09 Synthesis of Cholesterol Epoxide

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CHM25601 Epoxidation of Cholesterol 1 © Trustees of Purdue University. Permission for reproduction or further distribution must be obtained in writing from the Department of Chemistry. The Synthesis of Cholesterol Epoxide P RELAB R EADING /V IDEO : Review the Experimental Techniques listed below in Zubrick. Relevant videos for these techniques are posted on Brightspace. Review the relevant reactions in your lecture textbook. This is a 1+ week experiment. I NTRODUCTION Cholesterol is a member of a class of molecules called steroids . It is the principal component in gallstones; in fact, a common laboratory experiment for organic chemistry courses is the isolation of cholesterol from gallstones. You’ve undoubtedly heard about the molecule since it’s a primary cause of arteriosclerosis (hardening of the arteries). However, we also require a certain amount of cholesterol for good health (the average person has about 200 g, primarily in brain and nerve tissue), and the compound is synthesized naturally in the body. Some other examples of steroids are shown below. In this experiment, we will use 3-chloroperoxybenzoic acid (MCPBA) to epoxidize the alkene in cholesterol. This is called a Prilezhaev Reaction, after Nikolai Prilezhaev who first published the reaction in 1910.

CHM25601 Epoxidation of Cholesterol 2 © Trustees of Purdue University. Permission for reproduction or further distribution must be obtained in writing from the Department of Chemistry. The bond making and bond breaking steps are thought to be concerted: (from Brown, et.al. Organic Chemistry , 5 th ed. 2009 Chapter 11. Find this mechanism in your text and read about it!) Note that the reaction in this experiment is stereospecific; that is, the only product formed is the one where the epoxide group is trans to the OH group. This is because the methyl groups on the tetracyclic backbone sterically impede the approach of the 3-chloroperoxybenzoic acid from the “top” side of the molecule (if you’re having trouble visualizing this, make a model) . Note also that two different melting points are given for the product because this particular compound can crystallize in either of two different forms (i.e., either of two different crystal structures) depending on the conditions used to isolate the compound. P RE -L AB Q UESTIONS 1. What is the purpose of the lab? What specific techniques will you be using? 2. Provide the scheme and mechanism of the reaction you will be performing during the lab. 3. Provided below are the list of chemicals you will be using in this lab. Please fill in the table below.

CHM25601 Epoxidation of Cholesterol 3 © Trustees of Purdue University. Permission for reproduction or further distribution must be obtained in writing from the Department of Chemistry. Chemical Quant ity Used Molecular Weight Moles Density MP/BP Appearance Hazards Cholesterol m -chloroper- benzoic acid (MCPBA) Ethyl acetate sodium bicarbonate Product Calcium Chloride 4. What is the Limiting Reagent (LR) for the reaction (show calculation)? Calculate the theoretical yield based on the LR. 5. Draw the apparatus setup you will be using in this lab. 6. Does the epoxidation of trans -2-hexene produce a single enantiomer, a meso compound or a racemic mixture? Explain your reasoning and show the mechanism for the reaction of trans -2-hexene with MCPBA. 7. In steroids, the “face” (or “side”) of the molecule in which the methyl groups at the ring junctions are pointing is referred to as the β “ face ” ; the other “ face ” (or “ side ” ) of the molecule is referred to as the α “ face ” . Is the orientation of the epoxide group in cholesterol epoxide α , or β ? 8. What structural feature do all steroids have in common? List two different steroids (not including those listed in the Introduction to this experiment), draw their structures, and state their biological relevance. 9. What is the structural difference between a carboxylic acid and a percarboxylic acid? Why is commercial MCPBA often less than 75% pure, and what are the impurities? (Hint: Google is a very nice search engine.) 10. When the reaction mixture is worked-up, it is first washed three times with 5% sodium bicarbonate, and then with a saturated NaCl solution. Explain why. 11. What are the types of waste you are generating in this experiment and how are you disposing of them? 12. Please provide a flowchart of the procedure you will be following in this lab, highlighting the key steps.

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CHM25601 Epoxidation of Cholesterol 4 © Trustees of Purdue University. Permission for reproduction or further distribution must be obtained in writing from the Department of Chemistry. P ROCEDURE P ART 1: R UNNING THE R EACTION 1. In a 100-mL round bottom flask, place 1.0 g of cholesterol, 15 mL of ethyl acetate and a couple of boiling chips. Gently swirl the contents of the flask until the cholesterol is dissolved. Note that it may take several minutes of swirling for the cholesterol to dissolve. If swirling for 5 minutes and not completely dissolved, proceed to step 2 anyway. 2. Place 0.77 g of 70-75% m -chloroperbenzoic acid (MCPBA) in a 50-mL Erlenmeyer flask, add 10 mL of ethyl acetate, and gently swirl the contents of the flask until the solid has completely dissolved. Name, amount, and Physical description of reagents used: 3. Using a disposable pipet, add about half of a disposable pipet full of the MCPBA solution to the cholesterol solution, and swirl the 100-mL round bottom flask for about 1 min. Repeat this process, using about half of a disposable pipet full at a time and swirling for 1 min, until the MCPBA solution has been completely added to the cholesterol solution. 4. Attach a reflux condenser to the 100-mL round bottom flask, and gently reflux the solution for 35 min (time the reflux starting when reflux begins , not when you turn on the Thermowell). Note: • The flow of water through the condenser should be steady, but not so fast that the hoses can detach from the condenser. • Ethyl acetate has a low boiling point; start with the Thermowell on a low setting (30-40% of the maximum).

