Preparation and characterisation of oligosaccharides produced by nitrous acid depolymerisation of chitosans

Article abstract of DOI:10.1016/S0008-6215(01)00130-6

Two chitosans with widely different chemical composition (fraction of N-acetylated units (F_A) < 0.001 and F_A = 0.59), were degraded by nitrous acid, to obtain the reactive 2,5-anhydro-D-mannose- (M-) unit at the new reducing end. The

Full text of DOI:10.1016/S0008-6215(01)00130-6

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Carbohydrate Research 333 (2001) 137–144
Preparation and characterisation of oligosaccharides
produced by nitrous acid depolymerisation of chitosans
Kristoffer Tømmeraas,* Kjell M. Va˚rum, Bjørn E. Christensen, Olav Smidsrød
Norwegian Biopolymer Laboratory (NOBIPOL), Department of Biotechnology,
Norwegian Uni6ersity of Science and Technology (NTNU), N-7491 Trondheim, Norway
Received 8 January 2001; received in revised form 19 April 2001; accepted 11 May 2001
Abstract
Two chitosans with widely different chemical composition (fraction of N-acetylated units (FA)B0.001 and
FA=0.59), were degraded by nitrous acid, to obtain the reactive 2,5-anhydro- -mannose- (M-) unit at the new
D
reducing end. The fully N-acetylated and fully N-deacetylated oligomers were separated by size-exclusion chromatog-
raphy. Both the chemical structure and purity were studied by one- and two-dimensional 1H and 13C NMR methods.
The fully N-acetylated oligomers were found to be stable, whereas the N-deacetylated oligomers reacted intermolec-
ularly by a Schiff base reaction between the 2-amino group on the N-deacetylated units and the M-units, facilitating
the cleavage of the glycosidic bond next to the M-unit and the formation of 5-hydroxymethylfurfural (HMF).
© 2001 Elsevier Science Ltd. All rights reserved.
1
Keywords: Chitosan; Depolymerisation; Nitrous acid; H NMR; 13C NMR; Size-exclusion chromatography; MALDI-TOF MS
1. Introduction
weight distribution, the ionic strength and the
pH value, as the pKa-value of the amino-
group in chitosan has been determined as
6.5.2,3 It has previously been shown that the
A- and D-units are randomly distributed along
the chain in water-soluble chitosans.4,5 De-
polymerisation of chitosan by the use of ni-
trous acid (HONO) is a homogeneous
reaction where the number of glycosidic bonds
broken is roughly stoichiometric to the
amount of nitrous acid used.6,7 The mecha-
nism involves a deamination of a D-unit form-
Chitosan is a linear copolymer of b-(1“4)
bound 2-acetamido-2-deoxy- -glucopyranose
D
(GlcNAc, A-unit) and 2-amino-2-deoxy- -
D
glucopyranose (GlcN, D-unit) units produced
by alkaline N-deacetylation of chitin (homo-
polymer of b-(1“4) bound GlcNAc), a struc-
tural polysaccharide in the exoskeleton of
arthropods and in the cell-wall of many
fungi.1 The solution properties of chitosan are
governed by the relative content of acetylated
amino groups designated by the fraction of
N-acetylated units, FA, the distribution of A-
units, the molecular weight, the molecular-
ing 2,5-anhydro- -mannose (M-units) at the
D
new reducing end (see Scheme 1). Since the
M-residue is unstable, the standard procedure
has been to reduce to 2,5-anhydro- -mannitol
D
by the use of NaBH4. Depolymerisation of
chitosans, by use of an excess of nitrous acid
and subsequent reduction, has been used pre-
viously to study the distribution of N-acety-
lated units in partially N-acetylated chitosan.8
* Corresponding author. Tel.: +47-73-590662; fax: +47-
73-591283.
E-mail address: kt@chembio.ntnu.no (K. Tømmeraas).
0008-6215/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(01)00130-6

