Synthesis of a 3,4-di-O-substituted heptose structure: A partial oligosaccharide expressed in Neisserial lipooligosaccharide

Article abstract of DOI:10.1002/ejoc.200300651

We have synthesized a tetrasaccharide containing a 3,4-di-branched L-glycero-D-manno-heptose (Hep), β-lactosyl-(1→4)-[L-α-D-Hep- (1→3)]-L-α-D-Hep 19, by using a mannose (Man) derivative as an acceptor. Prior to the construction of the branched Hep, we confirmed that the 3,4-dibranched Man structure could be synthesized using a 3-branched Man 6 as an acceptor. Glycosylation of the acceptor 6 using hepta-O-acetyl-α-lactosyl trichloroacetimidate (7) gave the desired 3,4-dibranched structure, β-lactosyl-(1→4)-[α-Man-(1→3)]-α-Man 8. As expected, β-lactosyl-(1→4)-[L-α-D-Hep-(1→3)]-α-D-Man 14 was also obtained by glycosylating the 4-OH acceptor 13 with 7 in a similar manner. The Man residue of 14 was converted into the Hep unit by Swern oxidation, Grignard reaction, and oxidative cleavage followed by reduction. Thus, we constructed the 3,4-dibranched Hep structure 19 by using the 3-branched Man 13 as an acceptor. The current results demonstrate that the gauche orientation of the O-3 and O-4 units of the Man configuration does not prevent the formation of the 3,4-di-O-substituted structure. This approach should provide an alternative method to synthesize the 3,4-dibranched Hep structure expressed in LOS produced by pathogenic Gram-negative bacteria such as the Neisserial and Haemophilus species. Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004.

Full text of DOI:10.1002/ejoc.200300651

FULL PAPER
Synthesis of a 3,4-Di-O-substituted Heptose Structure: A Partial
Oligosaccharide Expressed in Neisserial Lipooligosaccharide
Hiroyuki Kubo,[a] Kazuyuki Ishii,[a] Hiroyuki Koshino,[b] Kenji Toubetto,[c]
Kentarou Naruchi,[a] and Ryohei Yamasaki*[a]
Keywords: Glycosylation / Neisseria / Oligosaccharides / Lipooligosaccharides / Lipopolysaccharides
We have synthesized a tetrasaccharide containing a 3,4-di-
branched -glycero- -manno-heptose (Hep), β-lactosyl- Thus, we constructed the 3,4-dibranched Hep structure 19
(1Ǟ4)-[ -α- -Hep-(1Ǟ3)]-
(Man) derivative as an acceptor. Prior to the construction of
nard reaction, and oxidative cleavage followed by reduction.
L
D
L
D
L-α-D-Hep 19, by using a mannose
by using the 3-branched Man 13 as an acceptor. The current
results demonstrate that the gauche orientation of the O-3
the branched Hep, we confirmed that the 3,4-dibranched and O-4 units of the Man configuration does not prevent the
Man structure could be synthesized using a 3-branched Man
6 as an acceptor. Glycosylation of the acceptor 6 using hepta-
O-acetyl-α-lactosyl trichloroacetimidate (7) gave the desired
3,4-dibranched structure, β-lactosyl-(1Ǟ4)-[α-Man-(1Ǟ3)]-α-
formation of the 3,4-di-O-substituted structure. This ap-
proach should provide an alternative method to synthesize
the 3,4-dibranched Hep structure expressed in LOS pro-
duced by pathogenic Gram-negative bacteria such as the
Neisserial and Haemophilus species.
Man 8. As expected, β-lactosyl-(1Ǟ4)-[
L-α-D-Hep-(1Ǟ3)]-α-
D-Man 14 was also obtained by glycosylating the 4-OH ac-
ceptor 13 with 7 in a similar manner. The Man residue of 14
( Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
was converted into the Hep unit by Swern oxidation, Grig-
Germany, 2004)
Introduction
the 3,4-dibranched Hep structure has been synthesized by
Oscarson’s group.[18] They used a 1,6-anhydro-heptose de-
rivative as an acceptor to build the 3-O-substituted Hep,
which was then employed to construct the 3,4-dibranched
Hep. The authors reported that glycosylation using either
Lipooligosaccharides (LOS) produced by Neisseria
gonorrhoeae are important antigenic and immunogenic
components of the outer membrane complex.[1Ϫ4] Recently,
we characterized an epitope defined by a monoclonal anti-
body 2C7 that potentially could be developed as a vaccine
target against the organism.[5Ϫ7] This 2C7-defined epitope
is expressed in the oligosaccharide (OS) moiety of LOS pro-
duced by strain 15253.[7,8]
As Figure 1 shows, the 15253 OS contains two heptose
(Hep) residues, HepI and HepII, within the conserved core.
Both of the Hep residues are dibranched; HepI and HepII
are branched at O-3 and O-4 and at O-2 and O-3, respec-
tively.[8] The 3,4-dibranched Hep, represented by HepI,
within 15253 LOS is a common structure among Neisserial
LOS[9Ϫ12] and it is also expressed in Haemophilus[13Ϫ15]
and Campylobacter LOS.[16,17] A trisaccharide containing
4
an O-3- or O-4-substituted Hep of C1 conformation as an
acceptor did not produce the desired 3,4-dibranched struc-
tures. In addition, they reported difficulties in synthesizing
the 3,4-dibranched mannose (Man) structure,[19] and these
unfruitful results were ascribed to steric crowding arising
from the gauche orientation of the O-3 and O-4 positions.[18]
Figure 1. The oligosaccharide structure of LOS produced by Neis-
seria gonorrhoeae strain 15253; Gal: -galactose; Glc: -glucose;
Hep: -glycero--manno-heptose; GlcNAc: N-acetyl--glucosa-
mine; KDO: 2-keto-3-deoxy--manno-octulosonic acid; the two
heptoses are designated as described previously[8]
[a]
Department of Biochemistry
University
&
Biotechnology, Tottori
Koyama-Minami 4-101 Tottori City, Tottori-Ken 680-8553,
Japan
Fax: (internat.) ϩ 81-857-31-5347
E-mail: yamasaki@muses.tottori-u.ac.jp
The Institute of Physical and Chemical Research (RIKEN)
Wako-shi,
Saitama 351-0198, Japan
Maruho Co Ltd., Central Research Laboratory
Shimogyo-ku, Kyoto 600-8815, Japan
[b]
[c]
In our present study, we examined glycosylation reactions
for the construction of the 3,4-dibranched Hep structure
using a Man derivative of 4C1 form as an acceptor and
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DOI: 10.1002/ejoc.200300651
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A Partial Oligosaccharide Expressed in Neisserial Lipooligosaccharide
FULL PAPER
found that the gauche orientation of the endocyclic hydroxy residue, which is almost identical to that (169 Hz) of the
groups does not disfavor the formation of the 3,4-di- α-ManI residue, and the linkage site was identified by the
branched structure. We constructed the 3,4-di-O-substi- distinctive downfield shift (∆δ ϭ 4.9 ppm) of the C-3 atom
tuted Hep expressed in 15253 LOS as follows: synthesis of of the ManI (Table 2) and was also confirmed by the
a disaccharide, α-Hep-(1Ǟ3)-Man, β-lactosylation at O-4 HMBC experiment. The minor product obtained from the
of the Man residue, and subsequent conversion the Man glycosylation above was found by 2D NMR spectroscopy
residue to the Hep residue.
to be a mixture of Man-(1Ǟ2)-Man and Man-(1Ǟ2)-[Man-
(1Ǟ3)]-Man. Although these products were not further
characterized, the yields of the disaccharide and the trisac-
charide were estimated to be 2.3 and 4.6%, respectively,
based on the ratio of integrals of the OCH3 signals. The α-
Man-(1Ǟ3)-Man 3 was acetylated with Ac2O/pyridine to
give the 2-O-Ac derivative 4 (99%), which was then hy-
drolyzed in aqueous 70% acetic acid (AcOH) to remove the
benzylidene acetal unit (93%). The resulting 4,6-diol 5 was
treated with tert-butyldiphenylsilyl chloride (TBDPSCl) in
Results and Discussion
First of all, we examined whether it is possible to synthe-
size a 3,4-dibranched Man using a 3-O-substituted Man as
an acceptor. As Scheme 1 shows, we prepared the acceptor
by coupling methyl 4,6-O-benzylidene-α--mannopyrano-
side (1)[20] and tetra-O-acetyl-α--mannopyranosyl trichlo-
roacetimidate (2).[21] Treatment of 1 with 1.1 molar equiva-
lents of 2 in CH2Cl2 in the presence of BF3·OEt2 for 2 h at
Ϫ10 °C and subsequent chromatographic purification of
the reaction mixture gave α-ManII-(1Ǟ3)-ManI 3 as a
major product (56%). The α-configuration of 3 was estab-
1
DMF to give the 4-OH acceptor 6 (93%). The H and 13C
NMR spectroscopic data of products 4Ϫ6 (Tables 1 and 2)
confirmed their structures.
Glycosylation of the 4-OH acceptor 6 with two molar
equivalents of hepta-O-acetyl-α-lactosyl trichloroacetimid-
ate (7)[25] using BF3·OEt2 as catalyst in CH2Cl2 for 2 h at
Ϫ10 °C, followed by chromatographic purification, gave the
desired 3,4-dibranched structure, β-lactosyl-(1Ǟ4)-[α-Man-
(1Ǟ3)]-α-Man 8 in 80% yield. The β-linkage of the Glc resi-
due was assigned from the 3J1,2 value (7.8 Hz; Table 1). The
downfield shifts of both C-4 (∆δ ϭ 4.3 ppm) and H-4 (∆δ ϭ
0.45 ppm) of the ManI residue (Tables 1 and 2) of 8 and
the HMBC experiment verified that the lactose was O-4-
linked to the ManI residue. The formation of the tetra-
saccharide containing the 3,4-di-O-substituted Man as a
major product showed that glycosylation of the 4-OH
group of the 3-O-substituted Man acceptor 6 is possible,
which demonstrated that the 3,4-gauche orientation of Man
would not disfavor the formation of the di-O-substituted
structure, in contrast to a previous finding by Oscarson et
al.[18]
1
lished from the JC-1,H-1 value[22Ϫ24] (168 Hz) of the ManII
After confirming that construction of the 3,4-dibranched
Man is possible using the 3-O-substituted Man 3 as an ac-
ceptor, we synthesized a 3,4-di-O-substituted Hep structure
expressed in 15253 LOS as follows: synthesis of α-Hep-
(1Ǟ3)-α-Man, lactosylation of the α-Hep-(1Ǟ3)-α-Man,
and conversion of the Man residue to the Hep residue
(Schemes 2 and 3).
