Concise preparation of biologically active chitooligosaccharides

Article abstract of DOI:10.1080/14786410902941006

Chitooligosaccharides (COSs) have demonstrated a diverse array of biological activities. Here we report a concise preparation method for tetra-N-acetyl-chitotetraose and penta-N-acetyl-chitopentaose. The FACE analysis showed that the partially N-acetylated COS mixture mainly contained glucosamine (GlcN) and some oligomers [(GlcN)n, n = 2-7]. The N-acetyl-D-glucosamine (GlcNAc) and peracetylated COSs [(GlcNAc)n, n = 2-7] were synthesised by treating the partially N-acetylated COS mixture with Ac₂O-NaOAc. The peracetylated chitotetraose and chitopentaose were obtained by isolation of peracetylated COS mixture. NaOMe in dry MeOH was used for the deacetylation of peracetylated chitotetraose and chitopentaose, to give the tetra-N-acetyl- chitotetraose and penta-N-acetyl-chitopentaose, respectively.

Full text of DOI:10.1080/14786410902941006

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Natural Product Research: Formerly
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Concise preparation of biologically
active chitooligosaccharides
G.L. Huang a
a College of Chemistry, Chongqing Normal University , Chongqing
400047, China
Published online: 10 Jul 2009.
To cite this article: G.L. Huang (2010) Concise preparation of biologically active
chitooligosaccharides, Natural Product Research: Formerly Natural Product Letters, 24:15,
1413-1417, DOI: 10.1080/14786410902941006
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Natural Product Research
Vol. 24, No. 15, 20 September 2010, 1413–1417
Concise preparation of biologically active chitooligosaccharides
*
G.L. Huang
College of Chemistry, Chongqing Normal University, Chongqing 400047, China
(Received 26 February 2009; final version received 31 March 2009)
Chitooligosaccharides (COSs) have demonstrated a diverse array of
biological activities. Here we report a concise preparation method for
tetra-N-acetyl-chitotetraose and penta-N-acetyl-chitopentaose. The FACE
analysis showed that the partially N-acetylated COS mixture mainly
contained glucosamine (GlcN) and some oligomers [(GlcN)n, n ¼ 2–7]. The
N-acetyl-D-glucosamine (GlcNAc) and peracetylated COSs [(GlcNAc)n,
n ¼ 2–7] were synthesised by treating the partially N-acetylated COS
mixture with Ac2O–NaOAc. The peracetylated chitotetraose and chito-
pentaose were obtained by isolation of peracetylated COS mixture.
NaOMe in dry MeOH was used for the deacetylation of peracetylated
chitotetraose and chitopentaose, to give the tetra-N-acetyl-chitotetraose
and penta-N-acetyl-chitopentaose, respectively.
Keywords: chitooligosaccharides;
N-acetyl-chitopentaose; preparation
tetra-N-acetyl-chitotetraose;
penta-
1. Introduction
Chitosan is a linear heteropolysaccharide composed of ꢀ-1,4-linked-D-glucosamine
(GlcN) and N-acetyl-D-glucosamine (GlcNAc) in varying proportions. Chitooligo-
saccharides (COSs) are known to be easily prepared by acidic or enzymatic partial
hydrolysis of chitosan. It has also been reported that lower COSs are water soluble
and biologically active (Huang, Mei, Zhang, & Wang, 2006; Suzuki et al., 1986).
Furthermore, the biological activity of COSs is known to depend on their structures
(Hahn, 1996). COSs having N-acetyl analogues are of special interest in the
agricultural and biomedical fields, because they exhibit strong bactericidal,
fungicidal (Hirano & Nagao, 1989) and antitumour activities (Suzuki et al., 1986).
Herein, we prepared and characterised the tetra-N-acetyl-chitotetraose and
penta-N-acetyl-chitopentaose using a partially N-acetylated COS mixture as starting
material. The method is simple, and it can be used to prepare the other pure
peracetylated COS.
*Email: huangdoctor226@163.com
ISSN 1478–6419 print/ISSN 1029–2349 online
ß 2010 Taylor & Francis
DOI: 10.1080/14786410902941006
http://www.informaworld.com

