Testing the Vibrational Theory of Olfaction: A Bio ... - ACS Publications

Laboratory Experiment pubs.acs.org/jchemeduc

Testing the Vibrational Theory of Olfaction: A Bio-organic Chemistry Laboratory Experiment Using Hooke’s Law and Chirality Rajeev S. Muthyala,* Deepali Butani, Michelle Nelson, and Kiet Tran Center for Learning Innovation, University of Minnesota Rochester, 111 S. Broadway, Rochester, Minnesota 55904, United States S Supporting Information *

ABSTRACT: Sense of smell is one of the important senses that enables us to interact with our environment. The molecular basis of olfactory signal transduction is a fascinating area for organic chemistry educators to explore in terms of developing undergraduate laboratory activities at the interface of chemistry and biology. In this paper, a guided-inquiry laboratory experiment is described to test the vibrational theory of olfaction according to which a molecule’s vibrations determine its odor. Two key predictions of this theory were tested in this experiment. The first was that deuterated odorants and their nondeuterated counterparts would have different odors. This is because application of Hooke’s law leads one to predict that the frequency of a C−H bond vibration is different compared to a C−D bond vibration. The second key prediction that students tested was that enantiomeric odorants should have the same odor since their individual bond vibrations should be identical. Contrary to these predictions, students observed that the enantiomeric odorants had different odors whereas deuterated acetophenone and its nondeuterated counterpart had the same odor. Therefore, students concluded that their empirical evidence does not support the vibrational theory of olfaction. A postlab evaluation showed student comprehension of the principles underlying the olfaction laboratory experiment. KEYWORDS: Bioorganic Chemistry, Laboratory Instruction, Organic Chemistry, Chirality/Optical Activity, Inquiry-Based/Discovery Learning, IR Spectroscopy, Isotopes

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INTRODUCTION Recent efforts to reform organic chemistry laboratory activities have spurred interest in the development of new discoverybased and guided-inquiry experiments.1,2 Designing activities to make seemingly abstract molecular phenomena more tangible to the average undergraduate student in introductory chemistry courses is challenging. Recourse is often taken to exploit visual cues, such as color changes, readily observable to the naked eye.3 Reliance on olfaction, another important sensory tool, in the undergraduate chemistry laboratory is sparse.4 Articles on the use of olfaction in undergraduate education include functional group recognition,5,6 determining end points in acid/base titrations,7 studying esterification kinetics,8 and distinguishing enantiomers.9−11 Herein, a guided-inquiry laboratory experiment is reported that combines Hooke’s law12 with the enantioselectivity of olfaction to test the vibrational theory of olfaction. Although there have been reports of pedagogical interventions to facilitate student learning of Hooke’s law (including interdisciplinary activities with physics faculty),12,13 we are unware of any reports of its use in the context of olfaction. Similarly, whereas there are excellent descriptions of the enantioselectivity of olfaction,9−11,14 to our knowledge, there are no reports demonstrating its utility as a tool for undergraduates to test a scientific theory. Through the application of chemical concepts to test the vibrational theory © XXXX American Chemical Society and Division of Chemical Education, Inc.

of olfaction, the laboratory experiment is intended to illustrate to undergraduates the multidisciplinary nature of contemporary scientific inquiry.

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BACKGROUND The laboratory experiment is based on a theory of olfaction called the vibrational theory according to which a molecule’s vibration determines its odor.15,16 First put forward in 1938 by Dyson,17 this theory did not gain much acceptance due to lack of adequate evidence.18 Further, the mechanism of transduction of a molecular vibration to activation of an odorant receptor (OR) was unclear. However, the theory garnered renewed interest recently due to the efforts of Luca Turin, who proposed inelastic tunneling between an odorant molecule and a receptor as a new mechanism for the transduction of a molecular vibration into activation of an OR.19 In designing this experiment, the goal was not, of course, to test the vibrational theory in its entirety. The concept of inelastic tunneling is beyond the scope of a first-year organic chemistry course. Our inspiration for conceiving this experiment was Keller and Vosshall’s work which described the use of psychophysical Received: December 19, 2016 Revised: May 21, 2017

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DOI: 10.1021/acs.jchemed.6b00991 J. Chem. Educ. XXXX, XXX, XXX−XXX

