Keto ester reduction with yeast

A representative set of a- and -keto esters was also tested as substrates (total 11) for each purified fusion protein (Figure 8.13b,c) [9bj. The stereoselectivities of -keto ester reductions depended both on the identity of the enzyme and the substrate stmcture, and some reductases yielded both l- and o-alcohols with high stereoselectivities. While a-keto esters were generally reduced with lower enantioselec-tivities, it was possible to identify pairs of yeast reductases that delivered both alcohol antipodes in optically pure form. These results demonstrate the power of genomic fusion protein libraries to identify appropriate biocatalysts rapidly and expedite process development. 

Scheme 65 summarizes Mori s synthesis of 44 [97]. Reduction of keto ester A with baker s yeast gave hydroxy ester B of about 98% ee. Methylation of the dianion derived from B diastereoselectively gave C, which was converted to 44. This process enabled the preparation of about 10 g of (lS,5R)-44. 

S Rodriguez Giordano. Baker s yeast (3-keto ester reductions whole cell biocatalysts with improved stereoselectivity by recombinant DNA techniques. Ph.D. thesis, University of Florida, Gainesville, FL, 2000. 

A Cyclic (3-Hydroxy Ester as a Building Block for Sporogen-AOl. Reduction of ethyl 2-oxocyclohexane-l-carboxylate with baker s yeast was first studied by Ridley in 1976 (12). Reduction of an analogous (3-keto ester 21 with baker s yeast yielded ethyl (lR,2S)-5,5-ethylenedioxy-2-hydroxycyclohexane-l-carboxylate (22) of 98.4% e.e. (23). 

Azides (175 X = OH) were resolved through microbial reduction of p-keto esters (181) with baker s yeast and provided the azido dienes (182) and (183) in about 70% ee in an approach to enantiocontrolled synthesis of pyrrolizidines (equation 55). In this series extensive racemization toc place prior to or during the vinylaziridine formation. A conversion of chlorobenzenediol (184) to lactone (185) and elaboration of this material to either enantiomer of trihydroxyheliotridane (187) constituted a fully enantiocontrolled approach to both enantiomeric series of highly oxygenated pyrrolizidine alkaloids (Scheme 41).2 ... 

Reductions. An enantioselective reduction of symmetrical diacetylarenes (e.g., 2,6-diacetylpyridine) is accomplished with baker s yeast. a-Functionalized ketones such as l-methanesulfonyl-2-alkanones and P-keto esters give chiral alcohols. Significantly, baker s yeast grown under limited oxygen effects reduction to selectively furnish o-hydroxy esters, whereas on slow addition of the keto esters to ordinary yeast in the presence of gluconolactone the L-hydroxy esters are produced. 

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. 

Dynamic kinetic resolution of a-alkyl-P-keto ester was conducted successfully using biocatalysts. For example, baker s yeast gave selectively syn(2R, 3S)-product [29a] and the selectivity was enhanced by using selective inhibitor [29b] or heat treatment of the yeast [29c]. Organic solvent was used for stereochemical control of G. candidum [29d]. Plant cell cultures were used for reduction of 2-methyl-3-oxobu-tanoate and afforded antialcohol with Marchantia [29e,f] and syn-isomer with Glycine max [29f]. 

Hydroxy esters have been obtained successfully with baker s yeast (Sac-charomyces cerevisidae), and this has shown a wide scope of application. The facial selectivity in the reduction of both isolated ketones and //-keto esters can be reliably determined by using Prelog s rule,8 which predicts that the hydrogen addition by the yeast will occur from the front face (Scheme 8-2). Anti-Prelog microbial reduction of a-ketones with Geotrichum sp. 38 (G38) has been introduced by Gu et al.9... 

K Nakamura, Y Kawai, S Oka, A Ohno. A new method for stereochemical control of microbial reduction. Reduction of (3-keto esters with baker s yeast immobilized by magnesium alginate. Tetrahedron Lett 2245-2246, 1989. 

