-1-
Experiment
Multi-step Synthesis -- Coenzyme Catalyzed Synthesis of Benzoin and
Derivatives
This experiment is basically complete. Modifications might still be made. Changes
will be made online or in class.
This multi-step experiment is divided into four sections:
•
•
•
•
Part 1: Synthesis of Benzoin
Part 2: Synthesis of Benzil
Part 3: Synthesis of Benzilic Acid
Part 4: Synthesis of Benzoic acid from Benzaldehyde
Each part is performed on successive days. The product from the previous day's
experiment is used as the reactant on the next day's experiment. It is vital that you do
the experiments on the days they are described. Part 4 is a "self-designed"
experiment where you devise a protocol to follow to prepare and purify benzoic acid
from benzaldehyde. You must design this experiment, and get the approval of your
instructor. You must then make a list of reagents you will need to fulfill this
experiment.
There are five laboratory periods devoted to this experiment. The first four lab
periods are used to make the chemicals listed for those days. The fifth day can be
used to finish up any incomplete analysis, including melt points, yield, etc.
Background
Vitamin B1, thiamine, as its pyrophosphate derivative (shown below), is a coenzyme
universally present in all living systems. It was originally discovered as an essential
nutrient required to prevent the human disease beriberi, which affects the peripheral
nervous system. Symptoms include pain and paralysis of the extremities, emaciation,
or swelling of the body. The disease is still common in the Far East.
-2-
In biochemical terminology, thiamine functions as a coenzyme, a biological molecule
that assists in enzymatic reactions. In most cases, coenzymes are directly involved in
the biochemical reaction that the enzyme catalyzes since they usually bind the
substrate (reactant) for the reaction. Without the coenzyme, no reaction will take place.
The enzyme, which is the biological catalyst, binds the substrate, controlling
stereochemistry, energetics, and entropic factors. As indicated above, the vitamin
(derived from VITal AMINe) thiamine is required for many enzymatic reactions. In
the current experiment, we will use thiamine to catalyze the reaction of benzaldehyde
into benzoin.
Thiamine, functioning as a coenzyme, can be used for (1) the nonoxidative
decarboxylations of a-keto acids, (2) the oxidative decarboxylations of a-keto acids,
and (3) the formation of a-hydroxy ketones. Most biochemical processes are no more
than organic chemical reactions carried out under special conditions. Like most
reactions in organic chemistry, many biochemical reactions can now be explained
using familiar reaction mechanisms. To enhance reactivity, and to be stereoselective,
enzymes are used to bind the substrate in a manner that allows only a single reaction,
with stereoselectivity to occur. In addition, enzymatic reactions can be carried out in
mild conditions and at moderate pH values. Reactions which involve hydrophobic
(lipid loving or water hating) conditions that might not otherwise be possible in an
aqueous, biological environment.
Part 1 of this experiment is designed to illustrate these types of processes. As a
biological reagent, the coenzyme thiamine in this reaction will be used to carry out an
organic reaction chemistry reaction without using an enzyme. The reaction is an
acylion condensation to benzaldehyde:
From a chemical point of view, many coenzymes have what we call "a business end"
to the molecule and the rest of the molecule. The reactive part of thiamine is the
thiazole heterocyclic ring (a 5-membered ring containing both a sulfur [thio] atom and
a nitrogen [azo] atom). This ring is the reactive portion of the coenzyme. The rest of
the molecule is important biochemically for enzyme associate, etc., but the thiazole
ring is the reactive portion. The rest of the molecule is important biochemically, but it
is not required for the reaction described here.
Experiments with the model compound 3,4-dimethylthiazolium bromide have
explained how thiamine-catalyzed reactions work. Part of the chemistry of thiamine is
the acidic proton located on the carbon between the sulfur and nitrogen atoms. Using
3,4-dimethylthiazolium bromide, it was found that there is a rapid exchange of the
-3-
C-2 proton for deuterium in the D2O solution. At a pD of 7 (No pH here!), this proton
was completely exchanged in seconds!