Journal of Chemical Education

Laboratory Experiment

whether the vibrational spectra of enantiomeric carvones would be the same or different and whether the enantiomers would have the same or different odors. Students determined that if the theory was correct, the enantiomers would have the same odor because they have identical vibrational spectra. This can be easily confirmed by examining the IR spectra of the enantiomeric odorants. Commercially available (R)- and (S)carvone as well as (R)- and (S)-limonene were used. Whereas the IR spectra of these enantiomeric odorants are identical, their odors are distinct. (Note: Care must be taken to ensure that the commercially available carvone or limonene samples are dried prior to acquiring their IR spectrasmall amounts of water in the samples could render their IR spectra nonidentical.) The (R) isomer of carvone has a strong spearmint odor, whereas the (S) enantiomer has a caraway-like odor.14 For the limonene enantiomers, the (R) isomer has a fresh citrus orange-like odor, whereas the (S) isomer has a lemony odor. It should be noted that enantiomeric odorants need not necessarily have different odors but many do.26 After completing their worksheets, students brainstormed ways to test the vibrational theory. They also helped design the experiment being performed with guidance. They tested the odors of pairs of compounds shown in Figure 1. The choice of

methods to test key predictions of the vibrational theory of olfaction.20

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EXPERIMENT DETAILS The intervention was implemented in the first semester of an organic first curriculum21−24 (where organic chemistry is taught in the freshman year instead of general chemistry), but it could be easily implemented in the first semester of a regular secondyear undergraduate organic chemistry course for chemistry majors or nonmajors. The students involved in this experiment were enrolled in a health science major at a health-care-focused four-year college. The laboratory experiment has been implemented over three different years for three separate cohorts with class sizes varying with 165 in the first year, 100 in the second year, and 167 in the third. Students in each cohort were divided into multiple sections with each lab consisting of 16−18 students. Three different instructors taught the lab sections each year, but all used the same worksheets and material. By the time the experiment was implemented, students were introduced to IR spectroscopy, Hooke’s law, and isotope effects on vibrational spectra in the classroom. Students were also familiar with the concept of chirality. The experiment was spread over two consecutive lab periods over 2 weeks. As part of their prelab assignment, students were asked to read the introduction section of two articles.19,25 They were also encouraged to peruse a link (see S5 in Supporting Information), which contains background information about the 2004 Nobel Prize in Physiology or Medicine jointly awarded to Linda Buck and Richard Axel for their contributions to our current understanding of olfaction. At the beginning of the experiment, the instructor provided an introduction to the molecular basis of olfaction, in particular, to the shape and the vibrational theories, which included watching a Ted Talk video featuring Luca Turin (see S5 in Supporting Information). Students were then divided into groups of four and asked to complete a worksheet (see S6 and S7 in Supporting Information), which was provided to facilitate/guide them toward designing experiments to test the vibrational theory. Students first discussed the answers to each question in the worksheet within each group before reporting to the entire class. Following is an example of a question designed to guide students to think about how deuterated odorants could be used to test the theory: “Based on the vibrational theory of olfaction with regards to the following two compounds, would you expect acetophenone-d8 to have the same odor as acetophenone? If they smell different, does this support the vibrational theory? Explain your answer.” To answer this question, students used the simple harmonic approximation to bond vibrations and applied Hooke’s law to predict and compare the stretching frequency of a C−D bond stretch versus that of a C− H bond (the latter bond stretching frequency is higher). They determined that deuterated compounds will have different vibrational frequencies compared to their hydrogen-atomcontaining counterparts, or isotopomers. The IR spectra of CHCl3 and CDCl3 were compared for students to illustrate the deuteration-induced shift in a C−H bond vibration (see S4 in Supporting Information). Therefore, they came to the conclusion that if the vibrational theory was correct, a deuterated compound will have a different odor compared to the nondeuterated compound. In another part of the worksheet, the focus was on using enantiomers to test the vibrational theory. Students predicted

Figure 1. Odorants used for testing the vibrational theory of olfaction.

odorants was restricted to compounds shown in Figure 1 because of their ready commercial availability and their nontoxic nature and because they are relatively inexpensive. Vials containing the odorants in Figure 1 were made available, with coded labels, to students to ensure the identity of the compounds remained unknown (A, B, C, etc.). Students were shown the proper wafting technique to determine if the odors were similar or different. They were asked to wait for 2 min prior to smelling a different sample. Students entered their data into a second worksheet indicating the odor, but they were not asked to identify the odors. The instructors then collected these worksheets, entered the data, and, after all the sections completed their work, provided students with the combined class data along with the names of compounds. The students analyzed the combined class data for evidence to test the vibrational theory of olfaction.

