Novel dialkoxytriazine-type glycosyl donors for cellulase-catalysed lactosylation

Article abstract of DOI:10.1039/c0ob00190b

Novel glycosidic compounds, 4,6-dialkoxy-1,3,5-triazin-2-yl β-lactosides (DAT-β-Lac), have been prepared directly in water from lactose. The reaction was carried out on a laboratory scale without protecting the hydroxy groups of lactose. The resulting triazine derivatives were found to be recognized by endo-β1,4-glucanase III from Trichoderma reesei (EGIII). The EGIII-catalysed transglycosylation of 4,6-dimethoxy-1,3,5-triazine derivative (DMT-β-Lac) with various glycosyl acceptors has successfully been demonstrated, affording the corresponding lactosylated products.

Full text of DOI:10.1039/c0ob00190b

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Novel dialkoxytriazine-type glycosyl donors for cellulase-catalysed
lactosylation†
Tomonari Tanaka, Masato Noguchi, Kazuhito Watanabe, Takuya Misawa, Masaki Ishihara, Atsushi Kobayashi
and Shin-ichiro Shoda*
Received 31st May 2010, Accepted 8th July 2010
DOI: 10.1039/c0ob00190b
Novel glycosidic compounds, 4,6-dialkoxy-1,3,5-triazin-2-yl b-lactosides (DAT-b-Lac), have been
prepared directly in water from lactose. The reaction was carried out on a laboratory scale without
protecting the hydroxy groups of lactose. The resulting triazine derivatives were found to be recognized
by endo-b1,4-glucanase III from Trichoderma reesei (EGIII). The EGIII-catalysed transglycosylation of
4,6-dimethoxy-1,3,5-triazine derivative (DMT-b-Lac) with various glycosyl acceptors has successfully
been demonstrated, affording the corresponding lactosylated products.
Introduction
The role of enzyme catalysts in stereo-selective synthesis of
oligosaccharides has become increasingly important in recent
years.1 In particular, much attention has been paid to glycosidase-
catalysed glycosylations2 because glycosidases satisfy strong re-
quirements as catalysts for industrial applications. Most glycosi-
dases are considerably stable and easily available. In addition, the
substrates for glycosidases are normally inexpensive.
In principle, glycosidase-catalysed transglycosylations utilise
activated glycosyl donors such as p-nitrophenyl glycosides,3 gly-
cosyl fluorides,4 or sugar oxazolines,5 the use of which greatly
contribute to the increase of transglycosylation yields. However,
the preparation of these glycosyl donors necessitates laborious
tasks including the protection of all of the hydroxy groups,
the regio-selective introduction of a bromine or chlorine into
the anomeric center under acidic conditions, the nucleophilic
substitution of these halogen atoms by, for example, p-nitrophenol
Scheme 1 Synthetic routes of glycosyl donors for enzymatic glycosylation
reactions. Lower: Conventional route that consists of (I) protection of
hydroxy group (᭹ = protecting group), (II) halogenation at the anomeric
position (X = Cl or Br), (III) introduction of a leaving group to the
anomeric position (LG = leaving group), and (IV) deprotection. Upper:
One-step route without using protecting groups (V).
or fluorine atom, and the removal of the protecting groups
(Scheme 1, Route I→II→III→IV).
Furthermore, in the case of employing oligosaccharides with
higher molecular weights or acid-labile functional groups as
starting materials, the cleavage of the inner glycosidic bonds by
an acid are often observed in the course of the introduction of
a leaving group at the anomeric center. Therefore, development
of a new class of glycosyl donors that can be prepared directly
from the corresponding unprotected sugars under mild reaction
conditions has been necessary in order to establish a practical
chemo-enzymatic glycosylating process (Scheme 1, Route V).
Recently, several attempts for direct anomeric activation of un-
protected sugars have been made in polar organic solvents such as
N,N-dimethylformamide.6 For example, p-nitrophenyl glycosides
have successfully been synthesized by using Mitsunobu coupling
between unprotected monosaccharides and p-nitrophenol without
using any protecting groups.6c However, it is extremely difficult
to apply this method to oligosaccharides with higher molecular
weights due to their poorer solubilities in organic solvents.
In this paper, we wish to report that 4,6-dialkoxy-1,3,5-triazin-
2-yl b-lactosides (DAT-b-Lac, 2a–d) can be directly prepared
starting from lactose in water without protecting the hydroxy
groups, and can be recognized by a cellulase catalyst (endo-b1,4-
glucanase).7 Based on these findings, a facile chemo-enzymatic
route for lactosylation via the one-step preparable DAT-type
glycosyl donor has successfully been demonstrated.
Results and discussion
Design and formation of 4,6-dialkoxy-1,3,5-triazine-type
b-lactosides
In general, a monosaccharide unit possesses three kinds of hydroxy
groups, the primary hydroxy group, the secondary hydroxy
groups, and the hemiacetal. The hemiacetal is more acidic than
other hydroxy groups, and can be easily deprotonated to give
the anomeric oxyanion. These facts make it possible for the
hemiacetal to increase its nucleophilicity towards an electrophile.
Department of Biomolecular Engineering, Graduate School of Engineering,
Tohoku University, 6-6-11-514 Aoba, Sendai, Miyagi, 980-8579, Japan.
E-mail: shoda@poly.che.tohoku.ac.jp; Fax: +81-22-795-7293; Tel: +81-22-
795-7230
† Electronic supplementary information (ESI) available: Copies of 1H and
13C NMR spectra. See DOI: 10.1039/c0ob00190b
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Consequently, a preferential attack of the hemiacetal over other
hydroxy groups would be achieved. On the other hand, the use
of water as reaction media would make the nucleophilic attack
of the primary and secondary hydroxy groups disadvantageous
as a result of competitive nucleophilic attack of hydroxy anions
to an electrophile. It is, therefore, extremely important to carry
out the reaction in an aqueous solution in order to restrict the
nucleophilic attack of the primary and secondary hydroxy groups
in the saccharide moiety. These hypotheses are partly supported by
the fact that the pKa value of water (15.7) is located between those
of the hemiacetal (12.2)8 and the primary and secondary hydroxy
groups (ca. 16). It was, therefore, postulated that a chemo-selective
nucleophilic attack with the hemiacetal towards an electrophilic
reagent would be possible in water without any protection of the
primary and secondary hydroxy groups, giving rise to a glycosyl
adduct that can be used as a glycosyl donor for glycosidase-
catalysed transglycosylation.9
We chose 2-chloro-4,6-dialkoxy-1,3,5-triazine derivatives as
appropriate candidates for the electrophiles due to the following
reasons: 1) 4,6-Dialkoxy triazine derivatives are considerably
soluble in water and facilitate the nucleophilic substitution re-
action. 2) The 4,6-dialkoxy triazine moiety can be constructed
by the reaction of cyanuric chloride and the corresponding
alcohols both of which are easily available and inexpensive,
and can be modulated by changing alcohols.10 3) The resulting
glycosyl adducts, 4,6-dialkoxy-1,3,5-triazin-2-yl glycosides (DAT-
glycosides), possess a partial structure of O–C N that could be
activated by protonation by an acidic amino acid in the catalytic
site of glycosidases, resulting in the formation of a glycosyl-enzyme
intermediate.
