In the Laboratory
Complete Assignment of Proton Chemical Shifts in Terpenes An Experiment Combining 2D NMR Spectroscopy with Molecular Modeling Nancy S. Mills Department of Chemistry, Trinity University, San Antonio, TX 78212-7200 The technology associated with NMR spectroscopy has become much more sophisticated with the introduction of Fourier transform techniques on spectrometers equipped with superconducting magnets and powerful computers. Unfortunately, most chemists tend to use NMR spectra in very traditional ways, collecting the data for 1H NMR and 13 C NMR spectra and using the information gained from chemical shifts, coupling, and integration to characterize a molecule. However, molecules with overlapping multiplets often defy interpretation. Two-dimensional (2D) methods allow the NMR experiment to illuminate relationships in molecules to make the interpretation easier, or even possible. For example, heteronuclear shift-correlation spectroscopy (HETCOR) allows identification of all carbon–proton pairs in a molecule, and nuclear Overhauser and exchange spectroscopy (NOESY) allows identification of nuclei that are in proximity to each other, accomplishing the same goal as an X-ray crystallographic structure, but at a fraction of the cost and time and without the requirement for a good crystal. In this experiment, correlation spectroscopy (COSY) will be used to make structural assignments in spectra with overlapping multiplets. This kind of NMR experiment would have been considered beyond the instrumentation and expertise of faculty at undergraduate institutions as recently as five years ago, but a number of institutions have purchased high field FT-NMR spectrometers in recent years. According to the CUR National Database on Undergraduate Research,1 123 institutions of the 226 reporting NMR instrumentation and included in the Directory of Undergraduate Research in Chemistry (1) have high field NMR spectrometers. The arsenal of techniques available to chemists for structure elucidation also includes molecular modeling using molecular mechanics calculations (2). These calculations, originally reserved for mainframe computers, are now routinely done on personal computers with inexpensive software (3). Recent articles in this Journal (4–6) describe such calculations and their effectiveness in the interpretation of experimental data. This experiment combines molecular modeling with a 2D NMR experiment, COSY, to make the complete assignment for the protons of a series of terpenes. In addition to utilizing modern methods, the compounds chosen reflect current work in the field (7, 8). We use this experiment in our instrumental analysis course, but it could also be used in the laboratory accompanying an advanced organic chemistry course. The Experiment The basic experiment involves obtaining the COSY spectrum for one of a series of bicyclic terpenes, verbenone, myrtenal, myrtenol, or α-pinene, and model-
1190
ing the terpene on a workstation or personal computer. CH3 CH3 CH3 H
CH3
R
H
H H
H
H H
H
O
H H
CH3
H
H
R=CHO, myrtenal =CH2OH, myrtenol =CH3, α-pinene
verbenone
These bicyclic[3.1.1]terpenes2 were chosen for examination because their rigidity limits the number of conformations that must be examined by molecular modeling and thereby increases the likelihood that students will be dealing with calculated data that approach reality. In addition, because of the rigidity of the carbon framework, the dihedral angle between several sets of protons is approximately 90°, which causes the coupling constant to be close to 0 Hz. The observation by students that not all adjacent protons possess observable coupling constants reinforces the importance of the Karplus relationship (9). Molecular modeling allows students to identify the protons whose dihedral angles would suggest no observable coupling. The rigidity of the bicyclic[3.1.1]terpene carbon framework also allows the observation of W-coupling between protons that are separated by four bonds, as shown below for the two bridgehead protons in α-pinene. Thus students are required to explain situations in which expected coupling doesn’t occur and unexpected coupling does occur. H
H CH3
Molecular Modeling The structure of the terpene to be examined is modeled using molecular mechanics calculations. While a variety of software exists for molecular mechanics calculations, either PCModel3 on a Macintosh or Sybyl4 on a Silicon Graphics workstation was used in these calculations. The output from the Sybyl calculation has a clearer three-dimensional representation of the molecule than does the output from the PCModel calculation and is easier to manipulate in space. However, the results of the PCModel calculation can be imported into Chem3D,5 which has superior visualization and manipulation capabilities. In addition, the Chem3D program will print a list of dihedral angles, eliminating the need to measure each angle individually.
Journal of Chemical Education • Vol. 73 No. 12 December 1996
In the Laboratory
Figure 1. 1 H NMR spectrum of verbenone.
Figure 2. COSY spectrum of verbenone.