As expected from the glycosylation results of 1 and 2,
treatment of 1 with 1.1 molar equivalents of 2,3,4,6,7-
penta-O-acetyl--glycero-α--manno-heptopyranosyl trichlo-
roacetimidate (9)[26] under conditions similar to those de-
scribed earlier gave the α-Hep-(1Ǟ3)-α-Man 10 in 70%
yield and the unreacted acceptor 1 (5%). Although a mix-
ture of two products, presumably Hep-(1Ǟ2)-Man and
Hep-(1Ǟ2)-[Hep-(1Ǟ3)]-Man were also obtained, we did
not carry out further structural characterization of these
two products. The structure of compound 10 was deter-
1
mined by the JC-1,H-1 value of the Hep residue (181 Hz)[18]
Scheme 1. a) BF3·OEt2, CH2Cl2, Ϫ10 °C, 56%; b) Ac2O, pyridine,
99%; c) AcOH (70% aq.), 60Ϫ70 °C, 93%; d) TBDPSCl, imidazole,
DMF, 0 °C Ǟ room temp., 93%; e) BF3·OEt2, CH2Cl2, Ϫ10 °C,
80%
and the shift of the 13C-3 satellite (Table 2) in a similar
manner described for 3.
Eur. J. Org. Chem. 2004, 1202Ϫ1213
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H. Kubo, K. Ishii, H. Koshino, K. Toubetto, K. Naruchi, R. Yamasaki
FULL PAPER
1
Table 1. H NMR spectroscopic data for compounds 1, 3Ϫ6, and 8
Compound[a]
Residue[b]
H-1
H-2
H-3
H-4
H-5
H-6a
H-6b
(3J1,2
)
(3J2,3
)
(3J3,4
)
(3J4,5
)
(3J5,6a
)
(3J5,6b
)
(2J6a,6b
)
1[c]
3[d]
4.76
(1.5)
4.77
(1.5)
5.13
(2.0)
4.67
(1.5)
5.19
(1.5)
4.68
(1.5)
5.19
(1.5)
4.65
(1.5)
5.20
(1.5)
4.70
(0.6)
5.10
(1.2)
4.84
(7.8)
4.49
(7.8)
4.05
(3.5)
4.06
n.d.
4.07
(9.0)
4.17
n.d.
5.38
(9.8)
4.30
(8.5)
5.19
(10.0)
4.06
(9.8)
5.21
(10.0)
4.06
(9.5)
5.23
(10.0)
4.13
(9.6)
5.24
3.92
(9.0)
4.17
n.d.
5.25
(9.6)
4.06
(9.5)
5.26
(10.0)
4.02
(9.0)
5.26
(10.0)
4.03
(9.8)
5.26
(10.0)
4.48
(9.6)
5.25
3.82
n.d.
3.82
n.d.
4.10
(2.0)
3.85
n.d.
4.08
n.d.
3.62
n.d.
4.08
(5.0)
3.57
n.d.
4.11
n.d.
3.50
n.d.
4.19
(1.8)
3.49
(6.0)
3.88
n.d.
3.83
n.d.
3.87
(4.4)
4.15
(6.4)
3.85
n.d.
4.11
(6.0)
3.88
n.d.
4.09
(6.5)
3.92
n.d.
4.10
(6.0)
3.87
(1.8)
4.13
(8.4)
4.25
(1.8)
4.06
(6.0)
4.29
n.d.
4.29
(10.4)
4.23
(12.2)
4.30
n.d.
4.28
(12.8)
3.88
n.d.
4.27
(13.0)
3.92
n.d.
4.27
(12.8)
3.99
(11.8)
4.24
(11.4)
4.46
ManI
ManII
Man I
ManII
ManI
ManII
ManI
ManII
ManI
ManII
Glc
5.43
(3.6)
5.33
(4.0)
5.35
(3.0)
5.20
(3.0)
5.32
(3.0)
5.18
(3.0)
5.36
(3.0)
5.23
(3.6)
5.39
n.d.
4[c]
5[c]
6[c]
8[e]
(10.2)
5.03
(9.6)
4.99
(10.2)
3.65
(9.6)
5.34
4.73
(9.6)
5.11
(11.8)
4.12
(11.8)
Gal
(10.2)
(3.0)
n.d.
[a] Data were acquired in CDCl3 at 20Ϫ23 °C. The 1H NMR spectroscopic chemical shifts (ppm) were determined by analyzing 2D NMR
spectroscopic data (DQF-COSY, HMQC and HMBC) comparatively, and the J couplings (Hz) were obtained by analyzing either the
[b]
DQF-COSY or 1D NMR spectra. The 1H NMR spectroscopic chemical shifts of other protons are listed in the Exp. Sect.
To
distinguish between the two Man residues, the one at the reducing end and the other at the non-reducing end are designated as ManI
[c]
[d]
[e]
and ManII, respectively. 500 MHz. 400 MHz. 600 MHz. n.d.: not determined.
Table 2. 13C NMR spectroscopic data for compounds 1, 3Ϫ6, and 8
sequent protection of the 6-OH group with TBDPSCl
(87%). The structures of 11Ϫ13 were confirmed by their
1H and 13C NMR spectroscopic data, which are shown in
Tables 3 and 4.
Glycosylation of the acceptor 13 with two molar equiva-
lents of the α-lactosyl trichloroacetimidate 7 (BF3·OEt2 in
CH2Cl2 for 3 h at Ϫ10 °C and then for 30 min at room
temperature) followed by flash column chromatography
gave the expected tetrasaccharide 14 in 59% yield and the
unreacted acceptor 13 (40%).
Compound[a] Residue[b]
C-1 C-2 C-3 C-4 C-5 C-6
1[c]
101.4 70.9 68.7 78.8 63.0 68.9
101.3 70.9 73.6 78.4 63.4 68.7
98.4 69.1 69.0 66.3 66.9 69.6
99.7 70.8 70.6 78.9 63.3 68.5
98.1 69.1 68.8 65.6 69.2 62.4
98.7 71.2 76.3 67.5 71.9 62.1
99.1 69.5 69.0 65.8 69.3 62.4
98.4 70.9 75.2 69.0 71.3 64.1
98.9 69.3 69.0 65.8 69.1 62.4
3[d]
4[c]
5[c]
6[c]
8[e]
ManI[e]
ManII[e]
ManI
ManII
ManI
ManII
ManI
ManII
ManI
ManII
Glc
Analysis of the HMQC spectrum (Figure 2, panels A and
98.6 71.5 72.1 73.3 71.8 60.9 B) identified the exo- and endocyclic protons and carbon
98.8 69.5 68.6 66.3 69.1 62.7
99.6 72.5 73.4 77.0 73.0 62.4
atoms of each residue of 14. Two sets of cross-relay peaks,
Hep H-1/Man C-3 and Man H-3/Hep C-1, Glc C-1/Man
101.3 69.0 71.1 66.7 70.5 60.7
Gal
H-4 and Man C-4/Glc H-1, in the HMBC spectrum (Fig-
[a] Data were acquired in CDCl3 at 20Ϫ23 °C. The 13C NMR spec-
troscopic chemical shifts (ppm) were determined by analyzing 2D
NMR spectroscopic data (DQF-COSY, HMQC and HMBC) com-
paratively. Only the data for the skeletal carbon atoms are pre-
sented, and those for other carbon atoms are listed in the Exp.
Sect. [b] The two Man residues are designated as ManI and ManII,
ure 2, panels C and D) confirmed that the lactosyl moiety
is linked to the 4-position of the Man residue. The J1,2
3
value (8 Hz) of the Glc residue (Table 3) verified that the
1
lactose is β-linked. In addition, comparison of the JC-1,H-1
values of α-Man-(1Ǟ3)-α-Man 3, α-Hep-(1Ǟ3)-α-Man 10,
[c]
[d]
[e]
1
as described in Table 1. 125 MHz. 100 MHz. The JH-1,C-1 and the tetrasaccharide 14 showed that the α-Hep residue
[e]
1
values are as follows: ManI, 168 Hz; ManII, 169 Hz. 150 MHz.
tends to give large JC-1,H-1 values (over 180 Hz) relative to
those of the α-Man residue (168 Ϫ175 Hz). In addition, the
1
The disaccharide 10 was converted into the 4-OH Man residue of 14 has the largest value of JC-1,H-1
acceptor 13 in a similar manner described earlier for 3 (175 Hz), which indicates that the degree of substitution
1
(Scheme 2): acetylation (89%), hydrolysis (88%), and sub- affects the JC-H,H-1 value.
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A Partial Oligosaccharide Expressed in Neisserial Lipooligosaccharide
FULL PAPER
Scheme 3. a) 1. MeOH/Et3N/water (2:1:1, v/v/v), room temp.; 2.
˚
BnBr, NaH, DMF, molecular sieves (4 A), DMF, 0 °C Ǟ room
Scheme 2. a) BF3·OEt2, CH2Cl2, Ϫ10 °C, 70%; b) Ac2O, DMAP,
pyridine, 89%; c) AcOH (70% aq.), 70 °C, 93%; d) TBDPSCl, imi-
dazole, DMF, room temp., 87%; e) BF3·OEt2, CH2Cl2, Ϫ10 °C Ǟ
room temp., 59%
temp., 69%; b) TBAF, THF, room temp. Ǟ 60 °C, 89%; c) 1.
(COCl)2, DMSO/THF, Ϫ60 °C, then Et3N; 2. CH2ϭCHMgBr,
THF, Ϫ70 °C, 78%; d) Ac2O, DMAP, pyridine, 82%; e) 1. OsO4/
NaIO4, Et2O/water (1:1, v/v), 30Ϫ35 °C; 2. NaBH4, DMF/MeOH
(2:1, v/v), 80%; f) 1. H2, Pd/C, EtOH/AcOH/water (7:5:5, v/v/v); 2.
Ac2O, DMAP, pyridine, 84%
The target compound, β-lactosyl-(1Ǟ4)-[α-Hep-(1Ǟ3)]-
α-Hep 17 was synthesized by converting the Man residue
of 14 to the Hep unit (Scheme 3) as described previously.[27]
Compound 14 was deacetylated in a mixture of triethyl- hyde with NaBH4, gave the tetrasaccharide 19 (80% yield)
amine/MeOH/water, and the resulting mixture was benzyl- having a Hep residue at the reducing end. Finally, hydro-
ated with NaH/benzyl bromide (BnBr) in DMF. Purifi- genation of 19 over Pd/C, followed by acetylation, provided
cation by flash column chromatography gave the perbenzy- the peracetylated β-lactosyl-(1Ǟ4)-[α-Hep-(1Ǟ3)]-α-Hep
lated product 15 in 69%. Although complete removal of the 20 in 84%. The structures of 17Ϫ20 were confirmed by their
TBDPS group of 15 did not occur when it was treated with NMR spectroscopic data, which are presented in Tables 5
tetrabutylammonium fluoride (TBAF) in THF for 2 days and 6. The stereochemistry of the C-6 center at the reducing
at room temperature, treatment of the reaction mixture at end of the Hep residue was determined as described pre-
60 °C for 14 h provided the de-O-silylated product 16 in viously to be the  form.[10,27,29]
89% yield. Oxidation of 16 with oxalyl chloride and di-
As described earlier, Oscarson et al. reported that glycos-
methyl sulfoxide (DMSO) in THF at Ϫ60 °C and sub- ylation of neither the 4-OH group of the 3-O-substituted
sequent Grignard reaction using vinylmagnesium bro- Hep nor the 3-OH group of the 4-O-substituted Hep was
mide[27,28] gave a tetrasaccharide 17 (78%) containing the successful,[18] and similar results had been experienced in
oct-7-enopyranoside moiety at the reducing end.
