Laboratory Experiment pubs.acs.org/jchemeduc
Investigation of Epimer Formation in Amide-Coupling Reactions: An Experiment for Advanced Undergraduate Students M. Jonathan Fray* School of Chemistry, University of Nottingham, University Park, Nottingham NG7 2RD, United Kingdom S Supporting Information *
ABSTRACT: An experiment is described to investigate how the choice of coupling agent and reaction conditions affects the ratio of epimers formed in the sensitive amidecoupling reaction between N-Boc- or N-benzoyl-(R)-phenylglycine and (S)-valine methyl ester. The experiment, which is suitable for third-year undergraduates, is designed to teach about important synthetic methods and reaction mechanisms and to develop skills in designing experiments, data analysis, and team work.
KEYWORDS: Upper-Division Undergraduate, Laboratory Instruction, Organic Chemistry, Collaborative/Cooperative Learning, Hands-On Learning/Manipulatives, Inquiry-Based/Discovery Learning, Amides, Amino Acids, Diastereomers, NMR Spectroscopy
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Scheme 1. General Reaction Scheme for Solution-Phase Peptide Synthesis
he value of investigational or research-driven laboratory projects (of varying length and complexity) has been discussed widely.1−3 Advantages are that students feel more engaged as projects are aligned to real-life problems,4 and there is not necessarily a “right” answer, as in real research. Indeed, it has been shown that the type of laboratory work affects views on the nature of scientific enquiry itself.5 Following the first two years’ laboratory experiments that teach techniques through following a set of traditional “recipe-following” laboratory experiments, a series of experiments for third-year students have been developed that are positioned somewhere between the “inquiry-led” (students define the problem, design experiments, and analyze the data generated) and the “researchbased” (working on a real problem with potential to advance knowledge)6,7 as preparation for extended fourth-year research projects. Another reason for embarking on this work was provided by a Higher Education Academy (HEA, United Kingdom) Physical Sciences survey of chemistry graduates in 20108 that highlighted underdeveloped skills. Top of the list were experiment design, team working, oral presentations, and time-management. Investigational projects could give muchneeded experience in these areas. The efficient formation of the amide bond between two αamino acid derivatives is crucial to the synthesis of peptides and proteins.9−12 Protecting groups (P1, P2) are normally employed to enable the desired reaction to take place (Scheme 1). Following selective protecting group removal, the amide coupling can be repeated to extend the peptide. The most © 2013 American Chemical Society and Division of Chemical Education, Inc.
common protecting groups are esters for carboxylic acids and carbamates for amines. A high yield and prevention of racemization of the activated carboxylic acid are desirable. The racemization sometimes occurs through base-promoted proton exchange in the oxazolone intermediate 1 (Scheme 2) formed by attack of the adjacent carbonyl group (illustrated with Boc). Subsequently, the amine nucleophile is acylated by 1 or its enantiomer 2, thereby leading to diastereomeric products 3 and 4. A wide range of coupling reagents has been developed, although their ability to suppress epimer formation varies.13−15 Some coupling partners present greater challenges than others. For example, sterically hindered amines slow the rate of coupling and increase the chance for the sensitive oxazolone to form. In the case of the carboxyl partner, the nature of P1 has a Published: November 14, 2013 136
dx.doi.org/10.1021/ed400255q | J. Chem. Educ. 2014, 91, 136−140
Journal of Chemical Education
Laboratory Experiment
Scheme 2. Mechanism of Epimer Formation in Amide Coupling
Figure 1. Available coupling reagents, bases, and solvents.
dramatic effect on the susceptibility to racemization. Though carbamate protecting groups, such as Boc, Cbz, and Fmoc, tend to inhibit cyclization to give 1, acyl derivatives are much more vulnerable, which is why coupling of segments in peptide synthesis traditionally involves glycyl as the carboxy terminus, rather than an amino acid with a stereogenic center.16 (For further discussion, see Supporting Information). New coupling methods have been developed to address this problem.
have approximately 40 h in the laboratory spread over four weeks; because each team can generally perform 12−25 experiments in the time available, experiments have to be designed carefully. Students prepare a plan (including a risk assessment for the chemicals) based upon a set of leading references and reviews, and they receive feedback on it before starting work. The project is designed to promote inquiry, and it is unlikely for two groups of students to do exactly the same experiments. The coupling partners were chosen deliberately for several reasons. First, an easy way to determine the isomer ratios 8:10 and 9:11, without resorting to very high-field NMR or highperformance liquid chromatography, was essential. N-Protected phenylglycyl is particularly valuable in this regard, as the phenyl group induces significant shifts in the proton NMR: Δδ = 0.09 ppm in the methyl ester singlet and Δδ ∼ 0.2 ppm in the isopropyl methyl groups (Table 1).20 Figure 2 shows the
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EXPERIMENTAL OVERVIEW This experiment investigates the extent of epimer formation in the coupling of Boc- and Bz-protected (R)-phenylglycines (517 and 618) with (S)-valine methyl ester 7 (Scheme 3).19 These Scheme 3. Reaction Under Investigation
Table 1. Methyl Signal 1H NMR Shift Differences for Diasteromers of Boc- and Bz-PhgValOMe
a
compound
δH OCH3a
(R,S)-BocPhgValOMe (8) (S,S)-BocPhgValOMe (10) (R,S)-BzPhgValOMe (9) (S,S)-BzPhgValOMe (11)
3.72 3.63 3.73 3.64
δH CHMe2a 0.73, 0.91, 0.71, 0.93,
0.66 0.86 0.64 0.87
Shifts are reproducible ±0.03 ppm.
