Preparation of p-nitrophenyl β-l-arabinofuranoside as a substrate of β-l-arabinofuranosidase

Article abstract of DOI:10.1016/j.carres.2013.10.005

Synthesis of p-nitrophenyl β-l-arabinofuranoside 1 as the substrate for novel β-l-arabinofuranosidase has been achieved by using both our inter- and intra-molecular glycosylation methodologies. Although the intermolecular glycosylation with l-Araf donors 3 and 4 resulted in a mixture of both α- and β-isomers, NAP ether-mediated IAD with 3 and 6 afforded the desired β-l-arabinofuranoside stereospecifically which was confirmed by NMR analysis on the ³J_H1-H2 coupling constant and ¹³C chemical shift of C1. As expected, 1 has been revealed to be an efficient substrate in the biological study of a novel β- arabinofuranosidase such as HypBA1 with higher apparent affinity compared with other reported substrates.

Full text of DOI:10.1016/j.carres.2013.10.005

Carbohydrate Research 382 (2013) 95–100
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Note
Preparation of p-nitrophenyl b-L-arabinofuranoside as a substrate
of b-L-arabinofuranosidase
Sophon Kaeothip , Akihiro Ishiwata , Tasuku Ito , Shinya Fushinobu , Kiyotaka Fujita d,
Yukishige Ito
a
a
b,
⇑
c
c,
⇑
a,b
ERATO Glycotrilogy Project, Japan Science and Technology Agency (JST), 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
Department of Biotechnology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8647, Japan
Faculty of Agriculture, Kagoshima University, Korimoto, Kagoshima 890-0065, Japan
b
c
d
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 22 August 2013
Received in revised form 30 September
Synthesis of p-nitrophenyl b-L-arabinofuranoside 1 as the substrate for novel b-L-arabinofuranosidase has
been achieved by using both our inter- and intra-molecular glycosylation methodologies. Although the
intermolecular glycosylation with -Araf donors 3 and 4 resulted in a mixture of both - and b-isomers,
NAP ether-mediated IAD with 3 and 6 afforded the desired b- -arabinofuranoside stereospecifically
which was confirmed by NMR analysis on the 3
H1–H2 coupling constant and C chemical shift of C1.
L
a
2
013
L
Accepted 7 October 2013
Available online 14 October 2013
13
J
As expected, 1 has been revealed to be an efficient substrate in the biological study of a novel b-arabin-
ofuranosidase such as HypBA1 with higher apparent affinity compared with other reported substrates.
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
b-
b-
L
-Arabinofuranoside
L-Arabinofuranosidase
PNP glycoside
Stereoselective synthesis
Enzyme substrate
Arabinofuranose (Araf) is one of the important constituents of
By contrast, several derivatives of
a-L-Araf are obtainable from
bacterial glycoconjugates, in which Araf residues are incorporated
commercial sources, while a variety of them are accessible from
1
10
in a
D
-enantiomeric form. On the other hand, plants solely employ
L
-arabinofuranose tetraacetate. By using them as substrates,
1
1
L-Araf. In either case, however, Araf residues mostly exist as
a-gly-
mechanisms of a-L-arabinofuranosidase have been well explored.
coside, with several important exceptions. For instance, non-
Because of the presence of cis-oriented 2-hydroxy group, prepara-
tion of b-arabinofuranoside is far less straightforward.12 Various
approaches toward stereoselective synthesis of b-Araf have been re-
ported for both bacterial and plant b-arabinofuranosides based on
reducing ends of mycobacterial cell wall arabinans are covered
2
by b-
L
-Araf, while in plants, oligosaccharide parts of hydroxypro-
3
4
line-rich glycoproteins (HRGPs), glycopeptide hormones, bio-
5
13
14,15
polymers comprise b-
the dinoflagellate algae Protoceratium reticulatum.
On the other hand, novel b- -arabinofuranosidases, HypBA2 and
L
-Araf, and so does glycoyessotoxin from
direct or intramolecular
glycosylation strategies (see Fig. 1).
6
To obtain 1,2-cis glycosides selectively, an approach based on
1
6
L
intramolecular aglycon delivery (IAD) is one of the most reliable.
HypBA1 have been recently isolated from human gastrointestinal
Among a number of variants, 2-(naphthyl)methyl (NAP) ether-
bacterium, Bifidobacterim longum.7 Both HypBA2 and HypBA1
,8
mediated IAD
15
has been shown to be most generic, which
were reported to show endo- and exo-b-
activity, which were categorized into new glycoside hydrolase
GH) families, GH121 and GH127, respectively. HypBA1 is in a do-
L
-arabinofuranosidases
achieved stereoselective glycosylation of various sugar residues.
Recently, the stereoselective synthesis of CLV3 glycopeptide hav-
(
ing hydroxylproline-linked b-
using IAD as the key reaction.
L
-Araf triose has been completed
1
4e,15c
main of unknown function (DUF) 1680 family, which is conserved
9
in many bacterial, fungi and plant species. However, facile evalu-
The initial attempt was made by use of 3,5-O-tetra-
isopropyldisiloxanylidene (TIPDS)-protected donors (3, 4), in light
ation of their enzymatic activities has been retarded by the diffi-
culty to obtain proper substrates.
of our experience in stereoselective synthesis of b-
D-arabinofur-
anosides. Tolyl 3,5-O-TIPDS-1-thio- -arabinofuranoside 2 was pre-
L
pared from
congener
L
3e
-arabinose as reported previously for the enantiomeric
(Scheme 1). It was converted to 2-O-triisopropylsilyl
⇑
Corresponding authors. Tel.: +81 48 467 9434; fax: +81 48 462 4680 (A.I.);
tel./fax: +81 3 5841 5151 (S.F.).
1
(
TIPS)-protected (3) as well as to 2-O-NAP protected derivatives
E-mail addresses: aishiwa@riken.jp (A. Ishiwata), asfushi@mail.ecc.u-tokyo.ac.jp
(
S. Fushinobu).
(4, 6).
