In the Laboratory
A Novel Target Synthesis Laboratory for Students C. Mark Smales and David R. K. Harding* Department of Chemistry, Massey University, Palmerston North, New Zealand; *
[email protected] In developing a new third-year course on drug design and synthesis with a large organic chemistry component, a totally research-based target synthesis laboratory course was introduced. We decided to avoid the recipe-type experimental roster that acts only to illustrate techniques, apparatus, and methods in favor of a “real” and “useful” synthetic target much along the lines of research projects that others have introduced elsewhere (1–4 ). It was necessary to choose a target that was both challenging and yet at the same time realistic so as not to dissuade the students. Relevance to the lecture course material was of course also taken into account.
students would understand that it is not simply a matter of undertaking a condensation reaction between two amino acids to form an amide bond, as is portrayed in many textbooks (5). A further advantage of this target molecule (Gly-Phe-LeuGly) was that there is a considerable volume of literature on the possible use of peptides as components of drug delivery systems (6–8). The students were therefore encouraged to make intelligent use of the literature as part of the shift from relying solely on laboratory manuals and laboratory staff to reading, understanding, and using research-based literature themselves.
Choice of the Target Molecule
Undertaking of the Target Synthesis by the Students
The students were all given the same synthetic target to achieve, as opposed to letting them choose their own or giving them a selection of target molecules. While both methods of laboratory assignment have advantages and disadvantages, we felt that giving students the same target molecule offered several advantages in this course over letting them choose different projects, which are outlined below.
The students were given an overview of a synthetic pathway (Scheme) likely to be successful, by which to synthesize the target molecule. They were, however, invited to consider alternate pathways to the target molecule. While none of the students attempted to follow an alternative synthetic pathway, possible synthetic routes differing from that provided, by which the same target molecule could be achieved, were discussed in the laboratory class. Students worked in pairs so that the level of cooperation between students could be assessed and the requirement for equipment and chemical reagents reduced. By working in pairs, the students could easily set up two different reactions and undertake all the required procedures to characterize their products (NMR, IR, melting points, etc.) within the laboratory sessions. Each student in a pair was individually responsible for a different reaction at each laboratory session. The students were encouraged to think for themselves and find out on their own how equipment and techniques operated. When questioned for help, we either suggested reference sources or “steered” the students in the correct direction without simply giving them
As the synthetic scheme proposed was linear and this was the first time many of the students had undertaken research-based chemistry, mistakes were bound to occur. We did not wish to penalize them for these. Students could carry on with the synthesis even in the event of “losing” all their product by using some of the product from other students. Less equipment and reagents were required than if students were all undertaking different syntheses. The students were able to discuss their results and findings together on the same level. It was much easier to objectively mark student effort, independence, thought processes, and reports on the same project than on different projects. We could observe how different students reacted to the same problem. Since the reactions undertaken had not been tested, laboratory staff could address problems as they arose with the whole group and discuss possible solutions. A synthetic target that was relevant to the lecture course could be chosen.
After looking at the lecture material, we selected the tetrapeptide glycyl-phenylalanyl-leucyl-glycine (see Scheme) as the target molecule. The lecture course for Drug Design and Synthesis contained a section on the chemical synthesis of peptides as therapeutic and diagnostic tools in medicine. The target tetrapeptide had the advantage of involving only bifunctional amino acids in this “first attempt” for us at this type of laboratory course. This meant that the overall cost and difficulty of the synthesis were less than if trifunctional amino acids were incorporated into the target molecule. The target tetrapeptide was also required in a research laboratory as part of a proposed drug delivery system. The expected procedure would involve protection and deprotection experiments along with the formation of peptide bonds. Additionally, we hoped that in performing the coupling reactions the 1558
Gly
Phe
Boc-Gly
Phe-OMe.HCl
Boc-Leu
p-TSA.Gly-OBzl
Boc-Gly-Phe-OMe
Boc-Leu-Gly-OBzl
Boc-Gly-Phe
TFA.Leu-Gly-OBzl
Boc-Gly-Phe-Leu-Gly-OBzl
Scheme: Proposed synthetic route for synthesis of the target tetrapeptide.
