Chemo-enzymatic synthesis of tetra-N-acetyl-chitotetraosyl allosamizoline

Article abstract of DOI:10.1016/j.bmcl.2006.03.016

A new compound 7, possessing a tetra-N-acetyl-chitotetraosyl moiety as a constituent, was synthesized by bacterial fermentation which used allosamizoline 6 as the initial acceptor.

Full text of DOI:10.1016/j.bmcl.2006.03.016

Bioorganic & Medicinal Chemistry Letters 16 (2006) 2860–2861
Chemo-enzymatic synthesis of tetra-N-acetyl-
chitotetraosyl allosamizoline
Gang-Liang Huang, Xin-Ya Mei, Hou-Cheng Zhang and Peng-George Wang*
State Key Laboratory of Microbial Technology (SKLMT), Shandong University, Jinan, Shandong 250100, China
Received 26 December 2005; revised 18 February 2006; accepted 7 March 2006
Available online 24 March 2006
Abstract—A new compound 7, possessing a tetra-N-acetyl-chitotetraosyl moiety as a constituent, was synthesized by bacterial
fermentation which used allosamizoline 6 as the initial acceptor.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Chitooligosaccharides (COS) have been attracting a
keen interest in their utilization because they have been
reported to possess physiological activities such as anti-
tumor activity1 and elicitor activity for plants.2 It is con-
sidered that the greatest physiological activities are
shown by COS with a degree of polymerization (dp)
greater than the chitopentaose.1 However, chitinases
and other enzymes rapidly cleave COS. Therefore,
improving the stability of the active COS is the key to
developing the biomedicines of COS.
The cyclopentadienylthallium 1 was alkylated and then
treated with thiourea and cationic methylene blue to
give the meso-diol 2. The optically active monoacetate
3 was derived from the meso-diol 2 by acetylation and
the action of the acetylcholinesterase from the electric
eel. The yield and the ee value of compound 3 were
89% and 95%, respectively. The carbamate 4 was formed
from the alcohol 3 by the action of benzyl chloroformate
followed by treatment with methanolic ammonia. The
diol 5 was obtained from the intermediate 4 by O-triflu-
oroacetylation, cyclization, O-methylation, exposure to
dimethylamine, hydroxylation with trifluoroperacetic
acid, and solvolysis with aqueous trifluoroacetic acid,
respectively. Catalytic hydrogenation of the diol 5 gave
allosamizoline 6.
Allosamidin, a Streptomyces metabolite, is an inhibitor
of family 18 chitinases and has a pseudotrisaccharide
structure consisting of two units of N-acetyl-D-allos-
amine and one unit of an aminocyclitol derivative, allos-
amizoline 6.3 And the allosamidin was synthesized
mainly by chemical methods.4
Bacterial fermentation was carried out as previously de-
scribed5 in 10 L bioreactors containing an initial culture
volume of 7 L. Compound 6 (1 g/L) was added to cul-
ture system. The Escherichia coli strain BL21(DE3) con-
taining a plasmid carrying the cloned nodC gene from
Mesorhizobium loti strain E1R6 was used as the source
of NodC protein. The culture time lasted 48 h.
The use of whole cells for the biotransformation of
organic substrates is a useful technique which has a
number of benefits over conventional, reagent-based
methods. With the aim of improving the stability of ac-
tive COS, herein we attempt to synthesize a new com-
pound 7 (Scheme 1) by bacterial fermentation. It is an
analogue of tetra-N-acetyl-chitotetraose with one unit
of an allosamizoline 6 at the reducing end as the
aglycone.
After centrifugation of the culture broth, chitooligosac-
charide was recovered exclusively in the pellet contain-
ing the bacterial cells. After disruption of the cells by
boiling, cell debris was removed by centrifugation and
the chitooligosaccharide was purified by activated char-
coal adsorption and aq ethanol (55%, v/v) elution. The
crude product was further purified by size-exclusion
chromatography on Biogel P4. The 1H NMR, 13C
NMR, and MS data confirmed the identification of
the obtained sample as the new compound 7.7 The yield
of 6 to 7 was 43%. Therefore, it indicates that 6 is clearly
Keywords: Tetra-N-acetyl-chitotetraosyl allosamizoline; Chemo-enzy-
matic synthesis; Allosamidin.
*
Corresponding author. Tel.: +86 531 8836 6078; e-mail:
hgl226@126.com
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.03.016

