Synthesis of α-lactosyl-(1 → 3)-L-glycero-α-D-manno-heptopyranoside, a partial oligosaccharide structure expressed within the lipooligosaccharide produced by Neisseria gonorrhoeae strain 15253

Article abstract of DOI:10.1016/S0008-6215(01)00282-8

The glycosyl donor, hepta-O-benzyl-β-lactosyl trichloroacetimidate (4) was prepared by treating hepta-O-benzyl-lactose with trichloroacetonitrile in the presence of potassium carbonate. The acceptor, methyl 2-O-benzyl-4,6-O-benzylidene-7,8-dideoxy-α-D-manno-oct-7-enopyranoside (8) was synthesized by hydrolysis of a 3,4-butane diacetal of methyl L-glycero-α-D-manno-oct-enopyranoside and subsequent benzylidenation. Glycosidation of the donor 4 with the acceptor 8 in 1,4-dioxane using Me₃SiOTf as a promoter for 1 h at room temperature gave methyl (2,3,4,6-tetra-O-benzyl-β-D-galactopyranosyl)-(1 → 4)-(2,3,6-tri-O-benzyl-α-D-glucopyranosyl)-(1 → 3)-2-O-benzyl-4,6-O-benzylidene-7,8-dideoxy-α-D-manno-oct-7- enopyranoside (9) as a major product (59%). The oct-enopyranoside moiety of the trisaccharide 9 was converted to a heptopyranoside (80%) by oxidative cleavage with OsO₄-NaIO₄ and subsequent reduction. Hydrogenolysis of the resulting trisaccharide and subsequent acetylation gave the peracetate of α-lactosyl-(1 → 3)-Hep. Deacetylation of the peracetate afforded the title trisaccharide.

Full text of DOI:10.1016/S0008-6215(01)00282-8

Carbohydrate Research 337 (2002) 11–20
www.elsevier.com/locate/carres
Synthesis of
a-lactosyl-(1“3)-
L
-glycero-a- -manno-heptopyranoside, a partial
D
oligosaccharide structure expressed within the lipooligosaccharide
produced by Neisseria gonorrhoeae strain 15253
Kazuyuku Ishii, Hiroyuki Kubo, Ryohei Yamasaki*
Department of Biochemistry and Biotechnology, Tottori Uni6ersity, Koyama-Minami 4-101, Tottori 680-8553, Japan
Received 10 July 2001; accepted 22 October 2001
Abstract
The glycosyl donor, hepta-O-benzyl-b-lactosyl trichloroacetimidate (4) was prepared by treating hepta-O-benzyl-lactose with
trichloroacetonitrile in the presence of potassium carbonate. The acceptor, methyl 2-O-benzyl-4,6-O-benzylidene-7,8-dideoxy-a-D-
manno-oct-7-enopyranoside (8) was synthesized by hydrolysis of a 3,4-butane diacetal of methyl
ranoside and subsequent benzylidenation. Glycosidation of the donor 4 with the acceptor 8 in 1,4-dioxane using Me3SiOTf as a
promoter for 1 h at room temperature gave methyl (2,3,4,6-tetra-O-benzyl-b- -galactopyranosyl)-(1“4)-(2,3,6-tri-O-benzyl-a-
glucopyranosyl)-(1“3)-2-O-benzyl-4,6-O-benzylidene-7,8-dideoxy-a- -manno-oct-7-enopyranoside (9) as a major product (59%).
L
-glycero-a-D-manno-oct-enopy-
D
D-
D
The oct-enopyranoside moiety of the trisaccharide 9 was converted to a heptopyranoside (80%) by oxidative cleavage with
OsO4–NaIO4 and subsequent reduction. Hydrogenolysis of the resulting trisaccharide and subsequent acetylation gave the
peracetate of a-lactosyl-(1“3)-Hep. Deacetylation of the peracetate afforded the title trisaccharide. © 2002 Elsevier Science Ltd.
All rights reserved.
Keywords: Lipooligosaccharide; Lipopolysaccharide; Neisseria; Vaccine; Oligosaccharide
on some of gonococcal LOS having OS on both Hep[1]
and Hep[2].7 This MAb 2C7-defined epitope requires
the OS structure of 15253 LOS as a minimum size, and
this 15253 OS has an unusual structure containing two
lactosides;5,7 one lactose is b-(1“4)-linked to Hep[1] of
the di-heptose, and the other is a-(1“3)-linked to
Hep[2]. This latter structure is not expressed on LOS or
LPS produced by other Gram-negative species except
for some gonococcal LOS,7 and its synthesis has not yet
been accomplished. To establish a synthetic methodol-
ogy for the 2C7-defined epitope, we undertook the
synthesis of this trisaccharide, a-lactosyl-(1“3)-Hep, a
partial OS structure of 15253 LOS.5
1. Introduction
Lipooligosaccharides (LOS) produced by Neisseria
gonorrhoeae are important antigenic and immunogenic
components of the outer membrane complex. The
oligosaccharide (OS) moiety of LOS consists of a struc-
turally variable region and a conserved inner core
linked to the lipid A moiety via the KDO bridge.1–7
Gonococci synthesize this variable region by elongating
a
conserved core trisaccharide, GlcNAc–Hep[2]–
Hep[1] to express two different types of OS elongation
(Fig. 1); OS elongates from Hep[1] only or from both
Hep[1] and Hep[2].3–5,7
In a recent publication, we characterized a murine
monoclonal antibody (MAb 2C7) whose epitope resides
2. Results and discussion
* Corresponding author. Fax: +81-857-31-5347.
E-mail address: yamasaki@muses.tottori-u.ac.jp (R. Ya-
masaki).
To accomplish a-lactosylation, we chose to use
hepta-O-benzyl-b-lactosyl trichloroacetimidate as
a
0008-6215/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(01)00282-8

12
K. Ishii et al. / Carbohydrate Research 337 (2002) 11–20
donor (Scheme 1). This donor was synthesized in three
steps (Scheme 1) from p-methoxyphenyl b-lactoside
(1).8 Benzylation of 1 with NaH and benzyl bromide in
DMF gave its hepta-O-benzyl ether 2 in 65% yield.