138
K. Tømmeraas et al. / Carbohydrate Research 333 (2001) 137–144
Preparation
of
fully
N-acetylated
oligomers.—Chitosan (FA=0.59, [p]=826
mL/g, 500 mg, HCl form) was dissolved in 30
mL 2.5% v/v AcOH. Dissolved oxygen was
removed by bubbling N2 gas through the solu-
tion for 5 min. After cooling to 4 °C, a freshly
prepared solution of NaNO2 (1.5 mmol) was
added, and the reaction was allowed to pro-
ceed for 12 h at 4 °C in darkness without
stirring. The product was centrifuged (10 min,
5000 rpm) and filtered (8 mm), to remove the
insoluble fractions of fully N-acetylated
oligomers before lyophilisation.
Preparation
of
fully
N-deacetylated
Scheme 1. The mechanism by which the nitrous acid reaction
oligomers.—A solution of chitosan (FAB
0.001, [p]=283 mL/g, 500 mg, HCl form) was
prepared and degraded by HNO2 as described
for the first sample, the only exception being
that the molar ratio of D-units to NaNO2 was
reduced to 0.45 (1.13 mmol NaNO2). All the
fully N-deacetylated oligomers were soluble at
low pH-values.
Chromatography.—The oligomers (500 mg)
were separated by gel-filtration on two 2.5×
100 cm columns connected in series packed
with Superdex 30, eluted with 0.15 M ammo-
nium acetate at pH 4.5. A flow-rate of 0.8
mL/min was used and the entire separation
took 16 h. The relative amounts of oligomers
were monitored by means of an on-line refrac-
tive index (RI) detector (Shimadzu RID-6A).
Fractions of 4 mL were collected and pooled
to provide the purified oligomers.
leads to chain cleavage resulting in a 2,5-anhydro-D-mannose-
(M-) reducing end.
The scope of this study has been to prepare
and characterise defined fully N-acetylated
and N-deacetylated oligomers with the intact
M-unit at the reducing end. The M-unit has
some advantages over a normal reducing end.
2,5-Anhydro- -mannose does not mutarotate
D
in solution and the aldehyde group is more
available for reactions (e.g., reductive amina-
tion) since it does not participate in in-
tramolecular
hemiacetals.
This
makes
chito-oligomers with an M-unit as the reduc-
ing end an interesting precursor in organic
synthesis of e.g., glycodendrimers.
NMR.—All samples were dissolved in D2O,
and transferred to 5-mm NMR tubes. The
measurements were performed on a Dpx 400
Bruker Avance spectrometer. All chemical
shifts were determined relative to internal TSP
(sodium 3-(trimethylsilyl)-propionate-d4 from
Aldrich Chemical Co., 5 mL added from a 1%
stock solution).11 Typical conditions for the
2. Experimental
Materials.—Two chitosans with fraction of
acetylation (FA) of 0.59 and B0.001 (as deter-
1
mined by H NMR spectroscopy), where used
in this study. Chitin was prepared from
shrimp shells.9 The chitosan with FA of 0.59
was prepared by homogeneous deacetylation10
of chitin, while the fully N-acetylated chitosan
was prepared by further heterogeneous
deacetylation of a commercial chitosan with
FA=0.01. Sodium nitrite and ammonium ace-
tate were obtained from Merck, D2O (99.96%
D atom) from Isotech Inc., DCl (11.7 M,
35%), HMF and NaOD from Sigma and
Superdex 30 (prep grade) came from Pharma-
cia Biotech.
1
acquisition of H NMR spectra: 400.13 MHz;
25 °C; size of spectral window, 8220 Hz; cen-
1
tre of the H NMR spectra, 1880 Hz; acquisi-
tion time, 3 s; actual pulse repetition time, 4 s;
number of scans, 64 and a 30° excitation
pulse-angle was used; data size, 32 K. Typical
conditions used for 13C NMR acquisition:
100.64 MHz; 25 °C; size of spectral window,
31,850 Hz; centre of the 13C NMR spectra,
12,700 Hz; acquisition time, 3 s; actual pulse

