Biological Reduction

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which shows chemoselectivity. Furthermore, molecular recognition and subsequent stereospecific addition of hydrogen across the carbon-oxygen double bond led to the formation of an enantiotopic product, (S)-ethyl 3-hydroxybutanoate, from an achiral substrate, which thereby shows enantioselectivity.

Other various methods for this conversion are possible and the most frequently employed is the use of complex metal hydride reagents such as lithium aluminum hydride (LiAlH4) and sodium borohydride (NaBH4). Of the two most common reducing agents, LiAlH4 is the more powerful, reducing most carbonyl-containing compounds compared to the more selective NaBH4.

If LiAlH4 was used to react with ethyl acetoacetate, it would cause reductions at both carbonyl groups of the starting material such that it will involve the formation of the two enantiomers, (S)-ethyl 3-hydroxybutanoate and (R)-ethyl 3-hydroxybutanoate. (Reduction of Ethyl Acetoacetate, 2007)

After letting the reaction proceed for a week, the resulting mixture became yellow-orange to brownish in color. To remove the yeast cells in the mixture, Celite filtration aid, which is made of silicon dioxide, was added to aid in filtration as it agglomerates used yeast cells so they would not come off with the solution.

After the mixture was filtered, the filtrate which now contains the product was added with sodium chloride to decrease the solubility of the product. The solution was then extracted with three portions of ethyl acetate. After extraction, the ester layer was dried by adding calcium chloride pellets. The solution was decanted into a distilling flask and ester was removed via distillation. The solvent used in extraction which is ethyl acetate has a boiling point of 77 deg C which makes separation from the product, which has a boiling point of 180-182 deg C, relatively easy to execute. However, a very small amount has remained after the reaction mixture reached about 80 deg C which is why the distillation was stopped at that point.

For the characterization tests, boiling point determination and ferric chloride test and Jones test for the starting material and product were done.

For the boiling point determination, an experimental BP was observed through distillation. Most of the solution has already evolved at about 75-80 deg C, which shows that most of the solution may be composed of ethyl acetate.

In the ferric chloride test, which is used to detect the presence of hydroxyl groups, both ethyl acetoacetate and the synthesized (S)-ethyl 3-hydroxybutanoate tested negative. Theoretically, the product was supposed to test positive. This may be accounted for by the possibly very small amount of product that was synthesized such that the reaction cannot be observed anymore.

In the Jones test, which is used to detect the presence of primary and secondary alcohols, aldehyde, enols and phenols, both the starting material and product tested positive. This may indicate that there is only a very small amount of product that was synthesized and that ethyl acetate was reacting to give a positive result.