A dimethoxytriazine type glycosyl donor enables a facile chemo-enzymatic route toward α-linked N-acetylglucosaminyl-galactose disaccharide unit from gastric mucin

Article abstract of DOI:10.1039/c2cc30946g

An efficient chemo-enzymatic process for construction of the α-linked disaccharide unit (GlcNAcα1-4Gal) found in gastric mucin has been developed. The process consists of a one-step preparation of a novel triazine type glycosyl donor in water and the subsequent transglycosylation to a galactose derivative catalysed by α-N-acetylglucosaminidase.

Full text of DOI:10.1039/c2cc30946g

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COMMUNICATION
A dimethoxytriazine type glycosyl donor enables a facile
chemo-enzymatic route toward a-linked N-acetylglucosaminyl-galactose
disaccharide unit from gastric mucinw
a
a
a
b
a
Masato Noguchi, Miwa Nakamura, Ayaka Ohno, Tomonari Tanaka, Atsushi Kobayashi,
a
c
c
c
Masaki Ishihara, Masaya Fujita, Akiko Tsuchida, Mamoru Mizuno and
a
Shin-ichiro Shoda*
Received 9th February 2012, Accepted 12th April 2012
DOI: 10.1039/c2cc30946g
An efficient chemo-enzymatic process for construction of the
a-linked disaccharide unit (GlcNAca1-4Gal) found in gastric
mucin has been developed. The process consists of a one-step
preparation of a novel triazine type glycosyl donor in water and
the subsequent transglycosylation to a galactose derivative
catalysed by a-N-acetylglucosaminidase.
of a-N-acetylglucosaminyl galactose derivatives by the combined
use of a novel dimethoxytriazine type glycosyl donor and an
a-N-acetylglucosaminidase (a-GlcNAcase).
Recently, we have developed novel glycosyl donors having a
4,6-dimethoxy-1,3,5-triazin-2-yl group on the anomeric carbons
with b-configurations (DMT-b-glycosides) for enzymatic glycosy-
7
lations. The DMT-b-glycosides could be directly prepared in
water by treating the corresponding unprotected sugars with a
water-soluble agent, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-
Oligosaccharides including an a-linked N-acetylglucosamine
(
GlcNAc) unit play biologically important roles in nature. The
8
morpholinium chloride (DMT-MM), without protection of the
a-N-acetylglucosaminyl galactose (GlcNAca1-4Gal), the
terminal disaccharide unit of gastric mucin, is closely related
1
to the infection of Helicobacter pylori in gastric mucosa. Since
sugar hydroxy groups. In the course of these studies, we found
that the reaction of GlcNAc and DMT-MM afforded an adduct 1
(
DMT-a-GlcNAc) with the opposite anomeric configuration
Scheme 1). We postulated that if 1 could be recognised by
compounds having the GlcNAca1-4Gal unit would be expected
to be potential inhibitors of the growth of Helicobacter pylori,
their efficient and practical synthesis in a stereospecific manner
is strongly demanded.
(
an a-N-acetylglucosaminidase that hydrolyses a-glycosidic
bonds with retention of configuration, a stereospecific enzymatic
synthesis of 1,2-cis-glycosides would be possible by using 1 as a
glycosyl donor substrate.
In general, stereoselective a-glycosylation (1,2-cis-glycosidic
bond formation) of N-acetylglucosamine is difficult because
undesired b-anomers (1,2-trans-isomers) are produced predo-
minantly as a result of neighbouring group participation of the
A nucleophilic substitution reaction took place between the
hemiacetal of GlcNAc and DMT-MM in the presence of
2,6-lutidine in water, giving rise to the corresponding adduct 1
in 84% yield accompanying a small amount of 1,6-disubstituted
2
-acetamido group in the GlcNAc unit. Much effort has so far
been devoted to the selective construction of an a-N-acetyl-
glucosaminide structure. A 2,3-cyclic carbamate-type protecting
2
group showed a complete a-selectivity in the N-acetylgluco-
saminylation of a galactose derivative. The use of glycosyl
3
donors possessing a 2-azide group also gave a-N-acetylgluco-
4
–6
saminide selectively.
