Báo cáo khoa học: Mechanism of mild acid hydrolysis of galactan polysaccharides with highly ordered disaccharide repeats leading to a complete series of exclusively odd-numbered oligosaccharides doc

Mechanism of mild acid hydrolysis of galactan
polysaccharides with highly ordered disaccharide repeats
leading to a complete series of exclusively odd-numbered
oligosaccharides
Bo Yang1, Guangli Yu1, Xia Zhao1, Guangling Jiao1, Sumei Ren1 and Wengang Chai1,2
1 Glycoscience and Glycoengineering Laboratory, School of Medicine and Pharmacy, Ocean University of China, Qingdao, China
2 Glycosciences Laboratory, Faculty of Medicine, Imperial College London, Harrow, UK

Keywords
acid hydrolysis; agarose; carrageenan; mass
spectrometry; polysaccharide
Correspondence
W. Chai, Glycosciences Laboratory, Faculty
of Medicine, Imperial College London,
Northwick Park & St Mark’s Campus,
Harrow, Middlesex HA1 3UJ, UK
Fax: +44 20 8869 3455
Tel: +44 20 8869 3255
E-mail:
G. Yu Glycoscience and Glycoengineering
Laboratory, School of Medicine and
Pharmacy, Ocean University of China,
Qingdao 266003, China
Fax: +86 532 8203 3054
Tel: +86 532 8203 1560
E-mail:
(Received 6 November 2008, revised 30
December 2008, accepted 4 February 2009)

Sulfated galactan j-carrageenan is a linear polysaccharide with a repeating
disaccharide sequence of alternating 4-sulfated 3-linked galactose and

4-linked 3,6-anhydrogalactose units. In contrast to many examples of
chemical hydrolysis of polysaccharides, mild acid treatment of j-carrageenan resulted in facile and highly specific cleavage. In this article, we
report the identification, by various MS and chromatographic techniques,
of an unexpected series of exclusively odd-numbered oligosaccharide fragments from its hydrolytic products. Detailed sequence analysis of the products indicated that all the oligosaccharide fragments have the 4-sulfated
3-linked galactose residues at both the reducing and the nonreducing
ends. Further detailed investigation and analysis suggested that these
odd-numbered oligosaccharides were derived from two-step cleavages of
the glycosidic bonds on either sides of the 3,6-anhydrogalactose residues.
Neutral galactan agarose also contains 3,6-anhydrogalactose and has a
similar backbone sequence, and exhibited similar results upon mild acid
hydrolysis. It is highly unusual to obtain exclusively odd-numbered oligosaccharides from polysaccharides composed of ordered disaccharide
repeats.

doi:10.1111/j.1742-4658.2009.06947.x

The diverse oligosaccharide sequences present in polysaccharides, glycoproteins, glycolipids and proteoglycans serve multiple functions. Acidic polysaccharide
glycosaminoglycans (GAGs) are ubiquitous in vertebrate tissues, and have important biological functions
through binding to various proteins. Marine-derived
polysaccharides are often of an anionic nature, and
these GAG-like molecules have been exploited for their
antiviral, antioxidant, anticoagulant and other signal-

ing activities [1–4]. Recent studies have shown that
marine polysaccharide carrageenans can inhibit the
attachment of several pathogenic viruses, e.g. herpes
simplex virus [5], dengus virus [6], and human papillomavirus [7,8], and hence they have become of considerable biomedical interest, owing to their antiviral
activities and therapeutic potential.
Carrageenans are highly sulfated galactans isolated
from marine red algae, with linear repeating sequences

Abbreviations
A, 4-linked a-3,6-anhydrogalactose; A2S, 4-linked 2-O-sulfated-a-D-3,6-anhydrogalactose; anGal, 3,6-anhydrogalactose; CID, collision-induced
dissociation; CTMS, chlorotrimethylsilane; D, 4-linked a-D-galactopyranose; DP, degree of polymerization; ELSD, evaporative light scattering
detector; Gal, D-galactopyranose; G, 3-linked b-D-galactopyranose; GAG, glycosaminoglycan; G2S, 3-linked 2-O-sulfated-beta-D-galactopyranose;
G4S, 3-linked 4-O-sulfated-b-D-galactopyranose; 5-HMF, 5-hydroxymethyl-furfural; MMB, 4-methylmorpholine borane; TFA, trifluoroacetic acid.

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Odd-numbered oligosaccharides from galactan polysaccharides

B. Yang et al.

of alternating 3-linked b-d-galactopyranose (b-Gal,
unit G) and 4-linked a-d-galactopyranose (a-Gal,
unit D), with unit D often occurring as its 3,6-anhydro
form (anGal, unit A). The classification of carrageenans
is based on the presence of a D or A form of the 4linked galactose, and the differing sulfate contents and
substitutions; for example, j-carrageenan, i-carrageenan
and k-carrageenan have different disaccharide building
blocks: -[3-linked 4-O-sulfated-b-d-galactopyranose
(G4S)-A]n-, -[G4S-4-linked 2-O-sulfated-a-d-3,6-anhydrogalactose (A2S)]n-, and -[3-linked 2-O-sulfated-b-dgalactose (G2S)-4-linked 2,6-O-sulfated-a-d-galactose
(D2S6S)]n-, respectively [9].
Detailed knowledge of these polysaccharide structures is necessary for an in-depth understanding of
their biological roles. However, their structural complexity causes considerable difficulties in sequence
analysis and assignment of structure–function relationships. Partial depolymerization by either chemical or
enzymatic means to obtain a range of oligosaccharide
fragments is a common strategy for detailed structural

