The Epoxidation of Cholesterol

Keithen Bailie Cast TA: Kristen Bert

Introduction: In this experiment Epoxidation is carried out on cholesterol to form 5, 6epoxycholestan-3B-ol. The purpose of this experiment is to convert a steroid alkene to an epoxide. An epoxide is a cyclic ether with three ring atoms. The peroxide-containing entity in this experiment is Chloroperbenzoic acid in dichloromethane solution. The steroid alkenes used was cholesterol. The stereochemistry of cholesterol and many other steroids are very rigid and stereospecific. The stereo specificity of steroid alkenes is key for the stereospecific nature of the Epoxidation mechanism. Stereospecificity is the property of a reaction mechanism that results indifferent stereospecific reaction products from different stereospecific reactants. Hormones are very common steroid compounds. An example of a hormonal steroid compound id testosterone and other growth stimulants such as ethyl testosterone (4)(3). Cholesterol is a very abundant compound amongst the animal kingdom. Cholesterol is found in mammalian cell membranes and plays a critical role is fluidity. Primarily found in the brain and nerve tissues, the average human contains 200g of cholesterol. This number may come as a surprise due to cholesterols negative reputation. Cholesterol is the principle component of gallstones and can be easily isolated via experimental procedures. High levels of cholesterol can lead to detrimental disease and further health degradation. Often high cholesterol results in hardening of the arteries, known as arteriosclerosis, which can constrict or completely block a blood pathway. If cholesterol prevents the flow of blood a stroke or heart attack can occur (4). In the Epoxidation mechanism of cholesterol the double bond is changed to the 5 alpha, 6 beta epoxide (2). The alpha designates the substituent on the back side, whereas

the beta indicates the substituent top side. Due to steric hindrance from the angular methyl from the angled methyl group the chloroperbenzoic acid cannot attack the top side. Consequently the chloroperbenzoic acid attacks the backside of the 5 alpha carbon. Thus the epoxide is exclusively formed on the back side (3). In this experiment, cholesterol is dissolved in dichloromethane and then added to a mixture of dichloromethane and 3-chloroperoxybenzoic acid. This reaction produces 3chlorobenzoic acid and 5, 6 Epoxycholestan- 3B-ol. This mixture is then pipetted into a chromatography column filled 1/2 full with alumina. T-butyl methyl ether is then eluted through the column. The 3-chlorobenzoic acid will be strongly absorbed by the alumina. This is because the 3-chlorobenzoic acid is polar and the relatively non-polar product is eluted easily by non-polar-ether. The ether can be eluted through gravity or the use of pressure from flash chromatography (4). The products of column chromatography will be eluted into a beaker using t-butyl methyl ether. The beaker will contain 5, 6 Epoxycholestan- 3B-ol and the ether solvent. Next the beaker is allowed to sit for 24 hours for t-butyl methyl ether to evaporate off. After evaporation concludes only 5, 6 Epoxycholestan- 3B-ol will remain in the beaker. This will allow percent yield to be calculated via equation 1. Percent Yield= (Actual yield/theoretical yield) 100 Equation 1

Results: Subsection 1, Compounds and Mechanism Figure 1, Structures of Relevant Compounds Formula C27H46O Structure

Cholesterol

5,6-epoxycholestan3B-ol

C27H46O2

3Chloroperoxybenzoic acid Dichloromethane

C7H5ClO2

CH2Cl2 (CH3)3COCH3

t-butyl methyl ether

Alumina Oxide

Al2O3

(1) Figure 2, Reaction Mechanism

Figure 3, One Step (actual Mechanism)

(1)(4) Subsection 2, Data Tables Table 1, Data collected during lab Mass in Grams Beaker (empty) 83.752 g Beaker with product 84.295 g Actual yield 0.543 g Theoretical yield 0.202 g Cholesterol 0.194 g 3-Chloroperbenzoic acid 0.114 g 90% cholesterol reacting 0.488 Table 1 shows experimental data collected and calculated during lab. The theoretical yield was calculated via equation 1; sample calculations are found in the sample calculations section of results. Subsection 3, Sample Calculations Cholesterol + (3-Chloroperbenzoic acid) 5, 6-epoxycholestan-3B-ol l + (3-Chlorobenzoic acid) 0.543g0.9 (amount actually reacting) =0.1746 Theoretical yield= 0.1746g/(386.66g/mol) 1:1 mol ratioanswer 402.66g/mol(5,6epoxycholestan-3B-ol)=0.202g of 5,6-epoxycholestan-3B-ol from 194 mg of cholesterol Percent Yield= 0.488 g/0.202g Percent Yield=241%