Scheme 2 Preparation of DAT-b-Lac 2 in water by the reaction of lactose
and DAT-chlorides 1 (a: R1 = R2 = CH3, b: R1 = CH3, R2 = CH2CH3, c:
R1 = R2 = CH2CH3, d: R1 = CH3, R2 = CH2CF3, e: R1 = R2 = CH2CF3)
accompanied by the formation of disubstituted compounds 3a–e.
yields (Table 1, entries 2, 3, and 4). The 2-chlorotriazine derivative
having two trifluoroethoxy groups 1e gave no glycosyl adduct due
to its poorer solubility in water (Table 1, entry 5).
In the presence of a base, lactose was treated with five kinds of 2-
chloro-4,6-dialkoxy-1,3,5-triazines (DAT-chlorides) (1a–e) which
had been prepared via the nucleophilic substitution reaction of
cyanuric chloride with the corresponding alcohols (R1OH and/or
R2OH) (Scheme 2). In case of using 2-chloro-4,6-dimethoxy
derivative (1a: R1 = R2 = CH3), the substitution reaction by the
hemiacetal of lactose took place smoothly in the presence of N-
methylmorpholine (NMM),11 giving rise to the corresponding 4,6-
dimethoxy-1,3,5-triazin-2-yl lactoside 2a in 82% yield (Table 1,
entry 1). When other 2-chlorotriazine derivatives, 1b (R1 = CH3,
R2 = CH2CH3), 1c (R1 = R2 = CH2CH3), and 1d (R1 = CH3,
R2 = CH2CF3) were employed as an electrophile, lactose could
be converted to the corresponding DAT-lactosides in moderate
It was assumed that the reactions proceeded by the ini-
tial attack of N-methylmorpholine to the 2-position of a
DAT-chloride, affording a 4-(4,6-dialkoxy-1,3,5-triazin-2-yl)-4-
methylmorpholinium chloride (DAT-MM) in situ. Then, the
hemiacetal of the lactose attacked the 2-position of the resulting
DAT-MM. These explanations were partiallysupported by thefact
that 4,6-dimethoxy-1,3,5-triazin-2-yl lactoside (DMT-Lac) could
be obtained when commercially available 4-(4,6-dimethoxy-1,3,5-
triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM)12 was
used as electrophile instead of DMT-chloride (Table 2). It has also
been found that the reaction with DMT-MM requires a general
base to enhance the nucleophilicity of the hemiacetal. Various
Table 2 Synthesis of DMT-b-Lac 2a by using DMT-MM in the presence
Table 1 Synthesis of DAT-b-Lac 2 by the reaction of lactose and DAT-
chlorides 1a
of basea
Entry Lactose (b/a) DMT-MM (equiv.) Base (equiv.)
Yield (%)
Entry
DAT-chloride
DAT-b-Lac
Yield (%)
1
2
3
4
5
6
7
8
9
74/26
74/26
74/26
10/90
74/26
74/26
74/26
74/26
74/26
2
2
2
2
2
2
2
2
2
2,6-lutidine (0.5) 71b
1
2
3
4
5
1a
1b
1c
1d
1e
2a
2b
2c
2d
2e
82b (40)c
42c
2,6-lutidine (1.0) 73b
2,6-lutidine (2.0) 70b
2,6-lutidine (2.0) 40c
45c
53d
NMM (1.0)
72c
67c
NDe
NaHCO3 (1.0)
a The reactions were carried out in water starting from b-enriched lactose
(b/a = 74/26) for 24 h at room temperature in the presence of 2
equivalent of DAT-chloride and 2 equivalent of N-methylmorpholine
(NMM). b Determined by 1H NMR by comparing the integrals of the
anomeric proton of the product and that of lactose in D2O. c Isolated yield
after crystallization from methanol. d Isolated yield after silica gel column
chromatography. e No product was detected by 1H NMR in D2O.
(i-Pr)2NEt (1.0) 66c
Et3N (1.0)
63c
62c
pyridine (1.0)
a The reactions were carried out in water for 18-24 h at r.t. b Isolated yield.
c Determined by 1H NMR by comparing the integrals of the anomeric
proton of the product and that of lactose in D2O.
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organic bases as well as sodium bicarbonate as an inorganic base
can be used to give to 4,6-dimethoxy-1,3,5-triazin-2-yl b-lactoside
(DMT-b-Lac) 2a by the reaction of lactose and DMT-MM.
In this reaction, the yield of the resulting DAT-b-glycosides 2
was strongly influenced by the anomeric ratio (b/a) of starting
lactose. When an a-anomerically-enriched lactose (b/a = 10/90)
was reacted with DMT-MM in the presence of 2,6-lutidine, the
yield decreased to 40% (Table 2, entry 4). In addition to the
formation of DAT-b-Lac 2, a disubstituted product 3 having
triazine rings at the 1 and 2 positions of lactose was produced
as by-product in 55% yield. The 1H NMR of the product showed
a doublet signal at 6.7 ppm with a coupling constant of 3.5 Hz
assigned to the anomeric proton. These results clearly indicate that
the stereochemistry of the anomeric center of the by-product is a-
type. Although at the early stage of this reaction, the formation
of a monosubstituted a-type product was observed by 1H NMR
spectroscopy, this compound could not be isolated because it
was converted to the disubstituted product immediately. The
nucleophilicity of the hydroxy group at the 2 position of the
monosubstituted product may be intramolecularly enhanced by
the action of the nitrogen atom in the triazine ring, resulting in the
formation of the disubstituted product.
changes to a half boat conformation in order that one of the
lone pairs on the ring oxygen could be in an antiperiplanar
position to the glycosidic bond connecting the anomeric carbon
and the DAT moiety. Then, the nitrogen atom (1 or 3 position)
in the triazine ring or the glycosidic oxygen are protonated by an
acidic amino acid in the catalytic site, and the DAT moiety was
liberated from the anomeric center, affording an oxocarbenium ion
intermediate or a covalent a-glycosyl-enzyme intermediate. The
resulting intermediate is then attacked by water at the anomeric
position to give the hydrolysate, lactose.
