4,6-dimethoxy-1,3,5-triazin-2-yl-d-glycosaminides: Novel substrates for transglycosylation reaction catalyzed by exo-d-glucosaminidase from amycolatopsis orientalis

Article abstract of DOI:10.1080/07328303.2012.698437

A novel sugar adduct, 4,6-dimethoxy-1,3,5-triazin-2-yl-d-glucosaminide (GlcN-DMT), has been prepared by the reaction of d-glucosamine (GlcN) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-morpholinium chloride (DMT-MM). The adduct was recognized by exo-d-glucosaminidase (GlcNase) from Amycolatopsis orientalis and transferred the GlcN moiety, giving rise to the corresponding glucosaminides. This chemo-enzymatic process was successfully applied to d-galactosamine (GalN). GalN-DMT prepared directly from GalN and DMT-MM behaved as an efficient glycosyl donor for transfer of the GalN moiety catalyzed by the same enzyme. The introduction of the 4,6-dimethoxy-1,3,5-triazin-2-yl leaving group to the anomeric center significantly enhanced transglycosylating ability, resulting in the efficient glycosidase-catalyzed synthesis of glycosaminides.

Full text of DOI:10.1080/07328303.2012.698437

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4,6-Dimethoxy-1,3,5-triazin-2-yl β-d-
glycosaminides: Novel Substrates for
Transglycosylation Reaction Catalyzed
by Exo-β-d-glucosaminidase from
Amycolatopsis orientalis
Tomonari Tanaka a b , Tomonori Wada a , Masato Noguchi a , Masaki
Ishihara a , Atsushi Kobayashi a , Takayuki Ohnuma c , Tamo Fukamizo
c , Ryszard Brzezinski d & Shin-ichiro Shoda a
a Department of Biomolecular Engineering, Graduate School of
Engineering , Tohoku University, 6-6-11-514 Aoba, Aoba-ku , Sendai ,
Miyagi , Japan
b Department of Biobased Materials Science, Graduate School
of Science and Technology , Kyoto Institute of Technology,
Matsugasaki , Sakyo-ku , Kyoto , Japan
c Department of Advanced Bioscience, Faculty of Agriculture , Kinki
University , 3327-204 , Nakamachi , Nara , Japan
d Département de Biologie, Faculté des Sciences , Université de
Sherbrooke , Sherbrooke , Quebéc J1K 2R1 , Canada
Published online: 31 Oct 2012.
To cite this article: Tomonari Tanaka , Tomonori Wada , Masato Noguchi , Masaki Ishihara , Atsushi
Kobayashi , Takayuki Ohnuma , Tamo Fukamizo , Ryszard Brzezinski & Shin-ichiro Shoda (2012) 4,6-
Dimethoxy-1,3,5-triazin-2-yl β-d-glycosaminides: Novel Substrates for Transglycosylation Reaction
Catalyzed by Exo-β-d-glucosaminidase from Amycolatopsis orientalis , Journal of Carbohydrate
Chemistry, 31:8, 634-646, DOI: 10.1080/07328303.2012.698437
To link to this article: http://dx.doi.org/10.1080/07328303.2012.698437
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Journal of Carbohydrate Chemistry, 31:634–646, 2012
ꢀ
C
Copyright Taylor & Francis Group, LLC
ISSN: 0732-8303 print / 1532-2327 online
DOI: 10.1080/07328303.2012.698437
4,6-Dimethoxy-1,3,5-triazin-2-
yl β-D-glycosaminides: Novel
Substrates for
Transglycosylation Reaction
Catalyzed by
Exo-β-D-glucosaminidase from
Amycolatopsis orientalis
Tomonari Tanaka,1,2 Tomonori Wada,1 Masato Noguchi,1
Masaki Ishihara,1 Atsushi Kobayashi,1 Takayuki Ohnuma,3
Tamo Fukamizo,3 Ryszard Brzezinski,4 and Shin-ichiro Shoda1
1Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku
University, 6-6-11-514 Aoba, Aoba-ku, Sendai, Miyagi, Japan
2Department of Biobased Materials Science, Graduate School of Science and
Technology, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto, Japan
3Department of Advanced Bioscience, Faculty of Agriculture, Kinki University,
3327-204, Nakamachi, Nara, Japan
4De´partement de Biologie, Faculte´ des Sciences, Universite´ de Sherbrooke,
Sherbrooke, Quebe´c J1K 2R1, Canada
A novel sugar adduct, 4,6-dimethoxy-1,3,5-triazin-2-yl β-D-glucosaminide (GlcN-β-
DMT), has been prepared by the reaction of D-glucosamine (GlcN) and 4-(4,6-
dimethoxy-1,3,5-triazin-2-yl)-4-morpholinium chloride (DMT-MM). The adduct was rec-
ognized by exo-β-D-glucosaminidase (GlcNase) from Amycolatopsis orientalis and trans-
ferred the GlcN moiety, giving rise to the corresponding glucosaminides. This chemo-
enzymatic process was successfully applied to D-galactosamine (GalN). GalN-β-DMT
prepared directly from GalN and DMT-MM behaved as an efficient glycosyl donor for
transfer of the GalN moiety catalyzed by the same enzyme. The introduction of the
Received January 25, 2012; accepted May 27, 2012.
Address correspondence to Shin-ichiro Shoda, Department of Biomolecular Engineer-
ing, Graduate School of Engineering, Tohoku University, 6-6-11-514 Aoba, Aoba-ku,
Sendai, Miyagi, Japan. E-mail: shoda@poly.che.tohoku.ac.jp
634

