Hydrobiologia 260/261: 641-645, 1993.

A. R. O. Chapman, M. T. Brown & M. Lahaye (eds), Fourteenth InternationalSeaweed Symposium. 641

© 1993 Kluwer Academic Publishers. Printed in Belgium.

A new chemical tool for characterization and partial depolymerization

of red algal galactans

A. I. Usov
N.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Leninsky Prospect 47,
Moscow 117913, Russia

Key words: agar, carrageenan, 3,6-anhydrogalactose, reductive hydrolysis, sulfated galactans, red
algae

Abstract

Complete acid hydrolysis of red algal galactans in the presence of borane - 4-methylmorpholine com-
plex has been shown to prevent the acid degradation of 3,6-anhydrogalactose derivatives by their
reduction to the corresponding 3,6-anhydro-galactitols, whereas all the other monosaccharides are lib-
erated essentially in the non-reduced form; the reductive hydrolysis products may be determined quan-
titatively using gas-liquid chromatography (GLC). The method is recommended for preliminary char-
acterization of the polysaccharide composition of red algal biomass. Partial acid hydrolysis of galactans
in the presence of the same reducing agent gives rise to reduced oligosaccharides having terminal 3,6-
anhydrogalactitol residues. Based on this reaction, the attribution of unknown galactans to the agar or
carrageenan groups is possible by partial reductive hydrolysis of small samples of algal biomass with
subsequent identification of agarobiitol or carrabiitol acetates by GLC. Sulfate groups are substantially
retained under partial reductive hydrolysis conditions; the isolation by liquid chromatography and elu-
cidation of structures of reduced sulfated oligosaccharides may be of great value for the structural
analysis of complex red algal galactans.