0
008-6215/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.carres.2013.10.005

9
6
S. Kaeothip et al. / Carbohydrate Research 382 (2013) 95–100
Glycosylation of 2-O-TIPS-protected donor 3 with p-nitrophenol
PNP–OH) under NIS-AgOTf conditions17 gave the desired product
b, however, only as the minor isomer (Scheme 2). Under similar
conditions, 2-O-NAP protected donor 4 gave similar results. Never-
theless, isolated b-isomers 7b and 8b were converted to the PNP
RO
NAPO
TolS
O
OR
O
O
(
7
a,b,c,d
g,h
Si(i-Pr)
2
L-arabinose
O
TolS
O
Si(i-Pr)
2
OR
R
R
b-
L
-arabinofuranoside 1, after deprotection in a standard manner.
To obtain the b-arabinofuranoside stereoselectively, we next
H
H
2
3
4
5
6
e
TIPS
NAP
TIPS
f
examined NAP-ether mediated IAD using the glycosyl donor 4
Scheme 3). DDQ treatment of 3 with PNP–OH under anhydrous
conditions gave the corresponding mixed acetal 9 (MALDI-TOF
MS: [M+Na]+ calcd for
Na Si 798.29, found
98.35). The latter was then treated with MeOTf in the presence
(
Scheme 1. Reagent and conditions: (a–d) Ref. 15c; (e) TIPSOTf, lutidine, CH
9%; (f) NAPBr, NaH, 84%; (g) TBAF, THF; (h) TIPSOTf, lutidine, CH Cl , quant. in two
steps.
2 2
Cl ,
8
2
2
C
41
H
53
N
1
1
O
8
S
1
2
,
7
1
8
of 2,6-di-t-butyl-4-methylpyridine (DTBMP) to give the desired
PNP 3,5-O-TIPDS-b- -arabinofuranoside 10 as a sole stereoisomer
TIPSO
PNPO
TIPSO
PNPO
a
O
O
L
O
O
3
4
+
+
in 53% yield after acidic work-up. Subsequent deprotection affor-
ded the pure b-isomer 1. An alternative donor 6 which is devoid
of 3,5-O-cyclic protection afforded the desired product 12 in 50%
yield at room temperature, which was also deprotected to afford 1.
Anomeric configuration of the arabinofuranoside 1 was con-
Si(i-Pr)
2
Si(i-Pr)
2
PNPOH
O
O
O
Si(i-Pr)
2
O
Si(i-Pr)
2
7
α
7β
b
1
NAPO
PNPO
NAPO
PNPO
1
9
a
O
O
firmed by NMR in comparison with methyl
L
-arabinofuranosides.
O
O
3
Si(i-Pr)
2
Si(i-Pr)2
As summarized in Table 1, both JH1–H2 coupling constants and d
C
PNPOH
O
O
values were in accordance with the stereochemical assignment of
the products.
O
Si(i-Pr)2
O
Si(i-Pr)2
8α
8β
c
To confirm the suitability of 1 as a substrate, its kinetic param-
1
eters for hydrolysis by b-
.2.1.185) were measured (Table 2). The K
were revealed to be 0.13 ± 0.03 mM, 16.6 ± 1.6 s
L-arabinofuranosidase HypBA1 (EC
Scheme 2. Intermolecular arabinofuranosylation with p-nitrophenol. Reagent and
conditions: (a) NIS, AgOTf, MS4A, CH Cl , 92%, :b = 6.1:1; (b) NH F, THF–MeOH
1:1), 40 °C, 28%; (c) i. TBAF, THF, 68%; ii. DDQ, CH Cl –H O; iii. Ac O, pyridine, 87%
3
m
, kcat, and kcat/K
m
values
and
ꢀ
1
2
2
a
2
4
(
2
2
2
ꢀ1
ꢀ1
1
30 ± 20 s mM , respectively. HypBA1 preferentially hydro-
lyzed substrate Ara -Hyp (b- -Araf-(1?2)-b- -Araf-Hyp derivative)
with K , kcat, and kcat/K values of 0.31 mM, 6.3 s and 20 s
respectively, which was compared with b- -Araf
-Araf-(1?2)-b- -Araf) and Ara-Hyp (b- -Araf-Hyp derivative)
shown in Table 2. These results indicated that PNP b- -arabinofu-
ranoside 1 was indeed an excellent substrate of HypBA1, having
–4 orders of magnitude higher values than other
Hyp-linked substrates.
In conclusion, we synthesized p-nitrophenyl b-
side 1 as the substrate for novel b- -arabinofuranosidase using
both our inter- and intra-molecular glycosylation methodologies.
Although the intermolecular glycosylation with -Araf donors 3
and 4 afforded b-glycoside as the minor isomer, NAP ether-medi-
ated IAD with 2-O-NAP- -Araf donors 4 and 6 resulted in the ste-
reospecific formation of desired b- -arabinofuranoside which was
in two steps; iv. NaOMe, MeOH, 81%.
2
L
L
ꢀ
1
ꢀ1
m
m
ꢀ1
mM
b-
,
L
2
1. Experimental
(
L
L
L
L
1.1. General procedure
2
kcat/K
m
Column chromatography was performed on silica gel 60 (EM
Science, 40–100 mesh). Reactions were monitored by thin-layer
chromatography (TLC) on Kieselgel 60 F254 (EM Science), and
the compounds were detected by examination under UV light
and by charring with 10% sulfuric acid in methanol. Solvents were
removed under reduced pressure at <40 °C. CH Cl was freshly dis-
L-arabinofurano-
L
L
2
2
tilled from calcium hydride under nitrogen prior to use. AgOTf was
co-evaporated with toluene (2–3 times) and dried in vacuo for
2–3 h directly prior to application. Molecular sieves (4 Å) were
activated at 170–200 °C for 2–3 h under vacuo prior to application.
L
L
3
confirmed by NMR analysis on the
J
H1–H2 coupling constant and
1
3
1
C chemical shift of C1. The molecule has been demonstrated to
All reactions were carried out under an argon atmosphere. H NMR
1
3
be useful as the substrate in the biological study of novel b-arabi-
nofuranosidases which is a more efficient substrate with higher
apparent affinity compared with other reported substrates.
and C NMR spectra were recorded with a Bruker Advance 500
1
spectrometer with a cryoprobe. H NMR spectra were referenced
to TMS at 0.00 ppm, CHCl3 at 7.24 and MeOH at 3.31 ppm.