4,6-Dimethoxy-1,3,5-triazin-2-yl β-D-glycosaminides
635
4,6-dimethoxy-1,3,5-triazin-2-yl leaving group to the anomeric center significantly en-
hanced transglycosylating ability, resulting in the efficient glycosidase-catalyzed syn-
thesis of glycosaminides.
Keywords: Amino sugar; Chemo-enzymatic synthesis; Glycosidase; Enzymatic trans-
glycosylation; Dehydrative condensing agent; DMT-glycoside
INTRODUCTION
One of the most practical tools for oligosaccharide synthesis is enzymatic trans-
glycosylation catalyzed by glycosidases. Glycosidases are generally more sta-
ble, easy to handle, and inexpensive compared to glycosyltransferases. Vari-
ous kinds of glycosidases such as β-glucosidases, β-galactosidases, cellulases,
chitinases, and endo-β-N-acetylglucosaminidases have extensively been em-
ployed as catalysts for stereoselective glycosylations.[1,2] Chitosanases are gly-
coside hydrolases that cleave the β-1,4-glycosyl bonds of chitosan, and are
classified into endochitosanases[3–8] and exo-β-D-glucosaminidases. Exo-β-D-
glucosaminidase purified from an actinomycete, Amycolatopsis orientalis, be-
longs to glycoside hydrolase family 2 and specifically hydrolyzes the β-1,4-
glucosaminyl linkage at the nonreducing end of chitosan.[9]
This enzyme showed a transglycosylating activity; however, yield of the
transglycosylation product was low because the glycosyl donors employed
were chitosan or a chitosan oligomer where the secondary alkoxy group at
the 4-position of the D-glucosamine (GlcN) moiety behaved as a poor leaving
group.[10–17] In order to increase the efficiency of chitosanase-catalyzed transg-
lycosylations, it is necessary to utilize an appropriate glycosyl donor having a
better leaving group at the anomeric center of the GlcN. In general, the most
frequently employed glycosyl donors are p-nitrophenyl glycosides and glyco-
syl fluorides. However, introduction of a p-nitrophenyl group or fluorine onto
the anomeric center of GlcN requires a laborious synthetic process including
protection and deprotection of the hydroxy groups.[18,19]
Recently, we have found that unprotected neutral sugars can be directly
converted to the corresponding 4,6-dimethoxy-1,3,5-triazin-2-yl glycosides by
using a dehydrative condensing agent, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-
morpholinium chloride (DMT-MM),[20] in water without protecting the hy-
droxyl groups. In addition, the resulting activated glycosides were found to be
recognized by the corresponding glycosidases and behaved as efficient glyco-
syl donors for glycosidase-catalyzed transglycosylation reactions. For example,
4,6-dimethoxy-1,3,5-triazin-2-yl β-lactoside prepared from unprotected lactose
via one-step reaction transferred the lactose moiety to a monosaccharide or
a disaccharide in the presence of cellulase, giving rise to the corresponding
lactosylated products.[21,22] We also demonstrated an enzymatic polyconden-
sation of 4,6-dimethoxy-1,3,5-triazin derivatives of oligoxyloglucan, affording

