A Drug-Discovery Experiment for the High School Chemistry or

Laboratory Experiment pubs.acs.org/jchemeduc

PharmaChemistry in the Classroom: A Drug-Discovery Experiment for the High School Chemistry or Biotechnology Classroom Emily Garcia Sega*,†,§ and Jewyl Clarke‡ †

Department of Chemistry, University of California, San Diego, La Jolla, California 92093, United States Eastlake High School, Chula Vista, California 91915, United States

‡

S Supporting Information *

ABSTRACT: In this laboratory experiment, students were exposed to a real-world approach to science through learning about and simulating the processes by which drugs are designed and discovered. The students first synthesized oil of wintergreen from aspirin and analyzed the physical properties of both the starting material (aspirin) and product (oil of wintergreen) to validate that their reaction was successful. Second, unique to this lab experiment, students further scrutinized the synthesis by performing a biological assay investigating the antibacterial properties of their samples. Through the use of these analyses, students were able to play the roles of chemists, biologists, and team members involved in the drug-discovery pipeline. The results revealed to the students that small changes in molecular structure caused significant changes in medicinal activity. Through data analysis, discussion, and problem sets, students evaluated various steps of the experiment as they simulated the drug-discovery process. KEYWORDS: High School/Introductory Chemistry, Interdisciplinary/Multidisciplinary, Laboratory Instruction, Hands-On Learning/Manipulatives, Applications of Chemistry, Biotechnology, Drugs/Pharmaceuticals, Medicinal Chemistry, Synthesis

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medical problem, performing a chemical synthesis to make a new molecule, characterizing the new molecule, and performing a biological assay comparing their starting material to their product. More specifically, this experiment involves the search for a “new” antibacterial agent. As an introduction to the experiment, students are taught about pathogenic bacteria and the potential health threat it poses. Students are then directed to use the natural product acetylsalicylic acid (aspirin) as a lead molecule. Next, students are guided through a chemical synthesis where they transform the lead molecule aspirin into methyl salicylate, more commonly known as oil of wintergreen. Unlike previously published articles on the synthesis of oil of wintergreen from aspirin,6,7 this experiment further investigates the starting material and product. On the basis of observations of physical properties before and after the synthesis, students see evidence that their reaction was successful and that their molecule has changed (solid to oil, bitter smell to minty smell, white color to colorless). The students then grow bacteria in the presence of the two different molecules and observe the effect each molecule has on bacterial survival by measuring the area around the molecule where bacterial growth is halted, also known as a zone of inhibition. Following the experiment, students convene to discuss their results and talk about the importance of those results in light of the drug-discovery process. When developing this experiment, the authors specifically chose the synthesis of oil of wintergreen from aspirin because

ncorporating a theme of drug discovery within a high school science curriculum provides opportunities for relevant experimentation and the potential to enhance student learning. It has been shown that including topics in the classroom that encourage connections between their lives and their science course content increases student interest.1 In addition, topics interesting to students result in increased motivation and academic performance.1,2 With this in mind, the authors sought to exploit the relevance and student interest of the medical field.3 As a result, an experiment was developed to incorporate various aspects of the drug-discovery process in high school chemistry or biotechnology classrooms. The discovery of new medicines is a complex process (Figure 1). First, scientists must recognize a medical problem and identify a target within the body, which, if hit, can lead to a treatment or cure. Chemists then synthesize and characterize molecules designed to hit the biological target. Small changes to these molecules may cause a large change in their therapeutic effectiveness. For this reason, an average of ten thousand new molecules are developed before an ideal drug candidate is found.4 Each newly made molecule must be tested in biological assays, animal testing, and human testing to ensure that the molecule is safe for use and that the desired effect is produced. Three of the drug-approval steps have been adapted for the high school classroom in this experiment.

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EXPERIMENTAL OVERVIEW

In the experiment presented here, students execute many roles of scientists within the drug-discovery pipeline: identifying a © 2013 American Chemical Society and Division of Chemical Education, Inc.

Published: November 18, 2013 1658

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discovery, which would require 20−30 min. If the experiment is run during a block schedule, it is possible to set up parts 1 and 2 on the same day. Analysis of antibacterial results could be completed on the following day. The experiment was performed with a typical class size of 30 students where students worked in groups of two. Further implementation strategies are available in the Supporting Information. Synthesis of Oil of Wintergreen from Aspirin Tablets

Students were provided with two tablets of 325 mg regular strength aspirin. After grinding the tablets using a mortar and pestle, students analyzed the physical properties of the powder, which would be compared with the product to verify a successful reaction. Next, students dissolved the active ingredient, acetylsalicylic acid, in methanol and filtered off the inactive ingredients. In the fume hood, students added two drops of the catalyst, concentrated sulfuric acid, to the test tube containing their sample, and placed it in a water bath set to 70 °C. After 30 min the transesterification, or Fisher esterification, reaction was complete and the sample was removed from the water bath (Scheme 1). The physical properties of this sample Scheme 1. Synthesis of Methyl Salicylate from Acetylsalicylic Acid

Figure 1. The drug-discovery process can be outlined in 5 main steps: discovery, lead molecule optimization, preclinical trials, clinical trials, and FDA review and approval.5

of its adaptability to the time, equipment, and chemical constraints of the high school classroom. Additionally, the drastic physical property changes observed provides confidence in the success of the reaction. Upon observation of smell, students recognize the familiar wintergreen scent further contributing to the relevance of the experiment to the students’ lives. Although oil of wintergreen is popularly known as a flavoring agent in gum, mints, and soft drinks, this experiment allows the students to “discover” the antibacterial properties of oil of wintergreen and speculation of its medicinal use in mouthwashes and topical analgesics is discussed.