Hyp
Hyp
O
OH
OH
O
OOH
O
Hyp
OH
PNPO
O
OOH
O
OH
OH β-L-Arafase
HypBA2
OH
1
HO
O
O
OH
O
β
OH
O
HO
OH
OH
HO
OH
O
O
O
OOH
OH
OH
OH
O
OH
O
O
β-L
-Arafase
HypBA1
HO
OH
OH
β
O
OH
O
α-
L-Arafase
HO
HO
OH
OH
α
HypAA (GH43 family)
HO
O
HO
OH
Hyp : hydroxyproline
Figure 1.

S. Kaeothip et al. / Carbohydrate Research 382 (2013) 95–100
97
chromatography using a gradient solvent system (hexane/ethyl
acetate = 100/1 to 50/1 to 25/1 to 10/1) to give the title compound
Naph
O
PNPO
PNPO
HO
2
6
1
a
O
b
d
O
O
PNPO
PNPO
O
3 (1.77 g, 89%): ½
a
ꢁ
D
ꢀ106° (c 1.0, CHCl
3
); H NMR (400 MHz, CD3-
4
6
Si(i-Pr)
Si(i-Pr)2
2
OD): d 0.92–1.20 (m, TIPDS, TIPS, 49H), 2.30 (s, Me, 3H), 3.90 (dd,
J = 12.4, 6.4 Hz, 5-H, 1H), 4.03 (dd, J = 12.4, 3.2 Hz, 5-H, 1H), 4.11
(td, J = 6.8, 3.2 Hz, 4-H, 1H), 4.23 (dd, J = 6.8, 4.0 Hz, 3-H, 1H),
PNPOH
O
O
TolS
O
9
Si(i-Pr)
2
O
Si(i-Pr)2
10
c
1
4.32 (dd, J = 4.0, 2.8 Hz, 2-H, 1H), 5.26 (d, J = 2.8 Hz, 1-H, 1H),
Naph
O
13
7
.07 (d, J = 8.4 Hz, Ar, 2H), 7.35 (d, J = 8.4 Hz, Ar, 2H); C NMR
HO
O
(
100 MHz, CDCl
3
): d 12.4, 12.9, 13.0, 13.65, 13.69, 17.02, 17.10,
a
OTIPS
OTIPS
O
1
7.16, 17.18, 17.24, 17.27, 17.34, 17.47, 18.0, 18.1, 21.1, 62.7
PNPOH
1
(
C5), 80.1 (C3), 82.3 (C2), 84.3 (C4), 93.5 (C1, JC1–H = 173.5 Hz),
TolS
OTIPS
OTIPS
+
1
O
29.6, 131.6, 137.1, 141.8; ESI-TOF MS: [M+Na] calcd for C33H
62-
1
1
12
+
e
5
1
S Si
3
Na
1
, 677.35, found 677.45; HRMS ESI-TOF: [M+Na] calcd
Si Na , 677.35235, found 677.3540.
1
for C33
H
62
O
5
S
1
3
1
Scheme 3. Intramolecular arabinofuranosylation with p-nitrophenol. Reagent and
conditions: (a) DDQ, MS4A, CH Cl ; (b) MeOTf, DTBMP, MS4A, CH Cl , 40 °C; then
TFA, CHCl , 0 °C, 53% (b only) from 4; (c) i. TBAF, THF; ii. Ac O, pyridine, 79%; iii.
NaOMe, MeOH, 81%; (d) MeOTf, DTBMP, MS4A, CH Cl , room temperature; then
TFA, CHCl , 0 °C, 50% (b only) from 6; (e) NH F, THF–MeOH (1:1), 40 °C, 83%.
2
2
2
2
1
a
.3. Tolyl 2-O-naphthylmethyl-3,5-di-O-triisopropylsilyl-1-thio-
3
2
-
L-arabino-furanoside (6)
2
2
3
4
To the solution of 3,5-O-TIPDS-protected
4
(588.0 mg,
0
.920 mmol) in THF (10 mL) was added 1.0 M solution of TBAF in
13C NMR spectra were referenced to the central peak of CDCl
7.0 ppm and CD OD at 49.0 ppm. Assignments were made by
standard pfg COSY and pfg HSQC or pfg HMQC. Optical rotations
were measured with a ‘Jasco DIP-370’ polarimeter. HRMS determi-
nations were made by the use of a JEOL AccuTOF JMS-T700LCK
at
3
THF (2.30 mL, 2.30 mmol) at room temperature. The mixture was
stirred for 18 h at room temperature and evaporated in vacuo.
The residue was purified by silica gel column chromatography
using gradient solvent system (CHCl /ethyl acetate = 20/1 to 10/1
to 5/1 to 3/1) to give tolyl 2-O-naphthylmethyl-1-thio-L-arabinofu-
7
3
3
mass spectrometer with CF
3
CO
2
Na as the internal standard. All
ranoside (5). Analytical data for Tolyl 2-O-naphthylmethyl-1-thio-
2
6
1
3
other reagents were purchased from the Wako Pure Chemical
Industries Ltd, Kanto Chemicals Co. Inc., Tokyo Chemical Industries
Co., Ltd, and Aldrich Chemical Co.
