Organic Chemistry III Laboratory
NMR Verification of Diastereoselective Reduction
1
of Substituted Cyclohexanones
Experiment 1
Weeks 1 & 2
Background Reading
th
Zubrick, J. W. The Organic Chem Lab Survival Manual, 5 edition, Wiley & Sons, Inc., New York, 2000.
IR Spectroscopy
NMR
nd
Jones, M. Organic Chemistry, 2 Ed., W. W. Norton, New York, 2000.
Coupling Constants, Complicated Splitting, Dynamic NMR; Sect 14.6-14.11, pp 670-690.
Background:
2-5
During the last decade, enzymes and microorganisms have become widely used for stereoselective synthesis.
These biocatalytic systems present some advantages because they can produce reactions under mild conditions
with high enantio- or diastereoselectivity. Obtaining purified enzymes is costly, however, microorganisms can be
easily obtained and exploited to carry out enzymatic reactions. A whole-cell system has the advantage that it is
inexpensive and contains all of the entities necessary for the transformation (enzymes, cofactors, metal ions, etc.)
within the cell. Common Baker’s yeast, Saccharomyces cervisiae, is an easily used, commercially available,
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inexpensive, and by far the most widely used microorganism for the asymmetric reduction of ketones. A large
number of different enzymes are present in yeast. Dehydrogenase enzymes catalyze oxidations and reductions
in which nicotinamide cofactors serve as the immediate two electron oxidants or reductants. Yeast contains
multiple dehydrogenases, which are able to accept a wide variety of unnatural substrates.
Oxidation and reduction reactions are important reactions in living systems. An example of an oxidation reaction
that takes place in animal cells is the oxidation of of ethanol to acetaldehyde, a reaction catalyzed by the enzyme
alcohol dehydrogenase (Figure 1). Ingestion of a moderate amount of ethanol lowers inhibitions and causes a
lightheaded feeling, but the physiological effects of acetaldehyde are not as pleasant. Acetaldehyde is
responsible for the feeling known as a hangover (flushing, nausea, dizzyness, sweating, headaches, decreased
blood pressure).
O H
H3C
H
Ethanol
H
H
O
NH2
N
R
H O
H
Alcohol
Dehydrogenase
O
NH2
N
NAD
R
NADH
+ H C
3
H
+ H
Acetaldehyde
Figure 1. Oxidation of Ethanol by the Enzyme Alcohol Dehydrogenase
Many biological redox reactions make use of nicotinamide adenine dinucleotide as the biological redox reagent
(Figure 2). We have oxidized alcohols previously with bleach and potassium permanganate. Neither of these
reagents are present in biological systems. Chemical hydride reducing agents include NaBH4 and LiAlH4 but
1
Clavier, J. W.; Fievet, J.; Geisler, V. Chem. Educator 2000, 5, 64-66.
Roberts, S. M.; Turner, N. J.;Willetts, A. J.; Turner, M. K. Introduction to Biocatalysis Using Enzymes and Microorganisms, Cambridge University Press, Cambridge, 1995, pp 1-33.
3
Faber, K. Biotransformations in Organic Chemistry; Springer-Verlag, Berlin, 1997.
4
Davies, H. G.; Green, R. H.; Kellt, Dr. R.; Roberts, S. M. Biotransformations in Preparative Organic Chemistry,
Academic Press, London, 1992.
5
Brooks, D. W. J. Org. Chem. 1982, 47, 2820.
6
Sih, C. J.; Chen, C. Angew. Chem. 1984, 23, 570-578.; MacLeod, R. Biochemistry 1964, 3, 838.
2
neither of these compounds is biologically available either. Nature uses nicotinamide adenine dinucleotide as
both a hydride acceptor (oxidizer) and hydride donor (reducer).