Introduction the acid degradation during hydrolysis. Recently

Acid hydrolysis is a common procedure for de-
termination of monosaccharide composition of
oligo- and polymeric carbohydrates. Most
monosaccharides are sufficiently stable to acids
under the conditions of glycosidic bond cleavage.
These monosaccharides are released by hydroly-
sis in a free state and may be easily identified and
quantified by different chromatographic methods.
But several types of monosaccharides, e.g. keto-
ses (including 3-deoxy-2-keto-octonic and sialic
acids), 3,6-dideoxyhexoses, and 3,6-anhydrohex-
oses, are acid-labile and require protection from
it was shown that reduction at the moment of
hydrolysis may be a method of choice for such
protection, and borane - 4-methylmorpholine
complex was recommended as a good reducing
agent for this approach (Garegg et al., 1988). Our
work is devoted to the application of reductive
hydrolysis to the structural analysis of red algal
galactans. A similar study was independently un-
dertaken by Stevenson & Furneaux (1991).
It is well known that red algae, a phylogeneti-
cally very old division of marine macrophytes,
differ from all the other plants in polysaccharide
composition (Painter, 1983; Usov, 1992). They
642
usually contain sulfated galactans as the main
polysaccharide components, but, in some species,
sulfated xylomannans or neutral xylans may pre-
dominate. Several representatives of galactans
known as agars or carrageenans have valuable
gelling and stabilizing properties and are prepared
from algae in large scale as products of great in-
dustrial importance. Red algal galactans usually
have a 'masked repeating' structure. Their carbo-
hydrate chains are essentially linear and based on
a common structural backbone of alternating
alpha- and beta-galactose residues substituted at
positions 4 and 3, respectively. The beta-galactose
residues always belong to the D-series, but the
alpha-galactose residues are D in carrageenans
and L in agars. Various hydroxyls may be sub-
stituted by ester sulfate or methyl groups, pyruvic
acid acetals and sometimes by additional
monosaccharides, as, for example, D-xylose or
4-0-methyl-L-galactose residues. A substantial
part of the alpha-galactose residues may exist in
the form of 3,6-anhydro derivatives. The proper-
ties of polysaccharides are influenced very much
by all these modifications, and high 3,6-anhydro-
galactose content is especially important for good
gelling ability.
Various chemical methods were developed to
analyze the composition and other structural fea-
tures of sulfated galactans, but they are, as a rule,
rather tedious and time- and substance-consum-
ing. Many of them are now substituted by 13C-
NMR spectroscopy. The anomeric carbon chem-
ical shifts were shown to be sensitive to the
absolute configuration of 4-0-linked galactose
derivatives, so the attribution of the polysaccha-
ride to the agar or carrageenan group may be
done at the very beginning of the structural in-
vestigation (Usov, 1984). The position of other
resonances are sensitive to the presence of
O-methyl and sulfate groups and 3,6-anhydroga-
lactose residues. In fact, the 13 C-NMR spectra of
many disaccharide repeating units usually present
in red algal galactans are now completely inter-
preted and may be used to identify the corre-
sponding structural elements in new polysaccha-
rides (Lahaye et al., 1989).
In spite of these achievements, it is desirable to
have some methods which may accelerate the
screening procedure and give the possibility to
characterize and to quantify polysaccharides
prior to their isolation from the algal biomass.
Improvement of methods of partial depolymer-
ization to obtain oligosaccharides, which are of
great value for determination of polysaccharide
structures, represents another very important field
in the study of red algal galactans. We have shown
that reductive hydrolysis may be effectively used
in both directions mentioned above.
Complete reductive hydrolysis
Hydrolysis of a mixture of model compounds,
namely, methyl glycosides of xylose, 3,6-anhydro-
galactose, and galactose (2 M CF 3 COOH,
100 ° C, 8 h) in the presence of inositol as an in-
ternal standard with subsequent conversion into
acetylated aldononitriles and GLC was shown to
afford a three-component mixture in the absence
of reducing agent (3,6-anhydrogalactose was
completely degraded), but four components were
found, when the reaction was carried out in the
presence of borane -4-methylmorpholine com-
plex. This additional compound was proved to be
3,6-anhydrogalactitol acetate, and good correla-
tion was observed between its peak area and the
quantity of methyl 3,6-anhydrogalactoside in the
starting mixture. It should be noted that practi-
cally no xylose or galactose was reduced under
conditions of reductive hydrolysis, since decom-
position of the reducing agent preceded the lib-
eration of these monosaccharides in appreciable
amounts. Hence, these sugars may also be deter-
mined quantitatively by the usual GLC procedure
as acetylated aldononitriles together with 3,6-
anhydrogalactitol acetate. The average molar re-
sponse factors were calculated from numerous
experiments, and the method was proved to be
satisfactorily accurate and reproducible (Usov &
Elashvili, 1991a).
When applied to several galactans of known
monosaccharide composition, the method gave
reasonable values for the main components, such
as 3,6-anhydrogalactose and galactose, as well as
 643

for some minor mono-O-methyl-galactoses in
usual agar or carrageenan samples. The proce-
dure of reductive hydrolysis permits 3,6-anhydro-
galactose and 3,6-anhydro-2-0-methylgalactose
to be determined separately, since these monosac-
charides are converted into different alditols. In
fact, the corresponding derivatives of all the
monosaccharides usually found in the hydrolyz-
ates of red algal biomass (besides components of
galactans, xylose, mannose, and glucose are often
formed as the hydrolysis products of xylans, man-
nans, and floridean starches, respectively) are well
resolved by GLC. The procedure is rather insen-
sitive to many non-carbohydrate components
present in the algal biomass, and therefore it may
be used to characterize the polysaccharide com-
position of algae without any pretreatment. The
analysis may be carried out rapidly and econom-
ically, using small amounts of starting material.
The advantage of this approach was demon-
strated by determination of monosaccharide
composition in hydrolyzates of 40 algal species
from Kamchatka coastal waters (Table 1). This
work was accomplished shortly after the collec-
tion of seaweeds in summer 1990 (Usov & Kloch-
kova, 1992).
Partial reductive hydrolysis
Hydrolysis of galactans with high 3,6-anhydroga-
lactose content in the presence of borane -
4-methylmorpholine complex at lower tempera-
ture and acid concentration (0.5 M CF 3 COOH,
65 C, 8 h) gave rise to reduced disaccharides
having terminal 3,6-anhydrogalactitol residue.
For example, agarobiitol (3,6-anhydro-4-0-,B-D-
galactopyranosyl-L-galactitol) was obtained after
partial reductive hydrolysis of agar, whereas car-
rabiitol (3,6-anhydro-4-0-fl-D-galactopyranosyl-
D-galactitol), carrabiitol 4'-sulfate, and carrabii-
tol 2,4'-disulfate were isolated as the main
hydrolysis products of undersulfated carrageenan
from Tichocarpus crinitus (Gmel.) Rupr., kappa-
carrageenan, and iota-carrageenan, respectively
(Usov & Elashvili, 1989). The latter result indi-
cates that partial reductive hydrolysis of galac-
tans may be carried out with substantial retention

