LABORATORY EXPERIMENT pubs.acs.org/jchemeduc
Student-Driven Design of Peptide Mimetics: Microwave-Assisted Synthesis of Peptoid Oligomers Nicola L. B. Pohl,*,† Kent Kirshenbaum,‡ Barney Yoo,‡ Nathan Schulz,† Corbin J. Zea,† Jennifer M. Streff,† and Kimberly L. Schwarz† ‡ †
Department of Chemistry, New York University, New York, New York 10001, United States Department of Chemistry, Iowa State University, Ames, Iowa 50011, United States
bS Supporting Information ABSTRACT: An experiment for the undergraduate organic laboratory is described in which peptide mimetic oligomers called “peptoids” are built stepwise on a solid-phase resin. Students employ two modern strategies to facilitate rapid multistep syntheses: solid-phase techniques to obviate the need for intermediate purifications and microwave irradiation to enhance reaction rates. The modular reaction protocol is compatible with a vast array of reagents, allowing students to design unique oligomer sequences and to get a sense of modern drug-discovery processes. Students compare reactions performed both with and without microwave irradiation for a collaborative learning experience. KEYWORDS: Second-Year Undergraduate, Laboratory Instruction, Organic Chemistry, Hands-On Learning/Manipulatives, Amides, IR Spectroscopy, Medicinal Chemistry, NMR Spectroscopy, Nucleophilic Substitution, Proteins/Peptides
T
he emergence of high-throughput automated chemistry protocols has spurred the development of new synthetic methods for the rapid synthesis and purification of diverse compounds.13 Solid-phase polymer-supported reactions have proved the most useful to date and serve as the basis for commercial automated peptide and nucleic acid synthesis. This article describes a laboratory module introducing second-semester organic chemistry undergraduates to polymer-supported multistep organic synthesis as well as to the use of microwave irradiation in a synthesis of a peptide mimetic. The lab exercise allows students to choose their own target molecule and compare reactions performed both with and without microwave irradiation for a collaborative learning experience. In addition, the experiments provide a window into the science of drug design and how reactions they have learned in organic chemistry are used in medicinal chemistry. Although peptides made by solid-phase synthesis have proven invaluable to drug-discovery efforts, their instability under physiological conditions has been a challenge. The search for alternatives to peptides in drug-discovery programs has led to the development of a class of biomimetic oligomers called peptoids.46 Peptoids, similar to peptides, can also be synthesized in modular fashion and therefore lend themselves to the design of combinatorial libraries.7 As distinct from peptides, the peptoid side chain is attached to the backbone nitrogen rather than carbon (Figure 1). This substituted amide linkage prevents the enzymatic hydrolysis reactions that normally degrade peptides. In addition, the asymmetric center is removed from the amino acid thereby eliminating worries of racemization side reactions during their synthesis. Peptoids are synthesized by a dehydrative amide coupling between bromoacetic acid and a Copyright r 2011 American Chemical Society and Division of Chemical Education, Inc.
Figure 1. Peptide versus peptoid structure. R can very depending on the amino acid or amine used in the synthesis.
substituted amine, followed by displacement of the bromide with the next amine (Scheme 1). This process can be performed stepwise using various amines to create a wide variety of peptoid oligomers with strict control over the sequence of the monomer units. The use of parallel and solid-phase synthesis techniques can directly facilitate the construction of compound libraries for screening applications.8 We previously described a laboratory module in which a small group of honors organic chemistry students synthesized a specific bioactive peptoid oligomer.9 Here, we extend the use of peptoids as a convenient platform for the expedient hands-on demonstration of multistep synthesis. This experiment establishes protocols to enable large numbers of students to design and synthesize a diverse set of peptoid products. Critical features include the use of microwave irradiation to speed reaction times.10,11 This permits six bond-forming steps to be performed in the course of synthesizing a peptoid trimer during one laboratory session.
