Mosher Ester Analysis - Department of Chemistry and Physics

Mosher Analysis of Enantiomeric Purity and Assignment of Absolute Configuratoin
by NMR spectroscopy.
References:
Patterson, J.; Sigurdsson, S.T. J. Chem. Educ. 2005, 82, 1049-1050.
Sullivan, G. R.; Dale, J.A.; Mosher, H.S. J. Am. Chem. Soc. 1973, 95, 512-519.
Dale, J.A.; Mosher, H.S. J. Org. Chem. 1973, 38, 2143-2147.
Prelab: Prepare your reagent table and procedure sheet, including the reaction. The
reaction is described below. Have them ready to do the experiment when you come into
the lab. You can find all of the reagents at www.sigmaaldrich.com. Also get NMR
spectra for the starting materials that you can use to compare with your crude product.
Background
While we often discuss chiral compounds as single enantiomers, it is common to
encounter these compounds as mixtures of enantiomers. This occurs both in the
laboratory and in nature. In the laboratory, this most commonly arises from the use of
less than enantiopure starting materials or reagents during synthesis or by racemization
during reactions. In nature, this can be due to competing enantomeric biosynthetic
pathways and by enzymatic or chemical racemization. Mixtures of enantiomers can be
racemic (50:50 mixture of enantiomers) or scalemic (any other proportion).
The analysis of enantiomeric mixtures can be divided into two objectives. The
first is the analysis of enantiomeric excess and the second is the establishment of absolute
configuration. Enantiomeric excess is the amount of one enantiomer over the other and is
usually expressed as a percentage value. Percent e.e. is calculated as:
% e.e. = (100%)|R – S|
R + S
Establishment of configuration is simply determining which enantiomer is R and
which is S, and determination of which is in excess. The latter is a difficult task as,
enantiomers are identical in all physical and spectral properties. Enantiomers do differ in
the sign of their optical rotation and certain compounds can be distinguished by
spectropolarimetry (the study of variation of rotation with wavelength, often referred to
as circular dichroism (CD) or optical rotatory dispersion (ORD)). However, this
approach is often limited to certain classes of compounds with large changes in their
rotation and it is not generally applicable.
In many cases both assignment of configuration and determination of e.e. can be
achieved by reacting the molecule in question with a chiral compound of known absolute
configuration and enantiomeric purity (preferably a single enantiomer). This produces a
mixture of diastereomers. The requirements for such a method are that the chemistry
involved does not cause the chiral centers of either of the reactants to racemize and that
the reaction is quantitative in reaction with both enantiomers of the compound you are
analyzing. That is, in some cases, one enantiomer reacts faster than the other. This is

called kinetic resolution. Usually this is avoided by using an excess of the enantiopure
reactant and using reactions that are fast and irreversible.
In this lab, you will prepare a chiral ester of a chiral secondary alcohol, 1phenylethanol.
The ester is derived from the chiral carboxlic acid -methoxy-trifluoromethylphenylacetic
acid (MTPA, Mosher’s acid), shown below.
F3C
MTPA
Diastereomers, unlike enantiomers, have distinguishable physical and spectral
properties because they are not mirror images. Diastereomers have two (or more) chiral
centers. The added chiral center(s) can be the same or opposite configuration as that of
the original molecule. The matched and mismatched diastereomers will have different
spectra and can thus be distinguished as shown below for one of the enantiomers of
MTPA and scalemic 1-phenylethanol.
F3C
O
OCH 3
OH
+
OH
CH 3
single enantiomer mixture of enantiomers
mixture of diastereomers
MTPA is a useful chiral acid for analyzing enantiomeric excess for several
reasons. First, both enantiomers are available in high enantiomeric purity. Second, the
chiral center is adjacent to the carboxyl bringing it in close proximity to the chiral center
of the alcohol when the ester is prepared. Also, the chiral center is not able to be
racemized as it is quaternary and thus contains no acidic protons. Finally, the presence of
the trifluoromethyl group allows for clean analysis of enantiomeric excess by 19 F NMR,
which is uncomplicated by signals from the chiral alcohol, unlike the 1 H NMR spectrum.
However, 1 H NMR can also be used if the signals are sufficiently resolved. This also
allows for assignment of configuration based on a preferred conformation of the ester. In
this experiment, you will team up with a partner. You will be given a sample of 1phenylethanol
of unknown enantiomeric composition. One of you will prepare the (R)-
(+)-MTPA ester of your 1-phenylethanol sample and the other will prepare the (S)-(-)-
MTPA ester of the sample. You will then compare your 19 F NMR and 1 H NMR data and
determine both the enantiomeric excess and the major enantiomer present according to
the published methods by Mosher.
We will prepare our esters using N,N’-dicyclohexylcarbodiimide (DCC) to
activate the acid toward substitution and 4-dimethylaminopyridine (DMAP) as a catalyst.
*
O
OCH 3
OH
F3C
F3C
O
OCH 3
O
+
OCH 3
H
O
H
O
CH 3
CH 3

