In the Laboratory
Glycosidation of Methanol with Ribose: An Interdisciplinary Undergraduate Laboratory Experiment Erin Simon Biogen Idec, 14 Cambridge Center, Cambridge, Massachusetts 02142 Katie Cook Molecular Medicine and Translational Science, Wake Forest University, School of Medicine, Winston-Salem, North Carolina 27157 Meredith R. Pritchard Musculoskeletal Sciences Research Center, Department of Orthopedic Surgery, SUNY Upstate Medical University, Syracuse, New York 13210 Wayne Stripe Theken Spine, 1800 Triplett Boulevard, Akron, Ohio 44306 Martha Bruch and Kestutis Bendinskas* Department of Chemistry, SUNY at Oswego, Oswego, New York 13126 *
[email protected] The chemistry of carbohydrates is complicated and beautiful, and this exercise offers a glimpse of it to the upper-division undergraduate chemistry students. In this experiment, methylation of an anomeric hydroxyl group of ribose is performed, and the kinetic and thermodynamic products of the reaction are identified using proton nuclear magnetic resonance (1H NMR) spectroscopy. The results of this acid-catalyzed reaction under two different sets of reaction conditions brings some surprises to be discussed with the class, challenges students to understand the subject of carbohydrates in depth, and brings an appreciation for the complexity of the system studied. The experiment can be performed as a single 3-h laboratory session or as a series of sessions if students collect their own NMR spectra and use longer reaction times. In this Journal and other educational literature, the subject of sugars in the laboratory has been discussed extensively. Potentiometric (1) and HPLC-electrochemical detection (2), basic Cu2þ-EDTA (3), and DNS reagents (4), among many, can be used for determination of sugar concentrations in different samples. Chemical and biochemical sugar detection methods have been recently compared for the detection of sugars in drinks (5). The biosynthesis of ethanol from starch is sometimes used as an example of carbohydrate chemistry (6, 7). Qualitative identification (8, 9), oxidation, reduction, and decarboxylation (9) of sugars can be used in teaching laboratories as well. Acetylation of cellulose by acetic anhydride is possible (9) and so is the production of glucose pentaacetates (10). A reaction of isopropanol with an acetylated glucal leads students, via modeling, toward an understanding that an SN1 mechanism is involved in glycosidation (11). Unlike any of the examples above, our laboratory exercise offers a possibility for students to follow the
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time course of a safe microscale glycosidation reaction using 1H NMR spectroscopy. Procedure Five milliliters of 0.1 g/mL ribose in HPLC grade methanol is allowed to react in the presence of an acidic catalyst, Dowex 50W X8 resin (12) for a specific time either at room temperature or at the temperature of refluxing methanol, 65 °C. The solvent is removed in vacuo, and the products are dissolved in D2O and analyzed by 1H NMR spectroscopy at 30 °C.1 The percentages of all detectable products are calculated using peak integrals. Each student runs a reaction at one or both temperatures, and the class data are pooled to get information for reactions performed for different times. The piece of equipment that might not be available in every chemistry department is a 300 MHz NMR spectrometer with a variable temperature probe. A rotary evaporator for every 4-6 students in the laboratory is desirable. Hazards Methyl alcohol is poisonous and flammable. Reflux reactions should be performed in hoods. Dowex 50W X8 resin and calcium chloride may cause irritation; handle them carefully. Deuterium oxide and D-ribose present no hazard. Results and Discussion Hemiacetal formation for D-ribose is shown in Figure 1. It yields five- or six-membered rings in either alpha or beta forms. The ribose monomer exists in all five forms, its linear form being
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r 2010 American Chemical Society and Division of Chemical Education, Inc. pubs.acs.org/jchemeduc Vol. 87 No. 7 July 2010 10.1021/ed100196w Published on Web 04/30/2010
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In the Laboratory
Figure 3. Glycosidation reaction run at room temperature. The data were collected by a class of 18 students. Nonmethylated riboses disappear; methyl ribofuranosides are formed as kinetic products.
Figure 1.
D-Ribose
in its linear and cyclic forms.
