Enantioselective Reduction of a Ketone Using Enzymes
carrot peels
O
O
H 2O
rt
O
OH
Background:
Enzymes are chiral biocatalysts. Some are capable of performing a reaction enantioselectively, to provide one stereoisomer as the major product. Enzymes have also been used
to selectively catalyze a reaction of one enantiomer of a substrate over another. The
separation of two substrates based on a difference in their reaction rates is known as “kinetic
resolution”. Lipases, which catalyze the formation and hydrolysis of esters, have been widely
used to resolve enantiomeric alcohols and esters.
Isolated and purified enzymes can be expensive and unstable. Often, whole cells (such as
baker’s yeast) are used as a convenient source of enzymes. In his week’s lab, an achiral
ketone will be reduced to a chiral alcohol using the enzymes found in carrots.
Hazards:
All steps involving chemicals are to be performed in a fume hood while wearing gloves and
goggles. Ethyl acetate, hexanes, and methanol are flammable liquids and must be disposed of
in the organic waste carboy.
Procedure:
To a 250-mL Erlenmeyer flask containing a large magnetic stir bar add 20 mg of
benzofuran-2-yl-methyl ketone followed by 75 mL of distilled water. Swirl the flask, and
then add 24 g of carrot peelings to it. Clamp the flask above a magnetic stirrer but not in
contact with it (Note 1) and adjust the stirrer to maintain a slow rate of stirring. Make a note
of the time.
Monitor the reaction by TLC. To do this, take aliquots (~1 mL) at 10, 30, 60, 100, and 120
minutes after stirring is initiated, transferring them to a centrifuge tube or test tube. Add ~1
mL of ethyl acetate, and tap the centrifuge tube repeatedly to thoroughly mix. Analyze the
top (organic) layer by TLC (Rf ≅ 0.2 (alcohol); 0.4 (ketone); 85:15 hexanes:ethyl acetate).
Visualize the spots using 254 nm UV light and circle the spots with a pencil. In addition, you
may use p-anisaldehyde reagent (“PAA”, a solution of anisaldehyde and sulfuric acid in
ethanol) as the detection agent. Quickly immerse your TLC plate into the PAA stain and
remove it; blot excess stain without touching the silica surface, then char on a hot plate until
spots just become visible. Make a note of the color of the spots.
Decant the liquid into a 250-mL Erlenmeyer flask; wash the carrot peels with an
additional 25 mL of distilled water and combine with the liquid in the Erlenmeyer flask.
Extract the aqueous solution with ethyl acetate (4 × 25 mL), dry the combined organics with
anhydrous Na2SO4, decant into a 250-mL round-bottom flask and concentrate in vacuo.
Purify the crude product by by column chromatography, using a Pasteur pipette halffilled with silica gel as the column. Elute your product with a mixture of 15 mL of hexanes
and 2.5 mL of ethyl acetate, collecting fractions in test tubes (ca. 1 column volume each
fraction). Pool the fractions that are pure in your product alcohol as judged by TLC,
concentrate them in vacuo, and obtain the mass of product. Obtain an IR spectrum of your
product.
Use a polarimeter to find the optical rotation of your 1-benzofuran-2-yl-ethanol.
Dissolve at least 30 mg of your compound in 12 mL of methanol (you may have to pool
material with other students in order to have enough sample). Fill the polarimeter cell with
your solution and obtain the optical rotation of your sample. Use the following formula to
calculate the specific rotation for 1-benzofuran-2-yl-ethanol:
[α]!"
! =
𝛼
𝑐∙𝑙
where α = measured optical rotation, c = sample concentration in g/mL, and l = the cell
length in dm.
Notes:
1) The Erlenmeyer must not be in contact with the stirrer plate, since heat development is
detrimental to the reaction progress. The yield dramatically drops off when the temperature
rises above 28 °C.