In the Classroom edited by
JCE DigiDemos: Tested Demonstrations
Ed Vitz Kutztown University Kutztown, PA 19530
Demonstration of Enantiomer Specificity of Proteins and Drugs submitted by:
Gretchen L. Anderson Department of Chemistry, Indiana University–South Bend, South Bend, IN 46634-7111;
[email protected] checked by:
Shallee T. Page Department of Chemistry and Biochemistry, University of Maine–Machias, Machias, ME 04654
Stereochemistry is important in both organic and biochemistry. In biochemistry, the interaction of chiral molecules with enzymes and other proteins has important implications that are often topics of discussion in undergraduate college courses for both nonscience majors and science majors. Textbook and online graphics can be supplemented by classroom demonstrations and activities, several of which have been described in this Journal (1–7). While these activities illustrate the properties of enantiomers, they do not go so far as to illustrate enantiomeric specificity of enzyme–substrate or protein–ligand interactions. I have expanded model work in the lecture classroom by using an inverted watch glass on a desk surface to represent the binding site of an enzyme or other protein. Three different colored Post-it Notes on the watch glass provide color-coded binding sites for tetrahedral models of portions of a substrate or other ligand. This approach not only serves as a graphic, three-dimensional demonstration of the ramifications of enantiomeric specificity of proteins or other molecules, but also provides a convenient extension exercise to illustrate prochirality. I have used this classroom exercise for liberal arts chemistry classes, health professional chemistry classes, and junior–senior level biochemistry classes. The exercise can be easily tailored to the sophistication and background of the students by asking appropriate questions at each stage of the classroom activity. The impact of the exercise is increased by using the models and watch glass to illustrate interactions of well-known drugs with human proteins.
and observing whether it is possible to align the colored balls of the two models. I accompany the discussion with examples of mirror images and ask the students whether their models are identical or reflections of each other. A definition of enantiomers and stereoisomers then ensues. A poll is taken to determine how many student pairs have identical models or stereoisomers and the results are displayed on the board. Depending on the level of the class, I may assign priority labeling for the colored balls, and ask the students to determine R or S configuration of their models. Student pairs with identical models attempt to modify one model to produce a stereoisomer with the minimum manipulation. Student pairs with enantiomers change their models so they are identical. A brief discussion follows to determine the necessary actions to produce the change. Classes for chemistry majors can discuss the production of an intermediate sp2 hybridized state and production of a carbocation intermediate. At this point, approximately half of the student pairs have identical models and half have stereoisomers. The number of groups with each type can be recorded and compared with the previous results. The results should be identical to the initial model building. The lesson for the students is that if just one of the students in the pair changes the model by one simple exchange, the models in the pair change from identical to different, and vice versa.
Enantiomers and Stereoisomers
The class is asked to assume that the combination of the watch glass and the desk models is an enzyme and the colored Post-it Notes model the binding site for a substrate. A yellow spot on the watch glass has affinity for yellow balls on the molecular models, and so forth. The students are then asked to align the colored balls of their models with the Postit Notes on their watch glass. A show of hands determines which student groups found both, one, or neither of their models “binds” to the enzyme. By recording the results according to whether they started with identical models or stereoisomers, students should be able to determine that student pairs with stereoisomers can bind only one of their isomers to the “enzyme”. Student pairs with identical models will either be able to bind both or neither to their watch glass enzyme. Students whose model substrates do not bind their watch glass enzyme can then move about to find someone else’s enzyme to which their model will bind. This illustrates