Figure 4. Percent composition of methyl ribosides for reactions run at 65 °C for extensive times. Every data point is run in triplicate; standard deviations are shown as error bars. Methyl furanosides are replaced by methyl pyranosides, thermodynamic products.
Figure 2. 1H NMR spectra in D2O at 30 °C of ribose methylation reaction that was run at 65 °C for 75 min. Signals of the anomeric hydrogen species present in the equilibrium mixture are shown.
of negligible quantity (R-ribofuranose-5.2%, β-ribofuranose17.4%, R-ribopyranose-11.6%, β-ribopyranose-65.8%).2 Under the acidic conditions used in this reaction, methanol attaches to the carbohydrate at the anomeric carbon in the alpha or beta position. Only the anomeric hydroxyl's modification by methanol is addressed throughout this article. There are eight species present in the reaction mixture, the four cyclic forms of ribose shown in Figure 1 and the four corresponding methyl ribosides being formed. This mixture can be quantitatively analyzed by 1H NMR spectrometry as shown in Figure 2. The reaction of methyl-glycosidation, as described in many texts (13), takes place as the following sequence: the protonation of an anomeric hydroxyl, the departure of water, the formation of a resonance-stabilized carbocation, the addition of methanol to an anomeric carbon, and the departure of the proton, yielding a methyl acetal of ribose. Considering this and also the overwhelming abundance of pyranosides in the equilibrium mixture 740
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before the reaction (77.4%), one might expect that most of the products early in the reaction would be methyl pyranosides. However, the results disprove this assumption. When the reaction is done at room temperature with relatively little quantity of catalyst, the kinetic products of this reaction are methyl furanosides, as shown in Figure 3. Methyl pyranosides are not detected. Different products are obtained when this reaction is run for a long time (hours versus minutes), indicating that the thermodynamic products are different from the kinetic products. The data shown in Figure 4 clearly demonstrate that the thermodynamic products of ribose methylation are methyl pyranosides. This part of the experiment cannot be run in a single laboratory session, thus, this data could be provided in the laboratory handout. Alternatively, students can set up runs to go for multiple days. To see the thermodynamic products of this reaction within one laboratory period, the process could be sped up by heating to reflux and adding more catalyst. The trends can be seen in the 1-h reaction (Table 1), although not as clearly as shown in Figure 4. The percentage of methyl ribopyranosides increases with time as methyl furanosides are converted to methyl pyranosides. The results provide the instructor an opportunity to discuss the concepts of kinetic control versus thermodynamic equilibrium with the class. It should be noted that stability of the carbohydrates is a function of annular strain similar to cycloalkanes but it also depends on multiple interactions among hydroxyl groups and hydrogen bonds among them. Overall, students are
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In the Laboratory
Students recommended the glycosidation experiment to be continued as part of the course in the future.
Table 1. Products of the Glycosidation Reaction a
Reaction Products (%) Methyl Methyl Methyl Methyl Time/min R-furanoside β-furanoside R-pyranoside β-pyranoside 30
21.4
51.5
4.82
12.6
60
20.0
47.7
6.90
18.1
Acknowledgment The authors thank Jim MacKenzie for his help and insight. Notes 1. At room temperature, the solvent peak overlaps with other signals. 2. The instructors measure the quantities of the ribose monomer and provide the data to the students.
a
Reaction was run in refluxing methanol at 65 °C. The data were collected by a class of 18 students. Methyl ribofuranosides are replaced by methyl ribopyranosides, thermodynamic products.