Based on these results obtained in the hydrolysis experiments,
we tried EGIII-catalysed transglycosylation reactions using DAT-
b-Lac 2 as glycosyl donors. Firstly, we investigated the influence
of pH on the transglycosylation yields by using DMT-b-Lac
2a as a glycosyl donor and phenyl l-thio-a-cellobioside 4 as a
glycosyl acceptor (Fig. 1).13 The yield of lactosylated product
reached a maximum when the reaction was performed in pH 5.5.
These results were in agreement with the previous report that
the optimum pH for the hydrolysis of nitrophenyl-b-cellobioside
catalysed by EGIII is 5.5.14
It is, therefore, necessary to utilize a b-anomerically-enriched
lactose (b/a = 74/26) as a starting material in order to increase the
yield. These results clearly indicate that both of the reaction rates
of the aromatic nucleophilic attack of the a-anomer and b-anomer
are considerably large and presumably comparable with that of the
anomeric interconversion between two anomers in water.
The 1H NMR spectrum of DMT-b-Lac 2a showed a singlet peak
at 3.91 ppm (6H) due to the 4,6-dimethoxy groups on the triazine
ring. A doublet peak at 5.81 ppm with the coupling constant of
8.1 Hz is ascribable to the anomeric proton of the reducing end of
the lactoside. These results clearly indicate that the 4,6-dimethoxy
triazine moiety is connected to the anomeric carbon of lactose
with a b-configuration.
Fig. 1 pH-Dependence of EGIII-catalysed lactosylation reaction for 3 h
at 30 ◦C using DMT-b-Lac 2a as a glycosyl donor and 4 as a glycosy
acceptor. (ꢀ: 200 mM acetate, ᭺: 40 mM citrate, ꢀ: 40 mM Mes, ᭛:
40 mM phosphate).
Various glycosyl acceptors 4–8 have been enzymatically trans-
glycosylated, affording the corresponding tetrasaccharides or
trisaccharides (Scheme 3 and Table 4). In the absence of the
enzyme catalyst, no transglycosylated products could be observed
by HPLC analysis, showing that the lactosylated products are
enzymatically transglycosylated.
EGIII-catalysed hydrolysis and transglycosylation using
DAT-b-lactosides as glycosyl substrates
All of the DAT-b-lactosides (DAT-b-Lac) 2a–d were found to
be smoothly hydrolyzed by the action of endo-b1,4-glucanase III
from Trichoderma reesei (EGIII), affording lactose and 2-hydroxy-
4,6-dialkoxy-1,3,5-triazine (4,6-dialkoxy-1,3,5-triazine-2-one) in
acetate buffer (pH 5.5). The enzymatic hydrolysis was monitored
by quantitating the amount of DAT-b-Lac by HPLC (UV at
214 nm). The Michaelis constants (Km) and the first-order rate
constants (kcat) for each substrate have been determined (Table 3).
The detailed mechanism for EGIII-catalysed hydrolysis of DAT-
b-Lac has not been made clear. It is assumed that the 4C1
conformation of the pyranose ring, located in the -1 subsite
The 13C NMR spectra of the resulting oligosaccharides show
signals at around 102 and 78 ppm due to the anomeric and
C4 carbon atoms of the newly formed glycosidic bonds, clearly
indicating that b1,4 glycosidic linkages were stereoselectively
constructed between the glycosyldonor andglycosylacceptors. No
signals derived from a b1,3 or b1,6 glycosidic bond were detected.
When the enzymatic reaction was carried out using DMT-b-Lac
2a as a glycosyl donor under the condition of donor : acceptor
ratio 1 : 1, the yield of tetrasaccharide 9 was 66% (Table 4, entry
1). The other sugar triazine derivatives 2b–d having an electron-
releasing ethoxy group or electron-withdrawing trifluoroethoxy
group on the triazine ring were also able to be used as glycosyl
donors (Table 4, entries 2–4). In the condition of donor : acceptor
ratio 1 : 0.4, the yield of 9 significantly increased to 95% on the
basis of acceptor (Table 4, entry 6). Interestingly, the lactosylating
yield of phenyl l-thio-a-cellobioside 4 was much higher than that
of phenyl 1-thio-b-cellobioside 5a (Table 4, entries 6 and 7).
Table 3 Michaelis–Menten kinetics for hydrolysis DAT-b-Lac 2 catalysed
by EGIII
Entry
Substrate
Km/mM
k
cat/s-1
1
2
3
4
2a
2b
2c
2d
12
13
15
13
1
3
3
1
5.4 0.2
5.6 0.2
4.2 0.4
12 1.6
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Table 4 EGIII-catalysed lactosylation using DAT-b-Lac 2 as glycosyl
donorsa
Entry Donor/Acceptor (mol/mol) Time/h Product Yield (%)b
1
2
3
4
5
6
7
8
2a/4 (1/1)
3
2
2
2
3
1
10
0.25
0.25
—
—
3
3
1
1
3
9
66
48
51
53
76
95
33
3
trace
NDc
NDc
21
47
45
2b/4 (1/1)
9
2c/4 (1/1)
2d/4 (1/1)
9
9
2a/4 (1/0.8)
2a/4 (1/0.4)
2a/5a (1/0.4)
2a/5b (1/0.4)
2a/5c (1/0.4)
2a/6a (1/0.4)
2a/6b (1/0.4)
2a/7a (1/0.4)
2a/7b (1/0.4)
2a/7c (1/0.4)
2a/7d (1/0.4)
2a/8 (1/0.4)
9
9
10a
10b
10c
—
—
11a
11b
11c
11d
12
9
10
11
12
13
14
15
16
39
16
a The reactions were carried out in 200 mM acetate buffer (pH 5.5) at 30 ◦C.
b Determined by HPLC based on acceptor. c No product was detected.
We have observed that the rate of disappearance for the glycosyl
donor 2a in the presence of 5a as glycosyl acceptor is much slower
than the case of using 4 as glycosyl acceptor. These results may be
explained by assuming a competitive inhibition between 5a and 2a
towards the donor site of the EGIII enzyme, because the structure
of these two compounds having a b-glycosidic linkage at their
reducing ends is very similar. In the case of the transglycosylation
by using 4 as glycosyl acceptor, the enzyme may not be inhibited
because the affinity of 4 towards the donor site of EGIII is
considered to be much lower due to the steric hindrance of the
axial SPh group of 4.
The present EGIII-catalysed lactosylation reaction was found
to be applied to monosaccharide acceptors having a b-glycosidic
bond including phenyl b-glucoside derivatives as well as a xyloside
(Table 4, entries 12–16). No product was obtained when a-glucosyl
acceptors 6a and 6b were used, because of the steric hindrance of
the axial group in the +1 subsite of EGIII enzyme (Table 4, entries
10 and 11).
