Synthesis of Chiral Molecules
The big picture:
Synthesis of enantiomericaly pure compounds is of increasing importance to the
pharmaceutical industry, as enantiomers interact differently with receptors. In the next
two experiments we will explore methods to synthesis and characterize chiral
compounds. In both experiments we will convert a diacid precursor to form a chiral
monoester taking two different approaches. The enantiomeric purity of the products will
be determined by measuring specific rotation and the diastereomeric excess will be
determined by NMR. The two experiments are run together to allow for efficient use of
time as is done in the ‘real world’. As you will see time management and organization are
important for successful research.
Experiment 1.
This experiment starts with the resolution of a reacmic mixture of diacid. Once the two
enatiomers are separated, the optical purity will be determined by its specific rotation
([α]D). At this point you are asked to find conditions that will favor the formation of a
monoester product. You will need to look in the literature and use your chemical
knowledge to decide on an esterification reaction conditions. The products will be
purified by column chromatography and identified by combination of IR and NMR.
COOH
COOH
resolution
COOH
COOH
determination of purity
by measuring [α]D
+
COOH
COOH
formation of monoester
chromatography
and charectarization
Experiment 2.
This experiment starts with a meso diacid that is converted to a diester. Stereo-selective
enzymatic hydrolysis of one of the ester groups will result in a monoester . To determine
the optical purity, a sample of the product is reacted with enantiomericaly pure alcohol
(therefore resulting in diastereomeric mixture). The ratio between the two diastereomers
is determined by NMR.
O
COOMe
1. MeOH
O
2. SOCl2
COOH
porcine liver
esterase
COOMe
O
COOMe
Ph
HO
O
Ph
O
Determining Enantiomeric
Excess by Direct NMR
H
COOEt
COOEt
COOMe
Tentative timetable:
Meeting
12 pm 1pm
1pm -2pm
2pm-3pm
resolution
(exp. 1)
recrystallization
(exp. 1)
esterific
ation
(exp. 2)
present
ations
[α]D
determination
(exp. 1)
chromatography
(exp. 2)y
preparation of
buffer
(exp. 2)
present
ations
monoesterification
(exp. 1)
6
DCC
reaction
(exp. 2)
workup of
monoesterification
(exp. 1)
time for
completing
exp. 1
7
chromato
graphy
1
2
3
4
5
DCC reaction
3pm-4pm
4pm-5pm
anhydride
opening
(exp. 2)
workup of
esterification
(exp. 2)
enzymatic
hydrolysis
(exp. 2)
work up of
enzymatic
hydrolysis
(exp. 2)
chromatography
(exp. 1)
workup of
DCC
reaction
(exp. 2)
Procedures for experiment 1.
Resolution of 2-phenylsuccinic acid
COOH
COOH
1. L-proline
2. HCl
COOH
COOH
+
COOH
COOH
Racemic phenylsuccinic acid (1.94 g, 0.01 mmol) is dissolved in isopropanol (50 ml) and
L-proline (1.15 g, 0.01 mol) is added. The mixture is refluxed for 30 minutes and then
cooled to about 300C. The precipitate is filtered and washed with acetone (2x10 ml). The
solid material is added into ice-cold 6N HCl (10 ml) and the solution is stirred for 5
minutes. The solution is filtered and the precipitate is washed with ice-cold water (1-2
ml). The solid is recrystalized from water (10 ml) and the crystals are dried in desicator.
Prelab assignment
1. Write the chemical reactions that are corresponding to each step of the resolution and
draw a flow-chart that show how the compounds are divided between the various
phases.
2. Write a step-by-step detailed procedure for the lab. Don’t forget to add notes that are
not a part of the procedure but are important such as which glassware to use.
Determination of specific rotation
Prepare a solution in acetone (~120 mg/1 ml) and measure the specific rotation using a
polarimeter. Recover your compound after the measurement.
Prelab assignment
Specific rotation can have higher values then 360 deg.cm2g-1 yet the observed rotation
can only have values between -90 and +90. Therefore a compound that has a specific
rotation of 386 o will appear as 26o. How would you know that the specific rotation that
you are measuring is the absolute value?
Monoesterification
This is a mini project were your mission is to maximize the formation of a monoester
from the diacid. Use the literature to find relevant information (don’t forget to add the
references to your lab report) and come with a synthetic plan. Consult your TA about
your plan and set the reaction. Isolate the products by column chromatography and
identify them.