1414
G.L. Huang
Chitoheptaose
Chitohexaose
Chitopentaose
Chitotetraose
Chitotriose
Chitobiose
Glucosamine
ANTS
Figure 1. FACE of partially N-acetylated COS mixture.
2. Results and discussion
2.1. FACE analysis of partially N-acetylated COS mixture
Fluorophore-assisted carbohydrate electrophoresis (FACE) is a high-resolution
polyacrylamide gel electrophoretic procedure that separates oligosaccharides on
the basis of size (Huang, 2008; Huang, Mei, & Wang, 2006; Huang & Wong, 2008).
The partially N-acetylated COS mixture was tagged at the terminal aldehyde with
the highly charged fluorophore 8-aminonaphthalene-1,3,6-trisulfonate (ANTS).
Then, the saccharide-ANTS adducts were separated on 32% CACR/2.4% CBIS
(Figure 1) polyacrylamide gels at alkaline pH value. It was indicated that the
partially N-acetylated COS mixture mainly contained GlcN and some oligomers
[(GlcN)n, n ¼ 2–7].
2.2. Preparation of tetra-N-acetyl-chitotetraose and penta-N-acetyl-chitopentaose
The partially N-acetylated COS mixture was acetylated with Ac2O–NaOAc to
maximise the yield of peracetylated COSs. The peracetylated COS mixture was
isolated on a silical gel 60 column to give peracetylated chitotetraose and
peracetylated chitopentaose, respectively. The peracetylated chitotetraose and
chitopentaose were deacetylated with NaOMe in dry MeOH at room temperature,
sequentially neutralised, desalted and evaporated, to give the corresponding tetra-
N-acetyl-chitotetraose and penta-N-acetyl-chitopentaose in the overall yields of
16 and 10%, respectively (Figure 2).
3. Experimental
3.1. General
Partially N-acetylated COS mixture was purchased from the Aoxing
Biotechnology Co., Ltd. (Zhejiang Province, China). N,N0-methylenebisacrylamide,

Natural Product Research
1415
Partially N-acetylated chitooligosaccharide mixture
Ac O/NaOAc
2
Peracetylated chitooligosaccharide mixture
Column chromatography
Peracetylated chitotetraose and peracetylated chitopentaose
a. NaOMe/MeOH
b. Neutralise, desalt, evaporate,
respectively
Tetra-N-acetyl-chitotetraose and penta-N-acetyl-chitopentaose
Figure 2. Preparation of tetra-N-acetyl-chitotetraose and penta-N-acetyl-chitopentaose.
N,N,N0,N0-tetramethylethylenediamine (TEMED), ammonium persulfate, 8-amino-
naphthalene-1,3,6-trisulfonate (ANTS) and sodium cyanoborohydride were pur-
chased from Sigma-Aldrich Chemical Company. Optical rotations were determined
1
at 25ꢀC with a Perkin–Elmer Model 241-Mc automatic polarimeter. 13C and H
NMR spectra were recorded using a Bruker DPX-300 spectrometer at 75 and
300 MHz, respectively. Mass spectra were recorded with a VG PLATFORM mass
spectrometer using the ESI mode. TLC was performed on silica gel plates (GF254
with detection by UV light or by charring with 10% H2SO4 in EtOH.
)
3.2. Electrophoresis of ANTS-labelled partially N-acetylated COS mixture
The resolving gel was 32% acrylamide/2.4% bisacrylamide in a 140 ꢁ 160 ꢁ 0.75 mm
glass cassette. For every 35 mL of resolving gel, 150 mL of 10% ammonium
persulphate and 15 mL of TEMED were added. The stacking gel was 8%
acrylamide/0.6% bisacrylamide containing 50 and 5 mL of ammonium persulphate
and TEMED, respectively, for every 6 mL of stacking gel. The running buffer and the
gel buffer were 0.025 mol Lꢂ1 Tris/0.192 mol Lꢂ1 glycine (pH 8.4) and 0.42 mol Lꢂ1
Tris (pH 8.5), respectively. Electrophoresis was run at a constant current of 15 mA
for 6 h in a cooled buffer system.
3.3. Preparation of target compounds
To boiling Ac2O (20 mL) in a three-necked flask, 200 mg of KOAc was added. Then,
200 mg of partially N-acetylated COS mixture was gradually added under vigorous
stirring. The solution was kept for 1 h at 140ꢀC, then cooled to room temperature.
The crude product directly was purified on a silical gel 60 column (eluant : AcOEt :
EtOH 4 : 1), and the peracetylated chitotetraose and peracetylated chitopentaose
were obtained, respectively. The peracetylated chitotetraose or peracetylated
chitopentaose were suspended in anhydrous MeOH to a concentration of
100 mg mLꢂ1 and deacetylated with an equal volume of 1 mol Lꢂ1 NaOMe at
room temperature for 60 min with continuous mixing. The sample was then
desalted and neutralised by addition of Amberlite IRC-50 (Hþ) ion-exchange resin
(40 mg mgꢂ1 sample) previously washed in MeOH. After 30 min of vigorous
shaking, the deacetylated sample was recovered and the IRC-50 ion exchanger was

