Isolation and structural confirmation of the oligosaccharides containing α-d-fructofuranoside linkages isolated from fermented beverage of plant extracts

Article abstract of DOI:10.1016/j.carres.2011.09.002

Fermented beverage of plant extracts was prepared from the extracts of approximately 50 types of vegetables and fruits. Natural fermentation was carried out mainly by lactic acid bacteria (Leuconostoc spp.) and yeast (Zygosaccharomyces spp. and Pichia spp.). Two oligosaccharides containing an α-fructofuranoside linkage were detected in this beverage and isolated using carbon-Celite column chromatography and preparative HPLC. The structural confirmation of the saccharides was determined by methylation analysis, MALDI-TOF-MS, and NMR measurements. These saccharides were identified as α-d-fructofuranosyl-(2→6)-d-glucopyranose, which was isolated from a natural source for the first time, and a novel saccharide β-d- fructopyranosyl-(2→6)-α-d-fructofuranosyl-(2?1) -α-d-glucopyranoside.

Full text of DOI:10.1016/j.carres.2011.09.002

Carbohydrate Research 346 (2011) 2633–2637
Contents lists available at SciVerse ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Note
Isolation and structural confirmation of the oligosaccharides containing
-D-fructofuranoside linkages isolated from fermented beverage of plant
extracts
a
a,
⇑
b
a
a
c
b
Hideki Okadac , Eri Fukushi , Akira Yamamori , Naoki Kawazoe , Shuichi Onodera , Jun Kawabata ,
Norio Shiomi
a
General Institute of Ohtakakohso Co., Ltd, 1-22-10 Sakura, Otaru 047-0193, Japan
Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan
Department of Food and Nutrition Sciences, Graduate School of Dairy Science Research, Rakuno Gakuen University, 582-1 Bunkyoudai-Midorimachi, Ebetsu 069-8501, Japan
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 20 June 2011
Received in revised form 25 August 2011
Accepted 3 September 2011
Available online 9 September 2011
Fermented beverage of plant extracts was prepared from the extracts of approximately 50 types of veg-
etables and fruits. Natural fermentation was carried out mainly by lactic acid bacteria (Leuconostoc spp.)
and yeast (Zygosaccharomyces spp. and Pichia spp.). Two oligosaccharides containing an a-fructofurano-
side linkage were detected in this beverage and isolated using carbon–Celite column chromatography
and preparative HPLC. The structural confirmation of the saccharides was determined by methylation
analysis, MALDI-TOF-MS, and NMR measurements. These saccharides were identified as
anosyl-(2?6)- -glucopyranose, which was isolated from a natural source for the first time, and a novel
saccharide b- -fructopyranosyl-(2?6)- -fructofuranosyl-(2M1)- -glucopyranoside.
Ó 2011 Elsevier Ltd. All rights reserved.
a-D-fructofur-
Keywords:
Fermented beverage of plant extracts
a-D-Fructofuranoside
Natural fructopyranoside
D
D
a
-D
a-D
Oligosaccharide
We had previously investigated the preparation of a fructopyr-
anoside series of saccharides from the fermented beverage of plant
b-
copyranoside, and b-
syl-(1?3)]-b- -glucopyranoside.
In this paper, we report the isolation and structural confirma-
tion of two oligosaccharides containing -fructofuranoside linkage
and fructopyranoside residue, which are unusual in the fermented
beverage of plant extracts (Fig. 1).