Each pair of students is given a portion of a molecular model kit with four different colored balls in addition to a central ball for the asymmetric center (alternatively, colored marshmallows and toothpicks could be used), three small Post-it Notes of different colors (matching the colors in the molecular model kit), and a watch glass. (I used model kits from Fisher Scientific with approximately 1-in. diameter balls, 12-cm diameter watch glasses, and 1.5-in. × 2-in. Post-it Notes.) Students are asked to place the watch glass, rim side down, on their desk or bench and to stick the three Post-it Notes on the glass in a triangle. Each student then builds a model of a carbon atom bonded tetrahedrally to four different molecular groups in the usual manner. Students within a pair compare their models and determine whether the molecules are identical or stereoisomers by rotating the models www.JCE.DivCHED.org
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Protein Specificity for a Single Enantiomer
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that an enzyme may bind only one enantiomer, but the other enantiomer may bind to a different enzyme. (Depending on the context, this can easily be extended to receptors and other proteins.) After establishing the stereospecificity of enzymes, the concept can be extended to show that this only occurs if the substrate molecule is chiral with an asymmetric center. This can be demonstrated by removing one colored ball from both enantiomers and substituting a different color, so that there are two groups of the same color in the model to generate an achiral molecule. The molecular models that were enantiomers are now identical molecules and all molecular models bind to all the watch glass active sites (assuming the three colors are the same as those on the watch glass). Practical Applications and Ramifications of Stereospecificity of Proteins This exercise lends itself nicely to illustrating the importance of chiral molecules in the pharmaceutical industry. The tetrahedral models represent various well-known drugs, and the watch glass on the desk represents molecules with which the drugs interact. Most students are familiar with ibuprofen,
of naproxen have been associated with liver damage, the dose of naproxen must be kept minimal. Since one enantiomer of naproxen, (S )-naproxen, is about 28 times more therapeutic than the other enantiomer, (R )-naproxen, this drug is sold as a single enantiomer to provide maximum therapeutic effect while minimizing toxic side effects. Students can reassign the groups on their models to represent naproxen (facilitated by assigning color codes to the groups around the chiral center). Students in pairs can then discuss how they might be able to separate a racemic mixture into pure enantiomers. Depending on the background of the student, this could range from very general (e.g., binding a single enantiomer in a mixture to a protein) to fairly specific (e.g., cyclodextran chromatography), but all the students should come to realize that the enantiomers likely have very similar chemical and physical properties but interact differently with other chiral molecules. Some racemic drugs interact with more than one protein. The classic example is thalidomide, asymmetric center O
asymmetric center O H
H N
asymmetric center
O
CH3
H3C CH
CH2 ibuprofen
the pain reliever found in Advil, Motrin, and Nuprin. By assigning colors to each group around the chiral center of ibuprofen, each student’s model represents one stereoisomer of ibuprofen. The watch glass on the desk now represents the appropriate protein that binds ibuprofen. Only one isomer binds to the receptor: it is the (S)-(+) form that is active as an analgesic. Ibuprofen is usually sold as the racemic mixture of both enantiomers. Although ibuprofen is available as the pure, active stereoisomer, (S )-ibuprofen, it is more costly, even when correcting for the smaller dose required. In addition, (S )-ibuprofen (active) is readily converted to (R )ibuprofen (inactive) in the body and vice versa, so that even administration of enatiomerically pure ibuprofen results in a racemic mixture (8). Naproxen is another analgesic drug, sold as Aleve, asymmetric center
CH
COOH
CH3 CH3
O naproxen
with a single asymmetric center. Because high concentrations
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O
(S)-thalidomide
CH COOH
H3C
O
N
O NH
NH O
O
(R)-thalidomide
