Laboratory Experiment Cite This: J. Chem. Educ. XXXX, XXX, XXX-XXX
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Scaffolding Students’ Skill Development by First Introducing Advanced Techniques through the Synthesis and 15N NMR Analysis of Cinnamamides Sara Shuldburg and Jennifer Carroll* California Polytechnic State University, 1 Grand Avenue, San Luis Obispo, California 93407, United States S Supporting Information *
ABSTRACT: An advanced undergraduate experiment involving the synthesis and characterization of a series of six unique cinnamamides is described. This experiment allows for a progressive mastery of skills students need to tackle more complex NMR structure elucidation problems. Characterization of the products involves IR spectroscopy, GCMS, and proton, carbon, and nitrogen NMR analyses. Understanding the principles of advanced NMR techniques, including nitrogen NMR spectroscopy, is highly relevant to advanced organic chemistry students. KEYWORDS: Upper-Division Undergraduate, NMR Spectroscopy, Amides, Organic Chemistry, Laboratory Instruction, Hands-On Learning/Manuipulatives
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until reaching the graduate level.10,11 Nitrogen-containing compounds are important to many fields of chemistry, biology, and medicine. This nucleus can be found in a wide variety of biologically important molecules, such as alkaloids, nucleic acids, proteins, cytokinins, and purine-based antitumor and antiviral drugs. Considering the intrinsic value of nitrogen and recent technical improvements in nitrogen-based NMR applications, we have recently begun to explore its coverage in undergraduate teaching.
INTRODUCTION The instruction of NMR spectroscopy typically focuses on hydrogen, carbon, and a variety of two-dimensional (2D) NMR techniques for the elucidation of both natural1−4 and synthetic5−7 compounds. As a mainstay of introductory and advanced organic chemistry curricula, these techniques give students valuable experience in the molecular characterization of synthetic and naturally occurring compounds. The typical series of experiments includes distortionless enhancement by polarization transfer (DEPT), heteronuclear single quantum correlation (1H−13C HSQC), heteronuclear multiple quantum coherence (1H−13C HMQC), homonuclear correlation spectroscopy (1H−1H COSY), heteronuclear multiple bond correlation (1H−13C HMBC), and nuclear Overhauser effect spectroscopy (NOESY). However, detailed 2D NMR analysis of complex natural products presents too great a challenge for introductory students. We have attempted to bridge this instructional gap with a series of straightforward elucidation challenges. In this paper, a laboratory experiment is introduced on the synthesis and characterization of a series of simple cinnamamides using proton, carbon, and nitrogen NMR spectroscopy. The main pedagogic goals of this experiment are to have students (1) synthesize and isolate one of six cinnamamides; (2) acquire and process their own NMR data; (3) interpret DEPT, HMQC, COSY, 1H−13C HMBC, and 1 H−15N HMBC data; and (4) fully elucidate the structure of their unknown compounds. The technique of nitrogen NMR spectroscopy is increasingly utilized to analyze organic, biological, and medicinal compounds; however, few examples of this technique can be found in the teaching literature.8,9 Undergraduate textbooks occasionally cover physical aspects such as the magnetic susceptibility and quantum factors, but 15N NMR spectra are rarely included © XXXX American Chemical Society and Division of Chemical Education, Inc.
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NITROGEN BEHAVIOR IN NMR SPECTROSCOPY The two main isotopes of nitrogen, 14N and 15N, are both NMR active, with spins of 1 and 1/2, respectively, and the chemical shifts of both isotopes are identical. However, due to the differing physical characteristics (Table 1), these isotopes behave quite differently.10,11 The high isotopic abundance of 14 N over 15N leads to a greater signal intensity; however, the extremely low gyromagnetic ratio of 14N causes nontrivial line broadening of tens to hundreds of Hz. Alternatively, the 15N line widths, which are typically less than 1 Hz, allow utility in Table 1. Selected NMR-Active Isotopes Isotope
Abundance (%)
Spin (I)
Gyromagnetic Ratio (γ)a
99.99 1.11 99.635 0.365
1/2 1/2 1 1/2
26.751 6.726 1.932 −2.711
1
H C 14 N 15 N 13
a
γ, rad T−1s
−1
.
Received: April 24, 2017 Revised: October 8, 2017
A
DOI: 10.1021/acs.jchemed.7b00279 J. Chem. Educ. XXXX, XXX, XXX−XXX
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NMR data analysis.12 The low isotopic abundance of 15N is typically overcome by enrichment. The negative gyromagnetic ratio of 15N is also interesting, indicating a counterclockwise precession of its spin angular momentum.12 A common challenge in the comparison of nitrogen NMR data in the literature is the previous lack of a standardized internal reference. Many internal chemical shift standards have been reported in the literature (Table 2), including CH3NO2,
this application of indirect (proton) detection with the aim of overcoming the intrinsically low sensitivity of 15N NMR measurements.