analysis and for use in activity assays. Enzymatic
depolymerization is generally more specific, with cleavage at selected glycosidic bonds without the risk of
modification of the native structures. However, suitable enzymes are not always available for all polysaccharides. Chemical hydrolysis is widely employed for
depolymerization of various types of carrageenan.
Enzyme digestion cleaves the 1,4-linkages, resulting in
even-numbered neocarra-oligosaccharides, -(A-G)n- or
-(D-G)n-, with G at the reducing terminus and A or D
at the non-reducing terminus [10–14]. For A-containing carrageenans (e.g. j-carrageenan and i-carrageenan), mild acid hydrolysis has been used, and is
considered to cleave the 1,3-linkages, producing evennumbered carra-oligosaccharides, -(G-A)n-, with A at
the reducing and G at the non-reducing terminus [15–
20]. It was surprising that acid hydrolysis selectively
cleaved the 1,3-linkage, giving oligosaccharides -(GA)n-, without affecting 1,4-linkages. Similar results
were obtained with 3,6-anGal-containing neutral galactan polysaccharide agarose, which has a similar linear
chain, -(G-A)n-, although unit A has an l-configuration rather than a d-configuration. However, the mechanism for the cleavage at the 1,3-linkage, the reducing
side of the anGal residue, is not yet known.
This highly specific and facile cleavage of galactans
by acid hydrolysis is unusual. However, Yu et al. have
recently observed pentasaccharides, heptasaccharides
and undecasaccharides among many other hydrolysis
products of j-carrageenan isolated by anion exchange
chromatography [21]. This prompted us to carry out a
detailed study of the acid hydrolysis of carrageenans.

Unexpectedly, we found that the j-carrageenan hydrolysis products are exclusively odd-numbered oligosaccharides, in contrast to the results of various previous
studies, in which even-numbered oligosaccharides were
found among the mild acid hydrolysis products of
3,6-anGal-containing galactans [15–17,20]. This is a
very unusual finding, as the polysaccharides are composed of highly ordered disaccharide repeats. Understanding the specificity and mechanism of the
hydrolysis process is key to its application, as this
knowledge will help us to determine the oligosaccharide sequences obtained from partial depolymerization,

in order to deduce the overall structure of the parent
polysaccharides and to derive structure–function relationships for biological function studies, such as in the
investigation of their potent inhibitory antiviral properties [5–8]. In this article, we report our detailed investigations on the mechanism of acid hydrolysis of the
3,6-anGal-containing galactans.

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Results and Discussion
Identification of a complete series of
odd-numbered oligosaccharides resulting from
mild acid hydrolysis of j-carrageenan
Polysaccharide j-carrageenan was hydrolyzed under
mild acid conditions using 0.1 m H2SO4 at 60 °C for
1.5 h. The hydrolytic product was fractionated by gel
filtration chromatography. As shown in Fig. 1A, a series of well-separated peaks with a regular pattern was
obtained. Each of the eight pooled fractions, K1–K8,
was analyzed by negative-ion ESI-MS. The presence of
multiple sulfates in each fraction gave rise to spectra in
which multiply charged ions dominated, and from
which the molecular mass and degree of polymerization (DP) of components were determined (Table 1).
In the mass spectrum of the slowest-eluting fraction
K1, the doubly charged ion at m ⁄ z 322.1 identified a
trisaccharide with a molecular mass of 646.2 Da, indicating a composition of two G4S units and one A unit
(Table 1), with a likely sequence of G4S-A-G4S. It
was surprising initially that the shortest fragment identified was a trisaccharide, and not the expected disaccharide as reported previously [15–17,20]. The
molecular mass of the adjacent fraction K2 was
386 Da higher than that of K1, suggesting a pentasaccharide with an additional A-G4S biose unit (Table 1).
A similar regular increment of 386 Da was determined
for each of the next six fractions, K3–K8, with DP7 to
DP17, respectively. The detailed sequences of oddnumbered j-carra-oligosaccharides were corroborated

by negative-ion ESI collision-induced dissociation

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B. Yang et al.

Odd-numbered oligosaccharides from galactan polysaccharides

Fig. 1. Gel filtration chromatography of j-carra-oligosaccharides
and agaro-oligosaccharides and their alditols. (A) j-Carraoligosaccharides resulting from mild acid hydrolysis. (B) j-Carraoligosaccharide alditols resulting from reductive hydrolysis.
(C) Agaro-oligosaccharides resulting from mild acid hydrolysis.
(D) Agaro-oligosaccharide alditols resulting from reductive hydrolysis. (E) i-Carra-oligosaccharides resulting from mild acid hydrolysis
(-S represents desulfated product).

K1

K2

K4

K3

K5

K8
K7
K6

A

K R1

K R2

K R3

K R4

K R8
K R7
K R6
KR5

B

A1

A2

A3

A4

A5

A8
A7
A6

C

(CID) MS ⁄ MS. Owing to the lability of the free acid
forms of the sulfated molecules, singly charged molecular ions [M ) Na]) of the fully sodiated forms were
selected as the precursors [22]. As an example, the
product-ion spectrum of trisaccharide K1, [M ) Na])
at m ⁄ z 667, is shown in Fig. 2A. A reducing or nonreducing terminal fragment ion was assigned on the basis
of the product-ion spectrum of its alditol after reduction, in which the reducing terminal ions would have a
2 Da increment [23]. The intense B2 and C2 ions [22]
clearly identified an internal A residue and two terminal G4S residues (Table 1).
There were no coeluting even-numbered oligosaccharides detected as minor components in any fraction.
Therefore, a complete series of exclusively odd-numbered oligosaccharides was obtained from mild acid
hydrolysis of j-carrageenan.
Identification of odd-numbered oligosaccharides
resulting from mild acid hydrolysis of agarose