This rather moderate yield was improved by benzylat-
ing 1 in the presence of tetra-n-butyl ammonium iodide,
and compound 2 was obtained in 95% yield. Treatment
of 2 with ceric(IV) diammonium nitrate gave a known
hepta-O-benzyl-lactose 3.9 Although the melting point
of compound 3 was higher than the one that had been
reported,9 the structure of 3 was fully characterized by
of each anomer was confirmed by both the J1,2 value
(7.0 and 3.5 Hz for b and a anomer, respectively) and
the 13C-signal of C-1 (98.26 and 94.50 ppm for b and a
anomer, respectively) of the Glc moiety (Tables 1 and
2). The b-trichloroacetimidate 4 was used as a donor as
will be described later.
As an acceptor, we selected methyl 2-O-benzyl-4,6-
O-benzylidene-7,8-dideoxy-a-D-manno-oct-7-enopyran-
oside (8) (Scheme 1). Compound 8 bearing a free 3-OH
was synthesized as follows. The 3,4-butane diacetal 613
of methyl
L
-glycero-a-D-manno-oct-enopyranoside was
1
analyzing its NMR data. We included the H and 13C
hydrolyzed in 9:1 TFA–water to give the 2-O-benzyl-
7,8-dideoxy-manno-oct-7-enopyranoside (7) as an oil
(87%). Treatment of 7 with benzaldehyde dimethylac-
etal and p-toluenesulfonic acid monohydrate in DMF
followed by chromatographic purification gave the ac-
ceptor 8 as a syrup in 79%. Its 4,6-O-benzylidene group
was confirmed by the typical downfield 13C shifts14 for
C-4 and C-6 compared with those of 7 (Table 2).
Glycosidation of the donor 4 with the acceptor 8 in
1,4-dioxane15 using trimethylsilyl trifluoromethanesul-
fonate (Me3SiOTf)16 as a promoter for 1 h at room
temperature and subsequent chromatographic purifica-
tion of the reaction mixture gave methyl (2,3,4,6-tetra-
data of 3 in Tables 1 and 2 as a reference because only
partial 13C-data10 had been available.
Treatment of compound 3 with trichloroacetonitrile
(CCl3CN) in the presence of potassium carbonate11 in
CH2Cl2 for 96 h at room temperature gave hepta-O-
benzyl-b-lactosyl trichloroacetimidate (4) and its a
anomer 5 in 62 and 6.4% yields, respectively. Similar
treatment of 3 with CCl3CN in the presence of 1,8-diaz-
abicyclo[5.4.0]undec-7-ene12 gave the a anomer 5 (a:b
2:1) as a dominant product, although the combined
yields (83%) of 4 and 5 were higher than those obtained
using potassium carbonate. The anomeric configuration
Fig. 1. N. gonorrhoeae synthesizes oligosaccharides of two different elongation patterns; R=(KDO)2-Lipid A. The Hep linked to
KDO is defined as Hep[1] and the other Hep linked to Hep[1] as Hep[2].
Scheme 1.

K. Ishii et al. / Carbohydrate Research 337 (2002) 11–20
13
Table 1
1H (500 MHz) NMR data for compounds 2–5, 7, 8
Compound Residue
H-1
H-2
H-3
H-4
H-5
H-6
H-7
H-8
2
Glc
Gal
Glc
Gal
Glc
Gal
Glc
Gal
4.88
4.47
5.15
4.34
5.77
4.45
6.42
4.34
4.70
4.82
3.69 3.65
3.80 3.46
3.50 3.85
3.75 3.35
3.67 3.67
3.76 3.40
3.68 3.93
3.75 3.34
3.68 3.78
3.81 4.05
3.99
3.95
3.93
3.89
4.08
3.91
4.77
3.91
3.95
4.12
3.52
3.40
3.98
3.33
3.54
3.37
3.91
3.35
3.46
4.05
3.84, 3.81
3.57, 3.36
3.83, 3.53
3.53, 3.37
3.85, 3.70
3.53, 3.36
3.87, 3.49
3.56, 3.37
4.46
3 a
4
5
7
8
5.97 5.35, 5.18
6.29 5.65, 5.57
4.81
J1,2
J2,3
J3,4
J4,5
J5,6 (6a) J5,6b
J6a,6b J6,7
J7,8a
J7,8b
J8a,8b
2
Glc
Gal
Glc
Gal
Glc
Gal
Glc
Gal
7.5
8.0
3.5
7.0
7.0
7.5
3.5
7.5
1.0
1.0
8.5
10.0
9.0
8.5
n.d. 9.0
9.5
9.8
10.0
3.5
9.0
2.5
8.8
n.d.
9.8
n.d.
8.5
n.d.
9.0
n.d.
9.5
n.d.
9.5
9.5
2.0
n.d.
3.0
n.d.
4.0
n.d.
3.0
8.5
2.5
n.d.
5.0
10.5
n.d.
n.d.
n.d.
11.0
10.0
10.8
n.d.
5.5
n.d.
n.d.
n.d.
1.5
5.0
2.0
3 a
4
2.5
9.5
2.5
9.5
9.5
5
5.0
7
8
17.5
18.0
10.5
11.0
n.d.
n.d.