synthesizing the 3,4-di-O-substituted Man structure.[19] The
The presence of the vinyl function of 17 was confirmed poor glycosylation results were ascribed to steric factors
by the distinctive chemical shifts of the corresponding pro- arising from the gauche-oriented OH units at the 3 and 4
tons (H-7, H-8a, and H-8b) and carbons (C-7 and C-8) positions of the Hep moiety. Our present results clearly
(Tables 5 and 6) as reported previously.[27,29]
indicate, however, that the gauche-oriented secondary OH
Compound 17 was acetylated with acetic anhydride/pyri- groups do not prevent the formation of the di-O-substituted
dine and then oxidative cleavage of the acetate 18 with product. This observation is also supported by a paper[30]
OsO4/NaIO4, followed by reduction of the resulting alde- that appeared during the final stages of preparation of this
Eur. J. Org. Chem. 2004, 1202Ϫ1213
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H. Kubo, K. Ishii, H. Koshino, K. Toubetto, K. Naruchi, R. Yamasaki
FULL PAPER
1
Table 3. H NMR spectroscopic data for compounds 10Ϫ16
Compound[a]
Residue
H-1
H-2
H-3
H-4
H-5
H-6a
H-6b
H-7a
H-7b
(3J1,2
)
(3J2,3
)
(3J3,4
)
(3J4,5
)
(3J5,6a
)
(3J5,6b
)
(2J6a,6b
)
(3J6,7a
)
(3J6,7b
)
(2J7a,7b
)
10[b]
Man
Hep
Man
Hep
Man
Hep
Man
Hep
Man
Hep
Glc
4.76
(1.5)
5.39
(1.5)
4.67
(1.2)
5.30
(1.6)
4.65
(1.6)
5.34
n.d.
4.60
n.d.
5.43
n.d.
4.70
n.d.
5.22
(1.5)
4.83
(8.0)
4.48
(8.5)
4.55
n.d.
5.63
n.d.
4.79
(8.0)
4.25
(8.0)
4.47
n.d.
5.67
n.d.
4.48
(8.0)
4.26
(8.0)
4.05
(3.0)
5.42
(3.5)
5.32
(4.0)
5.39
(3.2)
5.21
n.d.
5.33
(2.8)
5.18
(2.5)
5.38
(3.0)
5.22
(3.5)
5.43
(2.5)
4.70
(9.5)
5.10
(10.0)
3.73
(3.5)
4.28
(2.5)
3.22
(8.0)
3.69
(10.0)
3.70
(3.0)
4.28
(3.0)
3.23
(8.5)
3.70
(9.5)
4.11
(10.0)
5.34
(10.5)
4.22
n.d.
5.16
(10.4)
4.02
n.d.
5.15
(10.4)
4.00
(10.0)
5.19
(10.0)
4.08
(10.0)
5.23
(10.5)
5.02
(9.5)
5.00
(3.5)
4.18
(10.0)
3.91
(9.0)
3.39
(9.0)
3.25
(3.0)
4.14
(10.0)
3.92
(9.5)
3.57
(8.5)
3.25
4.20
(9.5)
5.29
(9.5)
4.07
(9.2)
5.30
(10.2)
4.02
n.d.
5.31
(10.4)
4.04
(10.0)
5.32
(10.5)
4.51
(10.0)
5.32
(10.5)
3.64
(12.0)
5.34
n.d.
3.82
(10.0)
4.15
(1.5)
3.85
n.d.
4.20
n.d.
3.60
n.d.
4.23
(1.6)
3.55
n.d.
4.23
n.d.
3.52
n.d.
4.28
n.d.
3.49
n.d.
3.89
(7.5)
3.38
n.d.
3.84
n.d.
3.35
(4.5)
3.20
(5.0)
3.40
n.d.
3.78
n.d.
3.43
n.d.
3.22
(5.0)
3.88
(4.5)
5.22
Ϫ
3.85
n.d.
5.22
Ϫ
3.87
n.d.
5.25
Ϫ
3.91
(4.5)
5.25
Ϫ
3.87
(1.0)
5.22
Ϫ
4.26
(2.0)
4.05
(6.5)
3.75
n.d.
4.12
Ϫ
3.62
n.d.
3.30
(9.0)
3.68
n.d.
4.08
Ϫ
3.68
n.d.
3.30
(9.0)
4.28
(10.0)
Ϫ
4.24
(6.5)
4.24
n.d.
Ϫ
n.d.
11[c]
12[c]
13[b]
14[b]
4.30
n.d.
Ϫ
4.21
n.d.
4.29
(6.8)
Ϫ
3.87
n.d.
Ϫ
4.27
(6.4)
4.27
n.d.
Ϫ
n.d.
3.91
n.d.
Ϫ
4.23
n.d.
4.30
(7.0)
Ϫ
(11.0)
(11.0)
3.98
(10.0)
Ϫ
4.18
(7.0)
4.28
n.d.
Ϫ
4.45
(11.5)
4.13
(11.5)
4.22
n.d.
Ϫ
Gal
15[b]
Man
Hep
Glc
4.65
n.d.
4.24
(10.0)
3.86
(9.0)
3.88
n.d.
3.74
(6.5)
3.85
n.d.
Ϫ
n.d.
n.d.
3.78
(11.0)
3.52
(9.0)
3.90
n.d.
Ϫ
Gal
16[b]
Man
Hep
Glc
4.30
n.d.
4.24
(9.5)
3.88
(9.0)
3.87
n.d.
3.69
(6.5)
3.80
n.d.
Ϫ
3.79
n.d.
3.50
(9.0)
Gal
(3.5)
[a] Data were acquired in CDCl3 at 20Ϫ23 °C. The 1H NMR spectroscopic chemical shifts (ppm) were determined by analyzing 2D NMR
spectroscopic data (DQF-COSY, HMQC and HMBC) comparatively, and the J couplings (Hz) were obtained by analyzing either the
1
[b]
DQF-COSY or 1D NMR spectra. The H NMR spectroscopic chemical shifts of other protons are listed in the Exp. Sect. 500 MHz.
400 MHz. n.d.: not determined.
[c]
manuscript: a similar 3,4-dibranched structure, α-Hep- LOS as follows: (1) synthesis of a α-Hep-(1Ǟ3)-α-Man con-
(1Ǟ3)-[β-Glc-(1Ǟ4)]-Hep was constructed after synthesiz- taining a free 4-OH group at the Man residue; (2) β-lactos-
ing the 4-O-substituted Hep. The fact that glycosylation ylation of the 4-OH, followed by elongation of the Man to
using the α-Hep-(1Ǟ3)-α-Man acceptor 10 gave the corre- Hep by Swern oxidation, Grignard reaction, and oxidative
sponding tetrasaccharide in lower yield than the one with cleavage followed by NaBH4 reduction. Our approach,
4
the α-Man-(1Ǟ3)-α-Man acceptor 3 may indicate that the which uses a 3-O-substituted Man derivative of C1 confor-
orientation of the endocyclic carbon atoms of a Hep residue mation as an acceptor, should provide an alternative ap-
could lead to steric crowding, which would affect the out- proach to build the 3,4-di-O-substituted Hep structure ex-
come of the glycosylation reaction. Comparative confor- pressed in LOS produced by pathogenic Gram-negative
mational analyses of an O-3 or O-4-substituted Hep with bacteria.
those of the Man counterparts may provide some insights
into whether the presence of the extra exocyclic carbon
would lead to steric crowding.
Experimental Section
Thus, we synthesized the 3,4-dibranched Hep structure
present as a partial structure of the OS moiety in 15253 measured using a HORIBA SEPA-200 polarimeter and a YANAG-
General: Optical rotations and melting points (uncorrected) were
1206
 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
Eur. J. Org. Chem. 2004, 1202Ϫ1213

A Partial Oligosaccharide Expressed in Neisserial Lipooligosaccharide
FULL PAPER
heptopyranosyl trichloroacetimidate (9; [α]2D1 ϭ ϩ25.5 (c ϭ 1.0,
Table 4. 13C NMR spectroscopic data for compounds 10Ϫ16
CHCl3), {ref.[26] [α]2D0 ϭ ϩ20.0 (c ϭ 1.0, CHCl3)}).
Compound[a] Residue C-1
C-2 C-3 C-4 C-5 C-6 C-7
Methyl (2,3,4,6-Tetra-O-acetyl-α-
D-mannopyranosyl)-(1Ǟ3)-4,6-O-
benzylidene-α- -mannopyranoside (3): A solution of BF3·OEt2 in
D
10[b]
11[d]
12[b]
13[b]
14[b]
Man[c] 101.2 71.0 73.2 78.7 63.3 68.7
CH2Cl2 (0.8 , 1.0 mL, 0.80 mmol) was added dropwise at Ϫ10 °C
to a mixture of methyl 4,6-O-benzylidene-α--mannopyranoside (1;
1.00 g, 3.54 mmol) and 2,3,4,6-tetra-O-acetyl-α--mannopyranosyl
trichloroacetimidate (2; 1.95 g, 3.96 mmol) in CH2Cl2 (10 mL) con-
Hep[c]
Man
Hep
98.6 69.8 68.8 64.9 69.2 67.0 61.5
99.5 70.6 70.2 79.0 63.1 68.5
98.2 69.0 68.9 64.3 68.8 66.9 61.1
98.6 70.8 74.4 67.7 71.9 61.6
98.8 69.3 69.1 64.3 68.8 67.0 61.4
98.5 69.4 73.7 70.3 70.7 64.5
98.9 70.6 69.2 64.5 68.9 67.2 61.5
98.5 71.6 71.9 73.3 71.9 60.9
99.1 69.5 68.8 65.0 68.8 67.2 61.2
99.5 72.4 73.4 77.1 73.1 62.4
101.4 69.0 71.1 66.7 70.6 60.8
99.6 77.8 73.6 75.1 72.6 61.6
98.9 75.7 80.2 74.5 71.9 74.5 70.1
103.5 82.4 83.3 76.7 75.2 68.5
102.4 79.7 82.2 73.5 72.6 67.8
99.6 77.1 73.4 74.9 72.3 60.7
98.9 75.4 79.9 74.4 71.9 75.0 68.9
103.5 82.4 83.1 76.7 75.0 68.9
102.4 79.8 82.2 73.4 72.5 67.8
Man
Hep
Man
Hep
˚
taining 4-A molecular sieves (2.5 g). After stirring at Ϫ10 °C for
2 h, triethylamine was added to the reaction mixture, which was
then extracted with CH2Cl2. Flash column chromatography
(CH2Cl2/acetone, 23:2 Ǟ 9:1) gave the title compound 3 (1.21 g,
56%) as a syrup, which was crystallized from EtOAc/hexane. M.p.