CHM25601 Epoxidation of Cholesterol 5 © Trustees of Purdue University. Permission for reproduction or further distribution must be obtained in writing from the Department of Chemistry. Physical description of the reaction mixture before and after reflux: P ART 2: W ORKING U P THE R EACTION 1. At the end of the reflux period, remove the thermowell and let the solution cool for 5 min. Transfer the solution to a 125-mL separatory funnel, and add 15 mL of 5% sodium bicarbonate solution. Gently shake the separatory funnel by repeated inverting, and be sure to vent the separatory funnel periodically . When the two layers have separated, transfer the ethyl acetate layer into a 50-mL Erlenmeyer flask. Place the aqueous layer in a beaker for later disposal in the appropriate waste container . (Do you remember which layer is which?) 2. Return the ethyl acetate layer to the separatory funnel, and wash this layer two more times using 15 mL of 5% sodium bicarbonate solution each time. Finally, wash the ethyl acetate layer once with 10 mL of saturated sodium chloride solution (brine). Be sure to vent the separatory funnel often! Name, amounts of each chemical added and observations from the work up: wood blocks (so you can quickly lower the heat source) Thermowell heater water IN water OUT Reflux Setup Discard disposable pipets in the GLASS / SHARPS WASTE BOX.

CHM25601 Epoxidation of Cholesterol 6 © Trustees of Purdue University. Permission for reproduction or further distribution must be obtained in writing from the Department of Chemistry. 3. Transfer the organic layer into a 50-mL Erlenmeyer flask and add just enough CaCl 2 pellets to cover the bottom of the flask. Swirl the contents of the flask, then let stand for 5 min. 4. Decant the organic solution from the Erlenmeyer into a 100 mL round bottom flask. ( decant = carefully pouring a solution from a container to leave any solids in the bottom of the original container.) Rinse the CaCl 2 pellets with 1 mL of ethylacetate and transfer the rinse to the round bottom flask. Remove the solvent using a rotary evaporator. Observation after addition of CaCl 2 and post-solvent evaporation: P ART 3: R ECRYSTALLIZING THE R EACTION P RODUCT 1. Add 8 mL of acetone to the 100-mL round bottom flask, clamp the flask to a ring stand, and heat the mixture just to boiling using a Thermowell. Then, carefully transfer the solution to a 50-mL Erlenmeyer flask. Rinse the round bottom flask with 1 mL of acetone and transfer the rinse to the Erlenmeyer flask. Add 1 mL of water to the Erlenmeyer flask, and again bring the solution just to boiling on a hotplate. Let the solution cool slowly to room temperature. Once the solution has cooled to room temperature, cool the mixture in an ice/water bath until crystals appear. Observations from Recrystallization: Place both the aqueous bicarbonate solution and the aqueous sodium chloride solution in the AQUEOUS WASTE JAR. Do not use ACETONE to rinse out the Separatory Funnel, Erlenmeyer Flask or Buchner Funnel- Use WATER Place the CaCl 2 pellets in the DRYING AGENTS WASTE JAR.

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CHM25601 Epoxidation of Cholesterol 7 © Trustees of Purdue University. Permission for reproduction or further distribution must be obtained in writing from the Department of Chemistry. 2. Collect the solid product by vacuum filtration using a Buchner funnel (see image below). Be sure to use thick-walled rubber tubing to connect the filter flask to vacuum and vacuum trap! Wash the solid with 1 mL of ice-cold, 90% acetone / 10% water. Let the solid dry on the funnel, while maintaining the vacuum, for 15 min. Physical description of the product: 3. Store your product in a labeled 50 mL beaker covered with a secured paper tissue in your lab drawer until the next lab period. P ART 4: I NFRARED S PECTROSCOPY /M ASS /M ELTING P OINT (R ECORDED IN THE NEXT I N L AB ) The following week, the crystals should be thoroughly dry and you will then be able to determine the mass, percent yield, melting point and infrared spectrum for the reaction product. You should also collect an IR spectrum of the starting material (cholesterol). Mass, MP of the product: Discard the filtrate (90% acetone / 10% water) in the ACETONE RINSE WASTE JAR. Allow the filter paper to dry in the hood, then discard the filter paper in the FILTER PAPER WASTE JAR. 3-prong clamps Vacuum regulator Filter flask Buchner funnel 2X filter Vacuum tubing (thick Vacuum trap Vacuum Filtration Set Up

CHM25601 Epoxidation of Cholesterol 8 © Trustees of Purdue University. Permission for reproduction or further distribution must be obtained in writing from the Department of Chemistry. D ISCUSSION Q UESTIONS 1. Based on the actual measured amount used in the experiment is the limiting reagent (LR) you identified in the prelab still the LR? How many moles of the LRs did you use in the experiment? 2. What is the theoretical yield (TY) based on the measured amount? Is it more or less than the TY you obtained in your prelab calculation? 3. How much product did you isolate and what does the final product look like? 4. What is the percent yield (PY) of the reaction? How do you explain the observed PY? 5. In addition to the cholesterol epoxide what other compound is formed during the reaction? How did you separate this compound from cholesterol epoxide? 6. What was the purpose of adding Calcium Chloride to the organic layer following the workup? 7. What was the experimentally determined melting point (and its range) for the product and how does it compare with the literature value for this compound? What does this tell you about the purity of your product? 8. Describe how the IR spectrum for the starting material (cholesterol) is similar to, or different from, the IR spectrum for the reaction product. List and identify any peaks that are different between the two spectra. What information, if any, does this comparison provide about the purity of the product? 9. Were you successful in accomplishing the purpose of this lab? Base your answer on your analysis of PY, IR, and MP. 10. Based on your experience doing the experiment, identify sources of error in your experiment and how they affected your results. (Human error is not an acceptable answer) What specific changes could you suggest to mitigate these errors in the future? After you have collected your data, discard the unknown solid in the PRODUCT WASTE JAR (SOLID JAR).