Carbon

Glucose Anomers

Conformational Analysis by NMR

Glucose in solution remains mostly in the cyclic pyranose form in two conformational anomers shown in the
figure above. These two forms interconvert via an aldehyde chain. The anomeric rotation can be observed
by monitoring the 1H NMR spectrum of the anomeric protons shown in red above. These protons inherit
two different characteristics due to their local environment: chemical shift due to shielding from the external
magnetic field and J-coupling (JH-H) from protons on the adjacent carbon (C2). The additional shielding of
the oxygen shifts the anomeric protons down field from the rest of the non­‐exchanging protons allowing
us to resolve them from the others, which occur between 3 and 4 ppm. In addition the equatorial (alpha)
anomeric proton resonates further down field (5.1 ppm) from the axial (beta) proton (4.5 ppm) making these
two anomer populations distinguishable by 1H NMR even at low field.
The spectrum below was collected on deuterium exchanged glucose to decrease the HDO signal and
heated to 90° C to shift it to 4.3 ppm. The spectrum shows the two anomers clearly resolved. The natural
distribution is 36% alpha and 64% beta, which is represented in the relative integrals of the two NMR peaks.

5.24

4.64

4.31

Beta
Alpha

HDO
0.37

0.63

6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5

f1 (ppm)
The second distinguishing characteristic is the spin-spin coupling (JH-H). Non-equivalent protons on the ad-
jacent carbon (C2) will couple to the anomeric proton creating a doublet. In rigid molecules where rotational
averaging is negligible, this coupling will not average out and will depend on the dihedral angle between the
two coupled protons. Martin Karplus presented this relation in the 1960s and would go on to win a Nobel
Prize for this work in 2013. In glucose the alpha anomer has a dihedral angle of 60 degrees and a coupling
constant of about 2.7 Hz, while the beta anomer has an angle of 180 degrees and a coupling constant of
about 7.2 Hz.

Beta
Alpha

7.0 Hz

2.7 Hz

5.8 5.7 5.6 5.5 5.4 5.3 5.2 5.1 5.0 4.9 4.8 4.7 4.6 4.5 4.4 4.3 4.2
f1 (ppm)
These couplings are also shown in the J-resolved NMR experiment below.

1D Spectrum

Projection of
J-Resolved
-12
-10
-8
beta -6
alpha -4
-2

f1 (Hz)
2.4 Hz 7.1 Hz 0
2
4
6
8
10
12
5.5 5.3 5.1 4.9 4.7 4.5 4.3 4.1 3.9 3.7 3.5 3.3 3.1 2.9 2.7
f2 (ppm)
Monitoring Reaction Kinetics by NMR

a -D-glucose HDO

b -D-glucose

5.6 5.4 5.2 5.0 4.8 4.6 4.4

f1 (ppm)

It is possible to monitor the mutorotation reaction by NMR because the two proton peaks that represent
the different anomers are spectrally resolved. About 80 mg of α-D-glucose was dissolved in 0.8 mL D2O
and then immediately transferred to an NMR tube and inserted into the Spinsolve NMR spectrometer. The
new reaction monitoring scripting procedure was used to collect a 4 scan average every two minutes for
two hours.
 The reaction starts instantly and can be ob-
served by watching the concentration of the
alpha anomer decrease (peak at 5.1 ppm) and
the concentration of the beta anomer increase
(peak at 4.45 ppm). The peak at 5.1 ppm was
integrated for each spectrum and plotted
against time. The data was fit to an exponential
function and a rate of 0.015 min­‐1 was meas-
ured at about 26°C, which agrees well with the
literature.

Going beyond
The reverse reaction can also be monitored starting with pure ß-D-glucose, but this anomer tends be
more difficult to find in a pure state.

The reaction can be run at other temperatures to determine the activation energy, but the reaction needs
to be run outside the magnet and sampled in aliquots.

The beta species can also be monitored by integrating the whole water peak and beta-anomeric proton
peak. The fit will pull out the water integral as a constant.

Reference:
Drake, E.N. and Brown C.E., J. Chem Ed. 54, 124 (1977).

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