able to explore the glycosidation reaction in its full complexity, observe that alpha forms are less stable than beta forms, and realize that the kinetic products are methyl furanosides, whereas thermodynamic products are methyl pyranosides. Additional Class Discussion Instructors may choose to discuss two additional advanced questions with the class. First, why are methyl furanosides and not methyl pyranosides the kinetic products of this reaction? Data collected by the students seem to indicate that methyl pyranosides may have a high-energy transition state, and thus, methyl furanosides are made first and then undergo a rearrangement reaction into methyl pyranosides. To provide a detailed answer to this question, one may need to consider the mechanism of the reaction and the relative stability of possible transition states, think about the open-form and cyclic carbocations, the gauche interactions among multiple groups of the ribose molecule, use models, and estimate the relative stability of species present in the reaction mixture if the appropriate software is available (11, 13-18). Second, biochemistry students may wonder why nature selects furanosides and not pyranosides as part of the structure of one of its most important group of biomolecules, ribonucleic acids (19), when these experiments prove that pyranosides are thermodynamic products of this reaction. One may suggest the importance of the flexibility of a structure as a possible answer (16, 20). Students should be encouraged to use research literature to answer both of these questions. Research literature in this area of science is extensive; however, mainly educational literature was reviewed in this publication. It is important to note that the use of a solid acidic catalyst in glycosidation (12), which can be quickly filtered off, made the study of this reaction feasible for the undergraduate laboratory. Summary This interdisciplinary laboratory experiment can be implemented in almost any upper-level chemistry course and provides students with the hands-on experience with the topics of glycosidation, hemiacetal and acetal formation, NMR spectroscopy, and kinetic and thermodynamic product formation. The students in the class stated that the laboratory handout introduction made material relevant, that the directions were presented in a clear manner, the session went as planned, and the interdisciplinary nature of investigation was evident (see the supporting information for the evaluation form and results).
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Literature Cited 1. Moresco, H.; Sanson, P.; Seoane, G. J. Chem. Educ. 2008, 85, 1091–1093. 2. Luo, P.; Luo, M. Z.; Baldwin, R. P. J. Chem. Educ. 1993, 70, 679–681. 3. Sandell, A. J. Chem. Educ. 1994, 71, 346. 4. Bittman, R. J. Chem. Educ. 1974, 51, 46–47. 5. Miloski, K.; Wallace, K.; Fenger, A.; Schneider, E.; Bendinskas, K. Am. J. Undergrad. Res. 2008, 7, 7–18. 6. Pelter, M. W.; McQuade, J. J. Chem. Educ. 2005, 82, 1811–1812. 7. Pavia, D. L.; Lampman, G. M.; Kriz, G. S.; Engel, R. G. Introduction to Organic Laboratory Techniques, 3rd ed.; Sauders College Publishing: Fort Worth, TX, 1999. 8. Militzer, W. E. J. Chem. Educ. 1941, 18, 25–28. 9. Zanger, M.; McKee, J. R. Small Scale Syntheses: A laboratory Textbook of Organic Chemistry; Wm. C. Brown Publishers: Boston, MA, 1995. 10. Mohrig, J. R. Experimental Organic Chemistry: A Balanced Approach, Macroscale and Microscale; W.H. Freeman and Company: New York, 1998. 11. Bedell, B. L.; Crouch, R. D.; Holden, M. S.; Martinson, H. E. J. Chem. Educ. 1996, 73, 1041–1042. 12. Podlasek, C. A.; Wu, J.; Stripe, W. A.; Bondo, P. B.; Serianni, A. S. J. Am. Chem. Soc. 1995, 117, 8635–8644. 13. Solomons, T. W. G.; Fryhle, C. B. Organic chemistry, 8th ed.; John Wiley and Sons: Hoboken, NJ, 2004. 14. Church, T. J.; Carmichael, I.; Serianni, A. S. J. Am. Chem. Soc. 1997, 119, 8946–8964. 15. Serianni, A. S.; Bondo, P. B. J. Biomol. Struct. Dyn. 1994, 11, 1133–1148. 16. Levitt, M.; Warshel, A. J. Am. Chem. Soc. 1978, 100, 2607–2613. 17. Bishop, C. T.; Cooper, F. P. Can. J. Chem. 1962, 40, 224–232. 18. Nowacki, A.; Blazejowski, J.; Wisniewski, A. J. Mol. Struct.: THEOCHEM 2003, 664-665, 217–228. 19. Voet, D.; Voet, J. G.; Pratt, C. W. Fundamentals of Biochemistry: Life at the Molecular Level, 3rd ed.; John Wiley and Sons: Hoboken, NJ, 2008. 20. Meints, G. A.; Karlsson, T.; Drobny, G. P. J. Am. Chem. Soc. 2001, 123, 10030–10038.
Supporting Information Available Student handout; instructor's notes; student evaluations. This material is available via the Internet at http://pubs.acs.org.
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