Pre-lab assignment – Due March 23rd, 5 pm to D2L Dropbox. 1 H-NMR Based Mono- and Disaccharide Configurational Analysis and Detection of β-Glucosidase Activity Provide precise but complete answers to the following questions. You must present spectra and structure in proper scientific (chemical) format with appropriate labeling and units for full credit. This exercise will guide you through a review of carbohydrate structures and how these structures are manifested in NMR spectra. You may use the Biochemistry course textbook or other resources to answer the following questions but make sure you understand the concepts. You are encouraged to work together on this assignment, but write out and turn in your own work. 1. Draw the two possible chair conformations of β-D-glucose. In your structures, include all –H and –OH attachments to carbon atoms. Identify the more stable conformation and justify your answer. Repeat for -D-glucose. 2. Redraw the two stable chair conformations for the alpha and beta forms that you have identified above (Only the more stable forms will appear in an NMR spectrum). a. Circle any protons directly bound to the anomeric carbon. Would you expect an NMR peak for these protons to appear at a higher or lower chemical shift than protons bound to other carbon atoms? Why? b. Place a box around any protons that you expect to couple with the “anomeric” protons and cause spin-spin splitting. What splitting pattern do you expect to observe for the signal associated with the “anomeric” proton? c. Note the relative positions, equatorial or axial, of the two interacting protons in each structure. Next to the “anomeric” proton, write the size of the splitting you expect for each, using the guideline that the coupling constants for “axial/axial” protons are expected to have splitting constants of 7-9 Hz, while axial/equatorial proton splitting should be 2-4 Hz. * d. Draw the 1H-NMR spectrum that you would expect to see in the anomeric region (considering that a sample of glucose, dissolved in water at room temperature and allowed to come to equilibrium, consists of a mixture of alpha and beta forms in a ratio of approximately 40:60. Assume that the chemical shift for a proton in the axial position is expected to have a somewhat lower chemical shift that a proton in an equatorial position. * 3. Draw chair conformers of cellobiose (glucose-β-1,4-glucose), and maltose (glucose-α1,4-glucose) as shown in the experiment worksheet but replace the wavy lines with the two different anomeric conformations possible at that carbon. (You will draw four structures in all; two each for cellobiose and for maltose.) a. On each of the four diagrams, circle any protons that you expect to appear in the anomeric region of a 1H-NMR spectrum. b. Given that the NMR spectrum for cellobiose will include signals from both anomers that you drew, indicate how many different peaks will appear in the anomeric region of the spectrum of cellobiose? What splitting pattern do you expect for each of these peaks? Answer the same questions for maltose. 4. Using structures show the hydrolytic breakdown of cellobiose and maltose. 5. Identify the type of glucosidase enzyme that is required for the breakdown of cellobiose and of maltose. Explain your answer in terms of enzyme specificity. * Gurst, J. E. NMR and the Structure of D-glucose. J. Chem. Educ. 1991, 68, 1003-1004.