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HAZARDS All odorants used in this study should be kept inside fume hoods. Carvone, limonene, acetophenone, and the deuterated odorants (acetophenone-d3 and acetophenone-d8) are irritants to the skin, eyes, and respiratory system. However, it is not known if they are carcinogenic, mutagenic, or teratogenic. B




Table 1. Summary of Student Data on Whether the Odorants Smell the Same or Different year 1 (N = 165)

a

year 2 (N = 100)

year 3 (N = 167)

odorants

same, %

different, %

same, %

different, %

same, %

different, %

(R)- and (S)-carvone (R)- and (S) -limonene acetophenone and acetophenone-d3 acetophenone and acetophenone-d8

14 28 67 N/Aa

86 72 33 N/Aa

34 15 69 N/Aa

66 85 31 N/Aa

16 36 50 60

84 64 50 40

These odorants were not used.

transduction of a molecular vibration to activate an OR was unlikely in a biological context.

Protective gloves and safety goggles should be worn at all times when handling the odorants. Students should be shown the proper wafting technique for odor detection to minimize exposure. Direct inhalation of any odorant should be avoided at all times.

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EVALUATION OF THE EXPERIMENT Prior to the experiment and subsequent to viewing Luca Turin’s TED talk (see S5 in Supporting Information), a majority of the students were inclined to support the vibrational theory of olfaction. Students’ sympathy for the vibrational theory stemmed from their view that it was an “underdog theory” pitted against the establishment’s theories.20 However, when asked after performing the experiment, “What data, if any, supports the vibrational theory?” a common sentiment was “Unfortunately, we did not find any data to support the vibrational theory.” To determine the impact of the experiment on students’ comprehension of its underlying principles, we examined their responses to a specific question (Figure 2) asked of two

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RESULTS AND DISCUSSION Data from three years of implementing the olfaction experiment are summarized in Table 1. Each year, students examined the data and answered a total of seven questions as part of their postlab worksheet (see Supporting Information) to determine if the evidence supported or contradicted the vibrational theory. In the first 2 years, the use of acetophenone and acetophenone-d3 proved to be adequate, although reported20 psychophysical testing of the vibrational theory relied on acetophenone and acetophenone-d8 as the isotopomeric pair. A consensus emerged that because (a) the enantiomeric carvones (and limonenes) have distinct odors and (b) acetophenone and acetophenone-d3 have identical odors, there is no experimental support for the vibrational theory just as Keller and Vosshall concluded in their 2008 paper.20 In the third year, however, students were split equally on whether acetophenone and acetophenone-d3 had identical or different odors. Such perceptual inconsistencies could arise due to peri-receptor events (such as the odorant undergoing an enzyme-mediated reaction) in the nasal mucus that could be sensitive to isotope effects as pointed out by Block et al.27 In our case, in the absence of a duo-trio test,20 we suspected that the perceptual difference between the third and the previous years could be due to the presence of trace impurities in one of the commercial samples of acetophenone-d3. Therefore, only in the third year we used acetophenone and acetophenone-d8 as an additional isotopomeric pair to test the vibrational theory. This additional test led to a majority (60%) of the students reporting that the two had the same odor, consistent with literature data.20 Overall, students in the third year reached the same conclusion as those in the previous two years: that available evidence does not support the vibrational theory of olfaction. We considered extending our test to other commercially available isotopomeric odorants such as benzaldehyde and benzaldehyde-d6. However, due to inhalation hazard concerns, chemical stability issues,28 and cost considerations, we confined ourselves to acetophenone and its isotopomers. It is noteworthy to mention that, instead of relying on behavioral tests, Block et al.27 tested the plausibility of the vibrational theory of olfaction at a molecular level using receptor-based assays. They found that a specific human muskrecognizing receptor (OR5AN1) failed to distinguish between muscone and muscone-d30 and several other related odorants and their isotopomers. An additional conclusion from this study is that the tunneling phenomenon proposed by Turin for the

Figure 2. Percent correct responses to an exam question before and after implementing the olfaction experiment.