K. Tømmeraas et al. / Carbohydrate Research 333 (2001) 137–144
3. Results and discussion
139
Preparation.—The
fully
N-acetylated
oligomers (A–[A]n–M) were prepared by
adding an excess of nitrous acid to a chitosan
(FA =0.59) in order to convert all D-units to
M-units. The fully N-deacetylated oligomers
(D–[D]n–M) were prepared by nitrous acid
depolymerisation of a fully N-deacetylated
chitosan (FAB0.001), where the molecular-
weight distribution of the oligosaccharides can
be controlled by the molar ratio of nitrous
acid to D-units.
Separation.—The oligomers were separated
by size-exclusion chromatography (SEC), and
the chromatograms of the fully N-acetylated
and fully N-deacetylated oligomers are given
in Fig. 1. For the N-acetylated oligosaccha-
rides 10–12 peaks were seen. The depoly-
merised chitosan contained an insoluble
fraction, which were probably the higher
oligosaccharides. Comparison of the reducing-
end intensities (at resonance 4.14, 4.36 and
5.01 ppm) with those of the H-1 (A-units)
signal at the glycosidic bonds (4.55–4.58 ppm)
of the different N-acetylated fractions (Fig. 2),
confirmed the separation to be according to
decreasing chain-length (i.e., the longest
chains were eluted first), and also the DP
(degree of polymerisation) as given in Fig. 1.
Similar results were seen for the N-deacety-
lated fractions.
Fig. 1. Size-exclusion chromatograms of the (a) fully N-acety-
lated; and (b) fully N-deacetylated chito-oligomers. (Superdex
30; two 2.5×100 cm columns in series, 0.15 M ammonium
acetate, pH 4.5, flow rate 0.8 mL/min.)
repetition time, 6 s; number of scans, 20,000;
30° excitation pulse-angle; data size, 32 K.
The spin-system was completely decoupled.
Conditions for acquisition of the COSY (2-D
homonuclear correlation spectroscopy) spec-
trum:13 memory size, 1024 (F2)×128 K (F1);
size of spectral window, 2400 (F2)×2400 Hz
(F1); centre of spectrum, 1881 Hz. Conditions
for acquisition of the HETCOR (2-D het-
eronuclear correlation spectroscopy) spec-
trum:14 memory size, 4096 (F2)×68 K (F1);
size of spectral window, 6040 (F2)×2400 Hz
(F1); centre of spectral windows, 8049 Hz for
F2 and 1873 Hz for F1. Both the COSY and
the HETCOR spectra were recorded in D2O
at 35 °C and pH* 5.0. The pH* (value ob-
tained when measuring with a pH-meter, cor-
rection for isotopic effect:12 pD=pH*+0.4)
were measured by use of a Mettler Toledo pH
electrode. The pH* value was controlled in all
NMR-samples.
MALDI-TOF MS.—Two samples, the N-
acetylated hexamer (A–A–A–A–A–M) and
the N-deacetylated tetramer (D–D–D–M)
obtained from the gel-filtration where solved
in deionised water (1 mg/mL) after lyophilisa-
tion. After mixing the oligomer solutions 1:4
with 2,5-dihydroxybenzoic acid (DHB) in
EtOH (100mM), 3 mL from each of the two
mixtures where dried on a target plate and
analysed on a Bruker MALDI-TOF Reflex III
mass spectrometer.
Chemical structure of the N-acetylated
oligomers.—The fully N-acetylated oligomer
fractions separated by SEC were characterised
1
13
using H and C NMR. The trimer 2-acetam-
ido-2-deoxy-b- -glucopyranosyl-(1“4)-2-
acetamido-2-deoxy-b- -glucopyranosyl-(1“
4)-2,5-anhydro- -mannofuranose (A–A–M)
D
D
D
was chosen to assign the reducing end reso-
nances by the use of 2-D homonuclear corre-
lation spectroscopy (COSY, see Fig. 3(a)), and
2-D heteronuclear correlated spectroscopy
(HETCOR, see Fig. 3(b)). In combination
with previously published spectra of fully N-
1
acetylated oligosaccharides,15 the H and 13C
chemical shifts of the reducing end of the
trimer (A–A–M) were assigned and the re-
sults are presented in Tables 1 and 2, respec-
1
tively. Neither the H nor the 13C spectra

140
K. Tømmeraas et al. / Carbohydrate Research 333 (2001) 137–144
1
Fig. 2. H NMR spectra of the fully N-acetylated oligomer fractions as they are collected from the peaks indicated in Fig. 1(a).
The spectra were recorded at 400.13 MHz, 25 °C and pH* 5.0.
Table 1
1H NMR (400.13 MHz) chemical shifts (ppm) and vicinal coupling constants (in Hz, given in parenthesis) for the reducing end
(M-unit) of the N-acetylated trimer (A–A–M) in D2O at 25 °C and pH* 5.0
H-1, gem diol
H-2
H-3
H-4
H-5
H-6a
H-6b
5.01
(6.1)
3.76
(4.5, 6.1)
4.35
(4.5, 4.5)
4.13
(4.3, 5.4)
3.98
(5.4, 9.5)
3.43
(9.5)
3.49
(9.5)
(Table 3), and the results from the former was
used in combination with the published spec-
tra of fully N-deacetylated oligosaccharides.16
contained the resonances expected for the free
aldehyde group of the M-residue (1H: ca. 9–
13
10 ppm and C: ca. 180 ppm13). The H-1 and
1
The H and 13C assignments of the reducing
C-1 resonances at 5.01 and 89.5 ppm, respec-
tively, indicated the presence of a hydrated
aldehyde where a water molecule has been
added to the carbonyl group (a so-called gem
diol), which seems to be the only existing form
of the reducing end in water. No major by-
products of the nitrous acid depolymerisation
reaction can be identified in the purified trimer
fraction. The assignments were almost identi-
cal for the N-acetylated oligomers, and are
not discussed further here.
Chemical structure of the N-deacetylated
oligomers.—The chemical shifts of the M-
residue resonances from the N-deacetylated
trimer (D–D–M) were generally shifted to
higher ppm-values (deshielded) relative to the
same resonances from the N-acetylated trimer
end resonances are presented in Tables 4 and
5, respectively. The H-1 proton (gem diol) of
the reducing M-unit is sensitive to whether the
neighbouring unit is a A- or a D-unit. This is
also seen in the previously published spectra4
of partially N-acetylated chitosan which were
depolymerised using nitrous acid, where the
Table 2
13C NMR (100.64 MHz) chemical shifts (ppm) for the reduc-
ing end (M-unit) of the N-acetylated trimer (A–A–M) in D2O
at 25 °C and pH* 5.0
C-1
C-2
C-3
C-4
C-5
C-6
91.86
88.13
79.18
88.03
85.01
61.8