This is an exceptionally mild method of carboxyl activation and is used extensively for
the synthesis of both esters an amides, particularly for peptide synthesis.
O
R OH
R'OH, DCC, DMAP
CH 2Cl 2
O
R OR'
The reaction proceeds in two steps. First, the the acid reacts with DCC, forming
an O-acylisourea. This species is similar to an anhydride and the carbonyl is highly
susceptible to substitution. In the second step, the alcohol attacks the carbonyl to produce
the ester and eliminates dicyclohexylurea as shown below.
Step 1.
O
R O
Step 2.
O
N
c-Hx
c-Hx
R O N
H
O-acylisourea
(similar to an anhydride)
O
H
+
N C N
Dicyclohexylcarbodiimide
(DCC)
O
HN
c-Hx
+
c-Hx
R OR'
O N
H
Dicyclohexylurea (DCU)
N
c-Hx
O
R O
O
R O
R' O H +
c-Hx
R' O +
R O N
H
+
+
O
H
R O
OR'
H
N C N
H
N C N
H-bond
O
R O
While this process can proceed by itself or with added bases such as
triethylamine, it is somewhat sluggish. In order to speed the process, a nucleophilic
N
c-Hx
N
H
H
N
c-Hx
c-Hx
N
H
c-Hx

catalyst, 4-dimethylaminopyridine (DMAP) is used. DMAP facilitates the process both
by acting as a base and by forming activated acylpyridinium derivatives (below) from
DCC and from the O-acylisourea. These are much more reactive as the pyridine is an
excellent leaving group. This accelerates the reaction severalfold over the uncatalyzed
reaction.
H 3C
N
CH 3
N
N
c-Hx
N
H
c-Hx
Procedure
Wear gloves. DCC and DMAP are very toxic.
You will be provided with a culture tube containing a scalemic solution of 6.0 L
of 1-phenylethanol in 1 mL of dry CH2Cl2. To this add a stir bar and 13 mg of (R)-(+)- or
(S)-(-)-MTPA as directed by your instructor. Be sure to record which enantiomer of
MTPA you use. Also be sure to cap the tube quickly to minimize any moisture entry
after each addition of reagent. A nitrogen balloon and septum can be used if you wish.
Cool the flask in an ice bath and add 12 mg of DCC and a spatula point (~1-2 mg) of
DMAP in sequence. Add the DCC to the reaction as soon as it is weighed as it tends to
pick up moisture in the air. Stir the solution for 5 minutes in the ice bath, and then 25
minutes at room temperature, during which time dicyclohexylurea will precipitate.
While you are waiting on the reaction to complete, experiment with different TLC
systems to determine if your reaction has finished. Spot 1-phenylethanol, DCC, DMAP,
MTPA, and your reaction separately on the TLC plate and develop. Visualize the plate
with UV light and circle the spots with a pencil. Begin with 1:1 hexane-ethyl acetate and
evaluate several mixtures until you find one that separates all of the components well,
especially the product. This mixture will be used for preparative TLC to purify the
product.
Also while you are waiting on the reaction to complete, prepare two large test
tubes for exraction of the reaction mixture. To one tube add 2 mL of 5% NaHCO3 and to
the second add 2 mL of saturated NaHCO3.
When the reaction is judged to be complete or 30 minutes passed, add 2 drops of
water and stir an additional 10 minutes. Then, add one mL of CH2Cl2 and 2 mL of 5%
acetic acid to the culture tube and swirl vigorously. Using a pipet, transfer the organic
layer to the test tube containing 5% NaHCO3 and swirl as before. Repeat this for the tube
containing saturated NaHCO3. Prepare a pipet drying column plugged with glass wool
and filled with ~3 cm of anhydrous Na2SO4. Clamp the tube in place and place a tared 10
mL round bottom flask below to collect the eluent. Add the organic phase from the last
tube and allow it to pass through the column and collect in the flask. Once all of the
organic phase has been added and the top of the liquid reaches the Na2SO4, wash the
column twice with 2 mL CH2Cl2. Remove the solvent by rotary evaporation and remove
any traces under high vacuum for no more than a minute or two (The instructor will show
R
O
N
N
CH 3
CH 3