T. Tanaka et al.
636
artificial xyloglucans having definite structures.[23] In the present paper, we
report a new approach to synthesize glycosaminides by a chemo-enzymatic
process, which consists of the following two reactions: (1) direct preparation
of novel glycosyl donors, 4,6-dimethoxy-1,3,5-triazin-2-yl derivatives of GlcN
and D-galactosamine (GalN), and (2) transglycosylation reactions catalyzed by
exo-β-D-glucosaminidase from A. orientalis.
RESULTS AND DISCUSSION
Synthesis and Kinetic Analysis of GlcN-β-DMT
When an aqueous solution of unprotected D-glucosamine hydrochloride
(GlcN-HCl) and 2,6-lutidine was treated with DMT-MM at rt for 24 h,
the corresponding glycosyl adduct of 4,6-dimethoxy-1,3,5-triazin-2-yl β-D-
glucosaminide hydrochloride (GlcN-HCl-β-DMT) was obtained in 43% yield
(Sch. 1). It is noteworthy that the anomeric center of unprotected GlcN was
activated in water by introducing the 4,6-dimethoxy-1,3,5-triazin-2-yl (DMT)
moiety without using any protecting groups. In order to evaluate the possibility
as a glycosyl donor substrate for chemo-enzymatic glycosylation, the resulting
GlcN-β-DMT was treated with a catalytic amount of GlcNase in acetate buffer
(pH 5.0) (Fig. 1). In the absence of GlcNase, the concentration of GlcN-β-DMT
did not considerably decrease, indicating that GlcN-β-DMT was considerably
stable in an aqueous solution (white circle in Fig. 1). Interestingly, when Glc-
Nase was added to the solution, the concentration decreased rapidly and GlcN-
β-DMT had been completely consumed after 3 h (black circle in Fig. 1). These
results clearly indicated that the GlcN-β-DMT moiety was accepted by the
catalytic site of GlcNase, affording GlcN and 2-hydroxy-4,6-dimethoxy-1,3,5-
triazine. In order to compare the hydrolysis activity of GlcN-β-DMT with that
of the conventional substrate, the hydrolysis rate of chitosan dimer (GlcNβ1-
4GlcN) was also measured. Approximately 75% of GlcNβ1-4GlcN remained
unreacted after 3 h, indicating that the glycosyl bond of GlcNβ1-4GlcN was
Scheme 1: Direct synthesis of GlcN-HCl-β-DMT from unprotected GlcN-HCl by using DMT-MM
in water.

4,6-Dimethoxy-1,3,5-triazin-2-yl β-D-glycosaminides
637
Figure 1: Enzymatic hydrolysis of GlcN-β-DMT and GlcNβ1-4GlcN in pH 5.0. •: GlcN-β-DMT,
GlcNase; ◦: GlcN-β-DMT, no enzyme; ꢀ: GlcNβ1-4GlcN, GlcNase; ꢁ: GlcNβ1-4GlcN, no
enzyme.
cleaved by the action of GlcNase much more slowly than that of GlcN-β-DMT
(black square in Fig. 1).
The kinetic analysis of GlcNase-catalyzed hydrolysis using GlcN-β-DMT
has been performed. The Michaelis constant (Km) and the unimolecular rate
constant (kcat) for GlcN-β-DMT were 2.5
0.6 mM and 73
5 s−1, respec-
tively. In order to obtain more information concerning the affinity of the β-
DMT moiety toward the +1 subsite of GlcNase, we also carried out a hydrolysis
experiment by using thiopyridyl derivative of chitosan dimer (GlcNβ1-4GlcN-
β-SPy).[24,25] The Km and kcat values for the hydrolysis of the inner glycosidic
bond of GlcNβ1-4GlcN-β-SPy were 0.43 mM and 48 s−1, respectively. These re-
sults indicated that the affinity of GlcN-β-DMT to GlcNase was not so high
presumably due to the steric repulsion between the methoxy groups on the tri-
azine ring and the amino acid residues in the vicinity of the +1 subsite. The
rapid hydrolysis of DMT moiety can be explained by the higher leaving ability
of the DMT group. Based on these results, we demonstrated a transglycosyla-
tion reaction catalyzed by GlcNase by using GlcN-β-DMT as glycosyl donor.
GlcNase-catalyzed Transfer of Glucosamine Unit by Using
GlcN-β-DMT as Glycosyl Donor
When GlcN-β-DMT was dissolved in a buffer and reacted with N,Nꢁ-
diacetylchitobiose (GlcNAcβ1-4GlcNAc) as acceptor in the presence of GlcNase,

