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Experiment 4 – enzymes specificity for enantioselective organic compounds

Introduction:

In biological process, enzyme plays the key role as they could acting as biological catalyst.
Moreover, enzymes may be brought of as highly efficient chiral organic reagents developed by
nature to carry out organic reactions in biological system. This practical is designed to use an
enzyme selectively reacts with N-acetyl-D, L-phenylalanine methyl ester in order to determine
the enantiomeric purity. Subtilisin Carlsberg is the enzyme used in this practical to hydrolyses
the carboxyl group of the L-enantiomer of derivatized amino acids. The completion of the
reaction is determined by thin-layer chromatography (TLC) plate using standard solutions and
monitoring with ninhydrin. Then the optical purity of L-enantiomer and D-enantiomer are
achieved by measuring the optical rotation with polarimetry.

Procedure:

Add 2.00 g phenylalanine methyl ester hydrochloride to 100 mL 10% sodium carbonate
solution to react with 50 mL dichloromethane in a separating funnel and then run off the
organic (bottom) layer into conical flask. The aqueous layer is further extracted with 25 mL
dichloromethane twice and the bottom layer was collected. Then the dichloromethane extracts
was dried over magnesium sulfate and filtration took place afterwards. The filtrate was
collected in a round bottom flask and then mixed it with 2.6 mL trimethylamine and 1.0 mL
acetyl chloride dropwise. After that, check the completion of reaction by analyzing the reaction
mixture and the starting material via a thin layer chromatography. Once the reaction was
completed, transfer the reaction mixture into separating funnel and added 50 mL 10% sodium
carbonate then run off the organic (bottom) layer with 50 mL of 3 M hydrochloric acid twice.
The organic layer was dried over magnesium sulfate and filtered the phase into a round bottom
flask afterwards, and then distilled off the dichloromethane. N-acetyl phenylalanine methyl
ester is collected in a 100 mL conical flask. Weighted and recorded the mass of N-acetyl
phenylalanine methyl ester.

All of the crude N-acetyl phenylalanine methyl ester made previously mixed with 25 mL
deionized water and 1 mL universal indicator in a 100 mL conical flask. Added 0.3 M NaOH
to adjust the pH to ~7.5. Then added 10 mg Subtilisin Carlsberg protease enzyme into the
mixture and then the reaction was placed into a thermos tatted bath. Stabilized the pH at 7.5
for 10 minutes by further adding 0.3 M NaOH dropwise into the mixture. Then the mixture
was extracted with 30 mL dichloromethane three time, the dichloromethane extracts (bottom
layer) were collected. The aqueous phase was acidified to pH 1 by adding 3 M hydrochloric
acid and use universal strips to check the pH value. Then extracted with 40 mL
dichloromethane for three times and collected the bottom layer. Two extracts were dried over
magnesium sulfate and filtered in to round bottom flask for evaporation later on. After the
evaporation, recorded down the mass of two solid products, then weighted 0.25 g of the each
solid product and mixed with ethanol up to the volume separately. Then the solutions were
transferred into a polarimetry cell and noted down the observed optical rotations of each
product.
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Results:

Yield of N-acetyl phenylalanine methyl ester = 1.1000 g

1.1000 𝑔
235.28 𝑔/𝑚𝑜𝑙
% yield of N-acetyl phenylalanine methyl ester = 2.0000𝑔 = 50.4%
215.68 𝑔/𝑚𝑜𝑙
Yield of the acid = 0.3316 g
0.3316 𝑔
221.254 𝑔/𝑚𝑜𝑙
% yield the acid = 1.10 𝑔 × 100 % = 64.1%
2
235.28 𝑔/𝑚𝑜𝑙

Yield of the ester = 0.3090 g

0.3090 𝑔
235.28 𝑔/𝑚𝑜𝑙
% yield the ester = 1.10 𝑔 × 100 % = 56.2%
2
235.28 𝑔/𝑚𝑜𝑙

Solvent front

6 cm
S: starting material
M: reaction mixture
P: product

Baseline
M S

Figure 1&2. The TLC plate for completed reaction.

5.3 𝑐𝑚
Rf value of N-acetyl phenylalanine = = 0.88
6.0 𝑐𝑚

Specific rotation of N-acetyl phenylalanine methyl ester = []

𝑜𝑏𝑠𝑒𝑟𝑣𝑒𝑑 𝑟𝑜𝑡𝑎𝑡𝑖𝑜𝑛 (𝑑𝑒𝑔𝑟𝑒𝑠𝑠)  −0.34°
= 𝑝𝑎𝑡ℎ 𝑙𝑒𝑛𝑔𝑡ℎ (𝑑𝑚)× 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 (𝑔/𝑚𝑙) = 𝑙 × 𝑐 = 0.25 𝑔 = −13.6°
1.0 𝑑𝑚 × 10 𝑚𝑙

% ee of N-acetyl phenylalanine methyl ester

[] 𝐷 𝑠𝑎𝑚𝑝𝑙𝑒 −13.6 °
=[ ] × 100% = × 100% = 63.55 % = 63.6%
 𝐷 𝑝𝑢𝑟𝑒 𝑒𝑛𝑎𝑛𝑡𝑖𝑜𝑚𝑒𝑟 −21.4 °
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Table 1. The observed rotation, specific rotation and % ee for the ester and acid

Observed rotation Specific rotation % ee

N-Acetyl-D phenylalanine −0.34° −13.6° 63.6 %
methyl ester
N-Acetyl-L-phenylalanine +0.54° +21.6° 47.0 %
methyl

Discussion:

• The phenylalanine methyl ester is used in this practical contains both R and S isomers.
If biologically-sourced phenylalanine was used to prepare the methyl ester for the
experiment, the isomers would exist in S isomers. If S-isomer was taken through the
experiment, the final product would be N-acetyl-L-phenylalanine after the enzyme
reaction.
• S represents starting material which is N-acetyl chloride, M is the reaction mixture
which phenylalanine, and P represents the product. From the figure 1&2, we can know
only product phenylalanine methyl ester presents in P.
• Acetyl chloride are likely to be other compounds in the reaction mixture. The reason
why extraction step was effective because magnesium sulfate was used to dehydrate,
and in addition, we used 10 % sodium carbonate solution plus 3 M hydrochloric acid
to wash the mixture, these two solutions would react with the impurities to produce
water-soluble products which would be separated from the oil phase due to different
density, hence, we could remove most of the impurities during the extraction process.
• The enzyme, Subtilisin carlsberg will only be activated around pH 7.5. An extreme pH
condition will kill the enzyme or the enzyme would be denatured (inactive).
• % enantiomeric excess(% ee) of ester was 63.6% and % ee of acid was 47.0%. This
means major enantiomer (S) is present in 64.95% or 78.74% excess over the minor
enantiomer (L).
• ninhydrin, if the reaction is completed, it would show a brown colored spot in the
reaction mixture which indicated the presence of amines. If the reaction was incomplete,
there would be a blue color spot on the TLC plate at the same position as the starting
material, and as for proline, it would only show a brown spot.

Conclusion:

In my conclusion, enzymes do react specifically with specific configurated enantiomers N-

acetyl phenylalanine methyl ester and it helped to determine the enantiomeric purity.
There are three products that we obtained from this experiment, Crude N-acetylphenylalanine
methyl ester, (R)-N-acetylphenylalanine and (S)-N-acetylphenylalanine, and the optical
purities of ester was 63.6% and acid was 47.0%.