Asymmetric oxidoreductions performed in isopropyl ether allow syntheses of optically active alcohols with ee >95% on a 1-10 mmol scale. Nakamura et al. investigated the effect of organic solvents on the reduction of ot-keto esters mediated by bakers yeast [140]. The reduction of ethyl 2-oxoheptanoate was tested in various solvents. Best results were achieved with benzene so further experiments were performed with benzene. Conversion only takes place in the presence of small amounts of water. The reduction of six ot-keto esters was examined regarding the stereoselectivity of the corresponding ot-hydroxy esters. The reactions were performed in aqueous systems as well as in benzene. In aqueous systems, the formed hydroxy esters show (S)-stereoselectivity while the stereochemistry of the reaction shifts markedly towards the production of (/ )-ot-hydroxy esters in benzene. 

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]. 

Ketones with useful heteroatomic functional groups containing nitrogen 199 2121, oxygen[163, 213 2171, phosphorus12181 and sulfur11 4, 18+1 219-2271 have been reduced by biocatalysts. For example, an intermediate in the synthesis of p-lactam antibiotics was obtained by microbial reduction of a P-keto ester as shown in Fig. 5-39(a)(1991, while yeast reduction of a p-keto dithioester afforded an easily separable mixture of p-hydroxy-dithioesters, the major component of which was converted enantiose-lectively into a sex attractant of the pine saw-fly as shown in Fig. 15-39(b)12191. 

A low enantiomeric excess of a reduced product can result from the inability of the oxidoreduc-tase to recognize the structural features of the substrate enantio- and diastereoselectively45. By far the more common case is the competition of two or more enzymes with different enantiose-lectivities and Michaelis constants for the substrate. Yeast cells not only contain yeast alcohol dehydrogenase but several enzymes that arc able to catalyze reductions. At least three different enzymes capable of reducing /i-keto esters have been purified, of which two have D- and one has L-selectivity62-65. Yeast alcohol dehydrogenase has a rather narrow selectivity for short-chain alcohols and aldehydes and certainly is not the catalyst in many of the reductions performed with yeasts. 

The selective enzymatic reduction of 2-substituted / -keto esters or /S-diketones is more interesting than that of unsubstituted compounds since two stereogenic centers can be introduced into the molecule in one step. The observation that baker s yeast reduction of these compounds results in predominantly one of the four possible diastereomeric products has been explained by a keto- enol equilibration of the enantiomeric / -dicarbonyls with the simultaneous removal of one of these substrates by an asymmetric reduction49. 

A bulkier 2-substituent leads to a reversal of the selectivity for baker s yeast reduction of (3-keto esters and gives a higher proportion of the cwti-diastcreomer (Table 5)1,7 121. For example, ethyl 2-benzy]-3-oxobutanoate leads to a 22% yield of ethyl 2-benzyl-3-hydroxybutanoate with a synjanti ratio of 35 65 while a 2-allyl substituent gives 84% yield of ethyl 2-allyl-3-hydroxy-butanoate with a synjanti ratio of 25 75. 

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. 

Another enzymatic approach to obtain the desired stereochemistry in the side chain is shown in Scheme 17.13. The keto ester precursor 10 of the side chain ethyl ester can be reduced to the hydroxy (2R,3S) ester 11 using either of the yeasts Hansenula polymorpha SC 13865 or Hansemla fabianii SC 13894. Screening a variety of strains from our culture collection revealed many other strains that could carry out the reduction reaction, but the best yields and ee s using whole cells were obtained with the two strains of Hansenula. Of four possible reduction products, the desired product 11 is obtained with 95 to 99% ee and 80 to 90% yield. Because of rapid ketone/enol tautomerism, the enzymatic reduction can work as a dynamic resolution and fix the stereochemistry at both the 2- and 3-positions. 

Bakers yeast is used almost exclusively for reduction, principally of ketones, by a dehydrogenase which usually follows the Prelog-Cram rule. As with chemical reductions, highest enantioselectivity obtains with aromatic aldehydes and aryl methyl ketones. /3-Keto esters arc also reduced with high canantiosclectivity by yeast. Some j8-kcto acids can also be reduced efficiently to (R)-jS-hydroxy acids. 

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