Experiment No.

6: Enzymatic Reduction of Methyl Acetoacetate

Beatrice Emmanuelle Garcia Marc Ralph Solomon Shaine Dominique Santos
Submitted on 09 May 2017
ABSTRACT
Reduction of organic compounds involve a decrease in the oxidation number of carbon atom. The reduction
process may or may not be stereospecific. Moreover, the stereochemistry and optical purity of the products can
be controlled through the use of catalysts. In this experiment, bakers yeast, which contains alcohol
dehydrogenase, was used to catalyze the reduction of methyl acetoacetate to S-(+)-3-hydroxybutanoate by
anaerobic fermentation. In addition, the racemic product of reduction of acetoacetate was synthesized with the
use of sodium borohydride. S-(+)-3-hydroxybutanoate was not obtained, but the R enantiomer was synthesized
which was confirmed by its IR spectra and specific rotation. The percent yield of the product was 99.73% and its
specific rotation is -12.038. The racemic product was successfully synthesized. Even though the expected
enantiomer was not obtained, enzymatic reduction through the use of fermented bakers yeast as a catalyst is
an effective technique to synthesize enantiomerically pure products. Synthesis of racemic mixture through
reduction with sodium borohydride is also effective.
Keywords: Enzymatic Reduction, Methyl acetoacetate, 3-hydroxybutanoate, polarimetry, enantioselective reaction

I. Introduction alcohol dehydrogenase which can be found in bakers

Reduction is a chemical process wherein an atom yeast.
or a molecule gains electrons. Reduction of organic
compounds involve a decrease in the oxidation
number of carbon atom It is usually the result of losing
an oxygen or a more electronegative atom such as Figure 1. Reduction of methyl aceteoacetate to methyl
halogens or bonding with a hydrogen atom. (S)-(+)-3-hydroxybutanoate. (Gilbert &Martin, 2015)

Reduction can easily be done with the use of

common reducing agents such as lithium aluminum
hydride (LiAlH4) or sodium borohydride (NaBH4). The
use of these reducing agents will produce a racemic
mixture. This is because the reaction is not
enantiospecific.
Enantiospecific reactions are those that favor the
production of one enantiomer over another. The
stereochemistry and optical purity of the products can
be controlled through the use of catalysts. Reduction
using achiral reagents, such as LiAlH4 or NaBH4
produces equal amounts of both enantiomers thus
making the mixture racemic. Enantiospecific
reactions can be done with the use of chiral reagents.
II. Methodology
Setup of Yeast Suspension
First, 40 g of glucose and 0.25 g of disodium
hydrogen phosphate were dissolved in 75 mL of
warm (40C) water in an Erlenmeyer flask. A (8 g)
packet of dry, active bakers yeast and a stirbar were
added to the flask. The apparatus for anaerobic
fermentation was then assembled.
In another tube, 3% barium hydroxide solution
was added along with a small amount of mineral oil,
enough to make a protective layer over the barium
hydroxide solution. The yeast mixture was then
stirred vigorously for about an hour to suspend the
yeast and initiate fermentation.

Reduction
In the experiment, methyl acetoacetate was
The stopper in the Erlenmeyer flask was removed
reduced to methyl-(S)-(+)-3-hydroxybutanoate using
and 2.5 mL of methyl acetoacetate was added. The

1 | Chemistry 35.1 Enzymatic Reduction

stopper was replaced and stirring was continued for
at least 48 to a week.
Enzymatic reduction of methyl acetoacetate was
performed using bakers yeast in order to produce an
Isolation of the Product
enantiomerically pure form of (S)-(+)-3-
A filter-aid of silica gel was prepared by mixing 10
hydroxybutanoate which is a chiral alcohol.
g of silica gel with 50 mL water and filtering the slurry
through a funnel with filter paper. Another 10 g of Reduction of carbonyl compounds is possible to
filter-aid was added to the yeast mixture. The mixture be carried out through specific reactions such as
was allowed to stand so that the solids will settle. The catalytic hydrogenation, reduction via hydride
mixture was then decanted through a bed of filter-aid reducing agents, and enzymatic reduction. However,
and was subjected to vacuum filtration. the first two mentioned reactions are not
stereospecific for they yield equal amounts of chiral
After filtering, the filter-aid was washed with water
alcohol and its enantiomer. This is due to the equal
and the combined filtrates were saturated with
chances of attacking either of the sides of the planar
sodium chloride. The resulting solution was extracted
carbonyl group.
three times with 5 mL portions of DCM. The bed was
also washed with 5 mL of DCM. Thus, in order to reduce a prochiral carbonyl
compound to its enantiomerically pure chiral alcohol,
The combined DCM layers was then dried with
the reduction reaction has to be stereospecific and
several spatulas full of anhydrous sodium sulfate.
enantioselective. This reduction is possible through
The solution was then decanted into a distilling glass
the use of enzymes which are chemospecific and
and was subjected to steam bath drying to remove
chemoselective in nature. In this experiment, a yeast
most of the DCM.
suspension was set up by adding disodium hydrogen
Analysis of the Product phosphate as a buffer, and maintaining the
The product was weighed to determine the in order to make the conditions
temperature at 40 C
percent yield of crude methyl-(S)-(+)-3- conducive for fermentation.
hydroxybutanoate. The IR spectra of the product was
Fermentation allows the conversion of sugar to
also obtained and compared to authentic samples.
alcohol and carbon dioxide through the 14 enzymes
serving as catalysts adenosine triphosphate (ATP),
thiamine pyrophosphate, magnesium ion, and
III. Results and Discussion
reduced nicotinamide dinucleotide (NADH). In case
6 12 2
of sucrose, it is hydrolyzed to produce glucose and
1.076 1
= 2.5 fructose which is eventually converted to pyruvic acid
116.12
1 118.13 that will be decarboxylated to form acetaldehyde.
= 2.7365
1 1 Acetaldehyde is then hydrogenated by alcohol