a
-
L
-arabinofuranoside (5): ½
a
ꢁ
ꢀ56° (c 1.0, CHCl ); H NMR
D
(400 MHz, CD OD): d 2.30 (s, Me, 3H), 3.57 (br s, OH, 1H), 3.76
3
(br dd, J = 12.4, 2.8 Hz, 5-H, 1H), 3.82 (br dd, J = 12.4, 3.2 Hz, 1-H,
1
H), 4.07–4.15 (m, 2-H, 4-H, 1H), 4.28–4.36 (m, 3-H, OH, 2H),
1
.2. Tolyl 3,5-O-tetraisopropyldisiloxanylidene-2-O-
4.74–4.84 (m, CH Naph, 2H), 5.49 (d, J = 3.2 Hz, 1-H, 1H), 7.04–
7.82 (m, Ar, 11H); C NMR (100 MHz, CDCl ): d 21.0, 60.9 (C5),
7
2
1
3
triisopropylsilyl-1-thio- -arabinofuranoside (3)
a-
L
3
1
2.4, 75.4 (C3), 81.9 (C4), 89.3 (C2), 89.9 (C1, JC1–H = 171.6 Hz),
To a solution of 2 (1.51 g, 3.04 mmol) in CH
added 2,6-lutidine (637 mL, 3.31 mmol) and TIPSOTf (980 mL,
.20 mmol) at 0 °C under an Ar atmosphere. The reaction mixture
was stirred at the same temperature for 2 h and quenched with
ice water, extracted with ethyl acetate, washed with sat. KHSO
aq, H O, satd NaHCO aq, and brine, dried over Na SO , and evap-
2
Cl
2
(10 mL) was
125.7, 125.9, 126.0, 126.8, 127.5, 127.8, 128.1, 129.6, 130.0,
+
132.2, 132.9, 133.1, 134.6, 137.6; ESI-TOF MS: [M+Na] calcd for
+
2
C23H24O Na S , 419.13, found 419.19; HRMS ESI-TOF: [M+Na]
4
1 1
calcd for C23H24O Na S , 419.1293, found 419.1304. To a solution
4
1 1
4
2 2
of 5 in CH Cl (5.0 mL) was added 2,6-lutidine (386 mL,
3.31 mmol) and TIPSOTf (593 mL, 2.20 mmol) at 0 °C under an Ar
atmosphere. The reaction mixture was stirred at the same
2
3
2
4
orated in vacuo. The residue was purified by silica gel column
Table 1
NMR comparison of PNP
a
and b-arabinofuranosides (1
-Arafb
a
, 1b)a
Atom number
Me
a
Me b-Arafb
PNP
a-Araf
PNP b-Araf
1H
13C
1H
13C
1H
13C
1H
13C
1
5.38
(1.2
4.84
4.84
4.68
4.36
110.5
168)
83.4
78.5
85.5
62.6
5.14
(4.3
103.9
170)
79.6
76.7
84.9
62.6
5.68
(1.6
4.31
4.05
4.05
3.78
3.48
107.7
178)
83.5
78.1
86.9
62.6
5.68
(4.3
4.20
4.09
3.88
3.65
3.48
101.1
180)
78.5
75.8
85.3
64.8
(J1H–2H, JC1–1H)
2
3
4
5
4.67
4.81
4.68
4.26
4.17
4
.23
a
In CD
Data in [D
3
OD.
b
5
]pyridine from Ref. 19.
Table 2
Kinetic parameters of HypBA1 activity on different substrates
cat (sꢀ1)
2.0 ± 0.20
6.3 ± 0.0084
k
cat/K
(mM
ꢀ1 ꢀ1
s )
Entry
1a
2
3a
Substrates
b- -Araf
K
m
(mM)
k
m
L
2
0.85 ± 0.13
0.31 ± 0.0013
2.3
20
a
b
L
-Araf -Hyp
2
L
-Araf-Hypb
0.43 ± 0.11
0.13 ± 0.03
0.0013 ± 0.0027
16.6 ± 1.6
0.030
130
4
PNP b-L-Araf(1)
a
The results are shown in Ref. 7.
cis Isomer of dansylated substrates were used in the reference.
b

9
8
S. Kaeothip et al. / Carbohydrate Research 382 (2013) 95–100
temperature for 1.5 h and quenched with satd NaHCO
tracted with ethyl acetate, washed with brine, dried over Na
and evaporated in vacuo. The residue was purified by silica gel col-
umn chromatography using a gradient solvent system (hexane/
ethyl acetate = 50/1 to 25/1 to 10/1 to 5/1) to give the title com-
pound 6 (0.652 g, quant.). Analytical data for tolyl 2-O-naphthyl-
3
aq, ex-
SO
and p-nitrophenyl 2-O-triisopropylsilyl-b-
L-arabinofuranoside
2
4
,
(6.8 mg, 27%). Analytical data for p-nitrophenyl 2-O-triisopropylsi-
1
3
lyl-b-L-arabino-furanoside: H NMR (400 MHz, CDCl ): d 0.97–1.32
(m, TIPDS, 21H), 2.10–2.40 (m, OH, 2H), 3.64 (dd, J = 12.4, 5.2 Hz,
5-H, 1H), 3.83 (dd, J = 12.4, 3.6 Hz, 5-H, 1H), 3.96–4.03 (m, 4-H,
1H), 4.38–4.45 (m, 2-H, 3-H, 2H), 5.53 (d, J = 3.6 Hz, 1-H, 1H),
1
3
methyl-3,5-di-O-triisopropylsilyl-1-thio-
L
-arabinofuranoside (6):
); H NMR (400 MHz, CD OD): d 0.92–1.15
m, TIPDS, TIPS, 49H), 2.33 (s, Me, 3H), 3.89 (dd, J = 11.2, 5.2 Hz,
7.08–7.13 (m, Ar, 2H), 8.15–8.20 (m, Ar, 2H); C NMR (100 MHz,
CDCl ): d 12.1, 17.8, 62.9 (C5), 75.1 (C3), 78.2 (C2), 82.7 (C4),
99.6 (C1), 116.3, 125.9, 142.5, 161.8; MALDI-TOF MS: [M+Na]
calcd for C20 Na Si , 450.19, found 449.77; ESI-TOF MS:
[M+Na] calcd for C20 Na Si , 450.19, found 450.16; HRMS
ESI-TOF: [M+Na] calcd for C20 Na Si , 450.1924, found
450.1924. Analytical data for 1: R = 0.40 (methanol/chloroform,
27
1
½
aꢁ
ꢀ44° (c 1.0, CHCl
3
3
3
D
+
(
5
2
-H, 1H), 3.99 (dd, J = 11.2, 4.8 Hz, 5-H, 1H), 4.12 (dd, J = 2.8,
.0 Hz, 2-H, 1H), 4.26 (td, J = 5.2, 4.8 Hz, 4-H, 1H), 4.46 (dd,
H
33
N
1
1
O
7
1
+
H
33
N
1
1
O
7
1
+
J = 5.2, 2.8 Hz, 3-H, 1H), 4.69 (d, J = 12.0 Hz, CH
2
Naph, 1H), 4.84
H
33
N
1
1
O
7
1
(
d, J = 12.0 Hz, CH Naph, 1H), 5.55 (d, J = 2.0 Hz, 1-H, 1H), 7.06–
2
f
1
3
25
D
1
7
1
2
1
1
3
.85 (m, Ar, 11H); C NMR (100 MHz, CDCl ): d 11.9, 12.3, 13.1,
1.5/8.5, v/v). ½
a
ꢁ
3
+153.9° (c 0.36, CHCl ); H NMR (400 MHz,
3.5, 14.1, 17.3, 17.4, 17.5, 17.6, 17.7, 17.91, 17.93, 17.98, 18.01,
1.0, 62.7 (C5), 72.4, 76.9 (C3), 85.1 (C4), 90.7 (C1,
71.7 Hz), 91.2 (C2), 125.76, 125.84, 126.0, 126.5, 127.6, 127.9,
CD OD): d 3.48 (dd, J = 12.0, 6.8 Hz, 5-H, 1H), 3.65 (dd, J = 12.0,
3
1
J
C1–H
=
3.6 Hz, 5-H, 1H), 3.88 (ddd, J = 9.2, 7.2, 6.8 Hz, 4-H, 1H), 4.09 (dd,
J = 8.0, 7.2 Hz, 3-H, 1H), 4.20 (dd, J = 8.0, 4.4 Hz, 2-H, 1H), 5.68 (d,
J = 4.4 Hz, 1-H, 1H), 7.20–7.23 (m, Ar, 2H), 8.17–8.19 (m, Ar, 2H);
28.0, 129.5, 131.5, 132.1, 133.0, 133.2, 135.0, 136.9; ESI-TOF
+
13
MS: [M+Na] calcd for C41
H64Na
1
O
4 1
S Si
2
, 731.40, found 731.50;
3
C NMR (100 MHz, CDCl ): d 64.8 (C5), 75.8 (C3), 78.5 (C2), 85.3
HRMS ESI-TOF: [M+Na]+ calcd for C41
found 731.3975.