Table 1. Polysaccharide composition of Northwestern Pacific red seaweeds as revealed by complete and partial reductive hy-
drolysis of biomass.

Order Number of Main poly- Group 3,6-Anhydrogalactose

(family) species saccharide attribution contentb)
(number of (number of of galactans
samples a)) species) C) d)

Bangiales 4 (5) Galactan Agars M H

Cryptonemiales 9 (9) Galactan (6) Agars or carrageenans L M
Glucan (3)
Gigartinales 6 (10) Galactan Carrageenans He)
Rhodymeniales 1(3) Galactan Agars L L
Palmariales 7 (14) Xylan (6) Agars L M
Glucan (1)
Ceramiales 2 (2) Galactan Agars L L
(Delesseriaceae)
Ceramiales 11(11) Galactan (10) Agars H -
(Ceramiaceae Glucan (1)
and Rhodomelaceae)
a) Samples of the same species differ in habitat or belong to different generations.
b Abbreviations: L: - low, M - moderate, H - high.
c) Before alkaline modification.
d) After alkaline modification.
') Low 3,6-anhydrogalactose content was found only in tetra-sporophyte of Iridaea cornucopiae P. et R.
644
of sulfate groups. It is interesting to note the char-
acteristic difference between pairs of G1 (ano-
meric carbon of /i-D-galactopyranose residue,
103.7 and 104.8 p.p.m.) and A4 (carbon 4 of 3,6-
anhydrogalactitol residue, 86.9 and 88.8 p.p.m.)
resonances in carbon-13 NMR spectra of agar-
obiitol and carrabiitol caused by different abso-
lute configuration of 3,6-anhydrogalactitol resi-
dues, whereas the positions of signals of other
carbon atoms are practically the same. The site of
sulfate may be easily determined from the large
low-field shift of the corresponding carbon reso-
nance in the 13 C-NMR spectra of oligosaccha-
rides.
Agarobiitol or carrabiitol, as the products of
partial reductive hydrolysis, may be unambigu-
ously identified as acetates by GLC. This fact
makes it possible to determine the absolute con-
figuration of 3,6-anhydrogalactose residues, using
this chromatographic technique alone, and to at-
tribute unknown galactans to the agar or carrag-
eenan group without isolation of polysaccharides,
according to the results of partial reductive hy-
drolysis of small amounts of algal biomass (Usov
& Elashvili 1991a).
It should be noted that the yields of reduced
disaccharides depend not only on the proportion
of 3,6-anhydrogalactose and galactose, but also
on the degree of regularity of polysaccharide mol-
ecules and on degree of sulfation, since agarobi-
itol or carrabiitol sulfates as well as higher oli-
gosaccharides are not registered by GLC. If the
ratio of 3,6-anhydrogalactose to galactose is
less than 0.1, the detection of disaccharides after
partial reductive hydrolysis of biomass may be
difficult. The situation may be improved by pre-
liminary alkaline treatment of the biomass, where
upon the 4-linked galactose 6-sulfate residues
in galactans are transformed into 3,6-anhydroga-
lactose residues (Rees, 1961). Based on this ap-
proach, it was possible to attribute galactans of
40 species of Kamchatka red algae to the agar or
carrageenan groups according to the partial re-
ductive hydrolysis data (Usov & Klochkova,
1992). Some interesting observations were made
concerning the relationship between the polysac-
charide content and taxonomic position of the
algae (Table 1). For example, representatives of
Palmariales differ from all the other orders, since
they contain xylans as the main polysaccharide
components. Cryptonemiales is the only order
where both agars and carrageenans were found in
different species. In Ceramiales representatives of
the family Delesseriaceae differ from those of two
other families in 3,6-anhydrogalactose content,
and so on. Some of these observations may be
significant for chemical taxonomy of red sea-
weeds.
Methylated derivatives of agarobiitol may be
formed besides agarobiitol itself after partial re-
ductive hydrolysis of highly methylated agars
present in several algae. For example, 2-0-
methyl-, 6'-O-methyl-, and 2.6'-di-O-methyl-
agarobiitol were identified in the partial hydrolyz-
ate of Gelidiella acerosa (Forssk.) Feld. et Hamel
biomass. Structures of these compounds were
confirmed using GLC - mass spectrometry (Usov
& Ivanova, 1992). Identification and quantifica-
tion of these methyl ethers together with fraction-
ation may be useful in the study of distribution of
methylated sugars in agar molecules.
The application of partial reductive hydrolysis
was especially effective in the structural investi-
gation of a complex sulfated galactan isolated
from Laurencia nipponica Yamada. Its composi-
tion and 13 C-NMR spectrum were characteristic
for non-regular structure. Among the partial re-
ductive hydrolysis products agarobiitol and its
2'-sulfate were shown to be the main compo-
nents. Also isolated were 2-O-methyl-agarobiitol,
two reduced disulfated tetrasaccharides of the
porphyran type, one reduced branched pentasac-
charide disulfate containing a xylose residue, and
a high-molecular fraction devoid of 3,6-anhydro-
galactose residues. Structures of higher oligosac-
charides were established using composition
analysis, secondary ion mass spectrometry, and
13
C-NMR spectroscopy. The tetrasaccharides
were shown to have identical sugar chains and
differ only in the position of one sulfate group (at
C-2 of internal or terminal /B-D-galactopyranose
residue, respectively). The pentasaccharide had
the same backbone and sulfation pattern with an
additional P-D-xylopyranose unit located at po-