you how to do this). Obtain a crude yield, dissolve the product in CDCl3 and obtain a 1 H
NMR spectrum of the crude ester.
Preparative Thin-Layer Chromatography
The Mosher ester will be purified by preparative thin-layer chromatography. This
is essentially the same as what you use for monitoring your reactions, only on larger
scale. We will use a large developing tank, and large, thickly coated plates. The plates
we are using are capable of separating around 10-20 mg, depending on separation
efficiency. Unlike flash chromatography, no specific Rf value is required. Simply
identify a solvent system which will adequately separate your compounds and develop
the plate. The technique does have limitations, however. The large surface area of the
silica, combined with its mildly acidic nature tend to promote air oxidation of sensitive
functionalities such as aldehydes, amines, and reactive double bonds. Also, the method is
rather expensive to perform on other than small scale separations. Nonetheless, it is of
great utility in difficult separations of small amounts of material, such as extracts of
natural products and separation of diastereomers. In the second part of this experiment,
you will separate the products of your Mosher ester reaction by preparative TLC and
characterize your product by 1 H and 19 FNMR.
Preparative TLC Procedures
1. Set up TLC Developing tank.
a. Examine TLC’s in various solvent systems (TLC hood) if you have not yet
determined an optimal composition.
b. Dissolve your sample in 1.0 mL CH2Cl2 or use the NMR sample directly.
c. Perform a TLC on your material using that system. (Don’t forget to cospot
with your starting material).
d. Make up 100 mL of your chosen solvent system and put it in the tank.
2. Prepare your TLC plate.
a. Set up the solvent streaker (Have the instructor help you.)
b. Draw a pencil line about 1” from the bottom of the plate.
c. Set the streaker up so that the tip of the capillary traverses this line as you
draw it along the benchtop.
d. Fill the streaker with your solution. The reservoir will only hold around
0.25-0.5 mL.
e. Streak the plate on the line you drew. Try to keep the thickness of your
band between 1-3mm. Wave your hand over the plate to assist drying.
f. Refill the reservoir again and repeat.
g. Clean the reservoir with acetone. Dry it under a nitrogen flow. Give it to
the next person to use. Be gentle with the streaker. We only have one and
if broken, it will end our experiment.
3. Develop your plate.
a. Place your plate in the developing tank.
b. Develop the plate until the solvent front reaches within one inch of the top.
This will take about an hour. Be sure to check the plate every 15 minutes
to see its progress.

c. Remove the plate from the tank and mark the solvent front with a pencil.
d. Wait for the plate to dry. You can assist this by fanning the plate with a
folder or using a flow of dry nitrogen. While you are waiting, you should
be cleaning glassware or something else productive. Do not wait an
excessive amount of time. Many compounds are sensitive to oxidation on
a TLC plate as the surface area is very large.
4. Isolate the product.
a. When your plate is dry enough, visualize the bands using UV light. Mark
them lightly with a pencil.
b. Using an exacto knife or the flat edge of a spatula, scrape the bands off
onto a lengthwise folded piece of clean, white paper.
c. Prepare a vacuum filtration using a fritted glass funnel as shown below.
d. Place the scrapings into a fritted glass funnel and press them into a pad.
e. Wash the compound off the silica into a round bottomed flask using 3 x 5
mL of TLC solvent.
f. Remove the solvent by rotary evaporation and determine the mass of your
product. While this is occurring have your sample analyzed by chiral GC
or be cleaning your glassware.
5. Characterize your product by 1 H and 19 FNMR.
Since one member of your team will prepare the R-MTPA ester and the other will
prepare the S-MTPA ester, compare your results and assign the stereochemistry of the
alcohol and the % e.e. according to the two Mosher references.
Questions:
1. Compare the % e.e. based on the integration of the 1 H and the 19 F spectra of the
Mosher ester you made. For 1 H, calculate for all resonances that are sufficiently
resolved to integrate reliably. Do they agree? If not, suggest a reason for the
discrepancy.
2. Calculate the % e.e. from your crude product and compare with that from the
purified sample. Do they agree? If not, suggest a reason for the discrepancy.
3. Compare the % e.e. you obtained to that obtained by your partner. Do they agree?
If not, suggest a reason for the discrepancy.
4. Suggest two alternate methods for determining the % e.e. of your alcohol.

Plate
20 cm x 20 cm
2.5 mm layer
thickness
Bench Guide/
Reservoir Holder
Streak line
~1" from bottom
1-3mm thickness
Glass Plate Cover
Developing chamber
Solvent
~3/4" from bottom
About 100 mL
TLC Plate Streaker
Bench
Preparative TLC Plate
Development
Reservoir
Capillary
TLC Plate
to aspirator
25 mL
Claisen
head