H64Na
(C4), 101.1 (C1, JC1-H = 180 Hz), 117.9, 126.6, 143.5, 163.6; HRMS
1
1
O
4 1
S Si
2
, 731.3962,
+
ESI-TOF: [M+Na] calcd for C11
94.0593. p-Nitrophenyl -arabinofuranoside (1
thesized from 7 . Analytical data for 1
CD OD): d 3.48 (dd, J = 12.4, 4.8 Hz, 5-H, 1H), 3.78 (dd, J = 12.4,
.2 Hz, 5-H, 1H), 4.01–4.09 (m, 3-H, 4-H, 2H), 4.31 (dd, J = 3.6,
1.6 Hz, 2-H, 1H), 5.68 (d, J = 1.6 Hz, 1-H, 1H), 7.20–7.24 (m, Ar,
H
13
N
1
Na
1
O
7
, 294.0589, found
) was also syn-
a: H NMR (400 MHz,
2
a-L
a
1
1
.4. p-Nitrophenyl 3,5-O-tetraisopropyldisiloxanylidene-2-O-
a
triisopropylsilyl- -arabinofuranoside (7 and 7b)
L
a
3
3
To a mixture of a thioglycoside 3 (300 mg, 0.458 mmol),
p-nitrophenol (81.9 mg, 0.94 mmol) and freshly activated molecu-
1
3
2H), 8.17–8.23 (m, Ar, 2H); C NMR (100 MHz, CDCl
3
): d 62.6
JC1-H = 178.4 Hz),
1
lar sieve (4 Å, 1.5 g) in CH
.685 mmol) and AgOTf (15.0 mg, 0.0584 mmol) at ꢀ40 °C under
an argon atmosphere. The reaction mixture was stirred at the same
temperature for 6 h and quenched with Et N. The suspension was
diluted with CHCl and filtered through a pad of Celite, the filtrate
was washed successively with 10% Na aq, saturated NaHCO
aq, and brine. The organic layer was dried through Na SO and con-
centrated in vacuo to give a residue. The residue was purified by
PTLC (ethyl acetate/hexane 10:1) to afford 7 (250.9 mg, 79%)
and 7b (40.9 mg, 13%) as colorless syrup ( :b = 6.1:1). Undesired
OD): d 0.92–1.20 (m, TIPDS, 21H),
Cl
2 2
(3.0 mL) were added NIS (153.6 mg,
(C5), 78.1 (C3), 83.5 (C2), 86.9 (C4), 107.7 (C1,
117.7, 126.2, 143.5, 163.4; ESI-TOF MS: [M+Na] calcd for
Na
, 294.06, found 294.12; HRMS ESI-TOF: [M+Na]+
calcd for C11 Na , 294.0589, found 294.0584.
+
0
C H
11 13
N
1
1 7
O
3
H
13
N
1
1 7
O
3
S
2 2
O
3
3
1.6. p-Nitrophenyl 3,5-O-(tetraisopropyldisiloxane-1,3-diyl)-2-
O-(2-napthyl)methyl- -arabinofuranoside (8)
2
4
L
a
A mixture of a thioglycoside 4 (400 mg, 0.62 mmol), p-nitrophe-
nol (130.8 mg, 0.94 mmol) and freshly activated molecular sieve
(4 Å, 0.8 g) in CH Cl (20.0 mL) was stirred under argon at room
2 2
temperature for 30 min. After the mixture was cooled to ꢀ20 °C,
NIS (2.11 mg, 0.94 mmol) followed by AgOTf (48.0 mg, 0.18 mmol)
was added. The reaction mixture was stirred at the same temper-
ature for 2 h. Upon completion, the reaction was quenched by
a
1
7
3
4
1
a:
H NMR (400 MHz, CD
3
.90 (dd, J = 13.2, 6.0 Hz, 5-H, 1H), 3.96–4.03 (m, 4-H, 5-H, 2H),
.33 (dd, J = 6.8, 5.2 Hz, 3-H, 1H), 4.60 (dd, J = 5.2, 2.0 Hz, 2-H,
H), 5.52 (d, J = 2.0 Hz, 1-H, 1H), 7.04–7.11 (m, Ar, 2H), 8.14–8.20
1
3
(
m, Ar, 2H); C NMR (100 MHz, CDCl
3
): d 12.2, 12.8, 12.9, 13.2,
1
7
1
3.6, 16.95, 17.02, 17.1, 17.2, 17.3, 17.4, 17.8, 17.9, 61.8 (C5),
the addition of Et
filtered through a pad of Celite, the filtrate was washed succes-
sively with 10% Na aq and brine. The organic layer was dried
with MgSO and concentrated in vacuo to give a residue. The resi-
due was purified by silica gel chromatography (ethyl acetate–hex-
ane gradient elution) to afford 8 in the title compound as colorless
3 3
N. The suspension was diluted with CHCl and
1
8.4 (C3), 83.2 (C2), 83.5 (C4), 105.8 (C1,
16.2, 125.8, 142.3, 161.7; ESI-TOF MS: [M+Na] calcd for C32
JC1–H = 177.9 Hz),
+
H
59-
2 2 3
S O