AR1

AR2

AR3

AR8
AR7
AR6
AR5
AR4

D

I1

I2

I3

I4

I5

I6

I8
I7

E

-S
-S

To investigate whether the unusual finding of oddnumbered oligosaccharides obtained from mild acid
hydrolysis was related to the presence of the 3,6-anGal
residue, neutral galactan agarose was also subjected to
hydrolysis under the same conditions. Not surprisingly,
gel filtration chromatography of the hydrolysate gave a
very similar pattern (Fig. 1C). Eight fractions, A1–A8,
were collected, and the molecular masses and the DPs
of these neutral oligosaccharides were determined by
positive-ion MALDI-MS (Table 2). A trisaccharide was
identified in fraction A1 with [M + Na]+ at m ⁄ z 509.

Odd-numbered agaro-oligosaccharides were identified
in fractions A2–A8, each having an additional agarobiose (A-G) with a mass increment of 306 Da (Table 2).
Negative-ion ESI-CID-MS ⁄ MS was used to confirm
their sequences, and the product-ion spectrum of agaropentasaccharide A2 ([M ) H]) at m ⁄ z 791) is shown in
Fig. 2B as an example. Clearly, a complete series of
odd-numbered oligosaccharide fragments was generated
by mild acid hydrolysis from the nonsulfated 3,6-anGalcontaining galactan agarose, with the 3,6-anGal residue
exclusively at internal positions.

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B. Yang et al.

Table 1. Negative-ion ESI-MS of j-carra-oligosaccharide fragments obtained from mild and reductive acid hydrolysis.
Assignment

Fractions

Ions founda
(charge)

Calculated
molecular
mass

DP

Sequences

Theoretical
molecular
mass

K1
K2
K3
K4
K5
K6
K7
K8
KR1
KR2
KR3
KR4
KR5
KR6
KR7
KR8

322.1
343.1
353.6
359.9
364.1

367.1
369.3
371.1
395.1
391.7
390.0
389.0
388.4
387.9
387.6
387.3

646.2
1032.2
1418.4
1804.5
2190.6
2576.7
2962.4
3348.9
792.2
1178.2
1564.2
1950.3
2336.4
2722.4
3108.8
3494.7

3

5
7
9
11
13
15
17
4
6
8
10
12
14
16
18

G4S-A-G4S
G4S-A-G4S-A-G4S
G4S-A-G4S-A-G4S-A-G4S
G4S-A-G4S-A-G4S-A-G4S-A-G4S
G4S-A-G4S-A-G4S-A-G4S-A-G4S-A-G4S
G4S-A-G4S-A-G4S-A-G4S-A-G4S-A-G4S-A-G4S
G4S-A-G4S-A-G4S-A-G4S-A-G4S-A-G4S-A-G4S-A-G4S
G4S-A-G4S-A-G4S-A-G4S-A-G4S-A-G4S-A-G4S-A-G4S-A-G4S
G4S-A-G4S-Aol
G4S-A-G4S-A-G4S-Aol
G4S-A-G4S-A-G4S-A-G4S-Aol
G4S-A-G4S-A-G4S-A-G4S-A-G4S-Aol
G4S-A-G4S-A-G4S-A-G4S-A-G4S-A-G4S-Aol
G4S-A-G4S-A-G4S-A-G4S-A-G4S-A-G4S-A-G4S-Aol

G4S-A-G4S-A-G4S-A-G4S-A-G4S-A-G4S-A-G4S-A-G4S-Aol
G4S-A-G4S-A-G4S-A-G4S-A-G4S-A-G4S-A-G4S-A-G4S-A-G4S-Aol

646.1
1032.1
1418.2
1804.2
2190.3
2576.3
2962.4
3348.4
792.1
1178.2
1564.2
1950.3
2336.3
2722.4
3108.4
3494.5

()2)
()3)
()4)
()5)
()6)
()7)
()8)
()9)
()2)
()3)

()4)
()5)
()6)
()7)
()8)
()9)

a

Major ion detected; other ions with different charge states and sodiated ion species were all present at much lower intensities and are
therefore not listed.

Analysis of monosaccharide degradation
products
It has been found in the past, during the investigation
of conditions for monosaccharide composition analysis
[24–30], that the hydrolyzed monosaccharide 3,6-anGal
is not stable at high temperature under strong acidic
conditions, e.g. 2 m trifluoroacetic acid (TFA) at
120 °C, typically used for complete hydrolysis of galactan into its constituent monosaccharides. Upon release,
it was readily destroyed and converted into 5-hydroxymethyl-furfural (5-HMF). However, the stability of a
3,6-anGal residue at the reducing terminal of an oligosaccharide under mild acid conditions is not known.
This prompted us to carry out further detailed analysis
of the hydrolysate.
Normal-phase HPLC was used for analysis of the
potential monosaccharide-related degradation products
Gal, 3,6-anGal and 5-HMF, which coeluted with the
large excess of salt in gel filtration chromatography
(Fig. 1), and the elution profiles of which are shown in
Fig. 3A. Gal and 3,6-anGal do not have a UV chromophore and can only be detected by an evaporative