Conclusions
Novel glycosidic compounds, 4,6-dialkoxy-1,3,5-triazin-2-yl b-
lactosides (DAT-b-Lac), can be directly prepared from lac-
tose in water by using 4,6-dialkoxy-1,3,5-triazine-type agents
in aqueous media. The resulting one-step preparable triazine-
type b-lactosides could be used as efficient glycosyl donors for
EGIII-catalysed transglycosylation reactions, affording the corre-
sponding lactosylated products. Compared with the conventional
chemo-enzymatic process, the present methodology requires fewer
tasks for preparation of glycosyl donors. In addition, the fact that
the DAT-derivatives can be prepared in aqueous media without
using any protecting groups enables us to develop a one-pot
procedure without isolating the DAT intermediate.7 The present
chemo-enzymatic process would pave a way which leads to an
efficient and practical synthesis of oligo- and poly-saccharides in
the synthetic field of glycochemistry.
Scheme 3 EGIII-catalysed lactosylation of glycosyl acceptors 4–8 by
using DMT-b-Lac 2a as a glycosyl donor to give lactosylated products
9–12.
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subsequently for 10 h at 60 ◦C. After a solid was removed by
filtration, the filtrate was concentrated in vacuo. The residue was
diluted with AcOEt, washed with water, dried over Na2SO4, and
concentrated in vacuo to give 2-chloro-4,6-diethoxy-1,3,5-triazine
(1.7 g, 8.3 mmol, 83%).
dH (500 MHz, CD3OD) 4.52 (4H, q, OCH2), 1.45 (6H, t, CH3);
dC (126 MHz, CD3OD) 172.6 (1C, N–C–Cl), 172.0 (2C, N–C–O),
65.4 (2C, OCH2), 14.2 (2C, CH3).
Experimental
General
All chemical agents and solvents were commercially available and
purchased. EGIII was expressed and purified by the previous
report.15 The specific activity of the EGIII was 13 U/mg. One unit
of enzyme activity was defined as the amount of enzyme that
released 1 mmol of 2-hydroxy-4,6-dialkoxy-1,3,5-triazine per min.
1H and 13C, NMR spectra were recorded on Bruker DRX-500 at
room temperature. Where applicable, assignments were based on
the combination of several 2D NMR techniques (H–H COSY,
HMQC, HMBC). ESI MS spectra were recorded on Bruker
Daltonics APEXIII. MALDI-TOF MS spectra were recorded on
Shimadzu AXIMA-CFR plus using 2,5-dihydroxybenzonic acid
as a matrix. Specific rotations were measured on Jasco DIP-1000
polarimeter using a 10 cm cell.
2-Chloro-4-(2¢,2¢,2¢-trifluoroethoxy)-6-methoxy-1,3,5-triazine
(1d). To a solution of 2,4-dichloro-6-methoxy-1,3,5-triazine
(2.2 g, 12 mmol) in acetone (60 ml) was added potassium
carbonate (2.2 g, 16 mmol) and 2,2,2-trifluoroethanol (1.1 ml,
16 mmol), and the resulting mixture was stirred for 5 h at
room temperature. After a solid was removed by filtration, the
filtrate was concentrated in vacuo. The residue was purified by
silicagel column chromatography (hexane–AcOEt = 19/1) to
give 2-chloro-4-(2¢,2¢,2¢-trifluoroethoxy)-6-methoxy-1,3,5-triazine
(2.1 g, 8.6 mmol, 72%).
Chemicals
dH (500 MHz, CD3OD) 4.85 (2H, q, OCH2), 4.11 (3H, s, OCH3);
dC (126 MHz, CD3OD) 173.0 (1C, N–C–O), 172.0 (1C, N–C–Cl),
171.0 (1C, N–C–O), 122.0 (1C, q, CF3), 64.2 (1C, OCH2), 56.0
(1C, OCH3).
2,4-Dichloro-6-methoxy-1,3,5-triazine. Cyanuric
chloride
(13.0 g, 70 mmol) was added to a solution of sodium hydrogen
carbonate (6.5 g, 77 mmol) in methanol (480 ml) at 0 ◦C and the
resulting mixture was stirred for 2 h at room temperature. After
a solid was removed by filtration, the filtrate was concentrated
in vacuo to give 2,4-dichloro-6-methoxy-1,3,5-triazine (10.0 g,
63 mmol, 82%).
dH (500 MHz, CD3OD) 4.10 (3H, s, OCH3); dC (126 MHz,
CD3OD) 172.0 (2C, N–C–Cl), 171.0 (1C, N–C–O), 56.2 (1C,
OCH3).
2-Chloro-4,6-bis(2¢,2¢,2¢-trifluoroethoxy)-1,3,5-triazine
(1e).
To a solution of cyanuric chloride (0.46 g, 2.5 mmol) in acetone
(20 ml) was added potassium carbonate (1.4 g, 10 mmol) and
2,2,2-trifluoroethanol (0.54 ml, 7.5 mmol), the mixture was
stirred for 4 h at room temperature. After a solid was removed
by filtration, the filtrate was concentrated in vacuo to give
2-chloro-4,6-bis(2¢,2¢,2¢-trifluoroethoxy)-1,3,5-triazine (0.68 g,
2.2 mmol, 88%).
dH (500 MHz, CD3OD) 4.95 (4H, q, OCH2); dC (126 MHz,
CD3OD) 173.0 (2C, N–C–O), 171.0 (1C, N–C–Cl), 122.0 (2C, q,
CF3), 64.0 (2C, OCH2).
2-Chloro-4,6-dimethoxy-1,3,5-triazine (1a). Cyanuric chloride
(228.7 g, 1.24 mol) was added to a solution of sodium hydrogen
carbonate (260.0 g, 3.0 mol) in methanol (2000 ml) at 0 C and
◦
the resulting mixture was stirred for 2.5 h at room temperature
and subsequently 2 h at 55 ◦C. After removal of a solid by
filtration, the filtrate was concentrated in vacuo. The residue was
crystallized from methanol to give 2-chloro-4,6-dimethoxy-1,3,5-
triazine (123.6 g, 0.70 mol, 57%).
dH (500 MHz, CD3OD) 4.08 (6H, s, OCH3); dC (126 MHz,
CD3OD) 172.6 (2C, N–C–O), 172.1 (1C, N–C–Cl), 55.4 (2C,
OCH3).
4,6-Dimethoxy-1,3,5-triazin-2-yl b-D-lactoside (DMT-b-Lac,
2a). [Method A] (Table 1, entry 1) 2-Chloro-4,6-dimethoxy-
1,3,5-triazine (1a, 527 mg, 3.0 mmol) was added to a solution
of b-D-lactose (513 mg, 1.5 mmol, containing 26% a-lactose) and
N-methylmorpholine (0.33 ml, 3.0 mmol) in water (15 ml), and
the resulting mixture was stirred for 24 h at room temperature.
