Stereospecific Reduction of Ethyl 4Chloroacetoacetate

Ethyl (R)-4-chloro-3-hydroxybutanoate formed through the reduction of ethyl 4-chloroacetoacetate with cells of S. salmonicolor showed low optical purity (8.5% ee) because the cells contained a significant amount of an enzyme(s) catalyzing the reduction of ethyl 4-chloroacetoacetate to ethyl (S)-4-chloro-3-hydroxybutanoate in addition to the aldehyde reductase forming ethyl (R)-4-chloro-3-hydroxy-butanoate. Because heat treatment and acetone fraction-ation of a cell-free extract of S. salmonicolor remove the (S)-isomer-forming enzyme(s) from the cell extract, the cell extract after these treatments was used as the source of aldehyde reductase. Commercially available glucose dehydrogenase, NADP + , and glucose were added as a cofac-tor regenerator. A practical preparation can be carried out in an organic solvent-water two-phase system, as shown

Figure 4. Conversion of ethyl 4-chloroacetoacetate to ethyl (R)-4-chloro-3-hydroxybutanoate in a two-phase reaction system by E. coli JM109 [pKAR, pKKGDH] cells. Glucose (100 mg/mL) was added to the aqueous phase periodically (arrows). Symbols: O, ethyl 4-chloroacetoacetate (CAAE); •, ethyl (R)-4-chloro-3-hy-droxybutanoate (CHBE). See Refs. 49-52 for details.

Figure 4. Conversion of ethyl 4-chloroacetoacetate to ethyl (R)-4-chloro-3-hydroxybutanoate in a two-phase reaction system by E. coli JM109 [pKAR, pKKGDH] cells. Glucose (100 mg/mL) was added to the aqueous phase periodically (arrows). Symbols: O, ethyl 4-chloroacetoacetate (CAAE); •, ethyl (R)-4-chloro-3-hy-droxybutanoate (CHBE). See Refs. 49-52 for details.

in Fig. 3a, because the substrate is unstable in an aqueous solution, and both the substrate and product strongly inhibit the enzyme reaction. In this system, most of the substrate is present in the organic phase; consequently, the ethyl 4-chloroacetoacetate is quite stable and cannot in-

Cl^^V-/- COOEt CAAE Organic phase

Cl^^V-/- COOEt CAAE Organic phase

Figure 3. Outline of the stereospecific reduction of ethyl 4-chloroacetoacetate by aldehyde reductase (AR) and glucose dehydrogenase (GDH) (a), and washed cells of E. coli JM109 [pKAR, pKKGDH] (b), as catalysts, in an organic solvent-water two-phase system. CAAE, ethyl 4-chloroacetoacetate; CHBE, ethyl 4-chloro-3-hydroxybutanoate. See Refs. 48-52 for details.

hibit the enzyme reaction occurring in the aqueous phase. «-Butyl acetate is regarded as the most suitable organic solvent for such a two-phase system because it shows high partition efficiencies with regard to both the substrate and product, and both enzymes are stable in the presence of this organic solvent. In a bench-scale two-phase reaction, 83.8 g/L of ethyl (,R)-4-chloro-3-hydroxybutanoate (86% ee) was produced from ethyl 4-chloroacetoacetate, with a molar yield of 95.4% (48,53).

Enzymatic Production of Ethyl (R)-4-Chloro-3-Hydroxybutanoate

The two-phase reaction was improved by using an Escherichia coli transformant. The aldehyde reductase gene from S. salmonicolor (38) and the glucose dehydrogenase gene from Bacillus megaterium (54) were transformed into the same E. coli strain; consequently, a transformant overproducing both aldehyde reductase and glucose dehydrogenase, E. coli JM109 [pKAR, pKKGDH], was obtained (49-52). The reduction reaction was carried out in an organic solvent-water two-phase system containing ethyl 4-chloroacetoacetate, glucose, and NADP + , and E. coli JM109 [pKAR, pKKGDH] cells as the catalyst (Fig. 3b). When the E. coli cells were incubated in the n-butyl acetate-water two-phase system, 300 mg/mL of ethyl 4-chloroacetoacetate was almost stoichiometrically converted to ethyl (^)-4-chloro-3-hydroxybutanoate (92% ee) in 16 h (Fig. 4). Because the use of E. coli transformant cells as the catalyst is simple and does not require isolation of the aldehyde reductase, which is necessary for S. sal-monicolor cells, it is highly advantageous for the practical synthesis of ethyl (^)-4-chloro-3-hydroxybutanoate.

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