Table 2. 15N Chemical Shift Reference Standards
Scheme 1. Synthesis of Cinnamamides
a
Standarda
Conc
δ (ppm)
NH3(l) CH3NO2 CH3NO2:CDCl3 urea NH4NO3 NH4Cl
neat neat 1:1 1 M in DMSO-d6 1 M in 1 M HNO3 2.9 M in 1 M HCl
0.0 381.7 379.8 77.0 21.0 23.6
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EXPERIMENTAL PROCEDURE In this reaction, cinnamoyl chloride reacts with either a primary or secondary amine to produce an amide and hydrochloride salt (Scheme 1).14 Prior to this experiment, students were
At 25 °C.
urea, NH4Cl, and NH3, the latter of which has been accepted as a standard in more recent literature.13 Characteristic nitrogen NMR chemical shifts for various groups have been summarized in Figure 1.13
familiarized with nucleophilic acyl substitutions, acid/base extractions, and one-dimensional NMR spectroscopy. A prelab worksheet on acid/base extractions was also completed before the reaction was begun. Students worked individually on this experiment during three laboratory sessions of 3 h, and then were given a week to complete their laboratory reports. During the first day of lab, students performed the cinnamamide synthesis and extraction. Separate vials containing cinnamoyl chloride (1.0 g) and unknown amine (1.5 mL) in THF (2.0 mL) were distributed and refluxed for 30 min. The chemically active extraction of products using methylene chloride and NaOH (5% w/v), followed by HCl (5% w/v), resulted in pure amides by thin-layer chromatography (TLC). Yields for this reaction were typically 70−80% for the six cinnamamide derivatives. The second lab period was used to introduce DEPT and 2D NMR concepts and to queue experiments on the NMR
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MODERN TECHNIQUES OF NITROGEN NMR SPECTROSCOPY A number of modern techniques for nitrogen analysis by NMR spectroscopy are present in the literature. High-sensitivity cryoprobes, low-volume microcells, and high-field magnets allow for 15N analysis of standard organic compounds with little to no enrichment. While this technique has advantages for both small molecule and protein samples, it remains a costly alternative for teaching laboratories. One-bond 1H−15N coupling, typically on the order of 90−100 Hz, can be measured with gradient-selected 2D HSQC and HMQC experiments.12 Most modern NMR experiments are based on
Figure 1. Characteristic 15N NMR chemical shifts for various groups. B
DOI: 10.1021/acs.jchemed.7b00279 J. Chem. Educ. XXXX, XXX, XXX−XXX
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autosampler. During the third lab period, students processed their spectra, collected GCMS and IR data, and began to elucidate the structures of their amides. Laboratory reports were due 6 days later, with fully annotated spectra, the structures of reactants and products, and an analysis of the results.
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INSTRUMENTATION NMR experiments were performed on a 300 MHz NMR instrument with a 16-slot autosampler. Students prepared samples for 13C and 2D NMR analysis that contained product (0.50 g) in CDCl3 (0.25 mL). Obtaining an acceptable signalto-noise ratio required the acquisition of 4 scans for 1H, 32 scans for 13C, 4 scans for COSY, and 8 scans for 1H−15N HMBC spectra. MNova NMR software was utilized by students to process spectra.15 FTIR spectra were run neat. GCMS data were acquired with amide solutions (1% w/v) in methanol. As high-field NMR instrumentation is not available at some institutions, a full set of spectra is in the Supporting Information.
Figure 2. 15N HMBC correlations for cinnamamide 1.
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HAZARDS Safety glasses, lab coats, and nitrile gloves should be worn for all portions of this laboratory experiment. Care should be taken to avoid contact with all solutions. Amines and solvents such as heptane, ethyl acetate, methanol, and THF are irritants to skin, eyes, and respiratory tract. Acyl chlorides are highly reactive and act as lachrymators. CH2Cl2 and CDCl3 are classified as carcinogens. The toxicological properties of the amides have not been fully investigated and may cause skin, eye, and respiratory tract irritation. Fume hoods should be used during the amide synthesis, extraction, and NMR sample preparation. Care should be taken to keep all equipment and personal items containing ferromagnetic material (e.g., steel, iron) at least 2 m away from the NMR spectrometer.
Figure 3. 2D NMR correlations.
chemical shift of 125.7 ppm (ref NH3(l)), which is characteristic of amide nitrogens.13 The 15N HMBC data connected the alkyl groups and alkene with the nitrogen. The nonequivalence seen in the alkyl groups of tertiary amides 1, 2, and 3 was a common stumbling block in this characterization. Figure 4 proved useful in illustrating two of the resonance structures that contribute to nonequivalent alkyl group positions “a” and ”b” seen in the 13C NMR spectrum.
