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400 Carbohydrates

trisaccharides (D–A–B and C–D–A) and one additional disaccharide or the aldobitol obtained by its reduction.
disaccharide (D–A). These compounds have one acyclic moiety (the aldonic
acid and the alditol, respectively), so that after hydrolysis
of the alkylated dimer, the unmethylated position in the
4. Ring Size and Position of Linkage
acyclic moiety will be the position of the glycosidic link-
The next stage in the structure elucidation of oligosaccha- age. If the disaccharide is nonreducing, the position of the
rides involves the determination of the ring size and the glycosidic linkage is known (C-1 for an aldose and C-2 for
position of linkage of the monosaccharide constituents. an ulose), and only one methylation experiment is needed
For nonreducing disaccharides, the position of linkage is (the unlabeled position is where the ring was attached).
by necessity between anomeric carbons; otherwise the dis- The use of labeling in structure elucidation is exem-
accharide would be reducing. If the nonreducing disaccha- plified by the methylation and hydrolysis of melibiose
rideiscomposedoftwoaldoses,thesetwomonomersmust to yield 2,3,4,6-tetra-O-methyl-D-galactose and 2,3,4-
be linked by an acetal oxygen bridge joining C-1 of one tri-O-methyl-D-glucose and by the oxidation of melibi-
aldoses to C-1 of the other, which is signified by (1 → 1); itose to melibionic acid or its reduction to melibiitol,
if the disaccharide is composed of two uloses (ketoses), then methylation of these, followed by hydrolysis of the
the oxygen bridge must link C-2 of one ulose to C-2 of methylated aldobionic acid or aldobiitol to yield 2,3,4,6-
the other (2 → 2); finally, if the disaccharide is composed tetra-O-methyl-D-galactose and 2,3,4-5-tetra-O-methyl-
of one aldose and one ulose, the linkage is (1 → 2) (with D-gluconic acid in the first case and 1,2,3,4,5-penta-O-
the oxygen bridge linking C-1 of the aldose to C-2 of the methyl-D-glucitol in the second (see Scheme 22).
ulose). Accordingly, when the monosaccharide compo-
nents of a nonreducing disaccharide are identified, there b. Partial hydrolysis. It is possible to use the struc-
is no uncertainty about the positions of linkage, but only ture of known disaccharides to determine the structure of
about the size of the rings (and the anomeric configuration, higher oligosaccharides. Thus, the fact that the trisaccha-
which will be discussed later). Reducing disaccharides, on ride raffinose (discussed above) affords on partial hydrol-
the other hand, must have their position of linkage and ring ysis melibiose and sucrose, whose structures are known
size determined. The position of linkage and/or ring size of to be 6-(α-D-galactopyranosyl)-D-glucopyranose and α-
the monosaccharide components of reducing and nonre- D-glucopyranosyl β-D-fructofuranoside, respectively, es-
ducing oligosaccharides can be determined by labeling or tablishes that the trisaccharide molecule is composed of an
by partial hydrolysis, as follows: α-D-galactopyranose ring attached through an α-glyco-
sidic bond to position 6 of an α-D-glucopyranose ring,
a. Labeling of free hydroxyl groups. The free hy- which in turn is attached glycosidically to the anomeric
droxyl groups in oligosaccharides are attached to carbon position of a β-D-fructofuranose ring. In other words,
atoms that are not involved in ring formation or in glyco- the trisaccharide must be 6-(α-D-galactopyranosyl)-α-D-
sidic bonds. These carbon atoms can be recognized if they glucopyranosyl β-D-fructofuranoside.
are marked with a suitable label, for example, by attach-
ing to their oxygen atoms permanent blocking groups that
5. Anomeric Configuration
will not be removed during the hydrolysis of the oligosac-
charide. Methylation is often used, since many of the par- a. By partial hydrolysis. The hydrolysis of raffinose
tially methylated monosaccharides that result from such to melibiose and sucrose, discussed earlier, suggests that
hydrolysis have been characterized by gas chromatogra- the linkage between the galactose and the glucose units in
phy. Methylation and hydrolysis of a reducing disaccha- the trisaccharide is α-D, and the linkage between glucose
ride afford two methylated monosaccharides. The first has and fructose is α-D for glucose and β-D for fructose.
two free hydroxyl groups (one at position 1 and one where
the ring was attached), and the second possesses three po- b. By enzymatic hydrolysis. The anomeric config-
sitions free (one at position 1, one where the ring was at- uration of the glycosidic bond of disaccharides can be de-
tached, and one where the glycosidic bond was attached). termined by enzymatic hydrolysis. For example, emulsin,
The vacant positions in the latter might create ambiguity, an enzyme obtained from bitter almonds, is known to
since it is not known which of the unblocked positions hydrolyze β-D-glucosidic linkages and not α-D linkages.
were due to the ring and which to the glycosidic bond. To Accordingly, if a glucose-containing disaccharide is hy-
avoid this confusion, it is necessary to carry out another drolyzed by emulsin, it can be concluded that it possesses
methylation on an acyclic disaccharide derivative, for ex- a β-D linkage. It is essential for structural work involving
ample, the aldobionic acid obtained by oxidation of the enzymes that the enzyme preparations be of the highest