Peptidomimetics from the Classroom to the Lab: Successful Research

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Chapter 9

Peptidomimetics from the Classroom to the Lab: Successful Research Outcomes from an “Upper-Level” Class at a Primarily Undergraduate Institution Danielle A. Guarracino* Chemistry Department, The College of New Jersey, 2000 Pennington Road, Ewing, New Jersey 08628, United States *E-mail: [email protected].

One of the challenges at a primarily undergraduate institution (PUI) is to afford meaningful research experiences to all students in the chemistry curriculum. With an ever-changing student population and those wishing to “try on” research, our department has creatively increased inclusion of all students in research through several possible upper-level classes. I will describe the incorporation of research into my Advanced Topics Chemical Biology course. Early on, students randomly choose a peptide from a list of new designs provided by the instructor. Throughout the semester, in the classroom and lab, they learn how peptides are made, synthesized and characterized, gaining experience at the bench. Near the conclusion of the course, they learn spectroscopic techniques to examine the secondary structure of their peptides. By this point they know the sequential and structural elements contributing to protein folding, what stabilizes natural and unnatural peptides, and can predict how their chosen peptide should behave. After testing the compounds, they describe the results in relation to their prediction, and compare across teams to determine the links between sequence and folding. Students write a communication-style final paper that encompasses their work. The lecture portion of the course has students applying the skills of chemical biology to novel problems. They prepare a final © 2018 American Chemical Society Gourley and Jones; Best Practices for Supporting and Expanding Undergraduate Research in Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 2018.


presentation on a unique area within epigenetics, teaching the class and writing a question for their final. In both iterations of the class that I have taught we were able to publish our results in peer-reviewed journals with the students as authors. Overall, students enjoy the course and materials and I plan to include some new areas of inquiry into future versions of the course, continuing this positive trajectory.

Development of the Course Advanced Topics in Biochemistry: Chemical Biology The College of New Jersey is a public liberal arts college located in central NJ across 289 acres. It is ranked as the number one public institution in the northern region of the country by U.S. News & World Report and boasts approximately 7,000 full-time students. The Chemistry Department is ranked in the top 4% of chemistry programs nationally in graduating American Chemical Society (ACS) certified bachelor degrees. Our chemistry department is “undergraduate only” and has about 110-160 chemistry majors, 12 full time faculty and approximately $1 million of research grade instrumentation. As the numbers of students interested in research is steadily increasing, we as a faculty, in conjunction with the standards of ACS certification, have developed a series of advanced topics classes for interested junior and senior students. All chemistry majors need to take 2-3 advanced topics classes for the ACS-certified degree and we offer a range of interesting options across the analytical, biochemical, inorganic, organic, and physical subdisciplines. In addition, we have had exciting forays into materials science, food science, forensics, and instrumentation, as well. I have twice taught the Chemical Biology course I designed, once in 2013 with 13 students and again in 2015 with 12 students. The course meets for two 80 minute lectures a week and one 3 hour lab and has the co- or pre-requirement of a biochemistry course (with a laboratory). I have built in several research components into the laboratory and lecture portions of the course across the general topics covered. Chemical Biology is a broad discipline that is defined as the use of chemical techniques and tools applied to understanding and controlling a biological system including fixing or altering a biological problem. In designing my course, I took inspiration from the topics covered in the Chemical Biology courses I took for my graduate work and prepared my own materials, updating and including interesting modern ventures in the field. The first course objective we go through in lecture is synthetic biology and biological synthesis, covering an array of techniques available. To connect the lecture and laboratory components, students perform solid phase peptide synthesis of short novel peptides by hand in the lab, and also learn how to synthesize non-natural β3-amino acids through several synthetic steps. This hands-on work ties into their second course objective, which is the study of peptidomimetics and disruption of protein-protein interactions. The third course objective covers common techniques and assays used in Chemical Biology, and the lecture covers several that, when brought to the lab, make use of 144 Gourley and Jones; Best Practices for Supporting and Expanding Undergraduate Research in Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 2018.


our instrumentation. The students use high performance liquid chromatography (HPLC) to assess the purity of their synthesis of their peptide and β3-amino acid, the liquid chromatography mass spectrometer (LCMS) to determine the identity of their compounds based on mass, and then proton nuclear magnetic resonance (1H-NMR) spectroscopy on the β3-amino acid for further identification. As part of this area, we discuss the bridge between biophysical characterization of peptidomimetic folding and methods that assess the disruption of protein-protein interactions. In the laboratory we focus efforts on predicting and assessing peptide folding by examination of their compounds by circular dichroism spectroscopy. And finally, the fourth course objective is the study of genomics and proteomics, modern methods and information. While we have not covered this “hands on” in the lab, students perform a final literature research project on epigenetics. By assimilating all of these topics into cohesive “research” projects, the entire class becomes a team working towards the same goals, each contributing a portion of new and interesting materials.