Man[e]
Hep[e]
Glc[e]
Gal[e]
Man
Hep
219Ϫ220 °C. [α]2D2 ϭ ϩ60 (c ϭ 0.5, CHCl3). H NMR (400 MHz,
1
CDCl3; data for the ring and the exocyclic protons are listed in
Table 1): δ ϭ 1.99 (s, 3 H, COCH3), 2.05 (s, 3 H, COCH3), 2.09 (s,
3 H, COCH3), 2.10 (s, 3 H, COCH3), 3.39 (s, 3 H, OCH3), 5.59 (s,
1 H, CH-Ph), 7.41Ϫ7.43 (m, 5 H, aromatic H) ppm. 13C NMR
(100 MHz, CDCl3; data for the skeletal carbon atoms are listed in
Table 2): δ ϭ 20.56 (COCH3), 20.66 (COCH3), 20.71 (COCH3),
54.9 (OCH3), 101.4 (CH-Ph), 1256.0, 128.1, 128.8, 137.2 (aromatic
C), 169.65 (COCH3), 169.70 (COCH3), 170.1 (COCH3), 170.7
(COCH3) ppm. C28H36O15 (612.58): calcd. C 54.90, H 5.92; found
C 54.66, H 5.85.
15[b]
16[b]
Glc
Gal
Man
Hep
Glc
Gal
[a] Data were acquired in CDCl3 at 20Ϫ23 °C. The 13C NMR spec-
troscopic chemical shifts (ppm) were determined by analyzing 2D
NMR spectroscopic data (DQF-COSY, HMQC and HMBC) com-
paratively. Only the data for the skeletal carbon atoms are pre-
sented, and those for other carbon atoms are listed in the Exp.
Sect. [b] 125 MHz. [c] The 1JH-1,C-1 values of 10: Man, 171 Hz; Hep,
A minor product (200 mg) was found to be a mixture of the follow-
ing products by 2D NMR spectroscopy: methyl (2,3,4,6-tetra-O-
acetyl--mannopyranosyl)-(1Ǟ2)-4,6-O-benzylidene-α--manno-
pyranoside and methyl (2,3,4,6-tetra-O-acetyl--mannopyranosyl)-
(1Ǟ2)-[2,3,4,6-tetra-O-acetyl--mannopyranosyl-(1Ǟ3)]-4,6-O-
benzylidene-α--mannopyranoside (2.3% and 4.6%, respectively).
1H NMR (600 MHz, CDCl3): δ ϭ 1.93Ϫ2.15 (each s, COCH3),
7.32Ϫ7.50 (m, aromatic H) ppm; ManII-(1Ǟ2)-ManI: δ ϭ 3.39 (s,
3 H, OCH3), 3.78 (m, 1 H, H-5I), 3.90 (m, 1 H, H-4I), 4.03 (m, 1
H, H-2I), 4.10 (m, 1 H, H-5II), 4.13 (m, 1 H, H-3I), 4.15 (m, 1 H,
H-6IIa), 4.23 (m, 1 H, H-6IIb), 4.26 (m, 1 H, H-6Ia), 4.79 (d,
[d]
[e]
1
181 Hz.
100 MHz.
The JH-1,C-1 values of 14: Man, 175 Hz;
Hep, 182 Hz; Glc, 165 Hz; Gal, 161 Hz.
IMOTO micro melting point apparatus, respectively. High-resolu-
tion fast-atom bombardment mass spectrometry (HR-FABMS)
was carried out as described previously.[29] Elemental analyses were
performed using a PerkinϪElmer 2400 Series II CHNS/O Ana-
lyzer. NMR spectra [400 MHz (JEOL JNM-A400), 500 MHz
(JEOL JNM-ECP 500), and 600 MHz (JEOL JNM-A600)] were
recorded in CDCl3 using tetramethylsilane as the internal standard,
and 2D NMR spectroscopic data (DQF-COSY, HMQC, and
HMBC) were processed in a manner similar to that described pre-
viously.[7,27,29] Silica gel 60 (E. Merck) was used for flash column
(0.040Ϫ0.063 mm) and open column (0.063Ϫ0.200 mm) chroma-
tography. Silica gel 60 F254 (E. Merck) was used for thin-layer chro-
matography (TLC), and compounds were detected under UV light
or by spraying with 10% concd. sulfuric acid in methanol and then
heating the plates at 120 °C for 5 min. The - and -glycero--
manno-heptoses were analyzed by high-performance anion-ex-
change chromatography (HPAEC) as reported previously.[11,29]
Glycosylation reactions were carried out under argon using dry
solvents. The glycosylation mixture was filtered through Celite, and
the filtrate was washed with water, dried over MgSO4 and concen-
trated to a syrup under diminished pressure below 40 °C, unless
otherwise stated. Methyl α--mannoside was purchased from
Sigma Chemical Co., and the following compounds were prepared
according to the literature procedures: methyl 4,6-O-benzylidene-
α--mannopyranoside (1; m.p. 155Ϫ156 °C, [α]2D3 ϭ ϩ64 (c ϭ 2.0,
CHCl3), {ref.[31] m.p. 146Ϫ147 °C, [α]D ϭ ϩ64.3 (c ϭ 2.1,
CHCl3)}), 2,3,4,6-tetra-O-acetyl-α--mannopyranosyl trichlo-
3J1 ,21 ϭ 1.0 Hz, 1 H, H-1I), 5.13 (d, 3J1 II ϭ 2.0 Hz, 1 H, H-1II),
1
II
,2
5.25 (m, 1 H, H-4II), 5.38 (dd, 3J2 II ϭ 3.4 Hz, 3J3 II ϭ 10.3 Hz,
II
II
,3
,4
1 H, H-3II), 5.43 (m, 1 H, H-2II), 5.63 (s, 1 H, CH-Ph) ppm;
{ManII-(1Ǟ2)-[ManIII-(1Ǟ3)]-ManI}: δ ϭ 3.38 (s, 3 H, OCH3),
3.77 (m, 1 H, H-5I), 3.89 (m, 1 H, H-6Ia), 3.95 (m, 1 H, H-5III),
4.04 (m, 1 H, H-2I), 4.08 (m, 1 H, H-5II), 4.09 (m, 1 H, H-6IIIa),
4.15 (m, 1 H, H-6IIa), 4.17 (m, 1 H, H-4I), 4.25 (m, 1 H, H-6IIIb),
3
I I
4.26 (m, 2 H, H-3, H-6Ib), 4.27 (m, 1 H, H-6IIb), 4.74 (d, J1 ,2
ϭ
1.5 Hz, 1 H, H-1I), 5.18 (m, 1 H, H-4III), 5.19 (s, 1 H, H-1II), 5.21
(m, 1 H, H-3III), 5.27 (d, J1
ϭ 2.0 Hz, 1 H, H-1III), 5.29 (m,
3
III III
,2
III III
1 H, H-4II), 5.32 (dd, J2
ϭ 2.9 Hz, 1 H, H-2III), 5.42 (m, 1
3
,3
H, H-2II), 5.43 (m, 1 H, H-3II), 5.66 (s, 1 H, CH-Ph) ppm. 13C
NMR (150 MHz, CDCl3): δ ϭ 20.6Ϫ20.8 (COCH3), 126.0Ϫ137.2
(aromatic C), 169.3Ϫ170.6 (COCH3) ppm; ManII-(1Ǟ2)-ManI:
δ ϭ 55.0 (OCH3), 62.7 (C-6II), 63.4 (C-5I), 66.4 (C-4II), 68.51 (C-
6I), 68.7 (C-3I), 69.02 (C-5II), 69.04 (C-3II), 69.17 (C-2II), 78.3 (C-
2I), 78.9 (C-4I), 99.8 (C-1II), 100.5 (C-1I), 102.1 (CH-Ph) ppm;
{ManII-(1Ǟ2)-[ManIII-(1Ǟ3)]-ManI}: δ ϭ 54.8 (OCH3), 62.5 (C-
6III), 62.8 (C-6II), 63.8 (C-5I), 66.3 (C-4III), 66.5 (C-4II), 68.3 (2 C,
C-3III, C-6I), 68.51 (C-3II), 69.02 (C-5III), 69.04 (C-5II), 69.15 (C-
2II), 69.3 (C-2III), 77.1 (C-3I), 77.3 (C-2I), 78.8 (C-4I), 97.4 (C-1II),
98.2 (C-1III), 100.5 (C-1I), 101.3 (CH-Ph) ppm.
roacetimidate (2; [α]2D3 ϭ ϩ51 (c ϭ 1.0, CHCl3), {ref.[21] [α]2D3
ϩ53 (c ϭ 1.01, CHCl3)}), 2,3,4,6-tetra-O-acetyl-β--galactopyr-
anosyl-(1Ǟ4)-2,3,6-tri-O-acetyl-α--glucopyranosyl trichloroaceti-
ϭ
Methyl (2,3,4,6-Tetra-O-acetyl-α-
acetyl-4,6-O-benzylidene-α- -mannopyranoside (4): A solution of 3
(2.74 g, 4.47 mmol) in pyridine/Ac2O (3:1, v/v, 4 mL) was stirred at
D-mannopyranosyl)-(1Ǟ3)-2-O-
D
midate (7; [α]2D6 ϭ ϩ47.4 (c ϭ 4.2, CHCl3), {ref.[25] [α]D ϭ ϩ48 (c ϭ room temp. for 16 h. The solution was quenched with water
4.2, CHCl3)}), and 2,3,4,6,7-penta-O-acetyl--glycero-α--manno-
(20 mL), stirred for 1 h, and then extracted with CH2Cl2 (2 ϫ
Eur. J. Org. Chem. 2004, 1202Ϫ1213
www.eurjoc.org
 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1207

H. Kubo, K. Ishii, H. Koshino, K. Toubetto, K. Naruchi, R. Yamasaki
FULL PAPER
Figure 2. Partial HMQC (panels A and B) and HMBC (panels C and D) spectra of compound 14 recorded in CDCl3 at 25 °C; only
partial 13C/1H cross-peaks (A and B) and cross-relay peaks (C and D) are labeled; each Roman numeral in panels AϪD corresponds to
that of 14 as shown in Scheme 2
30 mL). The extracts were washed with aqueous saturated
NaHCO3 and water, dried (MgSO4), and filtered and then the fil-
Table 1): δ ϭ 1.99Ϫ2.17 (5 s, 15 H, 5 COCH3), 3.37 (s, 3 H, OCH3)
ppm. 13C NMR (125 MHz, CDCl3; data for the skeletal carbon
trate was concentrated. The residual syrup was purified by flash atoms are shown in Table 2): δ ϭ 20.6Ϫ20.8 (5 C, COCH3), 55.0
column chromatography (CH2Cl2/acetone, 10:1) to give the title
compound 4 (2.90 g, 99%) as a white solid, which was crystallized
from EtOAc/hexane. M.p. 176Ϫ177 °C. [α]2D3 ϭ ϩ51.1 (c ϭ 1.0,
CHCl3). 1H NMR (500 MHz, CDCl3; data for the ring and the
exocyclic protons are shown in Table 1): δ ϭ 1.97Ϫ2.24 (5 s, 15 H,
5 COCH3), 3.39 (s, 3 H, OCH3), 5.61 (s, 1 H, CH-Ph), 7.31Ϫ7.42
(m, 15 H, aromatic H) ppm. 13C NMR (125 MHz, CDCl3; data for
the skeletal carbon atoms are shown in Table 2): δ ϭ 20.5
(COCH3), 20.6 (COCH3), 20.71 (COCH3), 20.73 (2 C, 2 COCH3),
55.0 (OCH3), 101.3 (CH-Ph), 125.9Ϫ137.0 (aromatic C), 169.7
(COCH3), 169.8 (COCH3), 169.9 (COCH3), 170.4 (COCH3), 170.6
(COCH3) ppm. C30H38O16 (654.62): calcd. C 55.04, H 5.85; found
C 55.02, H 6.00.