separate cohorts over two consecutive years. The first year the question was asked, students learned about Hooke’s law in the context of IR spectroscopy. They also learned about chirality and how enantiomers have identical physical properties except for their ability to rotate plane polarized light. However, these students did not conduct any specific laboratory experiment to test the vibrational theory of olfaction. Only 10% of this student cohort (cohort 1 in Figure 2) correctly answered the question, suggesting that others were unable to apply their knowledge of chirality in the context of olfaction. In the second year, another cohort of students who had participated in the experiment testing the vibrational theory of olfaction was asked the same question. This cohort did not have access to the question from the previous year. This time the percentage of correct answers C




T., Maelia, L. E., Eds.; American Chemical Society: Washington, DC, 2016; Vol. 1233, pp 123−147. (3) Popova, M.; Bretz, S. L.; Hartley, C. S. Visualizing Molecular Chirality in the Organic Chemistry Laboratory Using Cholesteric Liquid Crystals. J. Chem. Educ. 2016, 93 (6), 1096−1099. (4) Francl, M. Scents and sensibility. Nat. Chem. 2015, 7 (4), 265− 266. (5) Brower, K. R.; Schafer, R. The Recognition of Chemical Types by Odor. The Effect of Steric Hindrance at the Functional Group. J. Chem. Educ. 1975, 52 (8), 538−540. (6) Valentín, E. M.; Montes, I.; Adam, W. Counterion Effects in the Nucleophilic Substitution Reaction of the Acetate Ion with Alkyl Bromides in the Synthesis of Esters. J. Chem. Educ. 2009, 86 (11), 1315−1318. (7) Neppel, K.; Oliver-Hoyo, M. T.; Queen, C.; Reed, N. A Closer Look at Acid−Base Olfactory Titrations. J. Chem. Educ. 2005, 82 (4), 607−610. (8) Bromfield-Lee, D. C.; Oliver-Hoyo, M. T. An Esterification Kinetics Experiment That Relies on the Sense of Smell. J. Chem. Educ. 2009, 86 (1), 82−84. (9) Mannschreck, A.; Kiesswetter, R.; von Angerer, E. Unequal Activities of Enantiomers via Biological Receptors: Examples of Chiral Drug, Pesticide, and Fragrance Molecules. J. Chem. Educ. 2007, 84 (12), 2012−2018. (10) Mannschreck, A.; von Angerer, E. The Scent of Roses and Beyond: Molecular Structures, Analysis, and Practical Applications of Odorants. J. Chem. Educ. 2011, 88 (11), 1501−1506. (11) Kraft, P.; Mannschreck, A. The Enantioselectivity of Odor Sensation: Some Examples for Undergraduate Chemistry Courses. J. Chem. Educ. 2010, 87 (6), 598−603. (12) Hess, K. R.; Smith, W. D.; Thomsen, M. W.; Yoder, C. H. An Introductory Infrared Spectroscopy Experiment. J. Chem. Educ. 1995, 72 (7), 655−656. (13) Burke, J. T. IR Spectroscopy or Hooke’s Law at the Molecular Level - A Joint Freshman Physics-Chemistry Experience. J. Chem. Educ. 1997, 74 (10), 1213. (14) Murov, S. L.; Pickering, M. The odor of optical isomers. An experiment in organic chemistry. J. Chem. Educ. 1973, 50 (1), 74−75. (15) Roderick, W. R. Current ideas on the chemical basis of olfaction. J. Chem. Educ. 1966, 43 (10), 510−520. (16) Rinaldi, A. The scent of life. The exquisite complexity of the sense of smell in animals and humans. EMBO Rep. 2007, 8 (7), 629− 633. (17) Dyson, G. M. The scientific basis of odour. J. Soc. Chem. Ind., London 1938, 57 (28), 647−651. (18) Friedman, L.; Miller, J. G. Odor Incongruity and Chirality. Science 1971, 172 (3987), 1044−1046. (19) Turin, L. A Spectroscopic Mechanism for Primary Olfactory Reception. Chem. Senses 1996, 21 (6), 773−791. (20) Keller, A.; Vosshall, L. B. A psychophysical test of the vibration theory of olfaction. Nat. Neurosci. 2004, 7 (4), 337−338. (21) Muthyala, R. S.; Wei, W. Does Space Matter? Impact of Classroom Space on Student Learning in an Organic-First Curriculum. J. Chem. Educ. 2013, 90 (1), 45−50. (22) Coppola, B. P.; Ege, S. N.; Lawton, R. G. The University of Michigan Undergraduate Chemistry Curriculum 2. Instructional Strategies and Assessment. J. Chem. Educ. 1997, 74 (1), 84−94. (23) Ege, S. N.; Coppola, B. P.; Lawton, R. G. The University of Michigan Undergraduate Chemistry Curriculum 1. Philosophy, Curriculum, and the Nature of Change. J. Chem. Educ. 1997, 74 (1), 74−83. (24) Reingold, I. D. Bioorganic First: A New Model for the College Chemistry Curriculum. J. Chem. Educ. 2001, 78 (7), 869−871. (25) Hatt, H. Molecular and Cellular Basis of Human Olfaction. Chem. Biodiversity 2004, 1 (12), 1857−1869. (26) Bentley, R. The Nose as a Stereochemist. Enantiomers and Odor. Chem. Rev. 2006, 106 (9), 4099−4112. (27) Block, E.; Jang, S.; Matsunami, H.; Sekharan, S.; Dethier, B.; Ertem, M. Z.; Gundala, S.; Pan, Y.; Li, S.; Li, Z.; Lodge, S. N.; Ozbil,