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EXPERIMENTAL DETAILS

Materials

Methanol, sulfuric acid, salicylic acid, and ethanol were purchased from Sigma-Aldrich. Filter paper, large (90 cm diameter) Petri dishes, agar, and 6-well plates were purchased from Flinn Scientific. Aspirin (regular strength 325 mg tablets, noncoated, nonbuffered), cotton swabs, and bleach were purchased from a local grocery store. Gloves were purchased from the local hardware store. Mortar and pestles were purchased from Flinn Scientific. Bacteria stab used for making preplated lawns was provided by ScienceBridge.8 Paper discs were made from standard filter paper using a 1/2 in. hole punch. Sterile water was purified using a mega-pure water purification system from Flinn Scientific. The experiments require a fume hood, water bath, and a culture incubator.

were evaluated as the product. To fit into the time constraints, equipment availability, and approved chemical list of the school district, the lab was adapted accordingly from previously published procedures.9 Antibacterial Testing

Students were provided with agar plates and preplated colonies to create their own bacterial lawn, as well as solutions of aspirin and salicylic acid for preparation of paper discs for antibacterial testing. Students prepared their samples by placing 2−3 drops of these solutions onto the filter paper discs.10 While allowing the discs to dry, students created a lawn of bacteria on large agar plates using a cotton swab and spreading a colony of pregrown Escherichia coli bacteria onto their sample plate. After allowing 5 min for the paper discs to dry, students placed the paper discs onto appropriately labeled quadrants and placed them in an incubator set to 37 °C overnight. On day two, students took the agar plates out of the incubator and used a ruler to measure the zone of inhibition of samples from the paper discs.

Logistics

The authors suggest spreading out the experiment over three class sessions. Day 1 would cover an introduction and the synthesis of oil of wintergreen from aspirin tablets, which would require a 50 min session. Day 2 would involve the setup of bacterial plates for the biological assay, which would require 20 min. Day 3 would be reserved for the analysis of antibacterial results including a group discussion of the importance of drug 1659

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HAZARDS Sulfuric acid is corrosive and must be handled with care. Methanol is extremely flammable and toxic by inhalation, ingestion, or absorption through the skin. Therefore, gloves should be used. Acetylsalicylic acid and methyl salicylate are irritants. Bacteria strains used should be harmless, and students should be instructed to wash hands thoroughly after handling. Heating of methanol results in evaporation and should be carried out in a fume hood. All procedures should be carried out wearing safety glasses.

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RESULTS AND DISCUSSION

Comparing Physical Properties between Starting Material and Product

Figure 2. A sample student plate illustrating the antibacterial effects of the negative control, evaporated ethanol (upper left quadrant), the positive control, salicylic acid (upper right quadrant), the starting material, acetylsalicylic acid (lower right quadrant), and the reaction product, methyl salicylate (lower left quadrant).

The reaction of aspirin (acetylsalicylic acid) to form oil of wintergreen (methyl salicylate) is a convenient synthesis utilizing relatively common chemical reagents, heat, and a sulfuric acid catalyst. By analyzing the physical properties before and after the reaction, students were able to observe notable physical changes between starting material and product (Table 1). From class discussions and pre- and postlab questions,

properties. In completing their observations and participating in useful discussions, students came full circle in mirroring the processes used in drug discovery. Through further discussion, students were directed to analyze the active ingredients from the labels of hygienic products such as mouthwash and topical analgesics and were able to observe and discuss standard uses for methyl salicylate (Figure 3).11

Table 1. Sample Student Observations of Physical Properties before and after Chemical Synthesis Physical Property To Observe

Observations of Starting Material: Acetylsalicylic Acid (aspirin)

Observations of Product: Methyl Salicylate

Smella Color State

Bitter White Solid

Minty Colorless Liquid

a

The example of smell as a physical property is provided for the students, and they are responsible to fill in the others.

students studied the synthesis and were able to observe that small changes in a molecule can lead to substantial differences in physical nature. As students observed, acetylsalicylic acid was a white solid exhibiting a bitter odor and methyl salicylate was a colorless oil with a minty odor, indicating a successful synthesis. The synthetic procedure allowed students to play the role of a medicinal chemist in the search for new potential drugs.

Figure 3. Students discuss the use of methyl salicylate (oil of wintergreen) in common products. One example used is looking at the active ingredients of antiseptic mouthwash.