(OCH3), 169.8 (COCH3), 170.2 (COCH3), 170.3 (COCH3), 170.4
(COCH3), 170.7 (COCH3) ppm. C23H34O16 (566.51): calcd. C
48.76, H 6.05; found C 48.58, H 6.06.
Methyl (2,3,4,6-Tetra-O-acetyl-α-
acetyl-6-O-tert-butyldiphenylsilyl-α-
D
-mannopyranosyl)-(1Ǟ3)-2-O-
D-mannopyranoside (6): A solu-
tion of tert-butyldiphenylsilyl chloride (1.20 g, 4.35 mmol) in DMF
(4 mL) was added at 0 °C to a mixture of compound 5 (2.05 g,
3.63 mmol) and imidazole (0.54 g, 7.98 mmol) in DMF (8 mL).
The mixture was stirred at 0 °C for 2 h and then at room temp. for
18 h. The mixture was quenched with water (50 mL) and extracted
with EtOAc (4 ϫ 50 mL). Purification by flash column chromatog-
raphy (hexane/EtOAc, 2:1 Ǟ 3:2) gave the title compound 6 (2.71 g,
93%) as a foam. [α]2D3 ϭ ϩ39.3 (c ϭ 1.0, CHCl3). 1H NMR
Methyl (2,3,4,6-Tetra-O-acetyl-α-
acetyl-α-
4.27 mmol) in aqueous 70% AcOH (AcOH/H2O, 7:3, v/v, 80 mL)
was stirred at 60Ϫ70 °C for 4 h. The reaction mixture was concen- H, 5 COCH3), 2.78 (d, J
D-mannopyranosyl)-(1Ǟ3)-2-O-
D-mannopyranoside (5): A solution of compound 4 (2.80 g, (500 MHz, CDCl3; data for the ring and the exocyclic protons are
shown in Table 1): δ ϭ 1.10 [s, 9 H, C(CH3)3], 1.99Ϫ2.16 (5 s, 15
3
ϭ 2.0 Hz, 1 H, 4-OH), 3.30 (s,
4-OH,H-4
trated by co-evaporating with toluene and MeOH. The residual oil
was diluted with EtOAc (100 mL), washed with aqueous saturated
NaHCO3 (20 mL) and brine (20 mL), dried (MgSO4), and concen-
trated. The residue was purified by flash column chromatography
(CH2Cl2/acetone, 4:1) to give the title compound 5 (2.06 g, 93%)
3 H, OCH3), 7.39Ϫ7.73 (m, 10 H, aromatic H) ppm. 13C NMR
(125 MHz, CDCl3; data for the skeletal carbon atoms are shown
in Table 2): δ ϭ 19.1 [C(CH3)3], 20.49 (COCH3), 20.58 (COCH3),
20.67 (COCH3), 20.74 (COCH3), 20.8 (COCH3), 26.8 [C(CH3)3],
54.7 (OCH3), 127.7Ϫ135.6 (aromatic C), 169.8 (COCH3), 169.9
(COCH3), 170.0 (COCH3), 170.4 (COCH3), 170.6 (COCH3) ppm.
C39H52O16 (804.92): calcd. C 58.20, H 6.51; found C 57.96, H 6.48.
as a foam. [α]2D3 ϭ ϩ58.3 (c ϭ 1.0, CHCl3). H NMR (500 MHz,
1
CDCl3; data for the ring and the exocyclic protons are shown in
1208
 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Eur. J. Org. Chem. 2004, 1202Ϫ1213

A Partial Oligosaccharide Expressed in Neisserial Lipooligosaccharide
FULL PAPER
1
Table 5. H NMR spectroscopic (500 MHz) data for compounds 17Ϫ20
Compound[a] Residue[b] H-1
H-2
H-3
H-4
H-5
H-6a
H-6b
H-7a
H-7b
H-8a
H-8b
(3J1,2
)
(3J2,3
)
(3J3,4
)
(3J4,5
)
(3J5,6a
)
(3J5,6b
)
(2J6a,6b
)
(3J6,7a
)
(3J6,7b
)
(2J7a,7b
)
(3J7,8a
)
(3J7,8b (2J8a,8b
) )
17
18
19
20
Oct
4.46
(1.5)
5.58
n.d.
4.62
(8.0)
4.27
(7.5)
4.58
(1.5)
5.50
3.68
4.13
4.37
3.35
4.55
Ϫ
4.08
Ϫ
3.72
n.d.
3.28
(9.0)
5.67
Ϫ
4.06
Ϫ
3.23
n.d.
3.24
(10.0) (10.5)
4.04
Ϫ
Ϫ
Ϫ
Ϫ
Ϫ
3.80
n.d.
3.49
(9.0)
Ϫ
Ϫ
Ϫ
Ϫ
3.63
(12.0)
3.48
5.63
(5.0)
3.68
(6.0)
Ϫ
Ϫ
3.79
n.d.
Ϫ
Ϫ
4.96
4.98
(3.0) (9.5) (10.0) n.d.[c]
(17.0) (11.0) n.d.
Hep
Glc
4.30
(3.0) (9.5) (9.0) n.d.
3.23 3.60 3.93 3.46
(9.0) (9.0) (9.0) n.d.
3.69
n.d.
3.69
3.92
4.24
3.77
(9.3)
Gal
3.23
(3.0) n.d.
3.92 4.22
3.87
3.21
(4.5)
3.46
Oct
5.70
Ϫ
3.64
(6.0)
Ϫ
Ϫ
3.77
n.d.
Ϫ
Ϫ
4.96
5.05
(3.0) (9.5) (9.5) n.d.
4.27 4.09 4.24 3.77
(16.5) (10.0) (1.5)
Hep
Glc
(Ͻ1.0) (3.5) (9.5) (9.5) n.d.
(9.5)
4.33
(7.5)
4.29
(7.5)
4.44
3.23
3.60
3.78
3.31
(8.5) (9.0) (9.3) (5.0)
Gal
3.72
(10.0) (2.5) n.d.
3.70 4.12 4.35
3.31
3.89
3.25
(5.0)
3.42
HepI
HepII
Glc
Ϫ
Ϫ
Ϫ
Ϫ
3.80
(10.5)
3.51
(9.0)
Ϫ
Ϫ
Ϫ
Ϫ
4.25
(11.5)
4.13
n.d.
3.25
n.d.
3.67
(7.0)
3.39
(5.0)
3.78
n.d.
(Ͻ1.0) (2.5) (10.0) (10.0) n.d.
5.58 4.30 3.92 4.24 3.78
(Ͻ1.0) n.d. (9.0) (9.0) n.d.
3.61 3.91 3.45
(9.0) (9.0) (9.0) (4.5)
(11.5)
(9.5)
4.08
Ϫ
4.59
(8.0)
4.28
(8.0)
4.68
(1.5)
5.30
(1.5)
4.38
(8.0)
4.49
(8.0)
3.24
3.71
n.d.
3.32
(9.0)
5.27
Ϫ
5.21
Ϫ
4.44
(3.0)
4.03
n.d.
Gal
3.71
n.d.
5.20
3.25
(2.5) n.d.
4.02 3.99
3.87
3.21
(4.5)
3.76
HepI
HepII
Glc
4.19
(3.0)
4.27
n.d.
4.32
(6.0)
4.17
n.d.
(3.5) (9.5) (9.5) n.d.
5.42 5.14 5.30 4.21
(3.5) (10.0) (10.0) n.d.
4.66 5.03 3.71 3.54
(9.5) (9.5) (9.5) (2.5)
(11.0)
n.d.
Gal
5.10
4.99
5.32
3.89
n.d.
(10.0) (3.5) n.d.
[a]
1
Data were acquired in CDCl3 at 25 °C. The H NMR spectroscopic chemical shifts (ppm) were determined by analyzing 2D NMR
spectroscopic data (DQF-COSY, HMQC and HMBC) comparatively, and the J couplings (Hz) were obtained by analyzing either the
1
[b]
DQF-COSY or 1D NMR spectra. The H NMR spectroscopic chemical shifts of other protons are listed in the Exp. Sect. The oct-
7-enopyranoside residue is expressed as Oct. The two heptoses are designated as HepI and HepII, as described previously.[8]
not determined.
n.d.:
[c]
Methyl (2,3,4,6-Tetra-O-acetyl-β-
tri-O-acetyl-β- -glucopyranosyl)-(1Ǟ4)-[2,3,4,6-tetra-O-acetyl-α-
mannopyranosyl-(1Ǟ3)]-2-O-acetyl-6-O-tert-butyldiphenylsilyl-α-
D
-galactopyranosyl)-(1Ǟ4)-(2,3,6-
(COCH3), 170.7 (COCH3) ppm. C65H86O33Si (1423.44): calcd. C
54.85, H 6.09; found C 54.66, H 5.85.
