In the Laboratory
Use of 1H NMR in Assigning Carbohydrate Configuration in the Organic Laboratory
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John L. Sorensen,* Ross Witherell, and Lois M. Browne Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2G2; *
[email protected] Since the advent of NMR spectroscopy in the late 1950s, this instrumental technique has emerged as the organic chemist’s most important tool for structural elucidation at all levels of teaching and research (1, 2). Correspondingly, it has become increasingly important that students of chemistry be exposed to this spectroscopic technique as part of the undergraduate curriculum. The student can best become familiar and comfortable with NMR spectroscopy if he or she encounters it as an integral part of the laboratory experience. As such, the theory and application of NMR spectroscopy is the principal subject of the laboratory component of the first-term advanced organic chemistry course. Each week, the student is introduced to a new topic in NMR spectroscopy, by way of a short lecture that combines theory and practical examples to illustrate the concept. Following the lecture, the student performs a short organic chemistry experiment and the product is analyzed by high-resolution FT-NMR spectroscopy. The student is responsible for sample preparation and submission, as well as data processing and analysis, and thus gains an important practical appreciation for the NMR experiment. The chemistry experiments performed by the student are carefully chosen so as to be relevant to the NMR topics being introduced. In particular, the concepts of coupling constants and 2D NMR (1H–1H COSY) are introduced as part of an experiment involving simple transformations of carbohydrates. It has recently been demonstrated (3) that carbohydrate chemistry can be the basis of an instructive undergraduate experiment and that experiments can be devised that yield products quickly, cleanly, and in reproducible yield. Carbohydrates make excellent models for the introduction of coupling constants and 2D NMR, as these concepts are particularly useful in the structural determination of the products obtained by the students. As a result of the laboratory experience, the student becomes exposed to an important branch of organic chemistry and at the same time gains practical insight into modern spectroscopic techniques. This article describes the synthesis of a peracetyl sugar molecule and the use of 1H and 1H–1H COSY NMR spectroscopy to assign configuration.
are saved in a dedicated directory on the main NMR data server. Modern NMR processing software enables students to receive the raw FID data obtained from their samples, and to Fourier transform, process, and print their own spectra. The FID data from student 1H NMR samples are acquired on Varian Innova NMR spectrometers (300 or 400 MHz) and each student has remote access to the raw NMR data through dedicated terminals located in a computer lab near the organic chemistry teaching lab. This experiment was included at the end of the laboratory course, as it is intended that students should apply the cumulative knowledge obtained from previous lab classes in the analysis of their data. The tutorial session presented with this experiment expands upon the importance of the coupling constant and culminates with an illustration of the use of the Karplus (4) equation in conformational analysis of rigid systems. It begins with a review of the use of 1D 1H NMR in structure determination and conformational analysis and introduces the analysis of 2D NMR (1H–1H COSY). The remainder of the four-hour laboratory class involves student preparation of the peracetyl derivatives of either α-methyl galactopyranoside (1) or α-methyl glucopyranoside (2), followed by purification using flash column chromatography (5). Each student is given a sample (1 mmole) of either compound 1 or 2 as an unknown and performs an acetylation of this unknown using acetic anhydride and pyridine (Scheme I). The crude product of the reaction, a mixture of the peracetyl sugar and the products of incomplete acetylation, is purified by flash column chromatography.
HO OH
AcO OAc
1. Ac2O, pyridine
O HO
2. chromatography
HO OCH3 1 α-methyl galactopyranoside
O AcO AcO
OCH3 3 peracetyl α-methyl galactopyranoside
Background and Procedure OAc
OH
The first-term laboratory portion of the course includes ten four-hour sessions, and each session includes one hour of instruction in NMR interpretation. 13C NMR spectroscopic data are provided as part of the assignment for most lab work. Emphasis in the lab course is focused on the NMR experiment—the student is responsible for sample preparation, processing student FID data, and spectral interpretation. The raw FID data, acquired by spectral services staff in the chemistry department from student-submitted samples,
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HO HO
1. Ac2O, pyridine
O
2. chromatography HO
OCH3
2 α-methyl glucopyranoside
AcO AcO
O
AcO OCH3 4 peracetyl α-methyl glucopyranoside
Scheme I. Acetylation of compound 1 or 2 using acetic anhydride and pyridine.