1416
G.L. Huang
washed with MeOH. Then, the combined washings were evaporated to dryness
under reduced pressure at 45ꢀC. The tetra-N-acetyl-chitotetraose and penta-
N-acetyl-chitopentaose were obtained in the overall yields of 16 and 10%,
respectively. Tetra-N-acetyl-chitotetraose, [ꢁ]D ¼ ꢂ6.1 (c ¼ 0.5, H2O). 13C NMR
(75 MHz, D2O): ꢂ ¼ 177.00, 176.80 (C¼O NHAc), 104.21, 103.81 (C-1II, C-1III
,
,
C-1IV), 97.55 (ꢀ-C-1), 92.89 (ꢁ-C-1), 82.35-56.29 (C-2I–IV, C-3I–IV, C-4I–IV, C-5I–IV
C-6aI–IV, C-6bI–IV), 24.77, 24.63 (CH3 NHAc). 1H NMR (300MHz, D2O):
ꢂ ¼ 5.16 (d, 0.6H, ꢁ-H-1), 4.69 (d, 0.4H, ꢀ-H-1), 4.55 (m, 3H, H-1II, H-1III, H-1IV),
3.90–3.41 (m, 38H), 2.06, 2.02 (s, 12H, CH3 NHAc). ESI-MS: m/z ¼ 853,
[M þ Na]þ. Penta-N-acetyl-chitopentaose, [ꢁ]D ¼ ꢂ5.7 (c ¼ 0.5, H2O). 13C NMR
(75 MHz, D2O): ꢂ ¼ 175.57, 175.40 (C¼O NHAc), 102.39, 102.26 (C-1II, C-1III
C-1IV, C-1V), 95.75 (ꢀ-C-1), 91.39 (ꢁ-C-1), 80.65–54.60 (C-2I–V, C-3I–V, C-4I–V
,
,
C-5I–V, C-6aI–V, C-6bI–V), 23.09, 22.90 (CH3 NHAc). 1H NMR (300 MHz, D2O):
ꢂ ¼ 5.10 (d, 0.7H, ꢁ-H-1), 4.61 (d, 0.3H, ꢀ-H-1), 4.44 (m, 4H, H-1II, H-1III, H-1IV,
H-1V), 3.89–3.30 (m, 47H), 1.96, 1.93 (s, 15H, CH3 NHAc). ESI-MS: m/z ¼ 1056,
[M þ Na]+.
4. Conclusion
Tetra-N-acetyl-chitotetraose and penta-N-acetyl-chitopentaose were obtained in the
process of acetylation, column chromatography, deacetylation, neutralisation,
desalting and evaporation of partially N-acetylated chitooligosaccharide mixture,
respectively.
Acknowledgements
This work is supported by the Natural Science Foundation Project of CQ CSTC (No.
CSTC, 2009BB5238), Chongqing Education Commission Foundation (No. KJ080810),
and the Doctor Start-up Foundation of Chongqing Normal University (No. 07XLB025),
China.
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