After concentration in vacuo, the residue was crystallized from
methanol to give 4,6-dimethoxy-1,3,5-triazin-2-yl b-D-lactoside
(289 mg, 1.2 mmol, 40%).
[Method B] (Table 2, entry 2) 2,6-Lutidine (23 ml, 0.20 mmol)
was added to a solution of b-D-lactose (68.5 mg, 0.20 mmol,
containing 26% a-lactose) and 4-(4,6-dimethoxy-1,3,5-triazin-2-
yl)-4-methylmorpholinium chloride (110.7 mg, 0.40 mmol) in
water (1.0 ml), and the resulting mixture was stirred for 18 h at
room temperature. After concentrating the solution in vacuo, the
residue was crystallized from ethanol to give 4,6-dimethoxy-1,3,5-
triazin-2-yl b-D-lactoside (70.0 mg, 0.145 mmol, 73%).
2-Chloro-4-ethoxy-6-methoxy-1,3,5-triazine
(1b). 2,4-
Dichloro-6-methoxy-1,3,5-triazine (0.95 g, 5.3 mmol) was
added to a solution◦of sodium carbonate (0.56 g, 5.3 mmol) in
ethanol (8 ml) at 0 C and the resulting mixture was stirred for
10 h at 60 ◦C. After a solid was removed by filtration, the filtrate
was concentrated in vacuo. The residue was diluted with AcOEt,
washed with water, dried over Na2SO4, and concentrated in
vacuo to give 2-chloro-4-ethoxy-6-methoxy-1,3,5-triazine (0.75 g,
4.0 mmol, 75%).
dH (500 MHz, CD3OD) 4.49 (2H, q, OCH2), 4.05 (3H, s, OCH3),
1.40 (3H, t, CH3); dC (126 MHz, CD3OD) 172.6 and 172.1 (2C,
N–C–O), 172.0 (1C, N–C–Cl), 65.2 (1C, OCH2), 55.3 (1C, OCH3),
13.0 (1C, CH3).
d
H (500 MHz, D2O) 5.81 (1H, d, H-1, J1,2 = 8.1 Hz), 4.36 (1H, d,
H-1¢, J1¢,2¢ = 8.6 Hz), 3.91 (6H, s, OCH3), 3.86–3.81 (2H, m, H-6a,
H-4¢), 3.74–3.61 (7H, m, H-3, H-4, H-5, H-6b, H-5¢, H-6¢ , H-6¢ ),
3.60–3.50 (2H, m, H-2, H-3¢), 3.45 (1H, t, H-2¢, J1¢,2¢, J2¢,3¢ = 8.4 Hz);
dC (126 MHz, D2O) 173.3 and 171.9 (3C, triazine), 102.9 (1C, C-
1¢), 96.8 (1C, C-1), 77.5 (1C, C-4), 75.5 (1C, C-5), 75.3 (1C, C-5¢),
74.0 (1C, C-3), 72.5 (1C, C-3¢), 71.8 (1C, C-2), 70.9 (1C, C-2¢), 68.5
a
b
2-Chloro-4,6-diethoxy-1,3,5-triazine (1c). Cyanuric chloride
(1.9 g, 10 mmol) was added to a solution of sodium hydrogen
carbonate (1.9 g, 22 mmol) in ethanol (10 ml) at 0 ◦C and the
resulting mixture was stirred for 1 h at room temperature, and
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a
(1C, C-4¢), 61.0 (1C, C-6¢), 59.6 (1C, C-6), 55.9 (2C, OCH3); m/z
(ESI) 504.1434 (M+Na+. C17H27N3O13 requires 504.1436); [a]D23
-20.1 (c 1.0 in H2O); Decomposed at 163 ◦C without melting.
H-6a, H-4¢), 3.74–3.60 (7H, m, H-3, H-4, H-5, H-6b, H-5¢, H-6¢ ,
H-6¢ ), 3.59–3.53 (2H, m, H-2, H-3¢), 3.46 (1H, t, H-2¢, J1¢,2¢, J2¢,3¢
b
=
8.4 Hz), 1.26 (6H, t, CH3); dC (126 MHz, D2O) 172.7 and 171.8
(3C, triazine), 102.9 (1C, C-1¢), 96.7 (1C, C-1), 77.5 (1C, C-4), 75.5
(1C, C-5), 75.3 (1C, C-5¢), 74.0 (1C, C-3), 72.4 (1C, C-3¢), 71.7
(1C, C-2), 70.9 (1C, C-2¢), 68.5 (1C, C-4¢), 65.5 (2C, OCH2), 61.0
(1C, C-6¢), 59.6 (1C, C-6), 13.3 (2C, CH3); m/z (ESI) 532.1747
(M+Na+. C19H31N3O13 requires 532.1749).
4,6-Dimethoxy-1,3,5-triazin-2-yl
2-O-(4,6-dimethoxy-1,3,5-
triazin-2-yl)-a-D-lactoside (3a). 2,6-Lutidine (46 ml, 0.40 mmol)
was added to a solution of D-lactose monohydrate (72.1 mg,
0.20 mmol, b/a = 10/90) and 4-(4,6-dimethoxy-1,3,5-triazin-
2-yl)-4-methylmorpholinium chloride (110.7 mg, 0.40 mmol)
in water (1.0 ml), and the resulting mixture was stirred for
13 h at room temperature. After concentrating the solution,
the residue was purified by silicagel column chromatography
(CHCl3–MeOH = 5/1) to give 4,6-dimethoxy-1,3,5-triazin-2-yl
2-O-(4,6-dimethoxy-1,3,5-triazin-2-yl)-a-D-lactoside (67.7 mg,
0.109 mmol, 55%).
4-(2¢,2¢,2¢-Trifluoroethoxy)-6-methoxy-1,3,5-triazin-2-yl
b-D-
lactoside (2d). 2-Chloro-4-(2¢,2¢,2¢-trifluoroethoxy)-6-methoxy-
1,3,5-triazine (1d, 240 mg, 1.0 mmol) was added to a solution of
b-D-lactose (170 mg, 0.5 mmol, containing 26% a-lactose) and
N-methylmorpholine (0.11 ml, 1.0 mmol) in water (5 ml), and
the resulting mixture was stirred for 24 h at room temperature.