+
N
1
O
8
Si
3
Na
1
, 692.34, found 692.45; HRMS ESI-TOF: [M+Na] calcd
Si Na , 692.3446, found 692.3451; Desired 7b:
148° (c 1.0, CHCl
4
for C32
H
59
N
1
O
8
3
1
26
1
½
a
(
ꢁ
3
); H NMR (400 MHz, CD
m, TIPDS, 21H), 3.64 (dd, J = 12.0, 9.6 Hz, 5-H, 1H), 3.83 (dd,
J = 12.0, 4.0 Hz, 5-H, 1H), 3.95 (ddd, J = 9.6, 6.0, 4.0 Hz, 4-H, 1H),
3
OD): d 0.90–1.12
D
syrup (323.0 mg, 79%,
a
/b 4.2/1). Analytical data for 8
a
: R
f
= 0.62
);
): d 0.96–1.15 (m, TIPDS, 28H), 3.97 (m,
4-H, 5-Ha, 5-Hb, 3H), 4.35 (dd, J = 2.5, 6.5 Hz, 2-H, 1H), 4.43–4.46
(m, 3-H, 1H), 4.77 (d, J = 12.5 Hz, CH Naph, 1H), 4.90 (d,
J = 12.5 Hz, CH Naph, 1H), 5.56 (d, J = 2.5 Hz, 1-H, 1H), 6.80–8.15
2
4
(ethyl acetate/hexane), 1.5/8.5, v/v); ½
a
ꢁ
ꢀ54.79° (c 1.0, CHCl
3
D
1
4
1
.33 (dd, J = 7.6, 4.4 Hz, 2-H, 1H), 4.60 (dd, J = 7.6, 6.0 Hz, 3-H,
H), 5.42 (d, J = 4.4 Hz, 1-H, 1H), 7.04–7.10 (m, Ar, 2H), 8.14–8.19
H NMR (500 MHz, CDCl
3
1
3
(
m, Ar, 2H); C NMR (100 MHz, CDCl
3
): d 12.3, 12.6, 12.9, 13.3,
2
1
3.4, 16.9, 17.0, 17.2, 17.3, 17.4, 17.5, 17.8, 17.9, 65.8 (C5), 78.7
2
13
1
(
C3), 79.0 (C2), 83.1 (C4), 98.9 (C1,
J
C1–H = 178.3Hz), 116.3,
3
(m, Ar, 11H); C NMR (125 MHz, CDCl ): d 12.7, 12.9, 13.3, 13.8,
+
1
25.8, 142.3, 161.9; ESI-TOF MS: [M+Na] calcd for C32
H
59
N
1
O
8
Si3-
, 692.34, found 692.44; HRMS ESI-TOF: [M+Na] calcd for C32-
Si Na , 692.3446, found 692.3446.
17.1, 17.2, 17.3, 17.5, 17.6, 60.9, 73.5, 75.5, 81.8, 89.4, 104.1,
116.1, 125.7, 125.9, 126.2, 126.4, 127.1, 127.9, 128.0, 128.4,
133.2, 133.3, 135.0, 142.4, 161.7; HRMS ESI-TOF: [M+Na]+ calcd
+
Na
H
1
59
N
1
O
8
3
1
for C34
for 8b:
3 3
152.79° (c 1.0, CHCl ); H NMR (500 MHz, CDCl ): d 1.01–1.17
H
47
N
1
Na
1 8 2
O Si , 676.2738, found 676.2711. Analytical data
2
4
1
.5. Deprotection of 7b: p-Nitrophenyl b-
L
-arabinofuranoside (1)
R
f
= 0.50 (ethyl acetate/hexane, 1.5/8.5, v/v);
½ ꢁ
a
D
1
+
To a solution of 7b (40.9 mg, 0.0590 mmol) in MeOH–THF (1:1,
.0 mL) was added NH F (22.0 mg, 0.588 mmol) at room tempera-
ture under an argon atmosphere. The reaction mixture was stirred
(m, TIPDS, 28H), 3.68 (dd, J = 9.5, 11.5 Hz, 5-Ha, 1H), 3.84 (dd,
J = 3.5, 11.5 Hz, 5-Hb, 1H), 3.87–3.91 (m, 4-H, 1H), 4.17 (dd,
J = 4.5, 8.0 Hz, 2-H, 1H), 4.69 (dd, J = 6.0, 8.0 Hz, 3-H, 1H), 4.79 (d,
2
4
at 40 °C for 3 h and concentrated in vacuo. The residue was
J = 12.5 Hz, CH
(d, J = 4.5 Hz, 1-H, 1H), 6.85–8.20 (m, Ar, 11H);
2 2
Naph, 1H), 4.94 (d, J = 12.5 Hz, CH Naph, 1H), 5.32
13
purified by PTLC (CHCl
3
/MeOH = 10:1) to afford 1 (4.5 mg, 28%)
C NMR

S. Kaeothip et al. / Carbohydrate Research 382 (2013) 95–100
99
2
7
1
(
1
1
125 MHz, CDCl
3
): d 12.7, 13.0, 13.5, 13.7, 17.2, 17.3, 17.4, 17.5,
7.6, 17.7, 65.9, 73.7, 82.8, 84.5, 97.8, 116.7, 125.9, 126.0, 126.4,
26.5, 127.2, 127.9, 128.0, 128.5, 133.3, 135.1, 142.6, 161.9; HRMS
4.0/6.0, v/v); ½
a
ꢁ
D
3 3
+168.79° (c 1.0, CHCl ); H NMR (500 MHz, CDCl ):
d 1.93 (s, 3H, Ac) 2.14 (s, 3H, Ac), 2.15 (s, 3H, Ac), 4.11 (dd, 1H, J = 6.0,
11.5 Hz, 5-Ha), 4.23–4.27 (m, 1H, 4-H), 4.39 (dd, 1H, J = 4.5, 12.0 Hz,
5-Hb), 5.25 (dd, 1H, J = 4.5, 7.5 Hz, 2-H), 5.83 (dd, 1H, J = 5.5, 7.5 Hz,
3-H), 5.95 (d, 1H, J = 4.5 Hz, 1-H), 7.10 (d, 2H, J = 9.0 Hz, Ar), 8.20 (d,
+
47 1 1 8 2
ESI-TOF: [M+Na] calcd for C34H N Na O Si , 676.2738, found
6
76.2709.
