Enzymes reduction with bakers yeast

Enzyme reduction with Baker s yeast and enantioselective rule... 

Scheme 9 Enzyme-mediated chemical transformations. (A) Enatioselective enzymatic oxidation and lactonization (B) enzyme reduction with baker s yeast and enantioselective rule and (C) enzymatic hydrolytic desymmetrization.

The following experiment was conducted with a soluble starch in order to obtain solutions clear enough to permit determination of the optical activity. Reduction was determined with hypoiodite and calculated as degree of hydrolysis. Maltose and D-glucose were determined by fermentation with bakers yeast, Table II. The enzyme was quite... 

Li, Y.-N., Shi, X.-A., Zong, M.-H. et al. (2007) Asymmetric reduction of 2-octanone in water/organic solvent biphasic system with baker s yeast FD-12. Enzyme and Microbial Technology, 40, 1305-1311. 

Enzyme reductions of carbonyl groups have important applications in the synthesis of chiral compounds (as described in Chapter 10). Dehydrogenases are enzymes that catalyse, for example, the reduction of carbonyl groups they require co-factors as their co-substrates. Dehydrogenase-catalysed transformations on a practical scale can be performed with purified enzymes or with whole cells, which avoid the use of added expensive co-factors. Bakers yeast is the whole cell system most often used for the reduction of aldehydes and ketones. Biocatalytic activity can also be used to reduce carbon carbon double bonds. Since the enzymes for this reduction are not commercially available, the majority of these experiments were performed with bakers yeast1 41. 

Kometani et al. [71] reported that baker s yeast catalyzed the asymmetric reduction of acetol to (i )-1,2-propanediol with ethanol as the energy source. The enzyme involved in the reaction was an NADH-dependent reductase, and NADH required for the reduction was supplied by ethanol oxidizing enzyme(s) in the yeast. When washed cells of baker s yeast were incubated with 10 mg ml of acetol in an ethanol solution with aeration, (k)-1,2-propanediol was formed almost stoichiometrically with an optical purity of 98.2% e. e. 

Enzymes are natural biocatalysts that are becoming increasingly popular tools in synthetic organic chemistry [1]. The major areas of exploration have involved the use of hydrolases, particularly esterases and lipases [2]. These enzymes are readily available, robust and inexpensive. The second most popular area of investigation has been the reduction of carbonyl compounds to chiral secondary alcohols using either dehydrogenases (with co-factors) or a whole-cell system such as bakers yeast [3]. 

Highly enantioselective reduction of ethyl 6-benzyloxy-3,5-dioxohexanoate by ADH of Acinetohacter calcoaceticus has been reported (97 to >99% ee) [6]. Regi-oselectivity was not encountered, however, as was the case in the reduction of a variety of 3,5-dioxohexanoates A with baker s yeast [7]. The application of isolated enzymes in an anticipated regio- and enantioselective reduction of diketo esters A seemed most promising to us. 

Reduction of a cyclobutanone with baker s yeast yielded the diastereomeric alcohols 5 which could be separated by column chromatography.163 The optical purity was reported to be ca. 90%. Stereoselective reductions using isolated enzymes or complete organisms have also been reported for 6.82-276 278... 

An important step in the asymmetric synthesis of the angiotensin-converting enzyme inhibitor, benazepril HC1 132, was the reduction of the ketoester 128 (obtained from 127 by condensation with diethyl oxalate) with baker s yeast to give the chiral cr-hydroxy ester 129 in high yield and ee (Scheme 17). Direct formation of the 1//-1-benzazepin-2-one 131 from 129 proceeded in 42% yield (without racemization at C-3) or in 74% yield in two steps via 130, again with no racemization <2003TA2239>. 

In a different approach, instead of using a production enzyme together with an NADH-regenerating enzyme, baker s yeast was used to take over both objectives. Thus 3-keto esters were electrochemically reduced to give the optically active 3-hydroxy esters in the presence of baker s yeast and NAD" " using a viologen as redox catalyst to shuttle the electrons from the cathode to the yeast cells which then catalyze the NADH formation and the enzymatic reduction. In such an approach, usually the permeation in and out of the yeast cells is a limiting factor [54]. 

Several alternative methods with high ee s for various types of ketones are known reductions catalyzed by enzymes or baker s yeast [30] and microbial reagents [31], homogeneous hydrogenation (cf. Chapter 6.1), and stoichiometric reductions with chiral metal hydrides [32]. 