After concentrating the solution, the residue was purified by
silicagel column chromatography (CHCl3–MeOH = 3/1) to
dH (500 MHz, D2O) 6.73 (1H, d, H-1, J1,2 = 3.5 Hz), 5.25 (1H,
dd, H-2, J1,2 = 3.6 Hz, J2,3 = 10.1 Hz), 4.42 (1H, d, H-1¢, J1¢,2¢
=
give
4-(2¢,2¢,2¢-trifluoroethoxy)-6-methoxy-1,3,5-triazin-2-yl
7.8 Hz), 4.24 (1H, t, H-3, J2,3, J3,4 = 9.2 Hz), 3.99 (1H, m, H-5),
3.89 (1H, m, H-4), 3.87 (6H, s, OCH3), 3.82 (3H, m, H-6a, H-6b,
b-D-lactoside (90 mg, 0.27 mmol, 53%).
a
b
dH (500 MHz, D2O) 5.80 (1H, d, H-1, J1,2 = 8.1 Hz), 4.86 (2H,
m, OCH2), 4.34 (1H, d, H-1¢, J1¢,2¢ = 7.8 Hz), 3.91 (3H, s, OCH3),
3.81–3.78 (2H, m, H-6a, H-4¢), 3.73–3.60 (7H, m, H-3, H-4, H-5,
H-4¢), 3.80 (6H, s, OCH3), 3.69–3.63 (3H, m, H-5¢, H-6¢ , H-6¢ ),
3.58 (1H, dd, H-3¢, J2¢,3¢ = 10.0 Hz, J3¢,4¢ = 3.4 Hz), 3.49 (1H, m,
H-2¢, J1¢,2¢, J2¢,3¢ = 8.9 Hz); dC (126 MHz, D2O) 173.1, 173.0, 172.2,
and 171.3 (6C, triazine), 102.8 (1C, C-1¢), 92.4 (1C, C-1), 77.0 (1C,
C-4), 75.4 (1C, C-5¢), 75.1 (1C, C-2), 73.3 (1C, C-5), 72.5 (1C,
C-3¢), 70.1 (1C, C-2¢), 69.2 (1C, C-3), 68.5 (1C, C-4¢), 61.0 (1C, C-
6¢), 59.4(1C, C-6), 55.8 and 55.7 (4C, OCH3); m/z (ESI) 643.1816
(M+Na+. C22H32N6O15 requires 643.1818).
H-6b, H-5¢, H-6¢ , H-6¢ ), 3.59–3.51 (2H, m, H-2, H-3¢), 3.46 (1H,
t, H-2¢, J1¢,2¢, J2¢,3¢ = 8.4 Hz); dC (126 MHz, D2O) 173.5, 172.1, and
172.0 (3C, triazine), 122.5 (1C, q, CF3), 102.9 (1C, C-1¢), 97.0 (1C,
C-1), 77.5 (1C, C-4), 75.6 (1C, C-5), 75.3 (1C, C-5¢), 74.0 (1C, C-3),
72.5 (1C, C-3¢), 71.7 (1C, C-2), 70.9 (1C, C-2¢), 68.5 (1C, C-4¢), 63.8
(1C, q, OCH2), 61.0 (1C, C-6¢), 59.6 (1C, C-6), 56.1 (1C, OCH3);
m/z (ESI) 572.1310 (M+Na+. C18H26F3N3O13 requires 572.1308).
a
b
4-Ethoxy-6-methoxy-1,3,5-triazin-2-yl b-D-lactoside (2b). 2-
Chloro-4-ethoxy-6-methoxy-1,3,5-triazine (1b, 569 mg, 3.0 mmol)
was added to a solution of b-D-lactose (513 mg, 1.5 mmol,
containing 26% a-lactose) and N-methylmorpholine (0.33 ml,
3.0 mmol) in water (15 ml), and the resulting mixture was stirred
for 24 h at room temperature. After concentrating the solution
in vacuo, the residue was crystallized and washed from methanol
and dried in vacuo to give 4-ethoxy-6-methoxy-1,3,5-triazin-2-yl
b-D-lactoside (312 mg, 0.63 mmol, 42%).
Enzymatic hydrolysis of triazine-type b-lactosides by EGIII
A mixture of triazine-type b-lactoside and EGIII in 50 mM acetate
buffer was incubated at 30 ◦C. The reaction mixture was analyzed
by HPLC (column; Inertsil ODS-3 (f4.6 ¥ 250 mm, GL-Sciences),
eluent; MeCN–H2O = 5/95, flow rate; 1.0 ml min-1, column oven;
40 ◦C, detection; UV (214 nm)). The hydrolysis activity of EGIII
for substrates 2a–d was evaluated by measuring the amount of
unreacted substrates. The kinetic parameters and their standard
errors were calculated using the nonlinear regression analysis
program “KaleidaGraph 4.0J”.
d
H (500 MHz, D2O) 5.80 (1H, d, H-1, J1,2 = 8.1 Hz), 4.36 (1H, d,
H-1¢, J1¢,2¢ = 7.9 Hz), 4.37 (2H, m, OCH2), 3.91 (3H, s, OCH3), 3.82–
3.80 (2H, m, H-6a, H-4¢), 3.74–3.60 (7H, m, H-3, H-4, H-5, H-6b,
a
b
H-5¢, H-6¢ , H-6¢ ), 3.59–3.54 (2H, m, H-2, H-3¢), 3.46 (1H, t, H-2¢,
1¢,2¢, J2¢,3¢ = 8.4 Hz), 1.27 (3H, t, OCH2CH3); dC (126 MHz, D2O)
J
EGIII-catalysed lactosylation reactions
173.3, 172.7, and 171.9 (3C, triazine), 102.9 (1C, C-1¢), 96.8 (1C,
C-1), 78.3 (1C, C-4), 75.5 (1C, C-5), 75.4 (1C, C-5¢), 74.1 (1C, C-
3), 72.5 (1C, C-3¢), 71.8 (1C, C-2), 70.9 (1C, C-2¢), 68.5 (1C, C-4¢),
65.6 (1C, OCH2), 61.0 (1C, C-6¢), 59.7 (1C, C-6), 55.8 (1C, OCH3),
13.3 (1C, OCH2CH3); m/z (ESI) 518.1594 (M+Na+. C18H29N3O13
requires 518.1593); [a]D23 -4.9 (c 0.5 in H2O); Decomposed at 148 ◦C
without melting.
A mixture of glycosyl donor (2a–d, 0.3 mmol, final conc.: 30 mM),
glycosyl acceptor (4–8), and EGIII (1 ml, 0.4 mg ml-1) in buffer
was incubated at 30 C. The reaction mixtures were analyzed by
HPLC (column; TOSOH TSK-Gel Amide-80 (f4.6 ¥ 250 mm),
eluent; 80 vol.% MeCN aq., flow rate; 1.0 ml min-1, column
oven; 40 ◦C, detection; UV (214 nm)). The lactosylated products
were isolated by preparative HPLC (column; TOSOH TSK-Gel
Amide-80 (f21.5 ¥ 300 mm), eluent; MeCN–H2O = 7/3, flow rate;
7.0 ml min-1, column oven; 40 ◦C, detection; UV (214 nm)).