Begininng the Process: Laboratory Research Goals When the class premiered in 2013, I asked a very specific lab question, answerable by the work the students would perform: do idealized α-helical and 14-helical sequences translate to opposite molecules? There are specific sequences in proteins that have been found to confer α-helicity, the most common form of secondary structure, to the peptides that are comprised of these particular amino acids (1, 2). With that concept in mind, I designed one hexa-, one septaand two octa-peptides with helix-promoting sequences, which would segregate oppositely charged, polar groups from hydrophobic groups upon peptide folding (Figure 1, peptide sequences). The use of leucine as a hydrophobic group might also lead to a possible leucine zipper, across two helices, indicating higher level structure among the folded groups (Figure 1, PT3) (1). PT4 replaced those leucines with alanines to see what the effects would be to overall helicity (Figure 1, PT4). PT2 incorporated two non-natural aminoisobutyric acid (Aib) residues that have been known to positively influence helical structure (Figure 1, PT2) (3). The field of β-peptides emerged in the past two decades as non-natural folding oligomers. β-peptides, which are comprised of β-amino acids with an extra carbon along the amino acid backbone, can fold into what is known as the 14-helix when substitution is on the third carbon (Figure 1) (4, 5). The 14-helix resembles the α-helix however its notable differences include left-handedness, a reversed macrodipole placing a net partial negative charge on the N-terminus, and the approximate three residues per turn. It has been shown that arranging the three faces of the helix such that one has hydrophobic groups (usually valines that can interdigitate), one has salt-bridging (in line with the reversed macrodipole), and the final one is kept free for substitution, led to the development of an ideal peptidomimetic scaffold (7–9). Therefore, in the interest of creating the smallest possible motifs for comparison, BP1 represents the smallest scaffold of an idealized β-peptide, the minimal 145 Gourley and Jones; Best Practices for Supporting and Expanding Undergraduate Research in Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 2018.


epitope for binding (Figure 1). BP2-4 represent the beta equivalents of the alpha PT2-4 peptides, to test whether folding of sequences can translate between α- and β-peptides. PT1, tests this in the opposite direction; does an idealized 14-helix still have any relevance when the sequence is placed in an α-peptide? Students who worked on these peptides examined not just whether their peptide would be helical, but also the relationship of similar sequences in different types of peptides, whether it led to helicity. There was much comparing across groups.

Figure 1. Top: The four α-peptides, depicted in helical-wheel format with hydrophobic and charged residues highlighted. Middle: structures of the aminoisobutyric acid amino acid and a representative β3-amino acid. Bottom: The four β-peptides, depicted in helical net diagrams with hydrophobic and charged residues highlighted. Reprinted with permission from ref. (6). Copyright 2015 Taylor & Francis.

In 2015, on the heels of our inquiries from 2013, I instead branched the projects into three different directions. The first was taking the most folded α-peptide from the 2013 lab, PT3, and changing the potential salt bridging groups, examining different pairs. The original used lysine and glutamate arrangements, but now PT3R replaced lysine with arginine, whereas PT3D replaced glutamate with aspartate (Figure 2). Students who worked on these peptides would make comparisons to the original peptide as well as to each others’, and would research what might make one salt bridge more effective than another in helping a peptide fold (9–12). 146 Gourley and Jones; Best Practices for Supporting and Expanding Undergraduate Research in Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 2018.