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RESULTS AND DISCUSSION Rather than elucidate all six structures within this paper, dipropylcinnamamide (1) will be used as an example of the analysis to show how students process the data. The (M+) of 231 m/z and the base peak of 131 m/z for the [C6H5CH CHCO]+ fragment were readily apparent in the mass spectrum (Supporting Information, p S16). The IR spectrum of 1 (SI p S17) displayed a notable amide carbonyl signal at 1644 cm−1 and an amide CN stretch at 1442 cm−1. Using a combination of MS, 13C NMR (SI p S20), DEPT (SI p S21) and 1H NMR (SI p S18) integration data, the molecular formula of C15H21NO was determined. Further analysis of the DEPT data indicated the presence of two methyls, four methylenes, seven methines, and two quaternary carbons. The phenyl group and an E alkene (J = 15.30 Hz) were readily apparent in the 1H NMR spectrum, whereas the nitrogen attachments (δ = 49.51 and 48.28 ppm) and carbonyl (δ = 165.68 ppm) were detected in the 13C NMR spectrum. To aid in the elucidation, students utilized three-bond J H−H correlations from the COSY spectrum (SI p S19), in addition to JC−H and JN−H correlations from the HMBC data (SI pp S24 and S26), respectively (Figure 2). 13C HMBC experiments provided the connectivity from the phenyl ring to the E alkene, from the alkene to the carbonyl, and from the carbonyl to the N-alkyl groups (SI p S10). Three correlations were noted in the 15 N HMBC spectrum (Figure 3), corresponding to a 15N
Figure 4. Two resonance structures of dipropylcinnamamide resulting in nonequivalent alkyl positions “a” and “b”.
A total of 48 students have competed this lab to date. This laboratory experiment was first tested in an upper-division, advanced laboratory with 16 students, and in two sections of a second-year lab, each with 16 students. Students performed data analysis both in class and outside of class. Examples of student data tabulation can be found in the Instructors Notes in the Supporting Information. Students were able to complete the synthesis and analysis during three laboratory periods, with an additional week to complete their report. This procedure gave excellent yields, sufficient purity, and clearly elucidated data. Summative assessment tools used in gauging effectiveness of this experiment included in-class completion of NMR C
DOI: 10.1021/acs.jchemed.7b00279 J. Chem. Educ. XXXX, XXX, XXX−XXX
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correlation tables, as given in the Supporting Information, and formal reports including fully annotated spectra and characterization of their unknown. A similar amide spectroscopy problem was also included on their laboratory final exam. All but one of the 48 students identified their unknown amides correctly. The final exam amide spectroscopy problem averaged 62%. Averages for the formal reports ranged from 71% for the second-year group to 85% for the advanced upper-division group. Typically, errors in 2D NMR peak identification occurred when overlapping, or accidentally equivalent peaks occurred. Students reported that they utilized both 1D and 2D NMR spectra for their structure analysis, and that they found this experiment interesting and useful. Many of our students at the second-year undergraduate level have begun synthetic organic research and were pleased to have an introduction to more advanced NMR techniques. In addition, the choice of amine utilized makes this experiment highly adaptable for instructors.
CONCLUSION While a number of amide syntheses have been discussed in the recent literature,16−18 this report is the first to utilize 2D and 15 N NMR spectra for the characterization of synthetic products. This experiment is easily adaptable, provides excellent yields, and challenges students to think independently. Students are rarely ready to tackle a difficult natural product structural analysis when they are first introduced to HMBC, COSY, and HMBC data. The advantage of this approach is that simple amides are utilized to introduce advanced NMR analysis in an incremental manner so that students can gain confidence in these techniques before tackling more difficult NMR problems. In addition, this experiment increases the number of advanced NMR techniques to include 15N NMR data collection and analysis. This exposure benefits undergraduates in organic chemistry and biochemistry. ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available on the ACS Publications website at DOI: 10.1021/acs.jchemed.7b00279. Student laboratory protocols and worksheets (PDF, DOCX) Spectral data (PDF)
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REFERENCES
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Jennifer Carroll: 0000-0003-3172-176X Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors wish to thank Jan William Simek for assistance in development of the initial amide synthesis. NMR work was greatly facilitated by Kevin Dunham, and Celine DiBernardo provided essential laboratory stockroom assistance. S.S., a Summer 2016 Frost Research Fellow, was supported by the Bill and Linda Frost Fund at Cal Poly. D
DOI: 10.1021/acs.jchemed.7b00279 J. Chem. Educ. XXXX, XXX, XXX−XXX