◦
4,6-Diethoxy-1,3,5-triazin-2-yl b-D-lactoside (2c). 2-Chloro-
4,6-diethoxy-1,3,5-triazine (1c, 611 mg, 3.0 mmol) was added to
a solution of b-D-lactose (513 mg, 1.5 mmol, containing 26% a-
lactose) and N-methylmorpholine (0.33 ml, 3.0 mmol) in water
(15 ml), and the resulting mixture was stirred for 24 h at room
temperature. After concentrating the solution in vacuo, the residue
was crystallized from methanol to give 4,6-diethoxy-1,3,5-triazin-
2-yl b-D-lactoside (344 mg, 0.68 mmol, 45%).
Phenyl
glucopyranosyl)-b-D-glucopyranosyl)-a-D-glucopyranoside
dH (500 MHz, D2O) 7.47 and 7.29 (5H, Ph), 5.52 (1H, d, H-1,
J1,2 = 5.4 Hz), 4.44 and 4.43 (2H, d, H-1¢, H-1¢¢, J1¢,2¢, J1¢¢,2¢¢
8.0 Hz), 4.33 (1H, d, H-1¢¢¢, J1¢¢¢,2¢¢¢ = 7.8 Hz), 4.22 (1H, H-5),
3.90–3.50 (20H, m, sugar-H), 3.43 (1H, t, H-2¢¢¢, J1¢¢¢,2¢¢¢, J2¢¢¢,3¢¢¢
1-thio-4-O-(((4-O-b-D-galactopyranosyl)-4-O-b-D-
(9).
=
dH (500 MHz, D2O) 5.77 (1H, d, H-1, J1,2 = 8.1 Hz), 4.36 (1H,
d, H-1¢, J1¢,2¢ = 7.9 Hz), 4.36 (4H, m, OCH2), 3.83–3.78 (2H, m,
=
9.0 Hz), 3.27–3.23 (2H, m, H-2¢, H-2¢¢); dC (126 MHz, D2O)
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132.8, 132.3, 129.4, and 128.3 (6C, Ph), 102.9 (1C, C-1¢¢¢), 102.3
(2C, C-1¢, C-1¢¢), 88.9 (1C, C-1), 78.6 (1C, C-4), 78.2 and 78.0
(2C, C-4¢, C-4¢¢), 75.3 (1C, C-5¢¢¢), 74.8 (2C, C-5¢, C-5¢¢), 74.1 and
74.0 (2C, C-3¢, C-3¢¢), 72.9 and 72.8 (2C, C-2¢, C-2¢¢), 72.5 (1C,
C-3¢¢¢), 72.1 (1C, C-3), 71.4 (1C, C-5), 70.9 (2C, C-2, C-2¢¢¢), 68.5
(1C, C-4¢¢¢), 61.0 (1C, C-6¢¢¢), 59.9, 59.8, and 59.7 (3C, C-6, C-6¢,
C-6¢¢); m/z (MALDI-TOF) 782.1 (M+Na+. C30H46O20S requires
781.8).
and B. Pfannemueller, Carbohydr. Res., 1987, 160, 185–204; (e) M.
Kitaoka and K. Hayashi, Trends Glycosci. Glycotechnol., 2002, 14, 35–
50; (f) L. F. Mackenzie, Q. Wang, R. A. J. Warren and S. G. Withers,
J. Am. Chem. Soc., 1998, 120, 5583–5584.
2 (a) C. H. Wong and G. M. Whitesides, in Enzymes in Synthetic Organic
Chemistry, Pergamon, 1994; (b) K. G. I. Nilsson, in Modern Methods
in Carbohydrate Synthesis, ed. S. H. Khan and R. A. O’Neill, Harwood
Academic Publishers, 1996, pp. 518–547; (c) D. J. Vocadlo and S. G.
Withers, in Carbohydrates in Chemistry and Biology, ed. B. Ernst,
G. W. Hart and P. Sinay¨, Wiley-VCH, Weinheim, 2000, Vol 2, pp. 723–
844; (d) S. Shoda, in Glycoscience, ed. B. O. Fraser-Reid, K. Tatsuta and
J. Thiem, Springer-Verlag, Berlin, Heidelberg, New York, 2001, Vol. II,
pp. 1465–1496; (e) P. Bojarova´-Fialova´ and V. Krˇen, in Comprehensive
Glycoscience, ed. J. P. Kamerling, 2007, Elsevier, Oxford, 2007, Vol.
1, pp. 453–487; (f) J. Thiem, in Glycoscience, ed. B. O. Fraser-Reid,
K. Tatsuta and J. Thiem, Springer-Verlag, Berlin, Heidelberg, New
York, 2008, Vol. II, pp. 1387–1409.
3 (a) K. G. I. Nilsson, Carbohydr. Res., 1987, 167, 95–103; (b) J. Y. Winum,
A. Leydt, M. Seman and J. L. Montero, Farmaco, 2001, 56, 319–324.
4 (a) S. Shoda, K. Obata, O. Karthaus and S. Kobayashi, J. Chem. Soc.,
Chem. Commun., 1993, 1402–1404; (b) S. Shoda, M. Fujita and S.
Kobayashi, Trends Glycosci. Glycotechnol., 1998, 10, 279–289; (c) D. S.
Genghof, C. F. Brewer and E. J. Hehre, Carbohydr. Res., 1978, 61,
291–299.
5 (a) S. Kobayashi, T. Kiyosada and S. Shoda, J. Am. Chem. Soc., 1996,
118, 13113–13114; (b) S. Shoda, M. Fujita, C. Lohavisavapanichi,
Y. Misawa, K. Ushizaki, Y. Tawata, M. Kuriyama, M. Kohri, H.
Kuwata and T. Watanabe, Helv. Chim. Acta, 2002, 85, 3919–3936; (c) S.
Fujikawa, M. Ohmae and S. Kobayashi, Biomacromolecules, 2005, 6,
2935–2942; (d) B. Li, Y. Zeng, S. Hauser, H. Song and L. X. Wang,
J. Am. Chem. Soc., 2005, 127, 9692–9693.
6 (a) S. K. Sharma, G. Corrales and S. Penade´s, Tetrahedron Lett., 1995,
36, 5627–5630; (b) U. Huchel, C. Schmidt and R. R. Schmidt, 1995,
Tetrahedron Lett., 1995, 36, 9457–9460; (c) S. Shoda, A. Kobayashi
and S. Takahashi, PCT Int. Appl., WO 2006038440, 2006; (d) A. V.
Gudmundsdottir and M. Nitz, Org. Lett., 2008, 10, 3461–3463.
7 T. Tanaka, M. Noguchi, A. Kobayashi and S. Shoda, Chem. Commun.,
2008, 2016–2018.