However, these chemical processes
require a number of steps including protection and deprotection
of the hydroxy and amino groups. In this communication,
we report a two-step chemo-enzymatic route for preparation
a
Department of Biomolecular Engineering, Tohoku University,
6-6-11-514 Aoba, Aoba-ku, Sendai 980-8579, Japan.
E-mail: shoda@poly.che.tohoku.ac.jp; Fax: +81-22-795-7293;
Tel: +81-22-795-7230
Department of Biobased Materials Science, Kyoto Institute of
Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan
The Noguchi Institute, 1-8-1 Kaga, Itabashi-ku, Tokyo 173-0003,
b
c
Japan
1
13
C
w Electronic supplementary information (ESI) available: H and
NMR spectra of the transglycosylated products. See DOI: 10.1039/
c2cc30946g
Scheme 1 Reaction mechanism for selective formation of 1.
This journal is c The Royal Society of Chemistry 2012
5
560 Chem. Commun., 2012, 48, 5560–5562

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DMT derivative (less than 1%). These results showed that the
a-hemiacetal 2a reacted with DMT-MM at the 2-position of
the triazine ring almost selectively without protecting other
sugar hydroxy groups.
In this reaction, a-adduct 1 formed almost exclusively
and no b-adduct 3 was obtained. The initial ratio of GlcNAc
(
2a/2b) was 6/4, which has not been reflected in the ratio of the
resulting adducts. The exclusive formation of the a-adduct can
be explained as follows. In addition to the nucleophilic attack
of the a-hemiacetal 2a to DMT-MM, b-hemiacetal 2b also
reacts with DMT-MM to give the corresponding b-adduct 3.
It is assumed that the resulting 3 is very reactive and easily
cyclises to the oxazoline derivative 4.
The resulting oxazoline 4 is much less stable and immediately
9
Fig. 1 Time course of the enzymatic hydrolysis of 1 (closed circle)
and pNP-a-GlcNAc(open circle) by a-N-acetylglucosaminidase B1
from Bacteroides thetaiotamicron VPI5482(10 mg ml ) in PBS buffer
at 37 1C. The decrease of 1 and pNP-a-GlcNAc was evaluated by
normal phase HPLC and UV-VIS spectrometer, respectively.
hydrolysed to 2b which is in equilibrium with 2a in water. Since
ꢀ1
an excess amount of DMT-MM (2 equiv.) was used, a-adduct 1
can accumulate through the re-generation of 2b.
A typical procedure for preparation of DMT-a-GlcNAc 1 is
as follows: a mixture of GlcNAc (221 mg, 1.0 mmol), DMT-MM
Table 1 a-GlcNAcase-catalysed transglycosylation reaction by using 1
(
553 mg, 2.0 mmol) and 2,6-lutidine (230 ml, 2.0 mmol) in water
(6.25 ml) was stirred at room temperature for 24 h. After
confirming the disappearance of GlcNAc by TLC, the solvent
evaporated and the residue was purified by silica gel column
1
chromatography, giving rise to 1 (288 mg, 84%). The H NMR
spectrum of 1 showed a doublet peak at 6.4 ppm derived from the
anomeric proton with a coupling constant of 3.3 Hz, indicating
10
that the anomeric configuration of the product is a-type.
In order to know whether the resulting DMT-a-GlcNAc can
be recognised by the corresponding a-glycosidase, we demon-
strated hydrolysis experiments of 1 by using a-N-acetylgluco-
saminidase B1 (a-GlcNAcase B1) cloned from intestinal
a
Yield (%)
Entry
Acceptor
R
1
1
bacteria, Bacteroides thetaiotaomicron VPI5482. The hydro-
lysis rate of the conventional p-nitrophenyl a-N-acetylgluco-
saminide substrate (pNP-a-GlcNAc) was also measured as a
reference. In the absence of the enzyme, the concentration of 1
in PBS buffer slightly decreased at 37 1C, indicating that the
substrate was very stable in the buffer. When a-GlcNAcase
enzyme was added to the solution, the substrate was hydrolysed
to GlcNAc quantitatively within 3 h liberating 2-hydroxy-4,6-
dimethoxy-1,3,5-triazine (closed circle in Fig. 1). These results
clearly showed that the substrate 1 can be well recognised by the
catalytic site of the a-GlcNAcase to form an enzyme–substrate
complex. Interestingly, the most popular substrate for glyco-
sidases, pNP-a-GlcNAc, was not hydrolysed under the same
conditions (open circle in Fig. 1).