Saccharides 1 and 2 were isolated from fermented beverage of
plant extracts using carbon–Celite column chromatography, and
they were shown to be homogeneous by anion exchange HPLC
D
-galactopyranosyl-(1?1)-b-
D
-fructofuranosyl-(2M1)-
a-D-glu-
7
D-fructofuranosyl-(2M1)-[b- -glucopyrano-
D
1
8
extracts including b-
b- -fructopyranosyl-(2?6)-b-
pyranose, b-
D
-fructopyranosyl-(2?6)-
-glucopyranosyl-(1?3)-
-fructopyranosyl-(2?6)-[b- -glucopyranosyl-(1?3)]-
D
-glucopyranose,
D
D
D
D-gluco-
2
D
D
a
2
3
D
-glucopyranose,
b-D
-fructopyranosyl-(2?6)-
-fructofuranosyl-(2M1)-
-fructopyranosyl-(2?6)- -glucopyranosyl-
-fructofuranoside.3 The characteristics of b-
-fructopyr-
anosyl-(2?6)- -glucopyranose included non-cariogenicity and
D
-fructofuranose,
b-
D
-fructopyranosyl-(2?1)-b-
D
a-D
-gluco-
3
pyranoside, and b-
D
a-
D
(
1M2)-b-
D
D
D
low digestibility. This saccharide was selectively utilized by the
beneficial bacteria, Bifidobacterium adolescentis and Bifidobacterium
longum, but it was not utilized by the non-beneficial bacteria,
Clostridium perfringens, Escherichia coli, and Enterococcus faecalis,
which can produce mutagenic substances.4 Furthermore, these
novel saccharides were confirmed to be produced by fermentation
of the plant extract. In addition, we previously reported the synthe-
R
[t , sucrose (relative retention time; retention time of sucrose = 1.0,
5.24 min): 1.82 and 1.21, respectively]. The retention times of
saccharides 1 and 2 did not correspond with the following known
saccharides: glucose, fructose, sucrose, maltose, trehalose,
,5
Frufα
6
OH
OH
OH
sis of b-
and
-fructose using a thermal treatment process.6
Recently, we isolated and identified novel saccharides from the
beverage that possessed linkage features, including b- -glucopyr-
anosyl-(1?1)-b- -fructofuranosyl-(2M1)- -glucopyranoside,
D
-fructopyranosyl-(2?6)-
D
-glucopyranose from
D-glucose
O
O
O
D
HO
2
OH
Frufα
HO
OH
2
OH
2
O
OH
1
O
6
O
OH
D
O
HO
HO
OH
OH
OH
O
OH
D
a
-
D
HOHO
OH
Frupβ
Glcpα
2
1
Glcp
⇑
Corresponding author. Tel.: +81 134 54 7311; fax: +81 134 52 2610.
E-mail address: oelab@ohtakakohso.co.jp (H. Okada).
Figure 1. Structures of
b- -fructopyranosyl-(2?6)-
a
-
D
D
-fructofuranosyl-(2?6)- -glucopyranose (1) and
D
D
a-
-fructofuranosyl-(2M1)-a-D-glucopyranoside (2).
0
008-6215/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2011.09.002

2
634
H. Okada et al. / Carbohydrate Research 346 (2011) 2633–2637
D
-fructofuranosyl-(2?6)-
-fructosyl-(2M1)- -glucoside, respectively.
The structural conformations of saccharides 1 and 2 were deter-
D-glucose and D-fructopyranosyl-(2?6)-
laminaribiose, raffinose, 1-kestose, maltotriose, panose, nystose,
b-
D
-fructopyranosyl-(2?6)-
D
-glucopyranose,1 b-
D
-fructopyrano-
-glucopyranose,2 and b-
-glucopyr-
anose. The degree of polymerization of saccharide was
D
D
syl-(2?6)-b-
D
-glucopyranosyl-(1?3)-
D
D-
1
13
mined by H and C NMR analyses, followed by complete assign-
fructopyranosyl-(2?6)-[b-D-glucopyranosyl-(1?3)]-D
1
13
2
ment of H and C NMR signals for the two saccharides using 2D
NMR techniques. Each glucopyranose, fructofuranose, and fructo-
pyranose residue of saccharides 1 and 2 was represented as Glcp,
Fruf, and Frup in Figure 1.
