Enzymatic Reduction of Methyl Acetoacetate

Purpose To demonstrate the enantioselective reduction of a ketone with an enzyme. 

Preparation Sign in at www.cengage.com/login to answer Pre-Lab Exercises, access videos, and read the MSDSs for the chemicals used or produced in this procedure. Review Sections 2.11, 2.17, 2.21, and 2.29. 

Apparatus A 250-mL Erlenmeyer flask, separatory funnel, apparatus for magnetic stirring, anaerobic fermentation, vacuum filtration, simple distillation, and flameless heating. 

Reduction Remove the stopper in the Erlenmeyer flask, and add 2.5 ml of methyl acetoacetate. Replace the stopper and continue to stir the contents of the flask vigorously, under anaerobic conditions, and in a warm place, ideally at 30-35 °C, for at least 48 h, although one week is better.  

Wash the filter-aid with 25 mL of water and saturate the combined filtrates with solid sodium chloride. Extract the resulting solution three times with 25-mL portions of dichloromethane by genf/y shaking the separatory funnel to mix the layers. Do nof shake the funnel too vigorously, because a bad emulsion will form. If this occurs, pass the contents of the separatory funnel through a fresh bed of filter-aid by vacuum filtration this filtration will remove most of the fine particles that cause the emulsion. Wash the bed with 15 mL of dichloromethane. Transfer the filtrate and washing to the separatory funnel, and continue the extractions any fine emulsion that remains may be included with the aqueous phase. Dry the combined dichloromethane layers with several spatula-tips full of anhydrous sodium sulfate. Swirl the flask occasionally for a period of 10-15 min to facilitate drying add further small portions of anhydrous sodium sulfate if the solution remains cloudy. 

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The keto group of methyl acetoacetate may also be reduced selectively with sodium borohydride. Describe how the product of this reaction would differ from the product of the enzymatic reduction of methyl acetoacetate with baker s yeast. 

The NMR spectrum in Figure 17.21 is of a mixture of the chiral shift reagent 17 and a sample of methyl (S)-(+)-3-hydroxybutanoate that was produced by the enzymatic reduction of methyl acetoacetate using baker s yeast. Determine the enantiomeric excess of the methyl (S)-(+)-3-hydroxybutanoate contained in this sample. 

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