Experiment 3

Biotransformation Reactions –
Reduction of Carbonyls with Whole Plant Parts

adapted from Jorge Adum and Gustavo Seoane, Montevideo, Uruguay

Introduction

Organic reductions can be performed with whole plant parts, a method

that is more environmentally friendly and less expensive than traditional
methods. In this experiment, vegetables from Cambodia will be used for the
reduction of a carbonyl compound.
Biotransformation refers to changes in a compound that occur as a result
of enzymatic activity or other activity by a living organism. Biotransformations
often use enzymes in chemical synthesis to produce compounds of a specific
stereochemistry. Many oxidation and reduction reactions in organic chemistry
have been performed using biomaterials.
By using plants in organic reactions, we are practicing green chemistry.
Not only are biocatalysts better for the environment and the humans using them,
but also they will completely degrade, reducing or eliminating waste and toxic
substances released into the environment. Many other reagents are harmful to
the environment. Using plants for chemical reactions provides a new, cleaner,
way to do organic synthesis.
Scientists in developing countries should be very interested in
environmentally friendly and inexpensive chemistry because they do not have
the money for expensive catalysts or waste disposal. The goal of this experiment
is to understand which Cambodian plants are the best catalysts for reducing a
specific carbonyl compound.
As compared to other experiments that are typically used in a teaching
laboratory, these reactions are not fast, and will take several days to work. For
that reason, each section will be working on this experiment for 1 week, coming
into lab all three days of that week. During the other two weeks, you will not be
required to come into lab at all. Check the schedule.

Procedure

Monday
Each laboratory section will be reducing a different carbonyl compound,
and each group will be using a different vegetable. In this way, the class will be
able to get a lot of new data about the use of Cambodian vegetables for this
biotransformation. Possible carbonyl compounds include acetophenone,
benzaldehyde, cinnamaldehyde. During the lab period before this experiment,
each group will choose a vegetable to work with. It is recommended that you
choose a root or tuber, as the literature suggests these seem to work better.
Someone in your group must purchase this vegetable and bring it to lab on
Monday of your assigned week. Someone else should bring in a peeler from
home. We will only use that on Monday.
If your vegetable has a skin, peel it off. Then wash the whole vegetable
first with distilled water. Then soak in 5% sodium hypochlorite (Chlorox
bleach) for 10 minutes, 95% ethanol for 10 minutes and rinse again with
distilled water. The purpose of this step is to remove any bacteria on the
vegetable. Grate or peel the vegetable into small pieces and put 20 grams in a
250 ml Erlenmeyer flask. Add 70 mL of distilled water. Dissolve 100 mg of
your ketone or aldehyde in 1 mL of 100% ethanol in a small vial and add it to
the flask, saving a drop for the TLC analysis. Add a magnetic stirrer and begin
stirring, covered. Write down when your reaction has begun.
Cut a small TLC plate 7.5 cm in length. Spot your starting material and
develop the plate in 80% hexanes, 20% ethyl acetate. Use a small beaker with a
piece of filter paper wrapped in the inside. Cover. Observe your plate under uv
light, circle the spot and calculate the Rf value. You should draw a picture of
the resulting TLC plate in your notebook. Return your TLC solvent to the
appropriate bottle. Save your vial with the starting material for Wednesday.

Wednesday
On Wednesday come into lab and check the progress of your reaction.
Remove your reaction mixture from the stirrer and measure out 4 ml of the
aqueous solution. Return your Erlenmeyer to the stirrer, and continue stirring.
Transfer the 4 ml to a capped test tube and add 1 ml ethyl acetate. Invert about
20 times. (This is a mini-scale extraction.) Carefully remove the top layer with
a pipette and put it in another test tube. Add another ml of ethyl acetate to the
aqueous layer and invert 20 times again. Combine your two organic fractions
and dry with a small spatula full of anhydrous sodium sulfate. Let set for about
5 – 10 minutes. Take a TLC of this solution. Today you should spot several
spots – at least two, your starting material, and today’s reaction mixture. If the
product is available as a standard, you should also spot it.
Develop the TLC plate in the same solvent as previously. Observe under
uv-light, circle any spots and calculate the Rf values for these spot. We are
hoping to see the disappearance of the starting material, and the appearance of a
spot from the product.

Friday
Decant the water from the vegetable fragments, putting the aqueous
mixture into a clean Erlenmeyer flask. Wash the vegetables with another 25 mL
of distilled water and add to the same flask. Extract the aqueous solution with 4
x 25 mL of ethyl acetate. Combine the organic layers and dry with anhydrous
sodium sulfate. Gravity filter through a piece of cotton into a large round
bottomed flask and remove the solvent using the rotary evaporator in the organic
chemistry lab. Your instructor will help you with this. When this is finished,
add 1 ml of ethyl acetate to the round bottom flask, mix and transfer the solution
to a small vial. Add another ml of ethyl acetate, mix, and combine with the first.
Use TLC once more to compare your reaction mixture to the starting material
and perhaps a standard of the product. Has your reaction gone to completion?
At the end of this lab period, we will compare our results and determine
which vegetables worked best for our reduction reaction. These results will be
included in your report.

Waste Disposal
The carrots can be put in the trash, and the aqueous solution can be put
down the sink. We will collect the ethyl acetate from the rotary evaporation. It
can be used again.

Report

You will be required to submit a lab report for this experiment, in English.
It should include a title, a short introduction section, an experimental section
(how you did the experiment) and your results. Write it in your own words. .
 Experiment 3
Biotransformation Reactions –
Reduction of Carbonyls with Whole Plant Parts
Equipment Needed (Things in bold are not currently in 506)

Stirrer and magnetic stir bar (5 of them)

Balance

Supplies Needed (per group)

1 vegetable (brought in by students)

250 ml Erlenmeyer flask with stopper
2 ml glass vials with caps
100 ml graduate cylinder (1 for the class)
medium beaker (for washing vegetable)
TLC spotters
Silica gel – uv TLC plates (aluminum or plastic backed)
small beaker (for TLC)
piece of filter paper
petri dish for cover
10 ml graduate cylinder (2 for the class)
small capped test tube
another test tube
pipets and bulbs
spatula (for drying agent – 1 for the class)
125 ml separatory funnel
cotton
Funnel
round bottom flask

Chemicals Needed

5% NaOCl (Chlorox) – bought at larger Khmer markets (indoor), or Lucky

95% ethanol (Can buy from any pharmacy)
distilled water
several carbonyl compounds (acetophenone, benzaldehyde, cinnamaldehyde,
benzophenone)
100% ethanol (1 ml per group)
80% hexane / 20 % ethyl acetate (100 ml per week should be enough)