Figure 2. Left: The α-peptides with potential salt-bridging residues shown. Right top: The new β-peptide scaffolds. Right bottom: β-turn/β-sheet scaffolds. Reprinted with permission from ref. (13). Copyright 2017 Taylor & Francis. Additionally, on the heels of BP1’s success as a highly 14-helical, short βpeptide, I had other teams of students look into the idea of pi-pi stacking as a possibility for helping the 14-helix fold, in place of the valines, in peptide BP1Y (Figure 2). Additionally, on the helical face left free for substitution, I had them change the residues to leucines in an effort to target a particular protein-protein interaction that contributes to angiogenesis in certain cancers. The Hif1α helix has key leucines on one face of its structure, which mediates its binding to protein 147 Gourley and Jones; Best Practices for Supporting and Expanding Undergraduate Research in Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 2018.


CH1 (Figure 2, BPYHif) (14). Here, students would test if changing the residues of the scaffold, and attempting a new form of helical control, retained helicity, and if so, the helix could be further used in the future as a potential inhibitor. Finally, the last set of peptides were an α- and a β-peptide with same sequence, designed as a βturn/β-sheet model utilizing D-proline and glycine as turn initiatiors, threonine and tyrosine as potential intra-sheet points of interaction (HPA and HPB, respectively, Figure 2) (15–17). Students examining these would learn about spectroscopy signatures that indicate turn and sheet formations, as well as whether these minimal motifs can actually fold, followed by the comparison, again, between α- and βpeptides. The process begins with a “blind” sign-up, the first day of lab class. In the blind sign-up, each lab partnership chooses a β3-amino acid to synthesize, an αpeptide to synthesize and an α- or β-peptide to study with CD. The synthesized peptide and the peptide studied by CD may not be the same one for each group, which simply makes them study a larger variety of interesting compounds. I premake the peptides in question and purify about half the stock prior to the start of the semester, to ensure that there is enough peptide for them to perform the CD studies; their syntheses during the course are by hand and with the limited time may not yield much. Also, this ensures that there is compound available should they lose some as they work. I do not have them synthesize β-peptides in class as the building blocks are so precious. In the end, students choose their compounds of study in a random fashion, which works well as they have no preconceived notions as to whether their peptide will be “good” or “bad” at folding. This also makes for a fair distribution throughout the class; no one is sure which teams will have the “best” compounds. At this point, they cannot predict much since the choices are made at the beginning of the semester. Throughout the semester they will learn the contributing factors to folding, helicity, and sheet formation and will study peptidomimetics in the literature that were successful. From this, they can make predictions. Overall, their choices for β-amino acid and peptide synthesis will replenish our stocks. These processes will teach them how unnatural amino acids and peptides are made and can lead to further study.

Details of the Lab Modules Module 1: Synthetic Chemistry In the first module, which lasts about four lab periods of three hours each, students perform three synthetic steps to take a purchased α-amino acid, with necessary protecting groups on the N-terminus and side chain to a protected β3-amino acid (Figure 3). The steps can be broken into two reactions, the Arndt-Eistert homologation, which features the use of a specially-made glassware apparatus and the generation of the potentially explosive diazomethane, followed by the Wolff rearrangement, which, mechanistically, occurs via an interesting carbene intermediate (18). Between each step, students perform column chromatography to purify their product for the next reaction. As each team is working with a different β-amino acid, they likely experience unique polarities 148 Gourley and Jones; Best Practices for Supporting and Expanding Undergraduate Research in Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 2018.


and slightly differing results, therefore, while performing the same steps, their outcomes can differ and they need to use their insight to determine how to best purify their compounds. Additionally, in this module, students work on the solid phase peptide synthesis of their chosen peptide. Here, more than one group may have signed up for the same peptide, since they only work synthetically with the α-peptide designs. The general process has taken place with iterative steps by hand in a fritted syringe reaction vessel and is accomplished across three lab periods (Figure 3). Students complete the repetitive steps of deprotection and coupling protected amino acids, and end with capping the peptides with an alkyl group and cleaving them from the resin beads, followed by ether precipitation. The final products of both the β-amino acid and the α-peptide are freeze dried to a powder or oil prior to the next steps.

Figure 3. Top: Synthetic scheme to make an Fmoc-protected β3-amino acid. Bottom: Synthetic steps for solid phase peptide synthesis. 149 Gourley and Jones; Best Practices for Supporting and Expanding Undergraduate Research in Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 2018.


