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Exp't 205Conformational Analysis of Sugars by

1H NMR Spectroscopy and PolarimetryAdapted by R. E. Krull, M. Cocco, A. Freyer, and R. Minard from "Application of NMR to Biochemical Kinetics" by E. N. Drake and C. E. Brown, J. Chem. Ed., 54

(1977), 124-7. Revised 10/19/00

Introduction:The conversion of the six carbon sugar, α-D-(+)-glucose, to its "anomeric" form, β-D-(+)-glucoseproceeds as shown below:

O

H

HO

HO

HH

CH2OHH

H

OHOH

OH

H

HO

HO

HH

CH2OHH

O

H

OH

O

H

HO

HO

HH

CH2OHH

OH

HOH

H+

β: -OH equatorial

α: -OH axial

6

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32 1

a-D-(+)-Glucose(hemiacetal form)

[a]D = +112.2°

OpenAldehyde

Intermediate

β-D-(+)-Glucose(hemiacetal form)

[α ]D = +18.7°

At room temperature this interconversion of the α hydrogen from equatorial to axial occurs slowly in water,or rapidly if a small amount of base catalyst is added. You will be using two methods to observe theproduction of β-D-(+)-glucose from α-D-(+)-glucose; these are proton nuclear magnetic resonance (1HNMR) spectroscopy and polarimetry. 1H NMR Theory:This technique is used to provide information on: 1) the chemical environment surrounding a hydrogennucleus (chemical shift) and 2) other hydrogen nuclei connected through bonds (J coupling). The 200MHz NMR spectrum of D-(+)-glucose is shown below:

Downfield region Upfield Region

Electronic shielding and/or deshielding affects the magnetic environment around the hydrogen nucleus.For example, the electrons in a carbon-carbon bond will produce shielding (+) and deshielding (-) zones.(see next page) If the hydrogen nucleus of interest is positioned in a region of shielding (+), you wouldexpect it to have an upfield chemical shift. Similarly, if the hydrogen nucleus is in a deshielding region (-) itshould move the chemical shift downfield. This can help you to determine whether a hydrogen is in anaxial or equatorial position based on its chemical shift value.

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Additionally, if electronegative atoms such as oxygen are bonded to the carbon with the hydrogen ofinterest, they will deshield the hydrogen nucleus. For this reason, the α or β hydrogens in glucose will beshifted downfield from all of the other glucose 1H signals since the α or β H's are the only hydrogen nucleiare on a carbon which is bonded to two oxygens rather than one.

Another means to distinguish the axial and equatorial hydrogens comes from the coupling constants (Jvalues). Coupling is an interaction between nuclei which are connected through bonds. The effect ofcoupling is to split the NMR signal into n+1 peaks, where n is the number of hydrogen nuclei within fivebonds. The distance between the peaks (in Hertz) is the coupling constant and this value depends onboth the number of bonds separating the nuclei and their conformation. Since the C-1 hydrogen changesconformation during the interconversion of α- to β-D-(+)-glucose, the dihedral angle between the H on C-1 and the H on C-2 changes, and therefore the coupling constants change. For simple systems theKarplus equation can be used to estimate the conformation of two coupled nuclei. Shown below, a plotof the Karplus equation indicates that the coupling constant should have the smallest value at a dihedralangle of approximately 85o and the largest value at angles of 0o and 180o.

Coupling Angle, φ Calc. J ( Hz) Observed J

ax/ax 180 9 Hz 8 to 14 Hz

ax/eq 60 1.8 Hz 1 to 7 Hz

eq/eq 60 1.8 Hz 1 to 7 Hz

Exp't 205

HH

O

OHCHO

HO

H

O

H

CHO

φ = 180°

φ = 60°

βform

αform

View down C1-C2 bond:

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Experimental for NMR ( 2 team members do this, but all should observe):

If possible, the NMR analysis will be run on the 200 MHz FT-NMR in room 7 Chandlee Lab. Since thefirst spectrum should be taken as quickly as possible after dissolving the α-D-(+)-glucose, do not add theglucose to the D2O until the instrument has been adjusted.