The NMR spectra of saccharide 1 indicated that it was a disac-
charide containing Fruf and Glcp. It was an anomeric mixture of
Glcp and the anomer configurations of
and b-Glc (d 5.25 ppm, 8.1 Hz) were determined based on their J
values. The HSQC-TOCSY spectrum revealed the H and C signals
for each of the Fruf, Glcp , and Glcpb residues. The isolated meth-
ylene was assigned as H-1 and C-1 for Fruf. The COSY spectrum as-
signed the spin system of anomer units from H-4 to H-6 of Fruf,
from H-1 to H-3 and H-5 to H-6 of Glcpb, and from H-1 to H-4 of
1
+
established as 2 based on measurements of [M+Na] (m/z: 365)
by MALDI-TOF-MS (Fig. 2) and the same analysis of saccharide 2
established a degree of polymerization of 3 (m/z: 527). This was
supported by analyses of the molar ratios of D-glucose to D-fructose
in the acid hydrolysates. The relative retention times of the meth-
anolysates of the permethylated saccharides were investigated by
H
a- (d 4.64 ppm, 3.6 Hz)
gas liquid chromatography (GLC) analysis (t
ra-O-methyl-b- -glucoside = 1.0; actual retention time = 8.70 min).
The methanolysate of permethylated saccharide 1 produced four
peaks corresponding to methyl 1,3,4,6-tetra-O-methyl- -fructoside
, 1.09 and 1.31) and methyl 2,3,4-tri-O-methyl- -glucoside (t
.49 and 3.52, Table 1). The methanolysate of permethylated sac-
charide 2 exhibited eight peaks corresponding to methyl 1,3,4-
tri-O-methyl- -fructoside (t , 1.87, 2.50, 3.92, and 4.55), methyl
,3,4,5-tetra-O-methyl- -fructopyranoside (t , 0.94 and 1.43), and
methyl 2,3,4,6-tetra-O-methyl- -glucoside (t
R
of methyl 2,3,4,6-tet-
H
1
13
D
a
D
(t
R
D
R
,
2
Glcp
H H
a. However, H-3 (d 3.50 ppm) and H-4 (d 3.50 ppm) of the
Glcpb signals suggested overlapping with a proton. The corre-
sponding C signals were assigned using the HSQC spectrum.
The quaternary carbon was assigned as C-2 of Fru. Intra-residue
D
R
1
3
1
D
R
D
R
, 1.06 and 1.53, Table
H
HMBC correlation was found between the H-6 (d 3.91 ppm and
1). Based on these findings saccharides 1 and 2 were shown to be
%
%
1
00
100
A
B
5
27[M+Na]+
3
65[M+Na]+
5
0
50
3
00
400
500
600
700 800
300
400
500
600
700 800
m/z
m/z
Figure 2. MALDI-TOF-MS spectra of saccharides 1 (A) and 2 (B).
Table 1
Gas–liquid chromatographic analysis of methanolysates of permethylated saccharides 1 and 2
Methanolysate origin
Relative retention timea
Methyl 1,3,4,5-tetra-O-
methyl-
fructopyranoside
Methyl 1,3,4,6-tetra-O-
methyl-
fructofuranoside
Methyl 2,3,4,6-tetra-
Methyl 2,3,4-tri-O-
Methyl
1,3,4-
tri-O-
Methyl 3,4,6-tri-O-
methyl-D-fructoside
D
-
D-
O-methyl-
D-glucoside
methyl-
D-glucoside
methyl-D-
fructoside
Saccharide 1
Saccharide 2
1.09
2.49
3.52
1
.31
0.94
.43
1.06
1.53
1.87
2.50
1
3
4
.92
.55
b-Fp2-6Gb
0.99
.46
2.44
3.50
1
1
-Kestose
1.08
.28
1.08
1.49
2.65
3.96
1
Fructose
Levan
0.98
.47
1.06
1.28
1
1.90
2
3
4
.50
.92
.49
Methyl 2,3,4,6,-tetra-O-
1.00
methyl-b-D-
glucoside
a
Retention time of methyl 2,3,4,6-tetra-O-methyl-b-D-glucoside = 1.0; retention time, 8.70 min.