Published: May 05, 2011 999
dx.doi.org/10.1021/ed100763t | J. Chem. Educ. 2011, 88, 999–1001
Journal of Chemical Education Scheme 1. Synthesis of Peptoid on a Solid-Phase Resin: DIC = N,N0 -Diisopropylcarbodiimide and TFA = Trifluoroacetic Acid
’ SUMMARY OF THE EXPERIMENTAL PROCEDURE This procedure has been performed for three semesters in a second-year undergraduate organic laboratory course designated for chemistry and biochemistry majors. Typical enrollment for this course is 26 students who are supervised by 2 teaching assistants. Two or three 3-h laboratory periods were dedicated to completing the peptoid synthesis and characterization. An extra lab period allowed students to produce longer peptoids and do more purification and characterization experiments. Though the student handout suggests three units, advanced students (such as the chemistry and biochemistry majors) can easily synthesize a longer chain. A peptoid with more than six units is difficult to synthesize, and students are advised to repeat coupling steps if pursuing a large peptoid chain. A white board in the laboratory is used to track the identities of students’ chains to avoid exact peptoid duplication. The organic laboratory class was grouped into student pairs. Each pair of students designed their own unique trimer sequence from the available amines; one student used microwave-assisted reactions and the other did not. Both reaction schemes to make the peptoid trimer were completed within a normal 3-h lab period. For more advanced students, up to three additional amine units could be added in a second lab period. Students are expected to communicate frequently with the teaching assistants, especially prior to cleavage from the resin to ensure that a
LABORATORY EXPERIMENT
chain of appropriate length was prepared. Informal discussions of NMR concepts introduced in the lecture course and of the utility of solid-phase reagents and reactants in high-throughput chemistry filled the time between reaction setup and completion. If time allowed, discussions about aspects of medicinal chemistry, such as an introduction to the mechanism of common proteases (e.g., as serine proteases) and a subsequent discussion as to why peptoids would be resistant to protease cleavage, can be added. The intermediates and final product were characterized by IR and 1H NMR. To complete the peptoid synthesis, students need the following reagents: 25 mL plus necessary volume to prepare stock solutions of N,N-dimethylformamide; 10 mL of dichloromethane; 250 mg of bromoacetic acid; 2.4 mL of piperidine; 79 μL of N,N0 -diisopropylcarbodimide per coupling (up to 240 μL total); trace quantities of acetaldehyde and p-chloranil; 3 mL of trifluoroacetic acid; 50 mg of 4-(20 ,40 -dimethoxyphenylFmoc-aminomethyl)-phenoxy resin (Novabiochem, cat. no. 01-64-0013); 46 μL of aniline per coupling (up to 135 μL); 55 μL of benzylamine per coupling (up to 162 μL); and 63 μL of (S)-()-1-phenylethylamine per coupling (up to 191 μL). Additionally, students may need silica gel for an optional column and a silica gel thin-layer chromatography (TLC) plate (approximately 1 in. 3 in.).
’ HAZARDS All chemicals used are harmful to inhale, ingest, or contact directly and many are flammable, thereby realistically limiting this experiment to undergraduates with prior lab experience. Many carbodiimide coupling reagents are contact allergens. Piperidine, all amines, trifluoroacetic acid, and bromoacetic acid are corrosive to the eyes, skin, and mucous membranes and are incompatible with one another. Direct contact may result in serious irritation, burns, or blistering. The peptoid products should be presumed to be possibly bioactive, and therefore, direct contact with the products should be avoided. Gloves, goggles, and lab aprons should be worn at all times in the laboratory. All reactions should be performed in a functioning fume hood with the sash pulled down as low as possible. Proper syringe handling techniques must be demonstrated and enforced. ’ RESULTS AND DISCUSSION In the most recent execution of this procedure, all students demonstrated basic knowledge of solid-phase synthesis on the prelab quiz, but only 10 of 24 students could provide specific benefits of solid-phase synthesis, such as a reduced purification between couplings, reduction of side products, and easy cleavage of the peptoids from the resin beads. Half of the students in the most recent lab understood the structural difference between peptides and peptoids (the location of the substituent R group), but only 8 students could cite reasons as to why peptoids are more favorable for drug synthesis; the two most popular reasons being resistance to degradation by hydrolysis and lack of racemization due to the achiral alpha-carbon in the peptoid main chain. In each of the three different semesters in which this experiment was carried out, the experiment came after the separate lecture course had discussed amide-forming reactions. These experiments are an ideal way to show the relevance of both simple nucleophilic displacement reactions learned in the firstsemester undergraduate organic chemistry course and amideforming reactions to modern drug design. On the basis of the 1000