/
(
137
144
Fig. 3. The 2D NMR spectra used to elucidate the chemical shifts of the 2,5-anhydro-
D-mannose reducing end of the fully N-acetylated trimer: (a) homonuclear correlation
spectroscopy (COSY); and (b) heteronuclear correlated spectroscopy (HETCOR). The recordings were done at 400.13 MHz (1H), 100.64 MHz (13C), 25 °C and pH* 5.0.

142
K. Tømmeraas et al. / Carbohydrate Research 333 (2001) 137–144
Table 3
1H NMR (400.13 MHz) chemical shifts (ppm), vicinal coupling constants (Hz) for the reducing end (M-unit) on the
N-deacetylated trimer (D–D–M) in D2O at 43 °C and pH* 5.7
H-1, free aldehyde H-1, gem diol H-1, Schiff base H-2
H-3
H-4
H-5
H-6a H-6b
a
a
9.49
5.09
(5.3)
7.93–7.97
3.84
(5.3, 5.3)
4.44
(4.4, 5.3)
4.22
(4.7, 5.3)
4.13
(5.3, 9.9)
(9.9)
(9.9)
a Due to signal overlapping, assignment were not possible for these protons which resonate in the 3.7–3.9 ppm region.
H-1 protons of the M-units are seen as two
resonances at 5.01 (–A–M) and 5.09 ppm
(–D–M). In contrast to the N-acetylated
oligosaccharides, the aldehyde of the M-unit
exists both in its hydrated (ꢀCH(OH)2) and
free (ꢀCHO) form, and was also found to be
quite unstable as a result of the preparation
procedure. During the lyophilisation process
of the purified oligomers from the buffer (con-
taining ammonium acetate and acetic acid),
the pH value in the fractions changed from 4.5
to 8 as water and acetic acid evaporated first,
leaving ammonia. The pKa-value of the pri-
mary amino group of chitosan has been deter-
mined as 6.5,2,3 meaning that the amino
groups of the N-deacetylated oligomers were
deprotonated during lyophilisation to give a
strong nucleophile, which could react with the
aldehyde of the reducing end. This produced a
reversible imino bond with the release of a
water molecule, and as water evaporated dur-
ing the lyophilisation, the reaction was facili-
tated. This is a Schiff base reaction, similar to
the non-enzymatic browning reactions often
seen in foods (Maillard browning), where side
chain amino groups of proteins react with
mono- and oligosaccharides producing
melanoidin pigments.17,18 The occurrence of
the imino proton (ꢀCHꢁNꢀ) is clearly seen in
resonances and coupling constants of a nor-
mal, reducing end of a N-deacetylated dimer
(D–D) were identified in the spectrum (see
Fig. 4(b) and Table 5) when compared to
earlier work.16,21 HMF is a common by-
product of Maillard browning, produced by
two eliminations of water followed by chain
cleavage. According to earlier studies,22 the
rate-limiting step in the formation of HMF, is
the first b elimination leading to 3-deoxy-hex-
ulose. When the N-acetylated trimer (A–A–
M) was subjected to strongly acidic conditions
(pH* 1), only negligible amounts of HMF (ca.
1
1%) were detected by H NMR. Gottschalk
showed in 1952, that while fructose under
mild acidic conditions (2 M acetic acid) was
converted to HMF (63%) in the presence of
phenylalanine, only negligible amounts of
HMF could be detected without the presence
of the amino acid.23 Therefore it seems likely
Table 4
13C NMR (100.64 MHz) chemical shifts (ppm) for the reduc-
ing end (M-unit) of the N-deacetylated trimer (D–D–M) in
D2O at 25 °C and pH* 5.7
C-1, gem diol
89.8
C-2
C-3
C-4
C-5
C-6
86.5
77.0
85.6
82.6
63.1
1
the H NMR spectrum for the N-deacetylated
Table 5
trimer (D–D–M) shown in Fig. 4(a), with a
characteristic chemical shift at 8.1 ppm.19,20
When the pH* was reduced from 5.7 to 4.0,
the spectrum changed drastically, as shown in
Fig. 4(b). The intensity of the imino protons
had almost disappeared, and was replaced by
the resonances characteristic of 5-hydroxy-
methylfurfural (HMF), as indicated in Fig.
4(b) and Table 5. These were identical to the
resonances and coupling constants obtained
1H NMR (400.13 MHz) chemical shifts (ppm) and vicinal
coupling constants (Hz) confirming the presence of dimer
(GlcN)2 (D–D) and 5-hydroxymethylfurfural (HMF) in the
N-deacetylated trimer (D–D–M) fraction, after adjusting to
pH* 4.0 (43 °C, D2O)
HMF
H-1
9.50
H-3
7.52
(3.8)
H-4
6.66
(3.8)
H-6
4.68
(GlcN)2
H-1a
5.44
H-1b
4.92
1
when a H NMR spectrum of pure (commer-
(3.2)
(8.5)
cial) HMF was acquired. In addition, the

