Student Handout
Borohydride Reduction of Ketones: Stereochemistry of Estrone Reduction
Required Reading: Chapter 19 in Organic Chemistry by G. Marc Loudon (4th ed.; 2002, Oxford
University Press). Pay special attention to section 19.8.
Objective: In this experiment you will carry out the reduction of a ketone to an alcohol using
sodium borohydride. You will also study the stereochemistry of this reaction by reducing estrone
and analyzing the products of the reaction.
Scenario
You are a part of an organic synthesis team at a leading drug discovery research company. In a
recent meeting of division heads, the pharmacology and toxicology team proposed to carry out
metabolic studies on some synthetic anabolic steroids to find drug leads that can augment
breast cancer therapy. After the meeting your team has been assigned the task of synthesizing
stereochemically pure steroids for this project. Your project leader has asked you to evaluate the
stereochemical purity of steroids that can be obtained by utilizing various reagents for the
synthesis.
Steroids are a class of bioactive molecules that play a vital role in the body such as controlling
meiosis, carbohydrate metabolism, fat storage, muscle growth and immune function. Several
natural and synthetic steroids are used therapeutically in treating metabolic disorders, weight
loss and breast cancer.
Figure 3.1. Testosterone – a typical steroid.
Structurally, steroids contain a skeleton of four fused rings to which various functional groups are
attached. An important synthetic conversion of steroids that affects their metabolism is
methylation at the 17-position of the skeleton. Methylation at this position retards significantly the
metabolism of the steroid by the liver into waste products. Therefore, 17-methylated steroids
usually have a longer half life than their non-methylated counterparts. The basis of the retarded
metabolism is that the 17-methyl group prevents the steroid from fitting into the active site on the
various liver enzymes that process steroids. For this reason, most oral steroids are methylated
at 17-position.
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Figure 3.2. Examples of orally available synthetic steroids methylated at the 17-position.
In order to design an appropriate scheme to synthesize stereochemically pure steroids, your
team leader has asked you to investigate the feasibility of using steroids with a carbonyl group at
the 17-position as starting materials. An easy way to investigate the stereochemical course of
reduction is to carry out the reduction of estrone and determine whether the reaction leads to a
single stereochemically pure product or a mixture of stereoisomers.
Understanding the Experiment
Metal hydrides (sources of hydride anion H:–) of the group 3A elements, such as lithium
aluminum hydride (LiAlH4) and sodium borohydride (NaBH4), are widely used to reduce carbonyl
compounds to their corresponding alcohols. Lithium aluminum hydride reduces many
compounds containing carbonyl groups – aldehydes, ketones, carboxylic acids, esters and
amides. By contrast, NaBH4 is a weaker reducing agent and reduces only aldehydes and
ketones.
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The reduced reactivity of sodium borohydride and the fact that sodium ion is a weaker Lewis
acid than lithium ion requires that it be used in protic solvents such as alcohol. In contrast,
lithium aluminum hydride reacts rapidly with, and is thus destroyed by, protic solvents and
therefore must be used under anhydrous conditions in aprotic solvents free of any carbonyl
functionality. Ease of handling and optimal reactivity (and low cost) makes sodium borohydride
the reagent of choice for reducing aldehydes and ketones, not just in the laboratory but in large
scale industrial processes as well.
In this experiment, you will carry out the reduction of estrone to estradiol using sodium
borohydride in methanol as the solvent. Estrone and estradiol are classified as estrogens –
steroid compounds that function as female sex hormones. They promote the development of
female secondary sex characteristics, such as breasts, and are also involved in the thickening of
the endometrium and other aspects of regulating the menstrual cycle. Like all steroid hormones,
estrogens readily diffuse across the cell membrane; inside the cell, they interact with estrogen
receptors. Estrone and estradiol were first isolated from the urine of pregnant women. Besides
functioning as female sex hormones, natural and synthetic estrogens find their therapeutic
applications in hormone replacement therapy and treatment of breast cancer.
Q1. Examine the structures of 17-D-estradiol and 17-E-estradiol carefully and determine the
stereochemical relationship between the two isomers.
When estrone is treated with sodium borohydride (NaBH4), reduction of the carbonyl group takes
place to yield an alcohol. That is, estrone is converted to either 17-D-estradiol or 17-E-estradiol.
In order to predict the stereochemical course of the reaction, some additional structural features
of the estrone molecule should be considered. A steroid with all-trans ring junctions, such as
estrone, is fixed in a rigid, almost disc-like shape. The angular methyl group (see structure
below) is in an axial position, and is said by convention to occupy the E (beta) face of the steroid.
The other face —the “underside” of the steroid— is referred to as the D (alpha) face. When
sodium borohydride attacks the carbonyl group, it must attack the carbonyl S-orbitals. In
principle this can occur from either face of the steroid. From examination of the structure below,
however, one can see that the two different modes of approach of the BH4í are not equivalent.