D
D
-
-
D
mannopyranoside (8): A solution of 10% BF3·OEt2 in CH2Cl2 Methyl (2,3,4,6,7-Penta-O-acetyl-L-glycero-α-D-manno-heptopyr-
(0.65 mmol, 1.64 mL) was added dropwise at Ϫ10 °C to a mixture anosyl)-(1Ǟ3)-4,6-O-benzylidene-α-
D-mannopyranoside (10):
A
of 6 (2.60 g, 3.23 mmol) and the donor 7 (5.04 g 6.45 mmol) in solution of 10% BF3·OEt2 in CH2Cl2 (400 µL, 0.16 mmol) was ad-
˚
CH2Cl2 (32 mL) containing 4-A molecular sieves (3 g). After stir-
ded at Ϫ10 °C to a stirred mixture of compound 9 (890 mg,
˚
ring at Ϫ10 °C for 2 h, the reaction mixture was treated with tri- 1.58 mmol), 1 (404 mg, 1.43 mmol), and 4-A molecular sieves (1 g)
ethylamine (0.2 mL) and then extracted with CH2Cl2 (50 mL). in dry CH2Cl2 (8 mL). After stirring at Ϫ10 °C for 2 h, the mixture
Purification by flash column chromatography (CH2Cl2/acetone, was quenched with triethylamine (3 drops) and then extracted with
19:1 Ǟ 23:2 Ǟ 10:1), followed by crystallization from CH2Cl2/hex-
ane, gave the title compound 8 (4.27 g, 80%). M.p. 114Ϫ115 °C. [α]
CH2Cl2 (50 mL). After purifying twice by flash column chromatog-
raphy (CH2Cl2/acetone, 10:1, toluene/acetone, 3:1), the title com-
23
1
ϭ ϩ40 (c ϭ 1.0, CHCl3). H NMR (600 MHz, CDCl3; data for pound 10 was obtained (685 mg, 70%) together with the unreacted
D
the ring and the exocyclic protons are shown in Table 1): δ ϭ 1.11
[s, 9 H, C(CH3)3] 1.88, 1.97, 1.98, 2.04, 2.06, 2.06, 2.07, 2.11, 2.15,
acceptor 1 (20 mg, 5%). Although a mixture (130 mg) of two ad-
ditional products, presumably Hep-(1Ǟ2)-Man and Hep-(1Ǟ2)-
2.18, 2.21 (s, 36 H, 12 COCH3), 3.30 (s, 3 H, OCH3), 7.39Ϫ7.80 [Hep-(1Ǟ3)]-Man was obtained, their structural characterization
(m, 10 H, aromatic H) ppm. 13C NMR (150 MHz, CDCl3; data for was not carried out any further. Compound 10: [α]2D4 ϭ ϩ45.4 (c ϭ
1
the skeletal carbon atoms are shown in Table 2): δ ϭ 19.4
0.50, CHCl3). H NMR (500 MHz, CDCl3; data for the ring and
[C(CH3)3], 20.5Ϫ21.1 (COCH3), 26.6 [C(CH3)3], 54.7 (OCH3) the exocyclic protons are shown in Table 3): δ ϭ 1.98 (s, 3 H,
127.5, 128.2, 129.8, 130.3, 132.1, 133.4, 135.2, 136.1 (aromatic C), COCH3), 2.02 (s, 3 H, COCH3), 2.06 (s, 3 H, COCH3), 2.09 (s, 3
169.3 (COCH3), 169.6 (COCH3), 169.7 (COCH3), 169.8 (COCH3), H, COCH3), 2.13, (s, 3 H, COCH3), 3.38 (s, 3 H, OCH3), 5.59 (s,
170.07 (COCH3), 170.14 (COCH3), 170.3 (COCH3), 170.4
1 H, CH-Ph), 7.27Ϫ7.41 (m, 5 H, aromatic H) ppm. 13C NMR
Eur. J. Org. Chem. 2004, 1202Ϫ1213
www.eurjoc.org
 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1209

H. Kubo, K. Ishii, H. Koshino, K. Toubetto, K. Naruchi, R. Yamasaki
FULL PAPER
EtOAc). [α]2D4 ϭ ϩ45.3 (c ϭ 1.0, CHCl3). 1H NMR (400 MHz,
CDCl3; data for the ring and the exocyclic protons are shown in
Table 3): δ ϭ 1.98 (s, 3 H, COCH3), 2.02 (s, 3 H, COCH3), 2.06 (s,
3 H, COCH3), 2.11 (s, 3 H, COCH3), 2.14 (s, 3 H, COCH3), 2.17
(s, 3 H, COCH3), 2.66 (br. s, 1 H, OH), 3.37 (s, 3 H, OCH3) ppm.
13C NMR (100 MHz, CDCl3; data for the skeletal carbon atoms
are shown in Table 4): δ ϭ 20.5 (COCH3), 20.6 (COCH3), 20.61
(COCH3), 20.62 (COCH3), 20.7 (COCH3), 20.8 (COCH3), 54.8
(OCH3), 169.5 (COCH3), 169.96 (COCH3), 170.00 (COCH3), 170.1
(COCH3), 170.2 (COCH3), 170.3 (COCH3) ppm. C26H38O18
(638.57): calcd. C 48.90, H 6.00; found C 48.64, H 5.95.
Table 6. 13C NMR spectroscopic (125 MHz) data for compounds
17Ϫ20
Compound[a] Residue[b] C-1 C-2 C-3 C-4 C-5 C-6 C-7 C-8
17
18
19
20
Oct
Hep
Glc
Gal
Oct
Hep
Glc
Gal
HepI
HepII
Glc
99.5 77.0 73.4 75.1 73.9 69.0 137.9 115.2
98.9 75.5 79.9 75.0 71.8 75.0 69.8
103.4 82.4 83.1 76.6 75.0 68.8
102.4 79.8 82.1 73.5 72.5 67.8
99.2 75.5 73.9 74.4 73.4 72.2 133.1 118.4
99.2 76.4 80.1 74.6 71.9 75.1 69.8
102.8 82.5 83.1 77.0 75.4 68.9
102.6 79.8 82.5 73.3 72.6 67.8
99.7 76.8 73.4 75.1 72.9 64.4 67.2
98.9 75.4 79.9 74.4 71.8 75.0 69.8
103.4 82.3 83.1 76.6 74.9 68.9
102.4 79.8 82.2 73.4 72.6 67.8
98.7 71.0 72.3 72.0 69.2 68.0 62.0
99.4 69.3 69.0 64.8 69.1 67.2 61.4
99.2 72.2 73.4 77.3 73.1 62.6
Methyl (2,3,4,6,7-Penta-O-acetyl-L-glycero-α-D-manno-heptopyrano-
syl)-(1Ǟ3)-2-O-acetyl-6-O-tert-butyldiphenylsilyl-α-
D
-manno-
pyranoside (13): Compound 12 (250 mg, 0.391 mmol) was treated
with tert-butyldiphenylsilyl chloride (122 µL, 0.469 mmol) in dry
DMF (2 mL) in the presence of imidazole (67 mg, 0.978 mmol) for
20 h at room temp. After terminating the reaction by adding water
(3 mL), the mixture was extracted with EtOAc (3 ϫ 10 mL). The
combined extracts were washed with brine, dried (MgSO4), and
concentrated to a residue that was purified by flash column chro-
matography (hexane/EtOAc, 2:1 Ǟ 1:1). The title compound 13
was obtained as a syrup (297 mg, 87%). [α]2D4 ϭ ϩ34.3 (c ϭ 1.0,
CHCl3). 1H NMR (500 MHz, CDCl3; data for the ring and the
exocyclic protons are shown in Table 3): δ ϭ 1.09 [s, 9 H, C(CH3)3],
1.98 (s, 3 H, COCH3), 2.05 (s, 3 H, COCH3), 2.07 (s, 3 H, COCH3),
2.14 (s, 3 H, COCH3), 2.17 (s, 3 H, COCH3), 2.17 (s, 3 H, COCH3),
Gal
HepI
HepII
Glc
Gal
101.4 69.1 71.2 66.7 70.6 60.8
[a]
Data were acquired in CDCl3 at 25 °C. The 13C NMR spectro-
scopic chemical shifts (ppm) were determined by analyzing 2D
NMR spectroscopic data (DQF-COSY, HMQC and HMBC) com-
paratively. Only the data for the skeletal carbon atoms are pre-
sented, and those for other carbon atoms are listed in the Exp.
Sect. [b] The octenopyranoside residue is expressed as Oct. The two
heptoses are designated as HepI and HepII, as described pre-
viously.[8]
2.59 (d, 3J
ϭ 3.5 Hz, 1 H, 4-OH), 3.28 (s, 3 H, OCH3),
4-OH,H-4
7.40Ϫ7.72 (m, 10 H, aromatic H) ppm. 13C NMR (125 MHz,
CDCl3; data for the skeletal carbon atoms are shown in Table 4):
δ ϭ 19.1 [C(CH3)3], 20.56 (COCH3), 20.63 (COCH3), 20.7
(COCH3), 20.8 (COCH3), 20.9 (COCH3), 26.8 [C(CH3)3], 54.8
(OCH3), 127.8, 127.90, 129.93, 130.1, 132.6, 132.9, 135.5, 135.6
(aromatic C), 169.8 (COCH3), 169.9 (COCH3), 170.0 (COCH3),
170.2 (COCH3), 170.4 (COCH3), 170.6 (COCH3) ppm.
C42H56O18Si (876.97): calcd. C 57.52, H 6.44; found C 57.30, H
6.45.
(125 MHz, CDCl3; data for the skeletal carbon atoms are shown
in Table 4): δ ϭ 20.6 (COCH3), 20.7 (COCH3), 20.8 (COCH3), 54.9
(OCH3), 101.4 (CH-Ph), 126.0, 128.1, 128.9, 137.2 (aromatic C),
169.5 (COCH3), 169.7 (COCH3), 170.2 (COCH3), 170.3 (COCH3),
170.4 (COCH3) ppm. C31H40O17 (684.64): calcd. C 54.38, H 5.89;
found C 54.55, H 6.15.
Methyl (2,3,4,6,7-Penta-O-acetyl-L-glycero-α-D-manno-heptopyr-
anosyl)-(1Ǟ3)-2-O-acetyl-4,6-O-benzylidene-α-
D
-mannopyranoside
Methyl (2,3,4,6-Tetra-O-acetyl-β-
tri-O-acetyl-β- -glucopyranosyl)-(1Ǟ4)-[2,3,4,6,7-penta-O-acetyl-
glycero-α- -manno-heptopyranosyl-(1Ǟ3)]-2-O-acetyl-6-O-tert-
butyldiphenylsilyl-α- -mannopyranoside (14): A solution of 10%
D-galactopyranosyl)-(1Ǟ4)-(2,3,6-
(11): Compound 10 (600 mg, 0.876 mmol) was acetylated with pyri-
dine/Ac2O (3:1, v/v, 2 mL) in the presence of a catalytic amount of
4-dimethylaminopyridine (DMAP) over 16 h. Usual workup and
subsequent purification by flash column chromatography (CH2Cl2/
acetone, 15:1) gave the title compound 11 (569 mg, 89%). M.p.
190Ϫ193 °C (recrystallized from hexane/EtOAc). [α]2D4 ϭ ϩ23.9
(c ϭ 1.0, CHCl3). 1H NMR (400 MHz, CDCl3; data for the ring
and the exocyclic protons are shown in Table 3): δ ϭ 1.96 (s, 3 H,
COCH3), 2.05 (s, 3 H, COCH3), 2.09 (s, 3 H, COCH3), 2.09 (s, 3
H, COCH3), 2.17 (s, 3 H, COCH3), 2.25 (s, 3 H, COCH3), 3.39 (s,
3 H, OCH3), 5.60 (s, 1 H, CH-Ph), 7.30Ϫ7.40 (m, 5 H, aromatic
H) ppm. 13C NMR (100 MHz, CDCl3; data for the skeletal carbon
atoms are shown in Table 4): δ ϭ 20.57 (COCH3), 20.62 (COCH3),
20.67 (COCH3), 20.71 (COCH3), 20.8 (COCH3), 54.9 (OCH3),
101.3 (CH-Ph), 125.8, 127.9, 128.8, 136.8 (aromatic C), 169.51
(COCH3), 169.54 (COCH3), 169.7 (COCH3), 169.9 (COCH3),
170.1 (COCH3), 170.2 (COCH3) ppm. C33H42O18 (726.68): calcd.
C 54.54, H 5.83; found C 54.44, H 5.82.
D
L-
D
D
BF3·OEt2 in CH2Cl2 (250 µL, 0.10 mmol) was added to a stirred
mixture of 13 (285 mg, 0.325 mmol), 7 (761 mg, 0.975 mmol), and
˚
4-A molecular sieves (1 g) in dry CH2Cl2 (7 mL) at Ϫ10 °C. After
stirring for 3 h at Ϫ10 °C and then for 30 min at room temp., the
reaction mixture was quenched with triethylamine (4 drops) and
then diluted with CH2Cl2 (40 mL). The mixture was worked up as
described for compound 8 and purified by flash column chroma-
tography (CH2Cl2/acetone, 15:1 Ǟ 10:1) to give the title compound
14 (288 mg, 59%) and the unreacted acceptor 13 (114 mg, 40%).