light scattering detector (ELSD), whereas the furancontaining 5-HMF is readily detectable by UV. The
hydrolysis products from j-carrageenan and agarose
produced by 0.1 m TFA at various reaction time intervals were analyzed. The reaction products at 2 h are
shown in Fig. 3B, and those obtained at other time
intervals were similar to this, although the relative
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intensities of the peaks were different. The fraction
with high absorption at 280 nm was collected and further analyzed by GC-MS. It gave a GC peak at
15.65 min (Fig. 3D) and a mass spectrum (Fig. 3E)
identical to the library spectrum of 5-HMF. The
content of 5-HMF increased, whereas the content of
3,6-anGal decreased, with increasing reaction time
(data not shown).
It is highly likely, therefore, that a 3,6-anGal residue
at the reducing terminal of an oligosaccharide is unstable even under the mild acid condition. Following mild
acid hydrolysis, even-numbered oligosaccharides with
Gal at the nonreducing terminus and 3,6-anGal at the
reducing terminus were initially obtained. As the
reducing terminal 3,6-anGal is unstable, we believe
that it is immediately hydrolyzed from the even-numbered oligosaccharides originally formed, resulting in
odd-numbered oligosaccharides, and the cleaved
monosaccharide 3,6-anGal is further degraded to
5-HMF [25].
Complete series of even-numbered
oligosaccharide fragments resulting from
reductive hydrolysis
Various methods have been developed previously to
prevent the degradation of the unstable monosaccharide 3,6-anGal during monosaccharide composition
analysis [31]. Conversion of monosaccharides into their

alditols by reduction has been conventionally used for

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B. Yang et al.

Odd-numbered oligosaccharides from galactan polysaccharides

% relative intensity

A

% relative intensity

B

% relative intensity

C

Fig. 2. Negative-ion ESI-CID-MS ⁄ MS product-ion spectra. (A) j-Carra-trisaccharide. (B)
Agaro-pentasaccharide. (C) j-Carra-tetrasaccharide alditol. (D) Agaro-hexasaccharide
alditol.

% relative intensity

D

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B. Yang et al.

Table 2. Positive-ion MALDI-MS of agaro-oligosaccharides obtained by mild and reductive acid hydrolysis.
Assignment
Fractions

Found
MNa+

DP

Sequences

Theoretical
MNa+

A1
A2
A3
A4
A5
A6
A7
A8

AR1
AR2
AR3
AR4
AR5
AR6
AR7
AR8

509.4
815.2
1121.3
1427.5
1733.4
2039.4
2345.4
2651.2
655.2
961.3
1267.3
1573.4
1879.5
2185.0
2491.0
2797.2

3
5
7
9

11
13
15
17
4
6
8
10
12
14
16
18

G-A-G
G-A-G-A-G
G-A-G-A-G-A-G
G-A-G-A-G-A-G-A-G
G-A-G-A-G-A-G-A-G-A-G
G-A-G-A-G-A-G-A-G-A-G-A-G
G-A-G-A-G-A-G-A-G-A-G-A-G-A-G
G-A-G-A-G-A-G-A-G-A-G-A-G-A-G-A-G
G-A-G-Aol
G-A-G-A-G-Aol
G-A-G-A-G-A-G-Aol
G-A-G-A-G-A-G-A-G-Aol
G-A-G-A-G-A-G-A-G-A-G-Aol
G-A-G-A-G-A-G-A-G-A-G-A-G-Aol
G-A-G-A-G-A-G-A-G-A-G-A-G-A-G-Aol
G-A-G-A-G-A-G-A-G-A-G-A-G-A-G-A-G-Aol

509.1
815.2
1121.3
1427.4
1733.5
2039.6
2345.7
2651.8
655.2
961.3
1267.4
1573.5
1879.6
2185.7
2491.8
2797.9

this purpose [25,32]. We attempted a similar procedure
to stabilize oligosaccharides with 3,6-anGal residues at
the reducing termini. Hydrolysis was carried out in the
presence of the reducing agents sodium borohydride or
4-methylmorpholine borane (MMB); both reducing
agents gave identical results (Fig. S1). The hydrolysis
products from j-carrageenan were analyzed by PAGE;
the reductive hydrolysate gave a series of discrete bands
(Fig. 4A, lane 1) with different mobilities from those of
the bands obtained from the nonreductive hydrolysate
(Fig. 4A, lane 3). Similar results were obtained with
HP-TLC, in which the bands of the reductive hydrolysate (Fig. 4B, lane 2) had different mobilities from
those of the nonreductive hydrolysate (Fig. 4B, lane 1).

The products from reductive hydrolysis were fractionated by gel filtration chromatography, and eight fractions, KR1–KR8, were pooled (Fig. 1B). The retention
times of the fractions were clearly different from those
of the fractions obtained from nonreductive hydrolysis
(Fig. 1A). The molecular masses and DPs determined
by negative-ion ESI-MS (Table 1) unambiguously
identified a complete series of even-numbered alditols.
Negative-ion ESI-CID-MS ⁄ MS of the j-carra-tetrasaccharide alditol KR2, using its sodiated ion (m ⁄ z 813) as
the precursor, indicated the predicted sequence of G4SA-G4S-Aol (Fig. 2C). No reducing terminal monosaccharide anGal or its degradation product 5-HMF (and
their reduced forms) was detected with HPLC analysis
(Fig. 3C).
A complete series of even-numbered oligosaccharide
alditols was similarly obtained from 3,6-anGal-containing agarose. The reductive acid hydrolysate of agarose
also showed different mobilities from those of the
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nonreductive hydrolysate on HP-TLC (Fig. 4B, lanes 3
and 4). The identities of oligosaccharide fragments
(fractions AR1–AR8,
Fig. 1D),
following
their
fractionation by gel filtration chromatography, were
confirmed by positive-ion MALDI-MS (Table 2). The
sequences were unambiguously identified by ES-CIDMS ⁄ MS, as illustrated by the product-ion spectrum of
agaro-hexasaccharide AR2 ([M ) H]- at m ⁄ z 937). The
almost full set of sequence ions clearly indicated
a hexasaccharide G-A-G-A-G-Aol, with a reduced
terminal 3,6-anGalol (Fig. 2D).
The results indicated that the reducing terminal 3,6anGal is labile but can be stabilized by reduction.
Therefore, the primary acid hydrolysis products, evennumbered oligosaccharides, can be preserved as