This experiment indicates that the C-2 proton is much more acidic than one would
have expected. This hydrogen, bonded to the imine carbon has a pKa of 12.7, because
the carbanion formed when the proton is removed is stabilized by the adjadent
positively charged nitrogen, yielding the highly stabilized ylide. An ylide is a
compound, or reaction intermediate, with positive and negative formal charges on
adjacent atoms. This ylide can react with an aldehyde to produce an enamine:
The enamine which we will produce, using benzaldehyde, can react with a second
benzaldehyde molecule to produce the desired product, following the acyloin
condensation pathway. The enamine functions much like the enolate partner in an
acid-catalyzed aldol condensation. It can condense with a suitable
carbonyl-containing acceptor to form a new carbon-carbon bond. Decomposition of
the intermediate to regenerate the thiamine ylide yields the protonated acyloin,
benzoin, in this reaction. The final product depicted below just needs to undergo
deprotonation to produce benzoin.
-4-
The reaction pathway outlined above describes the pathway we will follow to produce
benzoin using thiamine as catalyst. The benzoin produced will be used for the
sequence of reactions which will be followed to produce benzil and benzillic acid.
Part 1: Synthesis of Benzoin
In this, the first step of our multistep reaction sequence, benzaldehyde will be
condensed, using the thiamine coenzyme as catalyst, to produce benzoin. The same
reaction can be performed using the cyanide ion (you should include a plausible
mechanism for cyanide catalyst and include it with your report). The reaction
mechanism for thiamine catalysis is described above. (It is imperative that the
benzaldehyde be from a newly opened bottle because of the ease of oxidation,
producing benzoic acid, which will interfer with the reaction. The thiamine
hydrochloride must also be from a newly opened bottle, although it is not quite as
critical as for the benzaldehyde.)
The overall reaction for conversion of benzaldehyde into benzoin is:
Procedure
Add 1.30 g of thiamine hydrochloride to a dry 50-mL flask. Dissolve the solid in 4.0
mL of water by swirling. Add 15 mL of 95% ethanol and cool the solution for a few
minutes in an ice bath. Very carefully and slowly, add 2.5 mL of 3 M NaOH
dropwise and mix by swirling, making certain that the temperature of the solution
never rises above 20oC. To the yellow solution, add 7.5 mL of pure benzaldehyde
and heat the mixture at 60oC for about 1.5 hour. (The temperature of this reaction
cannot go above 65oC. Contstant monitoring of temperature is paramount during
this part of the reaction and must be maintained between 60-65oC.) Alternatively,
the reaction mixture can be stored for 24-48 hours at room temperature, until the
beginning of the next lab period. (The rate of reaction usually doubles for every
10oC increment in temperature.)
Cool the reaction mixture in an ice bath. If crystallization does not occur
immediately, withdraw a drop of the solution on a stirring rod and let it dry to produce
a solid; then, rub it against the inside surface of the flask to induce
crystallization. Collect the product by vacuum filtration and wash it free of any
yellow mother liquor with a 1:1 mixture of 95% ethanol and water.
-5-
Weigh your product to determine an approximate yield, realizing that some water will
still be in your solid. This still moist product must be recrystallized using hot 95%
ethanol (you will need about 8 mL of ethanol per gram of product). Place your
product in the drying oven until the next lab period (if used for Part 2 on the same day,
continue to draw a vacuum through your funnel for 5-10 minutes to evaporate any
ethanol). Your chemical must be dry before beginning Part 2. Typical yield should
be about 5-6 g, although some students recover less than 4 grams. The product
should be colorless and of sufficient purity (mp 134-135oC) to use in the next
reaction.
Dispose of the liquid filtrates in the liquid waster container.
Part 2: Synthesis of Benzil
Starting with the α-hydroxyketone benzoin (prepared in Part 1), you will prepare an
oxidation product, benzil, which is an α-diketone.