b-Fp2-6G: b-D-fructopyranosyl-(2?6)-D-glucopyranose.
b
5

H. Okada et al. / Carbohydrate Research 346 (2011) 2633–2637
2635
was determined from the HMBC correlation between the C-2 of
Fru and that of H-6 (d 3.81 ppm, dd, 11.8 Hz and 5.7 Hz) of Glcpb
Fig. 3). This chemical shift position overlapped with the methy-
H
δC(ppm)
(
5
8
0
2
4
6
HSQC
lene proton of Glcp
was determined as the
a
-6 (d
3.86 ppm) and the H-6 of Fru. Fructose
-furanoside type. Therefore, this correla-
tion was not a correlation with a fructose intra-residue, so we were
able to judge the correlation of the C-2 of Fruf (d 109.20 ppm) and
the H-6 of Glcp (d 3.81 and 3.86 ppm).
These results indicated a Fruf2
?6Glcp linkage and all the 1
and 1 C NMR signals were assigned as shown in Table 2.
H
Fruf1
a
6
6
6
6
Glcpβ6
Fruf6
C
H
a
H
3
The NMR spectra of saccharide 2 were analyzed in the same
manner as that of saccharide 1. The 1D NMR spectra of saccharide
1
1
1
00
05
10
HMBC
3.4
0
2
showed that it was a trisaccharide containing Fru, Fru , and Glcp,
and not an anomeric mixture. The Glcp was determined as an
form based on the J value of H-1. The HSQC-TOCSY spectrum re-
a-
Fruf3
Glcp6Fruf1
Fruf2
1
13
0
vealed the H and C signals for each of the Glcp
residues. The isolated methylene was assigned as the H-1 and C-
of Fru. The remaining three methylene carbons were assigned
a, Fru, and Fru
δH(ppm) 4.4
4.2
4.0
3.8
3.6
1
Figure 3. Part of HSQC and HMBC spectra of saccharide 1.
to C-6 in these residues. The COSY spectrum assigned the spin sys-
tems of these residues from H-1 to H-6 of Glcp. Two signals at 3.85
and 3.73 ppm were assigned to H-6 of Fru using HSQC-TOCSY, and
the spin system of Fru was assigned to H-6 to H-3 by COSY. The
1
1
3
.81 ppm) of Glcpb and C-4. Moreover, the selected H– H coupling
1
3
corresponding C signals were assigned based on the E-HSQC
spectrum. The HMBC of C-3/H-1of Fru was used to assign this car-
bon. Fru was confirmed to be of the fructofuranose form based on
patterns of overlapping proton signals were extracted from the SPT
difference spectrum. The Fruf residue was then assigned. Isolated
methylene protons (d 3.80 and 3.72 ppm) showed HMBC of C-3
H
of Fruf. This was estimated as the H-1 of Fruf. These protons also
indicated HMBC to one quaternary carbon, which was assigned
as C-2 of Fruf. H-3 to H-6 of Fruf were assigned in reverse using
the HMBC correlation between C-2 and H-5 of Fru, and the
based on its chemical shift.