increased to 77%, thereby indicating that students understood the key ideas in the olfaction laboratory experiment. A majority of student responses from the postlab worksheet (see S9 in Supporting Information) also reinforced the above conclusion. For example, in response to the question: “If two enantiomers smell different, does this support the vibrational theory?” One student responded “No it does not because in the vibration theory, if we disregard shape and focus just on vibration, bonds are what affects vibrations and should be the same for both the R and S carvone. Thus, both should emit the same smell in accordance to the vibration theory.” Further, in response to another question in the worksheet whether the vibrational theory would be supported if acetophenone and acetophenone-d8 have different odors, a student responded as follows: “Yes, it would support the vibrational theory because it states that molecules of different vibrations will have different smells. The molecules have different vibrations, so if they smell different it will support the vibrational theory.” Therefore, multiple evaluations suggest that the experiment contributed to students’ learning the process of testing a scientific theory.

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SUMMARY Laboratory experiments in introductory organic chemistry courses are typically “recipe-based” and contribute little to meaningful learning for students. We developed a unique olfaction-based experiment which brings important findings from the realm of cutting edge research into the classroom by utilizing key concepts taught in introductory organic chemistry courses. The guided-inquiry experiment at the interface of chemistry and neuroscience introduces first- or second-year students to the importance of using empirical evidence to test a hypothesis. This experiment is suitable for both chemistry majors and nonmajors.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available on the ACS Publications website at DOI: 10.1021/acs.jchemed.6b00991. CAS registry numbers for odorants, Instructor notes, student handouts, pre- and postlab worksheets (PDF, DOCX)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Rajeev S. Muthyala: 0000-0003-0811-9877 Notes

The authors declare no competing financial interest.

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REFERENCES

(1) Schoffstall, A. M.; Gaddis, B. A. Incorporating Guided-Inquiry Learning into the Organic Chemistry Laboratory. J. Chem. Educ. 2007, 84 (5), 848−851. (2) Fahnhorst, G. W.; Swingen, Z. J.; Schneiderman, D. K.; Blaquiere, C. S.; Wentzel, M. T.; Wissinger, J. E. Synthesis and Study of Sustainable Polymers in the Organic Chemistry Laboratory: An Inquiry-Based Experiment Exploring the Effects of Size and Composition on the Properties of Renewable Block Polymers. In Green Chemistry Experiments in Undergraduate Laboratories; Fahey, J. D




M.; Jiang, H.; Penalba, S. F.; Batista, V. S.; Zhuang, H. Implausibility of the vibrational theory of olfaction. Proc. Natl. Acad. Sci. U. S. A. 2015, 112 (21), E2766−E2774. (28) Benzaldehyde undergoes autoxidation to benzoic acid when exposed to air at ambient temperature. See, for example van der Beek, P. A. A. The autoxidation of benzaldehyde. Recl. Trav. Chim. Pays-Bas 1928, 47, 286−300.

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Testing the Vibrational Theory of Olfaction: A Bio ... - ACS Publications

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