Weigh 40 mg α-D-(+)-glucose into a graduated reaction tube. Go to Chandlee lab with an instructorbringing along the D2O , a pipet, and an NMR tube. Once the spectrometer is ready, add the 0.75 mLD2O to the reaction tube. Mix the solution thoroughly by continuously pulling part of the solution up into apipet and then pushing it out until all of the sugar is dissolved. Record the time as the start of reaction.Pipet the solution into an NMR tube, and collect a 200 MHz spectrum as quickly as possible. Take aspectrum every 15 to 30 minutes for the next 2.5 hours. Since the most rapid change takes place at thestart of the process, it is important to follow one sample closely, obtaining an NMR spectrum every 15min., for the first hour. Then D2O can be added to the second sample and its NMR taken every 15 minfor an hour. Finally, obtain an NMR of the first sample run, and then of the second. Record the integrals ofthe α and β-D-(+)-glucose anomeric protons, which occur at 5.1 ppm and 4.5 ppm, respectively. Duringthe next lab period (2 or 5 days later) collect a final spectrum with the equilibrium integral values. Recordthe integrals, and calculate the mole fraction and the equilibrium constant. All team members should turn inan initial and a final spectrum with their lab reports.

Polarimetry

Be sure the polarimeter in 206 Whitmore has been on for at least 15 minutes so that it is stable. Put anote on it that you will be using it for the entire lab period. [NOTE: It would be appreciated if this analysiscould be done Mon-Fri AM or Fri. PM to avoid tieing up the polarimeter for an entire lab period.] As theinterconversion between the α form to the β occurs, the polarimeter readings indicate a steady movementtoward an equilibrium value. Therefore, the readings will be constantly fluctuating. However, you do needto obtain a reading just as quickly as possible after placing the solution in the polarimeter cell. Therefore,try to obtain your first reading as soon as the reading stabilizes at the tenths position, that is + 0.1°. Sincethe equilibrium solution is not a 50/50 mixture of the α and β forms, the final rotation is a weightedaverage from which one can calculate the mole fraction. Remember that the polarimeter readings have aconcentration correction factor (to convert readings to specific rotation) which is given in the polarimetry partof the Product Analysis section of your lab guide.

Experimental for Polarimetry (remaining team members do this, others observe):

Weigh out 480 mg of α-D-(+)-glucose in a small Erlenmeyer flask. Pipet 4.0 mL of D2O into the flask.Mix the solution thoroughly by continuously pulling part of the solution up into a pipet and then pushing itout until all of the sugar is dissolved. Record the time as the start of reaction. Pipet about 2 mL of thesolution into the polarimetry cell as described in your lab manual instrumentation section. Measure andrecord the rotation initially and then every 15 minutes for the next 2.5 hours. When finished for the day besure to rinse the polarimeter cell out carefully with water and again with methanol. Blow dry with N2stream. Store the remaining 2 mL of the solution in the original mixing flask until the next lab period. Atthat time you will measure and record the final equilibrium rotation. Then weigh out 240 mg of β-D-(+)-glucose and dissolve it in 2.0 ml. of D2O. Measure the rotation in the polarimeter as quickly as possible.Knowing the initial rotations of the α form and the β form, as well as the equilibrium value, one can calculatethe mole fraction of each as well as the equilibrium constant. When finished for the day, be sure to rinsethe polarimeter cell out carefully with water and again with methanol and dry.

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Final Report:

Include and analyze all team data taken by NMR and by polarimetry in your individual report. Study theNMR's and determine which protons are showing a change in chemical shift. Mark these on your finalspectrum. Do the direction of the chemical shifts and the coupling constants support the above discussionabout shielding and the Karplus correlation? Explain. One cannot see the aldehyde proton on the openaldehyde intermediate in these spectra; why not?

Draw the structures of the α and β forms of glucose. Explain why equilibrium favors the β form in solution.

Plot two curves on one graph with time as the X-axis and the mole fraction of β-D-(+)-glucose as the Yaxis. Label which curve comes from NMR data and which comes from polarimetry data. Also includedshould be the calculation of the equilbrium constant, Keq.Help:

mole fraction of β form = (integral of β anomeric proton) by NMR(integral of α + β anomeric protons)

and, using specific rotations,

mole fraction of β form = (initial rotation of α - observed rotation) by Polarimetry(initial rotation of α - initial rotation of β)