Analysis of the COSY Experiment The theory of 2D NMR spectroscopy is available in a number of references (10) and will not be discussed here. Nor will the details of running the experiment be discussed, since different spectrometers have different programs for running 2D experiments. A sample concentration of 5% (vol/vol) in CDCl3 is appropriate for the experiments. As is always true for the use of CDCl 3, care should be taken in preparing and disposing of the sample because of the known carcinogenicity of chloroform. The 1H NMR spectrum of verbenone is shown in Figure 1, and its COSY spectrum is in Figure 2. The calculated dihedral angles for all of the terpenes are shown in Table 1 and the proton assignments are in Table 2. The numbering systems for carbons and related hydrogens are shown below for the basic framework for myrtanol, myrtenal, and α-pinene. The stereochemical notations s/a (syn and anti) of the substituents refer to the position of the respective group relative to the gemdimethyl bridge. Verbenone differs by the presence of a carbonyl at C-4, rather than Ha and Hs. 9 10
C 3
C
2 4
8 6 C 1
5
7
CH3 R
Hs
CH3 H H
H Ha Ha
Hs
The assignment for verbenone will be discussed in detail and may be used as part of an introduction to 2D NMR, with the students responsible acquiring and interpreting the COSY spectrum for myrtanol, myrtenal, or α-pinene. The assignment begins by choosing a proton whose chemical shift assignment is unambiguous, such as the proton on the double bond, H-3 (5.72 ppm). The COSY spectrum clearly shows coupling between H3 and protons at 2.64, 2.43, and 2.02 ppm. Although there are no protons on adjacent carbons, examination of the structure shows that the protons that might show any coupling through the π-system would be H-1, H-5 and H-10. It is clear that the large singlet at 2.02 ppm belongs to the three protons of H-10. The furthest downfield shift (2.64 ppm) of the two remaining hydrogens belongs to the proton on the α-carbon of the carbonyl (H-5); H-1 has the remaining shift (2.43 ppm). H-5 is coupled to protons at 2.81 ppm and 2.43 ppm. There are two protons on C-7, the only adjacent carbon bearing hydrogens; but an examination of the calculated dihedral angles shows that the dihedral angle between H-5 and H-7a is 96°, which would show a very small coupling constant. The only other relationship that could give coupling is the W-geometry between H-5 and H-1. If H-1 is 2.43, as previously assigned, then H-7s is 2.81. H-1 should show the same types of coupling as seen for H-5, coupling with H-7s and W-coupling with the bridgehead proton, H-5. The proton at 2.81 ppm, H-7s, clearly shows coupling with three protons. Its three couplings are to protons at 2.64 ppm (H-5) and at 2.43 ppm (H-1) with the coupling at 2.08 ppm resulting from geminal coupling with H-7a. Consistent with this analysis, H-7a, which has dihedral angles of approximately 90° with the protons on adjacent carbons, shows only coupling with its geminal proton, H-7s. The resonances remaining, at 1.51 ppm and 1.01 ppm, are due to the bridgehead methyl groups. COSY analysis is unable to resolve this ambiguity. The assignment has been made using lanthanide shift reagents with related compounds (7). We have confirmed this assignment through NOESY spectroscopy (J. L. Malandra and N. S. Mills, unpublished data). In this experiment it is appropriate to ask the students to report ambiguity in the interpretation of the data and ask what experiments would be effective in removing that ambiguity. As revealed by Table 1, analysis of myrtenal, myrtenol, and α-pinene represents a new level of difficulty because of the additional coupling with the protons on C-4. If analysis of the spectrum of verbenone is done first, the analyses of the remaining compounds can be based on it. The complete analysis of myrtenal, myrtenol, and α-pinene will not be described in detail, but the most important aspects of the analysis will be outlined. The analysis of α-pinene begins with the vinyl proton on H-3 and is straightforward. A consideration of the relationship between the dihedral angles of H-7s and H1 and H-5 reveals that, as was true for verbenone, H-7a should only see coupling with H-7s, and should therefore be the only clean doublet in the spectrum, at δ1.16 ppm. A similar situation exists with myrtenal and myrtenol in which identification of the doublet for H-7a allows determination of the chemical shift for H-7s, and rapid assignment of the chemical shifts of all protons. One advantage of using myrtenol is that the spectral window for the aldehyde myrtenal is large enough so that the time required to collect the COSY spectrum is about 50% greater. Because the coupling between the
Vol. 73 No. 12 December 1996 • Journal of Chemical Education
1191
In the Laboratory
aldehydic proton and any other protons in the molecule is very small, one can reduce the time requirements by choosing a spectral window that does not include the aldehydic protons. On the Varian VXR-300, these COSY spectra take about 45 min for data collection with another 20–30 min for determining the optimal conditions for plotting and the actual plotting. This experiment is normally combined with one in which the 90° pulse for the proton is determined, both introducing the students to arrayed spectra and ensuring that the correct 90° pulse is used in the experiment. A lecture normally precedes the acquisition of data, and the explanation of the 2D experiment itself normally can be done in about 45 min, assuming some previous background in pulsed NMR theory. The time required for students to analyze the data varies but most students seemed to adequately assign the spectra in about 2 hours.
