Carbohydrate Analysis
265
less-well-defned
exchangeable
hydroxyl
protons and the vast excess (110 M) oF
H
2
O protons. Intramolecular hydrogen
bonding may be investigated by dissolving
the
carbohydrate
in
per-deuterated
dimethyl
sulFoxide
(DMSO)
when
the
hydroxyl
(and
amine)
protons
do
not
exchange with solvent and give useFul
inFormation. In some cases, hydrogen
bonding may be determined From proton
exchange in mixed H
2
O, HOD, and D
2
O
solvents where solvent exposure may be
quantifed From the diFFerential shiFts in
proton and carbon spectra.
Although
1D
proton
NMR
spectra
can be used as fngerprints to enable
structural determination where databased
structures exist, they are generally too
complex to assign Fully even For rela-
tively simple oligosaccharides.
13
Csp
e
c
-
tra are simpler but are also usually too
complex
to
use
without
the
availabil-
ity oF Fully assigned NMR spectra From
suitable known structures For guidance.
The structures oF unknown carbohydrates
generally demand a variety oF through-
bond (COSY), through-space (NOESY),
and
heteronuclear
13
C–
1
H2
DNM
R
techniques using high-feld spectrometers
(600 MHz) For their elucidation.
Complete assignment oF
1
HNMRand
13
C NMR resonances usually starts with
the well-resolved anomeric H-1 or C-1 peak
using 1D NMR. The proton resonances
can then be assigned sequentially using
through-bond techniques. The anomeric
conFormation may be confrmed From
the size oF the anomeric
1
J
C–H
coupling
constant, which is typically 170 Hz For
axial hydrogen and 160 Hz iF equatorial.
Structural conFormations may be given by
the
3
J
H–H
coupling constants, which obey
aKarplusrelationship(Eq.7).
3
J
=
A
cos
2
(θ)
B
cos
(θ)
+
C
(
7
)
where
A
,
B
and
C
are ftted parame-
ters derived From a number oF known
trainer molecules. The cosine nature oF
K
a
rp
lu
scu
r
v
e
sm
e
an
sth
a
ti
tg
i
v
e
son
e
to Four possible angles every 360
For a
given
3
J
(±ig. 7). Simple molecular mod-
eling can usually make a choice between
these, as oFten only one conFormation is
structurally reasonable. ±or example, the
Fig. 7
The Karplus relationship
3
J
=
A
cos
2
(θ)
B
cos
(θ)
+
C
.The
coupling constants involving the
glycosidic linking torsions (
3
J
COCH
)
A
,
B
,
and
C
are 7.6, 1.7 and 1.6 Hz
respectively. It can be noted that for any
experimental value of
3
J
,theremaybe
up to four possible torsional angles (e.g.
a
3
J
COCH
of 6 Hz may be derived from a
torsion of 28
,131
, 229
,or332
).
180
°
135
°
315
°
45
°
0
°
HCOC
torsion
90
°
225
°
270
°
3
J
HCOC
(Hz)
C
H
C
C
H
C
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