of dihydrocleavamine 18. Condensation with the 17-deacetyl-16-carboxyhydrazide derivative of vindoline (19) gave a binary indole-indoline product (20) (Scheme 4). The undesired C-16 con-... 

Onono, F.K., et al. (2010). A tagging-via-substrate approach to detect the farnesylated proteome using Two-dimensional electrophoresis coupled with western blotting. Mol Cell Proteom 9 742-751. 

In the build-couple-pair approach to library synthesis, the building blocks are first synthesized from commercial material (build phase), which are then coupled to form a substrate (couple phase) that can further react under certain conditions and pair itself intramolecularly to form the cyclic products depicting the scaffold framework [24]. It is in the last two steps that the domino reaction can intervene to yield a more complex scaffold and with higher ring diversity. 

Coupled-Enzyme Approach. The use of two independent enzymes is more advantageous (Scheme 2.112). In this case, the two parallel redox reactions - i.e., conversion of the main substrate plus cofactor recycling - are catalyzed by two different enzymes [721]. To achieve optimal results, both of the enzymes should have sufficiently different specificities for their respective substrates whereupon the two enzymatic reactions can proceed independently from each other and, as a consequence, both the substrate and the auxiliary substrate do not have to compete for the active site of a single enzyme, but are efficiently converted by the two biocatalysts independently. 

The application of the RCM approach to create a range of cyclic silyl ethers successfully expands the scope of reactions utilizing the temporary sihcon tether approach, and the novel concept of heteroatom activation of the sihcon from the coupling substrate itself Thus, in cases where homoallylic alcohols or mediumsized rings are the desired products, the aforementioned methods offer a facile and direct route for their synthesis. 

The coupling reaction may be either intermolecular, as for a synthesis of an intermediate in a synthesis of thyroxine 2.605 (Scheme 2.179), or intramolecular, as in a synthesis of a metalloproteinase inhibitor 2.607 (Scheme 2.180) (for a different approach to related peptides, see Scheme 10.47). It is notable in the thyroxine synthesis that the two iodine substituents of coupling substrate 2.602 are not affected and no Suzuki products are formed in the absence of palladium. 

Alcohol dehydrogenase-catalyzed regeneration of NAD(P)H by oxidation of alcohols certainly represents the most widespread regeneration method currently applied. Especially if the desired production reaction is an ADHsubstrate-coupled regeneration approach excels in simplicity, as only one biocatalyst has to be used for the whole reaction (Scheme 8.8). Another advantage of this methodology is that the nicotinamide cofactor does not have to leave the... 

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