1
2
3
5a
5b
5c
52
b
17
OMe
b
29
4
5d
5e
59
51
5
Based on these results, we tried a chemo-enzymatic
a-N-acetylglucosaminylation by using DMT-a-GlcNAc 1 as
glycosyl donor and various galactose derivatives as glycosyl
acceptors catalysed by a-GlcNAcase B1 (Table 1). The reaction
was carried out by pre-incubating a mixture of 1 and a glycosyl
acceptor in PBS buffer (pH 7.4) at 37 1C for 10 min. To this
mixture was added a PBS buffer solution of a-GlcNAcase B1
and the resulting product was purified by a preparative HPLC.
When p-methoxyphenyl b-galactopyranoside (5a) was used
as an acceptor, the corresponding disaccharide 6a was obtained in
a
b
Determined by HPLC. Isolated yield.
were analysed by HPLC (column: Inertsil ODS-3, eluent: 5%
CH CN to 15% CH CN gradient). The product 6a was purified
by HPLC (column: COSMOSIL, eluent: 8 vol% CH CN), and
freeze-dried.
3
3
3
1
The H-NMR spectrum of the resulting disaccharide
showed a doublet signal at around d 4.9 ppm ascribable to
0
5
2% yield (Table 1, entry 1). A mixture of 1 (50 mmol, final conc.:
0 mM), 5a (50 mmol, final conc.: 50 mM), and a-GlcNAcase B1
the H-1 proton with a coupling constant value of 3.6 Hz. The
3
1
5
C-NMR showed a signal due to the C4 at around 77.7 ppm.
All of these data clearly indicated that the glycosylation
proceeded in a regio- and stereo-selective manner, affording
(100 mg, 36.4 ml, PBS solution) in 100 mM sodium phosphate
buffer (pH 6.5) was incubated at 37 1C. The reaction mixtures
This journal is c The Royal Society of Chemistry 2012
Chem. Commun., 2012, 48, 5560–5562 5561

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1
2
an a-1,4 glycosidic bond. Methyl b-D-galactopyranoside (5b)
and isopropyl b-D-1-thiogalactopyranoside (5c) were also
a-glycosylated at the 4-positions, affording the corresponding
disaccharide derivatives 6b and 6c, respectively (entries 2 and 3).
We also demonstrated the transglycosylation reaction by
using N-acetyllactosamine derivative 5d and galacto-N-biose
derivative 5e as glycosyl acceptors. The glycosyl acceptor 5d could
be directly prepared from the corresponding unprotected sugars
Scientific Research from the Ministry of Education, Sports,
Science and Technology, Industrial Technology Research Grant
Program in 2006 from NEDO of Japan and International Center
of Research & Education for Molecular Complex Chemistry in
2007 from Tohoku University Global COE Program.
Notes and references
13
1 M. Kawakubo, Y. Ito, Y. Okimura, M. Kobayashi, K. Sakura,
S. Kasama, M. N. Fukuda, M. Fukuda, T. Katsuyama and
J. Nakayama, Science, 2004, 305, 1003.
according to the method described in the previous papers. The
glycosyl acceptor 5e was prepared in a similar manner as 1 by
treating galacto-N-biose with DMT-MM in water. The trans-
glycosylation reaction of the GlcNAc moiety with 5d and 5e
proceeded in a regio- and stereo-selective manner, giving rise
to the corresponding trisaccharide derivatives having a GlcNA-
ca-1,4Galb-1,4GlcNAc structure and a GlcNAca-1,4Galb-
2 K. Benakli, C. Zha and R. J. Kerns, J. Am. Chem. Soc., 2001,
23, 9461.
