Keto esters enzymic reductions

The enzyme recLBADH is the first catalyst that has been found to allow the highly regio- and enantioselective synthesis of 5-hydroxy-P-keto esters by reduction of the respective diketo esters. This enzymatic reaction is of enormous preparative value. The substrates are readily available by acylation of P-keto ester bisenolates and the reaction only requires a simple batch technique which is easy to scale up. Reduction of the chlorinated compound la has been performed routinely on a 75 g scale in our laboratory (8 L fed batch), yielding (S)-2a in an isolated yield of 84% [10]. 

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. 

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. 

Shafiee, A., Motamedi, H. and King, A., Purification, characterization and immobilization of an NADPH-dependent enzyme involved in the chiral specific reduction of the keto ester M, an intermediate in the s3mthesis of an anti-asthma drug, montelukast, from Microbacterium campoquemadoensis (MB5614). App/. Microbiol. Biotechnol., 1998, 49, 709-717. 

Table 9.1 Enzyme-catalysed stereoselective reduction of diketones/keto esters to keto alcohols/hydroxy esters...

The enzyme-catalyzed regio- and enantioselective reduction of a- and/or y-alkyl-substituted p,5-diketo ester derivatives would enable the simultaneous introduction of up to four stereogenic centers into the molecule by two consecutive reduction steps through dynamic kinetic resolution with a theoretical maximum yield of 100%. Although the dynamic kinetic resolution of a-substituted P-keto esters by chemical [14] or biocatalytic [15] reduction has proven broad applicability in stereoselective synthesis, the corresponding dynamic kinetic resolution of 2-substituted 1,3-diketones is rarely found in the literature [16]. 

In the use of whole cells severe problems may arise from strain specific activity of intracellular enzymes reacting with the product. For instance, the organism may use the substrate or the product as the carbon source or intracellular esterase activities may influence the yield of hydroxy esters formed by enzymatic reduction of keto esters. These problems may be avoided using isolated enzymes. These potential side reactions also define the purification grade of a technical enzyme sample because the complete separation of the disturbing activities must be ensured. 

Patel RN, McNamee CG et al (1992) Stereoselective reduction of P-keto esters by Geotrichum candidum. Enzyme Microb Technol 14 731-738... 

Glucose-grown cells of G. candidum SC 5469 have also catalyzed the stereoselective reduction of ethyl-, isopropyl-, and tertiary-butyl esters of 4-chloro-3-oxobutanoic acid and methyl and ethyl esters of 4-bromo-3-oxobutanoic acid. A reaction yield of >85% and e.e. of >94% were obtained. NAD+-depen-dent oxido-reductase responsible for the stereoselective reduction of P-keto esters of 4-chloro- and 4-bromo-3-oxobutanoic acid was purified 100-fold. The molecular weight of purified enzyme is 950,000. The purified oxido-reductase was immobilized on Eupergit C and used to catalyze the reduction of (39) to S-( - )-(40). The cofactor NAD+ required for the reduction reaction was regenerated by glucose dehydrogenase. 

Synthetic statins are important lipid regulating drugs for the treatment of atherosclerosis and other diseases related to hyperlipidaemia, especially coronary heart disease. As the pharmacophore, all synthetic statins contain a saturated or partially unsaturated syn-3,5-dihydroxy C7-carboxylate. An important building block for the synthesis of the side chain is represented by 4-chloro-3-hydroxybutanoate esters (CHBE). Both enantiomers can be obtained by enzyme-catalyzed reduction of the (1-keto ester. The group of Kataoka and Shimizu found that an aldehyde reductase of Sporobolomyces salmonicolor [161] and a carbonyl reductase of Candida... 

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]. 

The synthesis of the angiotensin-converting enzyme (ACE) inhibitor enalapril (1) incorporates the natural amino acids L-alanine and L-proline [1,2]. Although the compound can be thought of as a derivative of homophenylalanine, this part of the compound is prepared by a reductive amination with the keto ester (2), while the amino acids are coupled through an A-carboxy anhydride 3 (Scheme 1) [3-7]. 

Other prochiral units that can be trapped in rings are enolates. One famous application is the alkylation of [3-hydroxy acid derivatives available from the chiral pool (chapter 23), by asymmetric aldol reactions (chapter 27) and by asymmetric reduction of P-keto-esters either by catalytic hydrogenation (chapter 26) or by enzymes (chapter 29). Frater found that the alcohol 55, from the baker s yeast reduction of the ketoester 54, formed the enolate 56 held in shape by chelation. Alkylation occurred on the top face.8 This is not so much because the OH is down in 55 as that the methyl group is down in 56. 

For example, the low enantioselectivity in the yeast reduction of 3-keto ester was improved by addition of ethyl chloroacetate or methyl vinyl ketone as described in Fig. 15-13. The enzymes inhibited and those not inhibited were identified by enzymatic studies using purified enzymes [97l The mechanism of the inhibition is reported to be non-competitive. 

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. 

An illustrative example of the reactions of different enzymes is the yeast reduction of y-ehloro-/5-keto esters containing different ester groups. The S enantioselectivity observed on the reduction of the methyl ester is reversed to R when the octyl ester is used (Table 2)61,68,69. 

Selective inhibition of either the (S)- or the (7 )-alcohol-producing enzyme by addition of allyl alcohol91, of methyl vinyl ketone or several other a,/l-unsaturated ketones92, or of ethyl chloroacetate90 has been successfully used for the reduction of different /i-keto esters, although a decrease in chemical yield may result. 

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. 

With strains of Saccharomyces the (25,35)-isomer is produced predominantly, accompanied by some of the (2/J,3,S )-isomer. Similar to ft-keto esters, the formation of the undcsired isomer can be decreased by the addition of an a,/i-unsaturatcd carbonyl compound, e.g.. methyl vinyl ketone, or an allylic alcohol. These additives probably act as inhibitors for the enzyme which produces the (2/ ,35)-isomer202 203. More recently, the microbial reduction of a variety of simple 2,2-disubstituted cyclic 1,3-diketones of various ring size has been investigated204 205 206. In most cases one of the substituents in the 2-position is methyl. The configuration of the hydroxy group in the reduced product is always S, and the enantiomeric excess is often high (Table 7). 

Protease or lipase enzymes were useful for the regioselective aminoacylation of lobucavir. Lipase was also used for resolution of a synthon for the paclitaxel side chain. The paclitaxel side-chain ester was also prepared by reduction of a keto ester precursor. Enzymatic reduction of ketones to chiral alcohols is another reaction that has been widely applicable. C14-deacylase, ClO-deacety-lase, and C7-xylosidase were identified from microorganisms isolated from soil samples and were useful for converting complex mixtures of taxanes found in yew extracts primarily to 10-deacetyl baccatin III, a precursor for the semisynthesis of paclitaxel and analogs. 

Aromatic compounds can also be degraded anaerobically, via reductive pathways, which have been reviewed. The best-characterized example is the degradation of benzoic acid in Rhodopseudomonas palustris and Thauera aromatica via the pathway shown in Figure 8. Benzoyl-CoA is formed, and then a reductase enzyme is able to reduce the aromatic ring to a cyclohexadiene. Following two consecutive additions of water, and oxidation to a /3-keto ester, hydrolytic cleavage gives a linear 7-carbon CoA thioester, which can then be broken down via fatty acid /3-oxidation. 

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