1
3
2
H, J = 9.0 Hz, Ar); C NMR (125 MHz, CDCl
3
): d 20.7, 20.8, 20.9,
1
.7. 4-Nitrophenyl 3,5-O-(tetraisopropyldisiloxane-1,3-diyl)-b-L-
64.4, 74.8, 76.5, 79.74, 98.2, 116.8, 125.9, 143.1, 161.3, 170.3,
+
arabinofuranoside (10)
170.4, 170.5; HRMS ESI-TOF: [M+Na] calcd for
Na 10, 420.0906, found 420.0893. To a solution of 4-nitrophenyl
tetra-O-acetyl-b- -arabinofuranoside (40.0 mg, 0.10 mmol) in dry
17 19 1
C H N
1
O
2
,3-Dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) (85.0 mg,
L
0
0
.37 mmol) was added to a mixture of p-nitrophenol (53.0 mg,
.37 mmol), thioglycoside 4 (200.0 mg, 0.31 mmol), and freshly
methanol (1.0 mL) was added 1 M NaOMe till pH 10 was reached.
The reaction mixture was stirred for 1 h at room temperature, then
neutralized with Dowex (H ), filtered through a pad of Celite, and
+
activated MS (4 Å, 500 mg) in dry CH
2
Cl
2
(5 mL) at room tempera-
ture. The reaction mixture was stirred for 16 h under argon atmo-
sphere. The reaction mixture was quenched with aqueous
ascorbate buffer, and filtered through a pad of Celite. The filtrate
concentrated in vacuo. The residue was purified by column chroma-
tography on silica gel (methanol–chloroform gradient elution) to af-
ford the title compound as a colorless syrup (22.1 mg, 81%).
was extracted with CHCl
saturated NaHCO and brine. The organic layer was dried with
MgSO and concentrated in vacuo. The residue was purified by sil-
3
and the organic layer was washed with
3
1.9. p-Nitrophenyl 3,5-di-O-triisopropylsilyl-1-thio-L-
arabinofuranoside (12)
4
ica gel chromatography (ethyl acetate–hexane gradient elution) to
+
afford mixed acetal (179 mg, 74%). MALDI-TOF MS: [M+Na] calcd
Compound 12 was synthesized in 50% yield according to the
procedure for 11 from 6 and PNPOH except that the second intra-
molecular transfer reaction under MeOTf–DTBMP conditions has
53 1 1 8 1 2
for C41H N Na O S Si , 798.29, found 798.35. A mixture of the
mixed acetal (150.0 mg, 0.19 mmol), 2,6-di-tert-butyl-4-methyl-
pyridine (DTBMP) (158.7 mg, 0.77 mmol) and freshly activated
2
6
been carried out at room temperature. Analytical data 12: ½ ꢁ
a
D
1
molecular sieve (4 Å, 450 mg) in dry CH
under argon atmosphere at room temperature for 30 min. MeOTf
76 L, 0.67 mmol) was then added to the mixture and the mixture
2
Cl
2
(10.0 mL) was stirred
3 3
103° (c 1.0, CHCl ); H NMR (400 MHz, CDCl ): d 0.88–1.20 (m, TIPS
ꢂ2, 42H), 3.00 (d, J = 9.6 Hz, 2-OH, 1H), 3.75 (dd, J = 11.6, 3.6 Hz, 5-
Ha, 1H), 3.99 (dd, J = 11.6, 3.6 Hz, 5-Hb, 1H), 3.98 (dt, J = 4.8, 3.6 Hz,
4-H, 1H), 4.20 (td, J = 9.6, 4.8 Hz, 2-H, 1H), 4.46 (t, J = 4.8 Hz, 3-H,
1H), 5.70 (d, J = 4.0 Hz, 1-H, 1H), 7.10–8.18 (m, Ar, 4H); 13C NMR
(
l
was stirred for 48 h at 40 °C. Upon completion the reaction mixture
was cooled down, quenched with triethylamine, diluted with
CHCl
saturated NaHCO
in vacuo. The crude residue was dissolved in CHCl
cooled at 0 °C. TFA (0.5 mL) was added and kept stirring for
0 min at the same temperature. The reaction mixture was diluted
with CHCl washed successively with H O, saturated NaHCO and
brine. The organic layer was dried with MgSO and concentrated
in vacuo. The residue was purified by silica gel chromatography
ethyl acetate-hexane gradient elution) to afford compound as col-
orless syrup (70 mg, 71%). Analytical data for 10: R = 0.38 (ethyl
3
, and filtered through Celite. The filtrate was washed with
and brine. The organic layer was concentrated
(5 mL) and
(100 MHz, CDCl
(C3), 78.1, (C2) 86.2 (C4), 100.5 (C1,
53 1-
125.6, 142.4, 161.7; MALDI-TOF MS: [M+Na] calcd for C29H N
3
): d 11.8, 12.2, 17.75, 17.77, 18.0, 63.0 (C5), 76.4
1
3
JC1–H = 177.5 Hz), 116.5,
+
3
+
Na
1
O
7
Si
Na
for C29
2
, 606.33, found 606.90; ESI-TOF MS: [M+Na] calcd for C29-
+
3
H
53
N
1
1
O
7
Si
2
, 606.33, found 606.24; ESI-TOF MS: [M+Na] calcd
Si , 606.3258, found 606.3232.