1
3
4
S. Manabe, K. Ishii and Y. Ito, J. Org. Chem., 2007, 72, 6107.
T. Buskas and P. Konradsson, J. Carbohydr. Chem., 2000, 19, 25.
5 P. Wang, H. Lee, M. Fukuda and P. H. Seeberger, Chem. Commun.,
2007, 1963.
6
7
K. Goto and M. Mizuno, Tetrahedron Lett., 2010, 51, 6539.
(a) T. Tanaka, M. Noguchi, A. Kobayashi and S. Shoda, Chem.
Commun., 2008, 2016; (b) T. Tanaka, A. Kobayashi, M. Noguchi,
K. Kimura, K. Watanabe and S. Shoda, J. Appl. Glycosci., 2009,
1
,3GalNAc structure, respectively (entries 4 and 5). These structures
can be found in the oligosaccharides of mucin type glycoproteins.
The mechanism of the present enzymatic transglycosylation
has not been made clear, however, it is assumed that either the
glycosidic oxygen atom or the nitrogen atom (1 or 3 position)
of the triazine ring of 1 is protonated with the carboxylic acid
of an acidic amino acid located at the catalytic center of
a-GlcNAcase, affording a glycosyl-enzyme intermediate with
b-configuration, liberating 2-hydroxy-4,6-dimethoxy-1,3,5-
triazine. The resulting intermediate is then attacked by the 4-OH
of the galactose unit in the glycosyl acceptors to give the transgly-
cosylated products with double inversion of configuration.
5
6, 83; (c) A. Kobayashi, T. Tanaka, K. Watanabe, M. Ishihara,
M. Noguchi, H. Okada, Y. Morikawa and S. Shoda, Bioorg. Med.
Chem. Lett., 2010, 20, 3588; (d) T. Tanaka, M. Noguchi,
K. Watanabe, T. Misawa, M. Ishihara, A. Kobayashi and
S. Shoda, Org. Biomol. Chem., 2010, 8, 5126.
8
9
M. Kunishima, C. Kawachi, J. Morita, K. Terao, F. Iwasaki and
S. Tani, Tetrahedron, 1999, 55, 13159.
The use of a weak base of 2,6-lutidine is crucial for the selective
formation of a-adduct 1. Under more basic conditions, for
example, in the presence of triethylamine, the oxazoline 4 was
not hydrolysed to 2b, giving rise to a mixture of 1 and 4.
M. Noguchi, T. Tanaka, H. Gyakushi, A. Kobayashi and
1S. Shoda, J. Org. Chem., 2009, 74, 2210.
In conclusion, a novel compound, 4,6-dimethoxy-1,3,5-triazin-2-
yl a-N-acetylglucosaminide (DMT-a-GlcNAc), was found to be an
efficient glycosyl donor substrate for a-N-acetylglucosaminidase
from Bacteroides thetaiotaomicron (a-GlcNAcase B1), and could
transfer the GlcNAc unit to the 4-OH of various galactose
derivatives, affording the core disaccharide (GlcNAca1-4Gal) unit
of the oligosaccharide chain on mucin type glycoproteins. It is
noteworthy that this new glycosyl donor can be prepared in water
directly from GlcNAc without using any protecting groups. There
have been no reports on glycosyl donors that can transfer a
GlcNAc unit to an acceptor in an a-selective manner due to the
difficulty of their preparation and low reactivity in the catalytic site
enzymes. The DMT-a-GlcNAc is the first artificial substrate
that can be recognized by a-GlcNAcase B1 efficiently and can
be employed as the glycosyl donor for chemo-enzymatic
synthesis. The present synthetic process which consists of a
one-step preparation of the glycosyl donor and the subsequent
effective transglycosylation by a-N-acetylglucosaminidase will
be a practical method in synthetic organic chemistry for
production of the mucin core disaccharide derivatives and will
contribute to basic research in glycoscience by supplying key
10 H NMR(500 MHz, D
2
O): d 6.38(1H, d, H-1, J1,2 = 3.3 Hz),
.03(1H, dd, H-2, J1,2 = 3.3 Hz, J2,3 = 10.8 Hz), 3.89(6H, s,
OCH ), 3.83(1H, dd, H-3, J2,3 = 10.6 Hz, J3,4 = 9.9 Hz),
.74–3.66(3H, m, H-5, H-6a, H-6b), 3.51(1H, t, H-4, J3,4
4
–
3
3
=
13
J4,5 = 9.4 Hz), 1.85(3H, s, –COCH3). C NMR(126 MHz, D O):
2
d 174.7, 173.2, and 171.7(triazine, –COCH
3
), 94.3(C-1), 74.2(C-5),
0.4(C-3), 69.3(C-4), 60.1(C-6), 55.8(–OCH ), 52.8(C-2),
1.6(–COCH ). ESI-MS: m/z calcd for C13
83.1173, found: 383.1174.
