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Synthesis and Biological Testing of Penicillins: An Investigative Approach to the Undergraduate Teaching Laboratory Ragnhild D. Whitaker, Laura M. Truhlar, Deniz Yuksel, and David R. Walt* Department of Chemistry, Tufts University, Medford, Massachusetts 02155 *
[email protected] Mark D. Williams Department of Biology, Morehouse College, Atlanta, Georgia 30310
Most experiments in introductory undergraduate organic chemistry laboratories are based on following experimental procedures and obtaining an expected result, requiring a minimal degree of analysis and little critical thinking. This type of laboratory teaching format is widely used, but recently, emphasis has been placed on bringing a research-like environment to teaching laboratories, enabling students to develop critical-thinking skills, experience the atmosphere of a scientific research laboratory, and participate in more discovery-based research (1-4). In addition to the pedagogical advantage, research-based teaching laboratory experiments increase students' interest in scientific research (4, 5). A two-session laboratory experiment is presented that provides students with a discovery-lab experience that simulates realworld research. Experiment Development Two undergraduate students (coauthors L.M.T. and M.D.W.) were engaged in research to develop an undergraduate organic chemistry laboratory experiment with the goal of simulating reallife research. A project was selected that would resonate with the lab students in terms of having both a practical and positive outcome. In addition, the authors wanted the lab students to perform integrated interdisciplinary research spanning across traditional scientific boundaries, which is not typically accomplished in the undergraduate laboratory. With these goals in mind, a laboratory experiment that combined organic synthesis and biological testing of a library of penicillins was designed and developed. The logistics of the research and development of the lab are described in the supporting information. The experiment was further improved and refined by implementation in the organic chemistry laboratory class. Experiment Penicillins belong to the β-lactam class of antibiotics and are used to treat many Gram-negative and some Gram-positive bacterial strains (6-9). All penicillins have a common structural backbone (Figure 1 and can be synthesized using standard organic synthesis methods (10, 11) or enzymatic synthesis (12, 13). To overcome antibiotic resistance in bacteria and to increase antimicrobial activity, new penicillin derivatives have been steadily developed since the first resistant strain was discovered, mostly by 634
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changing the side chains of the penicillin nucleus to yield different penicillin derivatives (14-17). In the experiment, small subgroups of a larger organic chemistry laboratory class each synthesized a penicillin derivative through acylation of 6-aminopenicillinic acid (Scheme 1) (18-21). Fifteen acyl chlorides were available to the students, allowing each subgroup to choose one. The library of penicillins created by the class was subsequently tested in a bioassay to determine their efficacy (22-24). A broth-dilution method was used to estimate the IC50 (the concentration of penicillin that inhibits growth of 50% of bacteria in vitro) of each penicillin in the library. The synthesized penicillins were tested using Staphylococcus aureus Rosenbach (ATCC 25923), a Gram-positive bacterium. The viability of the bacteria was assessed by measuring the optical density (OD) of the bacterial broth at 600 nm after overnight incubation with different concentrations of the synthesized penicillin. By pooling and comparing results from the entire class, students were able to group all the penicillins according to efficacy (high, medium, and low or none) and to discuss structurefunction relationships based on the penicillins' effectiveness at inhibiting bacterial growth. The attractive feature of this experiment is that there was not a predetermined outcome. Furthermore, by including penicillins that are not commercially available, the experiment mimicked a drug-discovery process with the possibility to create and identify new penicillin derivatives. Implementation of the Experiment The penicillin synthesis and biological testing experiment was implemented over two sessions in the organic chemistry undergraduate laboratory. The synthesis was performed in the first session of the laboratory. The students could choose to synthesize commercially available penicillins or compounds that had not been previously reported. The resulting products were tested one week later during the next laboratory session. The logistics of the bioassay proved to be more complicated than the synthesis. Pregrown bacterial cultures were diluted and exposed to the synthesized penicillins at a minimum of four concentrations ranging from 0 to 1000 μg/mL, including an uninhibited control sample (0 μg/mL penicillin). Here, the students were introduced to the importance of control experiments in
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In the Laboratory
Figure 1. The penicillin backbone; the R group is varied to create a library of penicillin derivatives. Scheme 1. Acylation of 6-Aminopenicillinic Acid To Form the Penicillin Derivatives