alditols. It is interesting to note that reduction can be
carried out with both MMB and sodium borohydride.
The former is acid-stable, whereas the latter decomposes in an acidic medium. The fact that borohydride
can be used as an effective reducing agent under mild
acidic conditions despite its instability highlights the
fast rate of reduction.
Effect of acidity on acid hydrolysis
Acid hydrolysis of polysaccharides is conventionally
carried out in strong mineral acid and, as described
above, j-carrageenan and agarose can be readily
cleaved under mild conditions by H2SO4 (pKa: )3).
We further examined acid hydrolysis under the same
mild conditions with weaker organic acids, including
TFA (pKa: 0.23), oxalic acid (pKa1: 1.23), maleic acid
(pKa1: 1.83), phthalic acid (pKa1: 2.89), citric acid

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Odd-numbered oligosaccharides from galactan polysaccharides

A

B

C

D

Fig. 3. Analysis of monosaccharide degradation products. (A) HPLC of standard 5-HMF,
3,6-anGal and Gal detected by UV (280 nm)
and ELSDs, respectively. (B) Hydrolysate
obtained from j-carrageenan with 0.1 M TFA
at 60 °C for 2 h. (C) Reductive hydrolysate
obtained from j-carrageenan with 0.1 M TFA
at 60 °C for 2 h. (D) GC-MS total ion current
chromatogram of the HPLC fraction of j-carrageenan hydrolysate with high absorption
at UV 280 nm. (E) Mass spectrum of the
fraction at 15.65 min.

E

(pKa1: 3.13), formic acid (pKa: 3.75), succinic acid
(pKa1: 4.16), and acetic acid (pKa: 4.75). The hydrolysates from j-carrageenan were analyzed by PAGE
(Fig. 5), and selected fractions were analyzed by ESIMS (not shown). Clearly, even with these weaker
organic acids, the same odd-numbered j-carrageenan
oligosaccharides were obtained, indicating the uniquely
facile nature of the hydrolysis.
Effect of the 3,6-anGal form on the hydrolysis of
galactan
As the initial glycosidic cleavage by mild acid hydrolysis of galactan j-carrageenan and agarose takes place

at the reducing side, and subsequent cleavage at the
nonreducing side, of the 3,6-anGal residue to give oddnumbered oligosaccharide fragments, it is highly likely
that the 3,6-anGal form of the galactose has a major
effect on the specificity of the hydrolysis. It is important to compare directly, and investigate in detail, a
pair of polysaccharides or oligosaccharides with a similar galactan sequence but with Gal substituting for the
3,6-anGal residues. Unfortunately, such a pair is not

available. We selected k-carrageenan and carried out
desulfation to prepare a polysaccharide with a nonsulfated sequence of -(4Gala1-3Galb1)n-, similar to that of
agarose, -[4(3,6-anGal)a1-3Galb1]n-, apart from the
anhydro form in the latter. The structure of the

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A

B. Yang et al.

B

DP’

DP

1
DP
18
16
14

17
15

11

10

9

8

5

7
8

7
8

3

5
6

5
6

13

12

DP

3
4

DP’
3
4

DP

7

2
3
4
5
6
7

7

6

5

1

1

2

3

1

2

3

4

Fig. 4. Analyses of acid hydrolysis products obtained from j-carrageenan and agarose. (A) PAGE analysis: lane 1, reductive hydrolysate of j-carrageenan; lane 2, a mixture of reductive and
nonreductive hydrolysates of j-carrageenan; lane 3, nonreductive
mild acid hydrolysate of j-carrageenan. (B) HP-TLC analysis: lane 1,
mild acid hydrolysate of j-carrageenan; lane 2, reductive hydrolysate of j-carrageenan; lane 3, mild acid hydrolysate of agarose;
lane 4, reductive hydrolysate of agarose. Arrow indicates the origin.

DP
DP

19 18
17 16
15 14
13 12
11
9
7

19
17
15

13
11

10

9

8

7

6

5

5
4
3

1

2

3

4

5

6

7

8

9

10

Fig. 5. PAGE analysis of acid hydrolysis products obtained from
j-carrageenan with various acids. Lane 1: H2SO4 (pKa: ) 3). Lane 2:
H2SO4 under reducing conditions. Lane 3: acetic acid (pKa: 4.76).
Lane 4: succinic acid (pKa1: 4.16). Lane 5: formic acid (pKa: 3.77).
Lane 6: citric acid (pKa1: 3.13). Lane 7: fumaric acid (pKa1: 3.03).
Lane 8: phthalic acid (pKa1: 2.89). Lane 9: maleic acid (pKa1: 1.83).
Lane 10: oxalic acid (pKa1: 1.23). Lane 11: TFA (pKa: 0.23). Arrow
indicates the origin.

desulfated k-carrageenan was confirmed by 13C-NMR
and GC-MS (Figs S2 and S3, respectively).
Mild acid hydrolysis was carried out with the nonsulfated anGal-lacking k-carrageenan, without success.
2132

2

3

4

5

6

7

8

9

10

11

Fig. 6. HP-TLC analyses of acid hydrolysis products of agarose and
nonsulfated k-carrageenan polysaccharides and heptasaccharides.
Lane 1: agarose treated with 0.1 M H2SO4 at 60 °C for 1.5 h.
Lane 2: agaro-heptasaccharide. Lanes 3–5: agaro-heptasaccharide
treated with 0.1 M H2SO4 at 60 °C for 15, 30 and 45 min, respectively. Lane 6: nonsulfated k-carrageenan treated with 0.1 M H2SO4
at 80 °C for 6 h. Lane 7: nonsulfated k-carrageenan heptasaccharide. Lane 8: nonsulfated k-carrageenan heptasaccharide treated
with 0.1 M H2SO4 at 60 °C for 2 h. Lanes 9–11: nonsulfated k-carrageenan heptasaccharide treated with 0.1 M H2SO4 at 80 °C for 2, 4
and 6 h, respectively. Arrow indicates the origin.