4.0
5.0
1H-chemical shifts of 2–5, 7, 8 were determined by comparatively analyzing the 2D NMR data (DQF-COSY and HMQC), and
the coupling constants were obtained from the DQF-COSY experiment.
a A mixture of 20(a):1(b): The anomeric protons of the b anomer were as follows; l 4.61 (d, 1 H, J1 ,2 8.5 Hz, H-1I), 4.37 (d,
I
I
1 H, J1
8.0 Hz, H-1II); n.d.: not determined. Other protons of 2–5, 7, 8 are as follows: 2: l 7.38–7.14, 7.06–7.30, 6.83–6.79
II II
,2
(aromatic H), 5.08, 4.76, 5.02, 4.86, 5.00, 4.58, 4.85, 4.80, 4.75, 4.71, 4.51, 4.40, 4.36, 4.26 (PhCH2), 3.78 (PhOCH3); 3: l 8.66
[C(NH)CCl3], 7.35–7.10 (aromatic H), 5.06, 4.70, 4.97, 4.55, 4.86, 4.78, 4.79, 4.76, 4.71, 4.68, 4.56, 4.40, 4.35, 4.25 ( PhCH2); 4:
l 8.66 [C(NH)CCl3], 7.35–7.10 (aromatic H), 5.06, 4.70, 4.97, 4.55, 4.86, 4.78, 4.79, 4.76, 4.71, 4.68, 4.56, 4.40, 4.35, 4.25 (PhCH2);
5: l 8.57 [C(NH)CCl3], 7.36–7.09 (aromatic H), 5.02, 4.74, 4.98, 4.56, 4.81, 4.73, 4.74, 4.69, 4.72, 4.69, 4.51, 4.30, 4.36, 4.25
(PhCH2); 7: l 7.32–7.25 (aromatic H), 4.66, 4.58 (PhCH2), 3.26 (OCH3); 8: l 7.52–7.31 (aromatic H), 5.93 (acetal–PhCH), 4.74,
4.68 (PhCH2), 3.38 (OCH3).
O-benzyl-b-
benzyl-a- -glucopyranosyl)-(1“3)-2-O-benzyl-4,6-O-
benzylidene-7,8-dideoxy-a- -manno-oct-7-enopyran-
D
-galactopyranosyl)-(1“4)-(2,3,6-tri-O-
A minor component obtained in the above glycosida-
D
tion reaction was found to be a mixture of a b anomer
1
D
10 (Scheme 2) and an unidentified product by H and
13C NMR. Neither gel permeation chromatography
(Biobeads S-X1) nor flash-column chromatography sep-
arated these two products. Therefore, the b anomer 10
was characterized as its corresponding heptoside 12,
which will be described below.
oside (9) as a major product (59%) (Scheme 2). The
structure of 9 was determined by NMR spectroscopy.
Anomeric carbons and protons of 9 were confirmed by
the HMQC experiment (Fig. 2, Panel B), and other
carbons and protons were identified by analyzing its 2D
NMR data (DQF-COSY, HMQC, and HMBC). The
13C shift of the C-1 (at 97.25 ppm) and J1,2 value (4.0
Hz) of the Glc residue (Tables 3 and 4) confirmed that
the lactosyl moiety was a-linked to the oct-enopyran-
oside (Oct). The H-3, H-4 and C-3 signals of the Oct
moiety of the trisaccharide 9 were detected at lower
fields than those of the acceptor 8 (Tables 1 and 2),
which showed that the Glc component is linked to the
O-3 position of the Oct moiety. Also, the cross-relay
peaks, H-1(Glc)/C-3(Oct) and C-3(Oct)/H-1(Glc), in
the HMBC spectrum (Fig. 2, Panels C and D) con-
firmed the linkage site.
The Oct moiety of 9 was converted to the heptopy-
ranoside by oxidative cleavage with OsO4–NaIO4 and
subsequent reduction with NaBH4 as described previ-
ously,13 and compound 11 was obtained in 80% yield
after chromatographic purification. Periodate oxidation
of the diol formed from compound 9 was slow com-
pared with methyl manno-oct-7-enopyranoside13 due to
steric crowding, and warming the reaction mixture (30–
35 °C) was necessary for completion of the oxidation.
The structure of 11 was confirmed by analyzing its
NMR spectral data. As Fig. 3 shows, the 13C satellites

14
K. Ishii et al. / Carbohydrate Research 337 (2002) 11–20
of the vinyl carbons (C-7 and C-8 of 9, Panel A) were
absent in the 13C spectrum of 11 (Panel B), and the
DEPT and 2D NMR data (Tables 3 and 4) confirmed
conversion of C-7 to a primary carbon. The b anomer
of 9 was characterized as its corresponding heptoside 12
by treating the mixture of 10 and the unidentified
compound in a similar manner as described for 9 and
subsequent chromatographic purification. The yield of
10 was calculated to be 5.9% based on the isolated yield
of 12. The C-1 and H-1 chemical shifts and J1,2 (7.5 Hz)
of the Glc residue (Tables 3 and 4) showed that the
lactosyl moiety of 12 was b-linked, and the HMBC
experiment (data not shown) also confirmed the linkage
site.
Hydrogenolysis of 11 with Pd/C in DMF followed by
acetylation with pyridine and acetic anhydride in the
presence of 4-(dimethylamino)pyridine gave methyl
(2,3,4,6-tetra-O-acetyl-b-
(2,3,6-tri-O-acetyl-a- -glucopyranosyl)-(1“3)-2,4,6,7-
tetra-O-acetyl- -glycero-a- -manno-heptopyranoside
D
-galactopyranosyl)-(1“4)-
D
L
D
(13) as a colorless amorphous solid (73%). Deacetyla-
tion of 13 with NaOMe in MeOH followed by chro-
matographic purification gave the desired trisaccharide
14 (85%).
Table 2
13C (125 MHz) NMR data for compounds 2–5, 7, 8
Compound
Residue
C-1
C-2
C-3
C-4
C-5
C-6
C-7
C-8
2
3
4
5
7
Glc
Gal
Glc
Gal
Glc
Gal
Glc
Gal
102.88
103.04
91.46
103.05
98.26
102.67
94.50
102.77
98.71
81.67
80.07
79.24
80.07
80.28
79.86
78.33
79.78
77.93
78.43
83.10
82.67
80.09
82.56
82.81
82.48
79.61
82.44
71.60
69.17
76.94
73.67
76.76
73.87
75.89
73.67
75.73
73.64
68.08
74.48
75.45
73.15
70.47
73.26
76.01
73.06
76.14
73.04
73.25
65.58
68.48
68.21
68.25
68.37
67.56
68.14
67.45
68.09
70.54
74.15
138.12
131.52
115.75
120.58
a
8
99.18
13C-chemical shifts of 2–5, 7, 8 were determined by comparatively analyzing the 2D NMR data (HMQC and DQF-COSY).