T. Tanaka et al.
638
Scheme 2: GlcNase-catalyzed transglycosylation using GlcN-β-DMT as a donor.
Scheme 3: GlcNase-catalyzed transglycosylation using GalN-β-DMT as a donor.
the corresponding transglycosylated product was obtained (Sch. 2). The HPLC
chart of the reaction mixture after 40 min and 2 h from initiation showed
two peaks derived from GlcN, the hydrolyzate of GlcN-β-DMT, and the glu-
cosaminide at the retention time of 7.5 min and 12 min, respectively, in ad-
dition to the two major peaks for donor substrate (GlcN-β-DMT) and acceptor
substrate (GlcNAcβ1-4GlcNAc) (Fig. 2).
Effect of pH on the transglycosylation yield was investigated by using
GlcN-β-DMT as glycosyl donor and GlcNAcβ1-4GlcNAc as acceptor. The op-
timum pH of transglycosylation yield was pH 4.0 lower than that of hydrolysis
for GlcNβ1-4GlcN (pH 5.3).[16]
Figure 2: HPLC chart of GlcNase-catalyzed transglycosylation using GlcN-β-DMT as a donor
and GlcNAcβ1-4GlcNAc as an acceptor (donor/acceptor = 1/1 [mol/mol]) in pH 5.0. (a)
5 min, (b) 40 min, (c) 2 h.

4,6-Dimethoxy-1,3,5-triazin-2-yl β-D-glycosaminides
639
Table 1: GlcNase-catalyzed transglycosylation with various glycosyl acceptors by
using DMT-GlcN as a donor in pH 4.0
Entry
Acceptor
(Equiv.)
10
Yield (%)
1
2
GlcN
ND
ND
7
27
ND
9
39
25
ND
ND
GlcN-β-SPy
3
a
3
GlcN-β-DMT
4
GlcNβ1-4GlcN
GlcNAc
3
10
3
5
6
GlcNAc-β-SPy
GlcNAcβ1-4GlcNAc
GlcNAcβ1-4GlcNAc-β-SPy
Glc-β-SPy
7
10
3
8
9
3
3
10
Glcβ1-4Glc
DMT-glycoside (50 mM) and a glycosyl acceptor were incubated with GlcNase (92 μU) in
50 mM buffer solution (50 μL) at 37◦C.
ND: No product was detected.
a GlcN-β-DMT behaved as glycosyl acceptor as well as glycosyl donor under the concentration
of 500 mM, giving rise to a self-condensation product.
Effect of the donor/acceptor ratio on the yield was also evaluated. When
10 equivalent of GlcNAcβ1-4GlcNAc was used as glycosyl acceptor, the yield
reached 39%. When GlcN-β-DMT was reacted with 3 equivalent of acceptor,
the yield was 25%. It should be noted that the glucosaminide was obtained
only in 8% yield when GlcNβ1-4GlcN was used as glycosyl donor with 3 equiv-
alent of GlcNAcβ1-4GlcNAc. The GlcNase-catalyzed transglycosylation has
been demonstrated by using various kinds of glycosyl acceptors (Table 1). In
the case of using a monosaccharide as glycosyl acceptor, the transglycosyla-
tion product could not be obtained (entries 1 and 2). A self-condensation prod-
uct of GlcN-β-DMT was observed when the reaction was carried out under
a higher substrate concentration (entry 3). When the reaction was carried out
under normal concentration of GlcN-β-DMT, the self-condensation product was
not detected. The use of a disaccharide of GlcNβ1-4GlcN as acceptor afforded
the corresponding trisaccharide in a moderate yield (entry 4). A monosaccha-
ride of GlcNAc gave no transglycosylation product (entry 5); however, when
its anomeric configuration was fixed to β configuration by introducing an S-
pyridyl group or by using chito-oligosaccharide derivatives, the transfer prod-
ucts were obtained (entries 6–8). Glc-β-SPy and cellobiose (Glcβ1-4Glc) lacking
an amino group at the 2-position gave no transglycosylated products, showing
that the amino group at the 2-position was essential for the recognition by
GlcNase (entries 9, 10). These results suggest that the nitrogen atom at the
2-position of GlcN or GlcNAc interacts with an acidic amino acid through a
hydrogen bond.[26] The transglycosylation products were isolated by prepara-
1
tive HPLC and their structures were analyzed by NMR spectroscopy. The H
NMR spectra of the transglycosylated products showed a signal derived from

