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Experiment #4: ENZYMATIC REDUCTION OF METHYL ACETOACETATE

2018-04546, 2018-01441, 2018-01434, 2018-02050


Group 3, CHEM 36, LB2B, Mr. Mark Jeremiah Cleofas
October 24, 2019

I. ABSTRACT

Enzyme-catalyzed enantioselective reduction of ketones and keto-esters are vital in the


production of optically active alcohols that are used in various ​pharmaceutical and
agrochemical industries, especially in drug synthesis. In this experiment, Baker’s yeast
(specifically, its enzyme, invertase), a chiral reagent, was used to prepare sucrose ​by
breaking it down into fructose and glucose, with the latter being transformed into
glucose-6-phosphate, the enantiomer needed for the synthesis. An anaerobic yeast
suspension set-up was constructed and left to ferment for one week after the addition of
methyl acetoacetate, a keto-ester. To isolate the product a filter-aid made of silica gel was
prepared and dichloromethane or DCM was used to separate the crude product from the rest
of the yeast mixture. To confirm whether there were no more keto-esters in the solution, a
ferric ion test was performed. The DCM layer was dried, evaporated and weighed. The ferric
ion test for the sample tested negative indicating that there were no keto-esters in the
solution. ​The product obtained from the reduction of methyl acetoacetate weighed 0.0943 g
with a percent yield of 0.9655%. Methyl acetoacetate is reduced by the alcohol
dehydrogenase found in yeast, theoretically transforming into its chiral secondary alcohol
methyl-(S)-(+)-3-hydroxy- butanoate. In conclusion, further tests, such as IR and NMR
Spectroscopy, to confirm the production of the desired product may be administered to obtain
more accurate results.

II. KEYWORDS: ​Alcohol dehydrogenase, Baker’s yeast, chirality, fermentation, methyl


acetoacetate, stereoselective reduction

III. INTRODUCTION catalysts producing secondary alcohols with high


levels of enantiomeric purity​[2]​. Such reaction can
In chemical reactions, the gain of two be observed using enzymes that can perform such
hydrogen atoms or the loss of an oxygen atom or a enantioselective reductions.
halogen atom is called reduction that leads to an
important conversion of aldehydes and ketones, Enzymes are biological catalysts composed
being reduced to primary and secondary alcohols.​[1] of amino acids. (i.e. protein). Enzymes work by
The most frequently employed method is with lowering the activation energy for a reaction​[3]​. One
catalytic hydrogenation or with the use of complex particular reaction where enzymes are widely used
metal hydride reagents such as lithium aluminium is the conversion of ketones to their corresponding
hydride (LiAlH​4​) and sodium borohydride (NaBH​4​) optically active secondary alcohols​[4]​. Baker’s yeast
which gives equal amounts of the chiral alcohol and is a chiral reagent that acts as an enzymatic
its enantiomer. This is produced because these reducing agent and can introduce chirality to a
achiral reducing reagents attack both faces of the molecule. Transformation reactions involving these
plane of the organic substrate with equal organisms are beneficial since they occur at
probability.​[2] ambient temperature and pressure​[1]​. Most
enzymes create a chiral active site to yield a chiral
However, if the reducing agent or solvent is product.
chiral, the transition states leading to the two
isomers are different and become diastereomeric. For this experiment, the enzyme used was
Thus, the amounts of each will not be equal. An alcohol dehydrogenase, which is an important part
example may be, the reduction of ketones by of metabolic cycle glycolysis. In the case of Baker’s
catalytic hydrogenation in the presence of chiral Yeast, alcohol dehydrogenase not only changes
NADH to NAD​+ for energy production, but also Reduction. ​The stopper in the Erlenmeyer
changes the aldehydes and ketones into alcohols flask was removed and 2.5 mL of methyl
which is called fermentation. Both fermentation and acetoacetate was added. The stopper was replaced
glycolysis are processes that convert complex and the set up was left for one week to ferment in a
molecules into simple forms, however fermentation warm location.
uses bacteria or yeast in the process of conversion,
whereas glycolysis does not, In the reaction Isolation of the Product. ​The filter-aid was
mechanism of yeast with methyl acetoacetate, the prepared by filtering a slurry made from 10 g of
enzyme directs hydrogen addition from the top silica gel in 50 mL of water through a 10-cm
face​[3]​. Buchner funnel containing a piece of filter paper;
the bed was kept moist for the duration of the
filtering. The filtrate was discarded.