dx.doi.org/10.1021/ed100763t |J. Chem. Educ. 2011, 88, 999–1001
Journal of Chemical Education initial student feedback, the current version of the experiment includes more discussion of the mechanisms of each of the reactions involved to form the peptoids as well as the reactions involved in monitoring the reaction progress on solid phase. On the basis of analysis of 24 prelab quizzes in the most recent semester of the experiment, three-quarters of the class understood the mechanisms of each of the reactions and all of the students were able to correctly explain the difference between a peptide and a peptoid as well as the advantage of using solidphase chemistry methods. The experiment has worked well and the students received it very enthusiastically. As each pair of students made a different trimer, interactions among students in the course were frequent. Many students particularly commented that they appreciated the opportunity to work on something that seemed “real”—more in line with the kinds of research they might do after graduation. Students seemed to react positively to a procedure in which they were given the opportunity to design their own product and take ownership of the procedure. In all three semesters, students had previously worked with laboratory microwaves, and therefore, this lab served to reinforce their knowledge of the technique. In addition, microwave irradiation allowed students to construct longer and more complex peptoid units. Students particularly saw a difference with the microwave when using the relatively deactivated aryl amines compared to the alkyl amines. Student success in producing their desired peptoid was measured by TLC, IR spectroscopy, and 1H NMR spectra. Even with only a 60 MHz NMR spectrometer, the side chains showed distinct enough spectra to verify successful couplings. If cost constraints allow, HPLC and MS can also be valuable for characterization purposes.9 This experiment also would be appropriate for a more advanced undergraduate lab course, in which case a deeper discussion of solid-phase chemistry, combinatorial drug discovery, biomimetic chemistry, molecular recognition, polymer conformation, or molecular design could be added.12,13
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(3) Kirschning, A.; Monenschein, H.; Wittenberg, R. Angew. Chem., Int. Ed. 2001, 40, 650. (4) Simon, R. J.; Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 9367. (5) Barron, A. E.; Zuckermann, R. N. Curr. Opin. Chem. Biol. 1999, 3, 681. (6) Seo, J.; Barron, A. E.; Zuckermann, R. N. Org. Lett. 2010, 12, 492. (7) Figliozzi, G. M.; Goldsmith, R.; Ng, S. C.; Banville, S. C.; Zuckermann, R. N. Methods Enzymol. 1996, 267, 437. (8) Alluri, P. G.; Reddy, M. M.; Bacchawat-Sikder, K.; Olivos, H. J.; Kodadek, T. J. Am. Chem. Soc. 2003, 125, 13995. (9) Utku, Y.; Rohatgi, A.; Yoo, B.; Kirshenbaum, K.; Zuckermann, R. N.; Pohl, N. L. J. Chem. Educ. 2010, 87, 637. (10) Olivos, H. J.; Alluri, P. G.; Reddy, M. M; Salony, D.; Kodadek, T. Org. Lett. 2002, 4, 4057. (11) Gorske, B. C.; Jewell, S. A.; Guerard, E. J.; Blackwell, H. E. Org. Lett. 2005, 7, 1521. (12) Yoo, B.; Kirshenbaum, K. Curr. Opin. Chem. Biol. 2008, 12, 714. (13) Fowler, S. A.; Blackwell, H. E. Org. Biomol. Chem. 2009, 7, 1508.
’ ASSOCIATED CONTENT
bS
Supporting Information Student handout and additional information for laboratory instructors. This material is available via the Internet at http:// pubs.acs.org.
’ AUTHOR INFORMATION Corresponding Author
*E-mail:
[email protected].
’ ACKNOWLEDGMENT We thank the students and teaching assistants of Chem 334 L, as well as Allen Clague, for their enthusiasm and hard work. This material is based in part upon work supported by the National Science Foundation under CAREER Grant Nos. 0349139 and 0645361 and CHE No. 0911123 and by the Camille and Henry Dreyfus Foundation under the Special Grant Program in the Chemical Sciences. JMS acknowledges a Plagens Fellowship. ’ REFERENCES (1) Smith, R. A.; Griebenow, N. Methods Princ. Med. Chem. 2006, 35, 259. (2) Plunkett, M. J.; Ellman, J. A. Sci. Am. 1997, 276 (4), 68. 1001
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