T. Tanaka et al.
640
the anomeric hydrogen at around 4.7 ppm with a coupling constant of 8.3 Hz,
clearly indicating that the stereochemistry of the resulting glycosidic bond is
perfectly controlled to β type. The 13C NMR spectra showed a signal at around
76 ppm derived from the C4 that is connected to the anomeric carbon through
the newly formed glycosidic bond. These results showed that the transglycosy-
lation proceeded regio- and stereoselectively, leading to the specific formation
of the β1-4 glycosidic bond.
DMT-β-D-glucoside was not hydrolyzed by GlcNase, indicating that the
amino group at the 2-position is essential for the recognition. These results are
in good agreement with the previous report that three acidic amino acids play
important roles in the −1 subsite of GlcNase by interacting with the amino
group of GlcN.[26]
GlcNase-catalyzed Transfer of Galactosamine by Using
GalN-β-DMT as Glycosyl Donor
In order to have information concerning the substrate specificity of Glc-
Nase, we prepared a new substrate, 4,6-dimethoxy-1,3,5-triazin-2-yl β-D-
galactosaminide (GalN-β-DMT), and its hydrolysis behavior by GlcNase was
investigated. The substrate GalN-β-DMT could be synthesized starting from
unprotected galactosamine by the action of DMT-MM in water using ammo-
nia as base. Interestingly, the resulting GalN-β-DMT was found to be recog-
nized by GlcNase and consumed almost quantitatively after 210 min (Fig. 3).
Since there have been no appropriate GalN derivatives for investigation on
Figure 3: Enzymatic hydrolysis of GalN-β-DMT in pH 5.0. •: GlcNase; ◦: no enzyme.

4,6-Dimethoxy-1,3,5-triazin-2-yl β-D-glycosaminides
641
Figure 4: HPLC chart of GlcNase-catalyzed transglycosylation using GalN-β-DMT as a donor
and 3 equivalent of GlcNAc-β-SPy as an acceptor in pH 4.0.
GlcNase-catalyzed hydrolysis, it has been impossible to evaluate the substrate
specificity of the GalN derivative for GlcNase. It should be noted that the intro-
duction of a DMT group to GalN enabled us to acquire important information
concerning its substrate specificity for the first time.
Based on these results, we demonstrated a GlcNase-catalyzed transglyco-
sylation to GlcNAc-β-SPy as glycosyl acceptor and found that the enzymatic
reaction gave the transglycosylated product in 7% yield (Sch. 3 and Fig. 4).
The reaction proceeded in a regio- and stereoselective manner to form a β1,4-
glycosidic bond, which was confirmed by 1H and 13C NMR spectroscopy.
CONCLUSION
We successfully achieved an efficient enzymatic transglycosylation of amino
sugars by use of 4,6-dimethoxy-1,3,5-triazin-2-yl (DMT) D-glucosaminide and
D-galactosaminide as glycosyl donors catalyzed by GlcNase. Novel glycosyl
donors, GlcN-β-DMT and GalN-β-DMT, were directly synthesized starting
from unprotected amino sugars by using DMT-MM reagent without protection
in aqueous solution. The DMT derivatives were recognized as substrates and
efficiently hydrolyzed by GlcNase. The GlcNase-catalyzed transglycosylation
products, which had β1,4-glycosyl bonds between donor and acceptor units,
were obtained by using GlcN-β-DMT and GalN-β-DMT as glycosyl donors. Es-
pecially, an efficient GlcN unit transfer has been achieved when amino disac-
charide derivatives were employed as glycosyl acceptors. By using this method-
ology, the enzymatic transglycosylation of the GalN unit has been achieved for
the first time. The present reaction using the easily prepared glycosyl donors
would pave the way toward development of a general and promising method
for glycosylation of GlcN and GalN.