a drug that was widely prescribed outside North America to prevent morning sickness in pregnant women. However, the drug also caused severe birth defects including flipper-like appendages and poorly developed internal organs. It was later determined that one enantiomer of thalidomide, (R )-thalidomide, alleviated morning sickness, and the other enantiomer, (S)-thalidomide, caused the birth defects by interfering with blood flow to developing fetal tissue. Presumably, two different sets of proteins interact with the two enantiomers of thalidomide. To demonstrate this concept in the classroom, I assign a sequence of colors on the watch glass as a protein that affects morning sickness, and the reverse sequence as a protein involved with affecting blood vessel formation. Students then fit their model (now modeling one of the stereoisomers of thalidomide) to the appropriate active site. Although methods for separation or synthesis of enantiomerically pure thalidomide have been developed (9– 13), the drug is no longer used to treat morning sickness because of the interconversion of enantiomers in the body. However, many of the side effects of thalidomide, particularly its ability to alter immune responses, inhibit movement of white blood cells, and prevent formation of blood capillaries in tissues, may be beneficial in the treatment of diseases such as leprosy, AIDS, tuberculosis, lupus, and perhaps some forms of cancer (14). Before thalidomide is used therapeutically however, there must be a means of preventing the well-known induction of birth defects. Although drugs can be designed for specific target molecules, all drugs can be expected to have some side effects. Using enantiomerically pure preparations can minimize these,
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but even a pure stereoisomer may bind (albeit more weakly) to other biomolecules. Too little is understood to be able to predict side effects of drugs. Clinical testing may uncover some side effects before the drug is brought to market, but it is impossible to test for all known and unknown interactions. To minimize the appearance of unknown side effects, drug companies are increasingly developing enantiomerically pure drugs. Nearly 40% of all drugs sold worldwide in 2000 were of single enantiomers, accounting for $133 billion dollars in sales (15). Not all molecules are chiral. For example, diphenhydramine,
O
CH2
CH2
CH CH3
diphenhydramine
an antihistamine used in sleep aids such as Sleep-Eze, does not have an asymmetric center. In this case enantiomer purification is not necessary. To simulate this students remove a ball from their model and replace it with a different color so that the model has the same three colors as the watch glass binding site, but with two balls of one color. (I assign color codes for the phenyl, hydrogen, and ether groups of the molecule.) A poll is taken to demonstrate that all the molecular models bind to the watch glass binding site. All models with the same colored balls will be identical. Although there are no enantiomers of diphenhydramine, this drug most likely interacts with more than one protein; it is used not only to treat skin and nasal allergies, but is also the most commonly found active ingredient in nonprescription sleep aids. Students can move about the room to determine how many of the watch glass active sites fit their models of diphenhydramine. Prochirality I also use this exercise to illustrate prochirality. Once the students have made a tetrahedral model with two balls of one color (excluding the center ball), they have a prochiral molecular model: prochirality occurs when two chemically identical substituents to an otherwise chiral tetrahedral center are geometrically distinct (16). To demonstrate how a prochiral molecule can be converted into a single stereoisomer by an enzyme, the students are again asked to assume the watch glass and desk represent the active site of an enzyme. Each student’s prochiral model should bind to the pair’s watch glass active site. Each student simulates an enzyme reaction on this substrate by removing the vertical ball and replacing it with a colored ball so that there are four different colors on the product. For any watch glass or desk enzyme, only one enantiomer is produced. This can be confirmed by assigning priorities to each color and labeling the product as R or S. Enzymes generally produce a single enantiomer from prochiral molecules (16). A classic example is the formation of the single stereoisomer, (2R,3S )-isocitrate, from the de-
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−
prochiral center
−
COO
pro-S -arm
CH2 HO
C
COO −
COO
CH2 −
COO citrate
pro-R -arm
CH2 HO
C
−
COO
C HO
−
COO H
(2R, 3S)-isocitrate
This concept in turn forms the basis of several student exercises requiring tracking a labeled carbon atom through various metabolic pathways. Prochirality and its ramifications in biochemistry are difficult concepts for many students to grasp, even with the help of two-dimensional figures supplied in many biochemistry textbooks (16–21). However, I have found that most students readily come to understand prochirality with this classroom exercise.
CH3 CH
hydration and rehydration of citrate by aconitase.