Only at a higher temperature and with a longer reaction time (e.g. 80 °C, 6 h) was the polysaccharide
hydrolyzed and both odd-numbered and evennumbered oligosaccharides obtained, as identified by
HP-TLC (Fig. 6, lane 6) and MS (data not shown).
However, the anGal-containing agarose was readily
hydrolyzed under the mild conditions (Fig. 6, lane 1).
To compare further the difference in the hydrolysis,
heptasaccharide pairs derived from agarose and nonsulfated k-carrageenan were prepared (Fig. 6, lanes 2
and 7, respectively) and used for hydrolysis. Under the

mild conditions, agaro-heptasaccharide was readily
hydrolyzed (Fig. 6, lane 3). Further incubation did not
result in even-numbered oligosaccharides, but only
increased the content of monosaccharides and trisaccharides (Fig. 6, lanes 4 and 5). However, under these
conditions, no hydrolysis product was observed for the
desulfated k-carra-heptasaccharide (Fig. 6, lane 8).
Only under forcing conditions can hydrolysis of the
desulfated k-carra-heptasaccharide take place and both
odd-numbered and even-numbered oligosaccharides be
generated (Fig. 6, lanes 9–11). The identities of the
hydrolytic products dL2–dL6, together with that of the
parent heptasaccharide dL7, were confirmed by MS
analysis (Table 3). As the cleavage took place in a
random fashion, and the b1–4 and a1–3 linkages could
be similarly cleaved, both odd-numbered and even-

FEBS Journal 276 (2009) 2125–2137 ª 2009 The Authors Journal compilation ª 2009 FEBS


B. Yang et al.

Odd-numbered oligosaccharides from galactan polysaccharides

Table 3. Negative-ion ESI-MS of mild acid hydrolysis products obtained from a heptasaccharide of desulfated k-carrageenan.
Assignment
Fractions

Found
[M ) H])

DP

dL2
dL3
dL4
dL5
dL6
dL7

341.1
503.1
665.1
827.2
989.2
1151.4

2
3
4
5
6
7

G-D
G-D-G
G-D-G-D
G-D-G-D-G
G-D-G-D-G-D
G-D-G-D-G-D-G

Theoretical
[M ) H])

Sequences

numbered oligosaccharides were obtained with each
series in comparable amounts (Fig. 6, lanes 6, 9 and
10).
The results clearly demonstrated that the 3,6-anGal
residue has a profound effect on the hydrolysis of
galactan polysaccharides, and that the high specificity
of cleavage by acid hydrolysis is due to the 3,6-anGal
residue.
Effect of 2-O-sulfate on the stability of 3,6-anGal
We investigated the effect of sulfate substitution on
the 2-OH of 3,6-anGal (the only free hydroxyl group
of the residue) on the stability of the glycosidic bond
at the nonreducing side and, therefore, the effect on
the release of the reducing terminal 3,6-anGal residue.
The 2-O-sulfated 3,6-anGal occurs widely in carrageenans as the i-carrabiose unit -(G4S-A2S)-. The difference between j-carrageenan and i-carrageenan is the
additional 2-O-sulfation of 3,6-anGal in the latter.
Degradation of i-carrageenan under mild acid conditions required a longer time [25,29] (3 h) than that for
j-carrageenan (1.5 h). The hydrolysate was fractionated by gel filtration chromatography (Fig. 1E). The
oligosaccharide fractions (I1–I8) were analyzed by
ESI-MS and MS ⁄ MS (Fig. S4). The molecular masses
and sequences obtained were in agreement with those
of even-numbered i-carrageenan oligosaccharides of
the carra-series [-(G4S-A2S)n-]; for example, I1 was a
disaccharide, I2 a tetrasaccharide, and I8 a hexadecasaccharide. Some minor desulfation products were also
detected, owing to the effect of the prolonged reaction

time. It is well known that 2-O-sulfation stabilizes its
glycosidic bond (at the reducing side), so that stronger
conditions are required for its hydrolysis [25,29]. However, the results with i-carrageenan indicated that 2-Osulfation of 3,6-anGal also has a major stabilizing
effect on the glycosidic bond at the nonreducing side.
The 2-O-sulfated 3,6-anGal residue at the reducing
termini produced by the acid hydrolysis was stable,
and could not be released by cleavage of the glycosidic

D-G
D-G-D
D-G-D-G
D-G-D-G-D
D-G-D-G-D-G
D-G-D-G-D-G-D

341.3
503.4
665.6
827.7
989.9
1152.0

bond at its nonreducing side to give odd-numbered
oligosaccharides. The effect is very similar to that of
reduction.