Module 2: Instrumental Analysis For the following steps, students analyze the results from their earlier work, identifying and purifying their β3-amino acid and α-peptide. In one laboratory period, students will prepare small samples of their protected β-amino acid in inert solvents such as deuterated chloroform and will perform 1H-NMR spectroscopy. A full analysis of chemical shifts, splitting patterns and comparisons to expected outcomes are performed by each team in regard to their unique amino acid. In another lab period, each team prepares a small sample of β-amino acid and, separately, their α-peptides for HPLC and LCMS analysis by dissolving them in a mixture of acetonitrile and water. The HPLC used is from the Agilent Technologies 1260 Infinity series and a semi-preparative column (Grace Vydac, 218TP C18, 250 mm× 10 mm, 10–15 μm) was used. Analysis uses a gradient of approximately 5% acetonitrile in water to 95% acetonitrile in water over thirty minutes. Analytical HPLC is used to estimate the overall percent purity for their reactions in the preparation of both compounds. Additionally, the LCMS provides the students with a look into the accuracy of their synthesis of both compounds, helping them identify the peptide and β-amino acid by mass and what other impurities might be present (e.g. a peptide chain missing an amino acid, or a side product). For mass spectrometry, they use the Agilent Technologies 1260 Infinity with 6130 Quadrupole Liquid Chromatography Mass Spectrometer. Each sample is passed through the analytical column (Agilent poroshell 120, EC-C18, 2.7 μm, 4.6 mm × 50 mm). In a third lab period of this module, students receive the purified peptide I had prepared that they signed up for the first week. Here, they prepare buffer, stocks and dilutions for the upcoming spectroscopy experiments. Prior to lab, students use the online “protein calculator” from the Scripps Research Institute and input their sequences to get a molar absorptivity, ε, in M-1cm-1 for their peptide at 280 nm. In class, they prepare the provided samples and use UV/Visible spectroscopy to obtain an absorbance for their stock concentration, also at 280 nm. Therefore, using Beer’s Law (A = εcl) they can equate the absorbance they achieve (A) with the molar absorptivity they determined prior to class (ε), and the pathlength of the cuvette they use (l, usually 1 cm) to determine the concentration of their peptide in molarity (c). From here they perform a range of serial dilutions of their peptide from higher to lower concentration, in duplicate, so they will be able to examine the effects of concentration on their CD signal, over a range of values. Once prepared, all of their samples are refrigerated, with their crude samples frozen to replenish the stocks, and their purified (provided) samples in diluted form ready for spectroscopy. Module 3: Biophysical Characterization of Peptide Folding Using CD Spectroscopy In the third module, students focus on using CD spectroscopy to assess the degree of secondary structural pattern for their chosen peptide. Students learn about circular dichroism, how circularly polarized light travels through a chiral medium, such as a peptide in buffer, tracing out an ellipse versus a circle, due 150 Gourley and Jones; Best Practices for Supporting and Expanding Undergraduate Research in Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 2018.


to the different abosorbance of left- and right-hand components of the light. Alpha helices have a well described observable pattern with a double minmum in ellipticity found around 208 nm and 222 nm, and a calculatable maximum expected ellipticity based on the number of amino acids (19). 14-helices are generally described by a minimum ellipticity around 214 nm with a maximum in the 195 nm region. There are assumptions made about percent 14-helicity, such as fingerprints for short versus long sequences, however a definitive calculation about maxima is not established as for α-helices (8). The small folded turns, which resemble β-sheets, have a matachable signature as well; minimum ellipticity broadly around 216-218 nm and a maximum around 200 nm (20). Students begin this module with a solid background of the factors in peptide amino acid sequences that affect helicity and secondary structure. Therefore, their first activity is to revisit the peptide they chose the first week of lab and, based on its design, hypothesize if they think it will be helical and why, or why not. Additionally, they will discuss whether they think there are quaternary structures forming, therefore, whether they will see a concentration dependence in their CD trials. Also, as the work will contain a thermal denaturation scan as well, they will hypothesize whether their peptide should exhibit a cooperative melting transition or if it simply is not folded well enough to view this. This information becomes a part of their pre-laboratory assignment that is graded individually per student, out of ten points towards their final laboratory grade. Students accumulate data on their samples directly using our Jasco J810 spectropolarimeter. The raw data simply lists the CD readout for each wavelength assessed and students process it for each of the samples they measure. Each team typically has six samples, three different concentrations of peptide in buffer in duplicate for statistical purposes. Students calculate the mean residue ellipticity (MRE) by normalizing their data with a buffer blank, dividing by the number of amino acid residues, the concentration of their peptide in μM and the pathlength of the CD cuvette (8). In their teams, students work to best plot their data and draw conclusions. Their final reports are graded in their teams, with approximately half of the twenty points devoted to their write up of the results and the methods. The other half of their reports were dedicated to a full background description of what they were using CD to accomplish, what the data obtained explains and how it relates to their earlier hypotheses. I impressed upon the students that whether they saw helicity (or some form of β-sheet) was not as important as if they could describe why they were or were not seeing it. With the different iterations of the course we obtained different outcomes, as to be expected. Each has led to exciting revelations that have opened the doors to new inquiry.