Notes 1. CUR (Council on Undergraduate Research) is a society for the advancement of scientific research at primarily undergraduate colleges and universities. For more information contact John Stevens, National Executive Officer, University of North CarolinaAsheville, Asheville, NC (
[email protected]). 2. (–)-Myrtenol and (+)-α-pinene were purchased from Aldrich Chemical Company; 1-verbenone and (–)-myrtenal were purchased from Janssen Chimica. 3. PCModel is available from Serena Software, Dr. Kevin Gilbert, P. O. Box 3076, Bloomington, IN 47402. 4. Sybyl is available from Tripos Associates, Inc., 1699 S. Hanley Rd., St. Louis, MO 63144. 5. Chem3D is available from Cambridge Scientific Computing Computing, 875 Massachusetts Ave., Cambridge, MA 02139.
Literature Cited 1. Wenzel, T. J., Ed. Research in Chemistry at Primarily Undergraduate Institutions; 5 ed. Council on Undergraduate Research: Asheville, NC, 1993. 2. Sauers, R. R. J. Chem. Educ. 1991, 68, 816–818. 3. Bays, J. P. J. Chem. Educ. 1992, 69, 209–215. 4. Simpson, J. J. Chem. Educ. 1994, 71, 607–608. 5. Midland, M. M.; Beck, J. J.; Peters, J. L.; Rennels, R. A.; Asirwatham, G. J. Chem. Educ. 1994, 71, 897–898. 6. Lee, M.; Garbiras, B.; Preti, C. J. Chem. Educ. 1995, 72, 378–380. 7. Badhaj-Hadj, A. Y.; Meklati, B. Y.; Waton, H.; Pham, Q. T. Mag. Res. Chem. 1992, 30, 807–816. 8. Laihia, K.; Kolehmainen, E.; Malkavaara, P.; Korvola, J.; Mänttäri, P.; Kauppinen, R. Mag. Res. Chem. 1992, 30, 754, 759. 9. Karplus, M. J. Am. Chem. Soc. 1963, 85, 2870–2878. 10. Williams, K. R.; King, R. W. J. Chem. Educ. 1990, 67, A125–A137.
Acknowledgments I would like to thank James Malandra for helpful discussions and assistance in plotting the COSY spectrum and Michelle Bushey and her Instrumental Analysis class for testing the experiment.
Table 1. Calculated Dihedral Angles, in Degrees Compound
H1-H7a
H1-H7s
H3-H4s
H3-H4a
H4s-H5
H4a-H5
H5-H7a
H5-H7s
verbenone
–95.95
33.33
—
—
—
—
96.30
–33.00
myrtenol
–96.03
32.95
–58.24
60.22
60.47
–58.31
98.01
–31.22
myrtenal
–96.50
32.50
–58.35
60.06
60.50
–58.20
97.95
–31.30
α-pinene
–96.04
32.95
–58.49
59.97
60.56
–58.20
-97.97
–31.23
Table 2. Proton Chemical Shiftsa Compound
H2
H3
H4s
H4a
H5
H7s
H7a
H8
H9
H10
verbenone (exp) 2.43
—
5.72
—
—
2.64
2.81
2.08
1.51
1.01
2.02
Reference (1)
2.42
—
5.72
—
—
2.64
2.80
2.07
1.50
1.01
2.02
myrtenal (exp)
2.86
—
6.72
2.57
2.57
2.19
2.50
1.06
1.34
0.75
9.44
Reference (1)
2.86
—
6.71
2.56
2.56
2.19
2.48
1.05
1.34
0.74
9.44
Reference (2)
2.86
—
6.70
2.52
2.58
2.19
2.48
1.04
1.33
0.73
9.43
myrtenol (exp)
2.13
—
5.47
2.29
2.26
2.13
2.40
1.18
1.29
0.84
3.98 1.70 (OH)
Reference (1)
2.13
—
5.47
2.27
2.27
2.13
2.40
1.17
1.29
0.83
3.98 1.68 (OH)
Reference (2)
2.15
—
5.48
2.31
2.28
2.11
2.41
1.18
1.30
0.84
3.99
α-pinene (exp)
1.92
—
5.18
2.21
2.17
2.05
2.32
1.16
1.23
0.83
1.65
Reference (1)
1.93
—
5.18
2.21
2.17
2.06
2.33
1.15
1.26
0.83
1.65
Reference (2)
1.89
—
5.14
2.19
2.12
2.03
2.29
1.11
1.22
0.80
1.66
a
1192
H1
other
—
Spectra run in DCCl3, on a Varian VXR-300 NMR spectrometer. Sample concentration was 5% terpene/CDCl3 , vol/vol.
Journal of Chemical Education • Vol. 73 No. 12 December 1996