T. Tanaka et al.
642
EXPERIMENTAL
Materials
D-Glucosamine hydrochloride (GlcN-HCl), N-acetyl-D-glucosamine (Glc-
NAc), cellobiose (Glcβ1-4Glc), 2,6-lutidine, and 28% ammonia solution were
purchased from Nacalai Tesque Inc., Kyoto, Japan. D-Galactosamine hy-
drochloride (GalN-HCl) was purchased from Wako Pure Chemical In-
dustries, Ltd., Osaka, Japan. Chitosan dimer (GlcNβ1-4GlcN) and N,N’-
diacetylchitobiose (GlcNAcβ1-4GlcNAc) were purchased from Yaizu Suisank-
agaku Industry Co. Ltd., Shizuoka, Japan. DMT-MM and 2-pyridyl 1-
thioglycosides were synthesized according to the literature.[20,24,25] GlcNase
was prepared according to the literature.[16]
General Methods
NMR spectra were recorded by Bruker DRX-500 or DPX-400. ESI mass
spectra were recorded by Bruker APEXIII. High-performance liquid chro-
matography (HPLC) was conducted with JASCO PU-2089, HITACHI L-7100,
or GL Sciences PU-715 with JASCO UV-2075, SHIMADZU SPD-10AV UV
detectors, or SofTA Corporation 300S Evaporative Light Scattering detector
(ELSD).
Synthesis of GlcN-β-DMT
D-Glucosamine hydrochloride (GlcN-HCl, 1 g, 4.64 mmol) and 2,6-lutidine
(1.08 mL, 9.28 mmol) were added in water (19 mL) and the solution was kept
at rt for 1 day in order to equilibrate the α- and β-anomers. DMT-MM (2.57 g,
9.28 mmol) was added to this mixture and the resulting solution was stirred
for 24 h at rt. After removing the solvent in vacuo, the residue was puri-
fied by silica gel column chromatography (CHCl3/MeOH = 10/1→3/1) to give
4,6-dimethoxy-1,3,5-triazin-2-yl β-D-glucosaminide hydrochloride (GlcN-HCl-
β-DMT, 700 mg, 1.97 mmol, 43%). 1H NMR (500 MHz, D2O): δ (ppm) 6.10 (1H,
d, H-1, J1,2 = 8.76 Hz), 3.92 (6H, s, OCH3), 3.81 (1H, dd, H-6, J5,6 = 1.95 Hz,
J6a,6b = 12.4 Hz), 3.73 (1H, m, H-3), 3.67 (1H, dd, H-6, J5,6 = 5.29 Hz, J6a,6b
=
12.4 Hz), 3.62 (1H, m, H-5), 3.49 (1H, m, H-4), 3.38 (1H, m, H-2). 13C NMR
(126 MHz, D2O): δ (ppm) 173.3, 172.0 (triazine), 93.7 (C-1), 77.0 (C-5), 71.9 (C-
3), 69.2 (C-4), 60.0 (C-6), 56.0 (OCH3), 54.6 (C-2). ESI MS (M+Na)+: 341.1070
(calcd. 341.1067).
Synthesis of GalN-β-DMT
D-Galactosamine hydrochloride (GalN-HCl, 700 mg, 3.25 mmol), DMT-MM
(1.8 g, 6.5 mmol), and 28% ammonia solution (424 μL, 6.5 mmol) were added