This oxidation can easily be done with a variety of mild oxidizing agents, including
Fehling's solution (an alkaline cupric tartrate complex) or copper(II) sulfate in
pyridine. In addition, benzoin could be oxidized by sodium dichromate, but the yield
of benzil is lower because some of the benzoin is converted back into benzaldehyde
following cleavage of the bond between the two oxidized carbon atoms, which is
activated by the phenyl rings, producing benzoic acid as the final product. In this
experiment, due to ease of use and consistent results, we will use nitric acid as the
oxidizing agent.
Caution: Concentrated nitric acid is highly corrosive and causes severe burns if
spilled onto your skin. Nitrogen dioxide (NO2) fumes are highly toxic and can damage
the lungs due to inflammation. Do not breathe NO2 fumes, and perform this part of
the experiment in the hood.
Procedure
Heat a mixture of 4.0 g of benzoin (prepared in Part 1; save enough for analysis, but
proportion reagents as needed) into a round-bottom flask with 14.0 mL of
concentrated nitric acid. Add a stirring bar and attach a condenser to the top of the
flask. Set up a reflux in the hood to vent NO2 produced during the reaction. With
stirring, heat the reaction mixture. Begin timing the reaction when NO2 (red-brownish
colored gas) are visible above the reaction mixture and gas bubbles are present on the
-6-
stir bar. Reflux for at least 30 minutes, or until no more NO2 gas is apparent. Do not
stop the reaction until the reaction is complete. Stop the reaction by removing the
heating mantle, and letting the reaction mixture cool for about 5 minutes. Add about
75 mL of cold water to the reaction mixture, cool to room temperature, and swirl for a
minute or two to coagulate the precipitated product: collect and wash the yellow solid
using vacuum filtration. Continue drawing air through the crystals on the funnel by
suction for about 5 minutes to assist in drying the crystals.. The crude product (dry
weight 3.7-3.9 grams) need not be dried but can be crystallized at once from
ethanol. Dissolve the product in 10 mL of hot ethanol, add water dropwise to the
cloud point (mixture goes from clear to cloudy, or solids being formed), and set aside
to crystallize. Record the yield, crystalline form, color, and melting point of the
purified, and recrystallized, benzil.
Test for the presense of unoxidized benzoin: Dissolve about 0.5 mg of crude or
purified benzil in 0.5 mL of 95% ethanol or methanol and add one drop of 10%
NaOH. If benzoin is present the solution soon acquires a purplish color owing to a
complex of benzil with a product of autoxidation of benzoin. If no color develops in
2-3 min, and indication that the sample is free from benzoin, add a small amount of
benzoin, observe the color that develops, and not that if the test tube is stoppered and
shaken vigorously the color momentarily disappears; when the soluton is then let
stand, the color reappears.
Cleaning up: The aqueous filtrate (containing HNO3) should be neutralized with
sodium carbonate, diluted with water, and flushed down the drain. Ethanol used in
crystallization should be placed in the organic solvents container.
QUESTIONS
Show the correctly balanced oxidation-reduction reaction for this reaction.
Part 3: Synthesis of Benzilic Acid
In this experiment, benzilic acid will be prepared by causing a rearrangement of the
a-diketone benzil. (Preparation of benzil is described in Part 2 of this experiment.)
The rearrangement of benzil proceeds as follows:
-7-
The driving force for he reaction is provided by the formation of a stable carboxylate
salt (potassium benzilate). Once this salt is produced, acidification yields benzilic acid.
The reaction can generally be used to convert aromatic a-hydroxyacids. Other
compounds, however, also will undergo benzilicd acid type of rearrangement.
Procedure
Add 3.0 g of benzil and 9.0 mL of 95% ethanol to a 100-mL flask. Place a stirring
bar in the flask and attach a reflux condenser. Heat the mixture with stirring until the
benzil is dissolved. Add dropwise 7.5 mL of an aqueous potassium hydroxide
solution1 downward through the condenser into the flask. Gently reflux the mixture
for 15 minutes with stirring. After the mixture has dissolved and heated for a few
minutes, the mixture will turn blue-black in color. As the reaction proceeds, the
reaction product will turn brown and the solid may, or may not, be completely
dissolved. At the end of the reaction, remove the flask and let it cool.
