Carbonyl Group

EXER 4 BIOLOGICAL REDUCTION

Carbonyl group

Carbonyl compounds are those compounds that consist of a carbon and an oxygen atom connected by a double bond (Klein, 2015).

Ketones are one of the compounds that contain a carbonyl group which serves as the reaction site of the compound. The two faces of an unsymmetrical ketone are enantiotopic hence can undergo an enantioselective synthesis (Wade, 2013). Enantioselective synthesis or chiral synthesis is a chemical reaction in which one or more new elements of chirality are formed in a substrate molecule which produces the stereoisomeric products in unequal amounts. This process favors the formation of a specific enantiomer or diasteriomer (Wade, 2013). The favourable formation of one enantiomer is called asymmetric induction (Wade, 2013). 

Chiral catalysts are essential in promoting the progress of the enantioselective reaction.

In the experiment, (S)-ethyl 3-hydroxybutanoate was synthesized from ethyl acetoacetate through the action of the fermentation enzymes secreted by yeasts.

During the enzymatic reduction of (S)-ethyl 3-hydroxybutanoate, sucrose and Na2HPO4 were added in warm water which resulted in a turbid solution.

Baker's yeast is used to convert sucrose, ordinary table sugar, into ethanol and carbon dioxide with the aid of some 14 enzymes as catalysts, in addition to adenosine triphosphate (ATP), phosphate ion, thiamine pyrophosphate, magnesium ion, and reduced nicotinamide adenine dinucleotide (NADH), all present in yeast (Walker, 1998).

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The fermentation process-known as the Emden-Meyerhoff Parnas scheme-involves the “hydrolysis of sucrose to glucose and fructose, which, as their phosphates, are cleaved to two three-carbon fragments. These fragments, as their phosphates, eventually are converted to pyruvic acid, which is decarboxylated to give acetaldehyde. Acetaldehyde, in turn, is reduced to ethanol in the final step. Other ketones or aldehydes present can also be reduced by this enzyme. Each step requires a specific enzyme as a catalyst and often inorganic ions, such as magnesium and phosphate (Chen, 2007, p. 730)”.

 G. M. Walker, Yeast metabolism, in: Yeast Physiology and Biotechnology, John Wiley & Sons Ltd, 1998, p. 203-265.

R. R. Chen, Permeability issues in whole-cell bioprocess and cellular membrane engineering, Appl. Microbiol. Biotechnol. 2007, 74, 730-738.

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 In the experiment, these phosphates were provided by the addition of Na2HPO4 in the reaction mixture. Also, the Na2HPO4 was added to establish a buffered system which could resist drastic change in the pH of the mixture. Extremely high or low pH can cause denaturation of enzymes and death of the yeast cells.

 Warm water was used to favor the progress of the reaction. Lower temperature would slow down the fermentation process. High temperature, on the other hand, should not be too high since the yeast could die at a very high temperature.

Dry yeast was suspended throughout the solution. After the addition of yeast, the solution turned into a yellowish mixture.

        Baker's yeast uses sugars as their preferred carbon and energy sources. Baker’s yeast can reduce pro-chiral keto esters to the corresponding chiral hydroxy esters. Baker’s yeast is non-hazardous, requires only mild reaction conditions (close to room temperature and neutral pH) and is very cheap compared to chiral organometallic reducing agents.

G. M. Walker, Yeast metabolism, in: Yeast Physiology and Biotechnology, John Wiley & Sons Ltd, 1998, p. 203-265.

        The prochiral beta keto ester diffuses into the yeast cell where it is reduced by oxidoreductases to form the corresponding chiral alcohol. The overall process is a redox reaction: the carbonyl group is reduced to the alcoholic group, while at the same time the cofactor NAD(P)H is oxidized to NAD(P) + . Without regeneration of NAD(P)H, the reduction of beta keto esters to alcohols will eventually stop. Thus, a parallel reaction has to take place, through metabolism of a cosubstrate, to regenerate the cofactor. The cosubstrate is usually some carbon source such as sucrose or glucose, and the by-products formed from the metabolism are mainly ethanol and carbon dioxide (Chen, 2007).

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R. R. Chen, Permeability issues in whole-cell bioprocess and cellular membrane engineering, Appl. Microbiol. Biotechnol. 2007, 74, 730-738.

While the fermentation is progressing vigorously, ethyl acetoacetate was added to the reaction mixture.

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The hydride adds to the carbonyl carbon and not to the ester carbon since the acyl carbon is more electropositive due to the delocalization of the electron lone pairs in the oxygen atoms. The electrons in the carbon oxygen double bond of the ketone are not equally shared since there is only oxygen to pull the electrons. The pi electrons are pulled strongly to the electronegative atom oxygen thus making the carbonyl carbon more electropositive. This means that the carbonyl carbon is more susceptible to nucleophilic attack (McMurry, 2008).

        Ethyl acetoacetate may also react with LiAlH4 but it would not give a stereospecific reaction. it is given by the chemical equation:

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The flask used was equipped with rubber stopper with a hole in the middle. One end of the rubber tubing was inserted in the hole and the other was submerged in a beaker with half-filled water. This was done to trap the carbon dioxide in water to give carbonic acid. This is given by the reaction:

CO2 + H2O -> H2CO3

The mouth of the flask was sealed properly to prevent the oxygen from interfering in the reaction. Oxygen inhibits the fermentation process. Also, this prevents other microorganisms to be trapped in the reaction mixture. Given the warm environment of the reaction mixture, these microorganisms can thrive and propagate thus destroying the whole fermentation process.

The flask was stored in a warm place for 1 week to favour the fermentation process.

Yeast cells were removed through suction filtration with the aid of Celite filtration aid. The filtrate was a yellow solution which contains the product. The residue was brown solids. These solids were the yeast cells removed from the reaction mixture.

Celite is a diatomite (diatomaceous earth) which has an intricate shape which provides longer and more complicated pathways wherein the impurities can be trapped.