For example, the effect of cultivation time and different carbon sources on the enantioselectivity of the reduction of sulcatone by some anaerobic bacteria has been investigated [8 if Another example is the investigation on the effect of the medium concentrations for cultivation of Geotrichum candidum IFO 4597 on the enantioselectivity of the reduction of acetophenone derivatives. The yield of l -alcohol (the minor enantiomer) increased with the medium concentration therefore, the medium concentration was kept low, optimally to produce the S-enantiomer[82]. The effect of the aeration during cultivation on the enantioselectivity of bakers yeast production of 3-hydroxyesters has also been reported 86 Inducers such as a substrate analog may also induce the desired enzyme to improve the enantioselectivity. 

Long-chain 3-oxoalkanoates with a carbon chain length of 10,14 or 18 C-atoms are also reduced by baker s yeast in >97% ee, but low to moderate yields if the acids are used81-128. If the methyl esters are employed the reactions are much slower and less selective. In addition to substrate modifications, several other methods are known for improving baker s yeast reduction, namely by the selective reduction with or stimulation of one of the reducing enzymes in the cell90-92. 

In contrast, the latter. ryw-isomer, ethyl (TS,2R)-2-(1 -hydroxymethyl)-4-pentenoate. was recently prepared as the only product of a reduction with an enzyme fraction obtained from baker s yeast176. Introduction of a sulfur-containing functional group into the substrate increases stereocontrol in baker s yeast reduction of many ketonesI2e>. 2-Methylthio-3-keto esters are selectively reduced to the (3S)-3-hydroxy esters (Table 5)123,127. The 2-methylthio group is easily removed by 3-chloroperbenzoic acid oxidation to the corresponding sulfoxide followed by subsequent reduction with aluminum-mercury amalgam. Thus, these compounds can also be used for the preparation of optically pure 2-unsubstituted 3-hydroxy esters. 

Interestingly, reduction of ethyl 4-chloroacetoacetate with Baker s yeast gave predominantly the corresponding (5)-alcohol (i.e. the opposite configuration from that of the alcohol from ethyl acetoacetate itself) (7.103), but the corresponding octyl ester gave almost entirely the (/f)-alcohol. The stereochemistry of the reduction depends on the shape of the molecule and it is likely that the yeast contains at least two different oxidoreductase enzymes which produce the two enantiomeric alcohols at different rates. 

Prior to the widespread awdlabdity of recombiant carbonyl reductases enzymes, the use of microbial reductions using either actively growing or dormant cells was commonplace Bakers yeast in particular, was a readily available source of stereoselective carbonyl reductases enzymes. Even with the widespread knowledge of the power of recombinant CRED biocatalysts, the literature is still rife with wild-type whole-cell microbial reductions. The reductions presented have advanced well beyond the early Bakers yeast reduction and have an apphcation even today. When the whole-cell fermentation is developed and finely tuned, high titers of product alcohol are possible and Scheme 6.4 shows m example of a keto-amide 12 bioreduction performing at 100 g/L with more than 98% ee with multi-kg isolation [12]. The bioprocess was performed over 8 days at pH 7 using the yeast Candida sorbophila. 

Scheme 32 outlines a high yielding approach to the enantiopure appetite suppressant drug 77. The reduction of the unsaturated aldehyde 74 was reported to occur with modest enantioselectivity in normal fermenting conditions with baker s yeast [155]. When the biotransformation is performed at very low substrate concentration, the e.e. can be raised to more than 90%, suggesting that incomplete enantioselectivity is due to the action of enzymes operating on the same substrate with opposite stereochemical preference [115]. However, an efficient transformation can be performed if the substrate concentration is controlled with the addition of absorbing hydrophobic resins. At 5 g/L 97% recovery and 98% e.e. was obtained. The halohydrin 75 obtained was transformed into the epoxide 76 and finally into the enantiopure 2-/ -benzylmorpholine 77, the more active enantiomer with appetite suppressant activity [26]. 

Baker s yeast has been widely used for the reduction of ketones. The substrate specificity and enantioselectivity of the carbonyl reductase from baker s yeast, which is known to catalyze the reduction of P-keto ester to L-hydroxyester (L2-enzyme) [15], was investigated, and the enzyme was found to reduce chloro-, acetoxy ketones with high enantioselectivity (Figure 8.32) [24aj. 

An IL solvent system is applicable to not only lipase but also other enzymes, though examples are still limited for hpase-catalyzed reaction in a pure IL solvent. But several types of enzymatic reaction or microhe-mediated reaction have been reported in a mixed solvent of IL with water. Howarth reported Baker s yeast reduction of a ketone in a mixed solvent of [hmim] [PFg] with water (10 1) (Fig. 16). Enhanced enantioselectivity was obtained compared to the reaction in a buffer solution, while the chemical yield dropped. 

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