Ene Reductases

The biocatalytic counterpart for the stereoselective reductirai of alkenes is catalyzed by flavin-dependent ene-reductases, " EC 1.3.1.31], which are members of the old yellow enzyme family (OYE, Scheme 2.134) [968,969], first described in the 1930s [970]. These enzymes are widely distributed in microorganisms and in plants. Some of them occur in well-defined pathways, e.g., in the biosynthesis of fatty acids and secondary metabolites, such as morphine [971] and jasmonic acid [972]. Others are involved in the detoxification of xenobiotics [973], such as nitro esters [974] and nitro-aromatics [975] like trinitrotoluene (TNT) [976]. 

The catalytic mechanism of the asymmetric reduction of alkenes catalyzed by ene-reductases has been studied in great detail [977] and it has been shown that a hydride (derived from a reduced flavin cofactor) is stereoselectively transferred onto Cp, while a Tyr-residue adds a proton (which is ultimately derived from the solvent) onto Cot from the opposite side (Scheme 2.134). As a consequence of the stereochemistry of this mechanism, the overall addition of [H2] proceeds in a trans-fashion with absolute stereospecificity [978]. This reaction is generally denoted as the oxidative half reaction . The catalytic cycle is completed by the so-called reductive half reaction via reduction of the oxidized flavin cofactor at the expense of NAD(P)H, which is ultimately derived from an external H-source via another... 

Scheme 2.134 Asymmetric bioreduction of activated alkenes using flavin-dependent ene-reductases...

The following crude guidelines for the asymmetric bioreduction of activated alkenes using ene-reductases can be delineated ... 

The bioreductiOTi of citral using the ene-reductase OPR3 (12-oxophytodienoic... 

Scheme 2.135 Asymmetric bioieduction of enals and allylic alcohols using isolated ene-reductase...

In contrast to aldehydes, over-reduction is less prraiounced on a,p-unsaturated ketones (Scheme 2.136). Nonracemic levodione, which is a precursor for the synthesis of carotenoids, such as astaxanthin and zeaxanthin, was obtained in 80% yield and >95% e.e. via yeast-mediated reduction of ketoisophorone. Two other products arising from over-reduction of the carbonyl moieties were formed in minor amounts [1013]. In contrast, no trace of carbonyl reduction was observed using ene-reductase OPR3 (Scheme 2.136). 

The ultimate source of redox equivalents in microbial reduction reactions is usually a carbohydrate. Since the majority of it is metabolized by the cells and only a minor fraction (typically 0.5-2%) is used for the delivery of redox equivalents onto the substrate, the productivity of such processes is usually low and side-reactions are common. In order to avoid the undesired metabolism of the auxiliary substrate, nondegradable organic dye molecules such as viologens have been used as shuttles ( mediators ) for the electron-transport from the donor to the oxidized cofactor [1019]. Provided that the mediators are accepted by the ene-reductases and the recycling enzymes, the productivities were improved by one to three orders of magnitude. 

Ene-reductase Reduction of enones, a,p-unsaturated esters trans-reduction Large-scale applications State of the art... 

Recent breakthroughs in the cloning of oxygen-stable ene-reductases enables the asymmetric bioreduction of activated carbon-carbon double bonds for preparative-scale applications. NADH recycling is performed on industrial scale, for the more sensitive NADPH analog improvements would be desirable. 

The obseiwed strict NADPH specificity of A -SR is shared with the two others ene-reductases involved in sterol synthesis i.e. A -sterol A -reductase nd... 

The Wittig synthesis has also been successfully combined with oxidoreductase-catalyzed biotransformations in one-pot processes ranning in aqueous media, as demonstrated recently by the Groger and Hummel groups [49, 50]. The first example consists of an enantioselective two-step one-pot synthesis of allylic alcohols of type 61, which is based on an initial Wittig synthesis of the stabilized yhde 59 with aldehydes 54 and in situ reduction of the formed a,p-unsaturated ketones with an alcohol dehydrogenase (ADH) [49]. The desired allylic alcohols 61 were formed in up to 90% conversion and with excellent enantioselectivity (>99% ee) [49]. This one-pot process based on the use of an (S)-enantioselective ADH is shown in Scheme 19.20. Instead of C=0 double bond reduction, the C=C double bond can be reduced as well selectively when using an ene reductase instead of an ADH, and... 

The combination of a chemoselective enzymatic reduction step with another second enzymatic reaction is another opportunity to overcome limitations, for example, in the case of the enantiosdective reduction of prochiral unsaturated aldehydes by coupling a reduction step with an isolated ene reductase (OYE 2 or OYE3) together with an oxidation step with HLADH in a cascade system, which allowed both yields and enantioselectivities to be improved [136]. 

Brenna, E., Gath, F.G., Monti, D., Parmeggiani, F., and Sacchetti, A. (2012) Cascade coupling of ene reductases with alcohol dehydrogenases enantioselective reduction of prochiral unsaturated aldehydes. ChemCatChem, 4, 653-659. 

The very few ene reductases that do not belong to the OYE family are nortflavin-depmdent me redMctases[31],whichhavebeenrecognizedandisolatedonlyrecently from plants and mammalian cells (Table 3.1b) [32-34]. Remarkably, these biocatalysts were found to mediate both anti and syn addition of hydrogen, depending on the substrate. 

The second main class of enzymes capable of activated C=C bioreductions is that of enoate reductases (EC 1.3.1.31), which are also able to act on a, 3-unsaturated carboxylate anions, substrates typically not accepted by ene reductases. Up to now, only a few enoate reductases have been identified, all from anaerobic bacteria, mainly of the Clostridium species (Table 3.1c) [35,36]. They belong to the family of ferredoxins, containing an iron-sulfur cluster (Fe4-S4) that makes them extremely sensitive to molecular oxygen, and therefore much more difficult to isolate and employ in preparative chemical transformations. 

Figure 3.1 Mechanism of C—C double bond reduction mediated by OYE-like ene reductases (exemplified with OYEl) (a) classical binding mode (b) flipped binding mode.

The case of a-fluorodnnamyl alcohol [48,39] is a clear example of how ene reductases are superior in terms of chemoselectivity with respect to metal-based hydrogenations, since in the latter, in addition to the C=C double bond reduction, the cleavage of C—F bond also takes place. Chiral fluoroalcohols are important synthones in medidnal chemistry. 

So far, two strategies have been adopted to address some of these issues. The first one relies on the in situ SFPR technology cited earlier. The second one is based on the use of isolated ene reductases, allovhng in many cases almost quantitative conversions without the formation of side products. 

Recently, a substantial improvement of the low productivity of BY-mediated synthesis [49] of EEHP and EMHP was obtained by combining the in situ SFPR concept with an isolated ene reductase (Scheme 3.3) 61,62j. 

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