H
O
NH2
N
HO
HO
O
H
O
O
P
NH2
NH2
O
N
N
O P O
N
R
N
O
O
O
Abbreviated
N
+
Structure (NAD )
O
H
H
OH OH
Nicotinamide Adenine Dinucleotide
Figure 2. Full and Abbreviated Structure of Nicotinamide Adenine Dinucleotide
The reverse of the reaction shown in Figure 1 is the reduction of a carbonyl group by a dehydrogenase enzyme to
form an alcohol. Remember from CHM 114, that enzymes speed the attainment of equilibrium. That means that
the rates of both the forward and reverse reactions are increased. Thermodynamics determines the equilibrium of
a given reaction, but the microenvironment of the cell can manipulate the overall net direction of a reaction. We
will examine the reduction of cyclic ketones with both chemical and biological methods to compare the
diastereomeric ratios of the product alcohols (Figure 3).
O
R
Yeast
or
NaBH4
OH
OH
R
R
+
Mixture of
Diastereomers
Figure 3. Formation of Diastereomeric Alcohols From Ketones
In this laboratory exercise, you will reduce both 2-methyl- and 4-tert-butylcyclohexanone with baker’s yeast
(Saccharomyces cervisiae) and sodium borohydride (4 reactions total). The mixtures of cis- and trans-substituted
cyclohexanols that result from these reductions will be analyzed by NMR spectroscopy and gas chromatography
to determine the exact diastereomeric composition. It may be helpful to build the cis- and trans-cyclohexanols
using Spartan-Pro to get information relating to the dihedral angles which will influence the magnitude of the
1
coupling constants in the H-NMR spectrum. You will then compare and discuss the differences between the
product ratios from the different methods.
Understanding The Experiment:
This experiment takes place over two weeks. In the first week, the yeast reductions are set up and the sodium
borohydride reactions completed. The yeast reactions are carried out in Erlenmeyer flasks using one package of
commercial yeast (probably Fleischmann’s dry active baker’s yeast) and 0.5 grams of substituted cyclohexanone
suspended in warm water containing sucrose. The sodium borohydride reductions are carried out on microscale
1
(0.3 g cyclohexanone) in methanol and after workup should be sufficiently pure for direct characterization via H1
NMR. You will obtain a GC, H-NMR, and COSY spectra. The COSY spectrum is a 2-dimensional NMR
experiment that will identify coupling between hydrogens.
During the second week’s lab period, the yeast reactions will be worked up and the products isolated by
extraction and purified by filtering the mixture through a short column of silica gel. After removing the solvent, the
resulting product mixtures can be characterized by GC, 1H-NMR, and COSY spectra.
The carbonyl group of substituted cyclohexanones will have two distinct faces. Remember that cyclohexanes will
exist predominantly in the chair conformation and that when substituted, there are two different possible chair
conformations. One with the substituent group in an equatorial position and another with the group in an axial
position. 2-Methylcyclohexanone will be able to place the methyl group in either the axial or equatorial position
since the size of a methyl group is not too large (which of these two conformations will be the major form?). The
small borohydride ion will be able to attack either face of the ketone but will have a slight preference for attacking
from the more stericaly hindered axial face to place the hydroxy group of the product alcohol in the
thermodynamically favored equatorial position where it has more space. Think about why axial attack of the
hydride will be more sterically hindered.
H BH
3
H BH
3
O
O
CH3
H
H
CH3
H
H
OH
H
cis
OH
CH3
CH3
H trans
In 4-tert-butylcyclohexanone there is not the same conformational flexibility as in 2-methylcyclohexanone. The
tert-butyl group is too large to occupy the axial position in a cyclohexane ring. Therefore, the compound is
conformationally locked with the tert-butyl group in the equatorial position. The reduction using sodium
borohydride on 4-tert-butylcyclohexanone will give a good indication of the preference for axial attack of the
borohydride ion. Since there is no conformational mobility, the product ratio will accurately show the facial
preference. Notice that in 2-methylcyclohexanone there is no way to determine if the cis product is coming from
axial face attack of hydride on the cyclohexanone with an axial methyl group or from equatorial face attack on the
conformation with an equatorial methyl group.