Procedures for experiment 2.
O
1. MeOH
O
2. SOCl2
COOMe
COOMe
O
Do this reaction inside a capped vial:
Cis-1,2,3,6-tetrahydrophtalic anhydride (1 g, 6.5 mmol) is suspended in MeOH (2 ml)
and the solution is stirred for 24 h (in our case it will be longer…) until a clear solution is
formed. The solution is chilled to 0oC and thionyl chloride (0.475 ml, 6.5 mmol) is added
dropwise. After the addition, the solution is stirred at RT for 3 h. The solution is
concentrated, neutralized with sat. NaHCO3 and then diluted with CH2Cl2 (10 ml). The
organic phase is separated, dried over Na2SO4 and concentrated. The product is
chromatographed over silica with 9:1 CH2Cl2-hexane.
Prepare NMR sample in CDCl3.
COOMe
porcine liver
esterase
COOMe
COOH
COOMe
Dimethyl cis-1,2-cyclohex-4-enedicarboxylate (300 mg, 1.5 mmol) is added to a mixture
of phosphate buffer (30 ml of 0.05M KH2PO4 adjust to pH according to your assignment)
and acetone (3 ml). Esterase [20 µl (75 units) from porcine liver, sigma, containing 250
units/mg protein in 3.2 M (NH4)2SO4 buffer] is added and the solution is stirred at RT for
two days. The progress of the reaction is followed by TLC (2% MeOH-CH2Cl2).
Measure the pH at the end of the reaction.
The pH of the mixture is adjusted to pH=9 by addition of 2M NaOH and then the solution
washed with ether (2 x 10 ml). Subsequently, the solution is acidified to pH~2 with 6N
HCl. The solution is saturated with NaCl and then is extracted with ether (2x15 ml). The
organic phase is dried over Na2SO4 and the solution is concentrated.
Prepare NMR sample in CDCl3.
Determination of diastereomeric excess
O
COOH
Ph
+
COOMe
+ enantiomer
HO
H
COOEt
DCC
DMAP
Ph
O
COOEt
COOMe
+ diastereomer
(1R,6S)-6-methoxycarbonyl-3-cyclohexene-1-carboxylic acid (90 mg, 0.48 mmol),
Ethyl-(s)-mandelate (87.9 mg, 0.48 mmol) and dimethylaminopyridine (DMAP, 3 mg)
are dissolved in CH2Cl2 (5 ml, passed over basic alumina). The solution is cooled to 0oC
and dicyclohexylcarbodiimide (DCC, 100 mg, 0.48 mmol) is added. The solution is
stirred at 0oC for 30 minutes and then at RT for additional 2.5 hours. The precipitated
urea is filtered (trough a pipette with cotton wool). If additional precipitate is observed
the solution is filtered again. The solution is diluted with CH2Cl2 (15 ml) and washed
with 1N NaHCO3 (5 ml) and water (5 ml). The organic phase is dried over Na2SO4 and
concentrated.
Prepare NMR sample in C6D6.
Prelab assignment
1. What is the structure of DCC? Draw the mechanism for this reaction.
2. What is the structure of DMAP? What is the suggested mechanism for its catalytic
activity?
Synthesis of Chiral molecules-lab report
Experiment 1
1. Write chemical reactions that corresponds to each step of the resolution of 2phenylsuccinic acid by L-proline.
2. What was your yield and the [α]D?
3. Explain your synthetic route to the monoester.
4. Analyze your NMR.
Experiment 2
1. Analyze the NMR of the monoester product. What was your yield?
2. Explain why did we convert the monoester into a mandelate ester.
3. Determine the enantiomeric excess of the enzymatic reaction based on the
diastereomeric excess of the mandelate ester.
4. Compare your results to others. Is there a correlation between the pH of the
reaction and the selectivity of the enzyme?
5. What is the mechanism for the enzymatic reaction?
6. Prof. Carstenin Bolm (Institut für Organische Chemie der, RWTH Aachen,
Germany) described in several papers the enantioselective opening of Cis-1,2,3,6tetrahydrophtalic anhydride to the corresponding monomethyl ester. Look for
these papers and explain the proposed mechanism.