a The tertiary carbon of the benzylidene acetal was determined by the HMBC experiment. n.d.: not determined. Other carbons
of 2–5, 7, 8 are as follows: 2: l 155.31, 151.73, 139.17, 139.12, 138.84, 138.61, 138.57, 138.49, 138.17, 128.49–127.24, 118.5, 114.59
(aromatic C), 75.58, 75.40, 75.29, 74.83, 73.52, 73.19, 72.70 (PhCH2), 54.90 (PhOCH3); 3: l 139.42, 139.20, 138.92, 138.70, 138.31,
138.27, 138.17, 129.19–125.46 (aromatic C), 75.56, 75.35, 74.86, 73.67, 73.56, 73.26, 72.72 (PhCH2); 4: l 161.29 [C(NH)CCl3],
139.00, 138.94, 138.66, 138.49, 138.23, 138.15, 138.09, 128.36–127.11 (aromatic C), 90.99 [C(NH)CCl3], 75.26, 75.01, 74.68, 73.38,
73.01, 72.62 (PhCH2); 5: l 161.15 [C(NH)CCl3], 139.11, 139.04, 138.71, 138.51, 138.24, 138.12, 137.98, 128.34–127.03 (aromatic
C), 91.38 [C(NH)CCl3)], 75.38, 75.17, 74.67, 73.39, 73.12, 73.06, 72.52 (PhCH2); 7: l 137.71, 128.60, 128.05, 127.97 (aromatic C),
73.54 (PhCH2), 54.90 (OCH3); 8: l 137.90, 137.34, 129.10–126.43 (aromatic C), 95.85 (acetal–PhCH), 73.42 (PhCH2), 55.15
(OCH3).
Scheme 2.

Table 3
1H (500 MHz) NMR data for compounds 9, 11–14
Compound
Residue
H-1
H-2
H-3
H-4
H-5
H-6
H-7
6.26
H-8
a
9
Oct
4.72
5.44
4.29
4.69
5.41
4.29
4.67
4.55
4.43
4.75
5.11
4.49
4.63
5.12
4.35
3.80
3.44
3.70
3.81
3.43
3.70
3.82
3.41
3.72
5.23
4.68
5.13
3.96
3.50
3.44
4.31
3.88
3.32
4.28
3.88
3.32
4.26
3.52
3.38
4.15
5.31
4.96
3.74
3.80
3.55
4.51
3.84
3.88
4.57
3.82
3.88
4.50
3.98
3.88
5.32
3.68
5.36
3.93
3.56
3.81
4.09
3.81
3.30
4.16
3.82
3.31
4.11
3.17
3.31
3.92
3.99
3.93
3.50
3.83
3.62
4.74
5.60, 5.53
Glc
Gal
Hep
Glc
Gal
Hep
Glc
Gal
Hep
Glc
Gal
Hep
Glc
Gal
3.82, 3.60
3.52, 3.33
4.31
3.80, 3.61
3.51, 3.32
4.33
3.65, 3.46
3.49, 3.33
5.16
4.45, 4.18
4.13, 4.06
3.94
11
12
4.10–4.17
4.17–4.09
4.30, 4.22
3.64, 3.60
13
14 b
/
3.79, 3.74
3.69, 3.64
J1,2
J2,3
J3,4
J4,5
J5,6(6a)
J5,6b
J6a,6b
J6,7
J6,7b
J7a,7b
J7,8a
J7,8b
J8a,8b
9
11
Oct a
Glc
Gal
Hep
Glc
Gal
Hep
Glc
Gal
Hep
Glc
Gal
Hep
Glc
Gal
1.0
4.0
7.5
1.0
4.0
7.5
1.5
7.5
8.0
1.5
3.5
8.0
1.0
4.0
7.5
4.5
9.8
9.3
3.5
9.8
9.5
3.0
n.d
9.8
3.5
10.0
10.5
3.5
10.0
10.0
9.5
10.5
2.5
10.0
n.d.
2.5
10.0
9.0
2.5
10.0
10.0
3.5
9.8
10.0
3.0
10.3
n.d.
n.d.
10.0
n.d.
n.d.
10.0
9.0
6.0
n.d.
n.d.
6.0
n.d.
n.d.
n.d
4.0
n.d
2.0
1.5
4.0
1.0
2.0
8.0
4.5
n.d.
n.d.
n.d.
17.5
11.0
n.d.
n.d.
n.d.
3
n.d.
n.d.
5.5
n.d.
n.d.
7.0
n.d.
n.d.
11.3
11.0
n.d.
n.d.
10.0
n.d.
(
12
1.5
n.d.
11.0
10.5
2
n.d
13
10.0
10.0
n.d.
10.0
10.3
n.d.
6.0
8.0
12.0
11.0
14 b
6.5
5.5
4.5
4.0
12.0
11.8
1H-chemical shifts were determined by comparatively analyzing the 2D NMR data (DQF-COSY, HMQC and HMBC) except for 14 (DQF-COSY and HMQC), and the
coupling constants were obtained from the DQF-COSY experiment. n.d., not determined.
a The oct-enopyranoside residue is expressed as Oct.
b The chemical shifts are in D2O. Other protons of 9, 11–14 are as follows: 9: l 7.38–7.05 (aromatic H), 5.80 (acetal–PhCH), 4.95, 4.54, 4.95, 4.75, 4.85, 4.65, 4.71, 4.61,
4.68, 4.64, 4.52, 4.35, 4.50, 4.31, 4.32, 4.21 (PhCH2), 3.36 (OCH3); 11: l 7.37–6.96 (aromatic H), 5.70 (acetal–PhCH), 4.94, 4.54, 4.86, 4.62, 4.72, 4.62, 4.68, 4.63, 4.51, 4.35,
4.50, 4.32, 4.32, 4.21 (PhCH2), 3.34 (OCH3); 12: l 7.40–7.05 (aromatic H), 5.78 (acetal–PhCH), 5.02, 4.63, 4.94, 4.52, 4.77, 4.74, 4.73, 4.56, 4.73, 4.68, 4.65, 4.43, 4.32, 4.29,
4.22 (PhCH2), 3.31 (OCH3); 13: l 3.36 (OCH3), 2.24–1.97 (OAc); 14: l 3.27 (OCH3).