To the mixture containing the yeast cells,


another 10 g of silica gel was added and mixed
thoroughly. The solids were allowed to settle for a
few minutes and was decanted carefully through
the bed of filter-aid while applying gentle suction.
Figure 1. ​Reaction of Methyl acetoacetate with Baker’s After filtering the supernatant liquid, the residual
Yeast​[3] solid and liquid were poured into the funnel and
washed with 25 mL of water and 5 mL of
The objective of this experiment is to dichloromethane. The resulting solution was
demonstrate enantioselective reduction of methyl transferred to a separatory funnel and washed
acetoacetate using a chiral enzyme and have three times with 5 mL portions of dichloromethane.
ample knowledge on setting up anaerobic
fermentation setup. The combined dichloromethane layers were
obtained and dried with several spatulas of
IV. EXPERIMENTAL anhydrous sodium sulfate, swirling the beaker until
the mixture is less cloudy. The dry dichloromethane
Setup of Yeast Suspension. ​In an Erlenmeyer solution was decanted into a beaker and put into a
flask, about 20 g of sucrose and 0.25 g of disodium boiling water bath under the fume hood. When most
hydrogen phosphate was dissolved in 75 mL of tap of the dichloromethane evaporated, the remaining
water. One packet (8 g) of dry, active baker’s yeast solution was transferred to a pre-weighed glass vial
was subsequently added, and the apparatus was and replaced into the water bath to completely
assembled as shown in​ Figure 2. evaporate the liquid.

Analysis of the Product. T ​ he product was


weighed and computed for its percent yield of crude
methyl-(S)-(+)-3-hydroxybutanoate. Ferric ion test
was applied to know whether any ketoester
remained.

V. RESULTS

A. Reduction of Methyl Acetoacetate


Figure 2. ​Anaerobic Fermentation Setup[2]

The product obtained from the
reduction of methyl acetoacetate weighed
The ignition tube contained a 3% barium
0.0943 g with a percent yield of 0.9655%.
hydroxide solution and a layer of mineral oil to
(See Appendix A for calculation of
protect the solution from atmospheric carbon
theoretical yield and percent yield).
dioxide. The setup was allowed to ferment for one
hour in a warm location.
B. Ferric Ion Test

Table 1. ​Results for Ferric Ion test


Figure 3. ​Reduction of Aldehyde to Alcohol​ ​
[7]
Positive Deep
Control purple
Historically, biocatalytic ketone reductions
involved the use of Baker’s yeast.​[8] ​Among the
biocatalysts used in organic synthesis, baker’s
yeast (Saccharomyces cerevisiae) is the most
effective and preferable cell because of its
Product Light yellow
availability, milder reaction conditions, i. e. at room
brown
temperature in water under air atmosphere, higher
optical yield and chemoselectivity especially in the
reduction of saturated ketones.​[9] Furthermore,
The Ferric ion test is a test for confirming asymmetric reduction of carbon-carbon double
the presence of phenols. The test is positive if the bond and carbon-carbon bond forming reaction of
color of the solution ranges from intense blue to α, β-unsaturated carbonyl compounds are also
purple, red or green while the negative test is accomplished by baker' s yeast. The products thus
characterized by a yellow colored solution. The obtained are applied as chiral building blocks to the
product that underwent the Ferric ion test is synthesis of various natural products.
negative, therefore, there are no presence of
phenols in the sample.

VI. DISCUSSION

Owing to the importance of a broad range


of resulting chiral alcohol products in the field of
chiral drug synthesis, enantioselective catalytic Figure 4. ​Baker’s yeast structure[9]

reduction of ketones additionally gained
tremendous industrial interest. Notably, numerous Fermentation allows the conversion of
efficient catalytic routes have already been sugar to alcohol and carbon dioxide through 14
developed to date for enantioselective ketone enzymes that serves as catalysts - Adenosine
reductions. Enantioselective reduction of ketones triphosphate (ATP), thiamine pyrophosphate,
using alcohol dehydrogenases has been already magnesium ion, and reduced nicotinamide
comprehensively reviewed.​[5] dinucleotide (NADH). In the case of sucrose, it is
hydrolyzed to produce glucose and fructose which
Alcohol dehydrogenases (ADHs) constitute is eventually converted to pyruvic acid that will be
a large family of enzymes responsible for the decarboxylated to form acetaldehyde.
reversible reduction of aldehydes, ketones, and α​-, Acetaldehyde is then hydrogenated by alcohol
β​-, or γ-keto esters to the corresponding hydroxy hydrogenase to form ethanol.​[10]
compounds. This reduction is coupled with the
stoichiometric consumption of NADH or NADPH,
which needs to be regenerated simultaneously.
These enzymes show considerable diversity in
terms of substrate specificity, acting on numerous
aliphatic and aromatic aldehydes and ketones,
including sugars, steroids and xenobiotics.​[5,6]
Figure 5. Embden-Meyerhof-Parnas scheme​[11] FeCl​3 test was used to confirm that the
methyl acetoacetate was converted in the
The enzyme used, alcohol dehydrogenase, reduction. The result exhibits a brown color
plays a vital role in glycolysis, which provides because of the complexation of ester with Fe​3+​. The
energy to the cells under anaerobic conditions like results from the test confirmed that the methyl
yeast. Its active site has zinc atom serving as the acetoacetate reacted when reduction took place as
third element of three-point contact needed for the product observed has a lighter color than the
enantioselectivity. control. The reason for a lighter shade for the
product may be because of some unreacted methyl
acetoacetate, it could also be because of the
uncomplexed Fe​3+​.