Testing Oil of Wintergreen for Antibacterial Activity

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The biological assay was performed by plating the starting material (acetylsalicylic acid or aspirin), the product (methyl salicylate or oil of wintergreen), a positive control (salicylic acid, a known antibacterial substance), and a negative control (evaporated ethanol exhibiting no antibacterial effect) to show a comparison between antibacterial and nonantibacterial substances.9 After incubation, the students were able to observe whether a substance was antibacterial through the absence or presence of a zone of inhibition, or a clear area where bacterial growth is inhibited around the impregnated paper discs.4 Aspirin is not antibacterial and will therefore exhibit no zone of inhibition. Oil of wintergreen, however, is antibacterial and will exhibit a clear ring around the paper disc soaked with the compound. The diameter of the zone of inhibition can be compared to known antibiotics and will be proportional to the strength of antibacterial activity. In comparison with the positive and negative controls, students observed that oil of wintergreen killed bacteria, whereas aspirin did not (Figure 2). Students also compared data between groups to conclude that they created a new molecule with different medicinal

CONCLUSIONS The integration of medicinal chemistry in the high school classroom can be accomplished by taking students through various steps of the drug-discovery process. In this way, students are exposed to a real-world approach to science and to the integration of chemistry and biology so commonly found in many aspects of their lives. Through the use of a topic relevant to students’ lives, this experiment serves to promote interest and consequently improve student performance and achievement.1,12 Students were receptive to this laboratory. More than 400 high school chemistry students completed the lab. These students were sophomores, juniors, and seniors in college-prep general chemistry and honors chemistry courses. All but a few student groups successfully obtained the oil of wintergreen at the completion of the synthesis. Additionally, the majority of the students obtained the expected biological results. Following the experiments, students were asked to fill out a survey to gauge student interest and understanding. Overall, 67% of the 1660

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students surveyed found the experiment to be exciting.13 Students provided an average ranking of 3.65 out of 5.0 for the ability of the experiment to keep them engaged, providing further evidence of student interest. Inquiring about general understanding, students were asked what they thought they were supposed to learn from this science experiment, and 88% of students gave correct, relevant responses. The results of the survey reflected the influence of interest in overall student understanding. Unfortunately, more data is required to say with confidence that a positive correlation is found between student interest and understanding in this particular experiment. However, it has been shown that topics maintaining high levels of student interest, as observed here, consequently result in an increase in student achievement and future recall.14

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(8) Sciencebridge kit request. http://sciencebridge.ucsd.edu (accessed Oct 2013). (9) Although the sample is not purified, the physical properties after the synthesis are sufficient for validating that a new product has been made (Table 1). See implementation strategies section of the Supporting Information for adaptations from previously published procedures. (10) The sample solutions are made using ethanol and the product is diluted in ethanol after the reaction is complete, before antibacterial testing is performed. (11) To promote further student discussion and for more information, see powerpoint slides (available upon request). (12) Schwartz-Boom, R. D.; Halpin, M. J.; Reiter, J. P. J. Chem. Educ. 2011, 88 (6), 744−750. (13) Survey questions can be found as Science Activity Feedback Form in the Student Handout section of the Supporting Information. Of 123 students surveyed, 82 answered ‘Yes’ to the question “Do you feel the activity contained a “WOW” factor?” (14) Sandoval, J. Ann. Rev. Psychol. 1995, 46, 355−374.

ASSOCIATED CONTENT

S Supporting Information *

Material list, teacher preparation instructions, implementation strategies, student handouts, answer keys, and additional information. This material is available via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Present Address §

E.G.S.: Department of Chemical Sciences, Bridgewater State University, Bridgewater, Massachusetts, 02325, United States. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Jewyl Clarke’s high school chemistry students at Eastlake High School in Chula Vista, CA for their willingness to experiment with new procedures in the classroom. The authors also thank Thomas Hermann for support and use of lab space and ScienceBridge at UC San Diego (UCSD) for supplies and procedural assistance. The HASPI program is acknowledged for support of the Medical Chemistry class. Research was funded by the Socrates program at UCSD, a GK-12 grant (NSF 0742551). Special thanks also to Maarten Chrispeels and the Socrates 2010-2011 cohort and staff, especially Shelley Glenn Lee and Johnnie Lyman for their support and discussion throughout the development of the experiment.

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REFERENCES

(1) Hulleman, C. S.; Harackiewicz, J. M. Science 2009, 326 (5958), 1410−1412. (2) Kwiek, N. C.; Halpin, M. J.; Reiter, J. P.; Hoeffler, L. A.; Schwartz-Bloom, R. D. Science 2007, 317 (5846), 1871−1872. (3) Jenkins, E. W.; Nelson, N. W. Res. Sci. Technol. Educ. 2005, 23 (1), 41−57. (4) Turner, J. R. New Drug Development: Design, Methodology, and Analysis; J. Wiley & Sons: Hoboken, NJ, 2007. (5) Note that FDA review and approval takes place throughout the drug-discovery process, but has been simplified in this figure for the high school classroom. (6) Zanger, M.; McKee, J. R. J. Chem. Educ. 1988, 65 (12), 1106− 1106. (7) Hartel, A. M.; Hanna, J. M. J. Chem. Educ. 2009, 86 (4), 475− 476. 1661

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