16
K. Ishii et al. / Carbohydrate Research 337 (2002) 11–20
Fig. 2. Parts of HMQC (A, B) and HMBC (C, D) spectra of compound 9. The part of HMBC spectrum (C) was plotted at a
3
2
higher contour level to eliminate other relay peaks due to JC,H and JC,H. *, CDCl3.
Glycosidation of hepta-O-benzyl-b-lactosyl tri-
In the present study, we used the 4,6-O-benzylidene
manno-oct-enopyranoside 8 as the acceptor, instead of
Hep derivatives containing unprotected 3-OH.17–19 Be-
cause compound 8 can be prepared from the precursor
chloroacetimidate (4) with the manno-oct-enopyran-
oside 8, conversion of the oct-enopyranoside moiety of
the trisaccharide into the heptoside, and subsequent
removal of protecting groups gave a-lactosyl-(1“3)-
Hep, a partial oligosaccharide sequence expressed in
LOS produced by N. gonorrhoeae 15253.
6 for the synthesis of L
-glycero-manno-heptoside,13 our
approach would provide an alternative method to syn-
thesize 3-O-substituted heptoside.

K. Ishii et al. / Carbohydrate Research 337 (2002) 11–20
17
3. Experimental
were measured with a YANAGIMOTO micro melting-
point apparatus and are uncorrected. Elemental analy-
ses were carried out by using a Perkin–Elmer 2400
Series II CHNS/O Analyzer. High-resolution mass
General methods.—Optical rotations were measured
with a HORIBA SEPA-200 polarimeter. Melting points
Table 4
13C (125 MHz) NMR data for compounds 9, 11–14
Compound
Residue
C-1
C-2
C-3
C-4
C-5
C-6
C-7
C-8
a
9
Oct
100.14
97.25
103.13
100.28
97.26
103.14
99.98
102.16
102.55
100.00
96.64
78.21
78.21
79.92
78.31
78.16
79.93
76.44
81.67
79.84
70.57
70.89
68.79
70.20
71.93
71.43
73.54
79.32
82.42
73.57
79.34
82.46
75.99
83.33
82.43
72.99
68.69
70.96
79.61
71.92
73.00
74.31
76.95
73.62
73.75
77.00
74.24
72.93
76.22
73.65
66.47
75.83
66.67
65.77
78.68
69.03
66.06
71.02
73.02
65.78
71.04
73.04
65.97
75.48
72.93
68.17
68.97
70.47
71.43
71.60
75.82
74.24
68.33
68.14
74.24
68.38
68.16
74.24
67.81
68.06
66.67
62.07
60.74
69.12
60.31
61.51
131.51
120.45
Glc
Gal
Hep
Glc
Gal
Hep
Glc
Gal
Hep
Glc
Gal
Hep
Glc
Gal
11
59.23
59.25
61.74
63.27
12
13
100.24
101.27
100.78
103.27
14 b
13C-chemical shifts were determined by comparatively analyzing the 2D NMR data (DQF-COSY, HMQC and HMBC) except for
14 (DQF-COSY and HMQC). n.d., not determined.
a The oct-enopyranoside residue is expressed as Oct.
b The chemical shifts are in D2O. Other carbons of 9, 11–14 are as follows: 9: l 139.39, 139.03, 138.78, 138.49, 138.45, 138.34,
138.18, 138.13, 138.74, 129.02–126.36 (aromatic C), 95.82 (acetal–PhCH), 75.12, 75.01, 74.67, 73.53, 73.35, 73.18, 72.49, 70.91
(PhCH2), 54.88 (OCH3); 11: l 139.38, 139.04, 138.81, 138.49, 138.47, 138.18, 138.14, 137.40, 129.25–126.40 (aromatic C), 75.12,
75.01, 74.67, 73.62, 73.37, 73.21, 72.50, 70.93 (PhCH2), 54.99 (OCH3); 12: l 139.05, 138.80, 138.74, 138.56, 138.51, 138.22, 138.09,
137.57, 128.34–127.03 (aromatic C), 96.27 (acetal–PhCH), 75.11, 74.99, 74.70, 74.64, 73.34, 73.26, 72.93, 72.59 (PhCH2), 55.02
(OCH3); 13: l 170.25, 170.65, 170.46, 170.39, 170.35, 170.25, 170.18, 170.07, 169.26, 169.20, 168.99 (COCH3), 55.20 (OCH3),
20.77–20.49 (COCH3); 14: l 55.18 (OCH3).
Fig. 3. DEPT spectra of 9 (A) and 11 (B). Of the exocyclic carbons, only C-7 and C-8 are labeled.

18
K. Ishii et al. / Carbohydrate Research 337 (2002) 11–20
spectra were obtained with a JEOL JMS-600H mass
spectrometer as described previously.13 NMR spectra
[1H (500 MHz) and 13C (125 MHz), JEOL JNM-ECP
500] were recorded in CDCl3 or D2O. Me4Si was used
as an internal standard for CDCl3 and CH3CN (l 1.95
2,3,6-tri-O-benzyl-D-glucopyranose (3).—Ceric(IV) di-
ammonium nitrate (21.1 g, 38.4 mmol) was added to a
chilled (0 °C) solution of 2 (13.8 g, 12.8 mmol) in 4:1:1
MeCN–toluene–water (150 mL), and the mixture was
stirred for 3 h at 0 °C. Ethyl acetate (200 mL) was
added to the reaction mixture, and the aqueous solu-
tion was extracted with EtOAc (2×100 mL). The
combined organic extracts were washed with water
(3×150 mL) and brine (2×150 mL), dried (MgSO4),
filtered and concentrated to give a dark red syrup.