working with 6-aminopenicillanic acid, acetone, n-butanol, n-butyl acetate, potassium 2-ethylhexanoate, sodium bicarbonate, sodium sulfate, and sulfuric acid are also included in supporting information. S. aureus Rosenbach (ATCC 25923), a bacterium classified as biosafety level (BSL) 2 (25) was used to test the penicillins. BSL-2 bacteria present a moderate risk of infection and should be handled under BSL-2 guidelines from the Center for Disease Control (26). In addition to general safety precautions (goggles, gloves, lab coat), the students wore disposable aprons and worked close to flames from Bunsen burners to minimize aerosols when working with bacteria. All materials that had been exposed to S. aureus were disposed of as biowaste (see the supporting information). Conclusion
discovery-based research;something they do not usually learn in typical organic chemistry experiments. The control samples were used to identify a positive or a negative result. A penicillin having no antimicrobial effect (negative result) should have a measured OD600 similar to the uninhibited control sample, whereas a penicillin having maximal antimicrobial effect, resulting in no bacterial growth (a highly positive result), should have a measured OD600 close to that obtained when the sterile broth alone is measured. Any OD600 measurement between these two extremes indicates varying levels of antimicrobial activity. To avoid requiring students to come to the laboratory on two consecutive days, the students in each laboratory session performed the OD measurements for the group from the previous day. Concern was raised about this implementation because each person was not responsible for his or her own sample. To the contrary, we found that this solution created the impression that all the lab groups were working together toward a common goal;a characteristic of many research projects. This implementation demonstrated that sometimes in research one might not be able to follow a specific sample from synthesis through analytical testing. Upon reading all samples, each student plotted the OD values at the different penicillin concentrations using an Excel spreadsheet. After all the laboratory subgroups had obtained data from their own samples, data from the entire class were shared. A linear trend line was added to the individual plots, and R2 correlation values and equations for the trend lines were obtained. The students used these results to calculate the IC50 for each penicillin derivative. The students were then asked to group the different penicillins according to efficacy (high, medium, or low or none based on their IC50). Students were also asked to discuss any structure-function relationships that could be discerned about the different side chains from the efficacy of the penicillins.
The two-session laboratory experiment was successfully implemented in the undergraduate organic chemistry laboratory. The students synthesized a library of penicillins, tested these penicillins for antimicrobial activity, grouped them according to their effectiveness at inhibiting microbial growth, and finally determined chemical structure-function relationships of the various acyl side chains based on the biological effectiveness of the penicillins. The students reported that they found it exciting to work on a research-based laboratory problem and felt as if they were engaged in actual research where the answer or result was unknown. This laboratory experiment and all materials needed to implement it are available in the supporting information. The acyl chloride library can be expanded or shrunk to better fit the class size, and the data reporting and analysis can be altered to fit the needs of the institution. The experiment can also be modified to contain other antimicrobial agents, for example, cephalosporin antibiotics (27), allowing for expansion of the experiment and enabling added discovery-based research. Further discussion about implementation of this aspect of the laboratory experiment is available in the supporting information. Acknowledgment The research and activities described in this article were supported by a grant to Tufts University in support of David R. Walt from the Howard Hughes Medical Institute through the HHMI Professors' Program. The authors are grateful for the help and insightful feedback from the students and the teaching staff, in particular Sarah Iacobucci and Robert Stolow. The authors are also grateful for the support provided by program coordinator Meredith Knight. Literature Cited
Hazards Hazards regarding all materials used in these experiments are included in the student handouts (see the supporting information). Most of the acyl chlorides are corrosive and cause burns. Several acyl chlorides are harmful, and some are mutagens or toxic. A complete list of the hazards for the 15 acyl chlorides is provided in supporting information. Hazards involved with
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Supporting Information Available Laboratory handout and data sheet for penicillin synthesis and penicillin testing; student research report from development of penicillin synthesis and bioassay; notes for the teaching assistants; notes for the instructor; list of materials, CAS numbers, and hazards. This material is available via the Internet at http://pubs.acs.org.
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