axial face
attack
H BH
3
H
O
(H3C)3C
(H3C)3C
OH
H
H
OH
O
(H3C)3C
(H3C)3C
H
H O
H
trans product
H
H
cis product
NH2
N
Enzyme
In the yeast mediated reductions, the hydride source (NADH) is much larger than the borohydride anion. Axial
attack is no longer much of a possibility. Instead, the NADH is placed on the equatorial side. This is probably
governed by the position of the cyclohexanone when bound to the enzyme and not that the NADH can move to
either side of the carbonyl when it is on the enzyme. As a result, the selectivity of the reaction may be altered.
Borohydride reductions of 4-tert-butylcyclohexanone should give the trans diequatorial product as the major
product. If the axial face of 4-tert-butylcyclohexanone is not accessible to the enzyme-bound NADH, then yeast
will only be able to produce the cis product with the hydroxy group in the axial position. (See above)
1
H-NMR can distinguish between the cis and trans diastereomers produced in these reduction reactions. The
hydrogen on the carbon bearing the hydroxyl group will have a different chemical shift depending upon whether it
is in an axial or equatorial position. Coupling constants can be used to determine the location of this hydrogen.
Use the modeling results to determine the dihedral angle between the hydrogen of interest and its adjacent
hydrogens. Coupling constants, J, can be predicted using the Karplus correlation, where the J values are related
to the dihedral angle between the protons on vicinal (adjacent) carbons in conformationally restricted systems.
o
When the dihedral angle is 60 the coupling constant will be small (1-7 Hz, usually 2-3 Hz), but when the dihedral
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angle is 180° the coupling constant will be large (8-14 Hz, usually 8-10 Hz). Determine the coupling constants
for the pertinent proton resonances and correlate that with the modeled dihedral angles for the cis and trans
alcohols. An analogous type of analysis will then be used to determine the product ratio for the 2methylcyclohexanols.
Experimental:
Yeast Reductions. To a 250 mL Erlenmeyer flask, warm water (100 mL, 30° C), sucrose (10 g), and one package
of active dry granular baker’s yeast (7 g) is added. The resulting suspension is stirred with a stirring bar for 10
min. Then the 2-methylcyclohexanone (0.5 g) or 4-tert-butylcyclohexanone (0.5 g dissolved in 0.5 mL of ethanol)
is added dropwise over a 5 minute period. The reaction flask is loosely plugged with a piece of cotton, and the
mixture stirred at room temperature for one week. The fermentation broth is centrifuged and the supernatant
extracted with methylene chloride (3 x 50 mL). The combined organic extracts are washed with saturated NaCl
solution, dried over MgSO4, filtered and the solvent removed using a rotary evaporator. Finally, the substituted
cyclohexanol is purified by passing the material through a small silica gel column eluted with a hexane/ethyl
1
acetate (80/20) mixture. Characterize your product by IR, GC, and H-NMR.
Sodium Borohydride Reductions. Dissolve the substituted cyclohexanone (0.3 g) in methanol (1.25 mL) in a small
flask and cool the mixture in an ice bath. Slowly add sodium borohydride (50 mg) to the mixture. After the
vigorous reaction, remove the flask from the ice bath and stir the mixture for 15 min at room temperature. The
borate ester is decomposed by the addition of sodium hydroxide (3 M, 1.2 mL) solution. The product is extracted
with two 5 mL portions of methylene chloride, washed with saturated NaCl solution, and dried over MgSO4. The
methylene chloride solution is transferred to a tared flask and the solvent removed (rotary evaporator). The
1
product mixture that results should be sufficiently pure for direct characterization by IR, GC, and H-NMR.
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th
Silverstein, R. M.; Webster, F. X. Spectrometric Identification of Organic Compounds, 6 ed., Wiley, New York,
1998, pp 208-210.