In the Laboratory
Bromination and Debromination of Cholesterol: An Inquiry-Based Lab Involving Structure Elucidation, Reaction Mechanism, and 1H NMR
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Andrew Grant* Department of Chemistry, Mount Allison University, Sackville, NB, Canada, E4L 1G8; *
[email protected] Devin Latimer Department of Chemistry, University of Winnipeg, Winnipeg, MB, Canada, R3B 2E9
In discussions of structure, conformation, stereochemistry, and mechanism in upper-level organic chemistry courses, it is not enough to draw a reaction showing starting materials, products, and a possible mechanism without presenting some of the experimental data from which conclusions regarding the structure and mechanism were drawn. The laboratory offers students the chance to retrace for themselves the logic leading from experimental data to the proposed structures and mechanism (1). An upper-level organic experiment that incorporates all of the above ideas has been developed. The bromination (and subsequent debromination) of cholesterol (Scheme I) is performed; but rather than use the experiment in the context of a technique, such as isolation and purification of cholesterol (2), the emphasis is to focus on the reaction itself and ask “What is going on in this bromination reaction?” The objective is to get students to work out for themselves the correct structure of dibromocholesterol and then to propose a mechanism for its formation. The lab makes use of 1H NMR
21
18 25 17
11 19 1
9
H
2 10 5
HO
15
H
H 7
3 4
6
cholesterol Br2
Zn, H+
H H
H
HO Br
Br
dibromocholesterol Scheme I. The bromination and debromination reaction involving cholesterol.
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spectroscopy, in particular the approximate dihedral angle coupling constants, as the source for the experimental data. Rather than comparing a physical property of the product, such as melting point, to a known standard, students determine for themselves which structural isomer, out of four possible stereoisomers, to associate with a particular observed NMR spectrum. Our main contribution to the development of this lab is a series of instructions and questions designed to guide students through the inquiry-based analysis that leads them to the correct structure of dibromocholesterol. In so doing, the students become familiar with the logic required to connect 1 H NMR data to the three-dimensional shape of a complex molecule. Over a three-year period we have received very positive feedback about the lab from the students. Discussion Technically the lab is relatively easy and can be run over a two-week period using a procedure adopted from Fieser, and Fieser and Williamson (2) starting with 1 g of cholesterol. Students obtain the 1H NMR spectra of dibromocholesterol and cholesterol that has been resynthesized via zinc reduction. They then have to make assignments for the two downfield peaks in both spectra and account qualitatively, in terms of coupling constants, for the shape and overall widths of these peaks. A series of instructions and questions are provided that guide students through the conformational and mechanistic analysis and form the basis of a formal lab writeup. The downfield regions of the 1H NMR spectra for cholesterol and dibromocholesterol at 270 MHz are shown in Figure 1. Both peaks are essentially multiplets with little fine structure, but the overall widths of the peaks can be measured and used to qualitatively estimate the magnitude of the coupling constants that must be operating. By asking and answering the proper questions and making reasonable assumptions, the correct structure of dibromocholesterol and a mechanism for its formation can be proposed. The instructions and questions provided to the student to aid in their analysis and lab writeup are outlined below. Suggestions for answers are included in the Supplemental Material.W 1. Draw cholesterol in a 3D-like fashion using chair or halfchair conformations, clearly showing the axial and equatorial positions of important hydrogens. Assign the two downfield peaks in the 1H NMR of cholesterol to the correct H’s in the structure. Observe and explain the splitting
Journal of Chemical Education • Vol. 80 No. 6 June 2003 • JChemEd.chem.wisc.edu
In the Laboratory pattern of these peaks. Make a model, look at dihedral angles, and make a prediction as to what the approximate splitting pattern should be. Does your prediction match the actual spectrum? 2. Assign the two downfield peaks in the 1H NMR spectra of dibromocholesterol to the correct H’s in the structure. Measure the total peak widths. Comment on any changes in chemical shift. 3. Determine the stereochemistry of the two bromines. There are two possible approaches for making the assignments. The first would be to assume a particular mechanism, in this case trans-addition, then draw out the two trans products and see which one best explains the NMR data. The second approach would be to assume nothing regarding the mechanism, draw out all possible products (cis- and transdibromides) and see which stereoisomer best fits the approximate data gathered in steps 1 and 2. Pay particular attention to the orientation of the C3⫺H. Determine the structure of dibromocholesterol following the latter approach. 4. Having determined the correct structure of dibromocholesterol, propose a mechanism for its formation. Did the bromine molecule approach the double bond from the top (C10 methyl side) or bottom of the cholesterol ring plane? Propose an experiment which might prove which approach is taken. What would be the product if the mechanism went through a discreet carbonium ion? 5. What other features of the 1H NMR spectra can you explain? Can you account for all of the methyl groups in the two molecules? 6. Write out a mechanism for the zinc reduction of dibromocholesterol to cholesterol. If this reaction is a reduction, explain why the bromination of a double bond is an oxidation.
Materials This lab is conveniently carried out in a 25-mL Erlenmeyer flask in a fume hood. The following chemicals are needed for this experiment: cholesterol, bromine, acetic acid, sodium acetate, t-butyl methyl ether, zinc dust, water, 10% sodium hydroxide, saturated sodium chloride, methanol, calcium chloride (anhydrous), CDCl3. The bromine兾acetic acid兾sodium acetate solution is made up by an instructor prior to the lab. Hazards Bromine is highly toxic and corrosive. Acetic acid causes chemical burns. The vapors of both are irritating to the eyes, and can damage the eyes and respiratory tract. Gloves and protective eyewear should be worn, and the reaction carried out in a well-ventilated fume hood. Avoid contact with the acetic acid兾bromine solution and do not inhale the vapors.
Figure 1. Downfield portion of the 270 MHz 1H NMR spectra of (A) cholesterol and (B) dibromocholesterol.
Conclusions We assume that the topics on stereochemistry, conformation, and NMR required to answer the above questions have been covered or are being covered in class. Our objectives for the lab are to get students to think and draw threedimensionally, to emphasize the axial and equatorial nature of substituents, to gain an appreciation for dihedral angles and the approximate magnitude of coupling constants between vicinal protons, to relate structure with mechanism and vice versa, and to emphasize the fact that bromination of a double bond is actually an oxidation. In essence, this simple reaction serves as a physical organic experiment to probe a reaction mechanism and its stereochemical consequences. Additionally, the experiment can also serve as a means to place what we know about synthesis and reaction mechanisms in an historical context by reexamining the classic work of Barton from the pre-NMR era (3). W
Supplemental Material
Instructions for the students, suggested answers to questions, a note for the instructor, and CAS registry numbers for the required chemicals are available in this issue of JCE Online. Literature Cited 1. For recent examples of inquiry-based labs, see: Centko, R. S.; Mohan, R. S. J. Chem. Educ. 2001, 78, 77; Cabay, M. E.; Ettlie, B. J.; Tuite, A. J.; Welday, K. A.; Mohan, R. S. J. Chem. Educ. 2001, 78, 79. 2. Fieser, L. F. Org. Synth., Coll. 1963, IV, 195; Fieser, L. F.; Willianson, K. L. Organic Experiments, 8th ed.; Houghton Mifflin: Boston, MA, 1998; pp 241–245. 3. Barton, D. H. R.; Miller, E. J. Am. Chem. Soc. 1950, 72, 1066.
JChemEd.chem.wisc.edu • Vol. 80 No. 6 June 2003 • Journal of Chemical Education
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