M.p. 186Ϫ188 °C (crystallized from EtOH). [α]2D0 ϭ ϩ35.6 (c ϭ
1.0, CHCl3). 1H NMR (500 MHz, CDCl3; data for the ring and
the exocyclic protons are shown in Table 3): δ ϭ 1.11 [s, 9 H,
C(CH3)3], 1.89 (s, 3 H, COCH3), 1.97 (s, 3 H, COCH3), 1.97 (s, 3
H, COCH3), 2.04 (s, 3 H, COCH3), 2.05 (s, 3 H, COCH3), 2.06 (s,
3 H, COCH3), 2.06 (s, 3 H, COCH3), 2.06 (s, 3 H, COCH3), 2.09
(s, 3 H, COCH3), 2.15 (s, 3 H, COCH3), 2.15 (s, 3 H, COCH3),
2.18 (s, 3 H, COCH3), 2.23 (s, 3 H, COCH3), 3.30 (s, 1 H, OCH3),
7.39Ϫ7.79 (m, 5 H, aromatic H) ppm. 13C NMR (125 MHz,
Methyl (2,3,4,6,7-Penta-O-acetyl-
anosyl)-(1Ǟ3)-2-O-acetyl-α- -mannopyranoside (12): A mixture of
compound 11 (460 mg, 0.633 mmol) in 70% AcOH (3.5 mL) was
L-glycero-α-D-manno-heptopyr-
D
stirred at 70 °C for 4 h. After cooling down, the mixture was con- CDCl3; data for the skeletal carbon atoms are shown in Table 4):
centrated to a syrup, which was purified by flash column chroma-
tography (CH2Cl2/acetone, 2:1) to give the title compound 12
(355 mg, 88%). M.p. 194Ϫ195 °C (recrystallized from hexane/
δ ϭ 19.5 [C(CH3)3], 20.5 (COCH3), 20.52 (COCH3), 20.57
(COCH3), 20.62 (COCH3), 20.66 (COCH3), 20.71 (COCH3), 20.8
(COCH3), 20.9 (COCH3), 21.2 (COCH3), 26.7 [C(CH3)3], 54.9
1210
 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
Eur. J. Org. Chem. 2004, 1202Ϫ1213

A Partial Oligosaccharide Expressed in Neisserial Lipooligosaccharide
FULL PAPER
(OCH3), 127.5, 128.1, 129.8, 130.2, 131.9, 133.3, 135.0, 136.0 (aro-
Ph), 4.63, 5.04 (d, 2J ϭ 10.5 Hz, 1 H each, CH2-Ph), 4.67Ϫ4.72 (m,
matic C), 169.05 (COCH3), 169.1 (COCH3), 169.47 (COCH3), 4 H, 2 CH2-Ph), 4.74 (s, 2 H, CH2-Ph), 7.03Ϫ7.31 (m, 65 H, aro-
169.53 (COCH3), 169.65 (COCH3), 169.70 (COCH3), 169.91
matic H) ppm. 13C NMR (125 MHz, CDCl3; data for the skeletal
(COCH3), 169.96 (COCH3), 169.98 (COCH3), 170.10 (COCH3), carbon atoms are shown in Table 4): δ ϭ 54.6 (OCH3), 71.7 (CH2-
170.11 (COCH3), 170.17 (COCH3) ppm. C65H86O33Si (1423.44):
calcd. C 54.58, H 6.13; found C 54.87, H 6.33.
Ph), 72.35 (CH2-Ph), 72.40 (CH2-Ph), 72.42 (CH2-Ph), 72.8 (CH2-
Ph), 72.9 (CH2-Ph), 73.1 (CH2-Ph), 73.3 (CH2-Ph), 74.4 (CH2-Ph),
74.6 (CH2-Ph), 75.2 (CH2-Ph), 75.3 (CH2-Ph), 75.4 (CH2-Ph),
Methyl (2,3,4,6-Tetra-O-benzyl-β-
tri-O-benzyl-β- -glucopyranosyl)-(1Ǟ4)-[2,3,4,6,7-penta-O-benzyl-
glycero-α- -manno-heptopyranosyl-(1Ǟ3)]-2-O-benzyl-6-O-tert-butyl-
diphenylsilyl-α- -mannopyranoside (15): Compound 14 (346 mg,
0.231 mmol) was treated with a mixture of MeOH/triethylamine/
H2O (2:1:1, v/v/v, 8 mL) for 24 h at room temp. The reaction mix- glycero-α-
D-galactopyranosyl)-(1Ǟ4)-(2,3,6-
137.9Ϫ139.5, 126.6Ϫ128.3 (aromatic C) ppm.
(1882.22): calcd. C 54.38, H 5.89; found C 54.55, H 6.15.
C117H124O22
D
L-
D
D
Methyl (2,3,4,6-Tetra-O-benzyl-β-
tri-O-benzyl-β- -glucopyranosyl)-(1Ǟ4)-[2,3,4,6,7-penta-O-benzyl-
-manno-heptopyranosyl-(1Ǟ3)]-2-O-benzyl-7,8-dideoxy-
α-D-manno-oct-7-enopyranoside (17): Dimethyl sulfoxide (197 µL,
D-galactopyranosyl)-(1Ǟ4)-(2,3,6-
D
L-
D
ture was concentrated, by co-evaporating with toluene and MeOH,
to a residue that was dried overnight in vacuo over P2O5. The dried
residue was benzylated with benzyl bromide (427 µL, 3.60 mmol)
and NaH (60% in paraffin liquid, 240 mg, 6.01 mmol) in dry DMF
2.78 mmol) in THF (3 mL) was added to a solution of oxalyl chlo-
ride (121 µL, 1.39 mmol) in THF (1 mL) at Ϫ60 °C. After stirring
the mixture for 20 min, a solution of 16 (262 mg, 0.139 mmol) in
dry THF (5 mL) was added at Ϫ60 °C. The reaction mixture was
stirred for 1 h at Ϫ60 °C and then for 2.5 h at Ϫ50 °C. After adding
˚
(8 mL) in the presence of 4-A molecular sieves (0.2 g) for 30 min
at 0 °C and then for 7 h at room temp. The reaction was quenched
with water (10 mL) and then filtered through Celite. The filtrate triethylamine (0.78 mL, 5.56 mmol), the mixture was warmed to
was extracted with Et2O (4 ϫ 20 mL) and then the combined ex-
tracts were washed with brine (4 ϫ 20 mL) and dried. Purification
by flash column chromatography (hexane/EtOAc, 10:1 Ǟ 5:1) gave
the title compound 15 as syrup (338 mg, 69%). [α]2D4 ϭ ϩ19.6 (c ϭ
1.0, CHCl3). 1H NMR (500 MHz, CDCl3; data for the ring and
the exocyclic protons are shown in Table 3): δ ϭ 0.98 [s, 9 H,
room temp. and stirred for 30 min. The mixture was then cooled
to Ϫ70 °C and vinylmagnesium bromide (1  in THF, 1.39 mL,
1.39 mmol) was added. The mixture was stirred for 2 h at Ϫ70 °C
and then for 1 h at Ϫ50 °C. The reaction was terminated by the
addition of EtOH (2 mL) and, after adding saturated aqueous
NH4Cl (10 mL), the mixture was warmed to room temp. The mix-
C(CH3)3], 3.20 (s, 3 H, OCH3), 4.21, 4.29 (d, 2J ϭ 12.0 Hz, 1 H ture was extracted with Et2O (3 ϫ 20 mL), and the combined ex-
each, CH2-Ph), 4.22, 4.39 (d, 2J ϭ 12.0 Hz, 1 H each, CH2-Ph), tracts were washed sequentially with 5% sodium hypochlorite in
2
4.34, 4.44 (d, 2J ϭ 11.0 Hz, 1 H each, CH2-Ph), 4.34, 4.87 (d, J ϭ
water (2 ϫ 20 mL) and brine (2 ϫ 20 mL) and then dried (MgSO4).
11.0 Hz, 1 H each, CH2-Ph), 4.46, 4.53 (d, 2J ϭ 11.5 Hz, 1 H each, Flash column chromatography (hexane/EtOAc, 4:1 Ǟ 3:1) gave the
CH2-Ph), 4.50, 4.74 (d, 2J ϭ 10.0 Hz, 1 H each, CH2-Ph), 4.51,
title compound 17 as a syrup (207 mg, 78%). [α]2D0 ϭ ϩ21.9 (c ϭ
4.82 (d, 2J ϭ 12.0 Hz, 1 H each, CH2-Ph), 4.51, 4.95 (d, 2J ϭ 1.0, CHCl3). 1H NMR (500 MHz, CDCl3; data for the ring and
11.5 Hz, 1 H each, CH2-Ph), 4.62Ϫ4.72 (m, 4 H, 2 CH2-Ph), 4.62, the exocyclic protons are shown in Table 5): δ ϭ 3.16 (s, 3 H,
4.74 (d, 2J ϭ 12.0 Hz, 1 H each, CH2-Ph), 4.70, 5.01 (d, 2J ϭ OCH3), 4.31, 4.22 (d, 2J ϭ 12.0 Hz, 1 H each, CH2-Ph), 4.42, 4.23
2
2
11.0 Hz, 1 H each, CH2-Ph), 4.75 (s, 2 H, CH2-Ph), 7.03Ϫ7.34 (m, (d, J ϭ 12.0 Hz, 1 H each, CH2-Ph), 4.52, 4.43 (d, J ϭ 12.0 Hz,
71 H, aromatic H), 7.71Ϫ7.75 (m, 4 H, aromatic H) ppm. 13C 1 H each, CH2-Ph), 4.67, 4.49 (d, 2J ϭ 11.0 Hz, 1 H each, CH2-
2
NMR (125 MHz, CDCl3; data for the skeletal carbon atoms are
Ph), 4.39Ϫ4.77 (m, 10 H, 5 CH2-Ph), 4.80, 4.49 (d, J ϭ 11.5 Hz,
shown in Table 4): δ ϭ 19.4 [C(CH3)3], 26.6 [C(CH3)3], 54.5 1 H each, CH2-Ph), 4.88, 4.34 (d, 2J ϭ 11.0 Hz, 1 H each, CH2-
2
(OCH3), 71.4 (CH2-Ph), 71.8 (CH2-Ph), 72.48 (CH2-Ph), 72.48
Ph), 4.93, 4.52 (d, J ϭ 12.0 Hz, 1 H each, CH2-Ph), 5.06, 4.67 (d,
(CH2-Ph), 72.50 (CH2-Ph), 72.7 (CH2-Ph), 72.8 (CH2-Ph), 73.0 2J ϭ 11.0 Hz, 1 H each, CH2-Ph), 7.03Ϫ7.31 (m, 65 H, aromatic
(CH2-Ph), 73.3 (CH2-Ph), 74.7 (CH2-Ph), 75.1 (CH2-Ph), 75.2 H) ppm. 13C NMR (125 MHz, CDCl3; data for the skeletal carbon
(CH2-Ph), 75.4 (CH2-Ph), 126.6Ϫ139.7 (aromatic C) ppm. atoms are shown in Table 6): δ ϭ 54.5 (OCH3), 71.4 (CH2-Ph), 71.8
C
133H142O22Si (2120.62): calcd. C 54.38, H 5.89; found C 54.55,
(CH2-Ph), 72.38 (CH2-Ph), 72.42 (CH2-Ph), 72.8 (CH2-Ph), 72.9
(CH2-Ph), 73.1 (CH2-Ph), 73.3 (CH2-Ph), 74.4 (CH2-Ph), 74.6
(CH2-Ph), 75.1 (CH2-Ph), 75.30 (CH2-Ph), 75.34 (CH2-Ph),
126.6Ϫ128.3, 137.8Ϫ139.6 (aromatic C) ppm. C119H126O22
(1908.26): calcd. C 74.90, H 6.66; found C 74.64, H 6.59.
