Biological Reduction

EXER 4 POSTLAB

In the synthesis of (S)-ethyl 3-hydroxybutanoate, 16.241g of sucrose and 0.104g of NaH2PO4 were dissolved in 60mL tap water. Tap water obtained was at room temperature which is about 30-35 deg C. Temperature has a big effect on the conversion, though its effect on the enantioselectivity of the reaction was less significant. It was made sure that the reaction mixture was warm as it is the optimum temperature for the yeast cells to thrive, thus driving the reaction.

About 3.2g of dry yeast (Saccharomyces cerevisiae) was then added. In the presence of sugars, Saccharomyces cerevisiae will conduct fermentative metabolism to ethanol and carbon dioxide as the cells strive to make energy and regenerate the coenzyme NAD+ under anaerobic conditions. (Walker & Stewart, 2016) The NAD+ formed will then be reduced to produce NADH, the hydride source for the synthesis and the reducing agent in the reaction.

Fermenting yeasts acidify their growth environment through a combination of proton secretion during nutrient transport through the action of the plasma membrane proton-pumping ATPase, direct secretion of organic acids such as succinate and acetate, removal of buffering agents and carbon dioxide evolution and dissolution. The addition of sodium phosphate is to make the mixture neutral to slightly basic which is optimum for high conversion of ethyl acetoacetate and acceptable enantiomeric excess of the product.

After 15 minutes, while letting the fermentation proceed, 2.0g of ethyl acetoacetate was added. The resulting mixture was light brown to yellow in color. The flask was equipped with a rubber stopper with a hole in it where a rubber tubing was inserted with one end inside the reaction flask, not touching the mixture, and the other end submerged in a half-filled beaker. The mouth of the flask was sealed properly and the reaction was allowed to proceed for a week. The mechanism of the reaction is given by:

[insert mech]

Carbon dioxide was formed during the fermentation and was trapped in the water given in the reaction:

CO2 + H2O --> H2CO3

In the experiment, a fermenting mixture of sugar is prepared using Baker's yeast. While the yeast is actively metabolizing the sucrose, the reduced nicotinamide dinucleotide within the active site of the dehydrogenase is constantly being replenished to carry out the reduction of ethacetaldehyde to ethanol within the active site. The transfer of hydride by NADH is driven by the restoration of aromaticity to the pyridine ring within the molecule.

As the ethyl acetoacetate is added at the onset of fermentation, it can rapidly permeate across the cell membrane of the yeast and take the place of acetaldehyde as a substrate for alcohol dehydrogenase.

The enantiospecificity of this reaction becomes apparent as hydride can only be added to the face of the carbonyl recognized by the enzyme as the substrate. (Amos, n.d.)

Due to the highly specific nature of enzymatic reactions, the ester functionality within the molecule was left unreacted,