Discussion: The purpose of this lab was to convert cholesterol, a steroid alkenes, to an epoxide, 5, 6
Epoxycholestan- 3B-ol. The peroxide containing agent, 3-chloroperbenzoic acid, is added to cholesterol to produce both 5, 6 Epoxycholestan- 3B-ol and chlorobenzoic acid. In the epoxidation of cholesterol the double bond stereospecifically changes into a 5 alpha, 6 beta epoxide. The alpha

indicates that the substituent is on the backside, whereas the beta indicates the substituent is on the top side. The angular methyl group prevents the top side attack from the 3chloroperbenzoic acid, rendering only the backside attack possible. In order to separate 5, 6 Epoxycholestan- 3B-ol from 3-chlorobenzoic acid (the second product)
column chromatography was used. The column contains highly polar alumina oxide. The column was created using the slurry method. This is a method where the elutant, t-butyl methyl ether (mobile phase), and the alumina oxide (stationary phase) are mixed together in order to make a homogenous mixture to pour into the column. This method creates a more homogeneous stationary phase compared to directly adding alumina oxide to the column. If alumina oxide was added directly the stationary phase could have inadequacies in thickness, thus creating error in chromatography. The products of the preliminary reaction were placed into the column and eluted with t-butyl methyl ether. T-butyl methyl ether is a non polar solvent that has a high affinity for 5, 6 Epoxycholestan- 3Bol. Like dissolves like therefore 5, 6 Epoxycholestan- 3B-ol will elute down through the column. The 5, 6 Epoxycholestan- 3B-ol is non polar because of the peroxy functional group. Consequently 5, 6 Epoxycholestan- 3B-ol will be collected and thus be separated from 3-chlorobenzoic acid. 3chlorobenzoic acid will remain in the column because of its high affinity for the polar stationary phase. Alumina oxide is very polar; therefore highly polar 3-chlorobenzoic acid will stick together. 3-chlorobenzoic acid is very polar because it contains a carboxylic acid group. Carboxylic acid functional group is among the most polar functional groups. Structure of either compound has no significant role in the column except for extremely slow eluting process. The bulky 5, 6

Epoxycholestan- 3B-ol takes much longer to elute through the column than a small non polar compound would take. Once the column chromatography eluting process has concluded only t-butyl methyl ether and 5, 6 Epoxycholestan- 3B-ol remain in the beaker. T-butyl methyl ether is volatile and will evaporate over time. The beaker was placed in the hood to evaporate for 24 hours. Deductively after evaporation, only 5, 6 Epoxycholestan- 3B-ol remains in the beaker. The beaker was previously weighed and was weighed again. The weights were subtracted leaving 0.534 grams of 5, 6 Epoxycholestan- 3B-ol. However it must be taken into consideration that only 90% of the cholesterol reacts. Only 90% reacts because cholesterol has three other derivatives that closely mimic the structure of pure cholesterol. The theoretical yield was 0.202 grams. Using this data and equation 1 percent yield was calculated and found to be 268%. The percent yield is outrageously high and consequently must be contaminated. It is feasibly impossible to obtain more than 100% yield pure compound. Subsequently there is definitely error in the experimental process. The main instance of error was from the eluting process. The lab manual called for 30 ml of t-butyl methyl ether to elute the 5, 6 Epoxycholestan- 3B-ol down the column. In our lab the 5, 6 Epoxycholestan3B-ol was eluted after only 10 ml. consequently 20 ml moved through the column for no reason. These 20 ml of t-butyl methyl ether could have moved some 3-chlorobenzoic acid through the column because of gravity. The sheer amount of liquid moved down the column could have also displaced some alumina oxide particles, thus increasing the mass left in the beaker indirectly increasing yield.

Reference: 1. Reaxys. Cholesterol and 5, 6 Epoxycholestan- 3B-ol Reaxys. Elsevier Properties.2012

2. Smith B, Michael. Epoxidation. Organic Chemistry. HarperCollins Publishers: New York, NY, 2006: 152-154.

3. Vandichel, Matthias, Karen Leus, Pascal Van Der Voort, Michel Waroquier, and Veronique Van Speybroeck. 2012. "Mechanistic insight into the cyclohexene epoxidation with VO(acac)2 and tert-butyl hydroperoxide." Journal Of Catalysis 294, 1-18.

4. Williamson, Kenneth L.; Minard, Robert; Masters, Katherine M . Epoxidation of Cholesterol. Macroscale and Microscale Organic Experiments. 5th ed. Houghton Mifflin Company: Boston, 2007. 390-345.