Chromatographic purification (1:7“1:6“1:5 EtOAc–
toluene) and subsequent crystallization (EtOAc–hex-
ane) gave 3 as needles (5.87 g, 47%). Compound 3 was
found to be an anomeric mixture (a:b 20:9) by 1H
NMR spectroscopy; mp 115–116 °C; [h]2D5 +16° (c 1,
CHCl3), lit.9 mp 102–105 °C; [h]D +10° (c 1.2,
CHCl3).
1
for H; l 119.58, 1.30 for 13C) for D2O. 2D NMR data
were acquired at rt (20–23 °C) and processed in a
similar manner as described previously;2,5,7,13 DQF-
COSY spectra data: digital resolutions in ꢀ1 and ꢀ2
after zero filling were in the range of 1.11–3.14 Hz/
point; HMQC and HMBC spectra data: digital resolu-
tions in ꢀ1 and ꢀ2 after zero filling were in the ranges
of 10–25 and 1.66–2.95 Hz/point, respectively. Silica
Gel 60 (E. Merck) was used for flash column (0.040–
0.063 mm) and open-column (0.063–0.20 mm) chro-
matography. Silica Gel 60 F254 (E. Merck) was used for
thin-layer chromatography, and compounds were de-
tected under UV light or by spraying 10% concd H2SO4
in MeOH and then by heating the plates at 120 °C for
5 min. For gel chromatography, we used two gel
columns packed with Biobeads S-X1 (Bio-Rad, 200-400
mesh, 3×90 cm, elution with toluene) and Bio-Gel P-2
(Bio-Rad, B400 mesh, 1.6×6.0 cm, elution with
deionized water). The P-2 column was used for purifica-
tion of an unprotected oligosaccharide, and to monitor
its elution, we used a refractrometer (ERC-7522, Erma
2,3,4,6-Tetra-O-benzyl-i-
D
-galactopyranosyl-(1“4)-
2,3,6-tri-O-benzyl-i- -glucopyranosyl trichloroacetimi-
D
date (4).—Trichloroacetonitrile (CCl3CN, 4.8 mL, 47.8
mmol) was added to a stirred suspension of 3 (2.51 g,
2.58 mmol) and potassium carbonate11 (4.6 g) in
CH2Cl2 (30 mL) at rt, and after stirring for 96 h, the
mixture was filtered and concentrated to a syrup. TLC
(2:3:1 hexane–toluene–EtOAc, containing 1% Et3N)
showed the presence of two new products and unre-
acted 3. Chromatography (4:2:1 hexane–toluene–
EtOAc, containing 1% Et3N) of the syrup gave the
b-imidate 4 (3.28 g, 62%, syrup). TLC (2:3:1 hexane–
toluene–EtOAc, containing 1% Et3N): Rf 0.63; [h]D22
+13° (c 1, CHCl3).
8
CR. Inc., Tokyo) connected to an AKTA explorer 10S
(Amasham Pharmacia, Uppsala, Sweden). p-
Methoxyphenyl
b-
D
-galactopyranosyl-(1“4)-b-D-
glucopyranoside (1) was prepared according to the
literature procedure;20 mp 271–272 °C; [h]2D3 −20.8° (c
0.5, DMF), lit.8 [h]D −20° (c 0.5, DMF).
Eluted next was the a-imidate 5 (0.35 g, 6.4%, syrup):
TLC (2:3:1 hexane–toluene–EtOAc, containing 1%
Et3N): Rf 0.53; [h]2D2 +30° (c 1, CHCl3).
p-Methoxyphenyl
(2,3,4,6-tetra-O-benzyl-i-
D-
galactopyranosyl)-(1“4)-2,3,6-tri-O-benzyl-i-
D
-gluco-
pyranoside (2).—A solution of 1 (6.0 g, 13.4 mmol) in
DMF (240 mL) was added dropwise to a chilled (0 °C)
mixture of NaH (60% in oil 7.5 g, 187.3 mmol; pre-
washed with hexane) in DMF (60 mL). After stirring
the mixture for 30 min at 0 °C, Bu4NI (3.0 g) was
added and the reaction mixture was stirred for 1 h.
Benzyl bromide (16.7 mL, 140.7 mmol) was added, and
the mixture was stirred for 1.5 h at 0 °C and then
overnight at rt. The mixture was quenched with water
(100 mL) and extracted with EtOAc (300 mL). The
aqueous solution was extracted with EtOAc (2×150
mL), and the combined extracts were washed with
water and brine, dried (MgSO4), and concentrated to
Further elution gave the starting material 3 (0.73 g,
17%): TLC (2:3:1 hexane–toluene–EtOAc, containing
1% Et3N): Rf 0.20.
Compound 3 (410 mg, 2.58 mmol) in CH2Cl2 (3 mL)
was also treated with CCl3CN (0.42 mL, 4.20 mmol) in
the presence of 1,8-diazabicyclo[5.4.0]undec-7-ene
(DBU) (0.25 mL, 0.08 mmol) at 0 °C for 6 h. The use
of DBU gave the a anomer 5 as a major product (a:b
2:1), although the combined yields (83%) of 4 and 5
were higher than those obtained with potassium
carbonate.
Methyl
2-O-benzyl-7,8-dideoxy-h-D-manno-oct-7-
enopyranoside (7).—Compound 6 (1.73g, 4.08 mmol)
was hydrolyzed in 9:1 TFA–water (10 mL) for 15 min
at rt, and the hydrolysate was concentrated to an oil
which was purified by chromatography (1:1“2:1
EtOAc–hexane) to give 7 (1.10 g, 87%) as an oil; [h]D22
−1° (c 1, CHCl3); FAB-HRMS: Calcd for C16H23O6
[M+H], 311.1495; Found: 311.1527.
give
a white solid. Chromatographic purification
(5:4:1“2:2:1 hexane–toluene–EtOAc) gave a crys-
talline residue which was recrystallized from EtOAc
hexane to give 2 (13.8 g, 95%) as fine needles. Benzyla-
tion of 1 with NaH–BnBr in the absence of Bu4NI gave
2 in 65% yield. mp 118–120 °C; [h]2D0 −14° (c 1,
CHCl3); Anal. Calcd for C68H70O12 (1079.29): C, 75.67;
H, 6.54. Found: C, 75.82; H, 6.52.