Summary The advantage of the watch glass on the desk is that it offers an immobile three-dimensional model of a protein of approximately the same size ratio to the chiral small organic molecule. Although the watch glass is not strictly necessary, it adds enough topology to the desk top to convert it from a flat surface to a three-dimensional model. The main limitation to this classroom activity is the logistical problem of supplying a large class with the appropriate materials in an efficient manner. I have found that placing a series of boxes at the front of the classroom, each containing a single item (e.g., a box of yellow balls), provides a relatively quick way to disseminate the materials. On one side of the room I place boxes from which each student takes an item, and on the other side of the room I place materials for each student pair (e.g., a box of watch glasses). Students form a line and pick up their materials from the series of boxes. The bottle neck in this procedure occurs when students pick up the Post-it Notes. For large classes, this could be alleviated by providing watch glasses with Post-it Notes (or other colored markings) already attached. I have found that this classroom activity is well worth the time taken in class. I can integrate the lecture material at the appropriate level at various points in the class activity. Each step of the activity is relatively short, so students remain focused and on-task. Polling to compare students’ results is also a powerful way to illustrate when racemic mixtures or enantiomerically pure compounds result from the different molecular manipulations. In a relatively short time students develop a working knowledge of the importance of stereochemistry in biochemistry and its effect on drug design and metabolism. Literature Cited 1. Lujan-Upton, H. J. Chem. Educ. 2001, 78, 475–477. 2. Collins, M. J. J. Chem. Educ. 2001, 78, 1484–1485.
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10.
11. 12. 13.
Hart, H. J. Chem. Educ. 2001, 78, 1632–1634. Evans, G. G. J. Chem. Educ. 1963, 40, 438–440. Eliel, E. L. J. Chem. Educ. 1964, 41, 73–76. Beauchamp, P. S. J. Chem. Educ. 1984, 61, 666–667. Barta, N.; Stille, J. R. J. Chem. Educ. 1994, 71, 20–23. Evans A. M. Clinical Rheumatology 2001, 20, S9–S14. Alvarez, C.; Sanchez-Brunete, J. A.; Torrado-Santiago, S.; Cadorniga, R.; Torrado, J. J. Chromatographia 2000, 52, 455– 458. Van Overbeke, A.; Aboul-Enein, H. Y.; Baeyens, W.; Van der Weken, G.; Dewaele, C. Analytica Chimica Acta 1997, 346, 183–189. Swartz, M. E.; Mazzeo, J. R.; Grover, E. R.; Brown, P. R.; Aboul-Enein, H. Y. J. Chromatogr. A 1996, 724, 307–316. Reepmeyer, J. C. Chirality 1996, 8, 11–17. Eriksson, T.; Bjoerkman, S.; Roth, B.; Fyge, A.; Hoeglund, P.
Chirality 1995, 7, 44–52. 14. Wright, K. Discover 2000, 21, 31–34. 15. Stinson, S. C. Chem. Eng. News 2001, 79 (40), 79–97. 16. Voet, D.; Voet, J. G. Biochemistry, 2nd ed.; John Wiley & Sons, Inc.: New York, 1995. 17. Garret, R. H.; Grisham, C. M. Biochemistry, 2nd ed.; Saunders College Publishing: Fort Worth, TX, 1995. 18. Zubay, G. Biochemistry, 4th ed.; Wm. C. Brown Publishers: Dubuque, IA, 1998. 19. Mathews, C. K.; van Holde, K. E.; Ahern, K. G. Biochemistry, 3rd ed.; Addison Wesley Longman, Inc.: San Francisco, CA, 2000. 20. Nelson, D. L.; Cox, M. M. Lehninger Principles of Biochemistry; Worth Publishers: New York, 2000. 21. Berg, J. M.; Tymoczko, J. L.; Stryer, L. Biochemistry; W. H. Freeman and Company: New York, 2002.
The structures of a number of the molecules discussed in this article are available in fully manipulable Chime format as JCE Featured Molecules in JCE Online (see page 981).
Featured Molecules
an interactive modeling feature, Only@JCE Online
http://www.JCE.DivCHED.org/JCEWWW/Features/MonthlyMolecules
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