Carbohydrate Analysis
263
isomeric sugar residues and cannot easily
provide the linkage and stereochemical
information of NMR.
2.3.5
NMR Spectrometry
The nuclei of many atoms (e.g.
1
H,
13
C,
15
N,
31
P) act as spinning magnets that
can orient themselves with or against an
external magnetic Feld. The strength of
this imposed magnetic Feld determines
both the frequency at which the nuclei
rotate (at high Feld strengths, the
1
H
resonance frequencies are 400–600 MHz)
and the resolution of the NMR spectra.
The very small difference in energy be-
tween the orientations of the spin of a
nuc
leu
sisa
f
fec
tedbyloca
lva
r
ia
t
ionsin
the magnetic Feld due to neighboring
nuclei and can be used to differentiate
between the nuclei and to provide infor-
mation on neighboring nuclei and their
relative orientation. The differences in the
magnetic environment of the nuclei are
reported in terms of the relative shift, in
parts per million, of the applied magnetic
Feld due to the local molecular mag-
netic Feld relative to a known structure.
±or protons, shifts are usually reported
downFeld (i.e. higher ppm) from the
methyl protons of sodium 3-trimethylsilyl-
1-propanesulfonate.
1
H-NMR spectroscopy is an extremely
powerful and elegant technique for the
determination of the structure of glycans,
although it is between three and six or-
ders of magnitude less sensitive than mass
spectrometry. It can provide information
about the number and variety of the com-
ponent carbohydrate residues, on how they
are joined together, and can indicate the
presence or absence of noncarbohydrate
groups. Taken together with component
and methylation analysis,
1
H-NMR spectra
often allow the unambiguous assignment
of the complete molecular structures of
pu
r
iFedg
l
y
can
sandthecompo
s
i
t
iono
f
less-puriFed mixtures of glycans.
The key features of carbohydrate NMR
are the downFeld shifts owing to the
spatial proximity of oxygen atoms. Pro-
ton NMR carbohydrate spectra contain
a
mainly
unresolved
heap
of
signals
between 3.5 and 4 ppm with relatively
few resolved outlying peaks (±ig. 6, top).
These outliers are termed
structural reporter
groups
and contain generally well-resolved
anomeric signals (H-1) with greater shifts
(4.4–5.4 ppm). As the anomeric carbon (C-
1) is bonded to two oxygen atoms, these
anomeric
13
Cand
1
H atoms are easily dis-
tinguishable, providing important inroads
into the structural determination. Their
chemical shifts and coupling constants in-
dicate the sugar residues involved, together
with the types and anomeric character (i.e.
α
or
β)
of the glycosidic linkages and their
ring size (pyranose or furanose).
Other reporter groups also provide use-
ful well-resolved resonances. The methyl
protons and carbon (
13
C) atoms in de-
oxysugars are upFeld (i.e. at lower ppm)
and the carboxylate carbon (
13
C) atoms
in
uronic
acids
are
downFeld.
Thus,
the position of fucose residues may be
conFrmed
by
the
shifts
of
their
H-
5 (4.0–4.8 ppm) and methyl hydrogen
(
1.2 ppm) atoms. Signals due to man-
nose H-2 atoms (4.1–4.2 ppm) help con-
Frm the type of antennary structure in-
dicated by the anomeric proton shifts.
The shifts of both the axial (
1.7 ppm)
and equatorial (
2.7 ppm) H-3 atoms in
sialic acids are characteristic for the type
and conFguration of their glycosidic links
and may be indicative of their position
in the chain. The shifts of the hydro-
gen atoms in acetyl groups (2.0–2.1 ppm)
are very useful in indicating the struc-
ture local to
N
-acetylhexosamine and sialic
acid residues.
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