Introduction:
James
V. McCullagh
Department of Chemistry and Biochemistry, Manhattan College, Riverdale, NY
"The ability to produce enantiomerically pure compounds is crucial to the fine
chemicals industries (pharmaceuticals, agrochemicals, fragrances etc.). For
instance, the revenues generated in the pharmaceutical and agrochemical industries
by the production of enantiomerically pure products was estimated at $8 billon
in 2003. The ability to produce enantiomerically pure compounds is probably
most important in the pharmaceutical industry, where approximately 80% of the
drugs currently under development are chiral (2). Enantiomerically pure compounds
can be produced by one of two methods. The first method is by a process referred
to as resolution. This a process by which the two enantiomers of a racemic
mixture are separated. The second method is by asymmetric synthesis. In an
asymmetric synthesis, only one of the two possible enantiomers of the desired
compound is produced. Asymmetric synthesis involves the use of chiral catalysts
such as chiral transition metal complexes or enzymes in key steps of the synthesis.
The use of asymmetric synthesis has increased dramatically over the last few
decades, but resolutions are still the most heavily used method in the large-scale
production of enantiomerically pure compounds industrially (2).
In this experiment, you will examine the resolution process by isolating the medicinally active (S)-(+)- ibuprofen enantiomer from a racemic mixture. Ibuprofen belongs to a class of pharmaceuticals referred to as non-steroidal anti-inflammatory drugs or NSAIDS. It is the active ingredient of Motrin®, Advil® and Nuprin® along with their generic equivalents. Ibuprofen exhibits analgesic, antipyretic and anti-inflammatory properties. This makes it well suited for alleviating minor to moderate aches and pains and in fever reduction. Additionally it has found use in treatment of rheumatic fever, rheumatoid arthritis and osteoarthritis (3). Ibuprofen functions by strongly inhibiting the conversion of arachidonic acid into prostaglandin E2, which serves as a chemical signal for vasodilatation (inflammation) and pain perception. It has been shown for the aryl-substituted proprionic acid NSAIDS, compounds such as ibuprofen, naproxen and related compounds, that of the two enantiomers only one has the desired biological activity. For ibuprofen, the (S)-(+)-enantiomer has the desired pharmacological activity while the (R)-(-) –enantiomer is inactive. With ibuprofen the (R)-(-) –enantiomer has been shown to be non-harmful so currently most commercial ibuprofen is sold in a racemic form (2,3). However, recent research suggests that optically pure (S)-(+)-enantiomer of ibuprofen is more effective and therefore several pharmaceutical companies are developing methods to produce the enantiomerically pure form of ibuprofen and its derivatives (2,4,5). [NOTE: There is value in the R- isomer. It has been found that humans have an enzyme which isomerizes the Inactive R- form to the active S- isomer in vivo.]
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There are several different types of enantiomeric resolutions. The first type
of resolution discovered was the direct crystallization method. In this method,
the compound
in question, forms half the crystals containing predominantly the R enantiomer
and the other half containing predominantly the S enantiomer (2). The different
crystals, if carefully examined can be physically separated by differences
in their appearances. Alternately, if you have an enantiomerically pure crystal
this can be used as a seed crystal to precipitate the crystals of one enantiomer
preferentially. This process was first used by Louis Pasteur in his groundbreaking
resolution of tartaric acid. This first resolution greatly aided the development
of our understanding of the stereochemistry of organic compounds and led to
the
discovery that many organic molecules have isomers that are non-superimposable
mirror images (2,6). This type of resolution, however, only works with 5 to
10 percent of organic compounds. The remaining organic compounds tend to form
racemic
crystals containing equal amounts of both enantiomers, thereby precluding resolution
using the direct crystallization method.
The other types of resolutions belong to a class of resolutions referred to
as chemical resolutions. These work by temporarily converting the two enantiomers
to be separated into diastereomers. In these cases the racemic mixture to be
separated is reacted with a single enantiomer of second chiral compound (a
resolving
agent). The resolving agent is most often a single enantiomer of another chiral
organic molecule but in some types of resolutions it can be a biological agent,
such as an enzyme, or a transition metal catalyst. By reacting the two enantiomers
of the racemic mixture with the resolving agent, two diastereomeric compounds
will be formed. Unlike enantiomers, diastereomers do not have identical physical
properties, i.e. melting points, boiling points, solubility, chemical relativities
etc… If these differences in physical properties are large enough they
can be used to separate the two diastereomers. After separation the two diastereomers
are converted back to the two enantiomers by removal of the resolving agent.
See Figure 1 for a general overview of the process.
A good resolving agent has several requirements. First, it
must be chiral and available as a single enantiomer (not a racemic mixture).
Second, it must react with the compound to be resolved to form diastereomers
with significant differences in their physical properties. Third, the reaction
that formed the diastereomers must be reversible so that the compound being
resolved can be recovered at the end of the experiment. (Once the diastereomers
are physically separated from each other, the resolving agent must be removed
to yield the individual enantiomers.)"
References
(1) Palleros, D. R., Experimental Organic Chemistry, John Wiley & Sons.
Inc. New York, NY, 2000, p 242-249, 642.
(2) Ager, D., Ed. Handbook of Chiral Chemicals, 2nd ed.; CRC Taylor & Francis
Group: Boca Raton, FL, 2006, p 75-78, 81-82, 97-98.