7
2
3
3
+
3
20 4 8
H N O [M+Na] :
11 M. Fujita, N. Kobayashi, A. Tsuchida, K. Goto, K. Osumi,
M. Mizuno, T. Yamanoi, H. Ashida, K. Haneda and J. Nakayama,
Glycoconjugate J., 2007, 24, 326.
1
1
2
H NMR (500 MHz, CD
(
d, H-1, J = 7.6 Hz), 4.29 (1H, ddd, H-5 , J = 2.5, 4.0, 9.9 Hz), 4.04
3
OD): d 7.08 (2H, d, Ph, J = 9.1 Hz), 6.86
2H, d, Ph, J = 9.1 Hz), 4.95 (1H, d, H-1 , J = 3.6 Hz), 4.84 (1H,
0
0
0
(
1H, d, H-4, J = 2.8 Hz), 3.97 (1H, dd, H-2 , J = 3.6, 10.9 Hz),
0
3
H-6a, H-3 , H-6 b, OCH
.83 (1H, dd, H-6a , J = 2.2, 11.8 Hz), 3.79–3.71 (8H, m, H-2, H-5,
0
0
3
), 3.69–3.65 (2H, m, H-3, H-6b),
3.46 (1H, t, H-4 , J = 9.4, 9.5 Hz), 2.02 (3H, s, CH ). C NMR
0
13
3
(
126 MHz, CD
3
OD): d 173.8 (CO of Ac), 156.7 (Ph), 153.0 (Ph),
19.1 (Ph), 115.5 (Ph), 104.0 (C-1), 100.3 (C-1 ), 77.7 (C-4),
0
1
7
7
0
0
6.8 (C-5), 74.3 (C-3), 73.7 (C-5 ), 72.6 (C-3 ), 72.4 (C-2),
0
0
0
2.0 (C-4 ), 62.3 (C-6 ), 60.7 (C-6), 56.1 (OCH
of Ac).
3
), 55.5 (C-2 ), 22.7
(CH
3
1
3 (a) T. Tanaka, T. Matsumoto, M. Noguchi, A. Kobayashi and
S. Shoda, Chem. Lett., 2009, 458; (b) N. Yoshida, M. Noguchi,
T. Tanaka, T. Matsumoto, N. Aida, M. Ishihara, A. Kobayashi
and S. Shoda, Chem.–Asian J., 2011, 6, 1876.
1
4
oligosaccharide materials on gram-scale. Further application
as well as the elucidation of detailed transglycosylation mechanism
of the reaction is now in progress.
1
4 In the present chemo-enzymatic process, isolation of the glycosyl
donor 1 is indispensable. When the reaction of GlcNAc and DMC
was carried out in the presence of a-GlcNAcase, the enzyme was
inactivated due to interaction of nucleophilic moieties of the
enzyme and DMC.
We thank Dr Motomitsu Kitaoka (National Food Research
Institute, Ibaraki, Japan) for providing us with galacto-
N-biose. This work was supported by a Grant-in Aid for
5
562 Chem. Commun., 2012, 48, 5560–5562
This journal is c The Royal Society of Chemistry 2012