G.-L. Huang et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2860–2861
2861
CH2OBn
OH
Tl+
(i) PhCH2OCH2Cl
HO
(ii) O2, hv methylene blue, thiourea, MeOH, 33%
2
H2N
1
CH2OBn
OH
CH2OBn
OAc
(i) PhCOCl, pyridine
(ii) NH3, MeOH; (iii) K2CO3
O
HO
electric eel acetylcholinesterase
O
4
3
CH2OH
CH2OBn
OH
OH
O
(iv) TFAA, TFA, 60%; (v) MeOTf, Me2NH, 77%
(vi) CF3CO3H, TFA, TFA, 46%
O
H2, Pd/C
Me2N
Me2N
O
N
6
N
5
OH
OH
O
OH
8'
OH
O
9'
OH
O
OH
O
1'
chitooligosaccharide synthase
HO
HO
O
HO
O
3'
(nodC gene encode)
HO
HO
5'
N
NMe2
1''''
1
AcHN
2
AcHN
AcHN
UDP-GlcNAc
4
UDP
7
Scheme 1. Chemo-enzymatic synthesis of tetra-N-acetyl-chitotetraosyl allosamizoline.
5. (a) Samain, E.; Drouillard, S.; Heyraud, A.; Driguez, H.;
Geremia, R. A. Carbohydr. Res. 1997, 302, 35; (b) Huang,
G. L.; Zhang, D. W.; Zhao, H. J.; Zhang, H. C.; Wang, P.
G. Bioorg. Med. Chem. Lett. 2006, 16, 2042; (c) Huang, G.
L.; Zhang, D. W.; Zhao, H. J.; Zhang, H. C.; Wang, P. G.
Bioorg. Med. Chem. 2006, 14, 2446.
used as substrate by NodC in vivo. The most obvious
explanation for this phenomenon is that 6 does not
influence the binding affinity of NodC for the oligosac-
charide intermediate and therefore leads to an elonga-
tion with additional GlcNAc units. In our system, the
exclusive formation of pentamer is probably due to the
fact that the synthesis was carried out in growing E. coli
cells in which the physiological pool of UDP-GlcNAc is
maintained at a high level.
6. Kamst, E.; Pilling, J.; Raamsdonk, L. M.; Lugtenberg, B. J.
J.; Spaink, H. P. J. Bacteriol. 1997, 179, 2103.
7. Analytical data for 7: [a]D ꢀ14.2 (c 0.42, 0.1 M AcOH). 1H
NMR (300 MHz, D2O): d 5.30 (dd, 1H, H-10), 4.85 (m, 4H,
H-1, H-100, H-1000, H-10000), 4.53 (dd, 1H, H-50), 4.50 (dd, 1H,
H-60), 4.45–4.14 (m, 4H, H-3, H-300, H-3000, H-30000), 4.05–
3.70 (m, 23H), 3.17, 3.14 (s, 6H, NMe2), 2.40 (m, 1H, H-80),
2.14, 2.10 (each s, 12H, CH3 NHAc). 13C NMR (75 MHz,
D2O): d 22.8, 23.5 (4· CH3NHAc), 38.3 (NMe2), 54.6
(C-80), 55.4, 55.6 (C-2, C-200,C-2000, C-20000), 60.9 (C-10), 62.3
(C-90), 63.9 (C-6, C-600, C-6000, C-60000), 68.5, 69.1 (C-400, C-4000,
C-40000), 71.8, 72.8 (C-3, C-300), 75.3, 76.3 (C-5, C-500), 79.7
(C-4), 82.3 (C-60), 86.8 (C-70), 92.7 (C-50), 102.8, 103.7 (C-1,
C-100, C-1000, C-10000), 162.9 (C-30), 176.7 (C@O NHAc).
ESIMS: m/z 1051.0 [M+Na]+. Anal. Calcd for C41H68N6O24:
C, 47.86; H, 6.66; N, 8.17. Found: C, 48.00; H, 6.39; N, 8.01.
References and notes
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