Figure 6. ​Active site of alcohol dehydrogenase approaching


​ ​from
of the substrate into a favorable three-point contact [7]​
http://chemistry.bd.psu.edu/kociolek/images/%20Yeast.pdf Figure 7. ​Complex formed by reaction of methyl
acetoacetate with Fe3+​ ​, ​from
http://dept.harpercollege.edu/chemistry/chm/100
Methyl acetoacetate, an organic molecule that ​
/dgodambe/thedisk/qual/fecl3.htm.[12]
is relatively small to permeate through the yeast’s
cell membrane, replaces the acetaldehyde The reduced methyl acetoacetate percent
substrate. The reaction happens at the NADH and yield was less than the theoretical value, this is due
the base which are the active sites of the alcohol to the fact that there have been failure to properly
dehydrogenase. The two hydrogens at the isolate the product upon the last step of isolation
4-position of nicotinamide of NADH are with the glass vial being submerged into the water
distinguished. When it binds the coenzyme, B-side bath when it is supposedly should not be which is
of NADH is blocked causing the substrate to bind to the cause of repetition of the steps starting from
the A-side of the coenzyme and H​a hydride ion will DCM washing and decreased the product.
be transferred to the substrate. The tendency of
the pyridine to restabilize its aromaticity causes the Theoretically, the enantiomeric product
transfer of hydride by NADH​[12]​. produced from this experiment is 85%
methyl-(S)-(+)-3-hydroxybutanoate, but there had
been no further tests done to confirm this product.

VII. CONCLUSIONS AND


RECOMMENDATIONS

With a 0.9655% yield, the ketone methyl


acetoacetate was reduced into its chiral secondary
alcohol methyl-(S)-(+)-3-hydroxy- butanoate
Figure 6. ​Mechanism of hydride NADH transferred to the through the chiral enzyme alcohol dehydrogenase
substrate
found in baker’s yeast. This was confirmed through
the ferric ion test which tested positive by the
presence of a brown solution lighter than the 04 December 2007,
positive control confirming that methyl acetoacetate https://pubs.acs.org/doi/full/10.1021/ar700167a?src
reacted when reduction took place. The low percent =recsys.
yield of the product may be caused by the
submersion of the glass vial into the water bath i.e. [​9] Bubun Banerjee, ‘Baker’s yeast’, Research
the carelessness of the experimenter. In addition, Gate, August 2015, ​https://www.researchgate.net/
several factors such as the vacuum filtration and publication/281107693_Baker's_yeast
type of filter-aid used must also be considered.
[​10] Academia.edu (n.d.). ‘​Reduction of Ethyl
For a better yield, it is recommended to use Acetoacetate with Baker's Yeast​’.
a lesser amount of yeast than the researchers have https://www.academia.edu/6322215/Reduction_of_
used to prevent spillage during fermentation. Ethyl_Acetoacetate_with_Bakers_Yeast
Continuous stirring using a magnetic stirrer is also
suggested to prevent the death of yeast in the [11] Bruice, P. Y. (2016). ‘Organic chemistry’ (​ 8​th
process. Also, one should make sure that the ed.). United States of America: Pearson Education,
fermentation should occur under optimal Inc.
temperature for it has something to do with the
activity of the yeast. Further tests such as IR [12] Harper College. ‘FeCl​3 Test’. 23 October 2019.
spectroscopy and optical rotation measurement http://dept.harpercollege.edu/chemistry/chm/100
should be conducted to confirm the production of /dgodambe/thedisk/qual/fecl3.htm.
the desired product.

VIII. REFERENCES

[1] Fox & Whitesell, 3rd Ed. Chapter 12,


pg.572-584.

[2] Villarante, et. al. ​Laboratory Manual in Organic


Chemistry 36 - Organic Chemistry II.​ pg. 22

[3] D.J. Dyer & SIUC. ​Enzymatic Reduction. 4th Ed.


546. pg. 588

[4] Bawa, R.A., Ajjabou, F., & Shalfooh, E. (2008).


Enzymatic Reduction of Ketones to Optically Active
Secondary Alcohols. Journal of Physical Science,
19(2), 1–5. Retrieved from
https://pdfs.semanticscholar.org/bba8/3dd48b40
126e00e81c8bc87432ccbb43fc42.pdf

[5] Karlheinz Drauz, et. al., ‘Enzyme Catalysis in


Organic Synthesis’, 3rd Ed., (2012), p. 1072.

[6] Alcohol Metabolism, ‘Alcohol Dehydrogenase’,


23 October 2019,
https://sites.tufts.edu/alcoholmetabolism/the-biologi
cal-pathway/the-answer/.

[7] D.J. Dyer, ‘Enzymatic Reduction of Methyl


Acetoacetate’, 23 October 2019,
http://juliethahn.com/JMH-Enzyme.pdf.

[8] Jeffrey Moore, et. al., ‘Advances in the


Enzymatic Reduction of Ketones’, ACS Publication,