Gly-Phe-Leu-Gly
Journal of Chemical Education • Vol. 76 No. 11 November 1999 • JChemEd.chem.wisc.edu
In the Laboratory
the answer(s). None of the reactions in the proposed peptide sequence had been attempted by laboratory staff. Students were informed from the start that they would not be penalized in any way if they could not synthesize the correct compound (and intermediates along the way), as long as they could show the planning and reasoning behind their work. All of the students eventually successfully prepared the phenylalanine methyl ester hydrochloride salt (Phe-OMe?HCl, Scheme) and glycine benzyl ester p-toluenesulfonic acid salt (p-TSA?Gly-OBzl, Scheme) and produced the evidence required to confirm this (NMR spectra, IR spectra, melting point, etc.). Some students took several attempts to synthesize the required product(s). The most common problem was the addition of incorrect quantities of reagent(s) or the complete omission of essential reagents. Many of the students initially attempted to synthesize the phenylalanine methyl ester salt using glassware that was not completely dry, thus greatly reducing the yield of product. The preparation of the Gly-OBzl ester (p-TSA salt) involved an azeotropic distillation step and a Dean and Stark apparatus. Most of the students had not previously encountered this type of reaction or piece of apparatus and had difficulties at first in planning and designing this experiment. However, all eventually overcame this and completed the required reaction successfully. Some students lost a significant proportion of their yield (>50%) while undertaking recrystallizations of these products. All of the students attempted to couple Boc-Gly with Phe-OMe?HCl and Boc-Leu with p-TSA?Gly-OBzl amino acids using dicyclohexylcarbodiimide (DCC) as a carbonyl carbon activator. This was the first step that some students did not complete successfully. Once again many of the problems arose because of addition of incorrect amounts of reagents, although by far the most common mistake occurred during the final washing steps undertaken in a separating funnel. Some students simply threw their products away by discarding the organic layer and working with the incorrect aqueous layer. The removal of the methyl ester carboxyl protecting group on the Boc-Gly-Phe-OMe dipeptide by base treatment was successfully carried out by all of the students who attempted this. Similarly, removal of the Boc amino protecting group on the Boc-Leu-Gly-OBzl dipeptide by acid treatment was successfully carried out by most of the students. However, once again many lost large amounts of material during recrystallization steps. Several students synthesized the protected tetrapeptide Boc-Gly-Phe-Leu-Gly-OBzl in very small yield. No pair of students finished the total synthesis of the target glycylphenylalanyl-leucyl-glycine molecule. Writing of the Report The students were asked to prepare a report that was written in the style of a scientific publication. Any scientific style could be chosen provided that the particular journal format followed was stated in the report and that it was relevant to the work being presented. The report was required to contain an abstract, an introduction, a methods section, a results section, and a discussion section. Relevant literature was to be cited as appropriate for the journal style followed. Successful completion of the report required an in-depth analysis of the results (and any relevant literature), presentation of the appropriate evidence that the products at each step
had been attained, and an understanding of scientific notation and how to present scientific data. Students were required to prepare their own report independently of their partner in the laboratory. Discussion of the results and the interpretation of results and their relevance was encouraged between students and between students and the instructor(s). We observed that the class often met outside lecture or laboratory hours, without prompting from staff, to discuss the project and strategies. Almost all of the students produced reports that were vastly different from those of their experimental partner or others in the class. Most took great pride in the report and their work in the laboratory. The students often spent more time than required in the laboratory and on preparing the report, particularly as examinations approached. Some had to be reminded to leave the laboratory course behind and concentrate on their course work. General Comments The laboratory time available to the students to complete the target (six 3-h periods) synthesis was obviously insufficient, as none of the pairs of students