2.729 hydrogenase to form ethanol (see Fig.2).

% = 100% = . %
2.7365

2 | Chemistry 35.1 Enzymatic Reduction

 Methyl acetoacetate is an organic molecule
relatively small enough to permeate across the
yeasts cell membrane, replacing the the
acetaldehyde substrate. The reaction occurs at the
NADH and the base which are active sites of the
alcohol dehydrogenase. Here, the two hydrogens at
the 4-position of nicotinamide of NADH are
distinguished. When it binds the coenzyme, B-side of
NADH is blocked causing the substrate to bind to the
A-side of the coenzyme and Ha hydride ion will be
transferred to the substrate (see Fig.4). The tendency
of the pyridine to restabilize its aromaticity causes the
transfer of hydride by NADH (Bruice, 2016).
Figure 2. Emden-Meyerhof-Parnas scheme (Academia,
n.d.)

Alcohol dehydrogenase is the enzyme used for

the reduction of methyl acetoacetate. This enzyme
also plays a vital role in glycolysis, a metabolic cycle
that provides energy to the cells under anaerobic
conditions like yeast. It has a zinc atom within its
active site serving as the third element of three-point
contact needed for enantioselectivity (see Fig.3).

Figure 4. Mechanism of hydride on the 4-position of

nicotinamide transferred to the substrate

After the enzymatic reduction, the obtained

product were subjected to infrared (IR) spectroscopy
analysis. The following spectra was obtained:

Figure 3. Active site of alcohol dehydrogenase

showing approach of the substrate into a favorable three
point contact
(http://chemistry.bd.psu.edu/kociolek/images/%20Yeast.p
df)