Outcomes Results from 2013 The initial work from class this first time it was taught had notable issues. The addition of trifluoroethanol (TFE) to buffer to aid helicity in studying small peptides is fairly common, but we had instead just tried a simple 1X phosphate 151 Gourley and Jones; Best Practices for Supporting and Expanding Undergraduate Research in Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 2018.


buffered saline (PBS) solution. Additionally, our peptides all had a free amine and carboxylic acid at the termini, however it is noted in the literature that capping the termini with an acetyl (at the N-terminus) and an amide (at the C-terminus) can aid in stability and folding (21, 22). Student results, therefore, had large error bars across the two trials, and also showed some unprecedented concentration dependence in the β-peptides (Figure 4). Therefore, to continue the work, even after the class had ended, I worked with three research students from my lab, one of whom had been in the class, and we resynthesized the peptides with the notable termini changes and brought them up in buffer containing TFE. Our results were later published in the Journal of Biomolecular Structure and Dynamics, and each student in the class was acknowledged for their early work on the peptides (6).

Figure 4. Uncapped PT3 (left) and BP1 (right) in 1X PBS buffer with no TFE, CD spectroscopy results across three concentrations.

Our results were important for shaping the course for the next time it was taught and also brought new insight into the field regarding primary sequence and secondary structure. Figure 5 shows how the most α-helical peptide indeed was PT3, designed to be most helical, and it did not show concentration dependence, therefore most likely was not forming a higher order structure such as the leucine zipper. PT2, with the Aib residues, and PT4, with the alanine replacement of leucine, were marginally helical whereas PT1, which transplanted the sequence most likely to form a 14-helix in a β-peptide into an α-peptide, showed no discernable helicity. Figure 6 shows the results from the β-peptides examined. Clearly, BP1 is most helical, as its general scaffold was designed as such, but BP2’s small unprecendented 14-helicity showed a bit of “cross-talk” between the α- and β-peptides. BP3 and BP4, both with the sequences most α-helical, showed no discernable helicity when placed in β-peptides. Therefore, general conclusions 152 Gourley and Jones; Best Practices for Supporting and Expanding Undergraduate Research in Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 2018.


indicated that there is little overlap between the sequences that form the best α-helices and those that form the best 14-helices. Interestingly, demonstrated in Figure 7, when each of the peptides with some evidence of secondary structure was heated in a CD melt, the α-peptides did not show much evidence of unfolding, most likely due to their low helicity to begin with, wheras BP1 showed the typical linear decrease seen for well-folded β-peptides. BP2, however, showed an odd gain in structure at increasing temperatures, until the final decrease (Figure 7). This is a pattern we would see again for one of the α-peptides studied in the future, and we hypothesized, at the time, that the peptide was not very folded and had gained the kinetic energy to sample the proper folding formation before being completely denatured.

Figure 5. PT1-4 (capped) CD spectroscopy results across three wavelengths in 1X PBS with 10% TFE. Reprinted with permission from ref. (6). Copyright 2015 Taylor & Francis.

With new, short sequences of peptides developed to initiate folding into αor 14-helices, I was armed with information for the next iteration of the course, where we built upon these designs to test some new and unique peptides. 153 Gourley and Jones; Best Practices for Supporting and Expanding Undergraduate Research in Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 2018.