3
2
3
H
53
N
1
Na
1
O
7
2
4
1.10. Deprotection of 12: p-Nitrophenyl b-L-arabinofuranoside
(1)
(
f
24
1
acetate/hexane, 2.0/8.0, v/v); ½
a
ꢁ
+176.19° (c 1.0, CHCl
3
);
H
Compound 12 was deprotected according to the procedure for
the deprotection of 7b to give the title compound 1 (83%) concom-
D
NMR (500 MHz, CDCl ): d 0.93–1.14 (m, TIPDS, 28H), 2.98 (d,
3
J = 9.5 Hz, -OH, 1H), 3.71 (dd, J = 8.0, 11.5 Hz, 5-Ha, 1H), 3.92 (dd,
J = 3.5, 11.5 Hz, 5-Hb, 1H), 3.95–3.98 (m, 4-H, 1H), 4.36–4.39 (m,
itant with p-nitrophenyl 3-O-triisopropylsilyl-1-thio-L-arabinofu-
ranoside (17%) which were purified by PTLC (Hexane/
2
-H, 1H), 4.43 (dd, J = 6.5, 8.0 Hz, 3-H, 1H), 5.62 (d, J = 4.5 Hz, 1-
AcOEt = 1:3). Analytical data p-nitrophenyl 3-O-triisopropylsilyl-
1
3
27
D
1
H, 1H), 7.12 (d, J = 9.5 Hz, Ar, 2H), 8.21 (d, J = 9.5 Hz, Ar, 2H);
NMR (125 MHz, CDCl
C
1-thio-
(400 MHz, CDCl
L
-arabinofuranoside: ½
a
ꢁ
3
112° (c 0.10, CHCl ); H NMR
3
): d 12.7, 13.0, 13.4, 13.7, 17.1, 17.2, 17.3,
3
): d 0.88–1.34 (m, TIPS, 21H), 1.99 (br s, OH, 1H),
1
Araf
1
7.5, 17.6, 65.1, 78.2, 78.3, 83.1, 99.1 ( JC1–H = 174.6 Hz, C1 b ),
2.67 (br s, OH, 1H), 3.65 (dd, J = 12.4, 4.4 Hz, 5-Ha, 1H), 3.80 (dd,
J = 12.4, 3.2 Hz, 5-Hb, 1H), 4.05 (dt, J = 4.8, 3.2 Hz, 4-H, 1H), 4.24–
4.29 (m, 2-H, 1H), 4.39 (t, J = 4.8 Hz, 3-H, 1H), 5.72 (d, J = 4.4 Hz,
+
1
16.8, 126.0, 142.9, 161.3; HRMS ESI-TOF: [M+Na] calcd for C23-
H
39
N
1
Na
1
O
8
Si
2
, 536.2111, found 536.2097.
1
3
1
-H, 1H), 7.15–8.22 (m, Ar, 4H); C NMR (100 MHz, CDCl
3
): d
1
.8. p-Nitrophenyl b-
L
-arabinofuranoside (1)
12.2, 17.93, 17.96, 62.4 (C5), 76.3 (C3), 78.5, (C2) 85.9 (C4), 100.5
1
(
C1,
JC1–H = 176.9 Hz), 116.6, 125.9, 142.9, 161.1; ESI-TOF MS:
+
To a solution of 10 (84 mg, 0.13 mmol) in THF (2 mL) at 0 °C was
added a solution of tetrabutylammonium fluoride (0.26 mL,
.26 mmol, 1 M in THF) in THF–pyridine (2–0.5 mL). After stirring
[M+Na] calcd for C20H N Na O Si , 450.1924, found 450.1945.
33 1 1 7 1
0
1.11. Procedure for enzyme assay
for 2 h at 0 °C, the mixture was concentrated in vacuo. Crude product
was dissolved in dry pyridine (3 mL), cooled to 0 °C and treated with
acetic anhydride (1.5 mL) dropwise. The reaction mixture was al-
lowed to stir overnight at room temperature. The reaction mixture
Expression and purification of recombinant HypBA1 were car-
ried out according to the general procedure shown in Ref. 7. The
hydrolytic activity of HypBA1 was assayed using PNP b-L-Araf 1
was diluted with CHCl
3
, and washed with H
2
O, 1 N HCl, saturated
and
as the substrate. C-terminally histidine-tagged HypBA1 from
B. longum JCM 1217 expressed by pET23b vector (Novagen, Madi-
NaHCO , and brine. The organic phase was dried with MgSO
3
4
concentrated in vacuo. The residue was purified by column chroma-
tography on silica gel (ethyl acetate-hexane gradient elution) to af-
son, WI) was used for kinetic analysis. PNP b-
activity was determined in the assay mixture (100
50 mM sodium acetate buffer (pH 4.5), 10 mM DTT, 0.05–0.5 mM
PNP b- -Araf and the enzyme. The assays were initiated by the
addition of 0.05 mg/mL enzyme (5 L), which was preincubated
L
-Araf-hydrolyzing
l
L) containing
ford 4-nitrophenyl tetra-O-acetyl-b-
colorless syrup (43 mg, 79%). Analytical data for 4-nitrophenyl tet-
ra-O-acetyl-b- -arabinofuranoside: R = 0.38 (ethyl acetate/hexane,
L-arabinofuranoside as a
L
L
f
l

1
00
S. Kaeothip et al. / Carbohydrate Research 382 (2013) 95–100
with 10 mM DTT at 37 °C for 30 min. The reaction was carried out
at 37 °C for about 10 min, in which the linearity of the reaction rate
Biol. 2009, 5, 578–580; (c) Matsubayashi, Y. Plant Cell Physiol. 2011, 52, 5–13;
d) Matsubayashi, Y. Genes Cells 2012, 17, 1–10; (e) Okamoto, S.; Shinohara, H.;
Mori, T.; Matsubayashi, Y.; Kawaguchi, M. Nat. Commun. 2013, 4, 2191. http://
dx.doi.org/10.1038/ncomms3191.
(
was observed. At every 1 min interval, a portion (10
from the assay mixture and mixed with the equal volume of stop
solution (200 mM of Na CO ). The amounts of liberated PNP were
lL) was taken
5. For example see; Mohnen D Curr. Opin. Plant Biol. 2008, 11, 266–277.
6.
(a) Konishi, M.; Yang, X.; Li, B.; Fairchild, C. R.; Shimizu, Y. J. Nat. Prod. 2004, 67,
309–1313; (b) Souto, M. L.; Fernández, J. J.; Franco, J. M.; Paz, B.; Gil, L. V.;
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We thank Dr. Yoichi Takeda (JST, ERATO Ito Glycotrilogy Pro-
ject) for his valuable advice and help. We thank Dr. Hiroyuki Kosh-
ino (Collaboration Promotion Unit, RIKEN) and his staff for ESI
HRMS measurements. We also thank Ms. Akemi Takahashi and
Ms. Satoko Shirahata for their kind technical assistance. This work
was partly supported by a Grant-in-Aid for Scientific Research from
the Ministry of Education, Culture, Sports, Science, and Technology
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