Conclusions
Acid hydrolysis is a classic method for depolymerization of polysaccharides. Generally, for a polysaccharide with highly ordered disaccharide repeats (such as
the GAGs), if selective cleavage takes place, even-numbered oligosaccharides are normally produced. In the
case of random and nonspecific cleavage, both oddnumbered and even-numbered oligosaccharides with

the two different residues at both termini are generated. There has been no report that a complete series
of exclusively odd-numbered oligosaccharide fragments
can be produced from such a polysaccharide, and it is
highly unusual for this to happen. We have proposed
a two-step cleavage for the mild acid hydrolysis of 3,6anGal-containing galactans (Scheme 1): initial cleavage
of the a1–3 glycosidic bond at the reducing side of the
3,6-anGal residue to give even-numbered oligosaccharides with a Gal at the nonreducing terminus and 3,6anGal at the reducing terminus, followed by immediate
removal of the newly created unstable reducing terminal 3,6-anGal at the b1–4 bond to give odd-numbered
oligosaccharides with Gal at both termini. The labile
reducing terminal 3,6-anGal can be stabilized by conversion into alditol via reduction and by 2-O-sulfation.
Clearly, the 3,6-anGal residue has a profound effect
on the hydrolysis of galactan polysaccharides, leading
to highly specific and facile cleavage.

Experimental procedures
Materials
The polysaccharides j-carrageenan (type III, from Eucheuma cottonii), i-carrageenan (type V, Eucheuma spinosa),
and k-carrageenan (type IV, from Gigartina aciculaire), and
agarose (type I, low electroendosmosis), MMB, NaBH4,

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2133


Odd-numbered oligosaccharides from galactan polysaccharides

O

O

OH
O

O

OH

Polysaccharides

O
O

O
OR

OH
O

O

OH
O

O
O

O
OR

OR'

OR'

OR'
O

B. Yang et al.

OH

O
OR

O
OH
OR'

(R=H, SO3Na; R'=SO3Na)

H+

(R=H, SO3Na)

OH
O
HO

OH
O

O
O

O

OH

O

OH

O

O
O

O

OH

OR

H+

OH

OR'
OH
O

HO

OH
O

O
O

O

OH

O

O OH
CH2OH
OR

OR'
OH
O

O
OR'

O

OH

OR

OR'

(R=H)

H

O

OH
O

O

Even-numbered oligosaccharide alditols
OH
OR

(R=SO3Na)
+

O
O

BH4–

OR'

OR'

OR'
OH
O

O
O
O

O
OSO3Na

OH
O
OH

O
O

O

OH
OSO3Na

O
O CHO

O

Even-numbered oligosaccharides

OH

OH
H+
OR'

OR'
OH
O
O

OH

O

O
O

OH
O
O
OH

O

O
OH

+

O CHO
HO

OH

Odd-numbered oligosaccharides

3,6-anGal
(furanose)

HO-CH2

O

HO

O

OH
OH

3,6-anGal
(pyranose)

CHO

5-HMF
Scheme 1. Proposed mechanism for the mild acid hydrolysis of galactan polysaccharides.

5-HMF, galactose, 3,6-anhydrogalactose, and ion exchange
resin Amberlite IR 120 (H+ form), were purchased from
Sigma-Aldrich (Shanghai, China). Chlorotrimethylsilane
(CTMS) was from J&K Chemical (Beijing, China). The
Superdex 30 column (30 lm, 1.6 · 60 cm), Superdex
Peptide HR column (10 · 300 mm) and Q-Sepharose Fast
Flow ion exchange resin were from Pharmacia Bioscience
(Uppsala, Sweden). The Aminex HPX-87H column
(300 · 7.8 mm, 9 lm) and AG50W-X8 ion exchange resin
were obtained from Bio-Rad Laboratories (Hemel Hempstead, UK). Fused-silica capillary columns HP-5MS
(30 m · 0.32 mm, internal diameter 0.25 mm) and
DB-225MS (30 m · 0.32 mm, internal diameter 0.25 mm)
were purchased from J&W Scientific (Folsom, CA, USA).
Aluminum-backed silica gel 60 HP-TLC plates were from
Merck (Darmstadt, Germany). All other reagents and
solvents used were of analytical grade.

Mild acid hydrolysis
Large-scale (100 mg) acid hydrolyses of the polysaccharides j-carrageenan and agarose were carried out typically
with 0.1 m H2SO4 (10 mgỈmL)1) at 60 °C for 1.5 h,
2134

similarly to the published procedure using HCl [21].
Hydrolysis of i-carrageenan was extended to 3 h. Smallscale (10 mg) hydrolysis of j-carrageenan was also carried
out with various organic acids, including TFA, oxalic
acid, maleic acid, phthalic acid, fumaric acid, citric acid,
formic acid, succinic acid, and acetic acid, at 60 °C for
1.5 h. Reductive hydrolysis of j-carrageenan and agarose
was carried out on a large scale with addition of 0.2 m
MMB at 60 °C for 1.5 h or 0.2 m NaBH4 at 60 °C for

3 h. The reaction was terminated by neutralization with
2 m NaOH before analysis.

Analysis and preparation of oligosaccharides
For HP-TLC analysis, aliquots ( 0.4 lL) of samples were
applied to a TLC plate and developed in n-butanol ⁄ formic
acid ⁄ water (4 : 6 : 1, v ⁄ v ⁄ v). Plates were stained by dipping
them in diphenylamine ⁄ aniline ⁄ phosphoric acid reagent for
3 s, and then heating them at 105 °C for 5 min for color
development, as described previously [33].
For PAGE, continuous gel with 22% polyacrylamide was
used, and PAGE was performed on a vertical slab
(0.1 · 8 · 10 cm) gel system. The gel was loaded with

FEBS Journal 276 (2009) 2125–2137 ª 2009 The Authors Journal compilation ª 2009 FEBS


B. Yang et al.

Odd-numbered oligosaccharides from galactan polysaccharides

20–50 lg of sample, and subjected to electrophoresis at
200 V for 2 h. The gel was stained with Alcian blue (0.5%
in 2% AcOH) and destained with 2% AcOH [21,34,35].
For oligosaccharide preparation, the hydrolysates were
concentrated by lyophilization and subjected to gel ltration
ă
chromatography using the AKTA-FPLC (Pharmacia
Biotech, Sweden) system with a Superdex 30 column, as previously described [15]. Elution was carried out with 0.1 m
NH4HCO3 for both acidic and neutral oligosaccharides, at

a flow rate of 0.2 mLỈmin)1, and detected by a refractive
index detector. Fractions were pooled, and the volatile
buffer was removed by repeated lyophilization with water.