Figure 6. BP1-4 CD spectroscopy results across three wavelengths in 1X PBS with 10% TFE. Reprinted with permission from ref. (6). Copyright 2015 Taylor & Francis.

Figure 7. Left: Summary of the thermal denaturation of BP1, BP2, and PT2-4. Right: Wavelength temperature scans for BP2. Reprinted with permission from ref. (6). Copyright 2015 Taylor & Francis. 154 Gourley and Jones; Best Practices for Supporting and Expanding Undergraduate Research in Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 2018.


Results from 2015

Figure 8. Top left and right are the CD spectroscopy results for PT3R and PT3D, respectively. Bottom is the CD spectroscopy result for HPA. Reprinted with permission from ref. (13). Copyright 2017 Taylor & Francis.

The outcomes from the second iteration of the class were more immediately informative, with some new insights into secondary structural folding. Both trials of each concentration from the α-peptide groups, working on PT3R, PT3D and HPA, demonstrated evidence of secondary structure (Figure 8). The arginine substitution into PT3 to make PT3R improved helicity, and is hypothesized to do so due to the ability of arginine to make complex salt bridges (23, 24). The aspartate substitution to yield PT3D did not appreciably change helicity. Thermal denaturation of these peptides was analyzed in class, giving a slightly cooperative unfolding to PT3R, which indicates its higher original helicity (Figure 9). Once again, however, an odd increase in helicity upon heating was seen for a peptide, this time PT3D. I, personally, performed a temperature/wavelength scan on this peptide (Figure 9) and saw the minimum at approximately 220 nm “grow in” from 10 °C to about 40 °C, before the denaturation occurred. Here, we hypothesized, 155 Gourley and Jones; Best Practices for Supporting and Expanding Undergraduate Research in Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 2018.


based on the shape seen, that this could indicate an intermediate between a 310 helix on its way to the α-helix, as the unique shape of the curve, with large negative MRE at approximately 205 nm indicates 310-helicity (25, 26). HPA, our first short α-peptide sequence designed to fold into a β-turn/β-sheet showed some proper CD signature to indicate this was occurring. This was an exciting possibility from which to build upon for a new scaffold in the future (Figure 8).

Figure 9. Left: Thermal denaturation of PT3R and PT3D, at ~220 nm and 100 μM concentration. Right: PT3D temperature wavelength scans across 70 degrees. Reprinted with permission from ref. (13). Copyright 2017 Taylor & Francis.

We prepared these results for publication, with the six students who worked on these particular peptides and summarized their results as part of class as contributing authors. Upon review of the submission of the follow-up paper for the Journal of Biomolecular Structure and Dynamics, it was suggested I examine the contributions from TFE across several percentages (Figure 10) and I saw that only 50% TFE truly exacerbates the α-helicity of PT3R, whereas 10% and 50% added helicity to PT3D, however not in as high a quantity. TFE is thought to either directly hydrogen bond with the helix to help stabilize it, or to weaken the interaction of the peptide with water, therefore allowing the intramolecular hydrogen bonds to form more readily (27). Additionally, it was suggested that I take a look at the effects of guanidinium hydrochloride (GuHCl), which breaks up salt bridges, on the helicity of the peptides without TFE (28). Any inference of structure was quickly removed with the presence of GuHCl, which was expected due to the importance of the salt bridging in the helical formations (Figure 10). These results, accumulated by myself outside of class, were added to the final paper and it was published in early 2017. 156 Gourley and Jones; Best Practices for Supporting and Expanding Undergraduate Research in Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 2018.


Figure 10. Top left: PT3R CD spectroscopy trials with varying TFE. Top right: PT3R CD spectroscopy trials with varying GuHCl. Bottom left and right: same for PT3D. Reprinted with permission from ref. (13). Copyright 2017 Taylor & Francis. However, as this was a research-based project for a class, not all data from every team was fruitful. The attempts to control β-peptide 14-helicity using tyrosine aromatic stacking led to a complete loss of 14-helicity (Figure 11). We hypothesized that too many changes to the scaffold were made and that, in the future, we can work off of these results and the initial ones for BP1 and try to build a different scaffold. Additionally, the HPB peptide did not show any evidence of β-sheet formation, more than likely not folding (Figure 11). As the α-peptides, however, gave repeatable and unique data, we did publish the results as a follow-up paper in the same journal as prior, with six student authors all from the class with their data and some analysis included.