Methyl 2-O-benzyl-4,6-O-benzylidene-7,8-dideoxy-h-
D
-manno-oct-7-enopyranoside (8).—A solution of p-
2,3,4,6-Tetra-O-benzyl-i- -galactopyranosyl-(1“4)-
D
TsOH·H2O (0.30 mg, 1.59 mmol) in DMF (20 mL) was

K. Ishii et al. / Carbohydrate Research 337 (2002) 11–20
19
mmol) was added to a chilled solution (0 °C) of the
syrup in 1:1 1,4-dioxane–MeOH (30 mL), and the
mixture was warmed to rt and stirred for 16 h. The
mixture was extracted with EtOAc (50 mL), and the
aqueous solution was extracted with EtOAc (2×20
mL). The combined extracts were washed with water
(2×40 mL) and brine (40 mL), dried (MgSO4), and
concentrated to a syrup. Purification by flash-column
chromatography (1:1:2 EtOAc–hexane–toluene) gave
11 (0.80 g, 80%) as a syrup; [h]2D2 +32° (c 1, CHCl3).
Anal. Calcd for C83H88O17 (1357.60): C, 73.43; H, 6.53;
Found: C, 73.22; H, 6.47.
added to a stirred solution of 7 (4.92 g, 15.9 mmol) and
benzaldehyde dimethylacetal (7.13 mL, 47.6 mmol) in
DMF (55 mL) at rt. The reaction mixture was warmed
to 40–45 °C and stirred for 16 h. After addition of
Et3N (2 mL), the reaction mixture was concentrated to
a syrup. The syrup was dissolved in EtOAc, and the
solution was washed with satd NaHCO3 and brine,
dried, and concentrated to give a syrup. Purification by
flash-column chromatography (1:4 EtOAc–hexane)
gave the 4,6-O-benzylidene compound 8 (4.97 g, 79%)
as a syrup; [h]2D3 −81.4° (c 1, CHCl3). Anal. Calcd for
C23H26O6 (398.45): C, 69.33; H, 6.58; Found: C, 69.35;
H, 6.37.
Methyl
osyl)-(1“4)-(2,3,6-tri-O-benzyl-i-
(1“3)-2-O-benzyl-4,6-O-benzylidene-
(2,3,4,6-tetra-O-benzyl-i-
-glucopyranosyl)-
-glycero-h-D-
D-galactopyran-
Methyl
osyl)-(1“4)-(2,3,6-tri-O-benzyl-h-
(1“3)-2-O-benzyl-4,6-O-benzylidene-7,8-dideoxy-h-
-manno-oct-7-enopyranoside (9).—A solution of
(2,3,4,6-tetra-O-benzyl-i-D-galactopyran-
D
D
-glucopyranosyl)-
L
manno-heptopyranoside (12).—The mixture (311 mg)
containing the b anomer 10 and the unidentified
product was oxidized and then reduced in a similar
manner as described for the synthesis of 11. Purification
of the reaction mixture by flash-column chromatogra-
phy (3:1 toluene–EtOAc) gave 12 as a syrup (103 mg);
[h]2D4 +9° (c 1, CHCl3).
D
Me3SiOTf (1.1 mL, 0.06 mmol) in 1,4-dioxane (20 mL)
was added dropwise at rt to a stirred mixture of 8 (0.58
g, 1.28 mmol), 4 (1.70 g, 1.52 mmol) and molecular
,
sieves 4 A (1.0 g) in 1,4-dioxane (12 mL). The mixture
was stirred for 1 h at rt, treated with Et3N (0.3 mL) and
filtered through Celite. The filtrate was concentrated to
a syrup which was dissolved in CH2Cl2 (30 mL). The
organic solution was washed with water (20 mL), and
the aqueous solution was extracted with CH2Cl2 (2×15
mL). The combined extracts were washed with aq satd
NaHCO3 (20 mL) and brine (20 mL), dried (MgSO4),
and concentrated to give a syrup. Elution of a flash
Methyl
osyl)-(1“4)-(2,3,6-tri-O-acetyl-h-
(1“3)-1,2,4,6,7-penta-O-acetyl- -glycero-h-
(2,3,4,6-tetra-O-acetyl-i-
-glucopyranosyl)-
-manno-
D-galactopyran-
D
L
D
heptopyranoside (13).—A suspension of 11 (238 mg,
0.18 mmol) and Pd/C (10%, 350 mg) in DMF (4 mL)
was stirred vigorously for 10 days under a hydrogen
atmosphere. The mixture was filtered through Celite,
and the filtrate was concentrated to a syrup. The syrup,
dried over P2O5, was acetylated with pyridine and Ac2O
(2 mL each) with a catalytic amount of 4-(dimethy-
lamino)pyridine at rt overnight. The reaction solution
was co-evaporated with toluene several times to a
residue. The residue was dissolved in EtOAc (5 mL),
and the organic solution was washed with water (5
mL). The aqueous solution was extracted with EtOAc
(5 mL), and the combined extracts were washed with
brine (5 mL), dried (MgSO4), filtered and concentrated
to a syrup. Purification by flash-column chromatogra-
phy of the syrup (2:1 EtOAc–hexane) gave 13 as a
colorless amorphous solid (129 mg, 73%); [h]2D2 +46° (c
1, CHCl3). Anal. Calcd for C42H58O28 (1010.90): C,
49.90; H, 5.78; Found: C, 49.65; H, 5.68.
column with 2:3:6 Et2O–hexane–toluene gave
a
product of Rf 0.54. This product was found to be a
mixture of a b-(1“3) linked trisaccharide and an
unidentified product by 1H NMR spectroscopy, and
neither flash-column chromatography nor gel chro-
matography (Biobeads S-X1) separated the two prod-
ucts. The above b anomer was therefore characterized
after osmylation of the mixture (311 mg) as will be
described below. Further elution gave 9 as a syrup (1.03
g, 59%). TLC (1:2:1 EtOAc–hexane–toluene): Rf 0.50;
[h]2D4 +17.9° (c 1, CHCl3). Anal. Calcd for C84H88O16
(1353.31): C, 74.54; H, 6.55; Found: C, 74.27; H, 6.40.