The nonsulfated k-carrageenan was then hydrolyzed with
0.1 m H2SO4 (10 mgỈmL)1) at 80 °C for 6 h. The oligosaccharide products were fractionated and purified with a
Superdex 30 and a Superdex Peptide column, respectively.
Hydrolysis of heptasaccharides (250 lg) derived from
nonsulfated k-carrageenan and agarose was carried out
with 0.1 m H2SO4 (10 lgỈlL)1) at 60 °C and 80 °C, respectively. The hydrolysis products were analyzed by HP-TLC.
Following sample application with a Linomat V TLC applicator (Camag Scientific, Switzerland), the TLC plate was
developed and stained as described above.

MS
Detection and characterization of
monosaccharide degradation products
The hydrolysate (10 lL) was analyzed by HPLC (LC-10Ai;
Shimadzu, Kyoto, Japan) on an Aminex HPX-87H column
[36], with elution by 40% aqueous CH3CN containing
0.01 m TFA, at a flow rate of 0.6 mLỈmin)1. Detection was
by UV (280 nm) and ELSDs in series. The former was for
the detection of 5-HMF, and the latter for the detection of
3,6-anGal.
The identity of 5-HMF was confirmed by GC-MS analysis following its collection from HPLC [37]. An Agilent 6980 system equipped with an HP-5MS fused-silica
capillary column was used. The injector temperature was
set at 220 °C. Helium was used as carrier gas, at a flow rate
of 1.0 mLỈmin)1. The oven temperature was initially kept
at 50 °C for 4 min, then increased to 250 °C at a rate of
8 °CỈmin)1, and finally kept at 250 °C for 10 min. The ion
source temperature at 280 °C, and the ionization energy

was 80 eV. The mass spectrum acquired was compared with
the NIST library spectrum.

Desulfation of k-carrageenan and hydrolysis of
the desulfated product
Desulfation was performed essentially as previously
described [38,39], with some modifications. In brief, k-carrageenan (100 mg) was converted into its pyridinium salt
by cation exchange, and desulfation of the pyridinium salt
of k-carrageenan was carried out in anhydrous pyridine
with CTMS (CTMS ⁄ sulfate molar ratio 400 : 1) in a sealed
reaction vial at 100 °C for 8 h. The excess CTMS was
destroyed by hydrolysis with water, and the desulfated
product was recovered by dialysis. The desulfated product
was purified by anion exchange chromatography with an
ă
AKTA-FPLC system on a Q-Sepharose Fast Flow column
(1.5 · 6 cm). The column was eluted with 50 mL of H2O at
a flow rate of 0.5 mLỈmin)1. The eluant was lyophilized to
give a nonsulfated k-carrageenan, and its structure was confirmed by 13C-NMR and GC-MS [40] to have the repeating
disaccharide sequence -(4Gala1-3Galb1)n-.

Negative-ion ESI-MS was performed on Micromass Q-Tof
or Q-Tof Ultima instruments (Waters, Manchester, UK)
for the sulfated oligosaccharides, as previously described
[41]. Nitrogen was used as the desolvation and nebulizer
gas, at flow rates of 250 LỈh)1 and 15 LỈh)1, respectively.
The source temperature was 80 °C, and the desolvation
temperature was 150 °C. Samples were dissolved in
CH3CN ⁄ 2 mm NH4HCO3 (1 : 1, v ⁄ v), typically at a concentration of 5–10 pmolỈlL)1, of which 5 lL was loopinjected. The mobile phase (CH3CN ⁄ 2 mm NH4HCO3,
1 : 1, v ⁄ v) was delivered by a syringe pump at a flow rate

of 5 lLỈmin)1. The capillary voltage was maintained at
3 kV and the cone voltage was 50–120 V, depending on the
size of oligosaccharides.
For CID-MS ⁄ MS product-ion scanning, argon was used
as the collision gas at a pressure of 1.7 bar, and the collision energy was adjusted between 17 and 100 eV for optimal sequence information. Neutral agaro-oligosaccharides
were analyzed by positive-ion MALDI-MS with a
Tof Spec 2E instrument (Waters, Manchester, UK), with
1,2-diamino-4,5-methylene dioxybenzene as the matrix.

Acknowledgements
This study was supported in part by the International
Science and Technology Cooperation Program of
China (2007DFA30980), the National Basic Research
Program of China (2003CB716401), the OUC Luka
Program (1405-814147), and a UK Medical Research
Council research grant (G0600512).

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Supporting information
The following supplementary material is available:
Fig. S1. PAGE analysis of reductive acid hydrolysis
products from j-carrageenan.
Fig. S2. 13C-NMR spectrum of desulfated k-carraeenan.
Fig. S3. GC-MS analysis of desulfated k-carraeenan.
Fig. S4. Negative-ion ESI-CID-MS ⁄ MS product-ion
spectrum of i-carra-tetrasaccharide.
This supplementary material can be found in the
online version of this article.
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