4,6-Dimethoxy-1,3,5-triazin-2-yl β-D-glycosaminides
643
in water (13 mL) and the resulting solution was stirred for 6 h at rt. After re-
moving the solvent in vacuo, the residue was purified by silica gel column chro-
matography (CHCl3/MeOH = 10/1→5/1) to give 4,6-dimethoxy-1,3,5-triazin-2-
yl β-D-galacosaminide hydrochloride (GalN-HCl-β-DMT, 518 mg, 1.46 mmol,
45%). 1H NMR (500 MHz, D2O): δ (ppm) 5.71 (1H, d, H-1 , J1,2 = 8.57 Hz), 3.89
(6H, s, OCH3), 3.86 (1H, d, H-4, J3,4 = 2.98 Hz), 3.79 (1H, m, H-5), 3.67–3.65
(3H, m, H-3, H-6×2), 3.14 (1H, dd, H-2, J1,2 = 10.5 Hz , J2,3 = 8.59 Hz). 13C
NMR (126 MHz, D2O): δ (ppm) 173.3, 172.0 (triazine), 97.5 (C-1), 76.3 (C-5),
71.7 (C-3), 67.6 (C-4), 60.1 (C-6), 55.9 (OCH3), 52.1 (C-2).
Hydrolysis of GlcN-β-DMT and GlcNβ1-4GlcN by GlcNase
After preincubation for 5 min, 20 mM GlcN-β-DMT or GlcNβ1-4GlcN was
incubated with GlcNase (736 μU) in 50 mM acetate buffer (pH 5.0) at 37◦C. The
reaction mixture was analyzed by HPLC (column: Shodex Asahipak NH2P-50
4E [φ 4.6 × 250 mm]), eluent: MeCN/H2O = 3/1, flow rate: 1 mL/min, oven:
35◦C, detector: UV 214 nm [GlcN-β-DMT], ELSD [GlcNβ1-4GlcN]).
Hydrolysis of GalN-β-DMT by GlcNase
After preincubation for 5 min, 10 mM GalN-β-DMT was incubated with
GlcNase (920 μU) in 50 mM acetate buffer (pH 5.0) at 37◦C. The reaction mix-
ture was analyzed by HPLC (column: Shodex Asahipak NH2P-50 4E [φ 4.6 ×
250 mm], eluent: MeCN/H2O = 4/1, flow rate: 1 mL/min, oven: 30◦C, detector:
UV 214 nm).
Kinetic Analysis of Enzymatic Hydrolysis of GlcN-β-DMT
After preincubation for 5 min, GlcN-β-DMT was incubated with GlcNase
(92 μU) in 50 mM acetate buffer (pH 5.0) at 37◦C. The reaction mixture was
monitored by quantitating the amount of GlcN-β-DMT by HPLC (column:
Shodex Asahipak NH2P-50 4E [ꢀ 4.6 × 250 mm], eluent: MeCN/H2O = 3/1,
flow rate: 1 mL/min, oven: 30◦C, detector: UV 214 nm). The kinetic constants
Km and kcat of GlcNase were calculated using the nonlinear regression analysis
program KaleidaGraph 4.1J.
Transglycosylation Reaction by Using
4,6-dimethoxy-1,3,5-triazin-2-yl glycoside
A 4,6-dimethoxy-1,3,5-triazin-2-yl glycoside (50 mM) and a glycosyl accep-
tor were incubated with GlcNase (92 μU) in 50 mM buffer solution (pH =
3.5–5.0: acetate buffer; pH = 6.0–8.0: phosphate buffer) 50 μL at 37◦C. The
reaction mixture was analyzed by HPLC (column: Shodex Asahipak NH2P-50
4E [φ 4.6 × 250 mm], eluent: MeCN/H2O = 4/1, flow rate: 1 mL/min, oven:

T. Tanaka et al.
644
30◦C, detector: UV 214 nm or ELSD). The product was isolated by prepara-
tive HPLC (column: Shodex Asahipak NH2P-90 20F [φ 20 × 300 mm], eluent:
MeCN/H2O = 85/15, flow rate: 10 mL/min, oven: 30◦C, detector: UV 280 nm).
GlcNβ1-4GlcNAc-β-SPy
1H NMR (500 MHz, D2O): δ (ppm) 8.28 (1H, m, Py), 7.65 (1H, m, Py), 7.37
(1H, m, Py), 7.18 (1H, m, Py), 5.21 (1H, d, H-1, J1,2 = 10.5 Hz), 4.72 (1H, d, H-
1’, J1’,2’ = 8.32 Hz), 3.86–3.77 (4H, m, H-2, H-4, H-6, H-6’), 3.72 (1H, t, H-3, J =
9.70 Hz), 3.68–3.63 (3H, m, H-5, H-6, H-6’), 3.54 (1H, m, H-3’), 3.44–3.31 (2H,
m, H-4’, H-5’), 2.98 (1H, dd, H-2’, J = 8.45 Hz, 2.22 Hz), 1.86 (3H, s, CH3). 13
C
NMR (126 MHz, D2O): δ (ppm) 174.4 (C O), 155.0, 149.3, 138.5, 124.6, 122.2
(Py), 97.9 (C-1’), 83.7 (C-1), 78.6 (C-5), 76.4 (C-4), 76.3 (C-5’), 73.0 (C-3), 72.2
(C-3’), 69.5 (C-4’), 60.3 (C-6, C-6’), 55.8 (C-2’), 54.0 (C-2), 22.0 (CH3).
GlcNβ1-4GlcNAcβ1-4GlcNAc-β-SPy
1H NMR (400 MHz, MeOH-d4): δ (ppm) 8.33 (1H, dd, Py, J = 4.92 Hz,
0.80 Hz), 7.71 (1H, td, Py, J = 7.60 Hz, 1.84 Hz), 7.42 (1H, d, Py, J = 8.12 Hz),
7.19 (1H, dd, Py, J = 7.48 Hz, 5.92 Hz), 5.42 (1H, d, H-1, J1,2 = 10.7 Hz), 4.84
(1H, d, H-1”, J1”,2” = 8.36 Hz), 4.62 (1H, d, H-1’, J1’,2’ = 8.24 Hz), 4.03–3.33
(17H, m, H-2, H-2’, H-3, H-3’, H-3”, H-4, H-4’, H-4”, H-5, H-5’, H-5”, H-6×2, H-
6’×2, H-6”×2), 3.01 (1H, dd, H-2”, J1”,2” = 8.40 Hz, J2”,3” = 10.5 Hz), 2.05, 1.98
(3H × 2, s, CH3). 13C NMR (100 MHz, MeOH-d4): δ (ppm) 172.4, 172.1 (C O),
157.6, 148.9, 137.2, 122.9, 120.7 (Py), 101.6 (C-1’), 98.1 (C-1”), 83.5 (C-1), 79.3
(C-4), 79.2 (C-5), 77.3 (C-5”), 76.9 (C-4’), 74.8 (C-5’), 74.2 (C-3), 72.6 (C-3”), 72.2
(C-3’), 70.2 (C-4”), 60.9 (C-6’), 60.7 (C-6”), 60.2 (C-6), 56.1 (C-2”), 55.8 (C-2’),
53.6 (C-2), 21.7, 21.5 (CH3).
GalNβ1-4GlcNAc-β-SPy
1H NMR (500 MHz, D2O): δ (ppm) 8.32 (1H, m, Py), 7.69 (1H, m, Py), 7.41
(1H, m, Py), 7.22 (1H, m, Py), 5.25 (1H, d, H-1, J1,2 = 10.5 Hz), 4.74 (1H, d, H-1’,
J1’,2’ = 8.30 Hz), 3.90–3.87 (3H, m, H-2, H-4, H-4’), 3.83–3.77 (3H, m, H-3, H-3’,
H-6), 3.74–3.65 (5H, m, H-5, H-5’, H-6, H-6’×2), 3.17 (1H, dd, H-2’) 1.90 (3H,
s, CH3). 13C NMR (126 MHz, D2O): δ (ppm) 174.4 (C O), 155.0, 149.3, 138.5,
124.6, 122.3 (Py), 98.0 (C-1’), 83.8 (C-1), 78.6 (C-5), 76.2 (C-4), 75.8 (C-5’), 73.2
(C-3), 69.3 (C-3’), 67.5 (C-4’), 60.7 (C-6’), 60.4 (C-6), 54.0 (C-2), 53.5 (C-2’), 22.1
(CH3).
ACKNOWLEDGMENTS
This work was supported by a Grant-in-Aid for Scientific Research from the
Ministry of Education, Sports, Science and Technology, and Grant-in-Aid for
JSPS Fellows 21-2850.

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