When the apparatus is cool enough to handle, remove the condenser and transfer the
contents, including any solids, into a 150-mL beaker. Allow the mixture to cool to
room temperature (do not rush!). When the mixture is cooled, continue the cooling
in an ice-water bath for an additional 15 minutes, when crystallization should be
complete. Crystallization is complete when it appears that virtually the entire mixture
is solidified. If this does not occur in 15 minutes, allow the mixture to set overnight,
or until complete (it is possible to store in an ice bucket or in a refrigerator, if
necessary). Collect the crystals using vacuum filtration and wash the crystals
throughly with three 15-mL portions of ice-cold 95% ethanol. The solvent should
remove most of the color from the crystals.
Transfer the solid, which is mostly the potassium benzilate salt, to a 125-mL
Erlenmeyer flask containing 30 mL of hot water. Stir the mixture until all the solid
has dissolved or until it appears that the remaining solid will not dissolve. If solid still
remains in the flask, filter the mixture through a Hirsch funnel to remove any
particulate material. (If all the solid dissolved, then filtration is not required.)
-8-
With stirring, add dropwise 15 mL of 1 M HCl to the solution of potassium benzilate.
The pH should be about 2; if it is higher than this add a few more drops of acid and
check the pH again. Allow the mixture to cool to room temperature and then complete
the cooling in an ice bath. Let the solid form in the ice bath for at least 30 min, up to
about 60 min. If solid has not formed after an hour, you can store your sample until
the next lab period.
Collect the benzilic acid by vacuum filtration. Wash the crystals with 30-40 mL of
water to remove salts. Dry the product thoroughly in a desiccator until the next
laboratory period.
Weigh the dry product and determine percent yield. Determine the melting point. Pure
benzilic acid melts at 150oC. At the option of the instructor, determine the IR
spectrum of the benzilic acid in KBr. Turn in the entire sample to the instructor in a
labeled vial. Your grade will be based on the purity of your final product, and the
amount of benzilic recovered.
___________
1
The aqueous potassium hydroxide solution should be prepared for the class by
dissolving 27.5 g of KOH in 60.0 mL of water. This will provide enough solution for 8
student groupss, assuming little solution is wasted.
QUESTIONS
1. Show how to prepare the following compounds, starting from the appropriate
aldehyde, following the same procedure performed in this experiment.
2. Give the mechanisms for the following transformations
3. Interpret the infrared spectrum of benzilic acid
Part 4: Synthesis of Benzoic Acid from Benzaldehyde
In this experiment, you will design and perform an experiment to prepare benzoic acid
from benzaldehyde.
-9-
You will devise a procedure to perform this experiment and to fully characterize your
product. The maximum amount of benzaldehyde you can use is 2.0 g. Prior to doing
this part of the experiment, you must compose an experimental protocol in complete
detail.
Authorization to perform this experiment will be given only after your protocol has
been approved by your instructor (his initial on the Chemical Request Form is
required). You are responsible for requesting the appropriate chemicals, and for
performing the experiment safely and for characterizing your product fully. (You
might have a little reprieve, since other students will likely be requesting chemicals,
and if you forgot, you can still use the chemicals on the cart, even if requested by
another group.)
The chemical request sheet is available online: Chem211 Experiment 10, Part 4
Chemical Request Form
Compound
o
o
MW (g/mol) Amount mmol mp ( C)
bp ( C)
Density (g/mL) ηD
benzaldehyde
106.1238
-26
179
1.045
benzoin
212.2476
137
344
benzil
210.2318
95
346 - 348
benzilic acid 228.247
150 - 153
benzoic acid
122.1232
122.4
KMnO4
158.0256
240
249
1.08
2.703
1.5454