a
-form
0
The COSY spectrum was used to assign H-3 to H-6 of Fru . The
0
Fru protons indicated an HMBC with a methine carbon at d
C
the COSY technique. It is known that
chemical shifts compared with b-fructoside. Fruf was determined
as the -furanoside type, because the C-2 (d 109.20 ppm), C-3
81.15 ppm), C-4 (d 78.19 ppm), and C-5 (d 84.23 ppm) of
Fru exhibited larger chemical shifts compared with the C-2
106.4 ppm), C-3 (d 79.6 ppm), and C-4 (d 77.1 ppm) of b-
a-fructoside produces larger
9
6
9.22 and only one quaternary carbon (d
C
101.51), which was as-
0
signed as C-3 and C-2 of Fru , respectively. The isolated methylene
was assigned as the H-1 and C-1 of Fru . Therefore, Fru was con-
firmed as the fructopyranose form based on the HMBC between
a
C
0
0
(d
C
C
C
0
the C-2 and H-6 of Fru , and as the b-form based on its chemical
(d
C
C
C
D-
shift. The positions of the glycosidic linkage and fructosidic linkage
were analyzed as follows. One methylene carbon was unassigned,
fructofuranosyl-(2?6)-a-D-glucopyranosyl-(1M2)-b-D-fructofur-
anoside (neokestose).10 The connectivity of saccharide residues
Table 2
1
H and 13C NMR spectral data (da in ppm, J in Hz) of saccharides 1 and 2
Saccharide 1
Saccharide 2
d
C
d
H
J
HH
d
C
d
H
J
HH
d
C
d
H
J
HH
Frufa
1
59.29
3.80 d
.72 d
12.6
12.6
Fruf
a
1
59.48
Fruf
a
1
62.22
3.79 sb
3
2
3
4
5
6
109.20
81.15
78.19
84.23
62.05
2
3
4
5
6
109.01
81.31
78.05
83.94
2
3
4
5
6
110.33
81.12
78.55
84.62
61.88
4.19 d
3.5
6.2, 3.5
4.22 d
2.5
3.99 dd
4.09 mc
3.84 dd
4.06 dd
4.26 ddd
3.80 dd
3.68 dd
4.4, 2.5
7.4, 4.4, 3.5
11.7, 3.5
11.7, 7.4
4.07 m
12.2, 3.5
12.2, 5.4
3
.72 dd
Glcpb
1
2
3
4
5
6
96.81
74.89
76.57
70.64
75.30
60.98
4.67 d
3.27 m
3.50 m
3.50 m
3.62 m
3.91 m
8.1
Glcp
a
1
2
3
4
5
6
92.99
72.26
73.65
70.64
71.01
60.83
5.25 d
3.6
Frup
1
61.37
3.80 m
3.56 dd
3.73 dd
3.52 dd
3.99 m
3.86 m
10.1,3.6
10.1,8.4
10.8,8.4
2
3
4
5
6
101.51
69.22
70.38
69.89
64.94
3.91 m
3.91 m
4.00 m
3.85 d
3
.81 dd
11.8, 5.7
13.5
3.73 d
13.5
Glcp
a
1
2
3
4
5
6
91.89
71.96
74.16
70.25
73.43
61.29
5.36 d
4.0
3.57 dd
3.69 dd
3.39 dd
3.91 m
3.85 m
9.5, 4.0
9.5, 9.4
9.9, 9.4
3.73 m
a
The chemical shifts of 1H (d
) and 13C (d
2
67.40 ppm) in D O.
2
H
C
) in ppm were respectively determined relative to the external standard of sodium [2,2,3,3- H
4
]-3-(trimethylsilyl)propanoate in
D
2
O (d 0.00 ppm) and 1, 4-dioxane (d
H
C
b
s, singlet.
m, multiplet.
c

2
636
H. Okada et al. / Carbohydrate Research 346 (2011) 2633–2637
but it was estimated as the C-1 of Frup (Fig. 4). Its protons
indicated an HMBC with a methine carbon at d 69.22 and only
one quaternary carbon (d 101.51, Fig. 4), which was assigned as
1. Experimental
1.1. Materials
C
C
the C-3 and C-2 of Frup, respectively. The C-2 of Fruf had an HMBC
with the H-1 of Glcp. The C-2 of Frup correlated with H-6 of Fruf.
Glucose, fructose, sucrose, maltose, trehalose, laminaribiose,
raffinose, maltotriose, panose, and levan were purchased from
Sigma Chemical Co. (St. Louis, MO, USA). Crystalline 1-kestose
These results indicated a Glcp1aMa2Fruf6 b2Frup linkage and
1
13
all the H and C NMR signals were assigned as shown in Table 2.