H 6.15.
Methyl (2,3,4,6-Tetra-O-benzyl-β-D-galactopyranosyl)-(1Ǟ4)-(2,3,6-
tri-O-benzyl-β-
glycero-α- -manno-heptopyranosyl-(1Ǟ3)]-2-O-benzyl-α-
pyranoside (16): Compound 15 (205 mg, 0.097 mmol) was treated Methyl (2,3,4,6-Tetra-O-benzyl-β-
with tetrabutylammonium fluoride (1  in THF, 1.5 mL, 1.5 mmol) tri-O-benzyl-β- -glucopyranosyl)-(1Ǟ4)-[2,3,4,6,7-penta-O-benzyl-
-manno-heptopyranosyl-(1Ǟ3)]-6-O-acetyl-2-O-benzyl-
7,8-dideoxy-α- -manno-oct-7-enopyranoside (18): Compound 17
D-glucopyranosyl)-(1Ǟ4)-[2,3,4,6,7-penta-O-benzyl-
L-
D
D-manno-
D-galactopyranosyl)-(1Ǟ4)-(2,3,6-
D
L-
in dry THF (5 mL) for 2 days at room temp. and then at 60 °C for
14 h. The reaction mixture was diluted with water (5 mL) and then
extracted with Et2O (5 ϫ 10 mL). The combined extracts were
washed with brine and dried (MgSO4). Flash column chromatogra-
phy (hexane/EtOAc, 2:1) gave the title compound 16 as a syrup
(162 mg, 89%). [α]2D4 ϭ ϩ19.6 (c ϭ 1.0, CHCl3). 1H NMR
glycero-α-D
D
(454 mg, 0.24 mmol) was treated with pyridine/Ac2O (2:1, v/v,
3 mL) and a catalytic amount of DMAP for 15 h at room temp.
The mixture was co-evaporated with toluene to give a residue that
was diluted with CH2Cl2 (10 mL) and washed with water (5 mL).
(500 MHz, CDCl3; data for the ring and the exocyclic protons are The aqueous solution was extracted with CH2Cl2 (2 ϫ 5 mL). The
shown in Table 3): δ ϭ 2.00 (br. s, 1 H, 6-OH), 3.23 (s, 3 H, OCH3),
combined extracts were washed sequentially with 10% NaHCO3
2
4.22, 4.30 (d, 2J ϭ 11.5 Hz, 1 H each, CH2-Ph), 4.23, 4.44 (d, J ϭ (5 mL) and water (5 mL), and then dried (MgSO4). Purification by
2
11.5 Hz, 2 H, 1 H each, CH2-Ph), 4.36, 4.88 (d, J ϭ 11.0 Hz, 1 H flash column chromatography (hexane/EtOAc, 7:3) gave the title
each, CH2-Ph), 4.44, 4.52 (d, 2J ϭ 11.5 Hz, 1 H each, CH2-Ph), compound 18 (380 mg, 82%) as a syrup. [α]2D5 ϭ ϩ30 (c ϭ 1.0,
4.44Ϫ4.52 (m, 2 H, CH2-Ph), 4.48, 4.80 (d, 2J ϭ 12.0 Hz, 1 H each,
CHCl3). 1H NMR (500 MHz, CDCl3; data for the ring and the
CH2-Ph), 4.50, 4.68 (d, 2J ϭ 12.0 Hz, 1 H each, CH2-Ph), 4.52, exocyclic protons are shown in Table 5): δ ϭ 2.03 (s, 3 H, COCH3),
4.93 (d, 2J ϭ 11.5 Hz, 1 H each, CH2-Ph), 4.63Ϫ4.77 (m, 2 H, CH2-
3.21 (s, 3 H, OCH3), 4.18, 4.27 (d, J ϭ 12.0 Hz, 1 H each, CH2-
2
Eur. J. Org. Chem. 2004, 1202Ϫ1213
www.eurjoc.org
 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1211

H. Kubo, K. Ishii, H. Koshino, K. Toubetto, K. Naruchi, R. Yamasaki
FULL PAPER
2
Ph), 4.28, 4.36 (d, J ϭ 12.0 Hz, 1 H each, CH2-Ph), 4.36, 4.90 (d, overnight. The mixture was filtered through Celite and the filtrate
2J ϭ 11.5 Hz, 1 H each, CH2-Ph), 4.40, 4.43 (d, 2J ϭ 12.5 Hz, 1 H was concentrated to dryness. The residue was treated overnight at
each, CH2-Ph), 4.42, 4.53 (d, 2J ϭ 11.5 Hz, 1 H each, CH2-Ph), room temp. with pyridine/Ac2O (2:1, v/v, 0.9 mL) containing a
4.49, 4.80 (d, 2J ϭ 11.5 Hz, 1 H each, CH2-Ph), 4.52, 4.61 (d, J ϭ
catalytic amount of DMAP. The solution was co-evaporated with
2
11.0 Hz, 1 H each, CH2-Ph), 4.52, 4.93 (d, 2J ϭ 11.5 Hz, 1 H each, toluene to give a residue, which was purified by flash column chro-
CH2-Ph), 4.75Ϫ4.77 (m, 4 H, 2 CH2-Ph), 4.63, 4.68 (d, 2J ϭ
11.5 Hz, 1 H each, CH2-Ph), 4.71, 5.04 (d, 2J ϭ 11.0 Hz, 1 H each,
matography (EtOAc/hexane, 3:1) to yield the title compound 20
(32 mg, 84%). [α]2D4 ϭ ϩ8.0 (c ϭ 1.0, CHCl3), 1H NMR (500 MHz,
CH2-Ph), 4.75, 4.81 (d, 2J ϭ 11.0 Hz, 1 H each, CH2-Ph), CDCl3; data for the ring and the exocyclic protons are shown in
7.35Ϫ7.03 (m, 65 H, aromatic H) ppm. 13C NMR (125 MHz, Table 5): δ ϭ 1.98 (s, 3 H, COCH3), 2.01 (s, 3 H, COCH3), 2.04 (s,
CDCl3; data for the skeletal carbon atoms are shown in Table 6):
δ ϭ 21.1 (COCH3), 54.7 (OCH3), 71.4 (CH2-Ph), 72.0 (CH2-Ph),
3 H, COCH3), 2.05 (s, 3 H, COCH3), 2.07 (s, 3 H, COCH3), 2.08
(s, 3 H, COCH3), 2.09 (s, 3 H, COCH3), 2.14 (s, 3 H, COCH3),
72.4 (CH2-Ph), 72.5 (CH2-Ph), 72.6 (CH2-Ph), 72.89 (CH2-Ph), 2.15 (s, 3 H, COCH3), 2.18 (s, 3 H, COCH3), 2.21 (s, 3 H, COCH3),
72.90 (CH2-Ph), 73.3 (CH2-Ph), 74.4 (CH2-Ph), 74.6 (CH2-Ph), 2.23 (s, 3 H, COCH3), 3.34 (s, 3 H, OCH3) ppm. 13C NMR
75.2 (CH2-Ph), 75.2 (CH2-Ph), 75.3 (CH2-Ph), 128.3Ϫ126.7 (aro-
matic C), 137.9, 138.1, 138.27, 138.39, 138.44, 138.5, 138.7, 138.8
(125 MHz, CDCl3; data for the skeletal carbon atoms are listed in
Table 6): δ ϭ 20.59 (COCH3), 20.66 (COCH3), 20.69 (COCH3),
(2 C), 139.0, 139.1, 139.3, 139.4 (aromatic C), 170.1 (COCH3) ppm. 20.73 (COCH3), 20.77 (COCH3), 20.80 (COCH3), 20.9 (COCH3),
HR-FABMS: calcd. for C121H128O23Na [M ϩ Na]ϩ: 1971.8744;
found 1971.8718.
21.2 (COCH3), 169.4 (COCH3), 169.6 (COCH3), 169.9 (COCH3),
170.0 (COCH3), 170.2 (COCH3), 170.3 (COCH3), 170.4 (COCH3)
ppm. HR-FABMS: calcd. for C57H78O38Na [M ϩ Na]ϩ: 1393.4069;
found 1393.4102.
Methyl (2,3,4,6-Tetra-O-benzyl-β-
tri-O-benzyl-β- -glucopyranosyl)-(1Ǟ4)-[2,3,4,6,7-penta-O-benzyl-
-manno-heptopyranosyl-(1Ǟ3)]-6-O-acetyl-2-O-benzyl-
D-galactopyranosyl)-(1Ǟ4)-(2,3,6-
D
L
-
-
glycero-α-
D
D
L
glycero-α-
-manno-heptopyranoside (19): A solution of osmium
Acknowledgments
tetraoxide in H2O (1% w/v; 1 mL) containing sodium periodate
(0.32 g, 1.5 mmol) was added to a mixture of 18 (292 mg,
0.150 mmol) in Et2O/water (1:1, v/v, 8 mL). After stirring for 8 h
at room temp., sodium periodate (0.32 g, 1.5 mmol) was added, and
the reaction mixture was stirred vigorously for 3 days at 30Ϫ35 °C.
The mixture was extracted with Et2O (3 ϫ 10 mL) and the com-
bined extracts were then washed with water (2 ϫ 15 mL), dried
(MgSO4), and concentrated. After re-dissolving the residue in
DMF/MeOH (2:1, v/v, 15 mL), the mixture was treated with
NaBH4 (90 mg, 2.4 mmol). The mixture was diluted with Et2O
(15 mL) and washed with water (10 mL). The aqueous solution was
extracted with Et2O (2 ϫ 10 mL). Purification by flash column
chromatography (hexane/toluene/EtOAc, 2:2:1) gave the title com-
pound 19 (229 mg, 80%) as a syrup. [α]2D2 ϭ ϩ28 (c ϭ 1.0, CHCl3).
1H NMR (500 MHz, CDCl3; data for the ring and the exocyclic
protons are shown in Table 5): δ ϭ 3.18 (s, 3 H, OCH3), 4.41, 4.22
This work was supported by Grants-in-Aid for Scientific Research
(10306022 and 14360207) from the Ministry of Education of Japan.
We thank Masayuki Hosoe and Yasuaki Esumi for FABMS analy-
ses.
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D-galactopyranosyl)-(1Ǟ4)-(2,3,6-
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 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Eur. J. Org. Chem. 2004, 1202Ϫ1213

A Partial Oligosaccharide Expressed in Neisserial Lipooligosaccharide
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 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1213