The acceptor 8 was also recovered (0.13 g, 25%), and
the hydrolyzed donor was not further characterized.
Methyl
osyl)-(1“4)-(2,3,6-tri-O-benzyl-h-
(1“3)-2-O-benzyl-4,6-O-benzylidene-
(2,3,4,6-tetra-O-benzyl-i-
-glucopyranosyl)-
-glycero-h-D-
D-galactopyran-
D
Methyl (i-
D
-galactopyranosyl)-(1“4)-(h-D-glucopy-
L
ranosyl)-(1“3)-
L
-glycero-h- -manno-heptopyranoside
D
manno-heptopyranoside (11).—A solution of OsO4 in
water (1%, w/v, 3.0 mL) containing NaIO4 (4.8 g, 22.4
mmol) was added to a chilled mixture of 9 (1.00 g, 0.74
mmol) in 2:1 Et2O–water (24 mL). The mixture was
stirred vigorously at 30–35 °C for 3 days and diluted
with Et2O (20 mL) and water (20 mL). The aqueous
solution was extracted with Et2O (2×15 mL), and the
combined extracts were washed with water (3×30 mL)
and concentrated to give a syrup. NaBH4 (0.28 g, 7.40
(14).—Compound 13 (21 mg, 20.8 mmol) was treated
with 20 mM NaOMe in MeOH (2.0 mL) for 2 h, and
the solution was neutralized with Amberlite IR-120
(H+), filtered and concentrated to a residue. The
residue was purified by gel chromatography [Bio-Gel
P-2] to give 14 (9.7 mg, 85%) as a white powder; [h]D25
+124° (c 0.5, water); FAB-HRMS: Calcd for
C20H37O17 [M+H], 549.2031; Found: 549.2043.

20
K. Ishii et al. / Carbohydrate Research 337 (2002) 11–20
8. Kuyama, H.; Nukada, T.; Nakahara, Y.; Ogawa, T.
Acknowledgements
Tetrahedron Lett. 1993, 34, 2171–2174.
9. Koto, S.; Morishima, N.; Shichi, S.; Haigoh, H.; Hi-
rooka, M.; Okamoto, M.; Higuchi, T.; Shimizu, K.;
Hashimoto, Y.; Irisawa, T.; Kawasaki, H.; Takahashi,
Y.; Yamazaki, M.; Mari, Y.; Kudo, K.; Ikegaki, T.;
Suzuki, S.; Zen, S. Bull. Chem. Soc. Jpn. 1992, 65,
3257–3274.
This work was supported by Grant-in-Aid
(10306022) for Scientific Research of the Ministry of
Education of Japan. We thank H. Masayuki for
HRMS analyses.
10. Jansson, K.; Ahlfors, S.; Frejd, T.; Kihlberg, J.; Magnus-
son, G. J. Org. Chem. 1988, 53, 5629–5647.
11. Schmidt, R. R.; Michel, J.; Michael, R. Liebigs Ann.
Chem. 1984, 1343–1357.
12. Numata, M.; Sugimoto, M.; Koike, K.; Ogawa, T. Car-
bohydr. Res. 1987, 163, 209–225.
13. Yamasaki, R.; Takajyo, A.; Kubo, H.; Matsui, T.; Ishii,
K.; Yoshida, M. J. Carbohydr. Chem. 2001, 20, 171–180.
14. Salanski, P.; Descotes, G.; Bouchu, A.; Queneau, Y. J.
Carbohydr. Chem. 1998, 17, 129–142.
15. Demchenko, A.; Stauch, T.; Boons, G. J. Synlett 1997, 7,
818–820.
References
1. Gibson, B. W.; Webb, J. W.; Yamasaki, R.; Fisher, S. J.;
Burlingame, A. L.; Mandrell, R. E.; Schneider, H.;
Griffiss, J. M. Proc. Natl. Acad. Sci. USA 1989, 86,
17–21.
2. Yamasaki, R.; Bacon, B. E.; Nasholds, W.; Schneider,
H.; Griffiss, J. M. Biochemistry 1991, 30, 10566–10575.
3. Kerwood, D. E.; Schneider, H.; Yamasaki, R. Biochem-
istry 1992, 31, 12760–12768.
4. John, C. M.; Griffiss, J. M.; Apicella, M. A.; Mandrell,
R. E.; Gibson, B. W. J. Biol. Chem. 1991, 266, 19303–
19311.
5. Yamasaki, R.; Kerwood, D. E.; Schneider, H.; Quinn, K.
P.; Griffiss, J. M.; Mandrell, R. E. J. Biol. Chem. 1994,
269, 30345–30351.
6. John, C. M.; Schneider, H.; Griffiss, J. M. J. Biol. Chem.
1999, 274, 1017–1025.
7. Yamasaki, R.; Koshino, H.; Kurono, S.; Nishinaka, Y.;
McQuillen, D. P.; Kume, A.; Gulati, S.; Rice, P. A. J.
Biol. Chem. 1999, 274, 36550–36558.
16. Schmidt, R. R.; Grudler, G. Angew. Chem., Int. Ed. Engl.
1982, 21, 781–782.
17. Boons, G. J. P. H.; Hoogerhout, P.; Poolman, J. T.; van
der Marel, G. A.; van Boom, J. H. Bioorg. Med. Chem.
Lett. 1991, 1, 303–308.
18. Grzeszczyk, B.; Zamojski, A. Carbohydr. Res. 1994, 262,
49–57.
19. Garegg, P. J.; Oscarson, S.; Tiden, A. K. Carbohydr. Res.
1990, 200, 475–480.
20. Zang, Z.; Magnusson, G. Carbohydr. Res. 1996, 925,
41–55.