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Q2. On the basis of the structure given above, which face of the carbonyl group in estrone will
be attacked most rapidly by borohydride anion? How does this affect the orientation of the
hydroxy group in the product?
Q3. Predict the major diastereomer that will be formed by borohydride reduction of estrone.
Procedure
Add 5 mL of 95% ethanol and 40 mg (148 Pmol) of estrone into a 25-mL scintillation vial along
with a magnetic stir-bar. Adjust the hot plate to the power setting “3” (temperature of heating
block ~80 °C), place the reaction flask in a heating block on the hot plate and stir until estrone is
completely dissolved.
Solubility of estrone: One gram of estrone is soluble in 250 mL of 95% ethanol at 15°C.
However it requires only 50 mL of boiling alcohol to dissolve the same amount of estrone. It will
take roughly 5 minutes for estrone to be dissolved in the 95% EtOH.
Obtain a silica gel TLC plate and carefully draw a line with a soft pencil 1.0 cm from the bottom
of the plate to mark the origin. Delineate five separate lanes by making five small, evenly
spaced marks perpendicular to the origin line; number these 1 through 5. With a capillary,
remove a small sample of your estrone solution and apply it three times to LANE 1. Add 1 mL of
0.5 M ethanolic NaBH4 to the estrone solution and stir the reaction mixture for 15 minutes. Note
that the initial mixture will be fairly clear and may become cloudy after a couple of minutes. At
the end of this period, remove a sample of the reactions with a capillary tube and spot it in LANE
2 of the TLC plate.
Adjust the power setting of the hot plate to “4” (temperature of heating block ~90 °C) and boil the
reaction mixture while stirring. Turn off the hot plate after the reaction starts boiling and continue
the stirring for 5 minutes. Slowly add distilled water to the reaction mixture with a Pasteur
pipette. It will take roughly 10–12 mL of water to form a precipitate or cause the solution to
become turbid.
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The initial addition of water quenches the reaction. The borate esters, which are less soluble in
water than they are in the 95% ethanol, will precipitate initially out of solution. However, borate
esters react with water to yield alcohols and sodium borate that is soluble in water. If you add
water slowly, you will be able to see that the initial precipitate re-dissolves. The water added will
produce a mixed solvent for the crystallization of the product in the correct proportion.
Remove the reaction vessel from the magnetic stirrer and cool it in an ice bath for 15 minutes to
allow the product to crystallize. Collect the solid crystals by suction filtration on a Hirsh funnel;
wash the product with 2 mL of distilled water and continue the aspiration for at least 15 minutes.
Determine the weight of the dried product and then transfer a pinch of your product with a clean
spatula into a small conical vial. Add 2 drops of 95% ethanol and mix the isolated product with a
spatula. Remove a small sample with a capillary tube and spot onto LANE 3. Spot the standard
solutions of 17-E-estradiol onto LANE 4 and 17-D-estradiol onto LANE 5 respectively. Fill the
TLC developing chamber with 8 mL of developing solvent provided (acetone: dichloromethane:
hexane, 22 : 50 : 28 by volume). Make sure that the level of the solvent is below the origin line
on your TLC plate. After your TLC plate is developed, mark the solvent front and visualize the
results by dipping the TLC plate in vanillin solution. Keep the TLC plate in the oven for 23
minutes and identify the isomer that you obtain as your product. Are the results consistent with
your prediction? Calculate the percent yield of the final product. Determine the melting point of
the dried product to verify the stereochemical assignment of your product.
To obtain an IR spectrum, transfer your entire product after taking the melting point into a 3-mL
conical vial. Add 0.5 mL of diethyl ether to it and vigorously mix the contents with your spatula.
Wait for the contents to settle down. Put a drop of the supernatant ether solution on the center of
the IR salt plate and allow it to evaporate. Repeat this process 4-5 times to get a homogenous
film of your product on the salt plate. Analyze the IR spectra of your products and compare it
with the spectrum of the starting material.
Observation and Results
Report the yield of estradiol isomer and identify its stereochemistry on the basis of your
observed melting point and TLC. Report the Rf values of all the spots that you observe on TLC.
Analyze the IR spectrum of your product. How does it differ from the spectrum of the starting
material?
Compound
MW Mp (°C)
estrone
270.38 260–2
272.39 220–3
17-D-estradiol
272.39 176–80
17-E-estradiol
sodium borohydride
37.83 400(d)
d=decomposes on melting
Discussion
Your discussion should include a detailed mechanism of reduction of estrone by sodium
borohydride. The mechanism should clearly explain the stereochemical route followed in the
reaction. Comment on stereospecificity of this reaction and answer the following questions.
1.
Why is it necessary to use aprotic solvents while carrying out reductions with lithium
aluminum hydride? By using methanol as an example of a protic solvent, write a balanced
equation to show the reaction between lithium aluminum hydride and methanol.
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