Based on all of these findings, the saccharides 1 and 2 isolated
from fermented beverage of plant extracts were confirmed as oli-
(b-
pyranoside) and nystose (b-
furanosyl-(2?1)-b-
were prepared from sucrose using a Scopulariopsis brevicaulis en-
D
-fructofuranosyl-(2?1)-b-
D
-fructofuranosyl-(2M1)-
-fructofuranosyl-(2?1)-b-
-glucopyranoside)
a
-
D
D
-gluco-
D
-fructo-
gosaccharides containing an
fructofuranosyl-(2?6)- -glucopyranose, and the novel saccharide,
b- -fructopyranosyl-(2?6)- -fructofuranosyl-(2M1)- -gluco-
pyranoside (Fig. 1). No saccharide containing -fructofuranoside
a-D
-fructofuranoside linkage,
a
-D
-
D-fructofuranosyl-(2M1)-
a
-D
D
1
2
D
a-
D
a
-D
zyme.
pyranosyl-(2?6)-b-
and b- -fructopyranosyl-(2?6)-[b-
glucopyranose were prepared according to methods described
b-
D
-fructopyranosyl-(2?6)-
-glucopyranosyl-(1?3)-
-glucopyranosyl-(1?3)]-D-
D
-glucopyranose, b-
D-fructo-
a
D
D
-glucopyranose,
linkage is known to have been isolated from natural sources, with
the exception of these saccharides isolated from the fermented
beverage of plant extracts. The chemical synthesis of saccharide
D
D
1
,2
previously. All other chemicals used in this study were of analyt-
ical grade.
1
has already been reported,11 but NMR analysis was not con-
ducted. The present study performed complete assignment of the
H and C signals.
The fermented beverage was prepared from extracts of fruits
and vegetables and naturally fermented using yeast (Zygosacchar-
omyces psedrouxii and Pichia anomala) and lactic acid bacteria
1
13
The synthesis of the saccharides by the fermentation of plant
1
,8,13
extracts was investigated using high performance anion-
exchange chromatography (HPAEC). Most of the monosaccharides
were removed using charcoal from the fermented and unfer-
mented plant extract beverage in a batch method. Saccharides 1
and 2 were identified in the fermented beverage, but they were
not present in the unfermented one. Therefore, saccharides 1
and 2 were confirmed as fermentation products in the plant
extract beverage (Fig. 5).
(Leuconostoc spp.), as described in previous reports.
1.2. HPAEC
The oligosaccharides were analyzed using a Dionex Bio LC Ser-
ies system (Sunnyvale, CA) equipped with an HPLC carbohydrate
column (Carbo Pack PAI, inert styrene divinyl benzene polymer,
Sunnyvale, CA) and pulsed amperometric detection (PAD).1
The mobile phase consisted of eluent A (150 mM NaOH) and eluent
B (500 mM sodium acetate in 150 mM NaOH) with a sodium
acetate gradient as follows: 0–1 min, 0–5 mM; 1–20 min,
4,15
5
–100 mM; 20–22 min, 500 mM; 22–30 min, 0 mM. The flow rate
a
Glcp1
through the column was 0.5 mL/min. The applied PAD potentials
for E1 (500 ms), E2 (100 ms), and E3 (50 ms) were 0.1, 0.6, and
ꢀ
δC(ppm)
9
9
0
16
0.6 V, respectively, and the output range was 1
lC.
HSQC
Glcp1
5
1.3. Isolation of saccharides
1
1
00
00
Fermented plant extract beverage (1000 g) was loaded onto a
.5 ꢁ35 cm carbon–Celite column, [1:1; charcoal (Wako Pure
4
HMBC
Frup2
Fruf2
1
1
05
10
A
δH(ppm) 5.5 5.4 5.3 5.2 5.1 5.0
b
δC(ppm)
5
8
9
0
1
2
3
4
5
6
HSQC
Saccharide 2
Saccharide 1
B
5
6
6
6
6
6
6
6
Glcp6
Frup1
Fruf6
Fruf1
Frup6
Frup2
2
6
10
14
18 8
Retention time (min)
1
00
HMBC
3.4
Fruf6 Fruf6
Fruf3 Fruf4
Figure 5. High performance liquid chromatogram of fermentation products.