methyl ester region of a typical 1H NMR spectrum. The origin of the shift differences has been explained in terms of the preferred conformation of the dipeptide (Figure 3).21 Second, phenylglycine is very sensitive to epimer formation as the phenyl group increases the acidity of the oxazolone 1 (Scheme 2) compared to an alkyl group; the pKa of the base would also be predicted to be relevant. Third, the coupling is fairly hindered, thereby increasing the chances of epimer formation. Finally, it was desirable that this particular coupling had not been widely investigated so that students would be required to consider how to relate literature examples to it. (R)Phenylglycine was chosen because it is slightly less expensive than the (S)-enantiomer.
reaction partners were selected because they had not been widely investigated before and to assist the analysis of the diastereomers by 1H NMR (see below). The aims of the experiment are to introduce modern coupling reagents and methods and to devise a logical set of experiments designed to minimize epimer formation. Through an inquiry-led approach, students are thereby introduced to new concepts that extend their knowledge of amino acid chemistry beyond what is normally taught at undergraduate level. Working in teams of 4− 6, students design a logical set of experiments that generally give rise to mixtures of the dipeptide derivatives 8 and 10 and 9 and 11 (Boc- and Bz-PhgValOMe). The following parameters may be varied: a set of eight coupling reagents, five bases, six solvents (Figure 1), temperature, and stoichiometry. Students 137
dx.doi.org/10.1021/ed400255q | J. Chem. Educ. 2014, 91, 136−140
Journal of Chemical Education
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Laboratory Experiment
RESULTS AND DISCUSSION The experiment has been run successfully with a total of 21 students. As part of the assessment, each student wrote a report summarizing and rationalizing their team’s results. Results for three coupling reagents (CDMT, isobutyl chloroformate, and T3P) are tabulated in the Supporting Information and confirm the expected effect of the protecting groups. Part of these data is shown in Table 2. The data should be interpreted with Table 2. One Set of Student Results for the Coupling Reactiona
Figure 2. 270 MHz 1H NMR spectrum (methyl ester region).
protecting group
solvent
temp (°C)
yieldb (%)
isomer ratio (R,S): (S,S)
Boc Boc Boc Boc Boc Boc Bz Bz Bz Bz Bz Bz
EtOAc EtOAc EtOAc EtOAc MeCN MeCN EtOAc EtOAc EtOAc EtOAc MeCN MeCN
10 20 30 25 15 25 10 20 30 15 15 25
71 63 82 10 25 85 98 77 84 56 46 55
99: 1 97: 1 98: 1 >99: 1 >99: 1 >99: 1 97: 1 96: 1 96: 1 NDc >99: 1 NDc
Figure 3. Conformational explanation of the origin of the shift differences.
a
EXPERIMENT The amide-coupling reactions do not require a special apparatus or techniques. The recommended scale for reactions is 1 mmol, although more able students may be able to manage with less. Reactions may be performed in round-bottomed flasks or glass tubes equipped with a rubber septum and balloon of nitrogen. Most of the reactions require room temperature or ice bath cooling, although the mixed anhydride method can be performed from −30 °C (dry ice−2-propanol bath) to −10 °C (ice−salt bath). Reactions are monitored by thin-layer chromatography on silica gel plates. Aqueous workup, with both acid and base washes, removes starting materials and generally leaves product that is sufficiently pure for NMR analysis. Students submit samples of their products for grading, ideally containing a single diastereomer. This encourages careful assessment of their various product batches and selection of ones with the highest isomer ratio for recrystallization. However, as it is recognized that not all possible isomeric mixtures can be crystallized to purity, submission of a “clean” mixture is not penalized. If necessary, products may be readily purified by silica gel chromatography, although separation of the isomers is not possible.
caution because they refer to single experiments and yields correspond to unpurified material; yields may be high (due to solvent impurities) or low (due to spillage!). As long as the quality of the NMR spectrum is good (i.e., signal-to-noise ratio >300:1 and correctly phased), isomer ratios of, say, 97:1 and 99:1 can be readily distinguished. Indeed, it is important not to confuse the minor isomer methyl ester peak with the 13C satellite peaks from the major isomer. As indicated in Table 2, CDMT has given superb results, even with the benzoyl-protected starting material (yields typically >50% and isomer ratios >95:5), whereas T3P gave good results with the Boc-protected phenylglycine (yields typically >50% and isomer ratios >15:1), but much more modest selectivities with the Bz-protected phenylglycine (yields typically >50% and isomer ratios