Assessment of the Research Project Whether students would be published or not, properly predicted the results they achieved, were surprised by the results, or even received negative results, they each wrote a final paper at the end of the course using the Journal of the American Chemical Society (JACS) communication style. They were tasked with working in their lab teams to cover the entire semester’s materials, from synthesis to instrumentation to the biophysical analysis. This required students to find 157 Gourley and Jones; Best Practices for Supporting and Expanding Undergraduate Research in Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 2018.


common themes throughout their work, even if their β-amino acid and α-peptide did not coincide with the peptide they examined using CD. Also, it required students to discuss results across teams to make some generalizations about the sequence and structure connection. The final communication-styled paper was also an exercise in brevity as students used the proper JACS template and were only allowed as many figures, references and pages as a true communication. Therefore, they had to work on their quick communication skills and could not write everything from the full semester course but had to limit their reporting to what was imperative and important to include.

Figure 11. Top Left: CD spectroscopy results for BP1Y. Top Right: CD spectroscopy results for BPYHif. Bottom: CD spectroscopy results for HPB.

As mentioned earlier, we did successfully publish two research papers based on the work from each iteration of the course. While the first paper, published online in 2014, with full publication in 2015, reflected work begun in the 2013 class it was completed by myself and my research students so of the authors on the paper only one is a student from the class. However, our most recent publication, online as of January 2017, comes from results from the 2015 version of the course and features six students from the class, which amounted to half of the class! As 158 Gourley and Jones; Best Practices for Supporting and Expanding Undergraduate Research in Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 2018.

publication occurred long after the courses were completed, I kept that separate from any part of grading the students. Some of the students with top grades in the course were not actually authors on the paper, as the inclusion of students as authors had more to do with their successful work on the compounds they picked randomly, as well as their analyses, rather than their academic achievement.


“Research” Incorporated into the Classroom While much of the focus has been to discuss the laboratory outcomes of the course, in the interest of incorporating research aspects to the class across the board, the lecture portion of the course had a number of research aspects, as well. I taught from the primary literature, pulling on papers from the past couple of decades, including current work, to immerse the students in the field of Chemical Biology.

In-Class Assignments Throughout the semester, students had four in-class assignments where they would work in groups of 2-4 students and have about 20-25 minutes to answer open-ended questions based on the course materials. The best three out of four of these assignments would count towards their final grade, and they were generally out of 10 points with multiple, possible well-thought-out answers accepted. For example, in the 2015 edition of the course, students began with an assignment that revolved around a protein they had not heard about before, and were told to use a process they learned about, in the biological synthesis and synthetic biology section, to make up the protein, including the details of how they would use the process and all the steps. Then, they had to discuss the benefits of the process they chose. In a later assignment, students were provided a β3-peptide general scaffold with X written at all of the amino acid locations. They had just learned about a protein-protien interaction involved in the Severe Acute Respiratory Syndrome (SARS) virus fusion mechanism with a host cell and were tasked with designing two β-peptides that would inhibit this event, keeping in mind controlling both the helicity of the scaffold and recognition of the protein target. They needed to describe their reasons, as well, for placing the different residues where they put them on the scaffold. As this was actually unpublished work from my own graduate thesis, I was curious to see if they would come up with the very compounds I had made and studied, or if they would introduce new ones, which led to lively discussion! A third assignment involved students evolving an enzyme that could cleave a link between mRNA and puromycin in mRNA display, a technique covered in the common techniques and assays section of the class. With hints, I wanted them to focus on how to separate the mRNA that codes for the enzyme versus other, nonfunctional mRNAs in a library. Their final assignment involved yeast two hybrid screening, another assay and technique they had learned about. They were asked to describe what type of screen they would 159 Gourley and Jones; Best Practices for Supporting and Expanding Undergraduate Research in Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 2018.


choose, how it would react and how they would use it to generate an inhibitor to a particular well-studied protein-protein interaction involved in cancer. There were several options, leaving the answers open for students to combine assay and peptidomimetics work. These types of assignments were useful for students in that they applied knowledge from the course materials, posed novel “research” problems for students to work at solving, and also afforded them the opportunity to work in different groups. Often, I would hear student debating what they thought was a good answer, and why, and, as there were frequently more than one way to answer the questions, it was interesting to moderate such debates. Through this, students really “owned” the materials they were learning.

