Carbohydrate Analysis
269
information is commonly obtained using
the off-diagonal peaks apparent in two-
dimensional NMR (Fig. 6). Spectra can be
obtained in a number of ways, by such
techniques as COSY and NOESY after a
full assignment of the
1
Hand
13
Cspectra.
In particular, COSY techniques indicate
neighboring connected nuclei, whereas
NOESY techniques give indication as to
the closeness of groups through space, the
effects of both being due to perturbations
caused to each other’s atom’s local mag-
netic ±eld. Linkages can be determined
from the
3
J
CH
scalar coupling through and
across glycosidic bonds and from inter-
glycosidic
1
H NOE effects using NOESY,
which gives those protons that are in close
proximity (less than about 0.5 nm).
By
spreading
out
the
NMR
spec-
tra over two or more dimensions, all
t
h
es
p
e
c
t
r
a
lp
e
a
k
sm
a
yb
eu
n
am
b
i
g
u
-
ously
assigned
to
atoms
in
the
gly-
can, fully con±rming its structure. Such
spectra also allow the determination of
the positions of noncarbohydrate sub-
stituents
such
as
sulfate
and
phos-
phate groups.
Sequences may be determined from
through-space
connectivities.
However,
with increasing size above about 20 gly-
cosyl residues, it becomes increasingly
dif±cult to delineate branch locations un-
ambiguously. NOE effects are also dif±cult
to interpret in the presence of signif-
icant internal molecular motion, owing
to positional averaging. Although NMR
is dif±cult to apply to many polysaccha-
rides, the presence of repeating units
can be identi±ed from the NMR spectra
of oligomers and then quanti±ed in the
polymers.
Linkage positions may also be discovered
by determination of the position of the
free hydroxyl groups, giving information
similar to that obtained from methylation
analysis (Sect. 2.4.3), but nondestructively.
1
HNMRsp
e
c
t
r
ainH
2
O(
10% D
2
O)
and D
2
O are compared to identify the
position(s) of the hydroxyl groups.
The biological roles of oligosaccharides
and polysaccharides are related to their
conformations. This mainly depends on
the trans-glycosidic torsional angles con-
necting the carbohydrate units (Fig. 9).
Fig. 9
The torsions and possible
coupling constants involved in the
anomeric linkages in oligosaccharides
and polysaccharides. (a) Torsions
φ
(phi) and
ψ
(psi) are defned as the
clockwise torsions O
r
C
a
O
a
C
n
,and
C
a
O
a
C
n
C
n
1
respectively, where
r
,
a
,
and
n
represent ring, anomeric, and
structural number, respectively. The
relevant and useFul coupling constants
are
3
J
C
1OC4C3
giving
ψ
and
3
J
C
2C1OC4
giving
φ
and
3
J
C
1OC4H4
and
3
J
H
1 C1OC4
.
(b) Linkages to the terminal carbon
atom may involve an exocyclic link; the
additional linking torsion
ω
(omega) is
defned as the clockwise torsion
O
a
C
n
C
n
1
C
n
2
. The relevant coupling
constants are
3
J
C
1OC6C5
giving
ψ
and
3
J
H
1 C1OC6
,
3
J
C
1OC6H
6
0
1
,
3
J
C
1OC6H
6
0
2
,
3
J
C
2 C1OC6
,
3
J
H
6
0
1
C
6C5C4
,and
3
J
H
6
0
2
C
6C5C4
.
C
2
C
1
O
5
O
C
5
C
4
C
3
O
H
1
H
4
fy
(a)
C
2
C
O
5
H
1
O
CO
C
5
C
1
4
6
H
6
1
H
6
2
f
y
w
(b)
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