Phenyl 1-thio-4-O-(((4-O-b-D-galactopyranosyl)-4-O-b-D-gluco-
pyranosyl)-b-D-glucopyranosyl)-b-D-glucopyranoside (10a). dH
(500 MHz, D2O) 7.47 and 7.29 (5H, Ph), 4.7 (1H, H-1, included in
DOH), 4.41 (2H, d, H-1¢, H-1¢¢, J1¢,2¢, J1¢¢,2¢¢ = 7.8 Hz), 4.33 (1H, d,
H-1¢¢¢, J1¢¢¢,2¢¢¢ = 7.4 Hz), 3.90–3.45 (20H, m, sugar-H), 3.42 (1H, t,
H-2¢¢¢, J1¢¢¢,2¢¢¢, J2¢¢¢,3¢¢¢ = 8.5 Hz), 3.30–3.20 (3H, m, H-2, H-2¢, H-2¢¢);
dC (126 MHz, D2O) 132.8, 132.3, 129.4, and 128.3 (6C, Ph), 102.9
(1C, C-1¢¢¢), 102.2 (2C, C-1¢, C-1¢¢), 87.1 (1C, C-1), 78.7 (1C, C-4),
78.0 (2C, C-4¢, C-4¢¢), 75.6, 75.3, 74.8, 74.0, 73.9, 72.9, 72.8, 72.3,
71.4, and 70.9 (12C, sugar-C), 68.5 (1C, C-4¢¢¢), 61.0 (1C, C-6¢¢¢),
60.0 (3C, C-6, C-6¢, C-6¢¢); m/z (MALDI-TOF) 782.1 (M+Na+.
C30H46O20S requires 781.8).
Phenyl 1-thio-4-O-((4-O-b-D-galactopyranosyl)-b-D-glucopyra-
nosyl)-b-D-glucopyranoside (11a). dH (500 MHz, D2O) 7.47 and
7.29 (5H, Ph), 4.7 (1H, H-1, included in DOH), 4.41 (1H, d, H-1¢,
J1¢,2¢ = 8.0 Hz), 4.32 (1H, d, H-1¢¢, J1¢¢,2¢¢ = 7.8 Hz), 3.86–3.80 (3H,
a
m, H-6a, H-6¢ , H-4¢¢), 3.71–3.47 (12H, m, sugar-H), 3.42 (1H,
t, H-2¢¢, J1¢¢,2¢¢, J2¢¢,3¢¢ = 9.1 Hz), 3.30–3.21 (2H, m, H-2, H-2¢); dC
(126 MHz, D2O) 132.8, 132.3, 129.4, and 128.3 (6C, Ph), 102.9
(1C, C-1¢¢), 102.3 (1C, C-1¢), 87.1 (1C, C-1), 78.7 (1C, C-4), 78.0
(2C, C-4¢, C-5¢¢), 75.6 and 74.1 (2C, C-3¢, C-3¢¢), 75.3 and 74.8
(2C, C-5, C-5¢), 72.8 (1C, C-2¢), 72.5 (1C, C-3), 71.5 (1C, C-2),
70.9 (1C, C-2¢¢), 68.5 (1C, C-4¢¢), 61.0 (1C, C-6¢¢), 60.0 and 59.9
(2C, C-6, C-6¢); m/z (MALDI-TOF) 619.7 (M+Na+. C24H36O15S
requires 619.7).
8 J. Thamsen, Acta Chem. Scand., 1952, 6, 270–284.
9 (a) M. Noguchi, T. Tanaka, H. Gyakushi, A. Kobayashi and S. Shoda,
J. Org. Chem., 2009, 74, 2210–2212; (b) T. Tanaka, W. C. Huang, M.
Noguchi, A. Kobayashi and S. Shoda, Tetrahedron Lett., 2009, 50,
2154–2157; (c) T. Tanaka, T. Matsumoto, M. Noguchi, A. Kobayashi
and S. Shoda, Chem. Lett., 2009, 38, 458–459; (d) T. Tanaka, H. Nagai,
M. Noguchi, A. Kobayashi and S. Shoda, Chem. Commun., 2009, 3378–
3379.
Acknowledgements
10 (a) Z. J. Kamin´ski, Synthesis, 1987, 917–920; (b) Z. J. Kamin´ski, P.
Paneth and J. Rudzin´ski, J. Org. Chem., 1998, 63, 4248–4255; (c) Z. J.
Kamin´ski, B. Kolesin´ska, J. Kolesin´ska, G. Sabatino, M. Cheli, P.
Rovero, M. Błaszczyk, M. L. Gło´wka and A. M. Papini, J. Am. Chem.
Soc., 2005, 127, 16912–16920.
11 Use of other bases, 2,6-lutidine or triethylamine, resulted in a decrease
of the yield.
12 (a) M. Kunishima, C. Kawachi, J. Morita, K. Terao, F. Iwasaki and S.
Tani, Tetrahedron, 1999, 55, 13159–13170; (b) F. Iwasaki, M. Miharu,
N. Hirano, M. Saijyo, S. Tani, M. Kunishima and K. Terao, PCT Int.
Appl., WO 2000053544, 2000.
The authors thank to Prof. Yasushi Morikawa (Nagaoka Univer-
sity of Technology) for supporting expression of EGIII. This work
was supported by a Grant-in Aid for Scientific Research from the
Ministry of Education, Sports, Science and Technology, Industrial
Technology Research Grant Program in 2006 from NEDO of
Japan, and an International Center of Research & Education for
Molecular Complex Chemistry in 2007 from Tohoku University
Global COE Program.
13 Phenyl thio-a-cellobioside 4 was found to be the best acceptor for an
EGIII-catalysed lactosylation reaction using b-lactosyl fluoride as a
glycosyl donor in comparison with a-glucoside, b-glucoside, and b-
cellobioside, which will be reported elsewhere.
Notes and references
1 (a) O. Blixt and N. Razi, in Glycoscience, 2nd edn, ed. B. O. F. Reid,
K. Tatsuta and J. Thiem, Springer-Verlag, Berlin, Heiderberg, New
York, 2008, Vol. 2, pp. 1361–1385; (b) M. M. Palcic and O. Hindsgaul,
Trends Glycosci. Glycotechnol., 1996, 39, 37–49; (c) L. Liu, C. S.
Bennett and C. H. Wong, Chem. Commun., 2006, 21–33; (d) G. Ziegast
14 M. Sandgren, J. Sta˚hlberg and C. Mitchinson, Prog. Biophys. Mol.
Biol., 2005, 89, 246–291.
15 H. Okada, K. Tada, T. Sekiya, K. Yokoyama, A. Takahashi, H. Tohda,
H. Kumagai and Y. Morikawa, Appl. Environ. Microbiol., 1998, 64,
555–563.
5132 | Org. Biomol. Chem., 2010, 8, 5126–5132
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