sition 3 of a 4-linked a-L-galactopyranose 6-sul-
fate residue. It was concluded from these data
that the parent polysaccharide is really a xyloga-
lactan with hybrid backbone containing both aga-
rose and porphyran type segments, where sulfate
groups occupy mainly position 2 of 3-linked E-D-
galactopyranose residues (Usov & Elashvili,
1991b).
Conclusion
The reductive hydrolysis procedures were used in
the present work for two purposes. The first one
was the rapid preliminary characterization of the
polysaccharide composition for a large series of
red seaweeds. In this case small samples of algal
biomass were subjected to complete and partial
reductive hydrolysis. Monosaccharide composi-
tion of the biomass reflected the content of dif-
ferent types ofpolysaccharides. Chromatographic
evidence on the absolute configuration of 3,6-
anhydrogalactose residues permitted algal galac-
tans to be attributed to the agar or carrageenan
groups, and determination of 3,6-anhydrogalac-
tose to galactose ratio made it possible to predict
some practically valuable properties of these
polysaccharides without their isolation from the
algal biomass. The method provided a real pos-
sibility to collect data on the polysaccharide com-
position for use in the chemical taxonomy of red
seaweeds.
The second purpose was the partial fragmen-
tation of a complex agar-like galactan. Practically
complete splitting of 3,6-anhydrogalactosidic
bonds took place regardless of the position of
other substituents, giving rise to reduced oligosac-
charides where sulfate groups and even xylose
residues were retained. This splitting was much
more extensive than enzymatic hydrolysis with
beta-agarase. The resulting reduced oligosaccha-
rides were more stable and more convenient for
chromatographic separation than the products of
partial acid hydrolysis or methanolysis. Their
645
structures were elucidated using mass spectrom-
etry and 1 3C-NMR spectroscopy, affording valu-
able information on the structural features of the
parent polysaccharide.
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