completed the total synthesis. Although no pair of students actually completed the synthesis of the target molecule glycyl-phenylalanyl-leucyl-glycine, many students were so enthusiastic about the project that they wanted to complete the synthesis in their own time after the completion of the laboratory course. Several pairs of students did reach the BOC-Gly-Phe-Leu-Gly-OBzl stage. Most students were initially hesitant to start reactions and appeared to lack confidence in their ability to undertake a linear target synthesis without “cookbook” instructions. As a result, some of them tended to be overly careful and conservative with their approach and reactions took longer to set up and perform than expected. However, as the laboratory course proceeded, most of the students grew in confidence and were able to set up, undertake, and characterize a reaction in a much shorter time. At first it was common for students to obtain low yields from their reactions. Without cookbook instructions, many students were initially confused over the stoichiometry of a reaction and thus added incorrect amounts of reagents. However, the most common reason for low-yielding reactions was losses during recrystallization steps, when students tended to use large volumes of solvent many times in excess of those required. The students encountered few difficulties in obtaining and interpreting NMR and IR spectra, as all were taking a parallel spectroscopy course. Various report styles were presented. Most were of an acceptable level for third-year students and in the correct scientific style. Many of the weaker academic students in the class actually submitted a more thorough and well-presented document than some of the stronger students. Perhaps the most alarming feature of the reports was their poor standard of English. Most of the students also tended to write in the present tense rather than the past tense, or in some cases used a combination of both. Students initially commented that they were concerned that the chemistries in the laboratory course had not been tried and perfected by laboratory staff. They were concerned that as a result they might fail to produce any sort of product, owing to unexpected problems with the chemistry involved.
JChemEd.chem.wisc.edu • Vol. 76 No. 11 November 1999 • Journal of Chemical Education
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In the Laboratory
Most of the students admitted that they felt a lot more confident with the laboratory course once they began to obtain results. Many students remarked that the laboratory course held extra appeal over others they had completed because it was aimed at producing a compound that could be used elsewhere for further research. Since completing the laboratory course, one student has decided to continue on with postgraduate work in the area of peptide synthesis, and another has decided on a postgraduate program in a related area, protein chemistry. Conclusion Despite the fact that the chemistries in the proposed synthetic route were untried, all of the students synthesized and characterized at least four of the intermediates on the pathway toward synthesizing the target molecule. The strict criteria for presenting a scientific report and the flexibility in planning a target synthesis were a surprise to many of the students. We personally enjoyed this approach to undergraduate laboratory teaching, although a far greater input of work was required of us than for the traditional recipe-type experiments we had previously taught.
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The tetrapeptide model does not preclude using this approach for other nonpeptidic linear synthetic schemes. A research-based target synthesis is also a viable option for an upper-level laboratory course in the areas of organic chemistry and biochemistry. Literature Cited 1. Campbell, D. L. J. Chem. Educ. 1991, 68, 784–785. 2. Spector, T. I. J. Chem. Educ. 1993, 70, 146–148. 3. Mahaffy, P. G.; Newman, K. E.; Bestman, H. D. J. Chem. Educ. 1993, 70, 76–79. 4. Buckley, P. D.; Jolley, K. W.; Watson, I. D. J. Chem. Educ. 1997, 74, 549–551. 5. Stojanoski, K.; Zdravkovski, Z. J. Chem. Educ. 1993, 70, 134– 135. 6. Duncan, R.; Kopecková-Rejmanová, P.; Strohalm, J.; Hume, I.; Cable, H. C.; Pohl, J.; Lloyd, J. B.; Kopecek, J. Br. J. Cancer 1987, 55, 165–174. 7. Duncan, R.; Kopecková, P.; Strohalm, J.; Hume, I. C.; Lloyd, J. B.; Kopecek, J. Cancer 1988, 57, 147–156. 8. Rihova, B.; Bilej, M.; Vetvicka, V.; Ulbrich, K.; Strohalm, J.; Kopecek, J.; Duncan, R. Biomaterials 1989, 10, 335–342.
Journal of Chemical Education • Vol. 76 No. 11 November 1999 • JChemEd.chem.wisc.edu