Literature Research and the Final Exam As a final project, students were given the task to work in their lab partnerships outside of scheduled class time and research the literature on a subtopic within the area of epigenetics. They signed up for a subtopic (i.e. environmental epigenetics, epigenetic therapeutics, epigenetics and inheritance, cancer epigenetics, epigenetics and stem cells and gene regulation and chromosome biology) on our course website, so each team had a unique area, and were tasked with giving a presentation on their topic the final week of class. Students were given 20-25 minutes to “teach” the class about their area, building off of the previous teams if necessary, and introducing a question that they would answer throughout their presentation. This question needed to be test-type in nature and would be included on their final, therefore each team was responsible for putting effort into their communication of the information as well as how the information would be assessed. Overall, their presentations needed to go through the general background of their subtopic, definitions, how the topic is approached technically, and the broader impacts of the topic, with several refernces from the primary literature. The test questions the students came up with were large in scope, ranging from “describe an advantage or disadvantage for a specific technique,” to “choose a therapeutic strategy and describe what it does,” and “which modification is best” for a specific aim, as well as “describe a mode and mechanism for epigenetic inheritance.” In addition, I created some multiple-choice and fill-in-the-blank type questions based on their work, and overall the epigenetics portion of their final was approximately 15% of the total covered materials. They so enjoyed taking the reins, I would like to increase this in the future! In both iterations of the course students showed an immersion into their literature research and delighted in hypothesizing where the field would go next. A couple of students, who continued on to graduate work after graduating from TCNJ, actually mentioned how the Chemical Biology course, and specifically their review of epigenetics, gave them direction in their graduate research in the field!

160 Gourley and Jones; Best Practices for Supporting and Expanding Undergraduate Research in Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 2018.


Grading the Course Overall, grading for the course was divided into several subsections. The research-inspired in-class assignments were 15% of the total grade (with the best 3 of 4 counting towards that), the two class exams, that pulled information from lecture from the primary literature, were 25% of the grade in total, the class presentation based on the literature research in epigenetics was 10%, the laboratory component, with its unique research-based structure, was 25% and the final exam, which the students participate in writing, was 25%. For the lab component, I did one notebook check during each module, looking for proper data collection, hypotheses and predictions, safety concerns described, and at the close of each module students wrote up a lab report. In the end, recall, students also submitted a final JACS communication-styled paper that summarized their entire semester’s lab work. Research, whether the act of studying something new through hands-on experimentation or by reading the literature and making conclusions based on deep and directed thought on the topics, is a part of each of these components in the lab and classroom. Therefore, through this course, and others in similar style at TCNJ, a valuable research experience can be brought to student participants.

Summary and Future Directions Using research-based laboratory projects asking novel questions with unique outcomes is a hallmark of the Chemical Biology course’s laboratory work. In the classroom, open-ended in-class group work, and the literature research of the final topic that students present and teach the class also brings research out of the lab. Together, from these research-aspects incorporated into the course, students contribute to their final by not only assimilating knowledge on a new topic but developing a way in which they and their peers can be assessed. Additionally, as a result of both iterations of the course, experimental materials were published in a peer-reviewed journal, adding to the breadth of the field. Taken as a whole, these facets have led to a successful Chemical Biology course! In the future, I will extend the topics in the classroom, with new in-class assignments and perhaps more possibilities for such group work. I will have the student presentations and “final questions” count more to their final grade as their work on these has proven exemplary. For the lab, building off of the helical scaffolds, we will continue to search for new ways to control small 14-helical β-peptides, and revisit our protein-protein target involved in cancer. As for the α-helices, as well as β-turn motifs, we will develop these short sequences further, examining well-tolerated substitutions and move into assay screens that could easily accommodate a classroom of students, such as antimicrobial vetting. Students will continue to train on the newest departmental instrumentation and, at the close of the future course I would have students themselves come up with the directions for future class iterations. What do they think is important to peptidomimetics and how would they design new compounds and pose new questions to incoming students? 161 Gourley and Jones; Best Practices for Supporting and Expanding Undergraduate Research in Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 2018.

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Peptidomimetics from the Classroom to the Lab: Successful Research

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