Analysis of saccharides produced during fermentation was done by HPAEC. (A)
Plant extracts were fermented for 0 days. (B) Plant extracts were fermented for
1
05
Fruf2
110
1
80 days. The beverage (100 mL) fermented for 0–180 days was added to charcoal
(
10 g), stirred for 3 h. and filtered. The charcoal was extracted with 30% ethanol
4.4
4.2
4.0
3.8
3.6
δH(ppm)
(500 mL) three times. The ethanol extracts were combined, concentrated to
dryness, and solubilized with one ml of distilled water. The sugar solution was
analyzed by HPAEC.
Figure 4. Part of HSQC and HMBC spectra of saccharide 2.

H. Okada et al. / Carbohydrate Research 346 (2011) 2633–2637
2637
Chemical Industries, Ltd, Osaka, Japan) and Celite-535 (Nacalai Tes-
que Inc, Osaka, Japan)] and was successively eluted with water
14 L), 5% ethanol (30 L), and 30% ethanol (10 L). Most of the glu-
cose and fructose were eluted with water (4 l) before saccharide
was eluted with water (5–6 L). The water fraction containing sac-
charide 1 was concentrated in vacuo and freeze-dried to yield
13 mg. The 30% ethanol fraction containing saccharide 2 was con-
centrated in vacuo and freeze-dried to yield 894 mg. The water
fraction (10 mg) and 30% ethanol fraction (10 mg) were subse-
quently applied to an HPLC system (Tosoh, Tokyo, Japan) fitted
with an Amide-80 column (7.8 mm ꢁ30 cm, Tosoh, Tokyo, Japan),
which was operated at 80 °C and eluted with 80% acetonitrile at
1.8. NMR measurement
(
The saccharides (ca. 0.5 mg of 1, 0.8 mg of 2) were dissolved in
0.06 mL (saccharide 1) and 0.4 mL (saccharide 2) of D O. NMR
spectra were recorded at 27 °C using a Bruker AMX 500 spectrom-
2
1
1
13
eter ( H 500 MHz, C 126 MHz) equipped with a 2.5 mm C/H dual
probe (saccharide 1), a 5 mm C/H dual probe (1D spectra of saccha-
ride 2), and a 5 mm TXI probe (2D spectra of saccharide 2). Chem-
9
1
13
ical shifts of H (d
H C
) and C (d ) in ppm were determined relative
2
to an external standard of sodium [2,2,3,3- H
4
]-3-(trimethylsilyl)
0.00 ppm) and 1,4-dioxane (d 67.40 ppm)
propanoate in D
in D
TOCSY,
selected pulse sequences. The TOCSY mixing period (0.1 s) was
2
O (d
H
1
C
1
18,19
20
20
2
O, respectively. H– H COSY,
HSQC, E-HSQC, HSQC-
2
0,21
22,23
2
.0 mL/min, and a refractive index detector. The chromatographic
and HMBC
spectra were obtained using gradient
separation was repeated 60 times. Furthermore, saccharide frac-
tions 1 (24.3 mg) and 2 (83.8 mg) were purified using an HPLC sys-
tem with an ODS-100 V column (4.6 mm ꢁ25 cm, Tosoh, Tokyo,
Japan), which was operated at room temperature and eluted with
water at 0.5 mL/min. The purified saccharides 1 (5.0 mg) and 2
1
conducted using DIPSI-2. The coupling patterns of overlapped
were analyzed by SPT method.
H
2
4,25
References
(
3.5 mg) were obtained as white powders.
1.
2.
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4.
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.4. Hydrolysis
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.5. Methylation and methanolysis
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.7. MALDI-TOF-MS
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2
tos mass spectrometer (KOMPACT Probe) in the positive ion mode
using 2,5-dihydroxybenzoic acid as the matrix. Ions were formed
using a pulsed UV laser beam (nitrogen laser, 337 nm). Calibration
was conducted using 1-kestose as an external standard.