(3) Delgado, J. N.; Remers, W. A. Wilson and Gisvold’s Textbook of
Organic Medicinal and Pharmaceutical Chemistry, 9th ed.; J. B. Lippincott
Co. : Philadelphia, PA, 1991, p 656 -664
(4) Manimaran, T.; Impastato, F. J.; U.S. Pat. 5,015,764, 1991.
(5) Manimaran, T.; Stahly, G. P.; U.S. Pat. 5,248,814, 1993.
(6) Kauffman, G. B.; Myers, R. D.; J. Chem. Educ. 1975, 52, 777-781.
Procedure: (Budget 1.5 lab periods)
Select a partner. Both partners are to collaborate in completing Part A, and independently do either Parts B and D, or Parts C and D.
Ibuprofen
is only very slightly soluble in water, less than 1mg of Ibuprofen
dissolves in 1ml water (< 1 mg/mL).
Part A – Formation and Separation of Diastereomeric Salts
Add ~15 mmol of racemic ibuprofen (Show your calculation for grams to Dr. R. as part of the prelab.) and 30 mL of 0.5M KOH to a 125 mL erlenmeyer flask. Equip the 125 mL erlenmeyer flask with a magnetic spin bar. Place the erlenmeyer in a beaker of water on a hot plate/stirrer. Start stirring slowly. Support an alcohol thermometer (110 oC) so that the spin bar does not come in contact with the thermometer. Heat the water bath so that the reaction mixture is 75 oC - 85 oC. Most but not all of the ibuprofen will dissolve at this temperature. Next add one molar equivalent of S-(-)-α-phenethylamine slowly (dropwise) to the reaction mixture. Show Dr. R. you calculation for mass and volume of the optically active amine.
(Note: (1) S-(-)-α-phenethylamine is irritating to the skin. Wear gloves when using this compound. (2) S-(-)-α-phenethylamine reacts with the CO2 in the air. Recap bottle immediately after using. http://en.wikipedia.org/wiki/Phenethylamine)
A precipitate should form in a few minutes. Keep the solution at 75 oC - 85 oC for 1 hour. Remove the flask from the hot plate and allow it to cool to room temperature. Collect the precipitated salt by vacuum filtration. Wash the solid (termed the filtrand) with a small amount (2-3 mL) of ice cold water. Save all of the filtrate (i.e., the liquid in the filter flask). Be careful to not let aspirator water back up into the flask. Use the filtrate in part C. Use the solid salt filtrand in part B.
Part B – Recrystallization of Ibuprofen / Phenethylamine Salt
Place the salt obtained in part A in a 50 or 100 mL beaker with a boiling stone. Add 30 mL of 2-propanol, place a watch glass on top of the beaker and heat the solution to a boil. At this point all the solid should be dissolved. (Note: If all the solid does not dissolve, add additional 2-propanol in small portions.) Remove the solution from the hot plate and allow it to cool to room temperature, which takes about 10-15 minutes. Place the solution in an ice bath for an additional 15 minutes. Vacuum filter, wash the crystals with 2-3 ml of ice cold water in the Buchner funnel.
Recovery of (+)-Ibuprofen
Place the recrystalized salt into a 50 mL beaker. Add a stir bar and 25 mL of 2M H2SO4 to the beaker. Stir the solution for 5 minutes. The crystals should dissolve and leave behind thick oily droplets suspended in the solution. Extract the aqueous layer with 15 mL portions of MTBE (methyl-t-butylether) three times. Combine the organic layers and extract once with 15 mL of water and then once with 15 mL of a saturated NaCl solution. Dry the organic layer over anhydrous sodium sulfate. Use the rotoevaporator to remove the MTBE. The product at first will be a thick clear oil but should solidify on standing. If solid, determine the melting point. Weigh the product. Be sure to record its exact mass. Proceed to Part D.
Part C – Recovery of (-)-Ibuprofen from Filtrate
In a beaker, add 25 mL of 2M H2SO4 to the filtrate from Part A. Stir the solution for 5 minutes. The solution should become cloudy and insoluble oily droplets should form. Extract the aqueous layer with 15 mL of MTBE (methyl-t-butylether) three times. Combine the ether layers and extract the ether once with 15 mL of water and then once with 15 mL of a saturated NaCl solution. Dry the ether over anhydrous sodium sulfate. Use the rotoevaporator to remove the MTBE. The product, at first, will be a thick clear oil but it usually solidifies on standing. If solid, determine the melting point. Weigh the product. Be sure to record its exact mass. Proceed to Part D.
Part D– Polarimetry (Read Lab Text/Guide pp.54-57)
(Note: (1) DO NOT combine material from Parts B or C. Part D will be conducted separately and independently for each product. Once for the material from part B and once for the material from part C. (2) Be sure you have an accurate weight of the resolved ibuprofen (+) or (-) before continuing on with part D.)
Dissolve the respective sample of resolved ibuprofen (+) or (-) in 2 mL of ethanol and transfer the solution to a clean 5 mL volumetric flask. Rinse the beaker used with another 2 mL of ethanol and transfer the solution to the 5 mL volumetric flask. Carefully add enough ethanol to the volumetric flask to bring it exactly up to the 5 mL mark. Carefully mix the solution until it is homogeneous. Load the polarimeter cell. Record the observed optical rotation of the solution. Pour the solution into a vial. Label the vial to indicate which enantiomer of ibuprofen it contains and turn in to Dr. R.Calculate the optical purity (enantiomeric excess) of each enantiomer.