3 | Chemistry 35.1 Enzymatic Reduction

 IV. Conclusion
Methyl acetoacetate was reduced to R-(+)-ethyl-
3-hydroxybutanoate but the expected product was S-
(+)-3-hydroxybutanoate through anaerobic
fermentation with bakers yeast as an enzyme
catalyst with a 99.73% yield. This error may be due
to contamination. The racemic product was
successfully synthesized with sodium borohydride as
a reducing agent which had a _______% yield.
Figure. IR spectra of the product in comparison to ethyl
3-hydroxybutyrate Even though the target enantiomer was not
synthesized, the successful synthesis of an
Based on the above figure, the spectra of the
enantiomer of 3-hydroxybutanoate confirms the
obtained product resembles most closely to ethyl 3-
effectiveness of bakers yeast as a source of enzyme
hydroxybutyrate. The computed yield also turned out
catalyst for enantioselective reactions, and sodium
to be 99.73% (see Eq.1).
borohydride as a selective reducing agent for
aldehydes and ketones which produces racemic
6 12 2
1.076 1 products.
= 2.5
116.12 To obtain the expected product with higher
1 118.13
= 2.7365 percent yield and an accurate specific rotation, it is
1 1
2.729 recommended to avoid contamination, and stirring of
% = 2.7365
100% = . % (Eq. 1)
the yeast should be done continuously to prevent
death of the yeast and ensure the effectiveness of the
The optical purity of the product was also reduction process. In addition, obtain the NMR
determined using a polarimeter to measure the spectra of the obtained product for additional
optical rotation. This part of the analysis gave a confirmation of its identity
specific rotation of -12.038. This result implies that
product is most like to possess an R enantiomer.
Guide Questions
Another reduction of methyl acetoacetate was
done using sodium borohydride. The same 1) Define the term enantioselective.
processes were done like the former reduction. IR An enantioselective reaction favors formation
analysis showed the same result as that of the one enantiomer of compound than of another
enzymatic reduction; however, subjection to 2) The keto group of methyl acetoacetate may
polarimetry analysis confirmed that the product is also be reduced selectively with sodium
racemic. borohydride. Describe how the product of this
reaction would differ from the product of the
enzymatic reduction of methyl acetoacetate with
bakers yeast.
4 | Chemistry 35.1 Enzymatic Reduction
 Sodium borohydride selectively reduces hydrogen to the positively charged carbon of the
ketones and aldehydes to alcohol which produces a ketone. It also serves as the third contact point
racemic mixture of the enantiomers of 3- needed for enantioselectivity of the reaction
hydroxybutanoate. On the other hand, enzymatic
reduction with bakers yeast produces an excess of b. Show the biochemical mechanism of the
one enantiomer which is S-(+)-3-hydroxybutanoate. reduction process.
3) What nucleophile is supplied by the yeast?
The yeast provides H- from the enzyme
NADH.
4) What is the purpose of using sucrose in the
bakers yeast reduction of methyl acetoacetate?
The yeast coverts sucrose to ethanol and
CO2 using 14 different enzymes. Hence, sucrose
allows fermentation process to proceed by providing
energy. In addition, starch is hydrolyzed to give
glucose which replenishes the enzymes which allows
the reduction to proceed faster. (http://chemistry.bd.psu.edu/kociolek/images/%20Ye
5) What is an emulsion and what substances ast.pdf)
present in the reaction mixture could be 7) Discuss the differences observed in the IR and
responsible for the formation of an emulsion NMR spectra of methyl acetoacetate and methyl
during the work-up? (S)-(+)-3-hydroxybutanoate that are consistent
An emulsion is a colloidal suspension of with the reduction of the carbonyl group in this
immiscible liquids as droplets in another. This may experiment.
happen when the separatory funnel is shaken In the IR spectra of methyl acetoacetate, there
vigorously during the liquid liquid extraction. The two are 2 peaks at around 1730 cm-1 to 1750 cm-1 which
layers will not separate cleanly, which may result to indicates the presence of the ketone and ester,
losses. respectively. In the IR spectra of (S)-(+)-3-
6) Alcohol dehydrogenase is one of the active hydroxybutanoate, there is only one peak at around
enzymes in yeast. The active site in alcohol 1700 cm-1 to 1800 cm-1which indicates the presence
dehydrogenase contains a zinc ion, Zn2+ that is of the ester group and a band at 3500 cm-1 which
coordinated to the sulfur atoms of two cysteine
residues of the enzyme.

a. Speculate how the zinc ion might assist the

addition of hyride ion to a ketone such as methyl
acetoacetate.
Zinc drags two hydride ions from two NADH
and then forms zinc hydride. It transfers one
5 | Chemistry 35.1 Enzymatic Reduction
indicates the presence of alcohol. siganls was due to hydrogens added through the
reduction process

IR spectra of methy acetoacetae (Gilbert &

Martin,2011)

H NMR spectra of (S)-(+)-3-hydroxybutanoate

(Gilbert & Martin, 2011)

V. References
Academia.edu (n.d.). Reduction of Ethyl
Acetoacetate with Baker's Yeast. Retrieved from
https://www.academia.edu/6322215/Reduction_
of_Ethyl_Acetoacetate_with_Bakers_Yeast

Bruice, P. Y. (2016). Organic chemistry (8th ed.).

IR spectra of (S)-(+)-3-hydroxybutanoate
United States of America: Pearson Education,
Inc.
In the H-NMR spectra, of methyl
acetoacetate, there are 3 singlet signals which Gilbert, J. C., & Martin, S. F. (2015). Experimental
organic chemistry: A miniscale & microscale
corresponds to the 3 non-equivalent types of approach (p.600-615). Boston, MA: Cengage
hydrogen. Learning.

Klein, D. R. (2012). Organic chemistry (1st ed.).

United States of America: John Wiley & Sons, Inc.

Williamson, K. L., & Masters, K. M. (2011).

Macroscale and microscale organic experiments
(6th ed.). Belmont, CA: Brooks/Cole, Centrage
Learning.

H NMR spectra of methyl acetoacetate (Gilbert &

Martin, 2011)

On the other hand, the H-NMR spectra of (S)-(+)-3-

hydroxybutanoate has 5 signals. The additional
6 | Chemistry 35.1 Enzymatic Reduction
 I hereby certify that I have given substantial
contribution to this report.

________________________
Garcia, Beatrice Emmanuelle B.

________________________
Solomon, Marc Ralph M.

_________________________
Santos, Shaine Dominique C.

7 | Chemistry 35.1 Enzymatic Reduction