K. Tømmeraas et al. / Carbohydrate Research 333 (2001) 137–144
143
1
Fig. 4. H NMR spectrum (400.13 MHz, 43 °C in D2O) of the N-deacetylated trimer (D–D–M) after lyophilisation: (a) at pH*
5.7. The intensities of the imino protons (Schiff base) are indicated in the spectrum (8.1 ppm). (b) At pH* 4.0 (adjusted with 0.1
M DCl). The resonances of HMF and the reducing end of the GlcN-dimer (D–D) are indicated.
form, they have to be separated and dried in a
system where the pH value can be kept well
below the pKa-value of the 2-amino group. As
an additional confirmation of the chemical
structure of the separated oligomers, the N-
acetylated hexamer (A–A–A–A–A–M) and
N-deacetylated tetramer (D–D–D–M) were
analysed by matrix-assisted laser desorption/
ionisation time-of-flight mass spectroscopy
(MALDI-TOF MS). For the N-acetylated
hexamer (A–A–A–A–A–M), a major peak
was obtained at m/z 1199.38 which was inter-
preted as the hydrated [M+Na]+-ion of the
oligomer. For the N-deacetylated tetramer
(D–D–D–M), the two main peaks were the
[M+Na]+-ions of the N-deacetylated trimer
(D–D–D) and the tetramer (D–D–D–M) at
m/z 507.17 and 668.18, respectively. These
mass/charge ratios (m/z) are in agreement
with the NMR study.
to conclude that the formation of a Schiff
base facilitated the elimination reactions lead-
ing to the formation of HMF and the subse-
quent cleavage of the glycosidic bond (Scheme
2) in a similar manner as for b-carbonyl
groups (b elimination).23 The formation of
Schiff bases could only occur during the final
lyophilisation step, where the pH increased to
well above the pKa-value of the amino group
of the D-unit (6.5). During all the prior steps
the oligomers were kept at acidic conditions
(pH was never above 4.5), and the amino
group will only exist in the protonated form,
i.e., as a poor nucleophile. It is only as a
nucleophile (deprotonated) that the amino
group can react with the carbonyl group to
form a Schiff base. When the pH value is
reduced to well below the pKa-value of the
amino group, existing imino nitrogens are
protonated and the divalent bonds are either
dissociated reversibly to the precursors, or an
elimination reaction may occur to yield the
3-deoxy derivative of 2,5-anhydro- -mannose.
D
4. Conclusion
After this rate-determining step, HMF is
formed directly, or via its Schiff-base deriva-
tive, produced by elimination of the GlcN-dis-
accharide (D–D). If the N-deacetylated
oligomers are to be isolated in their pure
Fully N-acetylated low-molecular weight
chito-oligomers with hydrated aldehyde reduc-
ing-ends were produced by nitrous acid de-
polymerisation of a partially N-acetylated

144
K. Tømmeraas et al. / Carbohydrate Research 333 (2001) 137–144
tra, and Mr Suresh Gohil for doing the
MALDI-TOF MS analysis at the Department
of Chemistry, Swedish University of Agricul-
tural Sciences. This work was financed by the
Research Council of Norway (Grant No.
121887/112).
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We like to thank Professor Hans Grasdalen
for his assistance in interpreting NMR spec-