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In the Laboratory
Each student submits his or her own purified sample to the NMR technical staff for 1H NMR data acquisition. Within 24 hours, the unprocessed FID data are made available to the student via the chemistry department network, and the student is then responsible for the hands-on processing, printing, and interpretation of the 1H NMR. Paper copies of both the 2D COSY (300 MHz) and 13C NMR (125 MHz) spectra are provided to the student. Whereas the 1D 1H NMR data corresponds to each student’s individual sample, the 2D COSY and 13C NMR spectra are acquired from authentic commercial material; the students were not responsible for the online data processing for these experiments. Hazards Acetic anhydride is an irritant. Long-term exposure to pyridine can cause sterility in males. Sulfuric acid is corrosive. Dichloromethane can be narcotic at high concentrations and is a suspected carcinogen. Students should work in the fume hood and avoid all contact with reagents. Inhalation of silica gel should be avoided. Visualization of the TLC plates requires phosphomolybdic acid dip, which is extremely corrosive. Results and Discussion The procedure used in this experiment yields about 300 mg of pure peracetyl α-methyl glycoside. The 1 mmol scale is convenient for this experiment, producing a quantity of product that is easily manipulated without generating a large quantity of waste. The workup and purification steps were designed to reflect techniques used in the modern synthetic organic chemistry laboratory. Successive washes are carried out using a separatory funnel to remove first the pyridine and then any acetic acid that may be present. Purification by flash chromatography is an instructive alternative to the recrystallization procedures commonly encountered by students of organic chemistry at this level. Special care is taken in the disposal of the pyridinium sulfate generated in the experiment, separating it from other aqueous wastes with proper disposal handled by the waste management services. The remainder of the aqueous waste can be safely disposed down the drain. The primary goal of the student is to determine the identity of the unknown sugar, either the peracetyl galactose sugar derivative (3) or the peracetyl glucose derivative (4). This is accomplished by analysis of the coupling constants observed for the H4 proton. In the case of the glucose derivative, both protons that couple to H4 are in an axial–axial relationship and have relatively large coupling constants (ca. 10 Hz) as predicted by the Karplus equation. In the galactose derivative, where the relationship between adjacent protons is axial–equatorial, the coupling constant observed in the NMR spectrum is considerably smaller (< 3 Hz), again in accordance with the Karplus equation. On this basis the students are readily able to distinguish between the two isomeric sugar molecules. The key to solving the prob-
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lem is realizing that the anomeric proton is the only signal displayed as a doublet in the NMR spectrum. This identifies a ‘jumping off ’ point that allows for assignment of the rest of the proton resonances. As stated above, each student submits a product sample for 1H NMR analysis, affording an opportunity to assess the purity of the chromatographed product. One advantage of allowing the student to process his or her own 1H NMR spectrum is that the student is encouraged to identify which regions of the spectrum are of particular importance for the stereochemical assignment of the carbon bearing H4. The student can then generate the appropriate expansions for the accurate measurement of the relevant coupling constants. The 2D COSY facilitates the identity of the coupling partners, but is not entirely necessary since it is possible to make the assignment based on the value of the coupling constants. This allows for the introduction of the 2D technique without the outcome of the structure assignment depending on the interpretation of these complex spectra. The use of the peracetyl sugar derivative in this experiment eases the analysis of its 1D NMR spectrum since the signals of the hydrogens are downfield and spread over a larger range compared to the 1D NMR spectrum of the free sugar. The 13C spectrum of the acylated sugar product is also provided to the student, since this spectroscopic method is part of the course and laboratory curricula. The students are expected to be able to identify unambiguously, on the basis of chemical shift, which signals correspond to the anomeric carbon and the methoxy carbon. The students should also be able to identify groups of signals corresponding to the four ester carbonyl carbons, the four methyl carbons of the acetyl groups and the five remaining sp3-hybridized oxygen-bearing carbons. To receive full credit, the student must recognize that within these groups of signals, individual unambiguous assignments cannot be made without further spectroscopic data. This experiment could easily be adapted to incorporate 2D 13C–1H HMQC spectroscopy to accommodate further assignment of the five oxygen-bearing sp3hybridized methylene and methyne carbons, although this has not been undertaken at this institution. The identification of an unknown α-methyl pyranoside experiment has been successfully performed in our undergraduate laboratories for three years, by approximately 120 students each year, and is normally completed within the allotted 3 hours. Typically, the three hour lab commences with an overview of the experiment and a brief review of safety considerations. After the first hour of lab work 80–90% of the students have completed the acetylation and are working up the product in preparation for chromatography. After the second hour, the majority of students have collected the product fractions from the column, and the remaining hour is sufficient for the remaining tasks: isolation of purified product by rotary evaporation, preparation and submission of an NMR sample, acquisition of an FT-IR spectrum of the product, and cleanup. The students typically obtained about 300 mg of product, a quantity more than sufficient for the required spectroscopic analysis.
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In the Laboratory
Conclusions
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This experiment is a useful addition to the curriculum of a third-year advanced organic chemistry laboratory course. It offers a fast, convenient reaction along with a straightforward workup procedure. The product is easily purified using flash chromatography as it is the least polar product formed in the reaction. The experiment reinforces organic chemistry laboratory techniques and challenges the students to apply their newly acquired skills in the analysis of NMR spectra to determine the identity of the unknown sugar. This is readily accomplished using conformational analysis derived from 1D 1 H NMR spectra. Additional information obtained from interpretation of 2D 1H–1H COSY can be of assistance, but is not required.
Handouts from the student lab manual and from the TA manual and the various NMR spectra are available in this issue of JCE Online.
Acknowledgments Financial support for this work was provided by the University of Alberta. We thank Albin Otter (University of Alberta) for assistance in developing the remote access NMR system and spectral services at the University of Alberta for acquiring spectra. We also thank Ole Hindsgaul (University of Alberta) for helpful suggestions and discussions.
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Supplemental Material
Literature Cited 1. Alexander, C. W.; Asleson, G. L.; Doig, M. T.; Heldrich, F. J. J. Chem. Educ. 1999, 76, 1294–1296. 2. Sanders, J. K. M.; Hunter, B. K. Modern NMR Spectroscopy, 2nd ed.; Oxford University Press: Oxford, UK, 1993. Derome, A. E. Modern NMR Techniques for Chemistry Research; Pergamon Press: Oxford, UK, 1987. 3. Callam, C. S.; Lowary, T. L. J. Chem. Educ. 2001, 78, 73– 74. Norris, P.; Freeze, S.; Gabriel, C. J. J. Chem. Educ. 2001, 78, 75–76. Cunha, A. C.; Pereira, L. O. R.; de Souza, M. C. B. V.; Ferreira, V. F. J. Chem. Educ. 1999, 76, 79– 80. 4. Karplus, M. J. Chem. Phys. 1959, 30, 11–13. Silverstein, R. M.; Webster, F. X. Spectrometric Identification of Organic Compounds, 6th ed.; Wiley: New York, 1998; pp 185–187. 5. Still, W. C.; Kahn, M.; Mitra, A. J. Org. Chem. 1978, 14, 2923–2925.
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